Ambiguous Genitalia in the Newborn

ABSTRACT

Ambiguous genitalia in a newborn are the clinical sign of atypical sexual development of the external genitalia in utero. This condition is rare and can result from various underlying factors, including certain disorders with potentially severe consequences, such as cortisol deficiency due to congenital adrenal hyperplasia. Therefore, it is crucial to promptly determine etiology when ambiguity is observed. The formation of typical male or female external genitalia is a complex process involving a cascade of genetic and physiological events that begin with sex determination and progress through the differentiation of internal and external reproductive structures. When this process is disrupted and does not occur in the typical manner, it is referred to as a difference or disorder of sex development (DSD). Not all DSD cases present with ambiguous genitalia at birth; for example, complete androgen insensitivity syndrome does not, but all cases of ambiguous genitalia are the result of a DSD. This chapter focuses on genital ambiguity associated with DSD in newborns who have either a 46,XY or 46,XX chromosomal sex. However, DSD with genital ambiguity may also be observed in newborns with other combinations of sex chromosomes, such as 45,X/46,XY. The chapter offers a comprehensive overview of the evaluation and management of newborns with ambiguous genitalia. It emphasizes the importance of a structured medical assessment of the external genitalia to diagnose and determine the underlying cause of genital ambiguity. It includes tables for differential diagnosis and step-by-step workup algorithms to guide medical professionals in their evaluation. It also includes additional information on structured physical assessments of external genitalia and tables with normative values for hormonal measurements, which are recommended in the diagnostic process. As the etiology of genital ambiguity in newborns is diverse and can have significant implications for management, the authors stress that obtaining an accurate diagnosis through a professional medical workup is crucial. The chapter highlights the recommendation for newborns with ambiguous genitalia (DSD) to be cared for by highly specialized, interdisciplinary DSD teams. These teams are equipped with medical and psychosocial expertise, and specialized psychologists are available to support parents and caregivers. The chapter recognizes that having a child with ambiguous genitalia can be very stressful for parents. It underscores the importance of early education, access to expert care through DSD network teams, psychological support, shared decision-making, and promoting acceptance and inclusivity. By providing comprehensive support and guidance, families can better navigate the challenges and uncertainties they may encounter when caring for a newborn with ambiguous genitalia.

INTRODUCTION

Typical male and female development commences with the presence of the typical 46,XY or 46,XX chromosomes, which play a pivotal role in determining the indifferent gonads. Around the 6th week of gestation, these gonads follow pathways to develop into male-typical or female-typical gonads (Figure 1). This gonadal determination is intricately regulated by a complex interplay of multiple genes, which guide the male gonads to become testes and the female gonads to become ovaries.

Subsequently, the gonads initiate the production of various hormones, including anti-Müllerian hormone (AMH) and testosterone. These hormones are responsible for driving the differentiation of the embryologic Wolffian and Müllerian structures into sex-typical internal reproductive organs (Figure 1).

Figure 1. Typical male and female sex determination and differentiation occur during fetal life. The presence of the Y chromosome in the karyotype and the SRY gene (sex-determining region Y gene) are the primary determinants of male sex development. The SRY gene initiates a cascade that leads to the development of testes from bipotential gonads. Testosterone and Anti Mullerian Hormone (AMH) are secreted by testicular cells, which masculinize the Wolffian (mesonephric) duct and cause the regression of the Mullerian (paramesonephric) duct, respectively. Dihydrotestosterone (DHT), produced by the 5-alpha reduction of testosterone, plays a major role in male external genital development and prostate formation. The absence of the SRY gene on the Y chromosome is the primary factor for the induction of the female sex, and the development of the uterus, fallopian tubes, cervix and upper vagina from the Mullerian duct.

The development of external genitalia begins with a neutral anlage, including a genital tubercule, genital folds, and a urogenital sinus. These structures are subsequently differentiated into typical male external genitalia under the influence of androgens, particularly dihydrotestosterone (DHT). Conversely, for the development of typical female external genitalia, the absence of androgens is crucial to prevent virilization.

Therefore, ambiguous genitalia at birth can arise from either an excessive or insufficient androgen effect on the neutral external genital anlage in males or females, respectively (Figure 2).

Figure 2. External genital differentiation. The development of external genitalia begins with a bipotential anlage which includes genital tubercle, urethral fold, urethral groove, and genital swellings. In a typical 46, XY, androgen secretion from the testes triggers the fusion of urethral folds, allowing the enclosure of the urethral tube. This, together with the cells from the genital swelling, forms the shaft of the penis. Genital swellings fuse in the midline to allow formation of the scrotum and genital Tubercle expands to give rise to the glans penis. Female external genital development is ensured by the absence of testosterone, it is independent of ovarian endocrine activity. Urethral folds and genital swellings remain separate to form the labia minora and majora. Genital tubercle forms the clitoris.

CHARACTERISTICS OF TYPICAL MALE AND FEMALE EXTERNAL GENITALIA

A full-term male infant is typically expected to have bilateral testicles that are descended, complete formation of scrotal folds with midline fusion, and a typical-sized penis, which includes well-formed corporal bodies and a urethral meatus located at the tip. The average penile length for a full-term newborn is approximately 3.5 ± 0.4 cm, but this measurement can vary with gestational age (Table 1) (1, 2). If an infant presents with bilateral cryptorchidism, a bifid scrotum, hypospadias, or isolated penoscrotal hypospadias, further investigations for a DSD are recommended. However, it’s important to note that isolated micropenis, as long as both testes are descended and normal in size, is not typically considered a manifestation of ambiguous genitalia. Similarly, in males, distal hypospadias with no other atypical genital features usually does not indicate a DSD (3).

Table 1. Stretched Penile Length in Preterm and Term Newborns According to Gestational Age (2)
Gestational age (week)	Number of cases	Median (cm)	Mean (cm)	SD	Min (cm)	Max (cm)
26	30	1.9	1.9	0.32	1.1	2.5
27	31	2.1	2.0	0.28	1.1	2.6
28	32	2.0	1.9	0.31	1.3	2.9
29	29	2.3	2.3	0.39	1.3	2.9
30	31	2.4	2.4	0.32	1.6	3.0
31	33	2.4	2.4	0.38	1.8	3.3
32	32	2.9	2.6	0.44	2.0	3.2
33	28	2.9	2.8	0.45	1.9	3.4
34	30	2.9	2.8	0.43	2.0	3.6
35	29	3.1	2.9	0.61	2.0	4.1
36	31	3.1	3.0	0.50	2.2	4.0
37	29	3.1	3.0	0.47	2.2	4.1
38	44	3.1	3.1	0.54	2.1	4.5
39	82	3.2	3.2	0.55	2.0	4.2
40	65	3.5	3.4	0.53	2.2	4.4
41	29	3.6	3.5	0.47	2.6	4.3

A full-term female infant typically presents with bilateral separated labial folds, no palpable gonads, and distinct urethral and vaginal openings (Figure 2). The average clitoral length and width for a full-term infant girl are approximately 6.1 mm and 4.2 mm, respectively (1, 4). In cases where clitoromegaly (clitoral size ˃10 mm), labial fusion or palpable gonads are observed in otherwise typical female genitalia, further investigation for a DSD is warranted. It’s important to note that perceived clitoromegaly is not typically associated with an underlying DSD in the event that the newborn girl was born prematurely (1). Please be aware that perceived clitoromegaly or pseudo-clitoromegaly is often seen in preterm newborns, where the clitoral size falls within the normal range, but the labia majora cannot completely cover labia minora, creating a misleading impression of clitoromegaly. Genital edema may also be observed in sick preterm babies or cases of ovarian hyperstimulation, which can cause pseudo-clitoromegaly.

Table 2. Clitoral Length and Width in Preterm and Term Newborns According to Gestational Age (4)
Gestational age (week)	Number of cases	Measure	Mean (mm)	+1 SD (mm)	+2 SD (mm)	+3 SD (mm)
28-30	30	Width	3.28	3.87	4.46	5.05
28-30	30	Length	5.03	5.91	6.79	7.67
30-32	57	Width	3.32	3.68	4.04	4.4
30-32	57	Length	5.03	5.52	6.01	6.5
32-34	74	Width	3.41	3.77	4.13	4.49
32-34	74	Length	5.14	5.65	6.16	6.67
34-36	104	Width	3.69	4.17	4.65	5.13
34-36	104	Length	5.53	6.12	6.71	7.3
36-38	128	Width	4.08	4.5	4.92	5.34
36-38	128	Length	5.83	6.29	6.75	7.21
>38	187	Width	4.21	4.64	5.07	5.5
>38	187	Length	6.11	6.49	6.87	7.25

GENERAL THOUGHTS ON AMBIGUOUS GENITALIA AT BIRTH

The reported incidence of ambiguous genitalia in newborns is approximately one in 4,500. It is essential that a newborn with ambiguous genitalia and their parents are promptly referred to a specialized center for evaluation. This evaluation should be carried out by a multi-disciplinary team specializing in DSD in accordance with international recommendations on diagnostic and therapeutic strategies. This team should provide holistic care and treatment guidance, collaborating closely with relevant subspecialists and peer support groups (5). Key subspecialties involved in DSD teams typically include psychology, pediatric endocrinology, urology, neonatology, gynecology, andrology, nursing, social work, genetics, and medical ethics. It's worth noting that certain aspects of DSD management remain contentious or uncertain, resulting in recent changes in clinical practice that are ongoing. Notably, genital surgery has been banned in several countries, and it is increasingly avoided in young children to protect their rights to an open future and the integrity of their bodies.

Consequently, we can anticipate a growing number of children growing up with atypical-looking genitalia, requiring a careful assessment of the psychological impact (6). Providing psychological support to affected families as a standard component of care from the outset is, therefore, of utmost importance. Issues related to gender that may arise in the context of ambiguous genitalia should be openly and thoughtfully discussed.

A comprehensive evaluation of ambiguous genitalia in a newborn is essential for understanding the underlying cause and potential consequences. On one hand, this evaluation can lead to the detection of concomitant, treatable disorders, such as adrenal insufficiency that requires cortisol replacement. Also, it is important to note that in up to 30% of children with a Difference/Disorder of Sex Development (DSD), other organ system disorders may be identified, necessitating medical care and qualifying as syndromic DSD (7, 8). On the other hand, identifying the underlying cause of ambiguous genitalia forms the basis for making an informed decision regarding sex registration at birth, if necessary and desired by the parents. Gender assignment/sex registration at birth should take into account long-term satisfaction with the sex of rearing, sexual function, and fertility potential (1, 9, 10). Ideally, it should result from a shared decision-making process involving the child's parents and the DSD network team. This decision should be based on the individual's phenotype/genotype, existing knowledge from literature, databases, and patient representative experience (11-13). This process may require time, as parents need to grasp the complexity of their child's unique biology in order to participate in this decision. As a first step, the overall health of the child should be assessed, and parents should be educated about typical sexual development before explanations regarding the underlying cause and its implications for their child's ambiguous genitalia are provided. Even if the exact details of the underlying cause remain unresolved, it is essential to communicate what is known and what remains uncertain to the parents and make plans for the family's future in the best interests of the child.

Several websites are available for patients and their families to exchange information, share coping strategies, and discuss decision-making processes that can enhance outcomes for newborns with ambiguous genitalia. Such resources can be found on websites supported by organizations like the Accord Alliance (www.accordalliance.org), DSD families (www.dsdfamilies.org), AIS DSD Support Group (www.aisdsd.org), and the CARES Foundation (www.caresfoundation.org). However, the most current information will always be available from the responsible DSD team, which will also be aware of country-specific support services.

GENETICS OF SEX DETERMINATION AND DIFFERENTIATION

Sex development is a sequential process that involves the coordinated action of numerous genes and pathways. This process culminates in the development of functional gonads, differentiated internal sex organs, external genitalia, and typical secondary sexual characteristics that manifest after puberty. Prenatal sex development can be divided into two distinct processes: sex determination and sex differentiation (Figure 3). Sex determination is the initial step that occurs shortly after conception, approximately at 6-7 weeks gestation. During this phase, the undifferentiated gonads evolve into either testes or ovaries. By contrast, sex differentiation occurs subsequently and involves hormones produced after the formation of the sex-specific gonads. These hormones play a critical role in shaping the further development of the dependent embryonic structures, leading to the formation of male and female internal and external phenotypes.

Figure 3. The genes and transcription factors required for gonadal development. The formation of the bipotential gonad requires at least seven genes including NR5A1 (Nuclear Receptor Subfamily 5 Group A Member 1), WT1 (WilmsTumor1 Transcription Factor), LHX9 (LIM Homeobox 9), EMX2 (Empty Spiracles Homeobox 2), CBX2 (Chromobox 2), PBX1 (Pre-B-Cell Leukemia Transcription Factor 1) and GATA4 (GATA Binding Protein 4). Adapted from (14).

The Male Specific Pathway

In the male specific pathway, the expression of the SRY gene in pre-Sertoli cells serves as a pivotal switch that directs the fate of the gonads toward testicular development. SRY, in conjunction with NR5A1, activates the SOX9 signaling pathway, initiating testis development (Figure 3). Other important genes for the early testis development are NR0B1(Nuclear Receptor subfamily 0 group B member 1), AMH (Anti-Mullerian Hormone) and GATA4. AMH secreted by Sertoli cells, plays a crucial role in promoting the regression of Müllerian structures and supporting the development of Leydig cells in the testes. Subsequently, androgens produced by the testes facilitate the development of male internal organs (epididymis, vas deferens, seminal vesicle) and external genitalia (penis, scrotum). The production of sex steroids relies on an intact steroidogenic pathway, with all steroid hormones originating from cholesterol. The initial steps of sex steroid biosynthesis overlap with the synthesis of mineralo-corticosteroids and gluco-corticosteroids (Figure 4). It is important to note that certain steroid disorders, often referred to as congenital adrenal hyperplasia (CAH), can affect both adrenal and gonadal steroidogenesis or result in androgen excess or deficiency that may impact fetal genital development. INSL3, produced in Leydig cells, plays a role in guiding the descent of the testes from the abdomen to the scrotal folds.

The process of masculinization of initially sex-neutral, undifferentiated external genitalia begins around 8-9 weeks of gestation when the potent androgen DHT (dihydrotestosterone) is produced by the testes. DHT is crucial for the fusion of the urethral and labioscrotal folds, elongation of the genital tubercle, regression of the urogenital sinus, and the development of the prostate (15). The enzyme 5α-reductase is responsible for converting testosterone produced by fetal Leydig cells into DHT. Complete masculinization of the external genitalia through DHT is typically achieved by week 14 of gestation (16).

Figure 4. Steroidogenesis of the adrenals and gonads. Adrenal and gonadal steroidogenesis starting from cholesterol and showing key steroids and intermediates of the classical and alternative pathways is illustrated. Cortisol and androgen biosynthesis are under control of hypothalamic-pituitary-adrenal (HPA) axis in a negative feedback loop. Androgen synthesis occurs via classical or aberrant and alternative pathways (i.e., backdoor pathway and 11oxC19 androgen pathway). Aberrant or alternative pathways are particularly active whenever cortisol with or without aldosterone biosynthesis is impaired and androgenic precursors accumulate proximal to enzymatic block. Abbreviations: StAR; Steroidogenic Acute Regulatory Protein defect, CYP11A1; P450 side-chain cleavage deficiency, CYP21A2; 21α-hydroxylase, HSD3B2; 3β-hydroxy steroid dehydrogenase 2, CYP11B1; 11β-hydroxylase, CYP11B2; Aldosterone synthase, DOC; 11-deoxycorticosterone, DHEA; Dehydroepiandrosterone, A4; Androstenedione, T; Testosterone, CYP17A1; 17α-hydroxylase/17,20-lyase, b5; CYP19A1; Cytochrome P450 aromatase, Cytochrome b5, POR; P450 oxidoreductase, 17β-HSD; 17β-hydroxysteroid oxidoreductase or 17-ketosteroid reductase; SRDA1/2; Steroid 5α -reductase 1/2, 11oxC19; 11-oxygenated 19-carbon (11oxC19) steroids; Fdx; Ferredoxin, FdR; Ferredoxin reductase, 11βOHA; 11β-hydroxyandrostenedione, 11βOHT; 11β-hydroxytestosterone, Pdione; 5α-Pregnane- 17α-ol-3,20-dione, Pdiol; 5α-Pregnane- 3α,17α-diol 20-one, AKR1C2,4; Aldo-Keto Reductase Family 1 Member C2,4, DHT; Dihydrotestosterone, HSD17B5,3,6= 17β-hydroxysteroid dehydrogenase 5,3,6. Adapted from (17).

The Female Specific Pathway

By contrast, the female-specific pathway is initiated in the absence of SRY and requires the suppression of SOX9 (Figure 3). Activation of Rspo1/Wnt4/b-catenin and Foxl2 (Forkhead Box L2) signaling pathways leads to the formation of typical ovaries. The absence of AMH allows the Müllerian structures to proliferate, resulting in the formation of the typical fallopian tube, uterus, cervix and the upper part of the vagina. In the absence of both testosterone secretion and 5α-reductase activity, the sex-neutral external genitalia develop along the female pathway. Specifically, without DHT, the labioscrotal and urethral folds give rise to the labia majora and minora, respectively. The genital tubercle develops into a clitoris, and the urogenital sinus contributes to the formation of the urethral opening and anterior portion of the vagina (16).

Any disruption in the intricate genetic and hormonal processes involved can lead to atypical sex development and result in atypical external genitalia at birth. Figure 3 provides a summary of these processes and highlights some key genes and hormones essential for typical development.

DIFFERENCES/DISORDERS OF SEX DEVELOPMENT (DSD)

The discordance between genetic, gonadal, or anatomic sex is commonly referred to as DSD. In addition to candidate gene testing, the new era of molecular diagnostic tools, including whole exome/genome sequencing has uncovered numerous novel molecular etiologies in recent years. Accurate molecular diagnosis aids in managing affected individuals and provides families with information concerning prognosis and the risk of recurrence. This information is often sourced from literature and comprehensive data registries, such as the I-DSD (18-24). For clinical purposes, DSD (including ambiguous genitalia in newborns) is typically classified based on the affected individual's karyotype. The consensus statement addressing the approach and care of DSD (originally published in 2006 and updated in 2016) suggests the following broad classification for DSD: (A) Sex Chromosome DSD, (B) 46,XY DSD, and (C) 46,XX DSD (Table 3) (1, 20).

Table 3. DSD Classification According to the Chicago Consensus.
Sex Chromosome DSD	46,XY DSD	46,XX DSD
A. 45,X0 (Turner syndrome and variants)	A. Disorders of gonadal (testicular) development Complete gonadal dysgenesis (Swyer syndrome) or partial gonadal dysgenesis (e.g., SRY,SOX9, NR5A1, WT1, DHHgene mutations etc) Testicular regression (e.g., DHX37 gene mutation) Ovo-Testicular DSD	A. Disorder of gonadal (ovarian) development Ovo-Testicular DSD Testicular DSD (SRY (+)) Gonadal dysgenesis
B. 47,XXY (Klinefelter syndrome and variants)	B. Disorders of testicular hormone production or action Impaired testosterone production LH receptor mutations CAH (3βHSD2 deficiency, 17OHD, POR, StAR, CYP11A1 deficiency) HSD17B3 deficiency Impaired testosterone action 5α-reductase deficiency Androgen insensitivity syndrome (Complete/partial) Impaired AMH production or action Persistent Mullerian Duct Syndrome (AMH and AMHR2gene mutations)	B. Androgen excess Fetal CAH (21-hydroxylase, 11β-hydroxylase, 3βHSD2 deficiency) Glucocorticoid resistance Feto-placental Aromatase deficiency POR deficiency Maternal Luteoma, hilar cell tumors, arrhenoblastoma, lipoid cell tumors, Krukenberg tumors, androgen producing adrenal tumors, External androgen exposure
C. 45,X0/46,XY (Mixed gonadal dysgenesis, ovotesticular DSD)	C. Other Syndromic Smith-Lemli-Opitz, Cloacal anomaly, Aarskog, Robinow, Meckel, Joubert, Hand-Foot-Genital, popliteal pterygium, CHARGE, VATER/VACTERL, IMAGe etc. Drugs Flutamide, ketoconazole, progestagens Endocrine disruptors Phthalate, BPA, paraben Cryptorchidism INSL3, GREAT gene mutations etc. Isolated hypospadias MAMLD1, HOXA4 gene mutations etc.	C. Other Syndromic Cloacal anomaly etc. Mullerian anomalies Mayer-Rokitanski-Küster-Hauser, MURCS, McKusick-Kaufmnann syndrome and variants, MODY5
D. 46,XX/46,XY (chimeric, ovotesticular DSD)

Abbreviations: StAR; Steroidogenic Acute Regulatory Protein defect, CYP11A1; P450 side-chain cleavage deficiency, 3βHSD2; 3β-hydroxy steroid dehydrogenase 2, CYP17A1; 17α-hydroxylase/17,20-lyase deficiency, 17OHD; 17α-hydroxylase/17,20-lyase deficiency, POR; P450 oxidoreductase, HSD17B3; 17β-hydroxysteroid oxidoreductase or 17-ketosteroid reductase; SF1/NR5A1; steroidogenic factor 1.

The 46,XY Newborn With Ambiguous Genitalia

Ambiguous genitalia in a 46XY newborn can result from abnormal formation of the early fetal testes (testicular dysgenesis), reduced production of testosterone or dihydrotestosterone (5α-reductase deficiency), or the inability to respond to androgens (androgen insensitivity syndrome, or AIS) (Table 3). Depending on the extent of androgen production defect or resistance, affected newborns may exhibit a range of external genitalia phenotype, varying from those that appear typically female to those that appear typically male but with a small phallus, hypospadias, and a bifid scrotum, with or without palpable testes. Here we focus primarily on causes that lead to ambiguous genitalia. Broadly, four groups of underlying causes of 46, XY DSD with ambiguous genitalia can be identified: (a) partial gonadal dysgenesis, (b) partial testosterone biosynthetic defects, (c) partial 5α-reductase deficiency, and (d) partial androgen insensitivity syndrome or PAIS.

PARTIAL GONADAL DYSGENESIS

In cases of partial gonadal dysgenesis, it is presumed that a gene mutation leads to a partial abnormality in the development of the early urogenital ridge or gonadal anlage. This category of DSD can also involve mutations in genes such as SRY and SOX9, which are essential for the differentiation of bipotential gonads into testes (Figure 3). Partial gonadal dysgenesis is typically associated with incomplete masculinization of the external genitalia, along with varying degrees of maintenance of Wolffian ducts and inhibition of Müllerian ducts. For a summary of known genetic variations associated with gonadal dysgenesis, please refer to Table 4.

Leydig cell aplasia or hypoplasia represents a variation of gonadal dysgenesis, characterized by inadequate Leydig cell differentiation in the male gonad, resulting in impaired androgen production. Mutations in the LH receptor gene (LHCGR) as well as polymorphisms have been associated with this condition (25). The phenotype associated with Leydig cell hypoplasia includes incomplete masculinization of the external genitalia, along with incomplete development and maintenance of the Wolffian ducts.

Table 4. Monogenic Disorders of Gonadal Development/Differentiation and Associated DSD Presentations in Humans, Both 46,XY and 46,XX
Gene defect	Locus	Mode of transmission	Clinical presentation			Reference
			DSD phenotype		Other
			46, XX	46, XY
ARX	Xp22.13	XL		Testicular dysgenesis	Lissencephaly, epilepsy, intellectual disability temperature instability	(26)
ATRX	Xq13.3	XL		Testicular dysgenesis	Dysmorphic features, intellectual disability, α-thalassemia	(27)
CBX2	17q25	AR	Ovarian dysgenesis	Testicular dysgenesis		(28)
DHH	12q13.12	AR		Testicular dysgenesis	Minifascicular neuropathy	(29)
DMRT1/DMRT2	9p24.3	AD	Ovarian dysgenesis	Testicular dysgenesis	Hypotonia, developmental delay, impaired intellectual development	(30)
DHX37	12q24.31	AD		Testicular dysgenesis, testicular regression		(31)
EMX2	10q26.11		Ovarian dysgenesis	Testicular dysgenesis	Intellectual disability, kidney agenesis	(32)
ESR2	14q23.2-q23.3	AD,?	Ovarian dysgenesis	Testicular dysgenesis	Dysmorphic features, eye abnormalities, anal atresia, rectovestibular fistula	(33)
FGFR2	10q26.13	AD		Testicular dysgenesis, ovotesticular	Craniosynostosis	(34)
GATA4	8p23.1	AD		Testicular dysgenesis	Congenital heart defects (atrial septum defects, ventricular septum defects, tetralogy of Fallot), diaphragmatic hernia	(35)
HHAT	1q32.2	AR		Testicular dysgenesis	Nivelon-Nivelon-Mabille syndrome. Dwarfism, chondrodysplasia, narrow, bell-shaped thorax, micromelia, brachydactyly, microcephaly with cerebellar vermis hypoplasia, facial anomalies, hypoplastic irides and coloboma of the optic discs	(36)
LHX9	1q31.3	AD		Testicular dysgenesis	Limb anomalies	(37)
MAMLD1	Xq28	XL		Severe hypospadias	Myotubular myopathy	(38)
MAP3K1	5q11.2	AD		Testicular dysgenesis		(39)
NR0B1 (Duplications, inversion and upstream deletion)	X21.3	XL		Testicular dysgenesis	Congenital adrenal hypoplasia, hypogonadotropic hypogonadism, cleft palate, intellectual disability	(40)
NR2F2	15q26.2	AD	Ovarian dysgenesis, ovotesticular DSD		Congenital heart disease, somatic anomalies including blepharophimosis-ptosis-epicanthus inversus syndrome	(41)
NR5A1	9q33	AD	Ovarian dysgenesis, ovotesticular DSD	Testicular dysgenesis	Adrenocortical insufficiency, spermatogenic failure, primary ovarian insufficiency, asplenia, polysplenia	(42)
PPP2R3C	14q13.2	AR		Testicular dysgenesis	Dysmorphic facies, retinal dystrophy, and myopathy.	(43)
RSPO1	1p34.3	AR	Testicular/ ovotesticular DSD		Palmoplantar hyperkeratosis	(44)
SART3	12q23.3	AR		Testicular dysgenesis	Intellectual disability, global developmental delay and a subset of brain anomalies	(45)
SOX3 (Upstream deletion and Duplication)	Xq27.1	XL	Testicular/ovotesticular DSD		Intellectual disability, hypopituitarism	(46)
SOX8 (SNV, upstream duplication, inversion)	16p13.3	AD		Testicular dysgenesis	Variants may contribute to male and female infertility, primary ovarian insufficiency.	(47)
SOX9 (SNV, upstream duplication, inversion)	17q24.3	AD	Testicular/ovotesticular DSD	Testicular dysgenesis	Campomelic dysplasia, Cooks syndrome, Pierre Robin sequence	(48)
SOX10 (Duplication)	22q13.1	AD	Testicular/ovotesticular DSD		Peripheral and central demyelination, Waardenburg syndrome type IV, and Hirschsprung disease	(49)
SRY (SNV, deletion, Translocation)	Yp11.3	XL, YL	Testicular/ovotesticular DSD	Testicular dysgenesis		(50, 51)
TSPYL1	6q22.1	AR		Testicular dysgenesis	Sudden infant death syndrome	(52)
WT1	11p13	AD	Testicular/ovotesticular DSD	Testicular dysgenesis	Denys-Drash syndrome; Frasier syndrome; WAGR syndrome; Meacham syndrome, Nephrotic syndrome; Wilms tumor	(53, 54)

Abbreviations: AD; autosomal dominant, AR; autosomal recessive, XL; X-linked, YL; Y-linked

DEFICIENCY OF TESTOSTERONE BIOSYNTHESIS

The inability to produce testosterone stems from defects in the activity of any of the enzymes required for testosterone biosynthesis from cholesterol (Figure 4able 5). Identified defects encompass genetic variants in Steroidogenic Acute Regulatory Protein (StAR), P450 side-chain cleavage (CYP11A1), 3β-hydroxy steroid dehydrogenase 2 (HSD3B2), 17α-hydroxylase/17,20-lyase (CYP17A1), and P450 oxidoreductase (POR) (59). In addition, apparent lyase deficiency due to variants in cytochrome b5 (CYB5) and variants in AKR1C2/4 have been very rarely described (60-62). Moreover, 17β-hydroxysteroid dehydrogenase deficiency (also known as 17β–hydroxysteroid oxidoreductase or 17-ketosteroid reductase, HSD17B3) is another cause of 46,XY DSD (63). Furthermore, variants in steroidogenic factor 1 (SF1/NR5A1), which regulates the transcription of several genes involved in steroidogenesis, may lead to ambiguous genitalia. Notably, variants in HSD3B2, POR, and NR5A1 can result in ambiguous genitalia in both 46,XY and 46,XX newborns (64-66).

Similar to partial gonadal dysgenesis, a partial testosterone biosynthesis defect leads to ambiguous external genitalia and variable degrees of Wolffian duct development. However, unlike partial gonadal dysgenesis, Müllerian ducts are not retained in 46,XY newborns with partial testosterone biosynthesis defects due to intact Sertoli cell function and normal AMH production.

Table 5. Adrenal and/or Gonadal Causes of Impaired Sex Steroid Biosynthesis Associated with Ambiguous Genitalia in 46,XX and 46,XY Individuals
Disorder	Gene/ OMIM	Adrenal Insufficiency	46,XY Gonadal Phenotype (Testosterone Deficiency)	46,XX Gonadal Phenotype (E2 Deficiency)	Fertility	Other Features
Lipoid congenital adrenal hyperplasia (LCAH)	StAR 201710	YES	Classic form: 46,XY DSD, gonadal insufficiency; ambiguous genitalia Non-classic form: normal or NK	Classic: primary or secondary ovarian insufficiency (POI) Non-classic: NK or normal	Classic:Absent in 46,XY; variable in 46,XX
P450 side chain cleavage syndrome (CAH)	CYP11A1 118485	YES	Classic form: 46,XY DSD, gonadal insufficiency; ambiguous genitalia Non-classic form: normal	Classic: primary or secondary ovarian insufficiency (POI) Non-classic: NK or normal	Reported in 46,XX
3β-hydroxysteroid dehydrogenase II deficiency (CAH)	HSD3B2 201810	YES	46,XY DSD, gonadal insufficiency; ambiguous genitalia Non-classic form: normal, but premature adrenarche	46,XX DSD with atypical genital development; gonadal insufficiency; ambiguous genitalia Non-classic form: normal, but premature adrenarche	Absent in 46,XY; reported in 46,XX
21-hydroxylase deficiency (CAH)	CYP21A2 201910	YES	Classic form: normal Non-classic form: normal	46,XX DSD with atypical genital development; Non-classic form: premature adrenarche, virilization, PCO	Normal in both 46,XX and 46,XY, if treated	Cave: Testicular adrenal rest tumor (m>>f) CAH-X (when combined with Ehlers-Danlos syndrome with contiguous gene variants)
11-hydroxylase deficiency (CAH)	CYP11B1 202010	YES	Classic form: normal Non-classic form: normal	46,XX DSD with atypical genital development; Non-classic form: premature adrenarche, virilization, PCO	Normal in both 46,XX and 46,XY, if treated	Hypertension
Combined 17-hydroxylase, 17,20 lyase deficiency (CAH)	CYP17A1 202110	Rare	46,XY DSD, gonadal insufficiency; atypical genital development	Lack of pubertal development, POI	Possible in 46,XX with assisted fertility measures	Hypertension and hypokalemic alkalosis (not seen with isolated lyase deficiency)
P450 oxidoreductase deficiency (CAH)	POR 124015 201750	Variable	Mild to severe 46,XY DSD, gonadal insufficiency; atypical genital development	46,XX DSD with atypical genital development or premature adrenarche, virilisation, POI, PCO	Reported	Maternal virilization during pregnancy; Antley-Bixler skeletal malformation syndrome; changes in drug metabolism
Cytochrome b5 deficiency	CYB5A 613218	NO	46,XY DSD; atypical genital development	NK	NK	Methemoglobinemia
17β-hydroxysteroid dehydrogenase III deficiency / 17-ketosteroid reductase deficiency	HSD17B3 264300	NO	46,XY DSD; atypical genital development; progressive virilization and gynecomastia at puberty	Normal	Decreased or absent in 46,XY
5α-reductase II deficiency	SRD5A2 607306	NO	46,XY DSD; atypical genital development; progressive virilisation and gynecomastia at puberty	Normal	Impaired in 46,XY
3α-hydroxysteroid dehydrogenase deficiency	AKR1C2/4 600450 600451	NO	46,XY DSD; gonadal insufficiency; atypical genital development	Normal	NK
Aromatase deficiency	CYP19A1 107910	NO	Normal	46,XX DSD with variable degree of virilisation at birth (ambiguous genitalia), gonadal insufficiency, POI	Impaired in 46,XX	Overgrowth and metabolic anomalies in males
Steroidogenic factor 1	NR5A1/ SF1 184757	Rare	Mild to severe 46,XY DSD; gonadal insufficiency – very variable; ambiguous genitalia	POI or normal; rarely 46,XX DSD with atypical genital development	Mostly impaired in 46,XY; variable in 46,XX

Abbreviations: NK; not known, POI; primary ovarian insufficiency, PCO; polycystic ovary.

5-ALPHA-REDUCTASE-2 DEFICIENCY (SRD5A2 VARIANTS)

Deficiency of the 5α-reductase enzyme arises from variants in the steroid 5α-reductase type 2 (SRD5A2) gene, which can show single point mutations to complete gene deletions. Newborns affected by this condition possess functioning Leydig and Sertoli cells, but due to their inability to convert testosterone to DHT, they may exhibit varying degrees of under-masculinized external genitalia, including genital ambiguity at birth in some cases (16, 67-69). The Müllerian ducts in affected individuals regress as expected, due to normal Sertoli cell function and AMH production. During puberty, significant virilization becomes possible in affected individuals, and the testes are capable of supporting spermatogenesis, as DHT is not required for germ cell maturation. Therefore, fertility is attainable in less severely affected individuals (70-72) or with the use of intrauterine insemination. Recent evidence also suggests that SRD5A2 activity may be even influenced by genetic polymorphisms of SRD5A2 (50).

GENETIC DEFECTS OF ANDROGEN ACTION

Complete or partial androgen insensitivity syndromes (CAIS/PAIS) are caused by genetic mutations affecting androgen receptor (AR) function (73, 74), and serum testosterone levels are typically elevated. Internationally, incidence of AIS is reported in 1 in 20,400 live born 46,XY infants, with CAIS occurring at a higher rate than PAIS (75).

The human androgen receptor (AR) is encoded by a single gene (AR) composed of 8 exons located in the q11-12 region of the X chromosome. The AR gene mutation database includes hundreds of AR variants that lead in varying degrees of atypical sex differentiation of 46,XY fetuses, also classified as AIS type I (76-78). A small number of complete AR gene deletions have been reported, as well as deletions that start at exons 2, 3 or 4 and extend to the terminus of the gene. In addition, a limited number of mutations resulting from premature terminations, base deletions and terminations have been identified. However, the most common type of AR gene mutation results from base substitutions (79). When mutations result in partial inactivation of AR activity (PAIS), the phenotypic variability in under-masculinization can be substantial and depends on the residual activity of the AR. In some cases, this can lead to ambiguous genitalia in newborns with a 46,XY chromosomal sex.

Unlike in CAIS, the underlying genetic defect in PAIS is only found in the AR gene in about 40% of cases (80). However, the clinical and biochemical phenotype is similar, and in vitro functional tests demonstrate androgen resistance in all of them. Thus, individuals with AIS without AR mutations are classified as AIS type II (73). In cases of AIS type II without AR variants, largely unidentified regulators or cofactors of the AR are responsible for the impaired AR signaling, as revealed by an AR-dependent bioassay using genital skin fibroblasts and the targeted apolipoprotein D as a biomarker (81). Given that AR activity can be regulated at various levels, the potential mechanisms of AIS type II are diverse.Recently, altered DNA methylation of the AR promoter has been observed in some individuals with PAIS (82). Additionally, mutations in the Disheveled associated activator of morphogenesis 2 gene (DAAM2) that impair nuclear actin assembly at AR have been associated with AIS type II (83).

Overall, individuals with PAIS experience variable degrees of end-organ unresponsiveness to androgens, resulting in varying degrees of Wolffian duct development and external genital ambiguity. Within families with the same AR mutation, phenotypic variability can occur due to variable degrees of insensitivity to androgens (84, 85). Consequently, solely relying on genetic information, it is challenging to predict whether a newborn with PAIS will identify as male, intersex, or female in later life and respond to future testosterone therapy. Similarly, genetic counselling is challenging.

The 46,XX Newborn With Ambiguous Genitalia

Ambiguous genitalia in a chromosomal 46,XX fetus invariably results from androgen excess during fetal development, and the degree of virilization (Figure 2) often provides clues about the timing and severity of the underlying disorder.

Similar to a 46,XY fetus, a 46,XX fetus may experience partial gonadal dysgenesis or develop testicular or ovo-testicular DSD due to pathogenic variants in genes involved in early sex determination (Table 3 and 4). Ovo-testicular DSD occurs when both ovarian and testicular tissue develop in the same individual (1). Most newborns with ovo-testicular DSD possess 46,XX chromosomes and present with ambiguous genitalia, although some affected individuals have 46,XY chromosomes or 46,XX/46,XY mosaicism. Just like in 46,XY DSD, the degree of testicular development determines the extent of masculinization/virilization of the external genitalia, Wolffian duct development, and Müllerian duct regression in affected newborns (79). A summary of known genetic variations is given in Table 4.

In the majority of cases, ambiguous genitalia in a 46,XX fetus with typical ovarian organogenesis can be attributed to excessive androgen production/exposure early in utero, originating either from the fetus itself (Table 3 and Table 5) or from the mother or environment.

ABNORMAL FETAL ANDROGEN PRODUCTION BY CONGENITAL ADRENAL HYPERPLASIA (CAH)

The term congenital adrenal hyperplasia (CAH) encompasses several adrenal disorders, each linked to a mutation in one of the enzymes necessary for the biosynthesis of cortisol from cholesterol (86) (Figure 4 and Table 5). These abnormalities lead to increased ACTH secretion by the pituitary gland, which, in turn, results in the increased secretion of cortisol precursors, including adrenal androgens.

Deficiency of steroid acute regulatory protein (StAR) causes congenital lipoid adrenal hyperplasia, characterized by salt loss and a lack of cortisol, androgen, and estrogen secretion. Genetic 46,XX infants affected by StAR deficiency exhibit typical female external genitalia, while genetic 46,XY are born with ambiguous genitalia or may experience sex reversal in cases of severe loss-of-function variants. A similar phenotype is also observed with variants in CYP11A1.

Deficiency in 17α-hydroxylase/17,20-lyase leads to hypertension due to the hypersecretion of corticosterone and impaired androgen secretion. Similar to congenital lipoid adrenal hyperplasia, 46,XX fetus affected by 17α-hydroxylase/17, 20-lyase deficiency (CYP17A1) develop typical female external genitalia, while 46,XY fetus present with ambiguity.

Deficiency of 3β-hydroxysteroid dehydrogenase (HSD3B2) results in salt loss and impaired androgen synthesis. Affected 46,XX females may or may not exhibit minimal genital masculinization, whereas all genetic 46,XY males are variably under-masculinized (87).

CAH due to 11β-hydroxylase deficiency (CYP11B1) and 21-hydroxylase deficiency (CYP21A2) results in the most significant masculinization/virilization of external genitalia in 46,XX fetus compared to all other types of CAH. Additionally, 11β-hydroxylase deficiency can lead to hypertension in individuals of either sex in later life (88, 89).

21-hydroxylase deficiency CAH, caused by variants in CYP21A2, represents the most common form (accounting for more than 90% of cases) of CAH and is the most frequent genetic cause of DSD with ambiguous genitalia at birth in 46,XX newborns (59, 63). Cortisol deficiency necessitates replacement therapy in all classic forms of CAH. In the milder (simple-virilizing) presentation of classical 21-hydroxylase deficiency, salt loss is typically not an issue, whereas in the more severe (salt-wasting) form of classical 21-hydroxylase deficiency, salt-loss occurs and may pose a risk to the newborn shortly after birth if not treated with both mineralocorticoids and glucocorticoids (86, 90, 91).

The pathological mechanisms of 21-hydroxylase deficiency leading to 46,XX virilization in utero have been well described (92). It involves androgen excess due to lack of 21-hydroxylase activity during the critical time window around 6-12 weeks of gestation when the external genitalia are formed, and adrenal androgen production should be redirected to cortisol production to protect the female external genital anlage. In 46,XX CAH (Table 5), the resulting adrenal androgen excess exceeds the androgen metabolizing capacities of the fetal-placental unit, and alternative pathways of androgen production (Figure 4) are also activated, contributing to the overall androgen excess. Figure 5 illustrates in a scheme how the fetal adrenals, liver and placenta cooperate under normal conditions to metabolize fetal adrenal androgens into estrogens.

Figure 5. Steroid metabolic pathways in the fetal-placental-maternal unit, also known as the fetal-placental unit. The main function of the FZ of the fetal adrenal is the production of DHEA-S from cholesterol, which is transported to the placenta, desulfated to DHEA, and sequentially metabolized by placental 3β-hydroxysteroid dehydrogenase type 1 (3βHSD1), 17β-hydroxysteroid dehydrogenases (17βHSD) and cytochrome P450 aromatase (CYP19A1), to androstenedione (A4), testosterone (T) and estradiol (E2), respectively. E2 is then transported from the placenta into the maternal circulation. A4 is additionally also a substrate for placental CYP19A1, producing estrone (E1). Fetal adrenal DHEA-S is also metabolized by the fetal liver to 16α-hydroxy-DHEA-S by cytochrome P450 family 3 subfamily A member 7 (CYP3A7), which is metabolized to estriol (E3), the estrogen marker of pregnancy. Abbreviations: PREG, pregnenolone; 17OHPREG, 17α-hydroxypregnenolone; PROG, progesterone; DHEA, dehydroepiandrosterone; DHEA-S, dehydroepiandrosterone-sulfate; CYP11A1, cytochrome P450 cholesterol side chain cleavage; StAR, steroidogenic acute regulatory protein; CYP17A1, cytochrome P450 17α-hydroxylase/17,20-lyase; SULT2A1, sulfotransferase; CYP3A7, cytochrome P450 family 3 subfamily A member 7; CYP19A1, cytochrome P450 aromatase; HSD17B, 17β-hydroxysteroid dehydrogenase; HSD3B1, 3β-hydroxysteroid dehydrogenase type 1; HSD11B2, 11β-hydroxysteroid dehydrogenase type 2; STS, sulfatase.

FETAL AND PLACENTAL AROMATASE DEFICIENCY

During fetal development, the adrenals produce substantial amounts of DHEA/S. These are either converted to 16-hydroxy-DHEA/S in the fetal liver and then transported or directly transferred to the placenta, where they are converted into estrogens (Figure 5). In case of an aromatase enzyme deficiency in the fetus (caused by variants in the CYP19A1 gene), androgen precursors accumulate and lead to virilization of 46,XX fetuses, typically leading to ambiguous genitalia at birth (93). Androgen excess due to fetal aromatase deficiency may also induce maternal virilization during pregnancy, as indicated by a deepening of the voice, oily skin and hair, and excessive hair growth (hirsutism).

EXCESS MATERNAL ANDROGEN PRODUCTION

Excessive androgen production can negatively impact fertility due to anovulation, therefore cases of maternal androgen production during pregnancy are exceedingly rare. Nonetheless, androgens originating from the mother can travers the placenta and lead to the masculinization of a female fetus. These maternal androgens usually originate from the ovaries or adrenal glands. Androgen-producing ovarian tumors include hilar cell tumors, arrhenoblastomas/androblastomas, lipoid cell tumors, and Krukenberg tumors. In contrast, pregnancy luteomas, rare non-neoplastic ovarian lesions occurring during pregnancy, are believed to be caused by hormonal effects related to pregnancy. Although exceptionally rare, androgen-secreting tumors of the adrenals can also occur during pregnancy.

DRUGS ADMINISTERED TO THE MOTHER DURING GESTATION

In 1958, Wilkins et al. reported that certain synthetic progestins administered to pregnant women, such as 17α-ethinyl-19-nortestosterone, can masculinize the external genitalia of female fetuses (94). Similarly, diethylstilbesterol (DHE), a nonsteroidal synthetic estrogen, has been linked with urogenital anomalies in both 46,XX and 46,XY fetuses (95, 96). More recently, numerous studies have indicated that fetal exposure to various endocrine disrupting chemicals (EDCs) such as pesticides, fungicides, herbicides, and plasticizers may adversely affect genital development (97-99).

Syndromes Associated with Ambiguous Genitalia

As previously mentioned in sections of 46,XX and 46,XY DSD, ambiguous genitalia can be associated with syndromes characterized by multiple congenital malformations, as summarized in Table 4. These associations arise because many of the transcription factors involved in sex development and differentiation also have roles in other aspects of development (Table 4).

For example, mutations of the WT-1 gene (11p3) can lead to several syndromes, including WAGR, Denys-Drash or Frasier syndromes (70-72). Mutations in SOX-9 (17q24-25) can result in campomelic dysplasia (100).

Mutations in DMRT1/DMRT2 (9p24.3), EMX2 (10q25.3q26.13), ATRX (Xq13.3) and WNT-4 (1p35) are often associated with developmental delay (101, 102). SF-1 (9q33) and DAX-1 (Xp21.3) play roles in the early formation of the adrenals, anterior pituitary, and parts of the hypothalamus.

Consequently, mutations can lead to abnormalities of these organs, in addition to those of the urogenital system. Abnormal development of the lower abdominal wall with pubic diastasis can result in urogenital anomalies observed in the VA(C)TER(L) and CHARGE syndromes, as well as bladder and cloacal exstrophy (103).

These examples underscore the complexities of the anomalies that may be associated with ambiguous genitalia in newborns and emphasize the importance of conducting a thorough physical examination and involving an experienced DSD team when faced with a child presenting ambiguous genitalia.

WORKUP OF NEWBORNS WITH AMBIGUOUS GENITALIA

The etiology of genital ambiguity in newborns is diverse and can have significant implications for management. Therefore, obtaining an accurate diagnosis through a medical workup is crucial. Some investigations are urgent, such as ruling out or confirming and treating potentially life-threatening adrenal insufficiency with mineralocorticoid and glucocorticoid replacement therapy in children with underlying CAH. Additionally, associated organ anomalies may require immediate attention to ensure the child’s survival, such as cardiac, pulmonary, or kidney malfunctions.

When the newborn is shown to be capable of excreting urine and stool without problems, and adrenal and other organ functions are found to be intact, investigations to understand genital ambiguity become primarily a psychosocial “emergency” for the family and caregivers. As a result, psychological support and holistic care by a DSD team as early as possible are essential to guide the parents and make decisions in the child’s best long-term interest. Obtaining an accurate diagnosis of the underlying cause of genital ambiguity early on is necessary for decision-making processes regarded to sex registration, hormonal and surgical treatments, and outcomes such as potential fertility and risk of gonadal malignancy. Therefore, it is recommended practice that a thorough workup should be performed for every child born with ambiguous genitalia, and guidelines for such assessments are available (22, 104-107).

Overall Assessment of the Child with Ambiguous Genitalia

Figure 6 provides an overview of the initial workup for a newborn with ambiguous genitalia. Several aspects are considered, including pregnancy and family history, as well as postnatal adaptation (such as feeding, weight gain, alertness, excretion). In some cases, suspicion of genital ambiguity may arise during pregnancy through ultrasound surveillance. In such cases, first contact and information through a DSD team member (e.g., neonatologist, geneticist, pediatric endocrinologist) together with the DSD psychologist is recommended to guide the parents through pregnancy and alert the future care team early. Prenatal workup of ambiguous genitalia is generally not necessary if the fetus is otherwise developing normally without other organ anomalies that require attention. It is important to note that a fetus with CAH does not suffer from mineralocorticoid and cortisol deficiency in utero, as these hormones become essential only after birth (Figure 4). Genetic workup of a child with ambiguous genitalia during the prenatal period may only be recommended when there is a previous index case with a diagnosis, or when parental genetic data inform an abnormality, and when genetic counseling of the parents has led to a personalized action plan.

When taking the medical history of a newborn with ambiguous genitalia, asking specific questions can help guide the diagnostic workup and management. Figure 6 provides a list of important questions to consider. For example, androgenic drugs used in pregnancy may explain the virilization of a 46, XX newborns. Maternal virilization during pregnancy, for example, voice change and hirsutism may be important for the diagnoses of maternal virilizing tumors, P450 oxidoreductase (POR) deficiency, or placental aromatase deficiency. Questions about parental consanguinity are important, because many of the causes of ambiguous genitalia are recessively inherited. History of previously unexplained neonatal deaths, ambiguous genitalia, infertility or genital surgery in the family is important for identifying potential genetic etiologies.

In addition to the medical history, every newborn with ambiguous genitalia should undergo a comprehensive physical examination, with a special focus on signs of adrenal insufficiency (dehydration, lethargy, poor feeding) and any dysmorphic features (Figure 6). This examination may guide the diagnostic process.

Figure 6. Algorithm for initial workup of the child with ambiguous genitalia at birth.
Abbreviations: SDSST; Standard dose synthetic ACTH (synacthen) stimulation test, HCG; Human chorionic gonadotropin, WES; Whole exome sequencing, WGS; Whole genome sequencing, EMS; External masculinization score, EGS; External genital score, QF-PCR; quantitative fluorescence polymerase chain reaction, SRY FISH; SRY fluorescent in situ hybridization.

Physical Examination of the External Genital Area

Systematic assessment of the external genital area in newborns with ambiguous genitalia is crucial, and it should be carefully documented. Over the years, different scales and scoring systems have been used to evaluate the external genitalia. These assessments help provide a standardized way to document the physical characteristics, which is essential for comparing data over time and between different healthcare providers and patients. The use of these scales and scoring systems aids in follow-up and tracking of changes in the external genitalia.

Historically, the Prader or Quigley scales were commonly used for this purpose (108, 109) (Figure 7). Later, the external masculinization score (EMS) was introduced as a more comprehensive assessment tool (104) (Table 6). Nowadays, the external genital score (EGS) is recommended for evaluating and documenting the external genitalia (110) (Table 7). These tools provide a structured way to assess and record the physical findings, making it easier to monitor and communicate about the patient's condition.

The careful examination of the external genitalia in newborns with ambiguous genitalia provides valuable non-invasive, indirect information about prenatal exposure and response to DHT, and about the timing of this exposure. For instance, androgen exposure must have occurred in the first trimester to cause labial fusion.

During the genital exam, it’s important to measure the stretched phallic length, assess the quality of the corpora (erectile tissues), inspect of the labia, labio-scrotal folds, or scrotum, and document the position of the urethral opening (and vaginal opening, if applicable). Furthermore, the presence and location of palpable gonads should also be recorded. Any asymmetry in the gonads and external genitalia should be noted. Asymmetry in internal and/or external genital structures may suggest mixed gonadal dysgenesis or ovo-testicular DSD. All these observations can be documented using the external genital score (EGS), which provides a standardized way to assess and record the physical characteristics of the external genitalia (Table 7). In addition, the anogenital distance (AGD), the distance between the anus and the base of the genitalia, can be helpful in determining the extent of androgen exposure during fetal development, as a higher ratio is generally consistent with increased androgen exposure (111) (Table 8). In older infants, the anogenital index (AGI), which is the body weight standardized index of AGD [AGI/weight (mm/kg)], may be used for assessments to account for changes of the external genitalia through growth and postnatal development (111).

Overall, a thorough examination and documentation of the external genitalia provide important clinical information that can guide the diagnosis and management of newborns with ambiguous genitalia.

Figure 7. Prader and Quigley Scales for visual grading of external genital virilization. In Stage 3 a single urogenital orifice can be observed.

Table 6. External Masculinization Score (EMS)
External Masculinization Score (EMS).	Scrotal fusion	Micropenis	Urethral meatus	Right gonad	Left gonad
3	Yes	No	Normal
2			Distal
1.5				Labioscrotal	Labioscrotal
1			Mid	Inguinal	Inguinal
0.5				Abdominal	Abdominal
0	No	Yes	Proximal	Absent	Absent

An EMS score <11 out of 12 needs further clinical investigation for DSD in 46,XY.

Table 7. External Genital Score (EGS)
External genital score (EGS)	Labio-scrotal fusion	Genital tubercle size (mm)	Urethral meatus	Right gonad	Left gonad
3	Fused	≥31	Typical male
2.5		26-30	Coronal glandular
2			Penile
1.5	Posterior fusion	21-25	Peno-scrotal	Labio-scrotal	Labio-scrotal
1		10-20	Scrotal	Inguino-scrotal	Inguino-scrotal
0.5			Perineal	Inguinal	Inguinal
0	Unfused	≤ 10	Typical female	Impalpable	Impalpable

Median EGS (10th to 90th centile) in males < 28 weeks gestation is 10 (8.6-11.5); in males 28-32 weeks 11.5 (9.2-12); in males 33-36 weeks 11.5 (10.5-12) and in full-term males 12 (10.5-12). In all female babies, EGS is 0 (0-0).

Table 8. Reference Ranges for Anogenital Distance and Anogenital Index in Term Neonates
	5th	10th	25th	50th	75th	90th	95th
Males
AGD (mm)	18.47	19.44	21.00	22.68	25.20	27.80	29.08
AGI (mm/kg)	5.59	5.83	6.30	6.93	7.62	8.22	8.57
Females
AGD (mm)	9.40	9.85	10.55	11.65	12.60	13.45	14.10
AGI (mm/kg)	3.04	3.15	3.38	3.59	3.80	4.00	4.19

Anogenital index (AGI): the body weight standardized index of AGD [AGI=AGD/weight (mm/kg)] and found that using AGI as parameter has a better correlation with age.

Basic Auxiliary Investigations in the First Days

In the first few days of life, basic investigations are essential to evaluate adrenal and gonadal development and function in otherwise healthy newborns with ambiguous genitalia. Some key aspects to address include serum electrolytes and glucose levels. They should be monitored daily as cortisol deficiency can manifest as hypoglycemia in newborns affected by CAH. Body weight and feeding characteristics should also be monitored as excessive weight loss may indicate dehydration and electrolyte disturbances.

KARYOTYPE

Besides careful clinical evaluation, first-line investigation of an individual with DSD involves confirming the chromosomal sex using quantitative fluorescence polymerase chain reaction (QF-PCR) and karyotype (22). QF-PCR detects a series of markers on the sex chromosomes and has a turn-around time of about 1–2 days. This technique has largely replaced fluorescence in situ hybridization (FISH) methods. The analysis of karyotype is a cytogenetic technique (involving G-banding) and is crucial for the initial classification of a newborn with ambiguous genitalia (Table 3). Some centers have replaced karyotyping by array-comparative genomic hybridization (aCGH) or SNP array, with faster turn-around times of 5–10 days. But these techniques will not detect structural chromosomal rearrangements and may be less effective at detecting sex chromosome mosaicism.

HORMONE STUDIES

Detailed hormone studies may be indicated including serum gonadotropins (LH, FSH), androgens and androgen precursors (17-hydroxypregnenolone, 17-hydroxyprogesterone, androstenedione, testosterone, dihydrotestosterone), adrenal steroids and hormones (cortisol, aldosterone, and their precursors; ACTH, renin), and anti-Müllerian hormone (AMH) (1, 24).

Care must be taken when interpreting the results in premature babies, in whom these studies may need to be repeated at a later age and special normative value may apply. Even in term babies the timepoint and the clinical context (mode of delivery, drug effects, actual stress level etc.) need to be considered when evaluating hormonal data immediately after birth. Hormonal data change massively during delivery (e.g., surge) and within days thereafter. Additionally, it's essential to be aware that normative values for hormone levels can vary depending on the laboratory and the methods used for testing. The interpretation of hormonal data should be done by experienced healthcare providers and, if necessary, in consultation with a specialist in DSD.

IMAGING STUDIES

Ultrasound imaging of the abdominal-pelvic organs is a valuable diagnostic tool in the evaluation of newborns with ambiguous genitalia. It provides important insights into the internal reproductive and urinary structures, helping to determine the presence and characteristics of gonads, Müllerian structures (e.g., the uterus), and any associated abnormalities of the urinary tract. It can be performed at the first visit of a child with ambiguous genitalia. Of course, this investigation is not easy and may only be performed by an experienced ultra-sonographer to reveal meaningful results. Ultrasound can also detect abnormalities of the urinary tract (kidney, ureters, bladder) that sometimes occur in conjunction with genital ambiguity. Interestingly, data show that a child with a uterus and a female internal system that presents with ambiguous genitalia without gonads in the genital folds and has a 46,XX karyotype, will have in 80% of cases a diagnosis of a 46,XX CAH (most likely 21-hydroxylase deficiency). The information needed to come to this conclusion in a newborn with ambiguous genitalia can be available within 48 hours in institutions with an experienced DSD team.

In a second step, usually after months or years, additional imaging studies with MRI can be helpful in identifying both the type and extent of internal sex organ development in more details. An MRI may reveal further details on Müllerian structures (uterus remanent, fallopian tubes, upper portion of the vagina) than an ultrasound and can also help for localizing abdominal gonads. MRI may also be used for surveillance of the gonad at risk for cancer.

A genitogram had been used in the past (often early) to visualize the urinary tract, and to determine its position in relation to the vagina or vagino-utricular pouch but is no longer performed in most institutions.

However, in follow-up, cystoscopy and vaginoscopy may be used. These investigations are invasive procedures requiring general anesthesia, and therefore need to have a clear indication. Often, they are performed after complications such as urinary tract infections, to better understand the anatomy and its consequences, and in preparation for a surgical procedure (112).

Overall, the choice of imaging studies and procedures should be guided by the individual's clinical presentation, diagnosis, and the specific clinical questions that need to be addressed. In many cases, a multi-disciplinary DSD team collaborates to determine the most appropriate imaging approach to provide a comprehensive evaluation and guide treatment decisions.

GENETIC STUDIES

The rapid advancement of genetic technologies, particularly massive parallel sequencing methods like DSD panels, whole exome sequencing (WES), and whole genome sequencing (WGS), has significantly expanded our understanding of the genes involved in sex development. As a result, genetic workup has become an integral part of the recommended routine evaluation for individuals with DSD and thus ambiguous genitalia at birth (8, 22, 24, 113, 114).

While making a precise genetic diagnosis is still a challenge in some cases, the use of advanced genetic techniques has improved the diagnostic yield. Currently, a clear molecular-level diagnosis can be established in approximately half of individuals with 46,XY DSD who undergo genetic workup. However, as genetic technologies continue to evolve, and our understanding of sex development genes expands, it is likely that more cases, including those involving individuals with ambiguous genitalia at birth, will receive accurate and valid diagnoses. Therefore, re-review of genetically unsolved cases by a DSD team and genetic re-analysis by DSD-specialized teams is recommended to achieve a final diagnosis in as many as possible.

In summary, the integration of genetic workup, in combination with ongoing advancements in technology and genetic knowledge, is expected to enhance our ability to provide individuals with DSD, including those with ambiguous genitalia at birth, with more precise and comprehensive diagnoses in the future. This progress will lead to better-informed clinical decisions and management strategies for newborns with ambiguous genitalia.

DIFFERENTIAL DIAGNOSIS

Generally, the result from the karyotype plays a major role in the differential diagnosis and workup of DSD and thus ambiguous genitalia (Table 3). In most cases, the chromosomes in newborns with ambiguous genitalia will be either 46,XX or 46,XY. In rare instances, chromosomal aberration such as 45,X/46,XY or 46,XX/46,XY mosaicism may be the underlying cause explaining the phenotype. Additionally, measurements of specific hormones, such as serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), sex steroids, AMH, inhibin B at birth and at follow-up, in the so-called mini-puberty of infancy period, can provide valuable information for the differential diagnosis of DSD and ambiguous genitalia.

In the following we provide two flowcharts and give some general information for the step-by-step workup of newborns with ambiguous genitalia and either 46,XX or 46,XY chromosomes.

46, XX DSD

A 46, XX karyotype in a newborn with ambiguous genitalia indicates that the child is a genetic female who was exposed to excessive amounts of androgens during fetal life. A flowchart for differential diagnosis is given in Figure 8.

Marked elevation of plasma 17-hydroxypregnenolone, 17-hydroxyprogesterone, 21-deoxycortisol and androstenedione, along with male-typical levels of testosterone, are characteristic of 21-hydroxylase deficiency (CYP21A2). High values of corticosterone and 11-deoxycortisol, along with elevated androgens, indicate 11β-hydroxylase deficiency (biallelic mutations in CYP11B1). In 3beta-HSD deficiency (biallelic mutations in HSD3B2), calculation of ratios of delta-5 (pregnenolone, 17OH-pregnenolone, DHEA) over delta-4 (progesterone, 17OH-progesterone, androstenedione) steroid metabolites (see Figure 4) will point towards the genetic defect.

Overall, comprehensive steroid profiling from blood or urine using chromatographic, mass spectrometric methods allow the diagnosis of most steroidogenic defects causing androgen excess CAH in 46,XX newborns (115-117) (see also Table 5). In addition, they have led to the description of novel alternate pathways to produce active androgens in the intermediate metabolism through the backdoor or 11-oxygenated pathways, especially in disorders such as 21-hydroxylase or POR deficiencies, thereby enhancing our understanding and diagnostic possibilities (115, 117, 118)(Figure 4). However, these methods are not (yet) widely available, and the interpretation of these data is not trivial. In addition, instruments and methods vary from lab to lab. Thus, method validation is tedious and comparison between labs difficult so far (119-121).

When excess maternal androgen production is the underlying cause for masculinization of a female fetus, the source of these steroids is eliminated postnatally. Thus, the various steroids studied in affected newborns will be in the female-typical range despite masculinization of the external genitalia. Then, detailed examination of the mother may provide the explanation.

Likewise, in cases of mild POR deficiency or aromatase deficiency, in which the feto-placental unit was not able to metabolize the fetal androgens and led to ambiguous genitalia, the problem will not (always) be visible in the newborn’s steroid metabolome, and the diagnosis may only be made by genetic testing. If not, only in later life, when aromatase activity is needed for estrogen production in puberty, or when ovarian cysts hint an underlying problem, the diagnosis may be made (Figure 8).

In individuals with ovo-testicular DSD and 46,XX chromosomes, masculinization arises from androgens secreted by the testicular portion of the differentiated gonads. Androgen production is similar to that produced by testes in unaffected males except that the amount is usually smaller. The degree of masculinization of the genitalia is thus linked to the amount of functioning testicular tissue, which can be determined by a serum AMH measurement (Table 9) (122).

Very low values of AMH and female-typical values for LH/FSH (Tables 9 and 10) are expected in 46,XX newborns with masculinized/virilized genitalia that is attributed to CAH or excess maternal androgen production during gestation (122, 123). AMH is higher in newborns with ovo-testicular DSD, due to Sertoli cell development in the testicular portion of their gonads where AMH is mainly produced.

On occasion, translocation of the pseudo-autosomal part of the Y chromosome along with a mutated SRY gene to an X chromosome occurs. The result is partial masculinization of the genitalia in a 46,XX newborn. With maturity, the phenotype of affected individuals closely resembles that of boys and men with Klinefelter syndrome.

Figure 8. 46,XX DSD decision tree diagram.
Abbreviations: USG: Ultrasound; 17OHP: 17OH-Progesterone; DSD: Differences in sex development; CAH: Congenital adrenal hyperplasia; 21OHD: 21α-Hydroxylase deficiency; 11β-OHD: 11β-Hydroxylase deficiency; 3β-HSD2: 3β-Hydroxysteroid dehydrogenase type 2; PORD: P450 oxidoreductase deficiency.

*Table 9. Normal Values for AMH (ng/mL) in Fetuses, Newborns and Infants Under 1 Year (122)**
	Female			Male
	n	Median	5-95th percentile	n	Median	5-95th percentile
Fetal blood	25	<0.4	<0.4-0.5	23	44.4	13.4-86.2
Cord blood	53	<0.4	<0.4-1.1	48	30.7	7.4-610.7
1-30 days	70	<0.4	<0.4-1.3	73	57.2	23.8-124.0
31-120 days	31	0.8	<0.4-6.4	39	88.4	46.8-173.0
4-12 months	13	1.0	<0.4-2.7	53	124.7	67.4-197.0

* AMH/MIS enzyme immunoassay kit cat 2368, Immunotech-Beckman Coulter, France

Table 10. LH and FSH Values (means ± SD) From Female and Male Newborns. Modified from (123)
		Female			Male
Age (days)	Hormone	n	Median	Mean±SD	n	Median	Mean±SD
1-5	FSH (IU/L)	31	1.80	2.00 ± 1.37	30	0.85	0.96 ± 0.60
1-5	LH (IU/L)	31	0.20	0.48 ± 0.66	30	0.20	0.39 ± 0.48
6-10	FSH (IU/L)	17	1.40	2.44 ± 2.52	15	1.40	2.91 ± 4.38
6-10	LH (IU/L)	17	0.30	0.45 ± 0.33	15	0.30	0.45 ± 0.33
11-15	FSH (IU/L)	8	8.95	8.16 ± 4.27	17	3.00	3.71 ± 2.69
11-15	LH (IU/L)	8	1.60	1.58 ± 1.28	17	2.90	3.55 ± 2.84
16-20	FSH (IU/L)	6	1.90	1.62 ± 1.05	14	2.15	2.63 ± 1.45
16-20	LH (IU/L)	6	0.35	1.03 ± 1.39	14	3.65	4.13 ± 2.76
21-25	FSH (IU/L)	3	3.90	7.07 ± 5.92	7	2.10	2.50 ± 1.51
21-25	LH (IU/L)	3	0.50	0.46 ± 0.25	7	2.70	2.86 ± 1.51
26-28	FSH (IU/L)	8	6.15	9.74 ± 9.89	8	2.40	2.25 ± 0.81
26-28	LH (IU/L)	8	2.80	2.75 ± 2.39	8	1.40	2.22 ± 2.37

Abbreviations: LH, luteinizing hormone; FSH, follicle-stimulating hormone.

46, XY DSD

A 46, XY karyotype reveals that one is dealing with a genetic male who was under-masculinized during fetal development. A flowchart for differential diagnosis is given in Figure 9.

Laboratory findings of normal or elevated testosterone and DHT with normal AMH (Figure 9) indicate a diagnosis of AIS. If testosterone levels are normal but DHT levels are low, a diagnosis of steroid 5α-reductase deficiency can be made. Low levels of testosterone and DHT, along with marked elevation of some androgen precursors, indicate a deficiency of one of the enzymes required for androgen biosynthesis (Figure 9). For example, if the elevated precursors include androstenedione and 17-hydroxyprogesterone, then the defective enzyme is 17-ketosteroid reductase (HSD17B3). In all cases of testosterone biosynthetic defects, AMH levels are similar to those observed in healthy 46,XY male infants with male-typical external genitalia (Figure 9). With early defects of steroidogenesis that affect both adrenal and gonadal function, it is though important to evaluate adrenal function, as it might be impaired with deficiencies of STAR, CYP11A1, HSD3B2, CYP17A1 and POR (Table 5, Figure 9).

Finally, when all androgens and their precursors are below normal levels, one is dealing with gonadal dysgenesis or 46,XY ovo-testicular DSD. In these cases, AMH values should also be low. In contrast, for babies affected by Leydig cell hypoplasia, androgens and their precursors are low, while AMH values should be in the normal male range.

Figure 9. 46,XY DSD decision tree diagram.
Abbreviations: USG: Ultrasound; 17OHP: 17OH-Progesterone; DSD: Differences in sex development; AMH: Anti-Müllerian hormone; T: Testosterone; N: Normal; PMDS: Persistent Müllerian duct syndrome; AIS: Androgen insensitivity syndrome; SRD5A2: 5α-reductase A2; HCG: Human chorionic gonadotropin; DHT: Dihydrotestosterone; 3β-HSD2: 3β-Hydroxysteroid dehydrogenase type 2; CYP17A1: 17α-Hydroxylase/17-20 lyase; POR: P450 oxidoreductase; StAR: Steroidogenic acute regulatory protein; CYP11A1; Cholesterol side-chain cleavage enzyme; 17β-HSD3: 17β-Hydroxysteroid dehydrogenase type 3; AKR1C2/4: Aldo-keto reductase family 1 member C2/4; CYB5: Cytochrome b5.

Testing Early Postnatal Gonadal Function and Mini-Puberty

In cases of ambiguous genitalia where a diagnosis may not be reached through first line investigations, and where a gonadal malfunction is suspected, functional testing and/or re-analysis of gonadal function at the timepoint of mini-puberty may be helpful, especially also with respect to having information when making considerations towards sex of registration (e.g., in utero effect of sex hormones on the brain) and prediction of later pubertal development and fertility potential.

Mini-puberty is the time window at day 30-100 after birth, when the hypothalamic-pituitary-gonadal (HPG) axis is re-activated for a short time frame in infancy, before going to inactivity until puberty. Little is known about the purpose of mini-puberty, but as the HPG axis is active and stimulates the gonads, it is a time window of opportunity to assess gonadal function without the necessity to stimulate with human chorionic gonadotrophin (hCG).

As a principle, in mini-puberty, the typical ovaries of a 46,XX newborn show little functional activity, while the typical testes of a 46,XY newborn are quite (re)active in testosterone (T), DHT, AMH and inhibin B production. While androgens are produced in the Leydig cells of the testes, AMH and inhibin B originate from the Sertoli cells. Table 11 gives an overview of the hormonal values observed during normal mini-puberty in healthy 46,XX and 46,XY infants (124).

Table 11. Evaluation of Reproductive Hormones During Mini-Puberty in Infants. Normative Values of Different Serum Hormones in 46,XX Female and 46,XY Male Infants. Modified from (124)
		Females			Males
	Post-natal time (months)	n	GMean	2.5-97.5th percentile	n	GMean	2.5-97.5th percentile
LH (IU/L)	2-3.5	432	<LOD	<LOD-0.98	581	1.71	0.62-4.08
LH (IU/L)	3.5-5	110	<LOD	<LOD-1.25	166	1.40	0.54-3.32
FSH (IU/L)	2-3.5	435	3.98	1.23-17.4	578	1.19	0.41- 3.02
FSH (IU/L)	3.5-5	111	3.93	1.30-17.7	165	1.11	0.42-2.68
Testosterone (RIA,nmol/L)	2-3.5	74	<LOD	<LOD-0.40	592	3.04	0.69-7.60
Testosterone (RIA,nmol/L)	3.5-5	14	<LOD	<LOD-NA	168	1.97	<LOD-7.01
Testosterone (LC-MS/MS, nmol/L)	2-3.5	165	<LOD	<LOD-0.21	251	4.75	1.35- 11.3
Testosterone (LC-MS/MS, nmol/L)	3.5-5	125	<LOD	<LOD-0.17	175	2.25	0.32- 9.65
Estradiol (pmol/L)	2-3.5	455	29	<LOD-79	571	<LOD	<LOD-47
Estradiol (pmol/L)	3.5-5	113	31	<LOD-98	160	<LOD	<LOD-49
SHBG (nmol/L)	2-3.5	427	133	67-264	579	135	66-268
SHBG (nmol/L)	3.5-5	110	141	72-282	162	143	71-262
Inhibin B (pg/mL)	2-3.5	423	62	<LOD-184	571	379	229-631
Inhibin B (pg/mL)	3.5-5	106	67	<LOD-174	158	379	222-662
AMH (pmol/L)	2-3.5	339	11	<LOD-49	48	1013	425- 1810
AMH (pmol/L)	3.5-5	67	15	2-46	12	1183	797-NA

Abbreviations: LOD, Limit of detection; GMean, geometric mean; LH, luteinizing hormone; FSH, follicle-stimulating hormone; T, testosterone; RIA, radioimmunoassay; SHBG, sex-hormone binding globulin; AMH, anti-Müllerian hormone; LC-MS/MS, liquid chromatography-tandem mass spectrometry; NA, not available.

HCG Testing of Gonadal Function in a DSD Newborn

The human chorionic gonadotrophin (hCG) test can assess whether in a newborn with ambiguous genitalia, functioning Leydig cells are present. The test may therefore be useful for diagnosing testosterone biosynthesis defects such as HSD17B3 or SRD5A2 deficiency (Table 12). Several protocols for the hCG test exist, but all baseline serum samples are taken for T, DHT and precursors. Then, one to three intramuscular injections of high dose hCG (500-1500 IU) are given at 24 hours interval, and finally repeat stimulated samples are taken for androgen measurements after 72 or 24 hours, respectively, after the last injection. The following Table 12 shows how results of the test may be interpreted in various DSD conditions that may lead to ambiguous genitalia (125, 126).

Table 12. Interpretation of hCG Stimulation Test Results (125, 126)
Indication	Consideration	Abnormal result
Testosterone biosynthesis defects	T before and after hCG administration	T increase < 100-150 ng/dL (3.5-5 nmol/L)
5α-Reductase deficiency	T/DHT ratio after hCG administration	≥ 8.5 for minipuberty, ≥ 10 for prepuberty, ≥ 17 for puberty, > 20 in genetically proven cases using LC-MS/MS
17β-HSD3 deficiency	T/A4 ratio after hCG administration	< 0.8
AIS	T, SHBG before and after hCG administration	T increases but SHBG does not change significantly

Abbreviations: T, testosterone; DHT, dihydrotestosterone; HCG, human chorionic gonadotropin; A4, androstenedione; SHBG, sex-hormone binding globulin; AMH, anti-Müllerian hormone; LC-MS/MS, liquid chromatography-tandem mass spectrometry; 17β-HSD3 deficiency, 17β-hydroxysteroid dehydrogenase type 3 deficiency; AIS, androgen insensitivity syndrome

RISK OF MALIGNANCY OF THE DSD GONAD

Individuals with certain disorders of sex development (DSD), especially those involving Y chromosome material, have an increased risk of developing gonadal tumors. These tumors are mainly gonadal germ cell tumors (GGCTs), which occur in about 15 % of all DSD cases, with the risk varying depending on the age and specific underlying diagnosis. Gonadal stromal tumors and smooth muscle leiomyomas/hamartomas are less common and are found in only about 0.9% of DSD cases (127). Risk factors for development of gonadal malignancy in DSD are shown in Figure 10. Several factors contribute to the increased risk of gonadal malignancy in individuals with DSD, including genetic factors, the presence of Y chromosome material, and the type of DSD. Therefore, ongoing monitoring and appropriate management are essential to minimize this risk. While gonadectomy (surgical removal of the gonads) is not recommended for every case of 46,XY DSD, it's crucial to consider the risk of malignant tumors in the decision-making process (1, 69). The consensus statement on the management of DSD provides guidelines and recommendations regarding the timing of gonadectomy for specific patients. These recommendations aim to balance the potential benefits of gonadectomy with the associated risks and individual patient needs.

In individuals with DSDs, germ cells typically undergo a process of apoptosis (programmed cell death) as the body grows and develops. This process eliminates germ cells that are not needed for reproduction. However, in some cases, certain germ cells may not undergo the normal maturation process and remain in an immature state. These immature germ cells can continue to express embryonic markers such as octamer-binding transcription factor 3/4 (OCT3/4) encoded by POU5F1; stem cell factor (SCF), also known as c-KIT ligand (encoded by KITLG); and placental alkaline phosphatase (PLAP). These cells are susceptible to developing into cancerous lesions, starting as pre-invasive lesions (gonado-blastoma) or as precursors to invasive GGCT (dysgerminoma) (Figure 10).

DSD patients who have Y chromosome material in their gonadal karyotype are at risk of GGCTs regardless of the degree of testicular differentiation. Y chromosome material associated with gonadal tumors is GBY locus which is around the centromere of the Y chromosome and includes the gene for ¨testis-specific protein on the Y chromosome¨ (TSPY, located in Yp11.2). Other potentially related genes are SRY and DYZ3.

The risk of developing gonadal tumors in gonadal dysgenesis may also vary according to the underlying molecular etiology. Patients with genetic defects that lead to early blockage of gonadal development such as mutation or deletions of SRY or WT1 have a higher risk. The risk of developing gonadal tumors ranges from 40% to 60% in gonadal dysgenesis due to WT1 gene mutations (Frasier syndrome and Denys-Drash syndrome). Early bilateral gonadectomy is therefore recommended. Similarly, early bilateral gonadectomy is recommended in patients with complete or partial gonadal dysgenesis due to SRY gene mutations due to the 20% to 52.5% risk of gonadal tumor development. Gonadal dysgenesis due to MAP3K1 gene mutations is another indication for early gonadectomy (127). Gonadal tumor risk is also present, though somewhat lower in gonadal dysgenesis due to 45,X0/46,XY mosaicism. The exact time of malignancy development and the prevalence of tumors in other molecular etiologies require further research for a comprehensive understanding.

Histologically the risk of gonadal tumor development increases with gonadal immaturity. Fully developed gonads such as the testes, ovary or ovotestis have lower risk of malignancy than streak or dysgenetic testes (Figure 10). In ovo-testicular DSD, the risk of germ cell tumors is low (3%) (1). Newborns with PAIS are at high risk (50%) for developing gonadal tumors, and bilateral orchiopexy or gonadectomy is recommended at the time of diagnosis, if the testes are located in the abdomen (1, 128).

The risk of developing germ cell malignancy in newborns with CYP11A1, HSD3B2 or CYP17A1 deficiency is unknown. Newborns with 17-ketosteroid reductase deficiency have a fairly low risk for malignancy (1). Tumor risk in newborns with Leydig cell hypoplasia is unclear, and thus the recommendation for gonadectomy is not well established (1, 25, 129, 130).

Overall, it remains challenging to predict the personal risk of gonadal neoplasia (mostly GGCT) for a DSD individual, because it depends on many other factors besides genetics. Stimulating gonadotropins and gonadal hormones during mini-puberty and after puberty onset are also regarded as risk factors increasing gonadal tumor risk in patients with DSD. Similarly, gonads located intra-abdominal carry a higher tumor risk. The management of gonadal tumor risk in individuals with DSD is a complex issue that requires careful consideration and a personalized approach, taking into account the specific DSD subtype, genetic factors, and patient preferences. Regular monitoring and discussions with a healthcare team experienced in DSD management are essential to make informed decisions about gonadectomy. If gonads are located in the scrotum, then periodic examination of the testes may be advised as an alternative to gonadectomy, while an intra-abdominal gonad at risk may only be monitored by imaging methods such as ultrasound or MRI.

Figure 10. Risk of malignancy development in the DSD gonad.
Abbreviations: (OCT3/4); Octamer-binding transcription factor 3/4, TSPY; testis-specific protein on the Y chromosome, c-KITL; c-KIT ligand also called stem cell factor (SCF).

SEX REGISTRATION OF A NEWBORN

Assigning or registering the sex of a newborn with atypical external genitalia is a complex and important decision that requires careful consideration, consultation, and a patient-centered approach. The parents and DSD network team should not rush but take the time needed to come to a consensus in the best interest of the child in its environment. Not knowing whether the newborn is a girl or boy causes mostly a lot of distress and one is tempted to call it an emergency, but if there are no medical reasons to make it an emergency (like e.g., suspected adrenal insufficiency or additional organ anomalies), it is important to declare it accordingly and inform that the newborn is not at risk and actually does not care whether it is a male or female or intersex person at this moment. Thus, the stress of ambiguity of the newborn is actually at this moment “only” an issue of the parents and the care team who mostly wish to know ASAP whether the child can be assigned/registered a “correct” sex that might also fit later in life.

To consider sex registration, not only is it important to take into account the underlying cause and knowledge on the possible outcome of the specific, often unique condition, but also the family, cultural, and societal preconditions and aspects. Most societies have a binary sex registration obligation by legislation, but some societies have more recently introduced a third sex category besides male and female. Although this seems to make things easier, it has been discussed that such categorization in a third or other or unclassified group might also stigmatize a person in a society that is mostly binary. Thus, in real life practice, there is rumor that a third sex category is rarely chosen for a newborn with ambiguous genitalia by the parents and the DSD team. Overall, a personalized approach for every newborn with ambiguous genitalia involving the parents and the DSD network team is recommended following the shared-decision-making process (12, 13).

Most important is that the team around the newborn with ambiguous genitalia works together in the best interest of the child with the latest knowledge of the literature and expertise when taking the decision of sex registration, and still remains humble and open minded for the child’s future that may follow a different path leading to sex reassignment later in life. Therefore, continuous support of the family and the child by a DSD psychologist is extremely helpful to recognize early signs of gender dysphoria, inform the child about the condition in an age-appropriate manner and follow its will, if case sex reassignment is desired.

Generally, people believe that the sex chromosomes indicate a person’s “true sex” and laws exist supporting this idea, to the detriment of some individuals affected by DSD (131). Scientifically speaking, it is clear that the majority of genes on the X chromosome do not influence sex development and differentiation, although the AR gene is necessary for phenotypic masculinization. Concerning the Y chromosome, only the SRY gene contributes to testicular formation. In fact, most of the genes required for sex development and differentiation are found on the autosomes (1). Therefore, chromosomes do not dictate the sex of rearing in newborns affected by a DSD and are only one piece of a big puzzle (Figure 3).

Although there is no overall recommendation for sex registration at birth for a child with ambiguous genitalia, some helpful information is available from larger patients’ cohorts with a specific DSD diagnosis in the literature.

Newborns presenting with 46,XY chromosomes and female external genitalia at birth (thus actually not with ambiguous genitalia) due to complete androgen insensitivity syndromes (CAIS), complete gonadal dysgenesis (Swyer syndrome), or other complete loss of testosterone biosynthesis live successfully when assigned a female sex of rearing. Female assignment in such cases is widely accepted by patients throughout their lives despite challenges that they may experience regarding sexual dysfunction and infertility (1, 132-134).

For newborns with ambiguous genitalia affected by partial gonadal dysgenesis, no general recommendation can be made. Fewer procedures were historically required for surgical feminization compared to surgical masculinization of the genitalia. However, the functional outcome in individuals who received feminizing or masculinizing procedures is less than optimal (135, 136). Additionally, rates of satisfaction with sex of rearing are similar for affected individuals whether raised male or female (1, 20, 137). Thus, the decision should not be made on the basis of “surgical possibilities”, and overall, such surgical procedures should be executed with reservation. Clearly, further studies are needed to elucidate why some individuals with partial gonadal dysgenesis experience gender dysphoria, while others do not.

Likewise, no general recommendation can be made for patients with ambiguous genitalia due to a partial androgen biosynthesis defect, (e.g., partial 3βHSD2, CYP17A1, POR, StAR, CYP11A1 deficiencies). But some studies suggest that the degree of masculinization of the external genitalia may reflect the degree of virilization of the brain and thus the gender preference of this person (138). This might be true for 46,XY newborns with ambiguous genitalia that are manifesting with severe hypospadias, but not without exceptions. For patients with variants in the SRD5A2 or HSD17B3genes recognized with ambiguous genitalia at birth (or even missed because manifesting with a female-typical external genitalia), there is quite a strong body of data recommending male sex registration at birth as this is the preferred sex of these persons when asked in later life (139, 140). Normal virilization at puberty, coupled with intact fertility potential, are strong factors for this recommendation at birth. If registered as female at birth and diagnosed later, gender dysphoria and desire of sex reassignment later in life is very likely.

Similar to partial gonadal dysgenesis, consensus regarding an optimal sex of registration for newborns affected by PAIS does not exist. Although earlier data showed that fewer procedures were usually required for surgical feminization, compared to surgical masculinization, in a person with ambiguous external genitalia in the past, when larger series of genital surgery were analyzed, it is unclear if the functional outcome was better among newborns who received feminizing procedures compared to those who received masculinizing procedures. Rates of satisfaction with sex of rearing are similar for individuals with PAIS raised female or male (132, 141). While reports exist of impaired sexual function in people with PAIS raised male, it is highly suspected that sexual functional outcomes in affected people raised female are similarly poor (137). Reports of sexual satisfaction for people with PAIS who have not received genitoplasty in infancy, or at a later age, are limited. However, more information on outcome of newborns with ambiguous genitalia who had no genital surgery, at least not in infancy and childhood, will hopefully soon be added to the medical literature as current developments recommend abstaining from genital surgery.

By contrast, in 46,XX newborns with ambiguous genitalia due to CAH, it is generally recommended to register them a priori in the female sex, even if they are severely virilized (142-144). Masculinization due to 21-hydroxylase deficiency does not impair the development of the ovaries or the Müllerian ducts and thus fertility potential. Limited information is available about the medical and psychosexual outcome of 46,XX newborns affected by 21-hydroxylase deficiency and raised male (145, 146), although these patients do tend to have an increased risk of gender dysphoria compared to their female counterparts (147). Thus, a female sex of rearing is typically the decision for 46,XX newborns affected by 21-hydroxylase deficiency (145). Female sex of rearing in 46,XX newborns with ambiguous genitalia is also recommended for conditions resulting from maternal overexposure to androgens and placental aromatase deficiency.

FINAL REMARKS TO OVERALL MANAGEMENT

Only recently has the understanding of, and reaction to, having a child with ambiguous genitalia received systematic study. For some parents and caregivers, feelings of isolation and concern over what the future may hold for their affected child in terms of stigmatization and sexual dysfunction are paramount (148). One potential approach to ameliorate this issue is to provide information and management options to the family early in pregnancy or as soon as the child is born. Guidance by a DSD network team is therefore extremely important.

Some research has been done to examine the effects of prenatal diagnosis and treatment. In females with 21-hydroxylase deficiency CAH, one of the most common causes of ambiguous genitalia, dexamethasone treatment of the pregnant mother has been reported to reduce virilization of an affected female fetus by 80-85% (149), but this approach is no longer offered or recommended in guidelines because of potential adverse effects of dexamethasone on the developing fetus. Before the discovery of methods to measure fetal DNA in maternal blood, which enabled specific diagnosis of sex and whether the fetus inherited and was affected by the mutation, controversies existed because 7 unaffected fetuses would be treated unnecessarily (1:4 chance of inherited mutation and 1:2 chance of male vs female), So, on the one hand 7 unaffected fetuses and the mother would be exposed to an unnecessary drug, while on the other hand, even in affected fetuses long-term outcome showed adverse effects on neurocognitive parameters (150). Today, noninvasive prenatal diagnosis using massive parallel sequencing of cell-free fetal DNA can be used to determine a specific diagnosis, such as CAH, as early as 6 weeks of gestation (149, 151). Even preimplantation diagnostics is available (152). These techniques could be used to look at other autosomal dominant or X-linked conditions that cause ambiguous genitalia, especially when the fetus is known to be at risk (151). Further studies need to be done on this subject, but for families who already have an affected child or know that they are carriers, this may be an option that needs to be discussed with a DSD geneticist.

Patients for whom prenatal diagnosis and/or treatment is not an option, prognosis and management has improved significantly over time, but many open questions and controversies still exist. Our studies of parents of children with DSD reveal that the appearance of atypical genitalia can result in significant stress and maladaptive parenting strategies. For example, mothers of children with ambiguous genitalia experience greater stress if their child has not received “corrective” genital surgery (153) and some parents believe such surgeries eliminate stigmatization that may arise due to their child’s ambiguous genitalia However, as their children mature past infancy, some parents realize that their child’s DSD has not been ameliorated by genitoplasty, and concerns for their son or daughter resurface (148). To illustrate, among parents studied by our group, caregivers of adolescents with DSD experienced increased stress as their child matured despite the fact that many had received earlier surgery (154). Additionally, parents of children reared male reported the most perceived child vulnerability for their sons (155),and their reported levels of depression were directly associated with more atypical genital appearance for their sons (156). Overall, parents report that while early genitoplasty seems to “fix” some of their concerns for their child with DSD, this “fix” is not long-lasting. Instead, what needs to be emphasized to families is how to support their child who is different, but not damaged. Family centered, interdisciplinary care, with open and clear communication between patients, parents, and caregivers has been shown to be essential for optimal quality of life (157). In addition, networks of families who have the personal experience of parenting children with ambiguous genitalia can be an invaluable resource (158). Reservation towards early interventions needs to be considered,keeping in mind to preserve the personal right of the child to take his/her own decision towards body integrity. Any intervention that can be delayed without harming the newborn with ambiguous genitalia should be postponed until personal consent can be given. Finally, referring parents to support groups and introducing them to other caregivers of children born with ambiguous genitalia is extremely important to optimize parents’ understanding and acceptance of their child’s condition. With increased understanding and acceptance, optimal growth and development for children born with ambiguous genitalia may be obtained.

In conclusion, this Chapter give some insight into the current diagnostic evaluation and care of the newborn with ambiguous genitalia. We underscore the importance of a multidisciplinary and compassionate approach to the care of newborns with ambiguous genitalia, acknowledging the complex medical, psychological, and emotional aspects involved in such cases. We highlight the critical role of specialized teams and support in ensuring the well-being of both the child and his/her family.

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Fibromyalgia

ABSTRACT

Fibromyalgia is a clinical entity characterized by the combination of chronic widespread pain and other non-pain symptoms, including fatigue, poor sleep, and cognitive disturbances, which can exhibit symptom variation not only between different patients, but also in the same patient during the course of the disease. These symptoms are relatively common and non-specific. They can be encountered in other disorders that may overlap with fibromyalgia, often without having clear boundaries, while their nature makes them difficult to be objectively defined and quantified. These issues have led to significant controversy over the definition and the diagnostic criteria of fibromyalgia. It has been suggested that the markers of physical and psychological distress have a continuous distribution in the general population with fibromyalgia patients being at the extreme end of this continuum. Genetic predisposition in combination with environmental factors, are responsible for each individual’s position in this this distribution. In recent years more knowledge has been obtained to better understand the environmental factors that seem to be important in triggering fibromyalgia. Most of them act as stressors superimposed onto a deranged stress-response system leading to dys-regulation of the nociceptive system and the appearance of clinical symptoms. The aim of the therapy is to relieve pain and motivate the patients to become more physically active using a multimodal individualized therapeutic strategy that includes education, exercise, cognitive-behavioral approaches and medications. The response to current therapeutic modalities varies significantly, with some patients responding adequately, while others do not seem to experience any long-term benefit.

INTRODUCTION

Fibromyalgia is a clinical entity characterized by the combination of ill-defined symptoms including chronic widespread pain, with concomitant fatigue, sleeping disorders, and cognitive disturbances (1). The severity of these symptoms can vary significantly during the course of the disease. Fibromyalgia has been described as an arbitrarily created syndrome that lies at the extreme end of the spectrum of poly-symptomatic distress (2). The term poly-symptomatic was used to emphasize the variety of multiple different symptoms that can be found in fibromyalgia patients, while the distress can have a physical and/or a psychological component. This exact nature of fibromyalgia makes it difficult to be clearly defined, often overlapping with disorders that are characterized by similar symptoms. It is important to note that fibromyalgia is not an exclusion diagnosis as it can co-exist with other clinical conditions (3).

CLINICAL FEATURES

The main presenting complains of patients with fibromyalgia include chronic widespread pain (also called multisite pain), fatigue, and poor sleep. Usually the pain is initially localized, but eventually it involves many muscle groups. It is characterized as persistent with varying intensity, while it can often be described as a sensation of burning, gnawing soreness, stiffness, or aching. Excessive sensitivity to normally painful stimuli, such as pressure or heat (hyperalgesia) and painful sensation to normally non-painful stimuli, such as touch (allodynia) are significant features of fibromyalgia. Often patients complain of swollen joints and paresthesias without though the presence of any objective clinical findings during physical examination. Pain is often aggravated by cold and humid weather, poor sleep, physical and mental stress. Additionally the patients may have a variety of less well understood pain symptoms, including abdominal pain, chest wall pain, symptoms suggestive of irritable bowel syndrome, pelvic pain, and bladder symptoms of frequency and urgency suggestive of interstitial cystitis (4–9).

Fatigue is present in almost all patients with fibromyalgia, while many complain of non-refreshing sleep, frequent awakening during the night, and difficulty falling back to sleep. Sleep apnea and nocturnal myoclonus can also be present along with a sensation of light-headedness, dizziness, and faintness. In addition, cognitive difficulties such as short-term memory loss, groping for words and poor vocabulary, are common among patients with fibromyalgia. Mood disturbances, including depression, anxiety and heightened somatic concern, may often also occur. Headaches, either muscular or migraine type, are also commonly present (6,7). Other often co-existing conditions include multiple chemical sensitivity, “allergic” symptoms, ocular dryness, palpitations, dyspnea, vulvodynia, dysmenorrhea, premenstrual syndrome, sexual dysfunction, weight fluctuations, night sweats, dysphagia, restless leg syndrome, temporomandibular joint pain, chronic fatigue syndrome (systemic exertion intolerance disease), Raynaud`s phenomenon, autonomic dysfunction, and dysgeusia (6,8,9). These conditions cannot be used to support the diagnosis of fibromyalgia.

Approximately 40% of fibromyalgia patients have accompanying depression at the time of diagnosis, while 60% of patients have a lifetime history of depression. In addition, an anxiety disorder is present in 30% of the cases at the time of diagnosis while the lifetime prevalence of an anxiety disorder in fibromyalgia patients is approximately 60% (10–14). The levels of depression and anxiety in patients with fibromyalgia seem to be associated with the degree of cognitive impairment, as shown in a meta-analysis of 23 case-control studies (15). Based on the coexistence of depression and anxiety, fibromyalgia patients can be divided into 2 major groups. The first group comprises of patients without coexisting mood disorders, while the second of patients with concomitant depressive mood, often in combination with anxiety. According to the results of a study that intended to subgroup fibromyalgia patients based on: 1) mood status (evaluated by the Center for Epidemiologic Studies Depression Scale for depression and the State-Trait Personality Inventory for symptoms of trait-related anxiety), 2) cognition (by the catastrophizing and control of pain subscales of the Coping Strategies Questionnaire), and 3) hyperalgesia/tenderness (by dolorimetry and random pressure-pain applied at suprathreshold values), it was noted that fibromyalgia patients with depressive mood and anxiety are also ‘catastrophizing’. This term is used to indicate that such patients have a very negative, pessimistic view of what their pain is and what is causing, while they have no sense that they can control their pain. On the contrary fibromyalgia patients who are neither depressed nor anxious and therefore do not catastrophize, have a moderate sense that they can control their pain. These patients can be further divided into 2 subgroups based on the degree of hyperalgesia/tenderness, the first subgroup comprises of patients with high hyperalgesia/tenderness, while the second one of patients with moderate hyperalgesia/tenderness (11). In addition, it has been proposed that depressed fibromyalgia patients can also be divided into 2 subgroups, in the first one depression is a co-morbid condition, while in the second depression is the cause of fibromyalgia (16). All these fibromyalgia subgroups are illustrated in Figure 1.

Figure 1. Subgroups of fibromyalgia patients.

In another study fibromyalgia patients were classified as dysfunctional, inter-personally distressed, or adaptive copers, based on their responses to the Multidimensional Pain Inventory. The dysfunctional patients experienced more pain behaviors and overt expressions of pain, distress, and suffering, such as slowed movement, bracing, limping, and grimacing compared to the inter-personally distressed or the adaptive copers (17).

It is of interest that up to 25% of patients correctly diagnosed with a systemic rheumatic disease (e.g. rheumatoid arthritis, systemic lupus erythematosus) will also fulfill the classification criteria for fibromyalgia (18). This is also the case for many patients who experience persistent various forms of pain (including widespread myalgias, arthralgias, and headache), fatigue, neurocognitive dysfunction, and sleep disturbances after an infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that caused a mild to moderate coronavirus disease 2019 (long COVID) (19,20). The most commonly encountered comorbid conditions in fibromyalgia patients are shown in Table 1.

- Table 1. The Most Commonly Encountered Co-Morbid Conditions in Fibromyalgia
Sleep disorders	- Non restorative sleep (alpha-delta sleep anomaly) - Sleep apnea - Restless leg syndrome - Nocturnal myoclonus
Chronic fatigue syndrome (systemic exertion intolerance disease)
Psychiatric disorders	- Anxiety disorders - Depression - Obsessive compulsive disorder
Headache	- Tension type headache - Migraine
Irritable bowel syndrome
Musculofascial pain syndrome	- Temporomandibular disorders - Interstitial cystitis
Dysmenorrhea
Premenstrual syndrome
Non-cardiac chest pain
Raynaud’s phenomenon
Systemic autoimmune diseases	- Rheumatoid arthritis - Systematic lupus erythematosus - Sjögren’s syndrome - Ankylosing spondylitis and other seronegative spondylarthritis - Polymyalgia rheumatica
Long COVID

ASSESSMENT

Fibromyalgia is a chronic illness, with a variety of symptoms that can change during the course of the disease and after treatment. Therefore, its core symptoms should be evaluated both in clinical practice and in treatment trials. A working group within OMERACT (Outcome Measures in Rheumatology) reached a consensus regarding the domains that need to be assessed in clinical trials for fibromyalgia, using Delphi exercises within patients and expert clinicians (21). The fibromyalgia core symptom domains include pain intensity, tenderness, fatigue, sleep disturbance, multidimensional function (including health related quality of life and physical function), patient global impression of change, cognitive dysfunction, and depression. However at the present time, there is no consensus on how to evaluate these domains, in order to quantify fibromyalgia disease activity state and/or response (22).

Pain intensity can be assessed using visual analog scales. It has been proposed to use the wording “please rate your pain by circling on the number that best describes your pain on average” and has anchors that vary from “no pain” to “pain as bad as you can imagine” (23).

Tenderness can be measured by evaluating the alteration of the severity of pain at the tender points, using visual or analog scales, but not by the change in their number. The change in the number of tender points is poorly correlated with improvement in fibromyalgia treatment trials. It has been noted that the tender points measure the combination of tenderness and distress an individual has, rendering them inadequate for the evaluation of tenderness per se (24).

Fatigue can be evaluated by the Fatigue Severity Scale (FSS), which measures the functional outcomes related to fatigue (25). It has been shown that FSS has the most robust psychometric properties of 19 reviewed fatigue measures, while it had the best ability to act as an outcome measure sensitive to change with treatment, in chronically ill patients (26). Other instruments that have been used to assess fatigue in fibromyalgia patients include the Multidimensional Fatigue Index and the Multidimensional Assessment of Fatigue.

Sleep disturbance in fibromyalgia patients has been evaluated by the Medical Outcome Studies (MOS) sleep scale, the Functional Outcomes of Sleep Questionnaire (FOSQ), and the Jenkins Sleep Scale (JSS). Of these instruments MOS sleep scale lacks validity to assess changes in sleep symptoms in fibromyalgia treatment trials, FOSQ has not been adequately validated in fibromyalgia patients, while JSS has been criticized for possible high-recall bias, since it requires the patients to rate the frequency of their symptoms over a period of a month (23).

Multidimensional function, including health related quality of life and physical function, can be evaluated using the fibromyalgia impact questionnaire (FIQ). This represents a useful tool in assessing functional abilities in daily life and measures patient status, progress and outcomes. FIQ is self-administered and is highly sensitive to changes during the course of the disease. However, its functional items are orientated toward high levels of disability, resulting in a possible floor effect, while its physical function items are addressed to women living in affluent countries, generating gender and ethnic bias. To address these issues the Revised Fibromyalgia Impact Questionnaire (FIQR) was developed, having modified physical function questions and including questions on memory, tenderness, balance and environmental sensitivity, while keeping questions that evaluate overall impact and symptoms (27). Other tools for assessing overall function and quality of life include the Health Assessment Questionnaire, the Symptom Interpretation Questionnaire, the Western Ontario and McMaster Universities Osteoarthritis Index, the Patient Global Impression of Change scale (PGIC), and psychometric scales (28).

Patient global impression of change can be evaluated by the Patient Global Impression of Change scale (PGIC), as recommended by the Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT) (29).

Cognitive dysfunction can be evaluated by the Multiple Ability Self-report Questionnaire (MASQ), which is a self-report questionnaire measuring language, visuoperception, verbal memory and attention (30). However self-assessment is poorly correlated with objective measures of cognitive function and has poor discriminating ability for patients with mild cognitive impairment (31).

Depression can be evaluated using the Hospital Anxiety and Depression Scale (HADS) depression subscale (HADS-D). The use of the original Beck Depression Inventory (BDI) as well as the current BDI-II, should be avoided, since they tend to overestimate the presence of major depressive disorder in fibromyalgia patients by evaluating a number of non-depressive symptoms, which are often encountered in fibromyalgia (23).

DIAGNOSIS

As already mentioned, fibromyalgia is characterized by symptoms that can vary in number and intensity not only between different patients, but also in the same patient during the course of the disease. These symptoms are also common in other disorders that can overlap with fibromyalgia, often without having clear boundaries. Additionally, the nature of the symptoms of fibromyalgia makes them difficult to be objectively defined and measured. All these issues have led to significant controversy over the definition and diagnosis of fibromyalgia. The clinical entity of fibromyalgia was first described in 1904, under the term “fibrositis”, after focusing on clinical evidence of muscle sensitivity (32). It was not until 1977 that specific criteria for the diagnosis of fibromyalgia were introduced (33). Since then a number of different criteria have been proposed, based on a combination of tender point examination and the presence of symptoms. In 1986 a committee of the American College of Rheumatology (ACR) started a multicenter study, trying to provide a definition of fibromyalgia and establish classification criteria (34). In 2013, the Analgesic, Anesthetic, and Addiction Clinical Trial Translations Innovations Opportunities and Networks (ACTTION) public-private partnership with the US Food and Drug Administration (FDA) and American Pain Society (APS) initiated the ACTTION-APS Pain Taxonomy (AAPT) in an attempt to develop a diagnostic system that would be clinically useful and consistent across chronic pain disorders, including fibromyalgia (35).

In clinical practice either the 2016 revision of the 2010/2011 fibromyalgia diagnostic criteria or the AAPT criteria can be used to help physicians to diagnose fibromyalgia (36). However, these criteria should be viewed as an aid and not as a gold standard for diagnosing fibromyalgia in clinical practice. Good clinical judgment is necessary to interpret the findings of physical examination and to assess psychological factors and associated comorbidities so as to correctly identify the patients with fibromyalgia (37).

Relevant social, personal and family history can be helpful in establishing the diagnosis of fibromyalgia, since there is evidence that the symptoms of fibromyalgia can appear after a physical or emotional trauma, a medical illness, or asurgical operation, while a family history of fibromyalgia, makes the diagnosis of fibromyalgia more likely (38).

1990 ACR Classification Criteria

In 1990 the American College of Rheumatology (ACR) committee established criteria for the classification of fibromyalgia. According to these criteria fibromyalgia is defined as chronic widespread pain involving both sides of the body, above and below the waist, as well as the whole length of the spine, and excessive tenderness in the pressure of 11 of 18 specific muscle-tendon sites (9 pairs of tender points). The locations of the tender points are described in Figure2 and Table 2. Pressure equivalent of 4 kg/cm is applied to these points using the pulp of the thumb or the first two or three fingers. This can be accurately measured with a dolorimeter or it can be estimated, since 4 kg/cm is the pressure needed to be applied so as to whiten the examiner’s fingernail bed. These criteria specifically state that fibromyalgia is not an exclusionary diagnosis (34).

Figure 2. Fibromyalgia tender points.

Table 2. Description of the Location of Fibromyalgia Tender Points
Occiput:	at the insertions of one or more of the following muscles: trapezius, sternocleidomastoid, splenius capitus, semispinalis capitus
Trapezius:	at the midpoint of the upper border
Supraspinatus:	above the scapular spine near the medial border
Gluteal:	at the upper outer quadrant of the buttocks at the anterior edge of the gluteus maximus
Low cervical:	at the anterior aspect of the interspaces between the transverse processes of C5–C7
Second rib:	just lateral to the second costochondral junctions
Lateral epicondyle:	2 cm distal to the lateral epicondyle
Greater trochanter:	posterior to the greater trochanteric prominence
Knee:	at the medial fat pad proximal to the joint line

The tender points, which are examined in fibromyalgia, are not just areas that the patient feels pain. They are points that fibromyalgia patients are relatively more tender, compared to normal individuals, when pressure is applied on them. But fibromyalgia patients are more tender wherever you apply pressure, not only to some of these 18 specific tender points, including areas previously considered to be “control points” (39). There is evidence that these tender points are areas that everyone is generally more tender. In contrast, fibromyalgia patients are more tender not only to pressure but to other stimuli such as heat, cold and other sensory stimuli, most probably due to decreased pain threshold. The number of tender points an individual has is highly correlated with distress, as defined by the presence of anxiety, depression, sleep disturbance, fatigue and global severity. Tender points have been described as “a sedimentation rate of distress”. Consequently tender points measure the combination of tenderness and distress an individual has (24).

The use of the 1990 ACR classification criteria in clinical practice is surrounded by substantial controversy. Tender points were introduced as an objective physical finding. However, if the physician who performs the physical examination is not experienced enough, tender point counting is impossible to be performed accurately. Most of the physicians examining fibromyalgia patients did not know how to carry out the tender point examination. Consequently, tender point count was not routinely performed, and when performed it was performed inaccurately (40). Another shortcoming of the tender point counting is that it is not as objective as it was initially considered, since the physician can be biased by the patient’s interview that precedes the physical examination. These issues lead to a low inter-examiner reliability of the tender point count (41).

Other more sophisticated measures of assessing tenderness, such as applying stimuli randomly, when the individual cannot anticipate what the next stimulus is going to be, are equally abnormal in fibromyalgia patients, but do not correlate with distress (39). These methods require special training and are more time consuming than the trigger point count. Other alternative assessment methods include functional magnetic resonance imaging (fMRI) and nociceptive flexion reflex (NFR) testing, which documents abnormal pain processing in fibromyalgia. Functional MRI demonstrates similar brain activation in regions involved in pain processing in fibromyalgia patients and normal individuals. However fibromyalgia patients have increased pain sensitivity and brain activation during comparable stimulus (42). Nociceptive flexion reflexes are sensory-motor responses elicited by electrical noxious stimuli, which involve activation of spinal and supraspinal neuronal circuits, providing an objective and quantitative assessment of the function of the pain-controlsystem. It has been demonstrated that the NFR threshold in patients with fibromyalgia is significantly decreased compared with that in controls (43). Of these methods fMRI is expensive and complex compared to NFR testing that appears to be more easily accessible and convenient, since standard electromyographic equipment can be used. This test also seems to eliminate subjective bias and dissimulation (44).

The 1990 ACR classification criteria, define fibromyalgia in terms of pain rather than its other features. However, patients with fibromyalgia apart from tenderness and pain, also have a number of other somatic symptoms. Although non-pain symptoms are important, there is no evidence to support the notion that they are more important than hyperalgesia and allodynia, which are key symptoms of fibromyalgia (44). Many clinicians with experience in fibromyalgia did not feel that the 1990 ACR classification criteria were sufficiently reliable for the diagnosis of fibromyalgia in clinical practice and were considering other aspects of the disease in an attempt to reach a more accurate diagnosis (38).

2010 ACR Preliminary Diagnostic Criteria

To address the aforementioned issues the ACR in 2010 proposed the preliminary diagnostic criteria for fibromyalgia (Table 3), that were not meant to replace the 1990 ACR classification criteria, but to represent an alternative simple and easy method of diagnosis in clinical practice (45). These diagnostic criteria do not require a tender point count. Instead, they rely only on symptoms for the diagnosis of fibromyalgia. They introduced the widespread pain index (WPI), which counts the areas that the patient feels pain during one week preceding the examination, and the symptom severity (SS) scale, which describes the severity of fatigue, unrefreshing sleep, cognitive problems, and a number of associated somatic fibromyalgia symptoms. These symptoms need to be assessed and rated by a physician, therefore the 2010 ACR preliminary diagnostic criteria are in-adequate for patient self-diagnosis.

Two more conditions need to be fulfilled so as to diagnose fibromyalgia. The symptoms need to be present at a similar level for at least 3 months while alternate disorders that would otherwise explain the pain need to be excluded (Table 3). The authors of the 2010 ACR preliminary diagnostic criteria have clarified that the latter condition does not mean that fibromyalgia is an exclusion diagnosis according to these criteria. The diagnosis of fibromyalgia should not be made only when there is not another disease that could explain the pain that would otherwise be attributed to fibromyalgia. It should be noted that rheumatic diseases usually do not cause pain that can be confused with fibromyalgia (3).

Table 3. 2010 ACR Preliminary Diagnostic Criteria
Criteria: A patient satisfies diagnostic criteria for fibromyalgia if the following 3 conditions are met: 1) Widespread pain index (WPI) ≥7 and symptom severity (SS) scale score ≥5 or Widespread pain index (WPI) 3-6 and symptom severity (SS) scale score ≥9. 2) Symptoms have been present at a similar level for at least 3 months. 3) The patient does not have a disorder that would otherwise explain the pain. Ascertainment: 1) WPI Note the number of areas in which the patient has had pain over the last week. In how many areas has the patient had pain? (Score will be between 0 and 19)
	-Neck	-Upper arm, left	-Abdomen	-Upper leg, left
	-Jaw, left	-Upper arm, right	-Upper back	-Upper leg, right
	-Jaw, right	-Lower arm, left	-Lower back	-Lower leg, left
	-Shoulder girdle, left	-Lower arm, right	-Hip (buttock, trochanter), left	-Lower leg, right
	-Shoulder girdle, right	-Chest	-Hip (buttock, trochanter), right
2) SS scale score The SS scale score is the sum of the severity of the 3 symptoms (fatigue, waking unrefreshed, cognitive symptoms) plus the extent (severity) of somatic symptoms in general. (The final score is between 0 and 12) -For the each of the 3 symptoms below, indicate the level of severity over the past week using the following scale: 0 = no problem 1 = slight or mild problems, generally mild or intermittent 2 = moderate, considerable problems, often present and/or at a moderate level 3 = severe: pervasive, continuous, life-disturbing problems Fatigue (0-3) Waking unrefreshed (0-3) Cognitive symptoms (0-3) -Considering somatic symptoms in general, indicate whether the patient has: muscle pain, irritable bowel syndrome, fatigue/tiredness, thinking or remembering problem, muscle weakness, headache, pain/cramps in the abdomen, numbness/tingling, dizziness, insomnia, depression, constipation, pain in the upper abdomen, nausea, nervousness, chest pain, blurred vision, fever, diarrhea, dry mouth, itching, wheezing, Raynaud's phenomenon, hives/welts, ringing in ears, vomiting, heartburn, oral ulcers, loss of/change in taste, seizures, dry eyes, shortness of breath, loss of appetite, rash, sun sensitivity, hearing difficulties, easy bruising, hair loss, frequent urination, painful urination, and bladder spasms 0 = no symptoms 1 = few symptoms 2 = a moderate number of symptoms 3 = a great deal of symptoms

Table 3. 2010 ACR Preliminary Diagnostic Criteria

Criteria:

A patient satisfies diagnostic criteria for fibromyalgia if the following 3 conditions are met:

1) Widespread pain index (WPI) ≥7 and symptom severity (SS) scale score ≥5 or

Widespread pain index (WPI) 3-6 and symptom severity (SS) scale score ≥9.

2) Symptoms have been present at a similar level for at least 3 months.

3) The patient does not have a disorder that would otherwise explain the pain.

Ascertainment:

1) WPI

Note the number of areas in which the patient has had pain over the last week. In how many areas has the patient had pain?

(Score will be between 0 and 19)

-Neck

-Upper arm, left

-Abdomen

-Upper leg, left

-Jaw, left

-Upper arm, right

-Upper back

-Upper leg, right

-Jaw, right

-Lower arm, left

-Lower back

-Lower leg, left

-Shoulder girdle, left

-Lower arm, right

-Hip (buttock, trochanter), left

-Lower leg, right

-Shoulder girdle, right

-Chest

-Hip (buttock, trochanter), right

2) SS scale score

The SS scale score is the sum of the severity of the 3 symptoms (fatigue, waking unrefreshed, cognitive symptoms) plus the extent (severity) of somatic symptoms in general.

(The final score is between 0 and 12)

-For the each of the 3 symptoms below, indicate the level of severity over the past week using the following scale:

0 = no problem

1 = slight or mild problems, generally mild or intermittent

2 = moderate, considerable problems, often present and/or at a moderate level

3 = severe: pervasive, continuous, life-disturbing problems

Fatigue (0-3)

Waking unrefreshed (0-3)

Cognitive symptoms (0-3)

-Considering somatic symptoms in general, indicate whether the patient has: muscle pain, irritable bowel syndrome, fatigue/tiredness, thinking or remembering problem, muscle weakness, headache, pain/cramps in the abdomen, numbness/tingling, dizziness, insomnia, depression, constipation, pain in the upper abdomen, nausea, nervousness, chest pain, blurred vision, fever, diarrhea, dry mouth, itching, wheezing, Raynaud's phenomenon, hives/welts, ringing in ears, vomiting, heartburn, oral ulcers, loss of/change in taste, seizures, dry eyes, shortness of breath, loss of appetite, rash, sun sensitivity, hearing difficulties, easy bruising, hair loss, frequent urination, painful urination, and bladder spasms

0 = no symptoms

1 = few symptoms

2 = a moderate number of symptoms

3 = a great deal of symptoms

There is evidence that there is good agreement between the 1990 ACR classification criteria and the 2010 ACR preliminary diagnostic criteria (46–53). However, these criteria are expected not to agree completely, as the former are focused on the presence of tender points while the latter on the presence of symptoms. The 1990 criteria can diagnose fibromyalgia in patients who do not have sufficiently high symptom score according to the 2010 criteria, while the 2010 criteria can diagnose fibromyalgia in patients who do not have sufficient tender points according to the 1990 criteria.

The introduction of the 2010 ACR preliminary diagnostic criteria was surrounded by controversy too. In particular, they have been criticized for being completely symptom focused, ill-defined, and lucking some mechanistic features of fibromyalgia, such as hyperalgesia, central sensitization and dysfunctional pain modulation (54). Additionally, these diagnostic criteria are based on the subjective assessment of the patient’s somatic symptoms by the physician, adding ambiguity and influencing repeatability among different physicians (55). A self-reported version of the 2010 ACR preliminary diagnostic criteria was developed in 2011, so as to be used in survey research, and not in clinical practice (56). These criteria are known as the modified 2010 ACR preliminary diagnostic criteria or the 2011 ACR survey criteria. They introduced the fibromyalgia severity (FS) score (originally called fibromyalgianess scale) which is the sum of the self-reported WPI and SS score. This score can be used as an approximate measure of the severity of fibromyalgia. The FS score has also been called polysymptomatic distress (PSD) scale. It has been proposed that the markers of physical and psychological distress have a continuous distribution in the general population with fibromyalgia patients being at the extreme end of this distribution (57). The PSD scale could be useful to define the position of each individual in this continuum, without having to differentiate between patients with fibromyalgia and those without, as this distinction can sometimes be unclear if not arbitrary (58).

2016 Revisions to the 2010/2011 Fibromyalgia Diagnostic Criteria

A limitation of the WPI is the fact that it counts the number of painful areas without considering their distribution in the body. Patients with regional pain disorders can fulfill the 2010 ACR preliminary diagnostic criteria since pain can be located in 3 or more areas in the same region (59). To overcome this issue the 2016 revision of the diagnostic criteria require the pain to be generalized (multisite pain). The areas WPI assesses are divided in 5 regions (Table 4) and the diagnosis of fibromyalgia requires the distribution of pain in 4 out of 5 regions (60). The jaw, the chest and the abdomen area are problematic when they are used to define a region. In this way they are excluded from the definition of generalized pain (61). Since pain needs to be located in at least 4 areas according to the 2016 revision, the previous criterion for diagnosis, WPI of 3-6 and SS scale score ≥9 was changed to WPI of 4-6 and SS scale score ≥9.

The 2010 and 2011 ACR preliminary diagnostic criteria are extremely similar. Their difference is that the 2010 criteria are physician-based and can be used in clinical practice for the diagnosis of fibromyalgia, while the 2011 criteria are self-reported and can be used only in survey research. According to the 2010 criteria the SS scale assesses a wide range of somatic symptoms, which makes them impractical for use in questionnaires. With the 2016 revision the assessment of somatic symptoms that is included in the SS scale is limited to headaches, pain and cramps in the lower abdomen and depression. In this way, there is no longer need for different criteria for clinical practice and for survey research. The same criteria can be used in both settings having 2 different methods of administration.

One prerequisite for diagnosis of fibromyalgia according to the 2010 ACR preliminary diagnostic criteria is the patient not to have a condition that would otherwise explain the pain. The authors of these criteria clarified that this does not mean that the diagnosis of fibromyalgia is an exclusion diagnosis. However, this phrasing was not considered clear enough and caused significant misunderstanding. In this way this criterion was removed in the 2016 revision. The diagnosis of fibromyalgia can be valid even if there is another condition that can cause the pain that is attributed to fibromyalgia. According to this definition fibromyalgia can coexist with other clinically significant conditions that can cause pain.

Table 4. 2016 Revisions to the 2010/2011 Fibromyalgia Diagnostic Criteria
Criteria: A patient satisfies diagnostic criteria for fibromyalgia if the following 3 conditions are met: 1) Widespread pain index (WPI) ≥7 and symptom severity (SS) scale score ≥5 or Widespread pain index (WPI) 4-6 and symptom severity (SS) scale score ≥9. 2) Generalized pain: Pain must be present in at least 4 of 5 regions. Jaw, chest, and abdominal pain are not included in generalized pain definition. 3) Symptoms have been generally for at least 3 months. 4) A diagnosis of fibromyalgia is valid irrespective of other diagnoses. A diagnosis of fibromyalgia does not exclude the presence of other clinically important illnesses. Ascertainment: 1) WPI Note the number of areas in which the patient has had pain over the last week. In how many areas has the patient had pain? (Score will be between 0 and 19)
	Region 1: Left Upper Region -Jaw, left * -Shoulder girdle, left -Upper arm, left -Lower arm, left	Region 2: Right Upper Region -Jaw, right * -Shoulder girdle, right -Upper arm, right -Lower arm, right	Region 5: Axial Region -Neck -Upper back -Lower back -Chest * -Abdomen *
	Region 3: Left Lower Region -Hip (buttock, trochanter), left -Upper leg, left -Lower leg, left	Region 4: Right Lower Region -Hip (buttock, trochanter), right -Upper leg, right -Lower leg, right
	* Not included in generalized pain definition
2) SS scale score The SS scale score is the sum of the severity of the 3 symptoms (fatigue, waking unrefreshed, cognitive symptoms) plus the sum of the number of 3 symptoms (headaches, pain or cramps in lower abdomen, depression) (The final score is between 0 and 12) - For the each of the 3 symptoms below, indicate the level of severity over the past week using the following scale: 0 = no problem 1 = slight or mild problems, generally mild or intermittent 2 = moderate, considerable problems, often present and/or at a moderate level 3 = severe: pervasive, continuous, life-disturbing problems Fatigue (0-3) Waking unrefreshed (0-3) Cognitive symptoms (0-3) - During the previous 6 months indicate the number of the following symptoms the patient has been bothered by: - Headaches (0-1) - Pain or cramps in lower abdomen (0-1) - Depression (0-1) The fibromyalgia severity (FS) scale is the sum of the WPI and the SS scale

Table 4. 2016 Revisions to the 2010/2011 Fibromyalgia Diagnostic Criteria

Criteria:

A patient satisfies diagnostic criteria for fibromyalgia if the following 3 conditions are met:

1) Widespread pain index (WPI) ≥7 and symptom severity (SS) scale score ≥5 or

Widespread pain index (WPI) 4-6 and symptom severity (SS) scale score ≥9.

2) Generalized pain: Pain must be present in at least 4 of 5 regions.

Jaw, chest, and abdominal pain are not included in generalized pain definition.

3) Symptoms have been generally for at least 3 months.

4) A diagnosis of fibromyalgia is valid irrespective of other diagnoses. A diagnosis of fibromyalgia does not exclude the presence of other clinically important illnesses.

Ascertainment:

1) WPI

Note the number of areas in which the patient has had pain over the last week. In how many areas has the patient had pain?

(Score will be between 0 and 19)

Region 1: Left Upper Region

-Jaw, left *

-Shoulder girdle, left

-Upper arm, left

-Lower arm, left

Region 2: Right Upper Region

-Jaw, right *

-Shoulder girdle, right

-Upper arm, right

-Lower arm, right

Region 5: Axial Region

-Neck

-Upper back

-Lower back

-Chest *

-Abdomen *

Region 3: Left Lower Region

-Hip (buttock, trochanter), left

-Upper leg, left

-Lower leg, left

Region 4: Right Lower Region

-Hip (buttock, trochanter), right

-Upper leg, right

-Lower leg, right

* Not included in generalized pain definition

2) SS scale score

The SS scale score is the sum of the severity of the 3 symptoms (fatigue, waking unrefreshed, cognitive symptoms) plus the sum of the number of 3 symptoms (headaches, pain or cramps in lower abdomen, depression)

(The final score is between 0 and 12)

- For the each of the 3 symptoms below, indicate the level of severity over the past week using the following scale:

0 = no problem

1 = slight or mild problems, generally mild or intermittent

2 = moderate, considerable problems, often present and/or at a moderate level

3 = severe: pervasive, continuous, life-disturbing problems

Fatigue (0-3)

Waking unrefreshed (0-3)

Cognitive symptoms (0-3)

- During the previous 6 months indicate the number of the following symptoms the patient has been bothered by:

- Headaches (0-1)

- Pain or cramps in lower abdomen (0-1)

- Depression (0-1)

The fibromyalgia severity (FS) scale is the sum of the WPI and the SS scale

AAPT Diagnostic Criteria

In an attempt to improve the recognition of fibromyalgia in clinical practice, the AAPT fibromyalgia working group proposed new diagnostic criteria in 2018 (62). These criteria are similar to the ACR criteria as they require the pain to be generalized (multisite), require the presence of non-pain symptoms and require the symptoms to be present for at least 3 months (Table 5). These diagnostic criteria are more simple than the ACR criteria and they can be easily implemented in primary clinical practice, but some of their aspects have been criticized (63). According to the AAPT criteria the head, the abdomen and the chest are included in the areas that are assessed for the presence of generalized musculoskeletal pain. However, these regions are problematic since pain originating from the teeth, the heart and the bowel can be referred to these areas. Additionally, the AAPT criteria do not have the ability to quantify the severity of fibromyalgia as, apart from the generalized pain, they only assess the presence or absence of the 2 most common non-pain symptoms of fibromyalgia, abolishing all other somatic symptoms. Compared with the 2016 ACR diagnostic criteria, individuals with less symptom severity and fewer pain sites can be classified as fibromyalgia patients, when the AAPT diagnostic criteria are used (64).

Table 5. AAPT Diagnostic Criteria
Criteria: 1) Multisite pain defined as 6 or more pain sites from a total of 9 possible sites: - Head - Left arm - Right arm - Chest - Abdomen - Upper back and spine - Lower back and spine, including buttocks - Left leg - Right leg 2) Moderate to severe sleep problems or fatigue 3) Multisite pain plus fatigue or sleep problems must have been present for at least 3 months NOTE. The presence of another pain disorder or related symptoms does not rule out a diagnosis of fibromyalgia. However, a clinical assessment is recommended to evaluate for any condition that could fully account for the patient’s symptoms or contribute to the severity of the symptoms.

Table 5. AAPT Diagnostic Criteria

Criteria:

1) Multisite pain defined as 6 or more pain sites from a total of 9 possible sites:

- Head

- Left arm

- Right arm

- Chest

- Abdomen

- Upper back and spine

- Lower back and spine, including buttocks

- Left leg

- Right leg

2) Moderate to severe sleep problems or fatigue

3) Multisite pain plus fatigue or sleep problems must have been present for at least 3 months

NOTE. The presence of another pain disorder or related symptoms does not rule out a diagnosis of fibromyalgia. However, a clinical assessment is recommended to evaluate for any condition that could fully account for the patient’s symptoms or contribute to the severity of the symptoms.

EPIDEMIOLOGY

The prevalence of fibromyalgia depends on the criteria used to define it. Most studies use either the 1990 ACR classification criteria or the modified 2010 ACR preliminary diagnostic criteria (2011 ACR survey criteria) and the prevalence varies between 2-4% (65). Using the 2016 ACR diagnostic criteria the prevalence of fibromyalgia in the general population is 3-4% while with the AAPT diagnostic criteria the prevalence of fibromyalgia is 73% higher, ranging from 5% to 7% (64). In the general population the prevalence increases with age from 2% at the age of 20 to 8% at age of 70. Fibromyalgia appears more often in relatives of patients suffering from fibromyalgia (66), whereas there is a significant difference on the women to men ratio depending on the criteria used to define fibromyalgia. When the 1990 ACR classification criteria are used the women to men ratio is 7:1 while when the 2011 ACR survey criteria are used, that do not use the tender point count, the ratio ranges from 4:1 to 1:1 (58,67–69). Using the 2016 ACR or the AAPT diagnostic criteria there is no statistically significant difference in the prevalence of fibromyalgia between women and men (64).

DIFFERENTIAL DIAGNOSIS

Several conditions can mimic or overlap with fibromyalgia. In order to reach a differential diagnosis, careful history taking should be followed by a thorough physical examination. Careful neurologic and musculoskeletal examination needs to be performed in all fibromyalgia patients in order to exclude the presence of such conditions. Mood and functional impairment should also be evaluated. This can be easily performed using simple self-administered questionnaires. Patients with obvious mood disturbances should have a formal evaluation by a mental health professional. Baseline blood tests should be limited to a complete blood count, erythrocyte sedimentation rate, a comprehensive metabolic panel, and thyroid function tests. These tests are usually normal in fibromyalgia patients. Consequently, the identification of abnormalities in any of these examinations might suggest that a different condition is present. Additional tests are not recommended for a diagnosis, unless they are clinically indicated. The disorders that can mimic and/or overlap with fibromyalgia and the characteristic clinical features which differentiate them from fibromyalgia are described in Table 6. The clinical features of fibromyalgia, chronic fatigue syndrome (systemic exertion intolerance disease), depression, migraine, and irritable bowel syndrome often overlap being so interchangeable that some authors consider that these conditions should be approached as a “spectrum” of associated disorders (10). They are also considered as part of the spectrum of post-traumatic stress disorder (70,71).

Table 6. Disorders that can Mimic and/or Overlap with Fibromyalgia Along with Characteristic Clinical Features that Differentiate Them from Fibromyalgia
Disorders	Differentiating clinical features
Rheumatoid arthritis, Systematic Lupus Erythematosus, Sjögren’s syndrome	· Characteristic synovitis and systemic features of connective tissue disease, apart from musculoskeletal pain, fatigue, Raynaud phenomenon, dry eyes and dry mouth, are usually not features of fibromyalgia. · Routine serologic tests are not recommended because of low positive predictive value.
Ankylosing spondylitis, other inflammatory back conditions	· Generally, there is normal spinal motion in fibromyalgia. · Characteristic radiologic features of these disorders are not present in fibromyalgia.
Polymyalgia rheumatica	· Tender points are not always present in polymyalgia rheumatica. · Stiffness is more prominent than pain in polymyalgia rheumatica. · Most patients with polymyalgia rheumatica have increased erythrocyte sedimentation rate, while it is normal in fibromyalgia. · Patients with polymyalgia rheumatica respond extremely well to modest doses of corticosteroids, in contrast to fibromyalgia patients.
Inflammatory myositis, metabolic myopathies	· Myositis and myopathies can cause muscle weakness and muscle fatigue, but they are not usually associated with diffuse pain. · Patients with myositis or myopathies have abnormal muscle enzyme tests and specific histopathologic findings on muscle biopsy, in contrast to fibromyalgia patients (muscle biopsy should be limited to cases that there is clinical evidence suggestive of myositis or myopathy).
Statin myopathy	· Statin myopathy symptoms are limited to muscle weakness and pain without other symptoms associated with fibromyalgia. · Statin myopathy pain is temporally associated with statin therapy. · Statin myopathy can be associated with abnormal muscle enzyme tests.
Infection: chronic viral infection (e.g., infectious mononucleosis, HIV, HTLV, HBV, HCV, Lyme disease), long COVID	· In fibromyalgia patients there is no objective evidence of inflammation or organ system dysfunction
Hypothyroidism	· Although thyroid autoantibodies are common in fibromyalgia patients, thyroid function tests are usually normal.
Hyperparathyroidism	· Hypercalcemia is not present in fibromyalgia.
Cushing’s syndrome	· Cushing’s syndrome is associated with muscle weakness rather than pain. · The characteristic facial and skin signs of Cushing’s syndrome are not present in fibromyalgia.
Adrenal insufficiency	· Adrenal insufficiency causes severe exhaustion, while it is not typically associated with chronic widespread pain.
Hypophosphatasia	· Most hypophosphatasia patients have low alkaline phosphatase
Neurologic diseases: peripheral neuropathies, cervical radiculopathy, entrapment syndromes (e.g., carpal tunnel syndrome), multiple sclerosis, myasthenia gravis	· Multiple sclerosis and myasthenia gravis are associated with post-exercise muscle and generalized fatigue, but not with widespread pain. · Thorough neurologic examination can reveal neurologic signs characteristic of specific diseases.
Myofascial pain syndromes (they may include other common regional pain disorders such as tension headaches, occupational overuse syndrome, cumulative trauma disorder, work related musculoskeletal disorder,idiopathic low back and cervical strain disorders, chronic pelvic pain temporomandibular disorderand interstitial cystitis)	· In myofascial pain syndromes the pain and the tenderness is confined in one anatomic region
Chronic fatigue syndrome (systemic exertion intolerance disease)	· Criteria for the diagnosis of chronic fatigue syndrome: 1. According to the modified United States Centers for Disease Control and Prevention chronic fatigue syndrome is diagnosed when two criteria are fulfilled (72): i) Clinically evaluated, unexplained, persistent or relapsing fatigue that is of new or definite onset; is not the result of ongoing exertion; is not alleviated by rest; and results in substantial reduction in previous levels of occupational, educational, social, or personal activities ii) Four or more of the following symptoms that last six months or longer: - Impaired memory or concentration - Post-exertional malaise where physical or mental exertions bring on "extreme, prolonged exhaustion and sickness" - Unrefreshing sleep - Muscle pain - Arthralgia in multiple joints - Headaches of new kind or greater severity - Frequent or recurring sore throat - Tender cervical or axillary lymph nodes 2. According to the proposed diagnostic criteria of the United States Institute of Medicine the chronic fatigue syndrome (systemic exertion intolerance disease) is diagnosed when two criteria are fulfilled (73): i) All of the following symptoms: - A substantial reduction or impairment in the ability to engage in pre-illness levels of occupational, educational, social, or personal activities that persists for more than 6 months and is accompanied by fatigue, which is often profound, is of new or definite onset (not lifelong), is not the result of ongoing excessive exertion, and is not substantially alleviated by rest - Post-exertional malaise* - Unrefreshing sleep* ii) Two or more of the following manifestations: - Cognitive impairment* - Orthostatic intolerance - * The diagnosis should be questioned if patients do not have these symptoms at least half of the time with moderate, substantial, or severe intensity. · Chronic widespread pain is not included in the criteria for diagnosis of chronic fatigue syndrome
Psychiatric disorders: depression, anxiety disorders, posttraumatic stress disorder	· In fibromyalgia patients with a concurrent psychiatric disorder, the attribution of symptoms to fibromyalgia or the psychiatric disorder is not always possible.
Sleep disorders: obstructive sleep apnea, restless legs syndrome, periodic limb movement disorders	· Detail history can identify the majority of the primary sleep disorders. · Chronic widespread pain is uncommon in primary sleep disorders.
Irritable bowel syndrome	· According to the 2009 American College of Gastroenterology recommendations for the diagnosis of irritable bowel syndrome, it is defined by abdominal pain or discomfort that occurs in association with altered bowel habits over a period of at least three months (74).
Temporomandibular disorders	· Temporomandibular disorders are characterized by recurrent facial/jaw pain and/or limitation in jaw opening occurring in the past six months.
Tension – Migraine headache	· Tension – migraine headache is characterized by recurrent headaches (at least five for migraine, at least 10 for tension-type) lasting 30 minutes.
Interstitial cystitis	· According to the American Urological Association guidelines interstitial cystitis is defined as an unpleasant sensation (pain, pressure, discomfort) perceived to be related to the urinary bladder, associated with lower urinary tract symptoms of more than six weeks duration, in the absence of infection or other identifiable causes (75).

HIV: human immunodeficiency virus, HTLV: human T-lymphotropic virus, HBV: hepatitis B virus, HCV: hepatitis C virus, COVID: coronavirus disease

PATHOPHYSIOLOGICAL MECHANISMS

Pain sensitivity in the population occurs over a wide continuum, forming a classic bell-shaped curve, just like any other physiologic variable. Genetic predisposition in combination with environmental factors, determine the place that each individual takes in this continuum. People who are placed in the right end of this curve are very sensitive to pain and they can probably develop pain even without having any inflammation or damage in the peripheral tissues. This pain can be either regional or widespread (39).

In the past fibromyalgia was thought to be a primary muscle disease. However, controlled studies found no evidence of significant pathologic or biochemical muscle abnormalities that can be the cause of chronic widespread pain and tenderness. Most investigators believe that any muscle pathology is secondary to chronic pain and inactivity, rather than primary in nature (76–79). Current research suggests that altered central nervous system (CNS) physiology might underlie the symptoms of fibromyalgia. Abnormal central sensory processing of pain signals seems to play a significant role in the pathogenesis of fibromyalgia. This dysregulation of the nociceptive system can arise from a combination of interactions between neurotransmitters, cytokines, hormones, the autonomic nervous system, behavioral constructs, and external stressors.

Abnormalities in Sensory Processing

Fibromyalgia overlaps with several other similar syndromes including chronic fatigue syndrome (systemic exertion intolerance disease) and myophasial pain syndrome (Table 6). It has been proposed that these disorders should be considered as members of the central sensitivity syndromes (Table 7). These similar and overlapping syndromes are bound by the common mechanism of central sensitization that involves hyper-excitement of the second-order neurons, especially the wide-dynamic-range neurons (WDR) in the dorsal horns of the spinal cord, by various synaptic and neurotransmitter/neuromodulator activities (6). Central sensitization is clinically and physiologically characterized by hyperalgesia, allodynia, expansion of the receptive field (pain expanding beyond the area of the peripheral nerve supply, after the application of a nociceptive stimulus), prolonged electrophysiological discharge, and an after-stimulus unpleasant quality of the pain (e.g., burning, throbbing, tingling or numbness). Parallel to central sensitization, temporal summation takes place in the second-order neurons. It is characterized by a progressive increase in electrical discharges (and consequently increase in the perceived intensity of pain) in response to repetitive short noxious stimuli. Temporal summation involves the production of second pain, which is described as dull or burning, and leaves an after-stimulus unpleasant sensation (80).

Table 7. Central Sensitivity Syndromes
· Fibromyalgia · Chronic fatigue syndrome (systemic exertion intolerance disease) · Irritable bowel syndrome · Tension type headaches · Migraine · Temporomandibular disorder · Myophasial pain syndrome · Restless leg syndrome · Periodic limb movements in sleep · Primary dysmenorrhea · Interstitial cystitis · Posttraumatic stress disorder

Table 7. Central Sensitivity Syndromes

· Fibromyalgia

· Chronic fatigue syndrome (systemic exertion intolerance disease)

· Irritable bowel syndrome

· Tension type headaches

· Migraine

· Temporomandibular disorder

· Myophasial pain syndrome

· Restless leg syndrome

· Periodic limb movements in sleep

· Primary dysmenorrhea

· Interstitial cystitis

· Posttraumatic stress disorder

N-methyl-D-aspartate (NMDA) receptors are mostly responsible for escalation of hyperexcitability of the second-order nociceptive neurons. The role of the major neurotransmitters of the nociceptive system that participate in signal conduction at the level of the spinal cord is briefly illustrated in Figure 3 (6).

Figure 3. The role of the major neurotransmitters of the nociceptive system that participate in signal conduction at the level of the spinal cord. SP: Substance P, NGF: nerve growth factor, NMDA: N-methyl-D-aspartate, D: dopamine.

The second-order neurons have ascending projections to the thalamus, hypothalamus, the limbic system and the somatosensory cortex. These supraspinal structures are involved in the sensory, evaluative and affective dimensions of pain (e.g., unpleasantness, emotional reaction). Several descending pathways from the cortico-reticular system, locus ceruleus, hypothalamus, brain stem, and local spinal cord interneurons terminate to the dorsal horn cells. These pathways utilize neurotransmitters that include serotonin (5-HT), norepinephrine, γ-amino-butyric acid (GABA), enkephalins and adenosine (6). This descending system, once thought to be predominantly inhibitory, is now known to have a facilitatory potential (81). Evidence suggests that the 5-HT₃ receptor has a facilitatory function, while the 5H-T_1Areceptor is inhibitory. The ascending and descending pathways should not be considered as dichotomous in function. They are interactive and their functions are bidirectional. Both pathways can either facilitate or inhibit pain, depending on the site of action and the neurotransmitters that are used (6).

The dysregulation of the nociceptive system, either at the level of the dorsal horns of the spinal cord, or at the level of the ascending and descending pathways, can lead to its hyper-excitability. In other words, it can lead to central sensitization. Several factors may amplify and sustain central sensitization through interactive and synergistic actions. These factors are summarized in Table 8 (80). Central sensitization can become self-sustained, even when the event that triggered it no longer exists, due to long-term CNS plasticity.

Table 8. Factors that may Amplify and Sustain Central Sensitization
· Genetics · Sympathetic over-activity · Endocrine dysfunctions · Viral infection · Peripheral nociception generators (e.g., arthritis) · Poor sleep · Environmental stimuli (e.g., weather, noise, chemicals) · Psychological distress (e.g., adverse childhood experience)

Table 8. Factors that may Amplify and Sustain Central Sensitization

· Genetics

· Sympathetic over-activity

· Endocrine dysfunctions

· Viral infection

· Peripheral nociception generators (e.g., arthritis)

· Poor sleep

· Environmental stimuli (e.g., weather, noise, chemicals)

· Psychological distress (e.g., adverse childhood experience)

Neuroimaging studies provide moderate evidence for structural changes in the brain of patients with fibromyalgia. Gray matter volume appears to be reduced in areas related with pain processing, such as the cingulate, the insular, and the prefrontal cortices (82). Functional MRI studies reveal alterations in the functional connectivity of brain areas responsible for pain processing and provide support of functional dysregulation of the ascending and descending pain pathways in fibromyalgia patients (82–85). Additionally, alterations in neuronal activity between the ventral and the dorsal spinal cord have been demonstrated in fibromyalgia patients (86).

Although nearly all of the research on sensory processing in fibromyalgia has focused on the processing of pain, there are some data suggesting a more generalized disturbance in sensory processing. There is evidence that fibromyalgia patients have a hypersensitivity to unpleasant stimuli of other sensory systems. For example, many patients experience reduced tolerance to loud noises, bright lights, odors, drugs, and chemicals (87,88).

Neurotransmitters

The levels of Substance P (SP) in the cerebrospinal fluid (CSF) in patients with fibromyalgia are significantly increased compared to normal individuals, whereas CSF levels of serotonin metabolites are decreased, as are metabolites of dopamine and norepinephrine (89).

The first direct evidence that fibromyalgia patients may have abnormal dopamine response to pain originated from positron emission tomography (PET) competitive binding studies using the D₂/D₃ receptor antagonist [¹¹C] raclopride. It was shown that dopamine is released in response to tonic toxic noxious muscle stimulation, but not after non-painful muscle stimulation in healthy human subjects. In contrast the dopamine response in fibromyalgia patients did not differ between painful and non-painful muscle stimulation (87). There are indications that disturbances of the opioidergic system occur in fibromyalgia patients, as there is an up-regulation of opioid receptors in the periphery, with a reduction of the brain opioid receptors (90,91). This implies an increased baseline endogenous opioidergic activity. Opioids can activate glial cells, via a non-stereoselective activation of toll-like receptor 4 (TLR4). Glial cells in turn can mediate pain by releasing neuroexcitatory, pro-inflammatory products (92).

In a study where fibromyalgia patients were evaluated for cortical excitability and intracortical modulation using transcranial magnetic stimulation of the motor cortex, it was shown that there were deficits in intracortical modulation of GABAergic and glutamatergic mechanisms (93). Diminished inhibitory neurotransmission resulting from lower concentrations of GABA within the right anterior insula of patients with fibromyalgia was documented using proton magnetic resonance spectroscopy (94). Evidence for enhanced glutaminergic neurotransmission in fibromyalgia patients is derived from studies that used magnetic resonance spectroscopy. It was shown that fibromyalgia patients have significantly higher levels of glutamine within the posterior insula and in the right amygdala (95,96). The levels of brain-derived neurotrophic factor, which is involved in neuronal survival and synaptic plasticity of the central and peripheral nervous system, have been found to be increased both in the brain and in the plasma of fibromyalgia patients (97).

Cytokines

Although fibromyalgia is not considered an inflammatory disorder, the interaction of immunological mechanisms with pain physiology, has led to the identification of alterations in the levels of various cytokines (98). However, it is not clear whether cytokine changes are the cause of pain in these patients, or just its consequence.

The serum levels of interleukin 1 receptor antibody (IL-1Ra), IL-6, and IL-8, and the plasma levels of IL-8 are higher in fibromyalgia patients, compared to controls (99). Inflammatory cytokines such as IL-1β, IL-6 and tumor necrosis factor alpha (TNFα) have been detected in skin biopsies taken from fibromyalgia patients, possibly indicating an element of neurogenic inflammation (100). Lower levels of the anti-inflammatory cytokines IL-4 and IL-10 have been reported in fibromyalgia patients compared to healthy controls (101,102). However, the interpretation of cytokine levels is not always easy. Most cytokines are expressed in low levels and a sensitive bioassay is needed for their detection. Consequently, a negative result can merely be the ramification of a not sensitive enough method. Additionally, cytokine levels can be vigorously influenced by a number of factors, including circadian rhythmicity, physical activity, and co-morbid conditions, including depression.

Inflammatory cytokines like IL-1β, IL-6 and TNFα can elicit pain, induce hyperalgesia and they are associated with neuropathic pain (103), although they do not appear to be involved in “normal” pain. Although serum cytokines do pass the blood brain barrier the release of pro-inflammatory cytokines by immune cells in the body leads, in turn, to release of pro-inflammatory cytokines by glial cells within the brain and spinal cord (104).Inflammatory cytokines like IL-1β, IL-6 and TNFα can also cause activation of the hypothalamic-pituitary-adrenal (HPA) axis alone, or in synergy with each other. There is evidence to suggest that IL-6, which is the main endocrine cytokine, plays the most significant role in the immune stimulation of the axis, especially in chronic inflammatory stress (105). IL-6 can stimulate the hypothalamic secretion of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), leading to the increase of serum adrenocorticotropic hormone (ACTH) and cortisol levels (106).

Hypothalamic-Pituitary-Adrenal Axis

There is substantial data supporting an abnormal function of the HPA axis in fibromyalgia. However, the results from studies on the HPA axis function in fibromyalgia patients are relatively heterogeneous and partially contradictory (107). The 24-hour urinary free cortisol (UFC) has been found to be reduced or normal (108). Findings regarding alterations in diurnal variation of cortisol secretion are also inconsistent. Although normal diurnal patterns of ACTH and cortisol have been reported, there is data demonstrating flattened cortisol diurnal rhythm with normal morning peak and higher evening cortisol, levels (109). It has been demonstrated that there is a significant decrease in the rate of decline from acrophase (peak) to nadir for diurnal cortisol levels in fibromyalgia patients, compared to controls, while there is no change in the ACTH to cortisol ratio (108). This implies a decreased ability of the HPA axis to return to baseline after a physiologic stimulation by meals, several other activities or even pain (108). Decreased morning cortisol release and reduced frequency of cortisol pulses over 24 hours have also been reported. There is evidence to suggest that the reduced cortisol release in fibromyalgia patients is associated with depressive symptoms and experiences of childhood trauma (109).

In line with studies suggesting reduced adrenal output in fibromyalgia patients, reduced cortisol secretion has been reported in response to pharmacological challenge with synthetic ACTH_1-24 and insulin tolerance test (109). Patients with fibromyalgia exhibit increased ACTH, but normal cortisol response to CRH stimulation, compared to controls. This finding suggests a sensitization of the pituitary in combination with a degree of adrenal insufficiency (109). Arginine vasopressin (AVP), an ACTH secretagogue, has been found to be more increased in response to the postural challenge test in fibromyalgia patients, compared to controls (110). Alterations in the feedback regulation of HPA axis have also been reported in fibromyalgia patients, using the overnight dexamethasone suppression test (DST). Increased rates of non-suppression following the standard (1 mg) DST have been reported in fibromyalgia patients, compared to controls, but this finding was difficult to be interpreted as it was associated with depression. Interestingly, other studies have revealed lower rates of non-suppression in fibromyalgia patients (111).

There are indications that there is a dissociation between total and free cortisol levels in fibromyalgia patients, with normal salivary and plasma free cortisol despite diminished total cortisol levels. One possible explanation of this finding is a reduced concentration of the glucocorticoid binding globulin (CBG). Reduced levels of CBG have been reported in fibromyalgia patients compared to controls. It is of particular interest that chronic social stress might result in reduced CBG levels, whereas IL-6 and IL-1β that can also inhibit the production of CBG may contribute further (109). Apart from HPA axis abnormalities in fibromyalgia patients, abnormal levels of growth hormone have also been found in some, but not all reports (112). On the other hand the levels of sex hormones have not been clearly shown to differ between female fibromyalgia patients and controls (113).

Autonomic Nervous System

Sympathetic hyperactivity, often associated with sympathetic hypo-activity in response to stressors, or parasympathetic underactivity has been described in fibromyalgia. Attenuated sympathetic and parasympathetic activity was demonstrated in a study where fibromyalgia patients and healthy controls were assessed for 24 hours in a controlled hospital setting, including relaxation, a test with prolonged mental stress and sleep. The urinary catecholamine levels were found to be lower in fibromyalgia patients compared to controls. Patients with fibromyalgia had significantly lower adrenaline levels during the night and the second day of the study, and significantly lower dopamine levels during the first day, the night, and the second day. Furthermore, heart rate during relaxation and sleep was significantly higher in patients than in controls (114). In another study, plasma catecholamines, ACTH, and cortisol levels were reduced in 16 fibromyalgia patients compared to 16 healthy controls while performing static knee extension until exhaustion (115). Nocturnal heart rate variability indices have been shown to be significantly different in fibromyalgia women compared to healthy individuals, indicating a sympathetic predominance (116). In addition, orthostatic hypotension and increased pain in response to tilt table test have been described along with increased resting supine heart rate and decreased heart rate variability (117,118). IL-6 administration causes exaggerated norepinephrine responses and increases in heart rate, as well as delayed ACTH release, suggesting an incapacitated stress-regulating system (119). In-vitro testing of beta adrenergic receptor mediated cyclic AMP generation has revealed decreased responsiveness to beta-adrenergic stimulation (120).

Overall, it has been suggested that sympathetic dysfunction can not only cause diffuse pain, but also contribute to other symptoms like sleep disturbances, due to sustained nocturnal sympathetic activity, and fatigue, due to deranged sympathetic response to stress (6).

Psychological, Cognitive, and Behavioral Factors

Pain apart from a sensory-discriminative dimension, which includes the location and the intensity of pain, has a very significant psychological component. This includes the affective dimension of pain, the emotional valance of pain in other words, as well as attention and cognitive aspects, which are based on CNS mechanisms. Emotion and selective attention can enhance pain perception, with the involvement of the descending pathways that have a facilitatory effect on the spinal cord dorsal horn neurons (80). Catastrophizing has been shown to be related to decreased pain threshold and tolerance to heat stimuli in fibromyalgia patients. However there is a subgroup of fibromyalgia patients that is very tender, despite the fact that they do not catastrophize and they have a moderate control over their pain (11). A fMRI study has shown that although depression is associated with the magnitude of neuronal activation in brain regions that process the affective-motivational dimension of pain, neither the extent of depression nor the presence of comorbid major depression modulated the sensory-discriminative aspects of pain processing in fibromyalgia patients (121). Catastrophizing, has been associated with increased activity in brain areas related to anticipation, attention and the emotional aspects of pain, as shown by fMRI in response to pressure stimuli. This study also revealed an association between catastrophizing and increased activity in the secondary somatosensory cortex, indicating that the way patients think about their pain might actually influence its sensory processing (80).

Genetic Predisposition

It is currently well established that familial aggregation is a characteristic of fibromyalgia. First degree relatives of fibromyalgia patients are 8.5 times more likely to have fibromyalgia than relatives of patients with rheumatoid arthritis (12). As with other complex and multifactorial syndromes, the occurrence of familial aggregation in the case of fibromyalgia does not necessarily imply a genetic basis. Shared environmental factors and learned patterns of behavior that may evolve within families are equally valid targets of investigation.

Genome-wide association studies have shown significant differences in allele frequencies between fibromyalgia patients and controls. However many of the results are inconsistent, without being replicated. Additionally the small sample size of these studies limits the genetic variants that can be identified only to those with large effects (122–126). The coexistence of other comorbidities in fibromyalgia patients further obscures these results. In a large scale genome-wide association study of 26,749 individuals the overall estimated heritability of fibromyalgia was 14%. There was a significant difference between age groups, with the heritability in individuals less than 50 years of age to be 23.5%, while in those over 60 years of age it was only 7.5% (126).

Although no specific candidate gene has been identified, the following genes have been associated with fibromyalgia:

SEROTONIN TRANSPORTER (5-HTT) GENE

An increased frequency of the S/S genotype of the 5-HTT gene has been found in fibromyalgia patients compared to controls (127,128). However this putative association may be limited to patients with concomitant affective disorders, since it was not confirmed in fibromyalgia patients without depression or anxiety (129).

D₄ RECEPTOR GENE

Polymorphisms affecting the number of repeats in the third cytoplasmic loop of the dopamine D₄ receptor gene have been shown to be significantly decreased in frequency in fibromyalgia patients (130).

CATECHOL-O-METHYL TRANSFERASE (COMT) GENE

The homozygous low activity (met/met) and the heterozygous low activity (val/met) COMT genotypes occur more often in fibromyalgia patients than in controls, whereas the homozygous high activity (val/val) genotype is less frequent (131). However in a meta-analysis COMT gene val(158)met polymorphism was not associated with an increased risk for fibromyalgia (132). The met/met genotype has been associated with greater fibromyalgia illness severity across the domains of pain, fatigue, sleep disturbance, and psychological distress, while fibromyalgia patients with the met/met polymorphism experienced a greater decline in exhibiting a positive attitude on days when pain was elevated than did patients with the val/met or val/val genotype (133).

OPIOID RECEPTOR μ 1 GENE (OPRM1)

The 118G allele frequency has been described to be significantly lower in patients with fibromyalgia than in the control group (134).

ADRENERGIC RECEPTOR GENES

The presence fibromyalgia and its symptom severity is associated with various adrenergic receptor gene polymorphisms (135).

Other genes associated with the regulation of nociceptive and analgesic neuronal pathways

Specific variants of trace amine-associated receptor 1 (TAAR1) gene, regulator of G-protein signaling 4 (RGS4) gene, cannabinoid receptor 1 (CNR1) gene, and glutamate receptor, ionotrophic, AMPA 4 (GRIA4) gene, have been associated with fibromyalgia (123).

External Stressors

Almost all diseases are caused by a combination of genetic predisposition and the effect of environmental factors. We are now beginning to better understand the environmental factors that seem to be important in triggering fibromyalgia. Most of them act as “stressors” that when superimposed onto a deranged stress-response system can lead to the dysregulation of the nociceptive system.

PERIPHERAL PAIN SYNDROME

Pain due to damage or inflammation of peripheral tissues may trigger fibromyalgia. Additionally, small fiber neuropathy can be associated with fibromyalgia (136–138). Chronic localized – regional pain can lead to central sensitization and pain dis-inhibition, causing pain hypersensitivity and widespread pain. Systematic autoimmune diseases can be associated with fibromyalgia too. Approximately 20-25% of patients with rheumatoid arthritis, systemic lupus erythematosus and ankylosing spondylitis, have co-morbid fibromyalgia (18). In such cases, it is important to realize that many symptoms may be attributed to fibromyalgia rather than the underlying disorder. This recognition has significant clinical implications.

INFECTIONS

Various infections have been linked to the development of fibromyalgia and chronic fatigue syndrome (systemic exertion intolerance disease). Epstein-Barr virus, parvovirus, Lyme disease, Q fever, HIV and hepatitis C virus (HCV), have been suggested as triggers of fibromyalgia or chronic fatigue syndrome (systemic exertion intolerance disease), but more robust evidence is needed. The role of vaccination in precipitating fibromyalgia and related syndromes is still not clear (139,140).

PHYSICAL TRAUMA

Various forms of physical trauma have been considered as culprits of triggering the pathogenesis of fibromyalgia. Many patients report the initiation or the exacerbation of their symptoms after a traumatic event such as whiplash injury, while increased rates of fibromyalgia have been demonstrated among patients undergoing cervical trauma during motor vehicle accidents (141,142).

PSYCHOLOGICAL DISTRESS

It has been considered that psychological factors that give rise to chronic stress may initiate the chain of events that leads to fibromyalgia. The chronic stress can be a result of the accumulation of daily stress events. Emotional stress, catastrophic events such as war, job loss, marital discord and excess family responsibilities such as caring for sick elders, have been implicated as triggers of fibromyalgia (143). However the data that supports the notion that psychological stress and distress directly causes fibromyalgia is rather weak (39).

MANAGEMENT

The treatment of fibromyalgia is challenging because of our limited understanding of its pathogenesis and the poor response of patients to conventional pain treatments. The aim of the therapy is to relieve pain and increase function using a multimodal individualized therapeutic strategy which, in most cases, includes pharmacologic and non-pharmacologic interventions. Current clinical-based evidence supports the use of a multimodal program that includes education, exercise, cognitive-behavioral approaches and medications. The treatment should be individualized based on the symptoms, the comorbidities and the preferences of the patient, who should be encouraged to participate in the decision-making process of selecting the optimal therapies (144,145). Coexisting disorders are common in fibromyalgia patients. Their identification and effective treatment can have beneficial effects on fibromyalgia symptoms. It is also important to assure that adequate adherence to both pharmacological and non-pharmacological treatment is maintained, so as to achieve the optimal benefit from these treatments.

Non-Pharmacological Management

PATIENT EDUCATION

The first step should be the education of the patient. The patients with fibromyalgia need to understand their illness before any treatment modality is used (146). Providing a diagnosis, “labeling” the patient with fibromyalgia, may have beneficial effects. It has been shown that fewer symptoms and an improvement in health status is noted after the patients are informed of their diagnosis (147,148). The physician should clarify that fibromyalgia is a real illness and the symptoms the patient experiences are not imaginary. The role of neurotransmitters and neuromodulators in pain perception, fatigue, abnormal sleep and mood disturbances should be discussed, so as the patient to understand the rationale of the pharmacologic therapy, especially when antidepressant drugs are used. The significance of good sleep hygiene should be reviewed and poor sleep habits should be addressed. Fibromyalgia patients who are overweight or obese should be informed for the adverse effect of increase body mass index to fibromyalgia symptoms and quality of life (149). For these patients weight reduction should be encouraged. The patient also needs to acknowledge that fibromyalgia is a chronic relapsing condition without though being life-threatening nor deforming.

EXERCISE

Another potent non-pharmacological treatment for fibromyalgia is exercise. It has been reported that an exercise program incorporating aerobic, strengthening, and flexibility elements can lead to greater benefits than a relaxation program. Exercise in fibromyalgia patients should have two major components: strengthening to increase soft-tissue length and joint mobility, and aerobic conditioning to increase fitness and function, reduce fibromyalgia symptoms and improve quality of life (144,150–153). Exercise should be of low impact and of sufficient intensity so as to be able to change aerobic capacity (28). Successful interventions include fast walking, biking, swimming, water aerobics, tai chi, and yoga. Land and aquatic training appears to be equally beneficial (154). An improvement in the severity of fibromyalgia symptoms has also been achieved with web-based exercise programs (155). A gradual incremental increase in physical activity should be encouraged as it is common for fibromyalgia patients to initially perceive an aggravation of their pain and fatigue at the beginning of a training program. It has been suggested that in the presence of exercise-induced pain, the intensity and duration of exercise should be reduced, while its frequency should be maintained, so as to avoid any further decrease in exercise tolerance (144). The type and intensity of the exercise program should be individualized and should be based upon patient preference and the presence of any other cardiovascular, pulmonary, or musculoskeletal comorbidities.

COGNITIVE-BEHAVIORAL APPROACHES

One of the goals of the management should be to help patients understand the effect of thoughts, beliefs and expectations on their symptoms. This can help them to abolish the perception of helplessness and the catastrophizing thoughts that can adversely influence their condition. Patients with greater self-efficacy are more likely to have a good response to treatment programs and experience better outcomes. The beneficial effect of cognitive-behavioral therapies in fibromyalgia patients with anxiety and depression disorders is limited to a reduction of negative mood, while the rest of the patients also demonstrate a reduction of pain and fatigue. Psychologically based interventions, have been proven to be useful when they are compared to no treatment or treatment other than aerobic exercise (156). In a 2021 systematic review and meta-analysis there was high quality evidence that cognitive-behavioral therapy can significantly reduce the pain intensity in fibromyalgia patients for 3 months (157). Preliminary data from functional MRI studies suggest that cognitive-behavioral therapies have the ability to restore the alterations in the functional connectivity of brain areas responsible for pain processing observed in fibromyalgia patients (158,159).

COMPLEMENTARY AND ALTERNATIVE APPROACHES

Acupuncture

Acupuncture is the insertion of needles in the human body. There are different styles of acupuncture depending on the location and the depth the needles are inserted. The inserted needles can be stimulated by heat, electrical current (electro-acupuncture), mechanical pressure (acupressure), or laser (laser acupuncture). The most common type of acupuncture involves skin penetration without stimulation (manual acupuncture). Sham or fake acupuncture is a research tool to control the effects of real acupuncture. It can involve skin contact with the needles without actual penetration or needle insertion in areas other than the ones usually targeted.

In a high quality meta-analysis it was demonstrated that the effects of manual acupuncture on pain, sleep quality and global well-being did not differ significantly from the effects of sham acupuncture. On the contrary electro-acupuncture significantly reduced pain, fatigue, and stiffness, while it improved sleep quality and global well-being when compared to sham acupuncture. Additionally, electro-acupuncture significantly improved pain, stiffness, and global well-being when compared to non-acupuncture. The beneficial effects of acupuncture could be observed at 1 month after treatment, but they were not maintained at 6-7 months (160).

Other

The effectiveness of meditative movement therapies (qigong, yoga, tai chi) on sleep and fatigue improvement and of hydrotherapy on pain reduction has been supported by some studies (161,162). A number of other modalities has also been utilized for the treatment of fibromyalgia including biofeedback, chiropractic therapy, massage therapy, hypnotherapy, guided imagery, electrothermal therapy, phototherapeutic therapy, music therapy, journaling / storytelling, static magnet therapy, transcutaneous electrical nerve stimulation, and transcranial direct current stimulation. However there are no well-designed studies to advocate their general use (145).

Pharmacologic Treatment

A wide range of drugs has been used in the treatment of fibromyalgia including antidepressants, sedatives, muscle relaxants and antiepileptic drugs. The choice of medication is influenced by patient preference; prominence of particular symptoms, including fatigue, insomnia, and depression; potential adverse effects; patient tolerance of individual medications; cost and regulatory limitations on prescription choice (163,164). Nonsteroidal anti-inflammatory drugs and opioids, although often prescribed for fibromyalgia, are not an effective form of treatment (39,165). However analgesics and anti-inflammatory medications can be useful in case of coexisting conditions that cause regional pain, like arthritis, which can aggravate or trigger the fibromyalgia symptoms. Regarding opioids, with the exception of tramadol, apart from not being effective for the treatment of fibromyalgia symptoms, their long-term use also curries a dose-dependent risk for serious adverse effects, including overdose, abuse, fractures, myocardial infarction and sexual dysfunction (166). Additionally opioids in fibromyalgia patients can reduce the effectiveness of psychological therapy (167), while their long-term use can cause sleep disturbances (168).

Patients should be informed that for most pharmacologic therapies several weeks may be needed until they experience a benefit. Initially a single drug should be administered. However, in the case of non-responsiveness combination therapy should be considered. Since therapeutic responses are rarely durable, physicians should not be surprised when the initial efficacy of a medication is abolished. Successful treatment of fibromyalgia may require regular reassessment and possible rotation or combination of medications (169). Adequate dose prescription and patient adherence are significant for the effectiveness and tolerability of pharmacologic treatment (170). The doses of the most commonly used medications with strong and moderate evidence of effectiveness are shown in Table 9.

ANTIDEPRESSANTS

Tricyclic Antidepressants (TCAs)

TCAs are often used as initial treatment for fibromyalgia. Their analgesic effect is independent of their antidepressant action and is thought to be mediated by inhibition of norepinephrine (rather than serotonin) reuptake at spinal dorsal horn synapses, with secondary activity at the sodium channels. The most widely studied drugs of this group are amitriptylineand cyclobenzaprine. They should be administered at lower doses than those required for the treatment of depression, a few hours before bedtime, and their dose should be escalated very slowly. A clinically important improvement is observed in 25-45% of patients treated with TCAs compared to 20% in those taking placebo (171–175). However their use is limited by the fact that they are ineffective or intolerable in 60-70% of patients (144), while their efficacy may decrease over time (171,176).

Amitriptyline is more efficient compared to the serotonin-norepinephrine reuptake inhibitors duloxetine and milnacipran in reducing pain, sleep disturbance, and fatigue, without differences in acceptability, as it was shown in a systematic review and meta-analysis (177). In a 2022 network meta-analysis comparing amitriptyline, duloxetine and pregabalin it was shown that treatment with amitriptyline 25 mg was superior to duloxetine and pregabalin for the reduction of pain intensity for at least 50% (178). The combination of 20 mg of fluoxetine in the morning with 25 mg of amitriptyline at bedtime has been shown to be more effective than either medication alone (179). Side effects of amitriptyline include dry mouth, constipation, fluid retention, weight gain, difficulty in concentrating and possibly cardiotoxicity.

Cyclobenzaprine has a similar tricyclic structure and presumed mode of action with amitriptyline in fibromyalgia, but is thought to have minimal antidepressant effect (163). A meta-analysis of five placebo-controlled trials has revealed improvement of the global functioning, with a similar effect size as this reported for amitriptyline. The group that received cyclobenzaprine had a significant decrease in pain for 4 weeks, compared to those treated with placebo, but the decrease in pain was not significantly different after 8 and 12 weeks. Sleep was improved at all time points in both cyclobenzaprine and placebo groups, while no effect was noted on fatigue (171–173). It has been demonstrated that the use of very low-dose cyclobenzaprine (1 to 4 mg at bedtime) can improve the symptoms of fibromyalgia, including pain, fatigue, and depression, compared to symptoms at baseline and to placebo. Significantly more patients who received the very low-dose of cyclobenzaprine experienced improved restorative sleep, based upon analysis of cyclic alternating pattern of sleep by electroencephalography. The increase in nights with improved sleep by this measure correlated with improvements in fatigue and depression (180).

Desipramine has fewer anticholinergic and sedative effects than other TCAs, which can make it a possible alternative, although its efficacy is not well studied in fibromyalgia.

Serotonin-Norepinephrine Reuptake Inhibitors (SNRIs)

SNRIs are similar to TCAs in their ability to inhibit the reuptake of both serotonin and norepinephrine, but they differ from TCAs in being devoid of significant activity at other receptor systems, resulting in diminished side effects and increased tolerance. Venlafaxine, duloxetine and milnacipran have been shown to be effective in diminishing fibromyalgia symptoms (147,181,182). These drugs can be used in fibromyalgia patients who do not respond to a trial of low-dose TCAs or who have intolerable side effects. They can also be administered as an alternative to amitriptyline for initial therapy especially for patients with significant fatigue or depression. Of these medications duloxetine and milnacipranare better studied and they are preferred to be administered to patients with fibromyalgia. There are more limited data regarding the efficacy of venlafaxine for fibromyalgia, while withdrawal symptoms if a dose is missed occur more often, because of the short half-life of this medication (183). A meta-analysis has shown that fibromyalgia patients treated with duloxetine at 60mg daily are more likely to have more than 50% reduction in pain, compared to patients taking placebo (184). However, duloxetine at 30mg daily does not significantly reduce pain (185). The efficacy of duloxetine can be maintained at 3 and 6 months of treatment (186). In a 2018 systematic review and meta-analysis it was shown that duloxetine and milnacipran were not superior to placebo in the frequency of pain relief of at least 50%, but there was a benefit in reducing the pain at least by 30% and in the patient's global impression to be much or very much improved. Additionally, there was not a significant difference in the reduction of fatigue, in the reduction of sleep problems, nor in the improvement of health-related quality of life (187). Another meta-analysis has shown that duloxetine, pregabalin and milnacipran were superior to placebo for pain relief, while duloxetine and pregabalin were superior to milnacipran. These drugs also differed in their effects on sleep disturbances, depression and fatigue (188). A fMRI study in fibromyalgia patients treated with milnacipran and placebo demonstrated that pain reduction with milnacipran treatment was associated with decreased functional connectivity between the insular cortex and the rostral part of the anterior cingulate cortex as well as the periaqueductal gray, while these changes were not demonstrated with the placebo (189).Regarding their side effects, headaches and nausea are more common with duloxetine and milnacipran treatment, while diarrhea is more common with duloxetine treatment. Other side effects related to SNRIs include dry mouth, constipation, somnolence, dizziness and insomnia (188).

Monoamine Oxidase Inhibitors

Monoamine oxidase inhibitors block the catabolism of serotonin, increasing its levels in the brain. It has been indicated that pirlindole and moclobemide have a significant beneficial effect on pain, without a significant effect on sleep nor fatigue. Their use for the treatment of fibromyalgia patients is limited (190).

ANTICONVULSANTS

Antiepileptic medications useful for the treatment of fibromyalgia patients include pregabalin and gabapentin. Both of these medications are structurally related to GABA and they bind with high affinity to the alpha2-delta subunit site of cellular voltage-dependent calcium channels. Although their exact mechanism of action is unknown their therapeutic effects can be mediated by blocking the release of various neurotransmitters. They can be used in cases where other medications initially administered to the fibromyalgia patients become intolerable or ineffective, or as the initial treatment for patients with significant sleep disturbance in addition to pain.

Pregabalin has been reported to be efficient against pain, sleep disturbances and fatigue in fibromyalgia. In a recent meta-analysis a reduction of at least 50% in pain intensity was found in 22-24% of patients taking pregabalin 300-600 mg per day, approximately 9% higher compared to the placebo group. A reduction in pain intensity of at least 30% was found in 39-43% of patients on pregabalin 300-600 mg per day, compared to 28% of patients taking placebo (191). In addition to pain, pregabalin at doses 300-600 mg per day can improve sleep and patient function, as it is demonstrated in a 2018 review of clinical trials, meta-analyses, combination studies and post-hoc analyses (192). The improvement in pain and sleep can be apparent as early as 1-2 days after the onset of treatment (193). In a 2022 network meta-analysis it was shown that treatment with pregabalin 450 mg per day was superior to duloxetine 30 mg for the reduction of pain intensity of at least 30% (178). A randomized placebo-controlled neuroimaging study demonstrated that the reduction in pain intensity from pregabalin was associated with a reduction in connectivity between the posterior insula and the default mode network (DMN) and that pregabalin but not placebo can reduce the response of the DMN to experimental pain (194). It is of interest that baseline patterns of brain connectivity have been used in a machine-learning model to successfully distinguish fibromyalgia patients who have a favourable response to pain intensity after the treatment with milnacipran from those who achieve a reduction of pain intensity after the treatment with pregabalin (195). Common side effects of pregabalin include somnolence, dizziness, weight gain and peripheral oedema. Discontinuation due to side effects is approximately 10% higher in patients treated with pregabalin compared to placebo, while discontinuation due to inefficiency of treatment is 6% lower (191). The intensity of adverse effects and the frequency of discontinuation of the treatment due to adverse effects is dose dependent. It is important for pregabalin to be titrated to the maximally tolerated therapeutic dose for each patient (192).

Gabapentin has been shown to be efficient in treating fibromyalgia associated pain, while it was well tolerated (196). Side effects include dizziness, sedation, lightheadedness, and weight gain. Its efficacy and tolerability is not well studied in fibromyalgia patients, however it can be considered as an acceptable alternative in case pregabalin cannot be administered due to its cost or due to regulatory limitations (197).

SEDATIVE HYPNOTIC AGENTS

Zopiclone and zolpidem have been used in fibromyalgia. It has been suggested that they can improve the sleep and perhaps fatigue, without any significant effects on pain (144).

Sodium oxibate, a precursor of GABA with powerful sedative properties has been shown to improve pain, fatigue and sleep architecture in fibromyalgia (198). However, in view of safety concerns the European Medicines Agency and the US Food and Drug Administration have not approved it for use in fibromyalgia patients.

TRAMADOL

Tramadol has multiple analgesic effects, since it inhibits norepinephrine and serotonin reuptake, and its major metabolite binds weakly to opioid μ receptors (144). The use of tramadol (with or without acetaminophen) is both effective and well tolerated for the management of pain in fibromyalgia (199,200). There are some concerns regarding the long-term potential of abuse of tramadol, although the risk is less than that of more potent narcotic analgesics that have also been used in fibromyalgia.

Table 9. The Doses of the Most Commonly used Medications with Strong and Moderate Evidence of Effectiveness in Fibromyalgia
Drugs	Doses
Tricyclic antidepressants
Amitriptyline	Start 5-10 mg at bedtime, increase up to 25-50 mg
Cyclobenzaprine	Start 10 mg at bedtime, increase up to 30-40mg, decrease to 5mg if 10mg too sedating
Serotonin-norepinephrine reuptake inhibitors
Duloxetine	Start 10-15mg twice daily, gradually increased to 30 mg twice daily
Milnacipran	Start 12.5mg in the morning, gradually increase to 50mg twice daily
Venlafaxine	167 mg per day
Anticonvulsants
Gabapentin	Start 100mg at bedtime, increase to 1200-2400 mg per day
Pregabalin	Start 25-50mg at bedtime, increase to 300-450 mg/day
Other
Tramadol	37.5 mg four times daily

CONCLUSION

Fibromyalgia is a common disease that is often underdiagnosed. Genetic predisposition, in combination with exposure to external stressors may lead to dysregulation of the nociceptive system and to the appearance of clinical symptoms. Fibromyalgia patients do not form a homogenous group with some patients responding adequately to current therapeutic modalities, and some others not experiencing any long-term benefit. Patients treated by primary care physicians in the community have a much better prognosis, compared to patients treated in tertiary referral centers. Certain psychological factors, such as an increased sense of control over pain, a belief that one is not disabled, that pain is not a sign of damage, and behaviors like seeking help from others, decreased guarding during examination, exercising more and having pacing activities are associated with better prognosis. Conversely, catastrophizing is associated with increased awareness of pain and with worsening symptoms.

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Immune System Effects on the Endocrine System

ABSTRACT

Among the most important and complex systems in the human body are the endocrine and immune systems. Emerging research over the last decade has shed light on their remarkable interplay, revealing a multitude of bidirectional communication pathways and reciprocal regulation mechanisms. Endocrine diseases, such as autoimmune thyroiditis, diabetes mellitus type 1 and type 2, osteoporosis, and disorders of the hypothalamic-pituitary-adrenal (HPA) axis, as well as endocrine malignancies, such as thyroid cancer, are highly interconnected with dysregulations of the immune system.Thus, multiple cytokines, chemokines, and evolving inflammatory processes are involved in the pathogenesis of immune-related endocrine disorders, providing potential targets for immune-based therapeutic approaches. In this chapter, we provide a comprehensive overview of the molecular mechanisms underlying these complex endocrine-immune interactions, and discuss the implications of immune system function or dysfunction in endocrine disorders.

INTRODUCTION

The immune system is a host defense system that comprises numerous biological structures and processes to defend the human body against potentially harmful substances and invading pathogens. It functions through a series of coordinated mechanisms, including innate and adaptive immunity. The innate immune response consists of i) phagocytosis by macrophages, neutrophils, monocytes, and dendritic cells, and ii) cytotoxicity by natural killer cells, providing an immediate, nonspecific defense against a wide range of invaders by recognizing common patterns shared by many pathogens (1).

Adaptive immunity, on the other hand, is an acquired, specially designed defense system; it develops over time and utilizes highly specialized immune cells involving antibody-dependent complement or cell-mediated cytotoxicity produced by T cells that recognize injurious agents, such as heat shock proteins or microbial antigens. During adaptive immunity, antigens taken up by antigen-presenting cells (APCs) are presented to T cells through binding with major histocompatibility complex (MHC) molecules on the surface of these cells. Activated CD4 helper T cells stimulate the release of cytokines, such as interleukin (IL)-2, which i) induce T cell proliferation and activation, ii) stimulate killer cell activity by CD8 suppressor T cells, and iii) activate B cells to differentiate into plasma cells and produce antibodies. Naïve T cells differentiate mainly into two main subsets that produce a different set of cytokines and regulate distinct immune functions. T-helper 1 (Th1) cells produce mainly interferon-γ (IFN)-γ, tumor necrosis factor-α (TNF)-, and IL-12 to regulate cell-mediated responses, while T-helper 2 (Th2) cells secrete IL-4, IL-5, and IL-13, to stimulate antibody production. In addition, three other subsets of T helper cells have been identified: Th22, which secrete IL-22, Th17, which secrete IL-17, and Treg, which secrete transforming growth factor (TGF)- b, all of which also play a role in the pathogenesis of autoimmune diseases (2).

Multiple regulatory mechanisms are involved in maintaining central and peripheral T and B cell tolerance (1). Defects in the processes that ensure immune cell tolerance may induce a maladaptive immune response to a self-antigen and lead to the development of autoimmune diseases.

Emerging research of the last few decades has shed light on the remarkable interplay between endocrine and immune systems, revealing a multitude of bidirectional communication pathways and reciprocal regulation.

Autoimmune endocrine diseases, such as Hashimoto thyroiditis, diabetes mellitus type 1 (DM1), and Addison disease, as well as endocrine malignancies, such as differentiated, anaplastic, and medullary thyroid cancer (3), and other endocrine disorders, including diabetes mellitus type 2 (DM2), osteoporosis, as well as a dysfunctional hypothalamic-pituitary-adrenal (HPA) axis response to stress and inflammation, are highly interconnected with dysregulations of the immune system.

This chapter seeks to elucidate the interplay between the endocrine and immune systems, exploring their interconnections and highlighting the impact of their crosstalk on health and disease. We present a comprehensive overview of the molecular mechanisms underlying this interaction and discuss the potential therapeutic implications of targeting the immune system for the management of endocrine diseases.

IMMUNE SYSTEM AND THYROID DISEASE

Autoimmune Thyroid Disease

Autoimmune thyroid disease (AITD) results from a dysregulation of the immune system that leads to loss of tolerance to thyroid antigens and to an autoimmune attack on the thyroid gland. The most common clinical manifestations of AITD are Hashimoto's thyroiditis and Graves’ disease, while less prevalent manifestations are drug-induced thyroiditis, postpartum thyroiditis, or thyroiditis associated with polyglandular syndromes (i.e., autoimmune polyglandular syndromes type 1 and type 2). The underlying molecular mechanisms of AITD involve both circulating autoantibodies and T cell immune mechanisms, while genetic background, as well as cross-reactivity to external antigens (4-6), are also implicated.

There are three major thyroid autoantigens that are targeted during autoimmune thyroid attack and are critical for thyroid homeostasis, namely, thyroglobulin (Tg), thyroid peroxidase (TPO), and the thyrotropin receptor (TSHR) (Figure 1).

Figure 1. Thyroid proteins that serve as autoantigens. Thyroglobulin (Tg) function as storage protein in thyroid cells, playing a critical role in the synthesis and release of thyroid hormones. Thyroid peroxidase (TPO) catalyzes iodination of tyrosines in thyroglobulin, which attaches one or two iodine molecules to form monoiodotyrosine (MIT) or diiodotyrosine (DIT), respectively. In addition, thyroid peroxidase catalyzes the coupling of iodotyrosine residues to form triiodothyronine (T3) and thyroxine (T4) attached to thyroglobulin. Thyrotropin receptor (TSHR) is a transmembrane G-protein coupled receptor that upon stimulation by circulating TSH activates the expression of downstream effector genes to regulate thyroid growth, thyrocyte differentiation, and thyroid hormone synthesis. Sodium/iodide symporter (NIS) is a membrane glycoprotein, which actively cotransports two sodium cations per each iodide anion, using the electrochemical sodium gradient generated by the Na+/K+-ATPase. Pendrin is involved in the apical iodide efflux in thyroid cells. It can also exchange chloride and bicarbonate. [Modified by Boguslawska et al (7)].

Thyroglobulin is a soluble glycoprotein homodimer composed of two subunits of ~330 kDa in size and is the most abundant glycoprotein in the thyroid gland. It is the scaffold for the synthesis of thyroid hormones and the storage-form of thyroid hormones inside the gland. Recently, researchers described the first atomic structure of full-length Tg and identified its hormone-forming tyrosine residues (8). Anti-Tg antibodies (TgAb) act mainly through antibody-dependent cytotoxicity cells rather than through complement fixation (7). In AITD, the prevalent TgAb species recognize native rather than denatured antigens and bind to a number of overlapping epitopic domains located mainly in the central region and C-terminal end of Tg. Of note, TgAb in the serum of healthy subjects have a different epitopic pattern (9).

Another major thyroid antigen is TPO, a glycosylated heme-containing homodimer of two 107-kDa transmembrane subunits located in the apical membrane of thyrocytes (Figure 1). It catalyzes the iodination of tyrosyl residues in Tg and the coupling of iodotyrosine residues to form iodothyronines attached to Tg (10). Both humoral and cellular immune responses are directed against TPO. TPO autoantibodies (TPOAb) occur in almost all patients with Hashimoto thyroiditis and approximately 75% of individuals with Graves’ disease (11). In addition, TPOAb may be involved in autoimmune thyroid cell death via antibody-dependent cytotoxic cells and C3 complement-mediated cytotoxicity (7). Specific patterns of TPOAb recognition remain stable in an individual over time and are genetically transmitted through family lineages (12).

Thyrotropin receptor is primarily expressed on the basolateral membrane of thyrocytes (Figure 1) and belongs to the transmembrane G protein-coupled receptor family. The intracellular signaling pathway that is activated by the interaction of TSH with TSHR is indispensable for the synthesis of thyroid hormones and the proliferation of follicular epithelial cells. TSHR-stimulating antibodies (TSAb) act as TSHR agonists and stimulate thyroid growth and production of thyroid hormone in an autonomous and unregulated manner. On the other hand, TSHR-blocking antibodies (TBAb) act as antagonists that block the intracellular signaling of TSH, leading to decrease of thyroid hormone synthesis and subsequently hypothyroidism. Neutral antibodies to TSHR, which may also be detected in the serum of patients with Graves’ disease, bind to the receptor but do not alter its activity (13). The exact antigenic sites of TSHR-specific TBAb and TSAb overlap and are mostly directed to the extracellular A subunit of the receptor (Figure 1) (14,15).

Other thyroid antigens include the iodide transporters, sodium iodide symporter (NIS), and pendrin (Figure 1). The presence of NIS antibodies is increased in AITDs, especially in patients with Graves’ disease, whereas their expression in euthyroid individuals is rare (16). Pendrin is an apical membrane-bound iodide transporter, but the diagnostic value of antibodies against pendrin is rather low (16).

Although the above-described circulating autoantibodies are useful markers of thyroid autoimmunity, it is the T cell immune mechanism, i.e., the loss of immune self-tolerance, that is the core of AITD pathophysiology. Loss of immune self-tolerance may result from either: i) the loss of central tolerance (i.e., disturbed deletion of autoreactive T cells in the thymus), ii) dysfunction of peripheral tolerance (i.e., impaired apoptosis of self-reactive T cells and inhibition of the activity of T-regulatory cells), or iii) disturbed energy (i.e., disturbance of the functional inactivation that prevents the lymphocytes from activating an immune reaction against the antigen).

Patients with AITD express IFN-γ-induced MHC class II molecules, which promotes the presentation of thyroid autoantigens and activates T cells (4). The subsequent infiltration of the thyroid gland by APCs (dendritic cells and macrophages) may be triggered by inflammation resulting from either viral or bacterial infection or exposure to toxins. The common mechanism involved in the initiation and perturbation of the inflammatory processes in AITDs and in other autoimmune endocrine diseases (e.g., type 1 diabetes and Addison disease) is the Th1-cytokine/chemokine axis (17). Upon activation, Th1 lymphocytes produce IFN-γ -and TNF-α, which stimulate thyrocytes and retroorbital cells (in Graves ophthalmopathy) to secrete chemokines (CXCL10, CXCL9, and CXCL11). Chemokines, in turn, bind and activate the CXCR3 receptor on Th1 cells and further enhance IFN-γ and TNF-α secretion in a positive feedback loop which aggravates recruitment and activation of inflammatory cells in the affected organs (Figure 2). In advanced thyroiditis, the thyroid gland is infiltrated by B cells (representing up to 50% of the infiltrating immune cells), as well as cytotoxic T lymphocytes and CD4+ cells (7).

Figure 2. Depiction of the molecular mechanism involved in the inflammatory processes in autoimmune thyroid diseases. Activation of Th1 lymphocytes by antigen presenting cells produce INF-γ -and IL-2 and 12, which stimulate thyrocytes and retroorbital cells to secrete chemokines (CXCL10, CXCL9, and CXCL11). Chemokines bind and activate the CXCR3 receptor on Th1 cells, further enhancing IFN-γ and ILs secretion. APC, antigen presenting cells; IFΝ-γ, interferon gamma; IL-2, interleukin 2; IL-12, interleukin-12; CXCR3, C-X-C Motif Chemokine Receptor 3.

In Hashimoto thyroiditis, the humoral immune response is characterized by the presence of autoantibodies to TPO or Tg and is related to thyroid lymphocytic infiltration. These autoantibodies are themselves cytotoxic or may affect antigen processing or presentation to T cells. Th1 cells are the predominant T cell clones found in patients with Hashimoto thyroiditis: they promote apoptosis of thyrocytes through secretion of IL-12, TNF-α, and INF-γ, which activate cytotoxic T lymphocytes and macrophages (18). In addition, the Toll-like receptor-3 protein is overexpressed in human thyrocytes surrounded by immune cells in all patients with Hashimoto thyroiditis, but not in Graves’ disease or in euthyroid individuals (17).

In patients with Graves’ disease, the predominant antibodies are directed against the TSHR. T cells are activated through the presentation of TSHR peptides and, in turn, trigger B-cells and plasma cells that infiltrate the thyroid to produce autoantibodies directed against the TSHR. Activity of B-cells and plasma cells is also regulated by liver-produced insulin growth factor 1 (IGF1). In contrast to TPO and Tg, TSHR is widely expressed in extrathyroidal tissues and cells, including lymphocytes, adipose tissue, and fibroblasts. In consequence, the presence of TSHR antibodies contributes to the extrathyroidal manifestations of Graves’ disease, such as Graves ophthalmopathy, Graves dermopathy, and Graves-associated thymus hyperplasia (19).

Particularly in Graves ophthalmopathy, the TSHR autoantigen is presented by macrophages and B cells recruited to the orbit (20). Activated T cells, in turn, initiate an immunological attack on the orbital fibroblasts expressing TSHR. In response to the cytokines released by Th cells, orbital fibroblasts and adipocytes —both expressing TSHR— produce and deposit large amounts of glycosaminoglycans (e.g., hyaluronan), leading to increased osmotic pressure and water uptake, swelling of the extraorbital muscles, and increased accumulation of orbital adipose tissue. The process is enhanced by the IGF1/IGF1R signaling pathway in fibroblasts and adipocytes, as well as the crosstalk between TSHR and IGF1R intracellular signaling (21). A similar molecular pathophysiology may underlie Graves dermopathy (19).

NON-CODING RNAs

In recent years, non-coding RNAs, which are known to modulate gene transcription at the post-transcriptional level, have attracted a great deal of attention as potential biomarkers in various endocrine diseases. Although the study of microRNAs and circular RNAs is still in its infancy, these newly discovered non-coding, single-stranded RNA molecules have been implicated in the development and progression of AITDs.

A cluster of biomarkers consisting of miR-205/miR-20a-3p/miR-375/miR-296/miR-451/miR-500a/miR-326 has been reported to be differentially expressed in patients with Hashimoto thyroiditis (22,23). Similarly, multi-miRNA-based biomarkers, such as miR-762/miR-144-3p or miR-210/miR-155/miR-146, were differentially expressed in the serum of patients with Graves’ disease compared to healthy individuals. These dysregulated miRNAs can target key genes involved in the immune response and thyroid function. Regarding their prognostic role, higher miR-21-5p expression is associated with a worse prognosis for patients with Graves’ disease, whereas impaired expression of miR-155 correlates with the size of the goiter (22,24). Further understanding of the role of miRNAs in AITDs could provide valuable insights into disease mechanisms and potentially identify novel therapeutic targets. Data on other non-coding RNAs (such as long-noncoding RNAs and circular RNAs) are scarcer. Impaired expression of n335641, TCONS-00022357-xloc-010919, and n337845 was found in B cells of patients with Graves’ disease (25), while altered expression of 627 circRNAs in PBMCs of patients with Hashimoto thyroiditis has been tested for their potential prognostic value (26).

GUT MICROBIOME

Emerging evidence suggests that the composition and function of the gut microbiome may be involved in AITDs. The gut microbiome refers to the complex community of microorganisms residing in the gastrointestinal tract, which plays a vital role in various aspects of human health, such as prevention of intestinal colonization by pathogenic bacteria, fermentation/degradation of food debris, and production of nutrients. Studies have indicated that alterations in the gut microbiome can influence immune responses and contribute to the development of autoimmune diseases (7). In this context, dysbiosis, or an imbalance in the gut microbiota, has been observed in patients with Hashimoto thyroiditis and Graves’ disease, thus distinguishing them from healthy controls, while it was associated with the stage of disease, the level of thyroid autoantibodies, and the response to therapy.

Targeting the microbiome through dietary interventions or probiotics may represent a promising potential therapeutic avenue. It is therefore of interest to note that in hypothyroid patients treated with LT4, symbiotic supplementation for 8 weeks resulted in decrease of TSH concentration and LT4 dose (27). Moreover, the results of a large clinical study, INDIGO, reported a significant effect of LAB4 probiotics on the gut microbiota composition of patients with Graves’ disease and a temporary reduction in the serum level of IgG and IgA antibodies (28). Further research involving patients from different populations is required to fully understand the relationship between the gut microbiome and AITDs and to explore potential strategies for intervention.

Postpartum Thyroiditis

Postpartum thyroiditis may occur up to 12 months after delivery. Usually it presents as transient hyperthyroidism (median time of onset, 13 weeks post-delivery), followed by transient hypothyroidism (median time of onset, 19 weeks post-delivery). In the majority of patients, restoration of normal thyroid function occurs. Pregnancy triggers hormonal changes and immune system alterations, such as a shift from Th1 to Th2 cytokine production followed by a "rebound" shift back to Th1 after delivery, and fluctuations in transforming growth factor-beta1 TGF-β1) serum levels (29,30).

Anti-TPO and anti-Tg antibodies are found in almost all patients with postpartum thyroiditis. Notably, up to 50% of women who had anti-TPO antibodies at the end of the first trimester of gestation (i.e., before thyroid antibody titers start to decline during pregnancy) developed postpartum thyroiditis. Furthermore, there is evidence that the anti-TPO antibody titer at 16 weeks of gestation is related to the severity of postpartum thyroiditis (31), while activation of the complement is also involved in the pathogenesis (23).

Euthyroid Sick Syndrome

Euthyroid sick syndrome (ESS) is a condition characterized by alterations in thyroid hormone levels despite normal thyroid gland function. The characteristic laboratory abnormalities of the ESS include low T3 and/or fT3, elevated reverse T3 (rT3), normal or low TSH, and normal or low serum T4 or fT4 concentrations. Clinical conditions that trigger the development of ESS include systemic inflammation, myocardial infarction, starvation, sepsis, surgery, trauma, chronic degenerative diseases, malignancy, and every other condition associated with severe illness.

During illness or periods of severe physiological stress, the immune system releases various proinflammatory cytokines, such as in IL-6, TNF-α, IL-1β, IFN-γ, and TGF-β2. These cytokines disrupt the hypothalamic-pituitary-thyroid axis and interfere with the normal synthesis, secretion, and metabolism of thyroid hormones (Figure 3), while the more severe the illness, the more extensive the hormonal alterations.

Figure 3. Secretion of proinflammatory cytokines during severe illness or stress inhibits the activity of hepatic deiodinase type 1- suppressing the peripheral conversion of T4 to T3. They also suppress intrathyroidal hormone synthesis, TRH release and TSH secretion from the pituitary. IL-6, interleukin-6; IL-1, interleukin-1; TNF-α, tumor necrosis factor alpha; INF-γ, interferon gamma; TGF-β, tumor growth factor beta; TG, thyroglobulin; NIS, sodium/iodide symporter; TSH, thyroid stimulating hormone; TRH, Thyrotropin-releasing hormone.

Proinflammatory cytokines suppress the peripheral conversion of T4 to T3, resulting in low T3 levels and increase rT3 levels, by inhibiting the activity of hepatic deiodinase type 1, which promotes conversion of T4 to T3 and of rT3 to diiodothyronine (32). They also suppress TRH release and inhibit TSH response to TRH stimulation, leading to a decrease in TSH levels. Thus, the cause of the decreased T3 concentration in ESS is decreased T3 production, whereas the cause of the increased rT3 concentration is the result of impaired degradation. Prolonged and severe illness is marked by a decrease in circulating total T4 along with low T3 and high rT3, with very low T4 levels displaying a poor prognosis and having been associated with an increased mortality rate (32). In addition, cytokines reduce iodine uptake by inhibiting sodium-iodine symporter protein expression (25-27), and decrease thyrocyte growth (33), iodide organification (34,35), and thyroglobulin synthesis (36,37).

The central role of cytokines in the pathophysiology of ESS has been further elucidated in studies involving cytokine administration to humans. TNF-α administration in healthy volunteers caused a decrease in serum T3 and an increase in serum rT3 concentration (38). Unlike IL-6, serum TNF-α levels did not correlate with any of the typical thyroid parameters, such as low T3, increased rT3, or decreased TSH levels seen in ESS (39,40), suggesting that the changes of thyroid hormone profile following TNF-α administration might be indirect (i.e., through TNF-α increase in circulating IL-6 levels). Furthermore, both IL-6 and TNF-α can upregulate type 2 deiodinase in the anterior pituitary, affecting TSH release and contributing to the development of the non-thyroidal illness syndrome (41,42).

Leptin, a hormone primarily known for its role in regulating appetite and energy balance, has also been implicated in the development of EES. During acute illness or chronic inflammation, leptin levels are usually elevated. A link between leptin and the proinflammatory cytokines TNF-α and IL-6 in chronic inflammatory diseases, such as chronic obstructive pulmonary disease (43) and ankylosing spondylitis (44), respectively, has also been proposed previously.

The primary effect exerted by leptin on the hypothalamic-pituitary-thyroid axis is alteration of the setpoint for feedback sensitivity of hypophysiotropic TRH-producing neurons in the paraventricular nucleus of the hypothalamus to thyroid hormones (mainly T3) by lowering of the setpoint when leptin levels are suppressed during fasting (45). Two anatomically distinct and functionally antagonistic populations of neurons in the arcuate nucleus of the hypothalamus, α-melanocortin-stimulating hormone (α-MSH)-producing neurons that co-express cocaine- and amphetamine-regulated transcript and neuropeptide Y (NPY)-producing neurons that co-express agouti-related peptide (AGRP), are responsible for the effects of leptin on hypophysiotropic TRH. It has also been proposed that leptin directly affects hypophysiotropic TRH neurons (46). Leptin has been found to inhibit the conversion of T4 to T3 in peripheral tissues and increase the activity of the enzyme type 3 deiodinase, which converts T4 to rT3. These data suggest that leptin can disturb thyroid function in seriously ill patients via two different independent mechanisms (cytokine-dependent and directly).

Amiodarone-Induced Thyroid Disease

Amiodarone, a benzofuran derivative with a similar structure to that of thyroid hormones, is a highly effective antiarrhythmic agent widely used in the treatment of various types of tachyarrhythmias (supraventricular and ventricular arrhythmias). Amiodarone contains two iodine atoms per molecule, which is approximately 37.5% iodine by molecular weight (47).

Treatment with amiodarone may be related to an increase in lymphocyte subsets leading to an exacerbation of pre-existing autoimmunity (48,49). The relative proportion of patients developing either thyrotoxicosis or hypothyroidism depends on the iodine content of the local diet and pre-existing thyroid autoimmunity. In relatively iodine-replete areas, approximately 25% of patients with amiodarone-induced thyroid dysfunction become thyrotoxic, accounting for approximately 3% of amiodarone-treated individuals (50).

Amiodarone-induced hypothyroidism is attributed to an increased susceptibility to the inhibitory effect of iodide on thyroid hormone synthesis and/or to a failure to escape the Wolff-Chaikoff effect (49). Hashimoto thyroiditis is the most common risk factor for amiodarone-induced hypothyroidism and it is considered the most likely reason for the female preponderance of this clinical entity (51). Female patients with positive anti-TPO and anti-Tg autoantibodies have a relative risk of 13.5% for developing amiodarone-induced hypothyroidism compared to men without thyroid autoantibodies (20).

The pathogenesis of amiodarone-induced thyrotoxicosis is complex, although two distinct forms, type 1 and type 2, are recognized. Type 1 develops in patients with latent thyroid disease, predominantly nodular goiter, in whom the amiodarone iodine load triggers increased synthesis of thyroid hormones. Type 2 is the result of a destructive thyroiditis in a previously normal gland, with leakage of preformed thyroid hormones despite a reduction in hormone synthesis (47,50,52,53).

Differentiating between the two types of amiodarone-induced thyrotoxicosis is an essential step in their management, as treatment of each type is different (50). Type 1 usually responds to thionamide therapy, which blocks hormone synthesis, and perchlorate, which blocks active transport of iodine into the thyroid, whereas type 2 responds to high-dose glucocorticoids (50,53-55). Nevertheless, several studies now suggest that these two types should be treated concomitantly; thus, currently, patients with amiodarone-induced thyrotoxicosis receive both antithyroid drugs and prednisolone. In cases resistant to medical treatment and/or in patients with severe cardiac diseases who cannot interrupt amiodarone or require quick amiodarone reintroduction, total thyroidectomy may be offered after rapid correction of thyrotoxicosis following combination treatment with thionamides, KClO4, glucocorticoids, and a short course of iopanoic acid (56).

Thyroid Cancer

Thyroid cancer is the most common endocrine cancer, the incidence of which has steadily increased over the past few decades (57).

The association of chronic inflammation induced by Hashimoto thyroiditis and thyroid cancer has been long recognized (58). However, the immune response triggered against thyroid cancer and AITDs differs significantly. In thyroid cancer, the immune response is more tolerant and allows tumor growth, whereas in AITDs, the response is more aggressive, triggering cell destruction and reduction of the function of the gland (59). Hashimoto thyroiditis is considered both a risk factor for the development of thyroid cancer (60) and a favorable prognostic factor due to chronic lymphocytic infiltration, which can downregulate tumor aggressiveness (60,61). In Graves’ disease, the presence of a strong humoral immune response appears to be protective against thyroid cancer. Patients with increased anti-TPO and anti-Tg levels show lower distant metastasis rates than patients without thyroid autoantibodies (62,63), suggesting their potentially protective role (59).

Immune infiltrates in the tumor microenvironment differ between the different thyroid neoplasm subtypes (Figure 4). In general, differentiated thyroid cancer (DTC) has a higher number of tumor-associated lymphocytes and regulatory T cells (Tregs), while anaplastic thyroid cancer (ATC) and medullary thyroid cancer (MTC) display a high density of tumor-associated macrophages (64). The number of tumor-associated macrophages has been associated with dedifferentiation, lymph node metastases, and reduced survival (65). It is important to note, however, that most of the studies analyzing the immune milieu of DTC have used papillary thyroid cancer (PTC) tumor samples (66-69). Myeloid-derived suppressor cells are associated with aggressive characteristics of differentiated thyroid cancer and are related to poor prognosis (65).

Figure 4. Immune infiltrates differ in different types of thyroid cancer [Modified by Garcia-Alvarez et al, (70)]. TAM, tumor-associated macrophages; MDSC, myeloid-derived suppressor cell.

The dendritic cells, which play a critical role in antigen presentation, are increased in PTC, while neutrophils are found in more aggressive thyroid cancers (such as poorly differentiated or anaplastic). In addition, natural killer cells that play an important role in immunosurveillance are also increased in PTC and are negatively correlated with tumor stage, while lymphocytic infiltration is associated with better overall survival and low recurrence rate (71,72).

Cytokines, which may be produced by thyroid follicular cells and by immune cells infiltrating thyroid tumors, are also related to tumor development. IL-1 and IL-6 stimulate thyroid cell proliferation and tumor growth, while TGF-β, which is a suppressive cytokine, is overexpressed in aggressive cancers (73). In addition, multiple chemokines may be secreted by thyroid cancer or immune cells and affect chemiotaxis, angiogenesis, and lymphangiogenesis (73).

Immunomodulatory proteins, such as programmed death-ligand 1 (PDL1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), T cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), and T cell immunoglobulin and ITIM domain (TIGIT), which are considered major immune coinhibitory receptors and promising immunotherapeutic targets in cancer treatment, are also expressed in thyroid cancer, being associated with more aggressive tumor characteristics and a poor prognosis (64). Programmed death-ligand 1 staining by immunohistochemistry has shown higher expression in ATC than in other subtypes (70). Six cohort studies have been published to-date reporting positive PD-L1 expression, varying between 22% and 65%, this being higher compared to that detected in DTC and poorly differentiated thyroid cancer (74-79). TIM-3 expression was observed in 48% of patients with MTC, and in the majority of cases (84.4%) its expression was restricted to tumor cells (80). Other coinhibitory receptors, such as LAG-3 and T cell TIGIT, were observed in a lesser percentage of cases (approximately 3%) (80). Nevertheless, results from clinical trials with immunotherapy as monotherapy or combinations have shown limited efficacy (70). In one phase Ib KEYNOTE-028 trial assessing the efficacy of pembrolizumab in patients with PD-L1+ (membranous staining on ≥1%) locally advanced or metastatic follicular or papillary thyroid cancer, pembrolizumab achieved an objective response rate of 9% and a median progression-free survival of 7 months (81). Several clinical trials further investigating the efficacy of combination therapy of immune checkpoint inhibitors are currently ongoing.

IMMUNE SYSTEM AND DIABETES MELLITUS

The immune system plays a crucial role in the pathogenesis of both type 1 and type 2 diabetes.

Diabetes type 1, which is immune-mediated in more than 95% of cases, is an organ-specific autoimmune disease characterized by lymphocytic infiltration and inflammation that leads to pancreatic β-cell destruction and absolute insulin deficiency (82). The immune system’s attack on pancreatic β-cells is usually triggered by a number of factors, including genetic predisposition and environmental triggers such as viral infections (e.g., Coxsackie B4, mumps, and rubella) or dietary compounds (e.g., cow’s milk) (82). The process involves the activation of immune cells, particularly T cells, which recognize self-autoantigens in pancreatic β-cells and initiate an immune response.

On the other hand, in type 2 diabetes, the immune system also plays a different role from that in DM1. Chronic low-grade inflammation, often associated with obesity, leads to immune cell activation and the release of proinflammatory cytokines. These cytokines interfere with the normal functioning of insulin and promote insulin resistance. Macrophages infiltrate adipose tissue and release inflammatory molecules, further exacerbating insulin resistance with increasing adiposity. The immune system, thus, contributes to the development and progression of insulin resistance and eventually promotes the onset of DM2.

Understanding the intricate relations between the immune system and diabetes pathogenesis is essential for the development of effective treatments and interventions for the management of both types of diabetes.

Diabetes Type 1

The susceptibility to develop DM1 is associated with multiple alleles of the major histocompatibility complex MHC I and II locus. More than 90% of patients with DM1 express either HLA DR3, DQ2 or DR4, or DQ8 (83), whereas HLA haplotype DR2, DQ6 is protective against DM1 development.

The primary pathological presentation of DM1 is inflammation of the pancreatic islets, also known as insulitis, caused by infiltration of immune cells, including CD4 and CD8 T cells along with B cells (84-86). Although the initial events triggering autoreactive responses remain unclear, presentation of pancreatic islet autoantigens by the associated MHC class II molecules contribute to priming and expansion of pathogenic T cells.

CD4 T helper cells are required for the development of the autoimmune process in the pancreatic islets, while CD8 cytotoxic T cells are the cells responsible for β-cell destruction (Figure 5). T cell receptors recognize peptides bound to MHC molecules on the surface of antigen-presenting cells (B-cells, dendritic cells, and macrophages). Each T cell then generates a unique receptor for the recognition of an autoantigen presented in the MHC molecule. The interaction between T cell receptor/autoantigen/MHC leads to activation of the T cells.

Figure 5. T cell mediated destruction of β- cells in diabetes type 1. CD4+ effector T cells recognize an insulin or islet peptide presented by diabetes-risk conferring HLA-DQ8 or DR4 on APCs, migrate to the pancreas and promote pancreatic β-cell destruction. Inside the pancreatic islets CD8+ effector T cells recognize islet antigens presented by HLA-A2 leading to the destruction of the β-cells. [Modified by Mitchel et al (87)]. TCR, T cell receptor; APC, antigen presenting cell; HLA-A2, human leukocyte antigen-A2.

Three different mechanisms have been proposed to explain T cell activation in DM1. One mechanism is thought to involve molecular mimicry-activated T cell proliferation. The hypothesis for this mechanism is based on the assumption that epitopes of proteins expressed by infectious agents can be shared by unrelated molecules encoded by host genes (88). A second mechanism that may trigger molecular mimicry-activated T cell proliferation is “bystander” T cell proliferation. This mechanism involves the stimulation of non-antigen-specific T cells by various cytokines during infection, simply because they are in the area. The cytokines thought to be involved in this nonspecific stimulation are IFN-α and IFN-β (89). The 3rd mechanism might involve a superantigen-mediated T cell proliferation mechanism: this theory proposes that autoreactive T cells can be inappropriately primed to react against self-structures through an encounter with a superantigen (90).

Multiple autoantigens in the pancreatic islets have been identified, including non-specific islet cell autoantigens (ICA), insulin, glutamic acid decarboxylase 65 (GAD65), insulinoma antigen-2 (IA-2), heat shock protein (HSP), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), imogen-38, zinc transporter-8 (ZnT8), and the most recently identified pancreatic duodenal homeobox factor 1 (PDX1), chromogranin A (CHGA), and islet amyloid polypeptide (IAPP) (91). Autoantibodies against these autoantigens may be detected long before the onset of hyperglycemia and usually decline during the course of the disease (92).

Most patients diagnosed with DM1 have circulating islet cell autoantibodies directed against pancreatic islet autoantigens. However, although the detection of autoantibodies may be useful for DM1 diagnosis and prediction, it is the cellular immune system that eventually infiltrates the pancreatic islets and causes β-cell destruction. In turn, further β-cell destruction leads to more self-antigen presentation and ensuing amplification of the immune response (82,93,94).

Among several proinflammatory cytokines, IL-21, produced by CD8 T cells, is required for the development of DM1, while TNF-α may also be involved (95,96). IL-6 plays an important role in the pathogenesis of vitiligo-associated DM1 (97). In contrast, in vivo experiments with non-obese diabetic mice have shown that IL-4, produced by Th2 cells, may be protective against developing diabetes (95,96,98,99).

Understanding both the pathophysiology and the regulatory mechanisms involved in DM1 is a critical step towards the development of antigen-specific, β-cell-directed, immunomodulatory or cellular treatment modalities (100).

Investigations into the efficacy and safety of various immunotherapeutic strategies against the development of DM1 have been carried out in recent clinical trials and are still ongoing in current trials (101). Among them, T cell-directed therapies that aim at a favorable balance between effector T cell depletion and regulatory T cell preservation have shown the most promising results. Teplizumab, an anti-CD3-directed monoclonal antibody, was the first immunomodulatory agent to demonstrate a significant delay in disease progression in high-risk individuals before clinical onset (102) and has recently (November 17, 2022) been approved by the FDA as the first disease-modifying therapy for DM1 in adults and in children aged 8 years and older.

The Enigma of Pancreatic α-Cell Resistance in Diabetes Mellitus Type 1

Although both insulin-producing β-cells and glucagon-producing α-cells of the pancreatic islets share a similar embryonic origin and are directly exposed to the deleterious immune signals in DM1, it appears that the immune system selectively destroys β-cells, while α-cells survive even in long-term DM1 (103). α-Cells are located in close proximity to β-cells in human pancreatic islets (104), creating a closed communication loop that regulates their function and secretory capacity (105). Dysfunction of α-cells plays a significant role in the pathogenesis of both types of diabetes (106). It has long been proposed that the reduced functional β-cell mass in DM1 and the consequent hypoglycemia were the key mechanisms indirectly inducing dysfunction of α-cells in diabetes (107). Advanced molecular technology of the last decade has, however, challenged this notion, showing that dysfunction of α-cells in diabetes is not secondary to β-cell pathology but is instead directly immune-induced (108-110). Despite this dysfunction, α-cells exhibit a remarkable autoimmune resistance that enables them to survive over β-cells in long-standing diabetes (103,111). Recent studies using single-cell RNA sequencing (109,112) have demonstrated important differences between these two cell types in terms of expression of: i) anti-apoptotic genes, ii) endoplasmic reticulum (ER) stress-related genes, III) innate immune response genes, and iv) antiviral response genes, all of which render α-cells less immunogenic and more resistant to viral infections and ER stress (113). In addition, CD8+ T cells invading the islets in DM1 are reactive to preproinsulin but not to glucagon. Furthermore, although β-cells are essential to life (neither humans nor animal models can survive without them), mice with 98% α-cell ablation retain near-normal glucose homeostasis (114): this points to the knowledge gap we have, from an evolutionary point of view, regarding the question of why β-cells are more fragile than α-cells. Certainly, greater understanding of the underlying mechanisms responsible for the autoimmune resistance of α-cells is critical since it is likely to reveal intracellular pathways amenable to therapeutic interventions that would increase the resistance of the β-cells themselves to the immune attack of the host’s immune system.

Diabetes Type 2

DM2 accounts for 90% of cases of diabetes worldwide (115). The increasing prevalence of DM2 around the world has been largely attributed to an unhealthy lifestyle and resultant development of obesity and overweight (116). Obesity is strongly related to DM2 mainly through inducing insulin resistance in the insulin sensitive tissues in the periphery.

The concept that a smoldering inflammatory process plays an important part in the pathogenesis of DM2 (117) has attracted much attention and is supported by evidence of inflammation in islets, adipose tissue, liver, and muscle that can provoke insulin resistance and β-cell dysfunction (118-120). Adipose tissue is characterized by infiltration by macrophages and other immune cells that produce cytokines and chemokines and contribute to the development of local and systemic chronic low-grade inflammation, this inflammatory milieu being the link between obesity, insulin resistance, and diabetes mellitus (121,122).

Earlier in vivo studies demonstrated that levels of TNF-α (123-125), IL-6, C-reactive protein, plasminogen activator inhibitor, and other inflammation mediators were elevated in adipose tissue and plasma of obese mice (126,127). It was also observed that these inflammatory mediators, together with saturated free fatty acids and reactive oxygen species (ROS), inhibited serine phosphorylation of the insulin receptor substrates (IRS-1 and 2) (128-130) in insulin sensitive tissues, such as adipose tissue and the liver (131), promoting insulin resistance (Figure 6). TNF-α is associated with increased release of free fatty acids by adipose tissue and leads to impaired insulin secretion and signaling (123,132).

Figure 6. Insulin resistance and inflammation in diabetes type 2. Insulin binds to insulin receptor (IR) in insulin sensitive tissues, and autophosphorylates tyrosine molecules of IRS-1 and -2 substrates. In the presence of obesity, and hyperlipidemia, the influx of free fatty acids, inflammatory cytokines and glucose activates IKKβ and JNK, which are the mediators for stress and inflammatory. In turn IKKβ and JNK inhibit tyrosine phosphorylation of IRS1 and 2 and promote transcriptional activation of genes related to inflammatory and stress responses resulting in insulin resistance. [Modified by Berbudi et al (133)]. IRS 1 -2, insulin receptor substrates; ER, Endoplasmic reticulum; IKKβ, inhibitory kappa B kinase β; JNK1 and c-Jun N-terminal kinase I.

In line with these early in vivo studies, studies in humans have shown that elevated levels of i) nonspecific indicators of inflammation, such as white cell count, fibrinogen, and CRP levels (134-136), ii) markers of reduced fibrinolysis, such as plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator (tPA), iii) von Willebrand factor (vWf), which is a marker of endothelial injury, and iv) early markers of inflammation, such as monocyte chemotactic protein-1 (MCP-1), IL-8, and interferon-γ-inducible protein-10 (137), were predictive of DM2 development.

Furthermore, CRP and/or IL-6 were associated with the incidence of DM2 independently of adiposity or insulin resistance (136,138,139). Visceral adipose tissue appears to be a major source of circulating IL-6 in humans, and obese people with insulin resistance display high levels of plasma IL-6 concentration, also predictive of DM2 development (113). TNF-α is also increased in obese individuals with insulin resistance (124,140) and it plays a major role in the pathogenesis of obesity-linked DM2 (141).

At the cellular level, chronic exposure of adipocytes to low doses of TNF-α led to a dramatic decrease in insulin-stimulated auto-phosphorylation of the IRS 1-2 (142). Treatment of cultured murine adipocytes with TNF-α induced serine phosphorylation of IRS-1 and convert IRS-1 into an inhibitor of IR tyrosine kinase activity in vitro. TNF-α has also been shown to downregulate glucose transporter GLUT4 mRNA levels in adipocyte and myocyte cultures as well (125,143,144).

Oxidative stress, as a result of increased cytokine levels in DM2, is also thought to play an important role in activating inflammatory genes (145,146) (Figure 6). It is possible that oxidative stress markers do not adequately reflect the impact of increased ROS on β-cells or insulin signaling, while inflammatory, procoagulant or endothelial dysfunction markers are more specific to the pathophysiology of hyperglycemia (145,146). Hasnain et al. showed that islet-endogenous and exogenous IL-22 suppressed oxidative and ER stress caused by cytokines or glucolipotoxicity in mouse and human β-cells by regulating oxidative stress pathways. In obese mice, antibody neutralization of IL-23 or IL-24 partially reduced β-cell ER stress and improved glucose tolerance, whereas IL-22 administration modulated oxidative stress regulatory genes in islets, suppressed ER stress and inflammation, promoted secretion of high-quality efficacious insulin, and fully restored glucose homeostasis, followed by reinstitution of insulin sensitivity (147).

The chemokine system is also associated with obesity and insulin resistance. MCP-1, which acts on monocytes, macrophages, T cells and NK cells, is increased in obese compared to lean subjects and is related to non-alcoholic fatty liver disease and other lipid overload states (148-153). A short-term program of 4-month lifestyle modification significantly decreases MCP-1 levels, with favorable effects on the glycemic and lipid profile (151).

Collectively, these findings supported the investigation of new therapeutic approaches that target inflammation to ameliorate diabetes and its complications. Regarding the latter, it is important to note that multiple sources of evidence support a pathogenic connection between rheumatoid arthritis (RA) and the mechanisms of DM2, via formation of a vicious circle that is perpetuated by impaired glucose metabolism and inflammation. In this context, ongoing clinical studies have shown that the inhibition of interleukin IL-1 and IL-6 may allow the treatment of RA and concomitant T2D at the same time (154).

Treatment with anakinra, a recombinant form of human IL-1 receptor antagonist that works as a competitive inhibitor of IL-1β, achieved significant improvements of glycemia and secretory function of β-cells (155,156), which was maintained after anakinra withdrawal during a 39-week follow-up (157). Canakinumab, a monoclonal antibody against IL-1β, significantly reduced inflammatory proteins, such as CRP, IL-6, and fibrinogen, in patients with DM2 and high cardiovascular risk (158,159), while regarding glycemic control, it reduced values of HbA1c during the first 6–9 months of treatment, without, however, consistent long-term benefits (159). Antagonists of IL-6 receptor tocilizumab and sarilumab have also been investigated in patients with RA with or without concomitant DM2. Tocilizumab showed a positive effect on insulin resistance in some studies (160-162), while other studies failed to report any beneficial effect on glycemic control (163,164). The efficacy of sarilumab was assessed in a post hoc analysis (165) of three randomized clinical trials in patients with RA with or without DM2 (166-168). Sarilumab, as monotherapy or in combination, significantly reduced HbA1c compared to adalimumab monotherapy or placebo plus methotrexate/convectional DMARDs in patients with RA and DM2 (165).

Several studies have analyzed the effects of TNF inhibitors (i.e., adalimumab, etanercept, and infliximab) on glucose metabolism, demonstrating a potential favorable effect on insulin resistance and insulin sensitivity (169,170). In a meta-analysis combining data from 22 randomized controlled trials and three cohort studies (171), new-onset DM2 was delayed in RA patients treated with TNF inhibitors.

The Effect of Diabetes on the Immune System

Like a mirror image, chronic hyperglycemia in diabetic patients impairs the host’s immune response, which in turn fails to control the spread of invading pathogens, rendering diabetic patients more susceptible to infections (133,172). Both innate immune response defects and defective adaptive immune response are implicated in this incapacity of the immune system to defend against invading pathogens in patients with diabetes. Several mechanisms have been proposed by experimental and human studies including: i) leukocyte recruitment inhibition (173,174), ii) defective pathogen recognition (175), iii) dysfunction of neutrophils (176-178), macrophages resulting in impairment of phagocytosis (179), iv) functional defects in natural killer cells (180), and v) dysfunction of complement activation (181).

OSTEOPOROSIS AND THE IMMUNE SYSTEM

Osteoporosis is a clinical condition characterized by low bone mass and impaired bone microarchitecture associated with an increased risk of fragility fractures. Growing evidence of the last few decades has demonstrated the effect of the immune system on bone metabolism, leading to the emergence of the new field of osteoimmunology (182-185) and immunology of osteoporosis (named as immunoporosis) (184-188).

States of immune dysfunction such as immunodeficiency, inflammatory diseases, or immune response to infections are associated with increased osteoclastic bone resorption and, therefore, increased bone loss and increased fracture risk.

Bone Remodeling and Bone Cells

Bone remodeling is a dynamic and continuous process that is responsible for the maintenance of skeletal health throughout life. It involves three consecutive phases: i) osteoclast-mediated bone resorption; ii) the reversal phase, during which mesenchymal derived osteoblasts are recruited to the bone site of bone resorption; and iii) osteoblast-mediated bone formation. The two processes of bone resorption and bone formation are tightly coupled and under control of the matrix-embedded osteocytes, capable of sensing and integrating mechanical and chemical signals from their environment to regulate bone formation and resorption at the bone surfaces.

Osteoclasts originate from the same myeloid precursor that derives macrophage and dendritic cells and are specialized in bone degradation (186). Osteoblasts are the main bone-forming cells and are derived from mesenchymal stem cells. Osteoclast formation and differentiation is regulated by macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor-kB (RANK) ligand (RANKL) produced by osteoblasts and osteocytes (189,190). Osteocytes are a significant source of RANKL and its decoy receptor, osteoprotegerin (OPG), which binds to RANKL, preventing its interaction with RANK, while they also secrete the Wnt signaling inhibitor sclerostin, which regulates bone formation (189). RANKL is additionally expressed by fibroblasts and immune cells, including antigen-stimulated T cells and dendritic cells (191-193), while OPG is also produced by B lymphocytes and dendritic cells (194).

Inflammatory Diseases

Activated T cells increase the production of TNF-α and RANKL and stimulate osteoclastogenesis during inflammation (182-185,192-195). Multiple cytokines may promote osteoclastogenesis mainly by regulating the RANK/RANKL/OPG axis. TNF-α, IL-1, IL-6, IL-7, IL-11, Il-17, and IL-23 promote osteoclast differentiation, while IFN-α, IFN-γ, IL-3, IL-4, IL-10, IL-27, and IL-33 are considered anti-osteoclastogenic cytokines that protect bone integrity (195). Th17 cells are considered an osteoclastogenic subset of T cells as they enhance osteoclastogenesis by secreting IL-1, IL-6, Il-17, RANKL, TNF-α, and IFN-γ. Activation of Th2 leads to enhanced production of PTH and promotes the anabolic activity of osteoblasts in several inflammatory states. Furthermore, Th2 lymphocytes are associated with a low RANKL/OPG ratio and inhibition of bone loss (196). In addition, β-cells produce RANKL and OPG and may influence bone formation and absorption, while it has been observed that in HIV infected patients, there is an altered β-cell RANKL/OPG ratio that is inversely correlated with BMD (197).

Interleukin 6, produced by both stromal and osteoblastic cells (198) in response to stimulation by systemic hormones such as PTH, PTH-related peptide (PTH-rP), thyroid hormones, and 1,25-dihydroxyvitamin D3 and other cytokines (i.e., TGF-β, IL-1, and TNF-α), plays a major role in osteoclast development and function. Increased IL-6 levels contribute significantly to the abnormal bone resorption observed in patients with multiple myeloma (199), Paget’s disease of bone (200), rheumatoid arthritis (201), and Langerhans cell histiocytosis (202). Effects of increased osteoclast-induced bone resorption are not solely attributable to IL-6, but to all IL-6 family cytokines (203).

TNF-α has also been shown to induce bone resorption and plays an important part in inflammatory bone diseases (192). TNF-α promotes RANKL expression in osteoclast precursors and the formation of multinucleated osteoclasts in the presence of M-CSF. Furthermore, TNF-α increases RANKL and M-CSF expression in osteoblasts, stromal cells, and T lymphocytes, while RANKL can also enhance TNF-α mediated osteoclastogenesis (195). IL-1β increases RANKL expression and stimulates osteoclast formation and bone resorption while also promoting TNF-α induced osteoclastogenesis (204,205).

Postmenopausal Osteoporosis

Estrogen deficiency is a state of increased bone remodeling associated with an increased rate of bone resorption relative to bone formation, resulting in net bone loss. It has been shown that estrogen deficiency-induced bone loss has a complex mechanism mainly involving the immune system rather than a direct effect of estrogen on bone cells (206). Estrogen loss during menopause is associated with an expansion of T and B lymphocytes (207,208) leading to increased production of RANKL (209). In addition, an increased level of proinflammatory cytokines, including TNF-a and IL-1b, is observed in postmenopausal women (210,211). In addition, B lymphocytes may partially contribute to trabecular bone loss in postmenopausal osteoporosis (212). In the absence of estrogens, dendritic cells live longer, increasing their expression of IL-7 and IL-15. In turn, IL-7 and IL-15 induce IL-17 and TNF-a production in a subset of memory T cells, independent of antigen activation (213). These proinflammatory cytokines contribute to inflammation-induced bone loss in postmenopausal osteoporosis by activating low-grade inflammation. In contrast, Treg cells have a bone-protective role in postmenopausal osteoporosis (210). However, it has been shown that Th17/Treg balance is disturbed in estrogen deficiency. Treg cells lose their immunosuppressive function and convert to Th17 cells, which explains the imbalance of Th17/Treg in postmenopausal osteoporosis (214).

Senile Osteoporosis

Senile osteoporosis, on the other hand, is a low-bone turnover disease with decreased bone resorption and significantly reduced bone formation (215), which commonly occurs in older people, above 65, and affects both males and females. In recent years, it was demonstrated that aging is usually accompanied by systemic low-grade chronic inflammation and enhanced inflammatory mediators, such as IL-6 and TNF-a (216). A recent study found that senescent immune cells, such as macrophages and neutrophils, accumulate in bone marrow during aging in rats and mice (217). The senescent macrophages and neutrophils repress osteogenesis by promoting bone marrow mesenchymal stromal cell adipogenesis. In addition to directly inhibiting osteogenesis, the senescent immune cells contribute to chronic inflammation, thus leading to inflammatory bone resorption (217). Aging can tilt the balance of Th1/Th2 toward Th2 cells, resulting in an increased inflammatory response (218) and low-level chronic inflammation, ultimately leading to continuous bone loss.

Thyrotoxicosis-Induced Osteoporosis

IL-6 and IL-8 play a major role in thyrotoxicosis-induced osteoporosis and are increased in patients with thyrotoxicosis due to Graves’ disease or toxic multinodular goiter (219). In addition, patients with thyroid carcinoma on TSH suppressive therapy have significantly increased circulating levels of IL-6 and IL-8 compared to controls (219), which are tightly associated with serum T3 and fT4 concentrations. Both IL-6 and IL-8 have also been shown to be released by human bone marrow stromal cell cultures containing osteoblast progenitor cells in response to T3 (196). TNF-α elevations due to low TSH signaling in human hyperthyroidism also contribute to the bone loss that has traditionally been attributed solely to high thyroid hormone levels (220). Hyperthyroid mice lacking TSHR had greater bone resorption than hyperthyroid wild-type mice, demonstrating that the absence of TSH signaling contributes to low bone mass (221) in the hyperthyroid state.

Primary Hyperparathyroidism-Induced Osteoporosis

Bone resorption in primary hyperparathyroidism (PHP) also appears to be related to immune system effects. Circulating levels of IL-6 and TNF-α, which are significantly increased in patients with PHP, are strongly correlated with biochemical markers of resorption, returning to normal after successful parathyroidectomy (222). The hypothesis that IL-6 mediates the catabolic effects of parathyroid hormone (PTH) on the skeleton has been further strengthened by the finding that neutralizing IL-6 in vivo attenuates PTH-induced bone resorption in mice, while the resorptive response to PTH was also reduced in IL-6 knockout mice (223). Furthermore, it has been observed that transplantation of parathyroid from humans with hyperparathyroidism to mice lacking T cells was not associated with bone loss, suggesting a possible role of T lymphocytes in PTH-related osteoporosis (224). A direct action of PTH on T lymphocytes may also contribute, as deletion of the PTH receptor from T cells failed to induce bone loss (225). It has been proposed that PTH action on T cells results in secretion of TNF-α and, in combination with RANKL increase and OPG suppression, guides their differentiation to Th17 subsets, with subsequent IL-17 secretion and further RANKL amplification (226).

Drug-Induced Osteoporosis

The immune cells are also involved in drug-induced osteoporosis, such as glucocorticoid and chemotherapy-induced osteoporosis. A recent study demonstrated that glucocorticoid-induced osteoporosis could not be induced in T cell-deficient mice, while re-establishment was found to be possible by transferring splenic T cells from wild-type mice (227).

Cyclophosphamide, is a chemotherapy drug that causes immunosuppression and is associated with increased risk of osteoporosis (228-230). By improving the functional status of immune cells in an immunosuppressive mouse model induced by cyclophosphamide, bone loss was dramatically reduced (231), pointing to the possible contribution of immune cells in cyclophosphamide-induced osteoporosis.

EFFECTS OF THE IMMUNE SYSTEM ON THE STRESS SYSTEM

The Hypothalamic-Pituitary-Adrenal (HPA) Axis

The relations between the immune and the stress systems are complex and bidirectional, denoting that while stress can affect immune function, immune responses can also influence stress levels through various ways and mechanisms.

INFLAMMATION

During acute inflammation, the immune system is activated in response to infection or injury and releases proinflammatory cytokines and other inflammation-related factors into the central nervous system (CNS), plasma, and endocrine glands. Inflammatory cytokines, such as TNF-α, IL-1, and IL-6, produced by a variety of cells, including monocytes, macrophages, astrocytes, endothelial cells, and fibroblasts, activate the HPA axis leading to an increase of corticotropin-releasing hormone (CRH), adrenocorticotrophic hormone (ACTH) and, finally, glucocorticoids (38, 184). In turn, increased circulating levels of glucocorticoids exert suppressive effects on the inflammatory reaction, controlling the immune response and helping the organism to reach its prior healthy homeostasis (232). Similarly, in acute stress, the amplitude and synchronization of CRH secretory pulses is increased, and this is reflected in the levels of ACTH and cortisol in the systemic circulation (233).

The proinflammatory cytokine IL-1, especially its β form, is probably the most important molecule capable of modulating cerebral functions during systemic and localized inflammation. Systemic IL-1β injection activates the neurons involved in the control of autonomic functions, and neutralizing antibodies or IL-1 receptor antagonists are capable of preventing numerous responses during inflammatory stimuli (234). Similarly to IL-1β, intravenous IL-6 stimulates the hypothalamic-pituitary unit, leading to the secretion of cortisol by the adrenal glands and subsequent termination of the inflammatory cascade (235). All three inflammatory cytokines (IL-1, IL-6, and TNF-α) have the capacity to activate the HPA-axis, but it appears that IL-6 is the most critical component of this cascade. Studies in rats have demonstrated that immunoneutralization of IL-6 abolishes the effects of the other two cytokines on the HPA-axis (236). TNF-α and IL-1, on the other hand, stimulate the production of IL-6, which in turn stimulates the HPA-axis. The final end-product of HPA activation, glucocorticoids, inhibit IL-6 secretion at the transcriptional level through interaction of the ligand-activated glucocorticoid receptor with nuclear factor-kappa B, creating a negative feedback loop. In this way, IL-6 stimulates glucocorticoid release to control inflammation, and glucocorticoids subsequently inhibit IL-6 release through a negative feedback loop preventing uncontrolled and potentially harmful sequalae of inflammatory mechanisms, including tissue damage (237,238).

In an older study involving patients with Cushing disease studied before and after transsphenoidal adenectomy, plasma IL-6 concentration was increased when patients were hypocortisolemic, experiencing symptoms of glucocorticoid deficiency, as part of the “steroid withdrawal syndrome” (i.e., pyrexia, headache, anorexia, nausea, fatigue, malaise, arthralgias, myalgias, and somnolence of variable degree). Notably, IL-6 levels did not increase in patients who did not become hypocortisolemic after surgery, while following glucocorticoid replacement, a dramatic decrease of IL-6 levels concomitantly with relief of the observed symptoms was reported (239).

While acute stimulation with IL-6 activates the HPA axis mainly through the hypothalamic CRH neurons, chronic exposure to IL-6 may also directly stimulate the pituitary corticotropic cells and the adrenal cells via CRH receptor-independent mechanisms (240).

In vivo experiments have shown a stimulatory effect of IL-6 on cortisol production by the adrenal cortex in the absence of CRH and subsequent activation of the HPA axis (240) in cytomegalovirus-infected mice (241), as well as in murine colitis (242). In addition, experiments in mice deficient in CRH (CRH KO) and deficient in both CRH and IL-6 (CRH KO)/IL-6 KO) have demonstrated that IL-6 during prolonged immunological challenge may surpass CRH in augmentation of adrenal function (240). Protein expression of IL-6 and IL-6 receptor has been detected in primary cultures of human adrenocortical cells depleted of macrophages (CD68-positive cells), predominantly in the zona reticularis but also in the zona fasciculata and in single cells within the zona glomerulosa and the medulla (243). Moreover, IL-6 was able to induce adrenal steroidogenesis in vitro in a time- and dose-dependent manner in the absence of macrophages, suggesting that IL-6 may be a long-term stimulator of steroidogenesis but with no acute effects (243).

In humans, IL-6 may stimulate cortisol release directly at the level of the adrenal gland in long-term stress situations (244), and the same may also apply in chronic inflammatory diseases, although direct evidence is still lacking (245). In a recent clinical study, increased daily, and especially evening, saliva cortisol secretion, in the context of the acute viral infection COVID-19, appeared to be mostly driven by hypersecretion of IL-6, independently of ACTH (246). However, the hypothesis that IL-6 may partially replace ACTH when there is an acute requirement for increased cortisol secretion has yet to be tested.

PROLONGED OR CHRONIC STRESS AND INFLAMMATION

Acute versus chronic stress and inflammation are distinct conditions that exert extremely different effects on the immune system, each altering its function in a distinct manner. Chronic stress is associated with a blunted circadian cortisol rhythm, a suppressed inflammatory response, and a shift from Th1 to Th 2 and a Th reg to Th 17 immunity. It has clearly been shown that in chronic stress, endogenous glucocorticoids fail to terminate the stress response and, in fact, cause body composition changes reminiscent of those in hypercortisolism, such as visceral adiposity, sarcopenia, and osteoporosis (247-249).

On the other hand, following a period of intense stress, there may be glucocorticoid-induced suppression of the HPA axis (238). In this case, long-term “inadequate” cortisol secretion may unleash its inhibitory effect on immune system activation, resulting in immune dysregulation and expression of sickness-syndrome manifestations. In this case, the post-stress sustained HPA axis suppression and hypocortisolism is reminiscent of the clinical picture of the intrinsic hypocortisolemia of primary adrenal insufficiency (Addison’s disease), associated with immune perturbations due to failure of cortisol to appropriately suppress the increased secretion of proinflammatory cytokines (239,250).

The subsequent protracted lack of axis recovery due to a post-illness state of hypocortisolism in probably predisposed individuals may explain the underlying pathophysiologic mechanisms responsible for some post-viral infection sickness syndromes (232,251), such as long COVID syndrome (252).

AUTOIMMUNE DISORDERS

In some cases, chronic stress can contribute to the development or exacerbation of autoimmune disorders. Without appropriate cortisol regulation in cases of chronic stress and chronic inflammation, the organism fails to downregulate inflammatory processes, contributing to a vicious cycle where stress, inflammation, and compromised HPA axis function may result in the development of chronic immune-mediated inflammatory diseases, such as rheumatoid arthritis. Stress can potentially trigger or worsen these conditions by dysregulating the immune response.

Cortisol diurnal secretion displays a circadian rhythm in healthy individuals, with the highest levels in the morning and a gradual decline throughout the day, reaching the lowest levels around midnight (253). During acute stress (254-256) or infection (237,257), the rhythm is disturbed and the afternoon and/or midnight cortisol levels do not drop. A disturbed circadian cortisol rhythm with abnormally high afternoon and night cortisol levels was found in patients with even mild or moderate COVID-19 compared to healthy controls (246). However, although not systematically studied, the adrenal response to chronic versus acute exposure to proinflammatory cytokines appears to follow a different pattern.

In patients with rheumatoid arthritis, symptoms follow circadian rhythms with impaired function due to pain and joint stiffness being most severe in the early morning (258,259) because of increased proinflammatory cytokines, such as TNF-a and IL-6, that occur during late night hours (260,261). This explains the inverse relation between diurnal variation of circulating IL-6 and glucocorticoid levels (262). Increased endogenous nocturnal secretion of cortisol could alleviate these morning symptoms, but this is not the case in most patients with rheumatoid arthritis (263-270). To clarify this issue, several studies have been carried out reporting that the optimal time for delivery of glucocorticoid treatment would be during the night in order to target the effects of nocturnal proinflammatory stimuli (271-274).

PSYCHOLOGICAL WELL-BEING

The immune system also plays a role in maintaining overall psychological well-being. Chronic stress and its impact on the immune system can increase susceptibility to mental health problems such as anxiety and depression. Conversely, psychological well-being can improve the human body’s immune responses, enhance resistance to diseases (including infectious diseases), and improve mental health (275,276). Changes in psychological status of patients with Alzheimer’s induced significant differences in their immune response (277).

In addition, long-term practice of meditation was shown to decrease stress reactivity and exert a favorable therapeutic effect in chronic inflammatory conditions characterized by neurogenic inflammation (278), while joyful activities such as singing were able to boost the immune response in cancer patients and family members (279).

ADRENAL MEDULLA

The chromaffin cells of the adrenal medulla play a role in stress response by secreting catecholamines and various biologically active peptides (238). As the stress response starts, rapidly augmented secretion of norepinephrine and epinephrine is initiated, followed by activation of the HPA axis and increased release of CRH and ACTH and secretion of glucocorticoids (280). CRH and norepinephrine stimulate the secretion of each other through CRH-R1 and α1-noradrenergic receptors, respectively (281).

Cytokines TNF-α, IL-1, and IFN-γ act directly on chromaffin cells (282-285). It has also been demonstrated that cytokines regulate the secretion of various peptides that are co-secreted with catecholamines, such as vasoactive intestinal peptide (VIP), galanin and secretogranin II, enkephalin and neuropeptide Y (283,285). IL-6 directly modulates the secretion of catecholamines and neuropeptides by chromaffin cells and therefore influences the adrenal stress response. It has been hypothesized that medullary peptides may serve as paracrine modulators of glucocorticoid production (286). It has also been shown that IL-6 increases intracellular Ca2+ concentration and induces catecholamine secretion in rat carotid body glomus cells, a finding which has finally confirmed the relations between IL-6 and catecholamine secretion (287). Furthermore, IL-10 is a critical target downstream of epinephrine and norepinephrine which limits inflammation (288). On the other hand, norepinephrine may act directly on macrophages and dendritic cells to suppress inflammatory cytokine secretion through primarily the β2-adrenergic receptors that are expressed by both innate and adaptive immune cells (289,290).

Although scientific advances of the last decade have shed light on the previously unrecognized major role of the catecholamines epinephrine and norepinephrine in controlling the immune system, much remains to be discovered, and further revelations will reveal new therapeutic targets in the management of inflammation.

ACKNOWLEDGEMENTS

This chapter is an update of a previous chapter and the authors would like to thank Professor Gregory Kaltsas, an author of the prior chapter.

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Diabetes and Hypoglycemia in Children and Adolescence

Type 2 Diabetes In Children and Adolescents – A Focus On Diagnosis and Treatment

ABSTRACT

Nearly three decades have passed since the first publications on type 2 diabetes (T2D) in children and adolescents, and it is now well established as a global problem. As the prevalence of obesity continues to increase within the general population, diagnosing T2D in adolescents presents a multifaceted challenge. In this chapter, we shall delve into the epidemiological aspects, as well as the distinct characteristics inherent to various types of diabetes. Cohort studies with long-term follow-ups have illuminated our understanding of the alarming incidence of complications in adolescents diagnosed with T2D. Recent approvals of novel pharmaceutical interventions for teenagers have ushered in a new era of hope. Hopefully, in the forthcoming decade, we anticipate a decline in the prevalence of these complications. However, in the interim, a proactive and assertive approach remains essential for addressing the complications associated with T2D in adolescents.

SHORT HISTORY

Youth-onset type 2 diabetes (T2D) was initially described in Pima Indian children and adolescents (1). This tribe, also known as “the pathfinders,” has a notable history of obesity and early-onset diabetes. In the mid-nineties, studies from various clinics in the United States reported cases of T2D in children from diverse ethnic backgrounds, primarily non-Hispanic Blacks and Hispanics (2-4). While these reports were initially met with suspicion, it soon became clear that a new disease had emerged among children. Subsequently, similar reports emerged from various parts of the world. In 2000, the SEARCH for Diabetes Study was launched in the US. This prospective nationwide, multi-center study aimed at understanding the epidemiology of both type 1 diabetes (T1D) and T2D in children. A few years later, in 2004, the Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) study was initiated. The TODAY study was a prospective, randomized treatment trial, designed to explore treatment regimens and the clinical course of T2D in youth. Registries from Germany, Diabetes Prospective Follow-up Registry (DPV) (5), Hong Kong (6), China (7), India (Registry of People with Diabetes with Youth Age at Onset (YDR)) (8) and Israel (9), contributed to the current knowledge. Importantly, while the US is leading in the scope of the T2D epidemic, similar trends have been observed worldwide.

EPIDEMIOLOGY

Epidemiology in the USA

According to the most recent data from the SEARCH study published in 2023, the adjusted incidence of T2DM among children and adolescents nearly doubled, from 9.0 to 17.9 cases per 100,000 persons per year, from 2002-03 to 2017-18 (10).

EPIDEMIOLOGY ACCORDING TO ETHNICITY

The incidence of T2D varies largely based on ethnicity, the highest incidence of T2D, 50.1 per 100,000 children age 10-20 years, was recorded in non-Hispanic Blacks, followed by Pima Indians (46), Hispanics (25.8), and Asian/Pacific Islanders (16.6). Among non-Hispanic Whites, the incidence is only 5.5 per 100,000. An annual increase was observed in all groups, but the highest increase was observed in Asian/Pacific Islanders (8.92%), followed by 7.17% in Hispanics and 5.99% in non-Hispanic Blacks, compared to 1.83% in non-Hispanic Whites.

EPIDEMIOLOGY ACCORDING TO GENDER AND AGE

The current incidence in females is 21.6 per 100,000 and in males 14.2; the temporal increase was similar in both genders. The incidence increases with age. One of the most remarkable findings in the SEARCH study is that among individuals 15-19 years of age, the incidence of T2D in 2017-2018 exceeded that of T1D (19.7 vs. 14.6 per 100,000). This is the first time the incidence of T2D surpassed that of T1D among youth, sounding alarming for other countries, which may have a lower incidence than the USA, but are showing steady increases.

Epidemiology in Europe

Although the overall incidence of T2D in Europe is much lower than in the US, a similar change over time has been reported. In Germany, a three-fold increase in the prevalence of T2D was reported for 10- to 19-year-olds between 2002 and 2020 (3.4 to 10.8 per 100,000) (5). Similar to the US, the estimated standardized prevalence of T2D was 1.4 times higher among girls (12.8) than boys (9.0). In the UK, the number of children registered as having T2D and being treated in pediatric diabetes units has risen by more than 50% in the past five years (11).

Epidemiology in Asia

Data retrieved from the Hong Kong Childhood Diabetes Registry revealed a threefold increase in T2D in children, from 1.27 per 100,000 in 1997-2007 to 3.42 per 100,000 in 2008-2018 (6). Data from China demonstrated an average annual increase of 26.6% in youth aged 10-19 years (12). There was no statistically significant difference in incidence between boys and girls. However, the risk of T2D was 1.49 times higher in urban areas than in rural areas.

RISK FACTORS FOR T2D IN CHILDREN

The increased risk of developing T2D in children is associated with genetics, an obesogenic environment, and obesity, along with the interactions among these factors (Figure 1). This visual representation emphasizes the multifaceted nature of risk factors for T2D in children and adolescents, illustrating the intricate connections between genetics, environmental factors, obesity, and the eventual development of T2D.

Figure 1. Risk Factors for T2D in Children and Adolescents. Risk factors for T2D in children and adolescents can be visualized as a series of interconnected circles, illustrating the complex interplay of various influences:
Environment (Outer Circle): The outermost circle represents the broader environmental factors that encompass everything from the intrauterine environment to residential neighborhood.
Genetics (Second Circle): Within the environment circle, genetics forms an important inner circle. It signifies the genetic predisposition that some individuals may have towards developing obesity and, consequently, T2D.
Obesity (Third Circle): Nested within the genetics circle is the obesity circle. It is influenced by multiple factors such as immigration, sedentary lifestyle, diet, microbiome, medication use, depression, and abuse. Of note, not everyone exposed to the obesogenic environment will become obese, indicating the influence of other factors.
Type 2 Diabetes (Innermost Circle): The innermost circle represents the development of T2D. Importantly, not all obese individuals will go on to develop early-onset T2D, highlighting the complexity of these interrelated risk factors.

Obesity

Severe obesity stands out as one of the most significant risk factors for youth-onset of T2D. Obesity is closely linked to the worsening of insulin resistance, a primary feature of the pathophysiology of T2D, along with progressive β-cell and α-cell dysfunction (13). In a recent meta-analysis comprising data from 30 studies involving 4688 children and adolescents, the prevalence of obesity at the time of T2D diagnosis was found to be 77 % (95% CI, 71%-83%) (14). Male participants exhibited higher odds of obesity than their female counterparts (odds ratio, 2.10; 95% CI, 1.3-3.3). These findings were sustained after several sensitivity analyses that excluded studies with uncertain or unspecified T2D diagnostic criteria, studies involving individuals with positive pancreatic autoantibodies, individuals presenting with weight loss, or those with genetically proven monogenic diabetes. While acknowledging the limitations of using BMI-based measures as a surrogate for obesity and considering the retrospective nature of the included studies, these data suggest that approximately 20-25% of adolescents with diabetes but without obesity may still have T2D. Furthermore, the definition of obesity needs to consider body composition variations among certain populations, such as East Asian and South Asian populations.

Family History of T2D

Most adolescents with T2D have a family member with the disease. In the TODAY study, 59.6% of adolescents with T2D had a first-degree family member with a history of diabetes, and 89% had a grandparent affected with diabetes (15). This increased risk of T2D associated with family history reflects genetic influences, the impact of the environment, and the intrauterine environment.

Maternal Obesity and Maternal T2D During Pregnancy

Exposure to maternal obesity has been linked to an increased risk of childhood obesity in offspring (16) and 2.8-fold higher odds of T2D. Exposure to maternal gestational diabetes was associated with 5.7-fold higher odds of T2D (17). Moreover, individuals exposed to maternal diabetes during pregnancy were diagnosed with diabetes at a younger age and exhibited worse β-cell function. Exposure in utero to maternal diabetes and obesity accounted for 47% of the T2D risk in this population (17,18), and, importantly, it sets up a vicious cycle for future generations. Suggested mechanisms affecting the developmental programming of offspring towards T2D include epigenetic modifications, alterations in stem cell differentiation, variation in the metabolome and microbiome, and immune dysregulation (19). Of note, assistedreproductive technology was not found to be a risk factor for early-onset T2D (20).

Pregnancies complicated by pre-existing diabetes are associated with extreme birth weights. In a large study, small for gestational age (SGA) babies, defined as below the 10th percentile for birth weight, were reported in 14.1% of pregnancies of women with T2D, while 26.2% had newborns with a large birth weight, defined as above the 90th percentile (21). Both SGA and high birth weight are associated with an increased risk of a history of early-onset T2D among adults. Of note, a lower birthweight was associated with an increased risk of developing T2D independently of adult BMI and the genetic risk of T2D (22).

Pregnancies complicated by pre-existing diabetes are associated with preterm delivery and birth weight extremes. In a national cohort study involving over 4 million singletons born in Sweden from 1973 to 2014, preterm birth (occurring before 37 weeks) was associated with a 1.3-fold increased risk of developing T2D before age 18 years (23). Notably, the association between preterm birth and T2D was significantly stronger among females (23).

Mental Health and Treatment

The association between psychiatric disorders in adolescents and T2D is bidirectional, complex, and not well studied in the pediatric population (24). On the one hand, youth with chronic illnesses such as obesity are more likely to develop depression, depressive symptoms, and anxiety than those without chronic illnesses. On the other hand, youth who have mental health morbidities are at increased risk for isolation, sedentary lifestyle, weight gain, and the development of T2D. Furthermore, some psychotropic medications, particularly atypical antipsychotics, are associated with weight gain and increased risk for T2D in adults. These associations have not been systematically studied in youth, but clinical experience suggests a contribution.

Immigration

Globally, immigration is on the rise. Studies conducted among adults have indicated that immigrants, when compared to non-immigrants, exhibit higher rates of obesity, insulin-resistance, and hypertension. In a recent review (25) focusing on European countries, non-European migrant children face a greater risk of being overweight or obese compared to their native counterparts; the prevalence of obesity in migrant and native children ranged from 1.2 to 15.4% and from 0.6 to 11.6%, respectively. The increased rates of overweight and obesity among migrating children and adolescents render them more susceptible to developing T2D and other metabolic abnormalities. An Israeli study found that among adolescents of Ethiopian origin, the prevalence of overweight and obesity increased two-and-a-half fold and fourfold in males and females, respectively, during the study period, compared to a 1.5-fold increase among native Israeli-bornmales and females (26).

Socioeconomic Status

A discernible disparity exists in the socioeconomic status (SES) of children and adolescents with T2D. In developed countries, such as the United States, low SES is a recognized risk factor for the development of obesity and T2D. This is attributed to factors such as limited access to healthy foods and reduced physical inactivity. However, in developing countries, higher SES groups tend to have greater levels of physical inactivity and consume higher quantities of fat, salt, and processed foods compared to their lower SES counterparts (Figure 2). When data from 384 youth with T2D who completed a baseline research visit as part of the SEARCH study and 227 youth with T2D from the Registry of People with Diabetes with Youth Age at Onset (YDR) in India who completed a baseline visit were compared, only 23.6% of SEARCH youth belonged to a high SES group, in contrast to 88.5% of YDR youths (P < .001) (8).

Figure 2. Global Disparities in Type 2 Diabetes Risk Factors. This illustrative figure offers insights into the diversity in risk factors for T2D among children across the world. It highlights how these risk factors vary across different regions and populations.
Gender Disparities (Upper Part of Figure): In many Western countries, the risk of developing T2D in children is higher in girls compared to boys. However, this pattern is not consistent globally. In other parts of the world, an equal or even higher risk of T2D has been documented in males. The upper portion of the figure visually represents these variations in risk, depicting the female-to-male ratios in different regions.
Socioeconomic Status (SES) Variation: (Lower Part of Figure) SES plays a significant role in T2D risk. In Western countries, lower SES is associated with an increased risk of T2D in children. Conversely, in developing countries, a contrary trend is observed, with a higher risk of T2D observed in populations with higher SES. These complex socioeconomic variations are highlighted in the figure, showcasing how T2D risk is influenced by economic factors on a global scale.

Data from 747 children and youths with T2D under 19 years of age, collected between 2009 and 2016 (from the population-based National Pediatric Diabetes Audit, covering over >95% of diabetes cases in England and Wales), revealed that under half of those with T2D reside in the most disadvantaged areas of England and Wales, and 41% fall into the most disadvantaged SES quintile (27). In contrast, in China, in Zhejiang, one of the most economically prosperous coastal provinces marked by industrialization and urbanization, changes in diet and decreased physical activity have led to a higher incidence of pediatric obesity. The mean annual incidence was 2.32 per 100,000 person-years in urban area compared with 1.44 in urban and rural area (12).

COVID-19 Pandemic

In a study conducted in the United States, the incidence of T2DM at 24 diabetes centers during the first year of the COVID-19 pandemic was examined (28). The average number of new diagnoses per year in the two years before the pandemic was 825 but rose to 1463 during the first pandemic year. This increase of 77% is significantly higher than the 5% expected annual increase in incidence observed in the two previous years. Similarly, a retrospective cross-sectional review of youths (age ≤ 21) diagnosed with T2D during the COVID-19 pandemic (from May 1, 2020, to April 30, 2021) and the five years preceding it (from May 1, 2015, to April 30, 2020) at a tertiary diabetes center revealed an increase of 293% (29). Similar trends were reported from Germany (30). Data on T2D in adolescents during 2 years of the COVID-19 pandemic (2020–2021) were compared with the control period 2011–2019 in children aged 6 to <18 years were obtained from the DPV (German Diabetes Prospective Follow-up) Registry. The incidence of youth-onset T2D increased from 0.75 per 100,000 patient-years in 2011 to 1.25 per 100,000 in 2019, an annual increase of 6.8%. However, in 2021, the observed incidence was 1.95, significantly higher than expected. Of note, the observed incidence was significantly higher in boys (2.16), leading to a reversal of the sex ratio of pediatric T2D incidence (30). The surge in T2D incidence during the pandemic can be attributed to several factors. First, there was an increase in obesity among young people during the COVID-19 pandemic, accompanied by increased consumption of processed foods and reduced physical activity, both of which contribute to the risk of developing T2D (31). In addition, there was increased psychosocial stress, which is emerging as an important contributor to the risk of T2D. Furthermore, viral mediated non-autoimmune β-cell destruction, resulting in reduced β-cell function in predisposed adolescents, has been suggested as a contributing factor (28).

Genetics

The genetics of T2D in children and adolescents have largely remained unexplored. ProDiGY is a multiethnic collaboration encompassing three studies (TODAY, SEARCH, and T2D-GENES) involving 3,006 youth subjects with T2D, diagnosed at a mean age of 15.1±2.9 years, along with 6,061 diabetes-free adult control subjects. Association analyses were conducted on approximately 10 million imputed variants, employing a generalized linear mixed model incorporating a genetic relationship matrix to account for population structure. The analysis was further adjusted for sex. This comprehensive study identified seven genome-wide significant loci that included a novel locus in PHF2, along with TCF7L2, MC4R, CDC123, KCNQ1, IGF2BP2, and SLC16A11. A secondary analysis involving 856 diabetes-free youth subjects uncovered an additional locus in CPEB2, and consistent directional effects for diabetes risk were observed (32).

CLINICAL PRESENTATION

Diabetes Related Signs

The classical signs at the onset of diabetes - polydipsia and polyuria - are observed in about two-thirds of youth at the diagnosis of T2D. Only about one-third are diagnosed through routine screening of asymptomatic youth with obesity. Furthermore, the prevalence of diabetic ketoacidosis (DKA) among youth with T2D in the SEARCH study and the YDR in India was 5.5% and 6.6%, respectively. Importantly, the incidence of DKA at the onset of T2D was higher during the SARS-CoV-2 pandemic (33). Hyperglycemic hyperosmolar state is present at diagnosis in 2% of youth with T2D (34).

Seasonal Diagnosis of T2D

There is significant seasonal variation in the onset of T2D in children and young people in the United States; diagnoses increased in August. Possible explanations for an August peak in diagnoses include weight gain during the summer vacation and an increase in physical exams for school athletic programs that may detect asymptomatic hyperglycemia. The greater proportion of diagnoses made during routine health visits rather than following symptoms is consistent with this latter possibility. Similar seasonal variations in incidence are beginning to emerge in other countries.

Age of Onset of T2D

The incidence of T2D rises gradually during puberty, primarily due to the physiological insulin resistance characteristic of this period. However, illustrating the heterogeneity of T2D in children and adolescents, there was a significant difference between the mean age of onset of youth in the United States (Pediatric Diabetes Consortium, PDC) databases who were diagnosed at the mean age of 12 years compared to the mean age in Germany of 13 years (35).

PREPUBERTAL T2D

With the worldwide epidemic of childhood obesity on the rise, there are increasing reports of T2D occurring in prepubertal children. Initially, there were case reports on prepubertal children with T2D, including cases from Nigeria (36), and a 5-year-old Indigenous girl from Australia (37). Lately, several reports have described the onset of T2D in prepubertal children. A total of 42 children ≤ 10 years with T2DM were reported from Alabama (38), 12 from Australia (39), 35 prepubertal children from Houston, Texas (40), from San Antonio, Texas (41) and India (42). The common denominator of all reports of prepubertal children is extreme obesity, with a disproportionate impact on females and ethnic minorities as shown in Table 1. Additionally, there is a high prevalence of comorbidities.

Table 1. Characteristics of Prepubertal Children Diagnosed with T2D
	San Antonio, Texas N=20	Alabama, US N=42	Australia N=12	Houston, Texas. N=35	Chennai, Tamil Nadu India N=4
Age, years Range	8.1 4-9		7-10	10.6± 2.5
5-8	10	8
9-10	10	34
Ethnicity	Hispanic 80%	African American 88(%) Caucasian 12%	Aboriginal Australians 92 Maori 8	Hispanic 71 Black 23 Non Hispanic White 3%	India
Females (%)	75	88	75	51	100
BMI Z score	2.72 (1.7-5.0)	2.5 ± 0.4	2.38± 0.64	2.4±0.4	>97%ile
Hypertension (%)	NA	21	25	14
Dyslipidemia	NA	58	58%	100
NAFLD	NA			20
Microalbuminuria	NA		16.6	5

These reports suggest that there are unique features and pathophysiologic drivers distinct from the influence of pubertal hormones, which are often implicated in the mechanism underlying T2D in older children and adolescents. Furthermore, the emergence of T2D in prepubertal children and the high prevalence of comorbidities among them serve as a warning for an impending public health challenge.

Sex

In the Western world, youth-onset T2D is almost twice as common in girls as in boys, whereas Asian countries report no differences in incidence by sex. For T2D, SEARCH had a higher proportion of females, while studies from China show no difference between females and males, and there are reports of increased prevalence in males in the Middle East; in the UAE (43), the female to male ratio was 0.5:1, in Iraq (44) 0.6:1, and Kuwait (45) 0.8:1 (Figure 2). This may reflect the higher prevalence of obesity in males in these countries (46). Indeed, in the UAE, among children aged 11 to 14 years, the prevalence of obesity was reported as 24.3% and extreme obesity (BMI ≥99^th percentile) as 5.7%; the rate of extreme obesity was 9.6-fold higher in boys than girls (47). In Kuwait, 41.4% of boys were classified as obese, compared to 28.9% of girls of the same age (46).

DIAGNOSIS

Early diagnosis of T2D is likely to bring several benefits, enabling prompt multifactorial treatment and management of cardiovascular risk factors. Considering the increasing prevalence of T2D among children and adolescents, clinical symptoms have become less distinct, and age, sex, and weight are no longer definitive criteria for a clear diagnosis. Therefore, the most important diagnostic feature is the presence or absence of pancreatic autoantibodies in any child diagnosed with hyperglycemia; positive antibodies indicate T1D even if the child is overweight or has a family history of T2D. If the antibodies are negative, a genetic test for monogenic diabetes (previously referred to as Maturity-Onset Diabetes of the Young (MODY) should be considered if available.

TREATMENT

Initial treatment for T2D includes a focus on lifestyle changes, including healthy diet, increasing regular physical activity, achieving weight loss, and receiving emotional support. While lifestyle change is recommended as a practical approach,there is limited evidence that lifestyle intervention alone has a sustained impact on glycemic control in youth with T2D (48).

The Impact of Lifestyle Changes

The impact of lifestyle intervention, as determined by changes in diet and cardiovascular fitness, on glycemic control in youth with T2D was assessed in the TODAY clinical trial cohort spanning 15 centers across the United States. The study included 699 youth aged 10 to 17 years with T2D for less than 2 years. Those participants randomly assigned to the lifestyle intervention participated in a family-based behavioral program aimed at promoting weight loss. Each family was assigned a dedicated leader responsible for guiding them through their journey of physical activity and nutrition improvement. The intervention was structured across three stages, initially there were weekly meetings for 6 to 8 months, focusing on physical activity, setting individual calorie intake goals, self-monitoring, and problem-solving. In the subsequent stage, biweekly meetings were held for 12 to 16 months. Lastly, meetings were held monthly for 24 to 28 months, concentrating on maintaining a healthy lifestyle. Dietary data were collected through an interviewer-administered food frequency questionnaire, and cardiovascular fitness was assessed using a submaximal cycle ergometer test at baseline, 6 months, and 24 months (49). At 6 months, approximately 25% of females and 33% of males improved cardiovascular fitness. The authors concluded that a minority of youth improved fitness and/or diet over time, although those who did showed a beneficial impact on glycemic outcomes. However, the positive lifestyle behavior changes did not persist after 24 months, providing a sobering perspective on the challenges of implementing lasting lifestyle changes in this population.

Pharmacotherapy

The beginning of the third decade of the 21st century will be marked by the introduction of novel medications for children with T2D. There are several groups of medications for T2D, biguanides, sulfonylureas, GLP1 receptor agonists, SGLT2 inhibitors, dipeptidyl peptidase inhibitors, and combinations of the dual incretin analogs and the new triple G agents (dual incretin and glucagon). However, only biguanides, GLP1 receptor agonists, and SGLT2 inhibitors are currently FDA-approved for children. Doses and modes of administration are presented in Table 2.

Table 2. FDA-Approved Medications for Treating Type 2 Diabetes in Children Aged 10 Years and Older
Biguanide	Metformin	Glucophage	p.o daily	850, 1000. up to 3000 day
GLP receptor agonist	Liraglutide	Victoza	s.c. daily	0.6mg 1.2mg 1.8mg
	Exenatide	Bydureon	s.c weekly	2 mg
	Dulaglutide	Trulicity	s.c weekly	0.75mg/0.5mL 1.5mg/0.5mL 3mg/0.5mL 4.5mg/0.5mL
SGLT2 inhibitor	Empagliflozin	Jardiance	p.o daily	10 mg 25 mg
SGLT2 inhibitor	Dapagliflozin	Forxiga	p.o daily	5mg 10 mg

Five randomized control studies assessed the impact of these drugs in children with T2D. The inclusion criteria were consistent across all studies and included an age above 10 years, a BMI above the 85^th percentile, and minor variations in HbA1c levels (between 7.0 and 11.0%, or 6.5% and 10%). The characteristics of the participants and outcomes are detailed in Table 3. Figure 3 depicts HbA1c results vs. placebo for each medication, and Figure 4 depicts the percent of individuals with HbA1c less than 7% at the end of each study.

Table 3. Effects of Various Medications on Glycemic and Anthropometric Parameters in Adolescents with Type 2 Diabetes After 26 Weeks of Treatment
	Liraglutide		Exenatide		Dulaglutide 0.75 mg		Dulaglutide 1.5 mg		Empagliflozin		Dapagliflozin		Linagliptin		Sitagliptin
	Tx	Pl	Tx	Pl	Tx	PL	Tx	PL	Tx	PL	Tx	PL	Tx	Pl	Tx	Pl
Number	66	68	59	24	52	51	52	51	52	5	39	33	53	53	95	95
Age (years)	14.6	14.6	15	16	14.7	14.2	14.7	14.2	14.6	14.4	16.1	14.4	14.6	14.4	14.3	13.7
Baseline HbA1c )%(	7.9	7.7	8.1	8.1	7.9	8.1	8.2	8.1	8.0	8.05	7.95	7.85	8.05	8.05	7.4	7.6
HbA1c change	-0.64	0.46	-0.36	0.49	-0.6	0.6	-0.9	0.6	-0.17	0.68	-0.25	0.5	0.33	0.68	-0.01	0.18
HbA1c ETD	1.06		0.85		1.2		1.5		0.84		0.75		0.35		0.19
% HbA1c <7%	63.7	36.5	48	35.1	55	14	48	14	34.6	24.5	25	4	26.5	24.5	49.5	36.8
Baseline BMI (kg/m²)	34.6	33.3	36.9	35.4	33.6	34.3	34.3	34.3	35.5	36.1	31.3	33.6	36.5	36.1	0.0	-0.7
BMI ETD	-0.25	-0.21	ND	ND	-0.2	0	-0.1	0	ND	ND	-0.08	-0.11	ND	ND	-0.7
Weight change (kg)	-2.3	-0.99	-0.59	0.63	0.3	0.1	0.2	0.1	-0.79	-0.04	ND	ND	1.42	-0.04	-1.1	0.06
Weight ETD	-1.3		-1.2		0.2		0.1		-0.75				1.46		1.04
Systolic BP ETD	0.03		-2.9		3.8		2.5		-1.42		1.9		0.91
Diastolic BP ETD	-1.08		ND		-0.8		-2.6		0.02		-0.5		1.5

Pl=placebo, Tx= treatment, ETD = estimated treatment difference, ND= no data, placebo group for Empagliflozin and Linagliptin was the same.

Figure 3. Change in HbA1c percentage points in adolescents with T2D under medication treatment compared with untreated control group. The differences in HbA1c percentage points from the baseline for adolescents diagnosed with T2D who received medication treatment (depicted in black) compared to control groups (shown in white with data points). In the medication-treated group, there is a significant decrease in HbA1c levels, denoted by the downward trend in the black bars. Conversely, in the control groups of all the studies, HbA1c levels increased, as indicated by the upward trend in the white bars with data points.

Figure 4. HbA1c Levels below 7% in adolescents with T2D. The percentage of adolescents who achieved HbA1c levels below 7% at the end of the follow-up period. A comparison is drawn between those treated with medication (depicted in black) and the control group (depicted in white).

METFORMIN

Metformin is a biguanide antihyperglycemic used in conjunction with diet and exercise to control glycemia. Metformin is considered the preferred first-line agent for treating T2D in pediatric patients 10 or older (50). Its onset of action is around 1.5 hours, and its total duration of action is 16-20 hours. Metformin exerts its glucose-lowering effect through various mechanisms, including inhibiting hepatic gluconeogenesis, increasing glucose uptake in skeletal muscles, reducing adipogenesis, and activating brown adipose tissue, leading to enhanced thermogenesis. Additionally, metformin decreases the absorption of glucose from the intestine and downregulates inflammation.

Although it is the first line of treatment for children and adolescents with T2D, prospective, randomized, controlled, long-term studies are limited. In a randomized control study involving 82 children and adolescents, 42 received metformin at doses up to 2000 mg/day, and 40 received placebo (51). After 16 weeks, mean HbA1c levels were lower in the metformin group compared to the placebo (7.5% and 8.6%, respectively). The metformin group also experienced a mean weight decrease of -1.5 kg compared with a mean increase of 0.9 kg in the placebo group. The mean BMI changed by -0.5 units in the treatment group vs. -0.4 units in the placebo group. In another study, metformin (500–1000 mg twice daily) was compared to glimepiride, a sulfonylurea (1–8 mg once daily), for 24 weeks (52). Both metformin (-0.71%) and glimepiride (-0.54%) groups showed significant reductions from baseline HbA1c levels. A total of 48.1% of metformin-treated and 42.4% of glimepride-treated participants achieved HbA1c levels below 7.0% at week 24. While the incidence of hypoglycemia was similar in both groups, metformin resulted in less weight gain compared to glimepiride. In a large observational study (53), 927 youth aged 13.7±2.0 years old, who had been diagnosed with T2D for a median of 2 months and had a baseline HbA1c of 7.7±2.2%, were treated with metformin. After a median of 71 days, the mean HbA1c change was -1.33, and the mean weight change -0.43 kg (53,54).

Metformin-related side effects are gastrointestinal, including diarrhea, abdominal pain, bloating, nausea, and decreased appetite, which occur in about 50% of users. Gastrointestinal side effects are usually transient and improve over time. Another potential side effect of metformin is vitamin B12 deficiency, and regular monitoring is indicated, though no cases of vitamin B12 deficiency were identified in the TODAY study. Lactic acidosis is a concerning side effect associated with metformin use in adults. It occurs in the presence of hypoperfusion or hypoxemia; however, it has rarely been reported in children and adolescents.

GLUCAGON-LIKE-PEPTIDE 1 (GLP1)

Glucagon-like peptide-1 (GLP-1) is a peptide hormone primarily produced by intestinal enteroendocrine cells at low basal levels that rapidly increases within minutes of food consumption. GLP-1 plays a vital role in regulating meal-related glycemic excursions by enhancing insulin secretion and inhibiting glucagon secretion. Additionally, GLP-1 slows gastric emptying and reduces food intake through a central effect on appetite, which contributes to weight loss. However, GLP-1 has a short half-life of approximately 2 minutes, as it is rapidly degraded by dipeptidyl peptidase-4 (DPP-4). To enhance GLP-1 activity, medications such as DPP-4 inhibitors and GLP-1 receptor agonists have been developed.

The story of the development of GLP1 agonists is truly fascinating. It begins with the venomous Gila monster (Heloderma suspectum), native to New Mexico and Arizona. H. suspectum, a long-lived and reclusive species, spends a significant portion of its life underground. Although its venomous bite causes pain and weakness, it is rarely fatal to adult humans. In 1990, endocrinologist Dr. John Eng, while analyzing the venom to identify new hormones, identified a peptide named exendin-4. He discovered that exendin-4 had a remarkable ability to stimulate the synthesis and release of insulin from β-cells in the pancreas. Interestingly, exendin-4 closely resembled GLP-1. However, while GLP-1 remains active for about two minutes, the effect of exendin-4 persists for several hours. Preclinical studies demonstrated that a single daily injection of exendin-4 normalized blood glucose concentrations in mice with diabetes. Following extensive clinical testing, exenatide, an analog of exendin-4, was found to be safe and effective, leading to its FDA approval for adults in 2005.

Of note, in a 2-year rat carcinogenicity study involving prolonged-release exenatide, an increased incidence of thyroid adenoma and C-cell carcinoma was observed at doses ≥2-fold the human systemic exposure. The clinical relevance of these adverse findings is currently unknown. However, GLP-1 analogs are contraindicated in patients with a personal or family history of medullary thyroid carcinoma (MTC) or in patients with multiple endocrine neoplasia type 2 (MEN 2), as well as in patients with a serious hypersensitivity reaction. Patients should be counseled about the potential risk of MTC associated with the GLP1 receptor agonists, and it is important to inform them about the symptoms that may indicate thyroid tumors, such as a neck mass, dysphagia, dyspnea, and persistent hoarseness. Routine monitoring of serum calcitonin levels or use of thyroid ultrasound for the detection of MTC is of uncertain value.

Side effects include serious hypersensitivity reactions, including anaphylactic reactions and angioedema. Additionally, acute pancreatitis, including fatal and nonfatal hemorrhagic or necrotizing pancreatitis, and the acute onset of gallbladder disease, such as cholelithiasis or cholecystitis, have been reported in GLP-1 receptor agonists trials and post-marketing surveys (55).

Liraglutide-Victoza®

Children aged 10 to less than 17 years with T2D were randomly assigned 1:1 to receive subcutaneous liraglutide (up to 1.8 mg per day) or placebo for a 26-week double-blind period, followed by a 26-week open-label extension (56). At the 26-week visit, the mean HbA1c had decreased by 0.64 percentage points with liraglutide and increased by 0.42 percentage points with placebo, resulting in an estimated treatment difference of -1.06 percentage points. By 52 weeks, the difference had increased to -1.30 percentage points (Table 3). In the liraglutide group, 63.7% of patients achieved HbA1c levels of less than 7.0%, compared with 36.5% in the placebo group. On the other hand, there was no statistical benefit of liraglutide over placebo in lowering BMI or blood pressure (Table 3). The overall rate of adverse events, including gastrointestinal adverse events, was higher among patients receiving liraglutide. The U.S. Food and Drug Administration approved liraglutide injection for the treatment of pediatric patients 10 years of age or older with T2D. Liraglutide was the first non-insulin drug approved to treat T2D in pediatric patients since metformin was approved for pediatric use in 2000.

Mode of administration: The recommended initial dose is 0.6 mg subcutaneously once daily for the first week, followed by an increase to 1.2 mg once daily. It’s important to note that the initial dose of 0.6 mg once daily is primarily intended to mitigate gastrointestinal adverse effects and does not provide glycemic control. If adequate glycemic control is not achieved, the dose can be further increased to 1.8 mg once daily.

Exenatide-Bydureon

The effectiveness of once-weekly exenatide 2 mg (Bydureon AstraZeneca) in youth with suboptimally controlled T2D was evaluated (57). At 24 weeks, exenatide demonstrated superiority over the placebo in reducing HbA1c levels (a decrease of -0.36% with exenatide compared to +0.49% with placebo), resulting in a significant between-group difference of 0.85 percentage points. Notably, no major hypoglycemic events were reported. and the most frequently reported adverse events included gastrointestinal symptoms such as nausea, diarrhea, and vomiting, along with injection site reactions such as pruritus, erythema, and nodules. Clinically meaningful differences were not observed in change from baseline in BMI Z score, body weight, or waist circumference, nor were there significant differences in blood pressure. The prolonged-release suspension of exenatide administered via injection with a pre-filled pen has received approval from the European Union and the FDA for the treatment of T2DM in children and adolescents aged 10 years and older.

Mode of administration: Bydureon (2 mg per dose) should be administered once every 7 days. The dose can be administered at any time of day, with or without meals. Bydureon is not recommended as first-line therapy for patients with inadequate glycemic control on diet and exercise because of the uncertain relevance of the rat thyroid C-cell tumor findings to humans.

Dulaglutide-Trulicity

Dulaglutide (Trulicity) is a GLP-1 receptor agonist administered via subcutaneous injections once a week. In a randomized, double-blind, phase 3 trial involving youth with inadequately controlled T2DM, the efficacy and safety of dulaglutide treatment in reducing HbA1c levels at 26 weeks were assessed (58). Remarkably, there was 99% adherence to treatment in this trial. Treatment resulted in a significant reduction in HbA1c relative to placebo; at week 26, HbA1c decreased by 0.8 percentage points but increased by 0.6 percentage points in the placebo group (1.4 percentage point difference). Gastrointestinal symptoms were among the most common adverse events, but they were primarily mild and were most likely to occur soon after the initiation of therapy. There were no clinically meaningful differences in the incidence or annual rate of hypoglycemia between the dulaglutide groups and the placebo group. Body weight did not decrease significantly following treatment.

Mode of administration: The recommended initiating dose of Trulicity is 0.75 mg once weekly, administered subcutaneously. The dose may be increased to 1.5 mg once a week for additional glycemic control. Trulicity can be administered any time of day, with or without meals, and should be injected subcutaneously in the abdomen, thigh, or upper arm. The day of weekly administration can be changed, if necessary, as long as the last dose was administered 3 days (72 hours) or more before. Trulicity and insulin should not be mixed in the same syringe and must be administered as two separate injections at two different injection sites.

SODIUM-GLUCOSE TRANSPORT PROTEIN 2 - SGLT2 INHIBITORS

Sodium-glucose transport protein 2 (SGLT2) inhibitors belong to a class of medications that modulate sodium-glucose transport proteins in the nephron. These inhibitors primarily target the SGLT2 proteins, expressed in the renal proximal convoluted tubules, to reduce the reabsorption of filtered glucose and sodium (59). Apart from their role in controlling glucose concentrations, SGLT2 inhibitors have demonstrated substantial cardiovascular benefits, particularly reducing heart failure events, for adults with T2D. In adults, the FDA-approved indications for SGLT2 inhibitors include the reduction of major adverse cardiovascular events in individuals with T2D and established cardiovascular disease, as well as the reduction of the risk of eGFR decline in patients with chronic kidney disease at risk of progression. The most common reported adverse events associated with SGLT2 inhibitors include female genital mycotic infections, urinary tract infections (including urosepsis and pyelonephritis), as well as nausea and constipation. Most importantly, SGLT2 inhibitors are associated with an almost three-fold increased risk for DKA in adults. The risk for DKA is highest for canagliflozin, followed by empagliflozin and dapagliflozin. Furthermore, individuals may develop euglycemic DKA, i.e., a triad of increased anion gap acidosis, ketosis, and a serum glucose level below 250 mg/dL. The association between SGLT-2 inhibitors and euglycemic DKA appears to be secondary to their noninsulin-dependent glucose clearance, compensatory hyperglucagonemia, and volume depletion. Currently, euglycemic DKA has not been reported among youth with T2D taking SLGT2 inhibitors.

Empagliflozin - Jardiance®

A total of 105 children and adolescents were randomly assigned 1:1 to receive empagliflozin 10 mg or placebo (60). The adjusted mean change in HbA1c from baseline at week 26 was significantly greater in the empagliflozin group (–0·84%). Changes in body weight and blood pressure did not reach statistical significance. Adverse events occurred in 64% of participants in the placebo group and 77% in the empagliflozin group up to week 26, including severe adverse events in 4% of participants in the placebo group and 2% in the empagliflozin group. Mild and moderate hypoglycemia were the most frequently reported adverse events, with higher rates for those on active drug treatment compared with placebo; no severe hypoglycemic events were reported, and there were no episodes of DKA or necrotizing fasciitis associated with empagliflozin treatment. An increased incidence of bone fractures has been reported in adults, leading the American Diabetes Association to recommend avoiding SGLT-2 inhibitors in patients with a risk factor for fractures. Furthermore, SGLT2 inhibitors are not recommended for glycemic lowering in patients with T2D with an eGFR less than 30 mL/min/1.73 m².

Mode of administration: Empagliflozin is an oral medication dosed at either 10 mg daily or 25 mg daily. The recommended dose is 10 mg once daily in the morning, taken with or without food. If tolerated initially, dosing may increase up to 25 mg.

Dapagliflozin - Farxiga®

Participants aged 10–24 years with T2D and HbA1c concentrations ranging from 6.5 to 11% were given oral dapagliflozin 10 mg or placebo during a 24-week double-blind period (61); 39 participants were assigned to dapagliflozin and 33 were assigned to placebo. After 24 weeks, the mean change in HbA1c concentration was −0·25% for dapagliflozin and 0·50% for the placebo group; the between-group difference was −0·75%, which did not reach statistical significance. However, in a sub-analysis, excluding participants with poor compliance, the HbA1c change was -0.51%, while it was 0.62% for the placebo group, resulting in a between group significant difference of 1.13%.

Adverse events occurred in 69% of participants assigned to dapagliflozin and 58% of those assigned to placebo over a 24-week period. Over 52 weeks, adverse events occurred in 74% of participants who received dapagliflozin. Hypoglycemia was noted in 28% of participants assigned to dapagliflozin and in 18% of those assigned to placebo, none were considered serious adverse events. Notably, there were no reported episodes of DKA.

Mode of administration: To improve glycemic control the recommended starting dose is 5 mg once daily, taken in the morning. Increase dose to 10 mg once daily in patients tolerating 5 mg who require additional glycemic control. Dapagliflozin is not recommended for use in patients receiving loop diuretics or who are volume depleted, e.g., due to acute illness (such as gastrointestinal illness).

DIPEPTIDYL PEPTIDASE 4 (DPP 4) INHIBITORS

DPP-4 is a ubiquitous enzyme that degrades GLP-1 and GIP (gastric inhibitory peptide), resulting in a short half-life. By inhibiting the DPP-4 enzyme, DPP-4 inhibitors increase the levels of GLP-1 and GIP. This, in turn, enhances beta-cell insulin secretion, thus reducing postprandial and fasting hyperglycemia (62).

Linagliptin (Tradjenta)

105 children and adolescents were randomly assigned 1:1 to oral linagliptin (5mg daily) or placebo (60). The adjusted mean HbA1c change from baseline at week 26 was 0.33% for linagliptin and 0·68% for the placebo group; the difference was not statistically or clinically significant. None of the adverse events of special interest occurred with DPP-4 inhibitors, such as hypersensitivity reactions, angioedema, angioedema-like events and anaphylaxis, skin lesions, pancreatitis, or hepatic injury, were present. Linagliptin is not approved for adolescents with T2D (60).

Sitagliptin (Januvia)

A total of 190 children and adolescents were randomly assigned 1:1 to oral sitagliptin 100 mg daily or placebo (63). The adjusted mean change in HbA1c from baseline at week 20 was -0.01% for sitagliptin and 0·18% for the placebo group, resulting in a between-group difference of 0.19%, which was not significant. There was no significant difference in the percentage of participants with HbA1c below 7%, with 49.5% in the sitagliptin group and 36.8% in the placebo group. Notably, after 54 weeks, the HbA1c in the sitagliptin group decreased from 7.4% at baseline to 7.1%. Sitagliptin is not approved for adolescents with T2D (50).

ANTI-OBESITY PHARMACOTHERAPY FOR TREATMENT OF PEDIATRIC TYPE 2 DIABETES

As obesity is a significant risk factor for the development of T2D, the American Diabetes Association (ADA) recommends the use of anti-obesity medications as adjuvant therapy for adults with both T2D and overweight/obesity. In adults, adding anti-obesity medications to a diabetes regimen can improve glycemic control, reduce weight, and decrease the use of anti-diabetes medication (64). Semaglutide and tirzepatide have shown promise in reducing weight in adults with obesity and T2DM compared to liraglutide (65). Tirzepatide is a novel dual glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 receptor agonist. A study to evaluate tirzepatide in pediatric and adolescent participants with T2D, who are inadequately controlled with metformin, or basal Insulin, or both (SURPASS-PEDS) is currently ongoing.

Liraglutide 3.0mg/day

A higher dose of liraglutide of 3.0 mg/day (Saxenda^®) is FDA-approved for obesity in youth aged 12 years and older who weigh ≥ 60kg or have an initial BMI ≥ 30kg/m². The Satiety and Clinical Adipose-Liraglutide Evidence Trial in Adolescents with and without T2D (SCALE-Teens) was a double-blind, placebo-controlled RCT that randomized 251 adolescents to either liraglutide 3.0 mg/day or placebo. However, only two adolescents with T2D were enrolled. Consequently, no conclusive findings can be drawn regarding the impact of high-dose liraglutide on reducing BMI and improving HbA1c in young individuals with T2D. Since almost all adolescents with T2DM have overweight or obesity and could potentially meet the prescribing criteria, it is reasonable to speculate that the results might resemble those observed in adult trials for T2D, potentially leading to reductions in weight and BMI as well as improvements in hyperglycemia.

Semaglutide 2.4mg Weekly

Semaglutide in its once-weekly injectable GLP-1RA form at doses up to 2.4 mg weekly (Wegovy^®) has shown significant weight loss in obese children and has now been approved for treatment of obesity in adolescents aged 12 years and older (66). The mean change in BMI from baseline to week 68 was −16.1% with semaglutide and 0.6% with placebo. Of note, HbA1c decreased 0.4 percentage points in the treatment group compared with 0.1 in the placebo. However, the impact on youth with T2D has not yet been studied.

Semaglutide injection up to 2.0 mg (Ozempic^®) and oral semaglutide up to 14 mg (Rybelsus^®) are approved for adults with T2D, but studies in youth with T2D have not yet been reported.

The answer to this pivotal question hinges on the art of personalized medicine. First, it is crucial to recognize that 50% of youth with T2D can maintain good glycemic control on metformin monotherapy, making this agent still an excellent choice for initial therapy. However, several measures can predict the likelihood of glycemic deterioration, including decreased insulin secretion, impaired insulin processing (elevated proinsulin/insulin ratio), and HbA1c concentration after a few months on monotherapy. Longitudinally, a rising proinsulin and an increase in HbA1c of more than 0.5 percentage points over any 6-month period are predictive of loss of glycemic control on monotherapy (67).

If these parameters suggest a risk for deterioration, the next step is to consider the therapeutic objective(s). Is it primarily elevated HbA1c? If so, the best approach may involve selecting a medication known for its efficacy in reducing HbA1c. Is obesity the central concern for the patient? In such cases, exploring pharmaceutical interventions tailored for weight management may be prudent. Does the individual have concerns about needle-based treatments? In such instances, favoring orally administered medications could offer a more suitable solution. If there is an elevated risk of cardiac or renal complications, it would be wise to consider options from the SGLT2 inhibitor group or GLP1 agonist group. In essence, treatment needs to be tailored to the unique needs and circumstances of each individual, ensuring that treatment decisions align with their specific health challenges and objectives.

BARIATRIC SURGERY

Weight reduction plays a crucial role in the management of T2D because weight loss is associated with improved insulin resistance and glycemic status. Considering the limited efficacy of lifestyle and pharmacologic interventions in treating severe obesity and obesity-related T2D, along with the remarkable weight reduction and remission of T2D observed in youth who undergo bariatric surgery the ADA recommends considering bariatric surgery for adolescents with T2D who have a BMI >35 kg/m²(68). The two most common bariatric surgeries performed in adolescents with T2D are laparoscopic sleeve gastrectomy (LSG) and laparoscopic Roux-en-Y gastric bypass (LRYGB).

Impact of Bariatric Surgery on Glycemic Control

Studies have demonstrated improvement in HbA1c across all age groups. In one study, sixty-four pediatric patients diagnosed with morbid obesity and T2D underwent LSG surgery. The patients’ ages ranged from 5 to 14 years old. Their BMI decreased from 44.6±9.3 to 34.8 ± 9.6 kg/m².Their HbA1c decreased from 6.0±0.8% prior to LSG surgery to 5.4±0.4%, 12 months post-surgery, a change of 10.9% (p = 0.001) (69). There was no significant difference in post-operative HbA1c between the age groups.

Impact of Bariatric Surgery on T2D Remission

Bariatric surgery in adolescents with T2D has a better outcome compared to medical treatment with metformin alone or in combination with rosiglitazone or intensive lifestyle intervention with insulin therapy given for glycemic progression (70). In a multicenter, nonrandomized, retrospective study of 202 obese adolescents with T2D (before the approval of new drugs for adolescents with T2D), 109 adolescents underwent surgery, and 93 adolescents received nonsurgical treatment (71(. In the surgery group, the remission rate for diabetes was 76%, compared to 6.5% in the medical treatment group. The remission was sustained for the two years of follow-up. Of note, LRYGB had better effects on weight loss and glycemic control than LSG.

Impact of Bariatric Surgery on Kidney Outcomes

In a three-year longitudinal study, kidney outcomes in youth following bariatric surgery were assessed. Improvement in albuminuria and GFR was observed in those with pre-operative kidney impairment. Among participants with a preoperative eGFR < 90 ml/min/1/73m², eGFR improved from a mean of 76 ml/min/1.73m² to 102 ml/min/1.73m². Similarly, in those with an elevated albumin-to-creatinine ratio at baseline (ACR ≥30mg/g), the median ACR decreased from 74 mg/g to 17 mg/g. Estimated GFR and albuminuria remained stable in those without any evidence of pre-operative impairment. The effect of bariatric surgery on diabetic kidney disease was also investigated in adolescents with severe obesity and T2D five years after surgery compared to adolescents receiving medical management in the TODAY study (72). Elevated albuminuria was present in 21% of those receiving medical management at baseline, and it increased to 43% at 5 years. Conversely, albuminuria decreased from 27% prior to surgery to 5% at the 5-year follow-up. In adjusted analyses, the medical group had 27-fold higher odds of diabetic kidney disease at 5 years compared to Teen-LABS participants (72-73).

Impact of Bariatric Surgery on CVD Events

In another analysis, the risk for CVD events in two cohorts of adolescents with T2D and severe obesity undergoing medical or surgical treatment of T2D was assessed. Thirty adolescents who underwent bariatric surgery were matched to 63 who had received metformin alone or in combination with rosiglitazone or an intensive lifestyle intervention, with insulin therapy given for glycemic progression. The BMI of those who underwent bariatric surgery was 54.4±9.5 kg/m², and that of the medical group was BMI 40.5±4.9 kg/m². Although the baseline likelihood of CVD events was higher in the group that underwent bariatric surgery, one year after the surgery, the event risk was significantly lower and sustained at 5-year follow-up, whereas medical therapy was associated with an increase in risk among adolescents with T2D and severe obesity (74).

Adverse Effects of Bariatric Surgery

Bariatric surgery in adolescents can lead to various potential complications beyond the first postoperative month. The most commonly reported include gastroesophageal reflux, incisional hernia, treatment failure requiring operative revision, and nutritional deficits (50). Less frequent complications include stomach cancer, liver necrosis, gallbladder disorders, pancreatic disorders, acute kidney failure, neuromuscular complications, skin complications, and rarely mortality (51).

Summary

In summary, bariatric surgery currently outperforms medical management of T2D in adolescents and is the most efficacious treatment for youth-onset T2D, albeit with significant risks and economic implications. However, given the rapidly emerging medical therapies for weight reduction in adults and youth, many of which (e.g., dual-incretin analogs, triple-G agonists (75)) are now achieving degrees of weight loss approaching those seen following surgery, the individual roles of surgical and medical treatment for youth-onset T2D will need continued reassessment in the coming years.

COMPLICATIONS AND COMORBIDITIES AMONG ADOLESCENTS WITH T2D

Large cohort studies investigating the incidence of complications in children and teenagers with T2D unequivocally demonstrated a higher incidence of complications, both compared to the incidence of complications in T2D in adults and in T1D in children. In a multicenter observational study conducted from 2011 to 2020, the cumulative incidence of diabetic complications was assessed in 500 adolescents who had participated in the TODAY study (76). After only 13 years from diagnosis, higher complication rates were observed compared to those reported for pediatric patients with T1DM or for adults with T2D. The first striking finding from this report is the participants’ poor glycemic control despite participation in a well-resourced clinical trial. While glycemic control in adolescents with T1DM tends to improve with age, a gradual deterioration occurred in adolescents with T2D throughout the follow-up period. Approximately 45.0% had HbA1c of at least 10%, and an additional 20% were in the range of 8-10%. Their BMI remained consistently in the range of 35.0 to 37.5 kg/m2. It is important to note that poor glycemic control and obesity are associated with early complications. The complications associated with T2D in children affect various systems (Figure 5).

Figure 5. Comprehensive Overview of Type 2 Diabetes (T2D) Complications in Adolescents. A comprehensive visual representation of the diverse range of complications affecting major physiological systems that can arise in adolescents with T2D.

On average, thirteen years after the initial diagnosis of T2D, incidence rates for hypertension, dyslipidemia, diabetic kidney disease, and neuropathy were 67.5%, 51.6%, 54.8%, and 32.4%, respectively. Additionally, retinopathy was present in 51% of the participants. One-fifth of the cohort (21.3%) had two complications, and 7.1% had three. There were seventeen serious cardiovascular events, including myocardial infarctions, six cases of congestive heart failure, three cases of coronary artery disease, and four stroke events. Additionally, six deaths were reported. This rapid development of complications was associated with severe insulin resistance and poor socioeconomic circumstances.

Hypertension

Hypertension, defined as blood pressure ≥ 95^th percentile for age, sex, and height or systolic BP ≥ 130/80 mm Hg, is often present at the time of diagnosis in adolescents with T2D. In a study of 391 children from Hong Kong under 18 years of age, 22.5% had hypertension at diagnosis. In the TODAY study, 11.6% were hypertensive at the time of diagnosis (77) and the cumulative incidence of hypertension was 67.5% at a mean age of 26.4±2.8 years and 13.3±1.8 years from diagnosis (78). A systematic review and meta-analysis of 31 international studies on 4363 children with T2D aged 6.5 to 21 years at diagnosis found a pooled prevalence of hypertension of 25.33% (95% CI, 19.57%-31.53%) (79).Male participants had a higher hypertension risk than female participants (odds ratio 1.42 [95% CI, 1.10-1.83]).

Initial treatment involves lifestyle modification, aiming to not only enhance glycemic control but also incorporate a diet that is low in sodium and abundant in fruits, vegetables, whole grains, low-fat dairy products, and lean proteins. The Dietary Approaches to Stop Hypertension (DASH diet) is highly recommended for its effectiveness in reducing high blood pressure and supporting weight loss. In addition to lifestyle modification, the ADA recommends starting Angiotensin-Converting Enzyme inhibitors (ACEis) or Angiotensin Receptor Blockers (ARBs). Due to the potential teratogenic effects, the use of reliable contraception should be encouraged in individuals of childbearing age (68). There are no agents specifically approved for use in youth with T2D, however, a once-daily medication is recommended for better compliance (80,81).

Nephropathy

The primary microvascular diabetic complication in adolescents with T2D is diabetic kidney disease (DKD), which develops in 25–40% of T2D patients and is associated with rapid progression and poor prognosis. The severity and risk of rapid deterioration in young people with T2D are reflected in the fact that microalbuminuria is reported at diagnosis among adolescents with T2D. In a recent systematic review and meta-analysis (79) that included 14 studies of 2250 children and adolescents with T2D, the prevalence of albuminuria was 22.17% (95%CI, 17.34%-27.38%), and the pooled prevalence of macroalbuminuria among 730 children and adolescents was 3.85% (79). In the SEARCH study (82) after a duration of 8 years, the prevalence was 19.9% among adolescents with T2D compared with 5.8% in those with T1D.

The accelerated development of kidney damage is attributed to inadequate glycemic control and the presence of various risk factors, including obesity, dyslipidemia, insulin resistance, hypertension, elevated serum uric acid, female sex, and the presence of chronic inflammation (83).

While albuminuria is the earliest clinical sign of diabetic kidney disease (DKD), the natural history of DKD begins with hyperfiltration, characterized by an increase in glomerular filtration rate (GFR) >120 mL/min/1.73 m² as a consequence of obesity and impaired glucose tolerance (84). This increase predicts deterioration before other clinical signs appear. The second stage in the evolution of kidney dysfunction, still without clinical manifestations, is a mild reduction in GFR (60-89 mL/min/1.73 m²) (Figure 6). Structural changes of the kidney are typical at this stage, yet they are often reversible, making this a critical time for risk factor reduction (84).

Figure 6. Heat map depicting diabetes kidney disease (DKD) staging by GFR and albuminuria and suggested treatments. Adapted from Naaman SC, Bakris GLJDC. Diabetic Nephropathy: Update on Pillars of Therapy Slowing Progression. 2023;46:1574-86.

Treatment in these early stages involves achieving adequate glycemic control along with lifestyle modification, including increasing physical activity, improving sleep quality, and quitting smoking. Additionally, a healthy diet with lower salt and potassium intake, along with a low protein intake of 0.8 g/kg/day, is recommended (68). Pharmacological treatment includes control of blood pressure and managing dyslipidemia. Theoretically, medications that improve glycemic control would also provide benefits for preventing and/or treating DKD. GLP1 receptor agonists are reported to have a renal protective effect in adults with T2D. A recent meta-analysis of approximately 60,000 adults with T2DM treated with GLP-1 receptor agonist found a significant reduction in the composite kidney outcome (development of macroalbuminuria, doubling of serum creatinine, 40% or greater decline in eGFR, kidney replacement therapy, or death from kidney disease) when compared to placebo, as well as a trend towards a reduction in worsening kidney function (84). Similarly, the hemodynamic and natriuretic effects of SGLT2 inhibition are also postulated as protective mechanisms for DKD (85).However, studies in adolescents with T2D did not show an effect of systolic or diastolic blood pressure (Table 3). Of note, these studies involved relatively small groups of youth, and were short-term. Larger, long-term studies are needed, as well as additional therapeutic options for reducing the risk of DKD, such as mineralocorticoid receptor antagonists (MRAs), and endothelin receptor antagonists (ERAs).

Dyslipidemia

Screening for dyslipidemia should be conducted once optimal glycemic control has been achieved or within 3 months after T2D diagnosis. Initial cholesterol screening can be performed non-fasting. The recommended target values are triglycerides <150 mg/dL (1.7 mmol/L), HDL cholesterol >35 mg/dL (0.91 mmol/L), and LDL cholesterol <100 mg/dL (2.6 mmol/L). If these levels are outside the normal range, medical nutritional therapy (MNT) should be initiated, including restricting caloric intake from fat to 20–30% and daily cholesterol intake to <200 mg/day, avoiding trans fatty acids, limiting saturated fats to <7%, and controlling mono and poly unsaturated fats to 10-15%. If, after 6 months of dietary intervention, the LDL-C remains >130 mg/dL, statin therapy should be initiated, with a goal of achieving LDL-C <100 mg/dL. Currently, there are 7 approved statins for use in children and adolescents: Lovastatin, Simvastatin, Atorvastatin, and Fluvastatin [children ≥10 years old], Pitavastatin and Pravastatin [> 8 years], and Rosuvastatin [>6 years old]. It is important to note that due to potential teratogenic effects, individuals of childbearing age should receive reproductive counseling, and statins should be avoided in individuals of childbearing age who are not using reliable contraception. If triglyceride levels are greater than 400 mg/dL (4.7 mmol/L) in a fasting state or exceed 1,000 mg/dL (11.6 mmol/L) in a non-fasting state, it is imperative to optimize glycemic control and initiate fibrate therapy. This is essential for reducing the risk of pancreatitis.

In a study assessing whether clinicians are sufficiently aggressive in treating diabetes-related hyperlipidemia in youth, among 278 youth with T2D <10 years, 57% had LDL-C exceeding 100 mg/dL, 24% had LDL-C at or above 130 mg/dL, and 9% had LDL-C surpassing 160 mg/dL (86). Additionally, 29% had hypertriglyceridemia, and 44% had HDL-C levels below 40 mg/dL. However, only 5% of these youth were prescribed lipid-lowering medications.

Polycystic Ovarian Syndrome (PCOS)

There is a strong association between T2D in adolescent girls and polycystic ovary syndrome (PCOS). A systematic review and meta-analysis comprising 6 studies that involved 470 girls diagnosed with T2D, with an age range at diagnosis between 12.9 and 16.1 years, revealed that the prevalence of PCOS was estimated at 19.58% (87). PCOS is associated with a range of metabolic diseases, including hypertension and dyslipidemia. Furthermore, it is associated with a higher prevalence of cardiovascular risk factors, such as higher carotid intima thickness, β stiffness index, and reduced arterial compliance. Additionally, adolescent girls with PCOS have an increased likelihood of experiencing psychiatric comorbidities, such as anxiety and depression, which can negatively impact their health-related quality of life. A menstrual history should be taken on every girl with T2D at the diagnosis and at every follow-up visit. Metformin, in addition to lifestyle modification, is likely to improve menstrual cyclicity and hyperandrogenism in female individuals with T2D.

Pregnancy and Contraception

In the TODAY study, the maternal and offspring outcomes of young women with youth-onset T2D who experienced one or more pregnancies were evaluated (88). Over a span of up to 15 years, a total of 260 pregnancies were reported by 141 women. Their average age was 21.5±3.2 years, a mean BMI of 35.6±7.2 kg/m², and an average diabetes duration of 8.1±3.2 years (88). Complications during pregnancy were identified in 65% of the women. Elevated HbA₁c levels of ≥8% were observed in 31.9% of the pregnancies, and chronic hypertension complicated 35% of them. Pregnancy loss was observed in 25.3% and preterm birth occurred in 32.6% of pregnancies. Among the offspring, 7.8% of the babies were classified as small for gestational age, 26.8% as large for gestational age, and 17.9% fell into the macrosomic range. Other complications and congenital anomalies were noted in 10% of infants, including anencephaly, renal anomalies, and complications related to prematurity. The rate of known miscarriage for the entire study was 12.3%. The rate of known stillbirths in the cohort was 3%, which is more than triple the reported national rates.

The impact of T2D during pregnancy extends beyond the immediate prenatal and birth periods, exerting long-term effects on offsprings. Data pertaining to the diagnosis of diabetes in biological mothers were available for 621 participants in the TODAY study, of whom 301 had never been diagnosed with diabetes, 218 were diagnosed either before or during pregnancy, and 102 were diagnosed after pregnancy (89). For biological fathers, the data were available for 519 participants, with 352 having no history of diabetes and 167 having paternal diabetes. Notably, it was found that maternal diabetes, but not paternal diabetes, was associated with lower beta-cell function and deterioration of glycemic control over time. These effects remained significant even after accounting for variables such as age, sex, race/ethnicity, and household income (89).

Contraception is indicated in adolescent girls with T2D for several compelling reasons. Firstly, it is a recommendedtreatment for managing the symptoms of PCOS. Secondly, these young women are at increased risk of unintended pregnancy, which can lead to poor outcomes. Lastly, it is imperative for those treated with potentially teratogenic medications such as ACEis, ARBs, and statins. Unfortunately, despite the evident need, preconception counseling was reported only in 16.3% of women prior to their first pregnancy and only 14.9% used any method of contraception prior to the first pregnancy (88). This gap in reproductive health care highlights the importance of healthcare providers and support staff addressing this issue promptly and effectively.

Although in theory, adolescent girls with T2D without severe obesity, micro- or macrovascular disease, or other cardiovascular risk factors can consider a wide range of contraceptive methods, the practical reality often differs. A significant proportion of these individuals are afflicted by severe obesity, and a considerable number also exhibit elevated blood pressure. Consequently, when morbid obesity, severe hypertension, micro- or macrovascular disease, or multiple cardiovascular risk factors are present, it is advisable to prioritize nonhormonal or progestin-only contraceptive methods (Figure 7) (90).

Figure 7. Heat map of contraceptive treatment options for adolescent girls with T2D based on disease duration and complications. Adapted from Merino PM, Codner E. Contraception for Adolescents and Young Women with Type 2 Diabetes-Specific Considerations. Current diabetes reports 2022;22:77-84.

Diabetic Retinopathy

Diabetic retinopathy (DR) is one of the most important causes of visual loss worldwide. DR is often asymptomatic until the very late stages. Given the potential for a rapid rate of progression and the effectiveness of new therapies in slowing deterioration, it is imperative to routinely screen individuals with diabetes for the early detection of retinal changes. It is important to note that direct fundoscopy may be less sensitive than 7-field stereoscopic fundus photography for detecting retinopathy (91). Diabetic retinopathy (DR) is divided into two major forms: non-proliferative and proliferative, according to the presence of abnormal new blood vessels emanating from the retina. Non-proliferative DR consists of microvascular abnormalities (including microaneurysms, occluded vessels, and dilated or tortuous vessels) primarily in the macula and posterior retina, intraretinal hemorrhages, hard exudates; and the variable presence of nerve-fiber layer infarcts (cotton wool spots). Visual loss is mainly due to the development of macular edema. Proliferative DR is marked by the presence of neovascularization arising from the disc and/or retinal vessels, resulting in preretinal and vitreous hemorrhage, subsequent fibrosis, and traction retinal detachment resulting in visual loss.

In a meta-analysis that included 27 studies involving 5924 children and adolescents (91) with T2D duration between 6.5-21.0 years, 1.11% had DR less than 2.5 years after T2D diagnosis. The prevalence of DR increased over time, with rates of 9.04% at 2.5 to 5.0 years after T2D diagnosis, and 28.14% more than 5 years after T2D diagnosis. The global prevalence of diabetic retinopathy in pediatric T2D was found to be 6.99%. Optical coherence tomography (OCT)performed in adolescents with T2D in the TODAY study revealed that changes in retinal thickness correlated with HbA1c, fasting glucose, and blood pressure, illustrating the importance of intensive control of hyperglycemia, hypertension, and hyperlipidemia.(92)

Among the systemic strategies for the prevention and treatment of diabetic retinopathy, two randomized clinical trials, "The Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) Study and the Action to Control Cardiovascular Risk in Diabetes (ACCORD)-Eye Study, documented a reduction in the risk of non-proliferative DR progressing in individuals using fenofibrate (93). The protective effects of fenofibrate have been attributed to its antioxidative, anti-inflammatory, anti-apoptotic, and anti-angiogenic properties. Other treatments include pan-retinal photocoagulation, intravitreal anti-vascular endothelial growth factor (VEGF) therapy, integrin antagonists, and anti-inflammatory agents. Corticosteroids remain important second-line therapies for patients with macular edema.

Diabetic Neuropathy

DIABETIC PERIPHERAL NEUROPATHY

Diabetic peripheral neuropathy (DPN) is a cause of significant disability and poor quality of life. Signs and symptoms include sensory loss, paresthesia, and pain. However, subclinical signs of DPN may precede the development of frank neuropathic symptoms, and systematic screening is required to identify DPN in its earliest stages. Studies from USA (78,94), Canada (95), and India (96) reported the prevalence of DPN in adolescents with T2D assessed by examination for foot abnormalities, distal vibration perception, and ankle reflexes (97). In the SEARCH study, the prevalence of peripheral neuropathy assessed after 8 years in 258 adolescents with T2D was 22%, compared with 7% in T1DM (98). Data from the TODAY Study Group revealed that at baseline, 1.0% of the participants had nerve disease, and the cumulative incidence at 15 years was 32.4% (78). The risk factors associated with peripheral neuropathy in youth are longer duration of diabetes, older age, male sex, and smoking.

Calcium channel a2δ ligands (gabapentin, pregabalin= Lyrica), serotonin- norepinephrine reuptake inhibitors, and tricyclic antidepressants are the most widely used medications for painful DN in adults. These medications are used to treat various presentations of neuropathic pain in children, but there have been no clinical studies devoted to painful DPN and no agents are licensed specifically for painful DN in childhood and youth (99). “Fortunately, painful DPN is relatively rare in children and youth.

AUTONOMIC NEUROPATHY

Diabetic autonomic neuropathy (DAN) is a common form of neuropathy in individuals with diabetes mellitus characterized by dysfunction due to impairment of peripheral autonomic nerves, with a wide spectrum of manifestations (100). DAN that involves autonomic fibers of the enteric nervous system may cause upper and lower gastrointestinal symptoms, including gastroesophageal reflux disease, gastroparesis, and constipation. Additional manifestations include bladder and sexual dysfunction. DAN also involves the cardiovascular system. Cardiovascular autonomic neuropathy (CAN), secondary to the pathology of the autonomic nerve fibers that innervate the heart and blood vessels, results in resting tachycardia, exercise intolerance, orthostatic hypotension, syncope, and silent myocardial infarction and ischemia(101). A reduction in heart rate variability is an early sign of CAN and was reported in 47% of youth with T2D after a mean disease duration of only 1.7 years (102). In the SEARCH study, the prevalence of CAN assessed after duration of 8 years in 252 adolescents with T2D was 17% compared with 12% in T1DM (103). CAN was associated with elevated triglycerides and increased urinary albumin excretion.

Cardiovascular Complications

T2D is a major risk factor for cardiovascular disease (CVD), including myocardial infarction and stroke. Markers for early CVD include increased arterial stiffness, carotid intima-media thickness, an alteration in the vascular endothelium, and cardiac autonomic neuropathy.
Arterial stiffness prevalence was significantly higher among youth with T2D than those with T1D (104). Arterial stiffness (measured by pulse wave velocity from carotid-femoral, femoral-foot, and carotid-radial), was measured in 388 youth with T2D at a mean age of 21 years and a diabetes duration of 7.7±1.5 years (105). Higher arterial stiffness was associated with an adverse change in left ventricular diastolic function. Similarly, cardiac parameters were assessed in 177 individuals with T2D, with a mean age of 24.6±4.2 years and a disease duration of 10.3±3.5 years (106). Of these, 72% were females, and their HbA1c levels averaged 8.9±1.9. Their parameters were compared to those of individuals with T1D. The prevalence of increased atrial stiffness was significantly higher in those with T2D, at 75.7%, compared to only 23.4% in individuals with T1D. In addition, the prevalence of cardiac autonomic neuropathy was significantly higher in those with T2D, at 16.9%, compared to 9.0% in individuals with T1D. Participants with T2D exhibited a higher left ventricular mass index, lower systolic function, and lower diastolic function compared to those with T1D.

Adolescents with T2DM have increased carotid intima-media thickness (cIMT) compared to obese and normal weight groups; the group difference was detected early at 14 years of age (107).

Vascular endothelial function was lower in adolescents with T2D compared to normal weight and compared to adolescents with T1D (108).These changes are of importance as the rate of all adjudicated heart, vascular, and cerebrovascular events was 3.73 per 1000 person-years (78). There were 17 serious cardiovascular events, myocardial infarction [4 events], congestive heart failure [6 events], coronary artery disease [3 events], and stroke [4 events].

Decreased Bone Mineral Density (BMD)

In a study involving 17 adolescents newly diagnosed with T2D and 59 age, sex, and BMI-matched controls, bone mineral density (BMD) was assessed at multiple sites while accounting for potential confounding factors such as age, sex, Tanner stage, and BMI (109). The results revealed that BMD Z-scores for the femoral neck and bone mineral apparent density Z-scores of the lumbar spine were notably lower in individuals with T2D compared to their healthy counterparts.In another cross-sectional study of BMD in 180 youths aged 10 to 23 years, the participants were categorized into three groups: those with T2D were compared to those with obesity (n=226) and those with a healthy weight (BMI <85th percentile; n=238) (110). An age-dependent pattern emerged, wherein the BMD Z-score in children was higher in the T2D group compared to the obese group. However, in adolescents and young adults, the BMD Z-scores were lower in the T2D group when compared to the obese group. These findings suggest that T2D may have a detrimental impact on bone density, particularly around the age when individuals reach peak bone mass. Considering the elevated fracture risk observed in adults with T2D, the decrease in bone density at a young age raises concerns about potential long-term morbidity and fracture risk in adulthood.

Mental Health Comorbidity

DEPRESSION

As part of routine diabetes care, a total of 197 adolescents and youth diagnosed with T2D, with a mean age of 16.9 years and 57% male, completed the Patient Health Questionnaire (PHQ) (111). 19.3% reported elevated depressive symptoms (PHQ score ≥10). Furthermore, in a sample of 53, 18.9% acknowledged thoughts of self-harm. Despite the prevalence of depressive symptoms, only 50.0% of those with depressive symptoms had a documented referral for mental health treatment in the electronic health record after the positive screening outcome. Older age, shorter diabetes duration, a higher HbA1c level, more blood glucose checks per day, and being prescribed oral medications were significantly associated with more depressive symptoms. Results from the TODAY study revealed that older teen girls had the highest rates of clinically significant depressive symptoms (112). Increased number of stressful life events were associated with elevated depressive symptoms. Depressive symptoms were correlated with low adherence to diabetes treatment, lower psychosocial functioning, and impaired health-related quality of life (QOL). In another study comparing PHQ between adolescents with T2D and T1D, the median PHQ-9 score in females with T2D was significantly greater than in females with T1D, but did not differ between males with T2D and T1D (113). The association between depression and T2D has been reviewed elsewhere (114).

DISORDERED EATING BEHAVIORS

In the TODAY study, out of 678 adolescents with T2D at a mean age of 14.0 years, 6% had clinical and 20% had subclinical levels of binge eating (115). Moreover, 50.3% of adolescents with T2D who are receiving insulin therapy had disordered eating behaviors (DEB) compared with 21.2% of those with T1DM (116). Disordered eating behavior is associated with poorer clinical outcomes and psychosocial well-being.

Mortality

In the SEARCH study, over a median follow-up of 5.3 years, the mortality rate among individuals with T1D was 70.6 deaths per 100,000 patient-years, compared with 185.6 deaths per 100,000 patients-years for those with T2D (117).^.When compared to the general populations of US states, it was observed that the mortality rate for individuals with T2D was significantly higher than expected while this was not the case for individuals with T1DM. Females had higher mortality rates than males. Among adolescents with T2D, the leading underlying cause of death was attributed to transport/motor vehicle accidents followed by accidental poisoning, and intentional self-harm. According to a life expectancy model, it was predicted that youth with T2DM lose approximately 15 years (118).

CONCLUSION

A summary of the key points is presented in Table 4.

Table 4. Key Points
• The prevalence of type 2 diabetes (T2D) varies across different countries and regions globally, but there is a consistent upward trend in its occurrence. Large-scale national studies in countries like Sweden, Germany, and India have reported similar increasing trends. • Contrary to common perception, not all adolescents with T2D are severely obese. Only 70-80% fall into this category. • Socioeconomic status plays a significant role in the prevalence of T2D, with lower socioeconomic status being associated with higher incidence in Western countries, while the opposite is observed in low-income countries. • Although adolescents are at higher risk of developing T2D, there are growing reports of T2D cases in children younger than 10 years old. • The gender distribution of T2D varies, with a higher proportion of cases in females in Western countries but a higher proportion in males in the Middle East and China. • Promisingly, there are new medications designed for weight loss, which hold the potential to reduce the high rates of complications associated with T2D in children. • There are low prescription rates for statins and contraception in the management of T2D in adolescents, highlighting potential gaps in medical care. • Many complications of T2D can potentially be reversed if detected and treated aggressively in their early stages.

Table 4. Key Points

• The prevalence of type 2 diabetes (T2D) varies across different countries and regions globally, but there is a consistent upward trend in its occurrence. Large-scale national studies in countries like Sweden, Germany, and India have reported similar increasing trends.

• Contrary to common perception, not all adolescents with T2D are severely obese. Only 70-80% fall into this category.

• Socioeconomic status plays a significant role in the prevalence of T2D, with lower socioeconomic status being associated with higher incidence in Western countries, while the opposite is observed in low-income countries.

• Although adolescents are at higher risk of developing T2D, there are growing reports of T2D cases in children younger than 10 years old.

• The gender distribution of T2D varies, with a higher proportion of cases in females in Western countries but a higher proportion in males in the Middle East and China.

• Promisingly, there are new medications designed for weight loss, which hold the potential to reduce the high rates of complications associated with T2D in children.

• There are low prescription rates for statins and contraception in the management of T2D in adolescents, highlighting potential gaps in medical care.

• Many complications of T2D can potentially be reversed if detected and treated aggressively in their early stages.

ACKNOWLEGEMENTS

The authors extend their heartfelt gratitude to the gifted illustrator, Mrs Noah Levek, for her exceptional eye-opening illustrations.

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Diabetes in People Living with HIV

ABSTRACT

People living with HIV (PLWH) are living longer and also have unique risk factors for developing several metabolic diseases, including diabetes. This has been observed in both high income and low to middle income countries. Risk factors for diabetes in PLWH consist of specific antiretroviral therapies (ART), including older generation protease inhibitors and nucleoside reverse transcriptase inhibitors, lipodystrophy, and hepatitis C co-infection. In addition, obese and overweight states are common in PLWH, and an increased risk of incident diabetes has been noted with weight gain after ART initiation in PLWH, compared to individuals without HIV. Inflammation associated with HIV has also been linked to incident diabetes. This chapter reviews points to consider in the diagnosis and monitoring of diabetes in PLWH and discusses interactions that may occur between specific ART agents and glucose-lowering medications. Moreover, PLWH have risk factors for complications involving organ systems that are also affected by microvascular disease in diabetes. Because PLWH have a greater risk for cardiovascular disease (CVD) than individuals without HIV, modifiable risk factors of CVD should be addressed in the care of PLWH, considering that dyslipidemia, hypertension, and cigarette smoking are all highly prevalent in PLWH.

BACKGROUND

HIV infection is prevalent in 38.4 million people worldwide as of 2021 (1) and 1.2 million people in the United States as of 2019. The incidence of HIV infection has decreased from 2015 to 2019 in the United States but has remained stable in some demographic groups, including African-Americans and Latinos (2). Untreated, HIV infection can lead to opportunistic infections including cytomegalovirus disease and pneumocystosis (3) associated with AIDS, which is defined as a CD4+ cell count < 200 cells/mL. With advances in antiretroviral therapy (ART), however, HIV infection has been transformed from a disease strongly linked to AIDS and opportunistic infections into a chronic disease that is associated with several cardiometabolic consequences, including diabetes (4), heart disease (5), and other non-AIDS related illnesses such as osteoporosis (6). In part, this phenomenon is secondary to the fact that people living with HIV (PLWH) are living longer and are more susceptible to diseases of aging.

An important aspect to consider in the understanding of HIV associated cardiometabolic diseases is their growing global presence. The same factors that are associated with cardiometabolic disease prevalence in PLWH in high-income countries are also present in low-income countries, in addition to unique factors including industrialization, with resultant decreased physical activity and increased energy consumption (7) (8). As such, the care of PLWH needs to focus not only on ART but also on the management of chronic co-morbidities. This chapter will focus primarily on diabetes in PLWH.

EPIDEMIOLOGY AND RISK FACTORS FOR DIABETES IN PLWH

Epidemiology

PLWH have a unique set of risk factors that may increase their likelihood of developing diabetes. Illustrating the greater burden of diabetes in PLWH on ART, a study from 2005 on the Multicenter AIDS Cohort Study (MACS) of gay and bisexual men with (MWH) and without HIV (MWoH) found that the incidence of diabetes was found to be more than fourfold higher in MWH and that the prevalence of diabetes was 14% in MWH on ART and 7% in MWH not on ART, compared to 5% in MWoH. These differences were significant even after adjusting for age and body mass index (BMI). Of note, the majority of MWH on ART in the study were on first generation protease inhibitor (PI) therapy (discussed further in the section Protease Inhibitors) (9). Other studies have described incidence rates of diabetes in PLWH of 4.4 cases per 1000 person-years of follow-up in the Swiss HIV Cohort Study (10) and 5.72 cases per 1000 person-years of follow-up in the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) Study of participants from Europe, the US, Australia, and Argentina (11).

The effect of HIV disease on developing diabetes is also growing in low- and middle-income countries (LMIC), where the majority of the people with diabetes live (12). Prevalence of estimates of diabetes in PLWH in LMIC range from 6.8% in Chile (13) to 26% in Cameroon (14). PLWH and diabetes in LMIC are younger than those in high income countries (15). The wide range of prevalence estimates of diabetes in PLWH in LMIC may be in part because of differences in the definitions used to identifying diabetes. In addition to factors common to PLWH globally, urbanization may be a contributing factor to the development of diabetes in PLWH in LMIC (16).

Antiretroviral Therapy (ART)

PROTEASE INHIBITORS (PI)

One risk factor for developing diabetes in PLWH is PI use. In 1995, saquinavir became the first FDA approved PI (17). Incidence rates of hyperglycemia as high as fivefold have been reported in the setting of PI use (18). In addition, PIs, including ritonavir, have been associated with hypertriglyceridemia (19,20) and lipodystrophy (21). Mechanisms that may account for these effects are multifold. In one study, insulin sensitivity, as measured by glucose infusion rate during hyperglycemic clamp, decreased significantly after 12 weeks of PI therapy compared to baseline. A defect in beta cell function, in particular, a decrease in the disposition index, was also observed after PI therapy (22). An in vitro study demonstrated that indinavir may lower the function of the glucose transporter GLUT4 (23). Additional mechanisms may include changes in the hormone adiponectin, which is associated with improved insulin sensitivity, although in vivo and in vitro effects of PIs on adiponectin have differed (24,25).

The prescribing patterns of PI use have evolved. One study found that in the Veterans Affairs system, the prevalence of PI based regimen use decreased from 1997 to 2004 (26). In addition, the effects of all PIs, namely older generation versus newer generation PIs, including atazanavir and darunavir, on glucose homeostasis are not equal. In an in vitro study, atazanavir, which received FDA approval in 2003 (27), did not inhibit either GLUT1 or GLUT4 glucose transporters (28). In a clinical study of HIV- participants, atazanavir in combination with ritonavir had a significantly lower effect on insulin sensitivity compared to lopinavir/ritonavir, as measured by glucose disposal rate during hyperinsulinemic euglycemic clamp. (29). Moreover, the newer generation PIs have not been associated with an increased incidence of diabetes, with one study showing a lower risk of diabetes in individuals on these medications (30).

NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS (NRTIs)

In addition to PIs, certain nucleoside reverse transcriptase inhibitors (NRTIs) have been associated with diabetes. In the D:A:D study, incident diabetes was associated with exposure to the NRTIs stavudine and zidovudine, both thymidine analogs, and didanosine, after adjusting for age, sex, race, and BMI (11). NRTIs have also been implicated in mitochondrial dysfunction, which may be another mechanism by which NRTIs are associated with diabetes. One study found that one month of treatment with stavudine was associated with significant decreases in mitochondrial DNA from muscle biopsies and insulin sensitivity, as measured by the glucose infusion rate during a hyperinsulinemic euglycemic clamp (31). Because of serious adverse effects linked to the use of these NRTIs, stavudine, zidovudine, and didanosine are not advised for use in the United States (32).

Similar to the newer generation of PIs, the use of newer NRTIs, including emtricitabine, abacavir, and tenofovir, have been associated with a lower risk of diabetes (30). Supporting this finding, tenofovir disoproxil fumarate (TDF), in a study of participants without HIV or diabetes, did not significantly affect insulin sensitivity, as measured by hyperinsulinemic euglycemic clamp (33).

Tenofovir alafenamide (TAF) is a newer prodrug of tenofovir and is associated with less renal and bone toxicity than TDF (33a). However, TAF has been associated with weight gain in PLWH, compared with TDF (33b). Weight gain leading to an overweight state or obesity is a risk factor for the development of type 2 diabetes. In a retrospective study of the ADVANCE trial, which included ART naïve PLWH randomized to 3 treatment arms, 2 of which included TDF and 1 of which included TAF, an increase in the 10-year predicted risk of type 2 diabetes was observed for participants in the TAF arm, compared to participants in one of the TDF arms, although the risk calculator that was used had not been validated for the population in the study (33c). On the other hand, in a cohort study of more than 4,000 PWH on a TDF-based ART regimen for at least 6 months, of whom about 80% switched to a TAF-based ART regimen and about 20% continued on a TDF-based ART regimen, TAF was associated with a significant weight gain, compared to TDF, after 18 months, but it was not associated with incident diabetes.

INTEGRASE STRAND TRANSFER INHIBITORS (INSTIs)

Integrase strand transfer inhibitor (INSTI) based regimens are among the recommended initial ART regimens for PLWH (32) (34). Reasons for this include the effectiveness of and fewer adverse effects associated with INSTIs in studies of treatment-naïve patients (34-37). However, studies have discovered some metabolic effects of INSTIs. These include weight gain on average of 2.9 kilograms over 18 months, as observed in a retrospective study of patients who switched to an INSTI based regimen from previously being on a non-NRTI (NNRTI)-based regimen. This was in contrast to an average 0.7 kg weight gain in those participants who switched to a PI-based regimen and 0.9 kg weight gain in those who continued on a NNRTI-based regimen (38). In addition to weight gain, case reports of new onset diabetes have been reported in conjunction with INSTI use (39,40). One clinical trial compared the INSTI raltegravir to 2 boosted PI regimens and found that the increases in homeostasis model assessment-insulin resistance (HOMA-IR) in all 3 arms were not significantly different from one another, were noted by 4 weeks, and appeared to be independent of changes in visceral adipose tissue (41). However, other studies have not demonstrated an association between diabetes and INSTI use (41a) or have found that the association between INSTI use and incident diabetes varies by individual INSTI (41b).

The exact mechanisms by which INSTIs may be related to weight gain and diabetes are not known. One proposed mechanism by which INSTIs may cause hyperglycemia is their effect on magnesium, which is a necessary cation for insulin action (40). In mice, INSTI use leads to an increase in fat mass and decreases brown and beige adipocyte differentiation, leading to lower energy expenditure and weight gain (41c).

Lipodystrophy

Lipodystrophy associated with ART is characterized by either lipoatrophy in the face, arms, and legs, or lipoaccumulation that can lead to gynecomastia, dorsocervical fat tissue, and increased intra-abdominal fat, (42,43), or mixed lipodystrophy, in which lipoatrophy and lipoaccumulation occur together. As noted above, lipodystrophy has been associated with PI use (21) and with older generation NRTI use, including stavudine (44), but less commonly with the newer generation NRTI tenofovir (45). In a cross-sectional study, HIV+ men with lipodystrophy had a greater insulin resistance than men without lipodystrophy (21). Lipodystrophy has also been associated with incident diabetes (46,47).

In one study that measured body fat changes longitudinally using dual-energy x-ray absorptiometry (DXA) in PLWH on ART (specifically zidovudine and lamivudine or didanosine and stavudine in combination with nelfinavir, efavirenz, or both), 32% of participants had discordant changes in trunk and limb fat (48).

Lipodystrophy may also be persistent in patients with exposure to thymidine analogs. Although improvements in limb fat mass were reported in patients who switched from taking the thymidine analog zidovudine to the NRTI abacavir, no significant improvement was noted in self-assessment of dorsocervical fat (49).

Hepatitis C Co-Infection

25% of PLWH in the U.S. are co-infected with hepatitis C (50). Hepatitis C infection is associated with a higher risk of developing diabetes. Treatments for hepatitis C, including direct-acting antiviral treatment and pegylated interferon/ribivirin, have been found to lower the incidence of diabetes, with a larger effect in those patients with advanced fibrosis/cirrhosis. In addition among individuals who received treatment for hepatitis C, those with a sustained virologic response were less likely to develop diabetes than those without a sustained virologic response (51).

Weight Gain, Overweight, and Obesity

Weight gain that is observed after the initiation of ART in those patients with wasting associated with untreated HIV has been characterized as a “return to health” phenomenon. However, because of an emphasis on early ART initiation, the wasting that was previously seen with advanced HIV infection is less common in countries with access to ART (52). In one 2012 study conducted in Alabama, more than 40% of patients were overweight or obese at the time of starting ART. After 2 years on ART, the percentages of underweight and normal weight participants decreased significantly, and significant increases in the percentages of overweight and obese participants were observed. Having a lower baseline CD4 count and the use of a PI as a third drug were risk factors associated with a greater increase in BMI (53).

The consequences of weight gain in PLWH may be different from those in the general population. In a study of U.S. Veterans, the risk of incident diabetes with 10 or more pounds of weight gain during the first year after ART initiation in HIV+ Veterans was significantly greater than in HIV- Veterans (54).

Among cohorts of PLWH from different countries, prevalence estimates of an overweight state or obesity range from 25% to 68% (55,56). In one study of PLWH, risk factors for being overweight or obese in PLWH included increasing age and either no evidence of hepatitis C infection or evidence of cleared hepatitis C infection (57).

Inflammation

In addition to the risk factors for diabetes in PLWH outlined above, inflammation may play a role in the development of diabetes in PLWH. Systemic inflammation results from several factors: viral replication, immune activation, which leads to T cell depletion, as well as T cell loss specifically in the gastrointestinal tract, with associated translocation of microbial factors including lipopolysaccharide (58). Co-infection with viruses, including hepatitis C virus (as discussed above), hepatitis B virus, Epstein-Barr virus, and cytomegalovirus, perpetuate systemic inflammation (59).

Although ART reduces some inflammatory biomarkers in PLWH (60), there is evidence of residual inflammation, with levels of other inflammatory biomarkers in PLWH on ART that do not decrease to levels seen in HIV- individuals (61). Persistent inflammation measured months after ART initiation has been associated with incident diabetes in PLWH (62).

DIAGNOSIS OF AND MONOTORING DIABETES IN PLWH

The American Diabetes Association (ADA) recommends the use of a fasting plasma glucose, hemoglobin A1c, or a 2-hour plasma glucose after a 75-gram oral glucose tolerance test to establish the diagnosis of diabetes in the general population. However, there is a caveat that for certain populations of patients, including PLWH, fasting plasma glucose is preferable to hemoglobin A1c (63). Hemoglobin A1c has been found to underestimate glycemia in PLWH (64,65). One reason for this is the use of medications that cause hemolysis, including dapsone and trimethoprim-sulfamethoxazole, which are used for prophylaxis of Pneumocystis jiroveci, an opportunistic infection (65). However, another reason for the discrepancy between glucose levels and hemoglobin A1c is NRTI use. NRTIs, especially the thymidine analogs, are associated with an increased risk of macrocytosis (66), which can lower hemoglobin A1c. Finally, lower CD4 cell count (< 500 cells/mm³) was associated with a significant discordance between expected and measured hemoglobin A1c in MWoH in the MACS (67). As such, self-monitoring of blood glucose in PLWH may be preferable to using hemoglobin A1c for monitoring glycemic control (64).

The most recent Infectious Diseases Society of America guidelines on the primary care of PLWH recommends obtaining either a fasting plasma glucose and/or hemoglobin A1c at baseline and at 1 to 3 months after starting ART. In addition, hemoglobin A1c is preferred to fasting glucose for diagnosing diabetes because of the relative ease of obtaining a hemoglobin A1c over a fasting glucose. However, a hemoglobin A1c level ≥ 5.8% can be considered to diagnose diabetes in PLWH, instead of the ADA recommendation of ≥ 6.5%, as this improves the sensitivity of the test (68,69). Moreover, a hemoglobin A1c is recommended to be obtained every 6 months in PLWH and diabetes, with a goal hemoglobin A1c of < 7% (68).

TREATMENT OF DIABETES IN PLWH

Special considerations should be taken into account in the treatment of diabetes in PLWH. PLWH may not respond to treatment for diabetes in the same manner as HIV- individuals. Part of this observation may be because treatment responses were measured using hemoglobin A1c, which may be an inaccurate indicator of glycemia in PLWH (70), as noted above.

Interactions between several diabetes medications and ART are known. In addition, some diabetes medications may present advantages or disadvantages in PLWH. These conditions are described in further detail below and are organized by diabetes medication.

Lifestyle Interventions

Both the 2023 ADA Standards of Care on Pharmacologic Approaches to Glycemic Treatment and the 2023 updated American Association of Clinical Endocrinology algorithm of the management of type 2 diabetes highlight lifestyle interventions as part of the recommended treatment of type 2 diabetes (70a, 70b). Lifestyle interventions include not only a healthy diet and exercise but also smoking cessation and moderating alcohol intake (70b).

Physical activity has benefits for PLWH, including decreasing waist circumference (70c) and improvements in glucose and high-density lipoprotein cholesterol levels (70d). However, low physical activity has been reported in some studies of PLWH (70e, 70f).

Metformin

The 2023 ADA Standards of Care on Pharmacologic Approaches to Glycemic Treatment includes metformin as one first-line treatment to consider in patients in the general population with type 2 diabetes (70a) (71).

In PLWH with lipodystrophy and insulin resistance but without diabetes, 3 months of metformin 1000 mg twice daily was found to significantly decrease insulin area under the curve after an oral glucose tolerance test. Although no increased incidence in lactic acidosis was noted in the participants who received metformin, the study was not powered for this outcome, and liver and kidney dysfunction were exclusion criteria (72).

The commonly used integrase transfer strand inhibitor, dolutegravir, increases plasma levels of metformin, and thus adjusting the dose of metformin upon dolutegravir initiation has been recommended (73,74). However, one retrospective study found that there was no significant difference in glycemic control before and after starting dolutegravir in PLWH and diabetes on metformin (75).

In summary, there are no guidelines to suggest that metformin should not be a possible first-line treatment of type 2 diabetes in PLWH, after consideration of liver and kidney function and dolutegravir treatment.

Sulfonylureas

Sulfonylureas are a substrate of the CYP2C9 enzyme. The PIs ritonavir and nelfinavir are CYP2C9 inducers and can decrease sulfonylurea levels (76,77). In comparison with initial use of metformin in PLWH, no significant difference in glycemia after one year of therapy was noted with initial use of a sulfonylurea (78).

The 2023 ADA guidelines note that sulfonylureas are a high efficacy drug class for glucose management and are less cost-prohibitive compared to many other drug classes (70a) (71). However, adverse effects of sulfonylureas include hypoglycemia and weight gain. In PLWH, consideration should be made if a patient is on ritonavir or nelfinavir and the possible loss of efficacy of sulfonylurea treatment.

Thiazolidinediones

The effect of thiazolidinediones on glycemic control in PLWH with diabetes specifically has not been studied, although some trials found that rosiglitazone lowered serum insulin levels in PLWH without diabetes (79) and improved insulin sensitivity in PLWH with hyperinsulinemia (80). Several studies have focused on the effect of thiazolidinediones (TZDs) on body fat in PLWH with lipodystrophy, with equivocal findings. One 24-week study on the effect of rosiglitazone on PLWH, all of whom were on a PI, found no significant difference in arm fat between the treatment and placebo groups (81). However, the study did not have enough power to detect a difference in this outcome (82). On the other hand, other studies demonstrated that rosiglitazone increased visceral and subcutaneous abdominal fat in MWH with lipodystrophy over 6 months (83) and subcutaneous leg fat in PLWH with lipodystrophy over 3 months (80).

Adverse side effects of TZDs should be considered and discussed prior to initiation. These include fluid retention, osteoporosis, edema, and potential liver injury (84).

Dipeptidyl Peptidase 4 Inhibitors

Some clinical studies have demonstrated that dipeptidyl peptidase 4 (DPP4) inhibitors exert an anti-inflammatory effect in patients with type 2 diabetes (85,86). This idea was further examined in PLWH in a pilot study of 20 PLWH on ART randomized to either the DPP4 inhibitor sitagliptin or placebo for 24 weeks. A significant decrease was noted in the chemokine SDF-1α in the treatment group. In addition, an improvement in glucose tolerance was noted at week 8 in the treatment group, but the difference in glucose tolerance between the two groups was no longer significant at the end of the study (87). In another study of PLWH with impaired glucose tolerance, sitagliptin resulted in a significant improvement in glucose tolerance and decreases in the inflammatory markers hsCRP and CXCL10 from baseline after 8 weeks of treatment, compared to placebo (88). Similarly, in a larger study of 84 PLWH on ART with viral suppression and without diabetes who were randomized to 16 weeks of sitagliptin versus placebo, a significant decrease from baseline was seen at week 15 in CXCL10 in participants in the treatment group (89).

Glucagon-Like Peptide-1 Receptor Agonists

Several glucagon-like peptide-1 (GLP-1) receptor agonists have been shown to improve weight and cardiovascular outcomes, albeit in patient populations not specific to PLWH (90,91). Liraglutide, dulaglutide, and semaglutide have been shown to lower the risk of a composite outcome of nonfatal stroke, nonfatal myocardial infarction, and cardiovascular death in patients with type 2 diabetes (90,91). Liraglutide, semaglutide, and dulaglutide have indications from the Food and Drug Administration for lowering cardiovascular event risk in patients with cardiovascular disease and type 2 diabetes, and dulaglutide is also approved for lowering cardiovascular event risk in patients with multiple cardiovascular risk factors and type 2 diabetes. The 2023 ADA guidelines recommend the use of a GLP-1 receptor agonist as a first-line treatment in patients with cardiovascular disease and type 2 diabetes (70a).

One case report described a single PLWH who was able to discontinue insulin treatment (insulin glargine 60 units daily) and who experienced improvements in weight and hemoglobin A1c after starting liraglutide therapy (92). Another case report described improvements in hemoglobin A1c and fasting glucose on dulaglutide in a PLWH. Relatively scant literature exists on the effects of GLP-1 receptors agonists in PLWH and diabetes (93,94).

Sodium-Glucose Co-Transporter-2 Inhibitors

Similar to the GLP-1 receptor agonists, sodium-glucose co-transporter-2 (SGLT2) inhibitors have demonstrated reductions in adverse cardiovascular outcomes, including cardiovascular death and hospitalization for heart failure in general population studies (95,96). In addition, both dapagliflozin and canagliflozin have an FDA indication to reduce the risk of end-stage renal disease in patients with type 2 diabetes and diabetic nephropathy with albuminuria (96a). As such, the 2023 ADA guidelines on pharmacologic treatment of diabetes recommend that in those patients with type 2 diabetes and chronic kidney disease, a SGLT2 inhibitor that has been shown to reduce risk of renal disease progression should be used (70a).

SGLT2 inhibitors are also approved for reduction of adverse cardiovascular events and heart failure hospitalization in people with diabetes and have cardiovascular benefits for patients with either systolic or diastolic heart failure (96a). Diastolic dysfunction is more common in PLWH compared with people without HIV. As such, the 2023 ADA guidelines recommend the use of an SGLT2 inhibitor with demonstrated benefit in patients with type 2 diabetes and heart failure (70a).

One small trial studied canagliflozin for 24 weeks in 8 obese PLWH with type 2 diabetes and hemoglobin A1c > 7% and observed improvements in weight and hemoglobin A1c at the end of the study compared to baseline (97).

Adverse side effects of SGLT2 inhibitors include genital mycotic and urinary tract infections. In addition, 55 post-marketing cases of Fournier’s gangrene have been reported between March 2013 to January 2019 (98). A risk factor for Fournier’s gangrene is HIV infection (99-101), especially a CD4 cell count < 200 cells/μL (102).

Insulin

The 2023 ADA guidelines recommend considering insulin initiation in the patient 1) with a hemoglobin A1c > 11%, 2) with signs and symptoms of catabolism, 3) who has a presentation concerning for type 1 diabetes, and/or 4) whose hemoglobin A1c is not at goal despite taking 2 to 3 medications for diabetes, in addition to a GLP-1 receptor agonist.

As with all patients with diabetes, side effects of insulin therapy to consider in a discussion with a patient with HIV disease and diabetes include the risks of hypoglycemia and weight gain.

Sequence of Initiating Diabetes Medications in PLWH

There are no specific guidelines for the treatment of diabetes in PLWH. The 2023 ADA guidelines recommend the treatment of type 2 diabetes be tailored to the individual patient and consider factors including cost of medications, weight loss goals, and comorbidities including chronic kidney disease, atherosclerotic cardiovascular disease, and heart failure. Healthy lifestyle interventions should be incorporated alongside the pharmacologic treatment of type 2 diabetes.

For those patients with type 2 diabetes and ASCVD or who are at high risk for ASCVD, treatment with either a GLP-1 receptor agonist or SGLT2 inhibitor is recommended. If A1c is not at goal, then adding an additional agent, 1) such as a SGLT2 inhibitor, if the patient is already on a GLP-1 receptor agonist, or a GLP-1 receptor agonist, if the patient is already on a SGLT2 inhibitor, or 2) a thiazolidinedione, is recommended (70a). For patients with type 2 diabetes and heart failure, a SGLT2 inhibitor is recommended, as noted above. For patients with type 2 diabetes and chronic kidney disease, a SGLT2 inhibitor, or if contraindicated, a GLP-1 receptor agonist is recommended. Finally, for those patients with type 2 diabetes for whom weight management is a priority, a medication with weight loss or weight neutral effects can be considered. With regards to weight loss, semaglutide and tirzepatide have the most effect, whereas metformin and DPP4 inhibitors are weight neutral. Dulaglutide and liraglutide have a high weight loss efficacy, and SGLT2 inhibitors have intermediate weight loss efficacy (70a).

In PLWH, an additional factor to consider is the possible interaction of a diabetes medication with specific ART.

MICROVASCULAR COMPLICATIONS ASSOCIATED WITH DIABETES IN PLWH

Peripheral Neuropathy

HIV is associated with multiple peripheral neuropathies (PNs), including a distal, symmetric polyneuropathy (DSPN) (103). The prevalence of HIV-associated PN among PLWH varies < 10% to upwards of 50%, with the wide variability in prevalence estimates secondary in part to differences in methods used to assess PN (104). Risk factors for HIV-associated DSPN include use of the NRTIs zalcitabine, didanosine, and stavudine (105). In addition, HIV-associated DSPN has been observed in PLWH within 1 year of HIV transmission and is significantly associated with evidence of immune activation present in the central nervous system (106).

HIV-associated PN, in addition to the risk of developing PN secondary to diabetes, may place PLWH and diabetes at increased risk of sequelae including falls, particularly in those PLWH with a detectable viral load (107), foot ulceration, and amputations.

In summary, in addition to considering the risk of diabetic peripheral neuropathy, the risk of HIV-associated PNs should be kept in mind in the assessment and treatment of PLWH with diabetes.

Nephropathy

The prevalence estimates of chronic kidney disease (CKD) in PLWH vary depending on geographic region, with 7.9% of PLWH affected in Africa (with the highest prevalence among African regions in West Africa at 22%), compared to 3.7% in Europe. In addition, prevalence estimates of CKD in PLWH are significantly greater among those individuals with co-morbid diabetes (108). In the MACS, two independent risk factors associated with greater odds of proteinuria were HIV+ serostatus with ART use, compared to HIV- serostatus, and a history of diabetes (109). Moreover, glomerular hyperfiltration, or a supranormal estimated glomerular filtration rate, is more prevalent among HIV+ men without CKD than HIV- men without CKD (110). Glomerular hyperfiltration has been reported to be an initial state of dysfunction seen in patients with diabetic kidney disease and proteinuria (111).

Other risk factors for CKD in PLWH include the following: recurrent acute kidney injury, African-American race, in part because of risk variants of the APOL1 gene, and persistent inflammation, even in the setting of ART (112-114). In addition, an HIV-associated nephropathy (HIVAN), which is characterized by several insults to the kidney, including focal and segmental glomerulosclerosis, exists. Certain ART drugs within the NRTI, NNRTI, and PI classes are also associated with renal injury (114). Among these are the NRTI tenofovir, which is associated with adverse kidney disease outcomes independent of diabetes (115), and the PI indinavir (116).

Retinopathy

Ocular opportunistic infections in PLWH include CMV retinitis and ischemic HIV retinopathy (117). In addition, patients with a history of CMV retinitis should be monitored periodically for retinitis recurrence by an ophthalmologist (118). However, these ocular opportunistic infections are less common in the setting of ART. However, retinal disease has been noted in PLWH on ART as part of a larger syndrome of the HIV-associated neuroretinal disorder (117), which has an incidence of more than 50% at 20 years after a diagnosis of AIDS (119). One study found that among HIV+ men with a median duration of ART use of 12 years and suppressed viremia did have a significant difference in total peripheral retinal thickness from HIV- men, although the long-term clinical relevance of this is unknown (117).

There is limited literature on the effect of co-morbid diabetes on HIV-associated retinal disease.

MODIFIABLE FACTORS OF MACROVASCULAR COMPLICATIONS ASSOCIATED WITH DIABETES IN PLWH

PLWH are at increased risk of developing atherosclerotic cardiovascular disease (ASCVD) compared to individuals without HIV, despite controlling for traditional cardiovascular (CV) risk factors such as diabetes (5,120). In addition, PLWH have a greater burden of traditional CV risk factors (120). Other non-traditional risk factors include some ART such as older generation PIs (121) and inflammation (122). As such, calculators developed for the general population to calculate ASCVD risk may not accurately capture risk in PLWH (123).

Aspirin

Recent American College of Cardiology/American Heart Association (ACC/AHA) recommend the use of aspirin for primary prevention of ASCVD in general population patients age 40 to 70 years with high ASCVD risk (124), and similarly the American Diabetes Association (ADA) guidelines also recommend aspirin use in select patients with diabetes < 70 years of age with high ASCVD risk and low bleeding risk (125). However, evidence has shown that aspirin use is lower in PLWH with CV risk factors than in HIV- individuals (126), and PLWH are less likely to be prescribed aspirin for primary prevention than HIV- individuals (127).

Blood Pressure

The prevalence of hypertension globally in PLWH is substantial. PLWH have several risk factors for developing hypertension, including a greater prevalence of smoking (as noted below) (128,129) and ART use (130). The relationship of ART to hypertension is thought to be in part from its association to weight gain. Moreover, in the MACS, ART use was associated with greater systolic hypertension but not diastolic hypertension. A mechanism for this could be a change in arterial compliance as a consequence of ART use (131).

The 2023 ADA guidelines recommend a goal blood pressure of < 130/80 (132).

Cholesterol

Dyslipidemia is seen in both untreated PLWH and PLWH on ART (133,134). The decision to initiate a statin in a patient living with HIV for primary prevention should take into account the patient’s HIV serostatus, especially in those patients with a 10-year ASCVD risk of 5 to < 20% (124).

The primary prevention of atherosclerotic cardiovascular disease is a clinically important topic. The Evaluating the Use of Pitavastatin to Reduce Cardiovascular Disease in HIV-Infected Adults (REPRIEVE) study is a multicenter, international, randomized clinical trial on PWH on ART, in which participants with a low to moderate risk of ASCVD were randomized to either pitavastatin daily or placebo (134a). In March 2023, the Data Safety and Monitoring Board recommended premature closure of the study based on the observed efficacy of the study treatment to reduce the primary endpoint of major adverse cardiovascular events (MACE) by 35% relative to the placebo. The incidence of a major adverse cardiovascular event was 4.81 per 1000 person-years in the pitavastatin group and 7.32 per 1000 person-years in the placebo group (hazard ratio, 0.65; 95% confidence interval, 0.48 to 0.90; P = 0.002). Of note, similar to other studies the risk of diabetes was increased in the pitavastatin group (diabetes mellitus occurred in 5.3% in pitavastatin group and 4.0% in the control group. These results suggest that many PLWH should be on statin therapy.

Cigarette Smoking

Cigarette smoking is more prevalent in PLWH than in individuals without HIV (129), and life expectancy is estimated to be lower in HIV+ men and women who are current smokers at the time of HIV care initiation than in HIV+ men and women who are former or never smokers, with a 2.9 year gain in life expectancy at 10 years after HIV care initiation (135). The ADA guidelines recommend that people with diabetes not smoke cigarettes and that smoking cessation treatment be offered for those patients with diabetes who do smoke (136).

SUMMARY

In summary, PLWH have unique risk factors that increase their risk of diabetes. Factors to take into consideration in the treatment of PLWH include the following: type of ART, evidence of lipodystrophy, co-infection with other viruses, and overweight or obese state. A caveat is that HbA1c may be inaccurate in diagnosing and monitoring diabetes in PLWH. PLWH have additional risk factors for developing microvascular complications of diabetes. Some glycemic agents may interact with ART, and other glycemic agents may have unwanted effects, including weight gain, that should be addressed in a patient-provider discussion. Finally, additional modifiable cardiovascular risk factors, including hypertension and smoking, should be addressed in the comprehensive treatment of diabetes in PLWH.

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Dyslipidemia in Patients With Diabetes

ABSTRACT

Atherosclerotic cardiovascular disease (ASCVD) is a major cause of morbidity and mortality in both men and women with T1DM and T2DM. In patients with T1DM in good glycemic control, the lipid profile is very similar to the general population. In contrast, in patients with T2DM, even with good glycemic control, there are frequently lipid abnormalities (elevated TG and non-HDL-C, decreased HDL-C, and an increase in small dense LDL). In both T1DM and T2DM, poor glycemic control increases TG levels and decreases HDL-C levels with modest effects on LDL-C levels. Extensive studies have demonstrated that statins decrease ASCVD in patients with diabetes. Treatment with high doses of potent statins reduces ASCVD events to a greater extent than low dose statin therapy. Adding fibrates or niacin to statin therapy has not been shown to further decrease ASCVD events. In contrast, studies have shown that the combination of a statin and ezetimibe or a statin and a PCSK9 inhibitor result in a greater decrease in ASCVD events than statins alone. Studies have suggested that EPA, an omega-3-fatty acid, when added to statins also reduces ASCVD events but this result is controversial. In statin intolerant patients with T2DM bempedoic acid decreases ASCVD events. Current recommendations state that most patients with diabetes should be on statin therapy. In certain patients with diabetes ezetimibe, PCSK9 inhibitors, and bempedoic acid can play a role in reducing ASCVD.

INTRODUCTION

Atherosclerotic cardiovascular disease (ASCVD) is a major cause of morbidity and mortality in both men and women with diabetes (1-5). In addition to coronary disease, ASCVD includes stroke and peripheral arterial disease (PAD). PAD is common in diabetes, may be the first presentation of ASCVD and should be recognized as needing aggressive treatment of risk factors. The risk of ASCVD is increased approximately 2-fold in men and 3-4-fold in women (2-4,6,7). In the Framingham study, the annual rate of ASCVD was similar in men and women with diabetes, emphasizing that woman with diabetes need as aggressive preventive treatment as men with diabetes (2,6). In addition, several but not all studies, have shown that patients with diabetes with no history of ASCVD have a similar risk of having a myocardial infarction as non-diabetic patients who have a history of ASCVD, i.e., diabetes is an equivalent risk factor as a history of a previous cardiovascular event (8,9). The duration of diabetes and the presence of other risk factors or complications of diabetes likely determine whether a patient with diabetes has a risk equivalent to patients with a history of previous ASCVD events (10,11). In one study patients with T2DM who had the following risk factors within the target range, HbA1c, LDL-C, albuminuria, smoking, and blood pressure, the risk of an acute myocardial infarction or stroke was similar to individuals without diabetics (12). Moreover, numerous studies have shown that patients with diabetes who have ASCVD are at a very high risk of having another event, indicating that this population of patient’s needs especially aggressive preventive measures (1,8). This increased risk for the development of ASCVD in patients with diabetes is seen both in populations where the prevalence of ASCVD is high (Western societies) and low (for example, Japan) (2). However, in societies where the prevalence of ASCVD is low, the contribution of ASCVD as a cause of morbidity and mortality in patients with diabetes is relatively low compared to Western societies (2).

While the database is not as robust, the evidence indicates that patients with T1DM are also at high risk for the development of ASCVD (1,13-15). Interestingly, women with T1DM have twice the excess risk of fatal and nonfatal vascular events compared to men with T1DM (16,17). Additionally, developing T1DM at a young age increases the risk of ASCVD to a greater degree than late onset T1DM (17). Approximately 50% of patients with T1DM are obese or overweight and between 8% and 40% meet the criteria for the metabolic syndrome, which increases their risk of developing ASCVD (18).

While the development of diabetes at a young age increases the risk of ASCVD in patients with both T1DM and T2DM the deleterious impact is greater in patients with T2DM (19). Lastly, in patients with both T1DM and T2DM the presence of renal disease increases the risk of ASCVD (4,14). Of note is that the risk of developing ASCVD events in patients with diabetes has decreased recently, most likely due to better lipid and blood pressure control, which again reinforces the need to aggressively treat these risk factors in patients with diabetes (5,7,20).

ROLE OF RISK FACTORS IN ASCVD

Numerous studies have demonstrated that the traditional risk factors for ASCVD play an important role in patients with diabetes (2,4,5,110). Patients with diabetes without other risk factors have a relatively low risk of ASCVD (in most studies higher than similar non-diabetic patients), whereas the increasing prevalence of other risk factors markedly increases the risk of developing ASCVD (2). The major reversible traditional risk factors are hypertension, cigarette smoking, and lipid abnormalities (2,4,5,14,111). Other risk factors include obesity (particularly visceral obesity), insulin resistance, small dense LDL, elevated TG, low HDL-C, procoagulant state (increased PAI-1, fibrinogen), family history of early ASCVD, homocysteine, Lp (a), renal disease, albuminuria, and inflammation (C-reactive protein, SAA, cytokines) (2,4,5,110,111). In the last decade, it has become clear that to reduce the risk of ASCVD in patients with diabetes, one will not only need to improve glycemic control but also address these other cardiovascular risk factors. In the remainder of this chapter, I will focus on the dyslipidemia that occurs in patients with diabetes.

ROLE OF LIPIDS IN ASCVD

As in non-diabetic populations, epidemiological studies have shown that increased LDL-C and non-HDL-C levels and decreased HDL-C levels are associated with an increased risk of ASCVD in patients with diabetes (2,4,110,111). In the UKPDS cohort LDL-C levels were the strongest predictor of coronary artery disease (112). While it is universally accepted that elevated levels of LDL-C and non-HDL-C cause atherosclerosis and ASCVD the role of HDL-C is uncertain. Genetic studies and studies of drugs that raise HDL-C have not supported low HDL-C levels as a causative factor for atherosclerosis (113). Rather it is currently thought that HDL function is associated with atherosclerosis risk and that this does not precisely correlate with HDL-C levels (113). In patients with diabetes, elevations in serum triglyceride (TG) levels also are associated with an increased risk of ASCVD (4,111,114). With regard to TG, it is not clear whether they are a causative factor for ASCVD or whether the elevation in TG is a marker for other abnormalities (4,111,114,115). Recent Mendelian randomization studies have provided support for the hypothesis that elevated TG levels play a causal role in atherosclerosis (115,116). Unfortunately, as will be discussed later in this chapter lowering TG levels in patients on statin therapy has not decreased cardiovascular events.

LIPID ABNORMALITIES IN PATIENTS WITH DIABETES

In patients with T1DM in good glycemic control, the lipid profile is very similar to lipid profiles in the general population (110). In some studies HDL-C levels are modestly increased in patients with T1DM (117). In contrast, in patients with T2DM, even when in good glycemic control, there are abnormalities in lipid levels (118-121). It is estimated that 30-60% of patients with T2DM have dyslipidemia (5,122). Specifically, patients with T2DM often have an increase in serum TG levels, increased VLDL and IDL, and decreased HDL-C levels. Non-HDL-C levels are increased due to the increase in VLDL and IDL. LDL-C levels are typically not markedly different than in normal subjects but there is an increase in small dense LDL, a lipoprotein particle that may be particularly pro-atherogenic (123). As a consequence, there are more LDL particles, which coupled with the increases in VLDL and IDL, leads to an increase in apolipoprotein B levels (118-121). Additionally, the postprandial increase in serum TG is accentuated and elevations in postprandial lipids may increase the risk of ASCVD (118-121).

It should be recognized that the lipid changes in patients with T2D are characteristic of the alterations in lipid profile seen in obesity and the metabolic syndrome (insulin resistance syndrome) (124). Since a high percentage of patients with T2DM are obese, insulin resistant, and have the metabolic syndrome, it is not surprising that the prevalence of increased TG and small dense LDL and decreased HDL-C is common in patients with T2DM even when these patients are in good glycemic control. Obesity is also accompanied by increased systemic inflammation. The increasing prevalence of obesity/overweight in patients with T1D will likely result in an increased prevalence of dyslipidemia in this population.

Studies have shown that the anti-oxidant and anti-inflammatory functions of HDL isolated from patients with T1DM and T2DM are reduced (117,125). Additionally, the ability of HDL to facilitate cholesterol efflux is reduced in patients with T1DM and T2DM (126,127). Together these findings indicate that HDL-C levels per se may not fully reflect risk of ASCVD in patients with diabetes and that HDL function is perturbed in patients with diabetes.

In both T1DM and T2DM, poor glycemic control increases serum TG levels, VLDL, and IDL, and decreases HDL-C levels (119). Poor glycemic control can also result in a modest increase in LDL-C, which because of the elevation in TG is often in the small dense LDL subfraction. It is therefore important to optimize glycemic control in patients with diabetes because this will have secondary beneficial effects on lipid levels.

Lp(a) levels are usually within the normal range in patients with T1DM and T2DM (128). Some studies have observed no impact of diabetes mellitus on Lp(a) concentrations while other studies reported an elevation or a decrease in Lp(a) concentrations (128). The development of microalbuminuria and the onset of renal disease are associated with an increase in Lp (a) levels (129). Of note low Lp(a) levels are associated with an increased risk of developing T2DM (128). A recent very large case control study found that an Lp(a) concentration in the bottom 10% increases T2DM risk (130).

Table 1. Lipid Abnormalities in Patients with Diabetes
T1DM	Lipid profile is similar to controls if glycemic control is good
T2DM	Increased TG, VLDL, IDL, and non-HDL-C. Decreased HDL-C. Normal LDL-C but increase in small dense LDL, LDL particle number, and apolipoprotein B.
Poor glycemic control	Increased TG, VLDL, IDL, and non-HDL-C. Decreased HDL-C. Modest increase in LDL-C with increase in small dense LDL, LDL particle number, and apolipoprotein B.

EFFECT OF GLUCOSE LOWERING DRUGS ON LIPIDS

Some therapies used to improve glycemic control may have an impact on lipid levels above and beyond their effects on glucose metabolism. In reviewing the literature, it is often very difficult to separate improvements in glycemic control vs. direct effects of drugs. Additionally, many of the changes induced by drug therapy result in only small changes in LDL-C, HDL-C, and TG levels, are variable from study to study, and are of questionable clinical significance. Insulin, sulfonylureas, meglinitides, DPP4 inhibitors, and alpha-glucosidase inhibitors do not appear to markedly alter fasting lipid profiles other than by improving glucose control (there are data indicating that DPP4 inhibitors and acarbose decrease postprandial triglyceride excursions, but they do not markedly alter fasting lipid levels) (131). In contrast, metformin, thiazolidinediones, GLP1 receptor agonists, bromocriptine-QR, and SGLT2 inhibitors have effects independent of glycemic control on serum lipid levels (table 2).

Metformin may decrease serum TG levels and LDL-C levels without altering HDL-C levels (131). In a meta-analysis of 37 trials with 2,891 patients, metformin decreased TG by 11.4mg/dL when compared with control treatment (p=0.003) (132). In an analysis of 24 trials with 1,867 patients, metformin decreased LDL-C by 8.4mg/dL compared to control treatment (p<0.001) (132). In contrast, metformin did not significantly alter HDL-C levels (132). It should be noted that in the Diabetes Prevention Program 3,234 individuals with impaired glucose metabolism were randomized to placebo, intensive lifestyle, or metformin therapy. In the metformin therapy group no significant changes were noted in TG, LDL-C, or HDL-C levels compared to the placebo group (133). Thus, metformin may have small effects on lipid levels.

The effect of thiazolidinediones depends on which agent is used. Rosiglitazone increases serum LDL-C levels, increases HDL-C levels, and only decreases serum TG if the baseline TG levels are high (131). In contrast, pioglitazone has less impact on LDL-C levels, but increases HDL-C levels, and decreases TG (131). In the PROactive study, a large randomized cardiovascular outcome study, pioglitazone decreased TG levels by approximately 10%, increased HDL-C levels by approximately 10%, and increased LDL-C by 1-4% (134). It should be noted that reductions in the small dense LDL subfraction and an increase in the large buoyant LDL subfraction are seen with both thiazolidinediones (131). In a randomized head-to-head trial, it was shown that pioglitazone decreased TG levels and increased serum HDL-C levels to a greater degree than rosiglitazone treatment (135,136). Additionally, pioglitazone increased LDL-C levels less than rosiglitazone. In contrast to the differences in lipid parameters, both rosiglitazone and pioglitazone decreased A1c and C-reactive protein to a similar extent. The mechanism by which pioglitazone induces more favorable changes in lipid levels than rosiglitazone despite similar changes in glucose levels is unclear, but differential actions of ligands for nuclear hormone receptors are well described.

Treatment with SGLT2 inhibitors results in a small increase in LDL-C and HDL-C levels (131). In a meta-analysis of 48 randomized controlled trials SGLT2 inhibitors significantly increased LDL-C (3.8mg/dL, p < 0.00001), HDL-C (2.3mg/dL, p < 0.00001), and decreased TG levels (8.8mg/dL, p < 0.00001) (137). The mechanism for these increases in LDL and HDL cholesterol is unknown but could be due to a decrease in plasma volume. The decrease in TG levels could be secondary to weight loss.

Bromocriptine-QR (Cycloset) treatment decreases TG levels but has no significant effect on LDL-C or HDL-C levels (138,139). The decrease in TG levels is thought to be due to a decrease in hepatic TG synthesis, likely due to a decrease in adipose tissue lipolysis resulting in decreased blood free fatty acid levels and reduced delivery of fatty acids to the liver for TG synthesis (140).

Colesevelam, a bile acid sequestrant that is approved for glucose lowering, lowers LDL-C levels by 15-20% and has only a modest effect on HDL-C levels (101,141). The effect of bile acid sequestrants on TG levels varies (141). In patients with normal TG levels, bile acid sequestrants increase TG levels by a small amount. However, as baseline TG levels increase, the effect of bile acid sequestrants on TG levels becomes greater, and can result in substantial increases in TG levels (141). In patients with TG > 500mg/dL the use of bile acid sequestrants is contraindicated (141).

Finally, GLP-1 receptor agonists can favorably affect the lipid profile by inducing weight loss (decreasing TG and very modestly decreasing LDL-C levels) (131). In a review by Nauck and colleagues it was noted that GLP-1 receptor agonists lowered TG levels by 18 to 62mg/dL depending upon the specific GLP-1 receptor agonist while decreasing LDL-C by 3-8mg/dL and increasing HDL-C by less than 1mg/dL (142). Additionally, GLP-1 receptor agonists reduce postprandial TG by reducing circulating chylomicrons by decreasing intestinal lipoprotein production (131,142). DPP4 inhibitors have a similar effect on postprandial TG levels as GLP-1 receptor agonists while having minimal effects on fasting lipid levels (142).

In the SURPASS trials, tirzepatide studies TG levels were consistently decreased by 13-25% (83,143). In most studies with the exception of SURPASS 5, HDL cholesterol levels increased by 3-11% (83,143). Total cholesterol and LDL cholesterol levels were modestly decreased in most studies (83,143). Not unexpectedly given the decrease in TG levels small LDL particles were decreased. For details see the Endotext chapter Oral and Injectable (Non-Insulin) Pharmacological Agents for the Treatment of Type 2 Diabetes (83).

Table 2. Effect of Glucose Lowering Drugs on Lipid Levels
Metformin	Modestly decrease TG and LDL-C
Sulfonylureas	No effect
DPP4 inhibitors	Decrease postprandial TG
GLP1 analogues	Decrease fasting and postprandial TG, modestly decrease LDL-C
Tirzepatide	Decrease TG, modestly decrease LDL-C, increase HDL-C
Acarbose	Decrease postprandial TG
Pioglitazone Rosiglitazone	Decrease TG and increase HDL-C. Small increase LDL-C but a decrease in small dense LDL
SGLT2 inhibitors	Small increase in LDL-C and HDL-C
Colesevelam	Decrease LDL-C. May increase TG
Bromocriptine-QR	Decrease TG
Insulin	No effect

PATHOPHYSIOLOGY OF THE DYSLIPIDEMIA OF DIABETES

Figure 1. Pathophysiology of the Dyslipidemia of Diabetes

Multiple mechanisms account for the dyslipidemia seen in patients with T2DM, which are affected both by the level of glucose control and by factors such as obesity and inflammation that also contribute to dyslipidemia.

Increase in TG

There are a number of different abnormalities that contribute to the dyslipidemia seen in patients with T2DM and obesity (figure 1) (119-122,144-146).

OVERPRODUCTION OF VLDL BY THE LIVER

A key abnormality is the overproduction of VLDL by the liver, which is a major contributor to the elevations in serum TG levels. The rate of secretion of VLDL is highly dependent on TG availability, which is determined by the levels of fatty acids available for the synthesis of TG in the liver. An abundance of TG prevents the intra-hepatic degradation of Apo B-100 allowing for increased VLDL formation and secretion. There are three major sources of fatty acids in the liver all of which may be altered in patients with T2DM. First, the flux of fatty acids from adipose tissue to the liver is increased. An increased mass of adipose tissue, particularly visceral stores, results in increased fatty acid delivery to the liver. Additionally, insulin suppresses the lipolysis of TG to free fatty acids in adipose tissue; thus, in patients with either poorly controlled diabetes due to a decrease in insulin or a decrease in insulin activity due to insulin resistance, the inhibition of TG lipolysis is blunted and there is increased TG breakdown leading to increased fatty acid deliver to the liver. A second source of fatty acids in the liver is de novo fatty acid synthesis. Numerous studies have shown that fatty acid synthesis is increased in the liver in patients with T2DM. This increase may be mediated by the hyperinsulinemia seen in patients with insulin resistance. While the liver is resistant to the effects of insulin on carbohydrate metabolism, the liver remains sensitive to the effects of insulin stimulating lipid synthesis. Specifically, insulin stimulates the activity of SREBP-1c, a transcription factor that increases the expression of the enzymes required for the synthesis of fatty acids. Thus, while the liver is resistant to the effects of insulin on carbohydrate metabolism the liver remains sensitive to the effects of insulin stimulating lipid synthesis. Additionally, in the presence of hyperglycemia, glucose can induce another transcription factor, carbohydrate responsive element binding protein (ChREBP), which also stimulates the transcription of the enzymes required for fatty acid synthesis. The third source of fatty acids is the uptake of TG rich lipoproteins by the liver. Studies have shown an increase in intestinal fatty acid synthesis and the enhanced secretion of chylomicrons in animal models of T2DM. This increase in chylomicrons leads to the increased delivery of fatty acids to the liver. The increase in hepatic fatty acids produced by these three pathways results in an increase in the synthesis of TG in the liver and the protection of Apo B-100 from degradation resulting in the increased formation and secretion of VLDL. Finally, insulin stimulates the post translational degradation of Apo B-100 in the liver and a decrease in insulin activity in patients with T2DM also allows for the enhanced survival of Apo B-100 promoting increased VLDL formation.

DECREASED DEGRADATION OF TRIGLYCERIDE RICH LIPOPROTEINS

While the overproduction of triglyceride rich lipoproteins by the liver and intestine are important contributors to the elevations in serum TG levels in patients with T2DM, there are also abnormalities in the metabolism of these TG rich lipoproteins. First, there is a modest decrease in lipoprotein lipase activity, the key enzyme that metabolizes TG rich lipoproteins. The expression of lipoprotein lipase is stimulated by insulin and decreased insulin activity in patients with T2DM results in a decrease in lipoprotein lipase, which plays a key role in the hydrolysis of the TG carried in chylomicrons and VLDL. Additionally, patients with T2DM have an increase in Apo C-III levels, a key regulator of TG rich lipoprotein clearance. Glucose stimulates and insulin suppresses Apo C-III expression; thus, diabetes with hyperglycemia and either insulin deficiency or insulin resistance contribute to an increase in Apo C-III. Apo C-III is an inhibitor of lipoprotein lipase activity and thereby reduces the clearance of TG rich lipoproteins. In addition, Apo C-III also inhibits the cellular uptake of lipoproteins. Studies have shown that loss of function mutations in Apo C-III lead to lower serum TG levels and a reduced risk of ASCVD (147,148). Interestingly, inhibition of Apo C-III expression results in a decrease in serum TG levels even in patients deficient in lipoprotein lipase, indicating that the ability of Apo C-III to modulate serum TG levels is not dependent solely on regulating lipoprotein lipase activity (149). Lastly, insulin resistance is associated with an increase in Angptl3, an inhibitor of LPL (150). Thus, in patients with diabetes, a decrease in clearance of TG rich lipoproteins also contributes to the elevation in serum triglyceride levels.

Mechanism for the Increase in Small Dense LDL and Decrease in HDL

The elevation in TG rich lipoproteins in turn has effects on other lipoproteins. Specifically, cholesterol ester transfer protein (CETP) mediates the exchange of TG from TG rich VLDL and chylomicrons to LDL and HDL. The increase in TG rich lipoproteins per se leads to an increase in CETP mediated exchange, increasing the TG content of both LDL and HDL. The TG on LDL and HDL is then hydrolyzed by hepatic lipase and lipoprotein lipase leading to the production of small dense LDL and small HDL. Notably hepatic lipase activity is increased in patients with T2DM, which will also facilitate the removal of TG from LDL and HDL resulting in small lipoprotein particles. The affinity of Apo A-I for small HDL particles is reduced, leading to the disassociation of Apo A-I, which in turn leads to the accelerated clearance and breakdown of Apo A-I by the kidneys. Additionally, the production of Apo A-I may be reduced in patients with diabetes. High glucose levels can activate ChREBP and this transcription factor inhibits Apo A-I expression. Furthermore, insulin stimulates Apo A-I expression and a reduction in insulin activity due to insulin resistance or decreased insulin levels may also lead to a decrease in Apo A-I expression. The net result is lower levels of Apo A-I and HDL-C levels in patients with T2DM.

Role of Poor Glycemic Control

The above-described changes lead to the typical dyslipidemia observed in patients with T2DM (increased TG, decreased HDL-C, and an abundance of small dense LDL and small HDL). In patients with both Type 1 and T2DM, poor glycemic control can further adversely affect lipid and lipoprotein metabolism. As noted above the expression of lipoprotein lipase is stimulated by insulin. If insulin activity is very low the expression of lipoprotein lipase is severely suppressed and the metabolism of TG rich lipoproteins is markedly impaired. This leads to the delayed clearance of both chylomicrons and VLDL and elevations of TG rich lipoproteins. Additionally, insulinopenia results in a marked increase in lipolysis in adipose tissue, leading to the release of free fatty acids into the circulation. This increase in serum fatty acids results in the increased delivery of fatty acids to the liver, enhanced TG synthesis in the liver, and the increased production and secretion of VLDL. Whereas patients with T1DM who are well controlled and not obese or overweight typically have normal serum lipid profiles, if their control deteriorates, they will develop hypertriglyceridemia. In patients with T2DM deterioration of glycemic control will further exacerbate their underlying dyslipidemia resulting in greater increases in TG levels. If the synthesis of new VLDL is increased sufficiently this can result in an increase in LDL-C levels. HDL-C levels may decrease due to the formation of small HDL that are more susceptible to accelerated clearance. Improvements in glycemic control can markedly lower TG levels and may increase serum HDL-C levels. In patients with poorly controlled diabetes improvements in glycemic control may also lower LDL-C levels.

Role of Obesity and Inflammation

Most patients with T2DM and many patients with T1D are obese or overweight. Obesity is a pro-inflammatory state due to the macrophages that infiltrate adipose tissue. The cytokines produced by these macrophages and the adipokines that are produced by fat cells also alter lipid metabolism (151,152). The pro-inflammatory cytokines, TNF and IL-1, decrease the expression of lipoprotein lipase and increase the expression of angiopoietin like protein 4, an inhibitor of lipoprotein lipase. Together these changes decrease lipoprotein lipase activity, thereby delaying the clearance of TG rich lipoproteins. In addition, pro-inflammatory cytokines stimulate lipolysis in adipocytes increasing circulating free fatty acid levels, which will provide substrate for hepatic TG synthesis. In the liver, pro-inflammatory cytokines stimulate de novo fatty acid and TG synthesis. These alterations will lead to the increased production and secretion of VLDL. Thus, increases in the levels of pro-inflammatory cytokines will stimulate the production of TG rich lipoproteins and delay the clearance of TG rich lipoproteins, which together will contribute to the increase in serum TG that occurs in obese patients.

Obesity and the increase in pro-inflammatory cytokines may also affect HDL-C levels (153-155). First, pro-inflammatory cytokines inhibit the production of Apo A-I, the main protein constituent of HDL. Second, in many tissues pro-inflammatory cytokines decrease the expression of ABCA1 and ABCG1, which will lead to a decrease in the efflux of phospholipids and cholesterol from the cell to HDL decreasing the formation of mature HDL. Third, pro-inflammatory cytokines inhibit the production and activity of LCAT, which will limit the conversion of cholesterol to cholesterol esters in HDL. This conversion step is required for the formation of a normal spherical HDL particle and is crucial for the ability of HDL to increase the efflux of cholesterol from cells (including macrophages). Together these effects may lead to a decrease in HDL-C levels and a decrease in reverse cholesterol transport. Reverse cholesterol transport plays an important role in preventing cholesterol accumulation in macrophages and thereby reduces atherosclerosis.

Inflammation also decreases other important functions of HDL, such as its ability to prevent LDL oxidation (156). This reduction in the ability of HDL to protect from oxidation may be mediated in part by inflammation inducing lower levels of the enzyme paraoxonase, which is commonly seen in patients with diabetes (151,157). In parallel inflammation increases the oxidation of LDL and the amount of small dense LDL that is more susceptible to oxidation.

Role of Adipokines

Adipokines, such as leptin, adiponectin, and resistin, regulate lipid metabolism and the levels are altered in obese patients. Obesity increases serum leptin levels and leptin stimulates lipolysis in adipocytes which will increase serum free fatty acid levels (158). The circulating levels of adiponectin are decreased in subjects who are obese (159). Decreased adiponectin levels are associated with elevations in serum TG levels and decreases in HDL-C levels (159). This association is thought to be causal as studies in mice have shown that overexpressing adiponectin (transgenic mice) decreases TG and increases HDL-C levels while conversely, adiponectin knock-out mice have increased TG and decreased HDL-C levels (159). The adiponectin induced decrease in TG levels is mediated by an increased catabolism of TG rich lipoproteins due to an increase in lipoprotein lipase activity and a decrease Apo C-III, an inhibitor of lipoprotein lipase (159). The increase in HDL-C levels induced by adiponectin is mediated by an increase in hepatic Apo A-I and ABCA1, which results in the increased production of HDL particles (159).

Resistin is increased in subjects who are obese and the levels of resistin directly correlate with plasma TG levels (160). Moreover, resistin has been shown to stimulate hepatic VLDL production and secretion due to an increase in the synthesis of Apo B, TG, and cholesterol (160,161). Finally, resistin is associated with a decrease in HDL-C and Apo A-I levels (160).

EFFECT OF LIPID LOWERING ON ASCVD EVENTS IN PATIENTS WITH DIABETES

Monotherapy Studies

STATINS

The Cholesterol Treatment Trialists analyzed data from 18,686 subjects with diabetes (mostly T2DM) from 14 randomized trials (162). In the statin treated group there was a 9% decrease in all-cause mortality, a 13% decrease in vascular mortality, and a 21% decrease in major vascular events per 39mg/dL (1mmol/L) reduction in LDL-C. The beneficial effect of statin therapy was seen in both primary and secondary prevention patients. The effect of statin treatment on cardiovascular events in patients with diabetes was similar to that seen in non-diabetic subjects. Thus, these studies indicate that statins are beneficial in reducing ASCVD in patients with diabetes. Because of the large number of patients with diabetes included in the Heart Protection Study (HPS) and CARDS these two studies will be discussed in greater depth.

The HPS was a double-blind randomized trial that focused on patients at high risk for the development of cardiovascular events, including patients with a history of MIs, other atherosclerotic lesions, diabetes, and/or hypertension (163,164). Patients were between 40 and 80 years of age and had to have total serum cholesterol levels greater than 135mg/dL (thus very few patients were excluded because they did not have a high enough cholesterol level). The major strength of this trial was the large number of patients studied (>20,000). The diabetes subgroup included 5,963 subjects and thus was as large as many other prevention trials. The study was a 2x2 study design comparing simvastatin 40mg a day vs. placebo and anti-oxidant vitamins (vitamin E 600mg, vitamin C 250mg, and beta-carotene 20mg) vs. placebo and lasted approximately 5 years. Analysis of the group randomized to the anti-oxidant vitamins revealed no beneficial or harmful effects. In contrast, simvastatin therapy (40mg per day) reduced cardiovascular events, including MIs and strokes, by approximately 25% in all participants and to a similar degree in the diabetic subjects (total ASCVD reduced 27%, coronary mortality 20%, MI 37%, stroke 24%). Further analysis of the subjects with diabetes revealed that the reduction in cardiovascular events with statin therapy was similar in individuals with diabetes diagnosed for a short duration (<6 years) and for a long duration (>13 years). Similarly, subjects with diabetes in good control (HbA1c <7%) and those not in ideal control (HbA1c >7%) also benefited to a similar degree with statin therapy. Moreover, both T1DM and T2DM patients had a comparable reduction in ASCVD with simvastatin therapy. The decrease in cardiovascular events in patients with T1DM was not statistically significant because of the small number of subjects. Nevertheless, this is the only trial that included patients with T1DM and suggests that patients with T1DM will benefit from statin therapy similar to T2DM. In general, statin therapy reduced ASCVD in all subgroups of subjects with diabetes (females, males, older age, renal disease, hypertension, high TG, low HDL, ASA therapy, etc.) i.e., statin therapy benefits all patients with diabetes (note this study did not include patients with end stage renal disease but other studies have failed to show benefits of statin therapy in patients with diabetes and end stage renal disease (165)).

The CARDS trial specifically focused on subjects with diabetes (166). The subjects in this trial were males and females with T2DM between the ages of 40 to 75 years of age who were at high risk of developing ASCVD based on the presence of hypertension, retinopathy, renal disease, or current smoking. Of particular note, the subjects did not have any evidence of clinical atherosclerosis (myocardial disease, stroke, peripheral vascular disease) at entry and hence this study is a primary prevention trial. Inclusion criteria included LDL-C levels less than 160mg/dL and TG levels less than 600mg/dL. It is important to recognize that the average LDL-C in this trial was approximately 118mg/dL, indicating relatively low LDL-C levels. A total of 2,838 T2DM subjects were randomized to either placebo or atorvastatin 10mg a day. Atorvastatin therapy resulted in a 40% decrease in LDL-C levels with over 80% of patients achieving LDL-C levels less than 100mg/dL. Most importantly, atorvastatin therapy resulted in a 37% reduction in cardiovascular events. In addition, strokes were reduced by 48% and coronary revascularization by 31%. As seen in the HPS, subjects with relatively low LDL-C levels (LDL <120mg/dL) benefited to a similar extent as subjects with higher LDL-C levels (>120mg/dL).

HPS and CARDS, in combination with the other statin trials, provide conclusive evidence that statin therapy will reduce cardiovascular events in patients with diabetes. Importantly, the benefits of statin therapy are seen in patients with diabetes in both primary and secondary prevention trials.

Effect of Aggressive LDL-C Lowering with Statins

Studies have compared reductions of LDL-C to approximately 100mg/dL to more aggressive reductions in LDL-C on atheroma volume. The Reversal Trial studied 502 symptomatic coronary artery disease patients with an average LDL-C of 150mg/dL (167). Approximately 19% of the patients in this trial had diabetes. Patients were randomized to moderate LDL lowering therapy with pravastatin 40mg per day or to aggressive lipid lowering with atorvastatin 80mg per day. As expected, LDL-C levels were considerably lower in the atorvastatin treated group (pravastatin LDL= 110mg/dL vs. atorvastatin LDL= 79mg/dL). Most importantly, when one analyzed the change in atheroma volume determined after 18 months of therapy using intravascular ultrasound, the group treated aggressively with atorvastatin had a much lower progression rate than the group treated with pravastatin. Compared with baseline values, patients treated with atorvastatin had no change in atheroma burden (there was a very slight regression of lesions), whereas patients treated with pravastatin showed progression of lesions. When one compares the extent of the reduction in LDL-C to the change in atheroma volume, a 50% reduction in LDL (LDL-C levels of approximately 75mg/dL) resulted in the absence of lesion progression. This study suggests that lowering the LDL-C to levels well below 100mg/dL is required to prevent disease progression as measured by intravascular ultrasound. Other studies, such as Asteroid, have shown that marked reductions in LDL-C (in Asteroid the mean LDL-C levels were 61mg/dL) can also result in the regression of coronary artery atherosclerosis determined by intravascular ultrasound measurements (168). Additionally, the Saturn trial demonstrated that aggressive lipid lowering with either atorvastatin 80mg or rosuvastatin 40mg would induce regression of coronary artery atherosclerosis to a similar degree in patients with and without diabetes if the LDL-C levels were reduced to less than 70mg/dL (169). Together these trials indicate that aggressive lowering of LDL-C levels to below 70mg/dL can induce regression of atherosclerotic lesions.

The Prove-It trial determined in patients recently hospitalized for an acute coronary syndrome whether aggressively lowering of LDL-C with atorvastatin 80mg per day vs. moderate LDL-C lowering with pravastatin 40mg per day would have a similar effect on cardiovascular end points such as death, MI, documented unstable angina requiring hospitalization, revascularization, or stroke (170,171). In this trial, approximately 18% of the patients were diabetic. As expected, the on-treatment LDL-C levels were significantly lower in patients aggressively treated with atorvastatin compared to the moderate treated pravastatin group (atorvastatin LDL-C = approximately 62 vs. pravastatin LDL-C = approximately 95mg/dL). Of great significance, death or major cardiovascular events was reduced by 16% over the two years of the study in the group aggressively treated with atorvastatin. Moreover, the risk reduction in the patients with diabetes in the aggressive treatment group was similar to that observed in non-diabetics.

In the treating to new targets trial (TNT) patients with stable coronary heart disease and LDL-C levels less than 130mg/dL were randomized to either 10mg or 80mg atorvastatin and followed for an average of 4.9 years (172,173). Approximately 15% of the patients had diabetes. As expected, LDL-C levels were lowered to a greater extent in the patients treated with 80mg atorvastatin than with 10mg atorvastatin (77mg/dL vs. 101mg/dL). Impressively, the occurrence of major cardiovascular events was reduced by 22% in the group treated with atorvastatin 80mg (p<0.001). In the patients with diabetes events were reduced by 25% in the high dose statin group.

Finally, the IDEAL trial was a randomized study that compared atorvastatin 80mg vs. simvastatin 20-40mg in 8,888 patients with a history of ASCVD (174). Approximately 12% of the patients had diabetes. As expected, LDL-C levels were reduced to a greater extent in the atorvastatin treated group than the simvastatin treated group (approximately 81mg/dL vs. 104mg/dL). Once again, the greater reduction in LDL-C levels was associated with a greater reduction in cardiovascular events. Specifically, major coronary events defined as coronary death, nonfatal MI, or cardiac arrest was reduced by 11% (p=0.07), while nonfatal acute MI were reduced by 17% (p=0.02).

Combining the results of the Heart Protection Study, CARDS, Reversal, Saturn, Asteroid, Prove-It, TNT, and IDEAL leads one to the conclusion that aggressive lowering of LDL-C with statin therapy will be beneficial and suggests that in high-risk patients lowering the LDL to levels well below 100mg/dL is desirable. Moreover, the Cholesterol Treatment Trialists reviewed five trials with 39,612 subjects that were designed to determine the effect of usual vs. aggressive reductions in LDL-C (175). They reported that intensive control (approximately a 19mg/dL difference in LDL-C) resulted in a 15% decrease in major vascular events, a 13% reduction in coronary death or non-fatal MI, a 19% decrease in coronary revascularization, and a 16% decrease in strokes. As will be discussed below treatment guidelines reflect the results of these studies. Additionally, as described in detail below, studies of the addition of either ezetimibe or PCSK9 inhibitors to statins further demonstrates that aggressive lowering of LDL-C levels further reduces cardiovascular events

FIBRATES

The beneficial effect of monotherapy with fibrates (e.g., gemfibrozil, fenofibrate) on ASCVD in patients with diabetes is shown in Table 3. The results of these randomized trials suggest that monotherapy with this class of drug might reduce cardiovascular events in patients with diabetes, but the data is not very robust. The largest trial was the Field Trial (176). In this trial, 9,795 patients with T2DM between the ages of 50 and 75 not taking statin therapy were randomized to fenofibrate or placebo and followed for approximately 5 years. Fenofibrate therapy resulted in a 12% decrease in LDL-C, a 29% decrease in TG, and a 5% increase in HDL-C levels. The primary outcome was coronary events (coronary heart disease death and non-fatal MI), which were reduced by 11% in the fenofibrate group but did not reach statistical significance (p= 0.16). However, there was a 24% decrease in non-fatal MI in the fenofibrate treated group (p=0.01) and a non-significant increase in coronary heart disease mortality. Total ASCVD events (coronary events plus stroke and coronary or carotid revascularization) were reduced 11% (p=0.035). These beneficial effects of fenofibrate therapy on ASCVD were observed in patients without a previous history of ASCVD. In patients with a previous history of ASCVD no benefits were observed. Additionally, the beneficial effect of fenofibrate therapy was seen only in those subjects less than 65 years of age. The beneficial effects of fenofibrate in this study may have been muted by the increased use of statins in the placebo group, which reduced the differences in lipid levels between the placebo and fenofibrate groups. If one adjusted for the addition of lipid-lowering therapy, fenofibrate reduced the risk of coronary heart disease events by 19% (p=0.01) and of total ASCVD events by 15% (p=0.004). Thus, while the results of this large trial are intriguing they do not clearly show a benefit of fibrate therapy reducing ASCVD events. The number of patients with diabetes in the other fibrate trials are relatively small (table 3).

While the results of the monotherapy fibrate trials have been very heterogeneous it should be noted that fibrate trials in patients with elevated TG levels have reported a greater reduction of cardiovascular events (177). Additionally, subgroup analysis of several fibrate trials has also suggested that the benefit of fibrates was greatest in patients with elevated TG levels (177,178).

The mechanism by which fibrates may reduce cardiovascular events is unclear. These drugs lower serum TG levels and increase HDL-C, but it should be recognized that the beneficial effects of fibrates could be due to other actions of these drugs. Specifically, these drugs activate the nuclear hormone receptor PPAR alpha, which is present in the cells that comprise the atherosclerotic lesions, and it is possible that these compounds directly affect lesion formation and development. In addition, fibrates are anti-inflammatory. In fact, analysis of the VA-HIT study suggested that much of the benefit of fibrate therapy was not due to changes in serum lipoprotein levels (179,180).

To summarize, while in general the studies suggest that monotherapy with fibrates may reduce ASCVD in patients with diabetes, the results are not very robust or consistent as seen in the statin trials. Of note fibrate therapy appeared to be most effective in patients with increased TG levels and decreased HDL levels, a lipid profile typically seen in patients with T2DM. However, as will be presented in detail below (combination therapy section) the addition of fibrates to statins does not reduce ASCVD.

Table 3. Effect of Fibrate Monotherapy on Cardiovascular Outcomes
Study	Drug	#Diabetic subjects	%Decrease controls	% Decrease diabetics
Helsinki Heart Study (181)	Gemfibrozil	135	34	60*
VA-HIT (180)	Gemfibrozil	620	24	24
DIAS (182)	Fenofibrate	418	-	23*
Sendcap (183)	Bezafibrate	164	-	70
Field (176)	Fenofibrate	9795	-	11*

* Not statistically significant

NIACIN

A single randomized trial, the Coronary Drug Project, has examined the effect of niacin monotherapy on cardiovascular outcomes (184). This trial was carried out from 1966 to 1974 (before the introduction of statin therapy) in men with a history of a prior MI and demonstrated that niacin therapy reduced cardiovascular events. The results of this study were re-analyzed to determine the effect of niacin therapy in subjects with varying baseline fasting and 1-hour post meal glucose levels (185). It was noted that 6 years of niacin therapy reduced the risk of coronary heart disease death or nonfatal MI by approximately 15-25% regardless of baseline fasting or 1-hour post glucose challenge glucose levels. Particularly notable is that reductions in events were seen in the subjects who had a fasting glucose level >126mg/dL or 1-hour glucose levels >220mg/dL (i.e., patients with diabetes). Thus, based on this single study, niacin monotherapy reduces cardiovascular events both in normal subjects and patients with diabetes. However, as will be presented in detail below (combination therapy section) the addition of niacin to statins does not reduce ASCVD.

EZETIMIBE

A multicenter, randomized trial in Japan examined the efficacy of ezetimibe in patients aged ≥75 years with elevated LDL-C (≥140 mg/dL) without a history of coronary artery disease who were not taking lipid lowering drugs (186). Patients were randomized to ezetimibe (n=1716) or usual care (n=1695) and followed for 4.1 years. The primary outcome was a composite of sudden cardiac death, MI, coronary revascularization, or stroke. In the ezetimibe group LDL-C was decreased by 25.9% and non-HDL-C by 23.1% while in the usual care group LDL-C was decreased by 18.5% and non-HDL-C by 16.5% (p<0.001 for both lipid parameters). By the end of the trial 9.6% of the patients in the usual care group and 2.1% of the ezetimibe group were taking statins. Ezetimibe reduced the incidence of the primary outcome by 34% (HR 0.66; P=0.002). Additionally, composite cardiac events were reduced by 60% (HR 0.60; P=0.039) and coronary revascularization by 62% (HR 0.38; P=0.007) in the ezetimibe group vs. the control group. There was no difference in the incidence of stroke or all-cause mortality between the groups. Approximately 25% of the patients in this trial had diabetes and the beneficial effects were similar in the diabetic and non-diabetic subjects. It should be noted that the reduction in cardiovascular events was much greater than one would expect based on the absolute difference in LDL-C levels (121mg/dL in ezetimibe group vs. 132mg/dL). As stated by the authors “Given the open-label nature of the trial, its premature termination, and issues with follow-up, the magnitude of benefit observed should be interpreted with caution.” Nevertheless, this study provides suggestive evidence that ezetimibe monotherapy may reduce cardiovascular events in patients with diabetes.

BEMPEDOIC ACID

A multicenter study of bempedoic acid in statin intolerant patients with ASCVD or at high risk for ASCVD was recently reported (187). Patients were randomized to bempedoic acid, 180 mg daily (n=6992), or placebo (n=6978) and the primary end point was death from cardiovascular causes, nonfatal MI, nonfatal stroke, or coronary revascularization. Bempedoic acid therapy reduced LDL-C and hsCRP levels by approximately 22% compared to the placebo group. The primary composite endpoint was reduced by 13% in the bempedoic acid group (HR 0.87; 95% CI, 0.79 to 0.96; P = 0.004). The four individual components of the primary endpoint were also significantly reduced in the bempedoic acid treatment group. In this trial approximately 45% of the patients had diabetes. In an analysis of the patients without clinical ASCVD, (i.e., primary prevention) (bempedoic acid n = 2100 or placebo n = 2106), there was a 30% decrease in cardiovascular events (HR 0.70: 95% CI, 0.55-0.89; P = .002) (188). In this subgroup analysis 66% of the patients had diabetes. This study clearly indicates that monotherapy with bempedoic acid will reduce cardiovascular events.

OTHER DRUGS

With regard to PCSK9 inhibitors and bile acid sequestrants there have been no randomized monotherapy studies that have examined the effect of these drugs on cardiovascular end points in subjects with diabetes. In non-diabetic subjects, monotherapy with bile acid sequestrants have reduced cardiovascular events (102,103). Since bile acid sequestrants have a similar beneficial impact on serum lipid levels in diabetic and non-diabetic subjects one would anticipate that these drugs would also result in a reduction in events in the diabetic population. Additionally, bile acid sequestrants improve glycemic control (101). However, bile acid sequestrants can raise TG levels and therefore must be used with caution in hypertriglyceridemic patients. There are no outcome studies with PCSK9 inhibitor monotherapy in patients with diabetes but given that these drugs reduce LDL-C levels and in combination with statins reduce cardiovascular events one would anticipate that PCSK9 inhibitor monotherapy will also reduce cardiovascular events.

Combination Therapy

The studies with statins have been so impressive that most patients with diabetes over the age of 40 are routinely treated with statin therapy and younger patients with diabetes at high risk for ASCVD are also typically on statin therapy (see Current Guidelines Section). Therefore, a key issue is whether the addition of other lipid lowering drugs to statins will result in a further reduction in cardiovascular events. A difficulty with such studies is that the reduction in cardiovascular events induced by statin therapy is so robust that very large trials may be required to see additional benefit.

STATINS + FIBRATES

The ACCORD-LIPID trial was designed to determine if the addition of fenofibrate to aggressive statin therapy would result in a further reduction in ASCVD in patients with T2DM (189). In this trial, 5,518 patients on statin therapy were randomized to placebo or fenofibrate therapy. The patients had diabetes for approximately 10 years and either had pre-existing ASCVD or were at high risk for developing ASCVD. During the trial, LDL-C levels were approximately 80mg/dL in both groups. There was only a small difference in HDL-C with the fenofibrate groups having a mean HDL-C of 41.2mg/dL while the control group had an HDL-C of 40.5mg/dL. Differences in TG levels were somewhat more impressive with the fenofibrate group having a mean TG level of 122mg/dL while the control group had a TG level of 144mg/dL. First occurrence of nonfatal MI, nonfatal stroke, or death from cardiovascular causes was the primary outcome and there was no statistical difference between the fenofibrate treated group and the placebo group. Additionally, there were also no statistically significant differences between the groups with regards to any of the secondary outcome measures of ASCVD. Of note, the addition of fenofibrate to statin therapy did not result in an increase in either muscle or liver side effects. On further analysis, there was a possible benefit of fenofibrate therapy in the patients in whom the baseline TG levels were elevated (>204mg/dL) and HDL-C levels decreased (<34mg/dL). Finally, similar to what has been reported in other trials, fenofibrate had beneficial effects on the progression of microvascular disease (190,191).

The PROMINENT trial studied the effect of pemafibrate, a new selective PPAR-alpha activator, in reducing cardiovascular outcomes in 10,497 patients (66.9% with previous ASCVD) with diabetes (192). This was a double-blind, randomized, controlled trial, in patients with T2DM, with mild-to-moderate hypertriglyceridemia (TG level, 200 to 499 mg/dL), LDL-C < 100mg/dL, and HDL-C levels < 40 mg/dL) who received either pemafibrate (0.2-mg tablets twice daily) or placebo in addition to statin therapy (96% on statins). The primary end point was a composite of nonfatal MI, ischemic stroke, coronary revascularization, or death from cardiovascular causes. Baseline fasting TG was 271 mg/dL, HDL-C 33 mg/dL, and LDL-C 78 mg/dL. Compared with placebo, pemafibrate decreased TG by 26.2%, while HDL-C increased 5.1% and LDL-C increased 12.3%. Notably non-HDL-C levels were unchanged and Apo B levels increased 4.8%. The primary endpoint was similar in the pemafibrate and placebo group (HR 1.03; 95% CI 0.91 to 1.15). The increase in LDL-C and Apo B levels likely account for the failure to reduce cardiovascular events.

Taken together the ACCORD study and the PROINENT trial indicate that the addition of fibrate therapy to statin therapy will not result in a reduction in cardiovascular events in patients with diabetes.

STATIN + NIACIN

The AIM-HIGH trial was designed to determine if the addition of Niaspan to aggressive statin therapy would result in a further reduction in cardiovascular events in patients with pre-existing ASCVD (193). In this trial 3,314 patients were randomized to Niaspan vs. placebo. Approximately 33% of the patients had diabetes. On trial, LDL-C levels were in the 60-70mg/dL range in both groups. As expected, HDL-C levels were increased in the Niaspan treated group (approximately 44mg/dL vs. 38mg/dL), while TG were decreased (approximately 121mg/dL vs. 155mg/dL). However, there were no differences in the primary endpoint between the control and Niaspan treated groups (Primary endpoint consisted of the first event of death from coronary heart disease, nonfatal MI, ischemic stroke, hospitalization for an acute coronary syndrome, or symptom-driven coronary or cerebral revascularization). There were also no differences in secondary endpoints except for a possible increase in strokes in the Niaspan treated group. The addition of Niaspan to statin therapy did not result in a significant increase in either muscle or liver toxicity. Thus, this study does not provide support for the addition of niacin to statins. However, it should be recognized that this was a relatively small study and a considerable number of patients stopped taking the Niaspan during the course of the study (25.4% of patients discontinued Niaspan therapy). In addition, most of the patients included in this study did not have a lipid profile that one would typically consider treating with niacin therapy. In the subset of patients with TG > 198mg/dL and HDL-C < 33mg/dL niacin showed a trend towards benefit (hazard ratio 0.74; p=0.073), suggesting that if the appropriate patient population was studied the results may have been positive (194).

HPS 2 Thrive also studied the effect of niacin added to statin therapy (195). This trial utilized extended-release niacin combined with laropiprant, a prostaglandin D₂ receptor antagonist that reduces the flushing side effect of niacin treatment. HPS 2 Thrive was a very large trial with over 25,000 patients randomized to either niacin therapy or placebo. Approximately 32% of the patients in this trial had diabetes. The LDL-C level was 63mg/dL, the HDL-C 44mg/dL, and the TG 125mg/dL at baseline. As expected, niacin therapy resulted in a modest reduction in LDL-C (10mg/dL), a modest increase in HDL-C (6mg/dL), and a larger reduction in TG (33mg/dL). However, despite these lipid changes there were no significant differences in major cardiovascular events between the niacin and control group (risk ratio 0.96 CI 0.90- 1.03). It is unknown whether laropiprant, the prostaglandin D₂ receptor antagonist, might have effects that worsen atherosclerosis and increase event rates. Similar to the AIM-HIGH study, the group of patients included in the HPS 2 Thrive trial were not the ideal patient population to test for the beneficial effects of niacin treatment added to statin therapy. Ideally, patients with high TG and high non-HDL-C levels coupled with low HDL-C levels should be studied. Nevertheless, the results of the AIM-HIGH and HPS 2 Thrive trials do not provide support for the addition of niacin to statin therapy in patients with diabetes.

STATIN + EZETIMIBE

The IMPROVE-IT trial tested whether the addition of ezetimibe to statin therapy would provide an additional beneficial effect in patients with the acute coronary syndrome (196). This was a large trial with over 18,000 patients randomized to statin therapy vs. statin therapy + ezetimibe. Approximately 27% of the patients in this trial had diabetes. On treatment LDL-C levels were 70mg/dL in the statin alone group vs. 53mg/dL in the statin + ezetimibe group. There was a small but significant 6.4% decrease in major cardiovascular events (Cardiovascular death, MI, documented unstable angina requiring re-hospitalization, coronary revascularization, or stroke) in the statin + ezetimibe group (HR 0.936 CI (0.887, 0.988) p=0.016). Cardiovascular death, non-fatal MI, or non-fatal stroke were reduced by 10% (HR 0.90 CI (0.84, 0.97) p=0.003). These beneficial effects were particularly pronounced in the patients with diabetes (Primary endpoint hazard ratio, 0.85; 95% confidence interval, 0.78-0.94) (197,198). This trial provides evidence that the combination of a statin + ezetimibe that results in a greater reduction in LDL-C levels will lead to a larger decrease in cardiovascular events than statin alone. It should be noted that the observed reduction in events was in the range expected based on the decrease in LDL-C levels.

The RACING trial compared rosuvastatin 10 mg plus ezetimibe 10 mg (combination therapy) vs. rosuvastatin 20mg in 3,780 patients (1,398 patients (37.0%) with diabetes) at 26 centers in South Korea (199). In the patients with diabetes the baseline LDL-C levels was 74mg/dL and during the study the median LDL-C was 53mg/dL in the combination therapy group and 61mg/dL in the high-intensity statin group (P < 0.001). After a median follow-up of 3 years the rate of cardiovascular events in patients with diabetes was 10.0% in the combination therapy group and 11.3% in the high-intensity statin group (HR: 0.89; 95% CI: 0.64–1.22; P = 0.460). Interestingly the rate of discontinuation or dose reduction of the study drug due to intolerance was lower in the combination therapy group than in the high-intensity statin group (5.2 vs. 8.7%; P = 0.014). This study demonstrates that cardiovascular outcomes were comparable between those receiving combination therapy vs. high-intensity statin monotherapy and that combination therapy significantly reduced the rate of drug discontinuation or dose reduction due to intolerance.

STATIN + PCSK9 INHIBITORS

The FOURIER trial was a randomized, double-blind, placebo-controlled trial of evolocumab vs. placebo in 27,564 patients with atherosclerotic ASCVD and an LDL-C level of 70 mg/dL or higher who were on statin therapy (200). Approximately 40% of the patients had diabetes (201). The primary end point was cardiovascular death, MI, stroke, hospitalization for unstable angina, or coronary revascularization and the key secondary end point was cardiovascular death, MI, or stroke. The median duration of follow-up was 2.2 years. Baseline LDL-C levels were 92mg/dL and evolocumab resulted in a 59% decrease in LDL-C levels (LDL-C level on treatment approximately 30mg/dL). Evolocumab treatment significantly reduced the risk of the primary end point (HR 0.85; 95% CI 0.79 to 0.92; P<0.001) and the key secondary end point (HR 0.80; 95% CI 0.73 to 0.88; P<0.001). The results were consistent across key subgroups, including the subgroup of patients in the lowest quartile for baseline LDL-C levels (median, 74 mg/dL). Of note, a similar decrease in cardiovascular events occurred in patients with diabetes treated with evolocumab and glycemic control was not altered (202). Further analysis has shown that in the small number of patients with a baseline LDL-C level less than 70mg/dL, evolocumab reduced cardiovascular events to a similar degree as in the patients with an LDL-C greater than 70mg/dL (203). Finally, the lower the on-treatment LDL-C levels (down to levels below 20mg/dL), the lower the cardiovascular event rate, suggesting that greater reductions in LDL-C levels will result in greater reductions in ASCVD (204).

The ODYSSEY trial was a multicenter, randomized, double-blind, placebo-controlled trial involving 18,924 patients who had an acute coronary syndrome 1 to 12 months earlier, an LDL-C level of at least 70 mg/dL, a non-HDL-C level of at least 100 mg/dL, or an Apo B level of at least 80 mg/dL while on high intensity statin therapy or the maximum tolerated statin dose (205). Approximately 29% of the patients had diabetes. Patients were randomly assigned to receive alirocumab 75 mg every 2 weeks or matching placebo. The dose of alirocumab was adjusted to target an LDL-C level of 25 to 50 mg/dL. The primary end point was a composite of death from coronary heart disease, nonfatal MI, fatal or nonfatal ischemic stroke, or unstable angina requiring hospitalization. During the trial LDL-C levels in the placebo group was 93-103mg/dL while in the alirocumab group LDL-C levels were 40mg/dL at 4 months, 48mg/dL at 12 months, and 66mg/dL at 48 months (the increase with time was due to discontinuation of alirocumab or a decrease in dose). The primary endpoint was reduced by 15% in the alirocumab group (HR 0.85; 95% CI 0.78 to 0.93; P<0.001). In addition, total mortality was reduced by 15% in the alirocumab group (HR 0.85; 95% CI 0.73 to 0.98). The absolute benefit of alirocumab was greatest in patients with a baseline LDL-C level > than 100mg/dL. In patients with an LDL-C level > than 100mg/dL the number needed to treat with alirocumab to prevent an event was only 16. It should be noted that similar to the FOURIER trial the duration of this trial was very short (median follow-up 2.8 years) which may have minimized the beneficial effects. Additionally, because alirocumab 75mg every 2 weeks was stopped if the LDL-C level was < 15mg/dL on two consecutive measurements the beneficial effects may have been blunted (7.7% of patients randomized to alirocumab were switched to placebo).

It should be noted that that the duration of the PCSK9 outcome trials were relatively short and it is well recognized from previous statin trials that the beneficial effects of lowering LDL-C levels takes time with only modest effects observed during the first year of treatment. In the FOURIER trial the reduction of cardiovascular death, MI, or stroke was 16% during the first year but was 25% beyond 12 months. In the ODYSSEY trial the occurrence of cardiovascular events was similar in the alirocumab and placebo group during the first year of the study with benefits of alirocumab appearing after year one. Thus, the long-term benefits of treatment with a PCSK9 inhibitor may be greater than that observed during these relatively short-term studies.

Additional support for the benefits of further lowering of LDL-C levels with a PCSK9 inhibitor added to statin therapy is seen in the GLAGOV trial (206). This trial was a double-blind, placebo-controlled, randomized trial of evolocumab vs. placebo in 968 patients presenting for coronary angiography. Approximately 20-21% of the patients had diabetes. The primary efficacy measure was the change in percent atheroma volume (PAV) from baseline to week 78, measured by serial intravascular ultrasonography (IVUS) imaging. Secondary efficacy measures included change in normalized total atheroma volume (TAV) and percentage of patients demonstrating plaque regression. As expected, there was a marked decrease in LDL-C levels in the evolocumab group (Placebo 93mg/dL vs. evolocumab 37mg/dL; p<0.001). PAV increased 0.05% with placebo and decreased 0.95% with evolocumab (P < .001) while TAV decreased 0.9 mm3 with placebo and 5.8 mm3 with evolocumab (P < .001). There was a linear relationship between achieved LDL-C and change in PAV (i.e., the lower the LDL-C the greater the regression in atheroma volume down to an LDL-C of 20mg/dL). Additionally, evolocumab induced plaque regression in a greater percentage of patients than placebo (64.3% vs 47.3%; P < .001 for PAV and 61.5% vs 48.9%; P < .001 for TAV). The results in the patients with diabetes were similar to the non-diabetic patients.

Taken together these trials demonstrate that further lowering LDL-C levels with PCSK9 monoclonal antibodies in patients taking statins will have beneficial effects on ASCVD outcomes. A study of the effect of inclisiran on ASCVD endpoints is currently in progress.

The results of the ezetimibe and PCSK9 trials have several important implications. First, it demonstrates that combination therapy may have benefits above and beyond statin therapy alone. Second, it provides further support for the hypothesis that lowering LDL per se will reduce cardiovascular events. Third, it suggests that lowering LDL levels to much lower levels than usual will have significant benefits. These results have implications for determining goals of therapy.

STATINS + LOW DOSE OMEGA-3-FATTY ACIDS

ORIGIN was a double-blind study in 12,536 patients at high risk for ASCVD who had impaired fasting glucose, impaired glucose tolerance, or diabetes (207). Patients were randomized to receive a 1-gram capsule containing at least 900mg of ethyl esters of omega-3 fatty acids (EPA 465mg and DHA 375mg) or placebo for approximately 6 years. Greater than 50% of the patients were on statin therapy. The primary outcome was death from cardiovascular causes. TG levels were reduced by 14.5mg/dL in the group receiving omega-3-fatty acids compared to the placebo group (P<0.001), without a significant effect on other lipids. The incidence of the primary outcome was not significantly decreased among patients receiving omega-3-fatty acids as compared with those receiving placebo. The use of omega-3-fatty acids also had no significant effect on the rates of major vascular events, death from any cause, or death from arrhythmia.

A Study of Cardiovascular Events in Diabetes (ASCEND) was a randomized, placebo controlled, double blind trial of 1-gram omega-3-fattys acids (400mg EPA and 300mg DHA ethyl esters) vs. olive oil placebo in 15,480 patients with diabetes without a history of ASCVD (primary prevention trial) (208). Approximately 75% of patients were on statin therapy. The primary end point was serious vascular events (non-fatal MI, non-fatal stroke, transient ischemic attack, or vascular death). Total cholesterol, HDL-C, and non-HDL-C levels were not significantly altered by omega-3-fatty acid treatment (changes in TG levels were not reported). After a mean follow-up of 7.4 years the composite outcome of a serious vascular event or revascularization occurred in 882 patients (11.4%) on omega-3-fatty acids and 887 patients (11.5%) on placebo (rate ratio, 1.00; 95% CI, 0.91 to 1.09). Serious adverse events were similar in placebo and omega-3-fatty acid treated groups.

Taken together these studies indicate that low dose omega-3-fatty acids do not reduce cardiovascular events in patients with diabetes. Studies in non-diabetics have also found little effect of low dose omega-3-fatty acids on ASCVD (209).

STATINS + HIGH DOSE OMEGA-3-FATTY ACIDS

The Japan EPA Lipid Intervention Study (JELIS) was an open label non-placebo controlled study in patients on statin therapy with total cholesterol levels > 254mg/dL with (n= 3664) or without ASCVD (n=14,981) who were randomly assigned to be treated with 1800 mg of EPA (Vascepa) + statin (n=9326) or statin alone (n= 9319) with a 5 year follow-up (210). Approximately 16% of the patients had diabetes. The mean baseline TG level was 153mg/dL. The primary endpoint was any major coronary event, including sudden cardiac death, fatal and non-fatal MI, and other non-fatal events including unstable angina pectoris, angioplasty, stenting, or coronary artery bypass grafting. On treatment total cholesterol, LDL-C, and HDL-C levels were similar in the two groups but plasma TG were modestly decreased in the EPA treated group (5% decrease in EPA group compared to controls; p = 0.0001). In the EPA + statin group the primary endpoint occurred in 2.8% of the patients vs. 3.5% of the patients in the statin alone group (19% decrease; p = 0.011). Unstable angina and non-fatal coronary events were also significantly reduced in the EPA group but in this study sudden cardiac death and coronary death did not differ between groups. Unstable angina was the main component contributing to the primary endpoint and this is a more subjective endpoint than other endpoints such as a MI, stroke, or cardiovascular death. A subjective endpoint has the potential to be an unreliable endpoint in an open label study and is a major limitation of the JELIS Study. The reduction in events was similar in the subgroup of patients with diabetes. In patients with TG levels >150mg/dL and HDL-C levels < 40mg/dL there was a 53% decrease in events (211). In the EPA group, small increases in the occurrence of bleeding (1.1% vs. 0.6%, p=0.0006), gastrointestinal disturbance (3.8%% vs. 1.7%, p<0.0001) and skin abnormalities (1.7 vs. 0.7%, p<0.0001) were seen.

The Reduction of Cardiovascular Events with EPA – Intervention Trial (REDUCE-IT) was a randomized, double blind trial of 2 grams twice per day of EPA ethyl ester (icosapent ethyl) (Vascepa) vs. placebo (mineral oil) in 8,179 patients with hypertriglyceridemia (135mg/dL to 499mg/dL) and established ASCVD or high ASCVD risk (diabetes plus one risk factor) who were on stable statin therapy (212). Approximately 60% of the patients in this trial had diabetes. The primary end point was a composite of cardiovascular death, nonfatal MI, nonfatal stroke, coronary revascularization, or unstable angina. At baseline, the median LDL-C level was 75.0 mg/dL, HDL-C level was 40.0 mg/dL, and TG level was 216.0 mg/dL. The median change in TG level from baseline to 1 year was a decrease of 18.3% (−39.0 mg/dL) in the EPA group and an increase of 2.2% (4.5 mg/dL) in the placebo group. After a median of 4.9 years the primary end-point occurred in 17.2% of the patients in the EPA group vs. 22.0% of the patients in the placebo group (HR 0.75; 95% CI 0.68 to 0.83; P<0.001), indicating a 25% decrease in events. The beneficial effects were similar in patients with and without diabetes. The number needed to treat to avoid one primary end-point event was 21. The reduction in cardiovascular events was noted after approximately 2 years of EPA treatment. Additionally, the risk of cardiovascular death was decreased by 20% in the EPA group (HR 0.80; 95% CI, 0.66 to 0.98; P=0.03). The cardiovascular benefits of EPA were similar across baseline levels of TG (<150, ≥150 to <200, and ≥200 mg/dL). Moreover, the cardiovascular benefits of EPA appeared to occur irrespective of the attained TG level at 1 year (≥150 or <150 mg/dL), suggesting that the cardiovascular risk reduction was not associated with attainment of a normal TG levels. An increase in hospitalization for atrial fibrillation or flutter (3.1% vs. 2.1%, P=0.004) occurred in the EPA group. In addition, serious bleeding events occurred in 2.7% of the patients in the EPA group and in 2.1% in the placebo group (P=0.06). There were no fatal bleeding events in either group and the rates of hemorrhagic stroke, serious central nervous system bleeding, and serious gastrointestinal bleeding were not significantly higher in the EPA group.

These results demonstrate that EPA treatment reduces ASCVD events. Of note the reduction in TG levels is relatively modest and would not be expected to result in the magnitude of the decrease in ASCVD observed in the JELIS and REDUCE-IT trials. Other actions of EPA, such as decreasing platelet function, anti-inflammation, decreasing lipid oxidation, stabilizing membranes, etc. could account for or contribute to the reduction in cardiovascular events (213). It is likely that the beneficial effects of EPA seen in the JELIS and REDUCE-IT trials are multifactorial.

The Statin Residual Risk Reduction with Epanova in High Risk Patients with Hypertriglyceridemia (STRENGTH) trial was a randomized, placebo controlled, double blind trial of 4 grams per day of omega-3-fatty acids (Epanova) (carboxylic acid formulation of EPA and DHA) vs. placebo (corn oil) in 13,000 patients on statins with hypertriglyceridemia (180-500mg/dL), optimal LDL-C levels (< 100mg/dL or on maximal statin therapy), low HDL-C (<42mg/dL in men and < 47mg/dL in women), and either ASCVD or high risk for ASCVD (214). The primary outcome was major atherosclerotic cardiovascular events (cardiovascular death, MI, stroke, coronary revascularization or hospitalization for unstable angina). The primary end point occurred in 785 patients (12.0%) treated with omega-3 CA vs 795 (12.2%) treated with corn oil (HR, 0.99: [95% CI, 0.90-1.09]; P = .84) (215). Thus, in contrast to EPA alone this omega-3-fatty acid formulation failed to show benefits despite reducing TG levels (18% decrease) to a similar degree as in the REDUCE-IT trial.

Whether EPA has special properties that resulted in the reduction in cardiovascular events in the REDUCE-IT trial or there were flaws in the trial design (the use of mineral oil as the placebo) is uncertain and debated. It should be noted that in the REDUCE-IT trial LDL-C and non-HDL-C levels were increased by approximately 10% (LDL-C by approximately 9mg/dL and non-HDL-C by approximately 10mg/dL) in the mineral oil placebo group (212). Additionally, Apo B levels were increased by 7% (6mg/dL) by mineral oil (212). Finally, an increase in hsCRP (20-30%) and other biomarkers of atherosclerosis (oxidized LDL-C, IL-6, IL-1 beta, and lipoprotein-associated phospholipase A2) were noted in the mineral oil group (212,216). In the STRENGTH trial there were no differences in LDL-C, Non-HDL-C, HDL-C, Apo B, or hsCRP levels between the treated vs. placebo groups (215). Whether EPA has special properties compared to DHA leading to a reduction in cardiovascular events or the mineral oil placebo resulted in adverse changes increasing ASCVD in the placebo resulting in an artifactual decrease in the EPA group is debated (217,218). Ideally, another large randomized cardiovascular trial with EPA ethyl ester (icosapent ethyl) (Vascepa) using a placebo other than mineral oil would resolve this controversy.

CURRENT GUIDELINES FOR SERUM LIPIDS

There are several different guidelines for treating lipids in patients with diabetes. While they all focus on lowering LDL-C there are differences between the various guidelines.

American Diabetes Association Guidelines

The 2023 American Diabetes Association (ADA) recommends that adult patients with diabetes have their lipid profile determined at the time of diabetes diagnosis and at least every 5 years thereafter or more frequently if indicated (219). This profile includes total cholesterol, HDL-C, TG, and calculated LDL-C. A lipid panel should be obtained immediately prior to initiating statin therapy. Once a patient is on statin therapy testing should be carried out 4-12 weeks after initiating therapy and annually thereafter to monitor adherence and efficacy. Lifestyle modifications including a reduction in saturated fat, trans fat, and cholesterol intake, weight loss if indicated, an increase in omega-3-fatty acids, viscous fiber, and plant stanols /sterol intake, and increased physical activity is indicated in all patients with diabetes. A focus on a Mediterranean style diet or Dietary Approaches to Stop Hypertension (DASH) diet should be encouraged. In patients with elevated TG levels glycemic control is beneficial and dietary changes and lifestyle changes including weight loss and abstinence from alcohol should be undertaken. Secondary disorders and medications that raise TG levels should be evaluated. Optimize glycemic control to improve TG and HDL-C levels. The recommendations for lipid lowering therapy are shown in table 4. If one follows these recommendations almost all patients with diabetes over the age of 40 will be on statin therapy and many under the age of 40 will also be treated with statins. The addition of ezetimibe should be considered to further lower LDL-C levels in high-risk primary prevention patients. In very high-risk patients with ASCVD if the LDL-C level on statin therapy is greater than 70mg/dL the use of ezetimibe or a PCSK9 inhibitor should be considered. The use of fibrates or niacin with statins were generally not recommended as there is no evidence of benefit. However, in patients with ASCVD or other cardiovascular risk factors on a statin with controlled LDL-C but elevated TG levels (135-499mg/dL) the addition of icosapent ethyl can be considered. Finally, in patients with fasting TG levels greater than 500mg/dL an evaluation for secondary causes of hypertriglyceridemia should be initiated and consideration of drug therapy to reduce the risk of pancreatitis.

Table 4. ADA Recommendations for Lipid Lowering Therapy

Primary Prevention

Age 20-39: With additional risk factors may be reasonable to initiate statin therapy

Age 40-75: Use moderate-intensity statin therapy* in addition to lifestyle therapy

Age 40-75: If at higher cardiovascular risk, including those with one or more ASCVD risk factors, it is recommended to use high intensity statin therapy to reduce LDL cholesterol by >50% and to target an LDL-C <70 mg/dL

Age 40-75: If at higher cardiovascular risk, especially those with multiple ASCVD risk factors and an LDL-C >70 mg/dL, it may be reasonable to add ezetimibe or a PCSK9 inhibitor to maximum tolerated statin therapy

Age > 75: Initiating moderate intensity statin therapy is reasonable after discussion and in patient already on statin therapy it is reasonable to continue statin therapy

Secondary Prevention

All ages: High intensity statin therapy**/maximally tolerated stain

For people with diabetes and ASCVD, treatment with high intensity statin therapy is recommended to target an LDL-C reduction of >50% and an LDL-C l goal of <55 mg/dL. Addition of ezetimibe or a PCSK9 inhibitor is recommended if this goal is not achieved

*Moderate intensity statin- atorvastatin 10-20mg, rosuvastatin 5-10mg, simvastatin 20-40mg, pravastatin 40-80mg, lovastatin 40mg, Fluvastatin XL 80mg, pitavastatin 3-4mg.

**High Intensity statin- atorvastatin 40-80mg, rosuvastatin 20-40mg.

American College of Cardiology and American Heart Association Guidelines

The 2018 American College of Cardiology and American Heart Association (ACC/AHA) guidelines recommend the following (220). “In patients 40 to 75 years of age with diabetes mellitus and LDL-C ≥70 mg/dL (≥1.8 mmol/L), start moderate-intensity statin therapy without calculating 10-year ASCVD risk. In patients with diabetes mellitus at higher risk, especially those with multiple risk factors or those 50 to 75 years of age, it is reasonable to use a high-intensity statin to reduce the LDL-C level by ≥50%.” In patients with diabetes and ASCVD they recommend “In patients with clinical ASCVD, reduce LDL-C with high-intensity statin therapy or maximally tolerated statin therapy. The more LDL-C is reduced on statin therapy, the greater will be subsequent risk reduction. Use a maximally tolerated statin to lower LDLC levels by ≥50%. In very high-risk ASCVD, use an LDL-C threshold of 70 mg/dL (1.8 mmol/L) to consider addition of non-statins to statin therapy. Very high-risk includes a history of multiple major ASCVD events or 1 major ASCVD event and multiple high-risk conditions. In very high-risk ASCVD patients, it is reasonable to add ezetimibe to maximally tolerated statin therapy when the LDL-C level remains ≥70 mg/dL (≥1.8 mmol/L). In patients at very high risk whose LDL-C level remains ≥70 mg/dL (≥1.8 mmol/L) on maximally tolerated statin and ezetimibe therapy, adding a PCSK9 inhibitor is reasonable, although the long-term safety (>3 years) is uncertain and cost effectiveness is low at mid-2018 list prices.” With regards to testing they recommend “Assess adherence and percentage response to LDL-C–lowering medications and lifestyle changes with repeat lipid measurement 4 to 12 weeks after statin initiation or dose adjustment, repeated every 3 to 12 months as needed”. Finally, there are several diabetes specific risk enhancers that are independent of other risk factors that should be considered in deciding the risk of cardiovascular events in a patient with diabetes (Table 5).

Table 5. Diabetes Specific Risk Enhancers That are Independent of Other Risk Factors in Diabetes

Long duration (≥10 years for type 2 diabetes mellitus or ≥20 years for type 1 diabetes mellitus

Albuminuria ≥30 mcg of albumin/mg creatinine

eGFR <60 mL/min/1.73 m2

Retinopathy

Neuropathy

ABI <0.9

ABI indicates ankle-brachial index

American Association of Clinical Endocrinologists/American College of Endocrinology Guidelines

The American Association of Clinical Endocrinologists and American College of Endocrinology guidelines consider individuals with T2DM to be at high, very high, or extreme risk for ASCVD (221,222). Patients with T1DM and a duration of diabetes of more than 15 years or two or more risk factors, poorly controlled A1c, or insulin resistance with metabolic syndrome should be considered to have an equivalent risk to patients with T2DM (221). The recommended treatment goals are shown in Table 6.

Table 6. ASCVD Risk Categories and Treatment Goals
Risk Category	Risk Factors/10-year risk	LDL-C mg/dL	Non-HDL-C mg/dL	Apo B mg/dL	TG mg/dL
Extreme Risk	Diabetes and clinical ASCVD	<55	<80	<70	<150
Very High Risk	Diabetes with one or more risk factors	<70	<100	<80	<150
High Risk	Diabetes and no other risk factors	<100	<130	<90	<150

European Society of Cardiology and European Atherosclerosis Society Guidelines

The European Society of Cardiology (ESC) and European Atherosclerosis Society (EAS) has 2019 guidelines for the treatment of lipids in patients with diabetes (223). These guidelines classify patients with diabetes as very high risk, high risk, or moderate risk (table 7). The recommended goals of therapy based on risk classification are shown in table 8. As with other guidelines intensification of statin therapy should be considered before the introduction of combination therapy. If the goal is not reached, statin combination with ezetimibe should be considered next.

Table 7. ESC/EAS Classification of Risk in Patients with Diabetes

Very High Risk- target organ damage, or at least three major risk factors, or early onset of T1DM of long duration (>20 years)

High Risk- without target organ damage, with DM duration >10 years or another additional risk factor

Moderate Risk- Young patients (T1DM <35 years; T2DM <50 years) with DM duration <10 years, without other risk factors. Calculated SCORE >1 % and <5% for 10-year risk of fatal CVD

Table 8. ESC/EAS Goals of Therapy in Patients with Diabetes
	LDL-C	Non-HDL-C	Apo B
Very High Risk	>50% reduction and <55mg/dL (<1.4mmol/L)	<85mg/d;	<65mg/dL
High Risk	>50% reduction and <70mg/dL (<1.8mmol/L)	<100mg/dL	<80mg/dL
Moderate Risk	<100mg/dL	<130mg/dL	<100mg/dL

European Society of Cardiology Guidelines

The ESC has updated their guidelines in 2023 (224). An important new recommendation is that in patients with T2DM without symptomatic ASCVD or severe target organ damage, it is recommended to estimate 10-year CVD risk using SCORE2-Diabetes (225). This has resulted in a new classification of risk in patients with T2DM (table 9). The LDL-C goals for each category are shown in table 9.

Table 9. Cardiovascular Risk Categories and Goals in Patients with Type 2 Diabetes
Very high risk	Clinically established ASCVD or Severe target organ damage or 10-year risk of CVD> 20% using SCORE2-Diabetes	LDL-C < 55mg/dl Non-HDL-C <85mg/dL
High risk*	10-year risk of CVD 10% to < 20% using SCORE2-Diabetes	LDL-C < 70mg/dL Non-HDL-C <100mg/dL
Moderate risk*	10-year risk of CVD 5% to <10% using SCORE2-Diabetes	LDL-C < 100mg/dL
Low Risk*	10-year risk of CVD <5% using SCORE2-Diabetes	no recommendations

*Patients not meeting the very high-risk category.

Severe target organ damage is defined as eGFR <45 mL/min/1.73 m2 irrespective of albuminuria; or eGFR 45–59 mL/min/1.73 m2 and microalbuminuria (UACR 30–300 mg/g; stage A2); or proteinuria (UACR>300 mg/g; stage A3), or presence of microvascular disease in at least three different sites [e.g., microalbuminuria (stage A2) plus retinopathy plus neuropathy].

My Guideline Recommendations

Thus, different organizations have proposed somewhat different recommendations for the treatment of lipids in patients with diabetes. Despite these differences it is clear that the vast majority of patients with diabetes will need to be treated with statins regardless of which guidelines one elects to follow.

The approach I use is to combine these recommendations (Tables 10 and 11). In patients with diabetes who have pre-existing ASCVD I initiate intensive statin therapy. I prefer LDL or non-HDL-C goals over percent reduction goals. Given the extensive data showing that the lower the LDL-C the greater the reduction in cardiovascular events most secondary prevention patients would benefit from the addition of ezetimibe to maximize LDL-C lowering without markedly increasing costs (226). In patients with diabetes 40-75 years of age without pre-existing ASCVD I calculate the 10-year risk of developing ASCVD (http://www.cvriskcalculator.com/) and identify risk enhancing factors (Table 5) and other factors that increase risk that are not included in the calculator (for example family history, inflammatory disorders, etc.). I initiate intensive statin therapy if the 10-year risk is > 7.5% or if there are multiple risk factors/risk enhancers. I initiate moderate statin therapy if the risk is < 7.5% without multiple risk factors/enhancers. Four to twelve weeks after initiating statin therapy I obtain a lipid panel to determine if the LDL and non-HDL-C levels are at goal. In patients with pre-existing ASCVD or multiple risk factors/risk enhancers (i.e., very high-risk patients) my goal is an LDL-C < 55mg/dL and a non-HDL-C < 80mg/dL. In patients that are at high-risk the goal my goal is an LDL-C < 70mg/dL and a non-HDL-C < 100mg/dL. In patients with moderate risk an LDL-C goal of < 100mg/dL and a non-HDL c < 130mg/dL is appropriate. If the levels are not at goal, I first adjust the statin dose until the patient is taking the maximally tolerated statin dose and then consider adding additional medications. In patients with diabetes who are less than 40 years of age I initiate statin therapy if the patient has overt ASCVD, long standing diabetes, or risk factors/risk enhancers for ASCVD and the LDL and non-HDL-C levels are not at goal. In these younger patients I also calculate the life time risk of ASCVD events to start a discussion of beginning early therapy given the abundance of data indicating that initiating LDL-C lowering therapy early has great potential in markedly lowering ASCVD risk (226,227). In patients over 75 years of age with a reasonable life expectancy I begin moderate statin therapy and adjust based on response. When there is difficulty classifying a patient’s risk, I will obtain a coronary calcium score and use the score to help stratify the patient’s risk. In all cases the benefits and risks of lipid lowering therapy needs to be discussed with patients and the patient’s personnel preferences taken into account.

Table 10. ASCVD Risk Categories and Treatment Goals
Risk Category	Risk Factors/10-year risk	LDL-C mg/dL	Non-HDL-C mg/dL
Very High Risk	Diabetes and clinical ASCVD, multiple risk factors, 10-year risk > 20%	<55	<85
High Risk	Diabetes with one or more risk factors, 10-year risk >7.5% to <20%	<70	<100
Moderate Risk	Diabetes and no other risk factors. 10-year risk <7.5%	<100	<130

Table 11. Drug Therapy According to Risk Category that is Typically Required
Very High Risk	Intensive statin therapy + ezetimibe. Add PCSK9 if not close to goal
High Risk	Intensive statin therapy. Add ezetimibe if not at goal
Moderate Risk	Moderate statin therapy. Increase to intensive statin therapy if not at goal

TREATMENT OF LIPID ABNORMALITIES IN PATIENT WITH DIABETES

Life Style Changes and Weight Loss

Initial treatment of lipid disorders should focus on lifestyle changes. There is little debate that exercise is beneficial and that all patients with diabetes should, if possible, exercise for at least 150 minutes per week (for example 30 minutes 5 times per week). Exercise will decrease serum TG levels and increase HDL-C levels (an increase in HDL-C requires vigorous exercise) (124). Exercise increases fitness and improves insulin resistance even with limited weight loss; reductions in obesity are even more beneficial. It should be noted that many patients with diabetes may have substantial barriers to participating in exercise programs, such as comorbidities that limit exercise tolerance, risk of hypoglycemia, and presence of microvascular complications (visual impairment, neuropathy) that make exercise difficult.

Diet is debated to a greater extent and for detailed information on nutrition therapy for adults with diabetes see the consensus report by the American Diabetes Association (228). Everyone agrees that weight loss in obese patients is essential (124). But how this can be achieved is hotly debated with many different "experts" advocating different dietary approaches. The wide diversity of approach is likely due to the failure of any approach to be effective in the long term for the majority of obese patients with diabetes. If successful, weight loss will decrease serum TG levels, increase HDL-C levels, and modestly reduce LDL-C (124,229). To reduce LDL-C levels, it is important that the diet decrease saturated fat, trans fatty acids, and cholesterol intake. Increasing soluble fiber is also helpful.

It is debated whether a low fat, high complex carbohydrate diets vs. a high monounsaturated fat diet is ideal for obese patients with diabetes (124). One can find "experts" in favor of either of these approaches and there are pros and cons to each approach. It is essential to recognize that both approaches reduce simple sugars, saturated fat, trans fatty acids, and cholesterol intake. The high complex carbohydrate diet will increase serum TG levels in some patients and if the amount of fat in the diet is markedly reduced serum HDL-C levels may decrease. In obese patients, it has been postulated that a diet high in monounsaturated fats, because of the increase in caloric density, will lead to an increase in weight gain. Both diets reduce saturated fat and cholesterol intake that will result in reductions in LDL-C levels. Additionally, both diets also reduce trans-fatty acid intake, which will have a beneficial effect on LDL and HDL-C levels and simple sugars, which will have a beneficial effect on TG levels. Very high levels of TG (>1000mg/dL), require diets that are very low in fat.

The available data do not indicate that any particular diet is best for inducing weight loss and it is essential to adapt the diet to fit the food preferences of the patient. Ultimately no weight loss diet will be successful if the patient cannot follow the diet for the long term and therefore the diet needs to be tailored to the specific preferences of the patient. For more detailed information on the effect of diet on lipid and lipoprotein levels and cardiovascular disease see the Endotext chapter “The Effect of Diet on Cardiovascular Disease and Lipid and Lipoprotein Levels” (229).

While it is widely accepted that lifestyle changes will decrease ASCVD events it should be recognized that the Look Ahead trial failed to demonstrate a reduction in ASCVD events (230). In this trial, over 5,000 overweight or obese patients with T2DM were randomized to either an intensive lifestyle intervention group that promoted weight loss through decreased caloric intake and increased physical activity or to a group that received diabetes support and education (control group). After a median follow-up of 9.6 years there was no difference in cardiovascular events (hazard ratio in the intervention group, 0.95; 95% CI 0.83 to 1.09; P=0.51). A major limitation of this study was that while the weight difference between groups was impressive during the first year of the trial, over time the differences greatly narrowed such that at the end of the trial the intensive group had a 6.0% weight loss while the control group had a 3.5% weight loss. This very modest weight difference demonstrates the difficulty in sustaining long term lifestyle changes. Thus, while weight loss and diet therapy are likely to be beneficial in reducing cardiovascular events, in clinical practice they are seldom sufficient because long-term life style changes are very difficult for most patients to maintain.

In contrast to the failure of lifestyle therapy in the Look Ahead trial to reduce cardiovascular events, the PREDIMED trial employing a Mediterranean diet (increased monounsaturated fats) did reduce the incidence of major ASCVD events (231,232). In this multicenter trial center trial, carried out in Spain, over 7,000 patients at high risk for developing ASCVD were randomized to three diets (primary prevention trial). A Mediterranean diet supplemented with extra-virgin olive oil, a Mediterranean diet supplemented with mixed nuts, or a control diet. Approximately 50% of the patients in this trial had T2DM. In the patients assigned to the Mediterranean diets there was 29% decrease in the primary end point (MI, stroke, and death from ASCVD). Subgroup analysis demonstrated that the Mediterranean diet was equally beneficial in patients with and without diabetes. The Mediterranean diet resulted in only a small but significant increase in HDL-C levels and a small decrease in both LDL-C and TG levels, suggesting that the beneficial effects were not mediated by changes in lipids (233).

The CORDIOPREV study was a single center randomized trial that compared a Mediterranean diet to a low-fat diet in 1,002 patients with ASCVD (234). Approximately 50% of the patients had diabetes. The Mediterranean diet contained a minimum of 35% of the calories as fat (22% monounsaturated fatty acids, 6% polyunsaturated fatty acids, and <10% saturated fat), 15% proteins, and a maximum of 50% carbohydrates while the low-fat diet contained less than 30% of total fat (<10% saturated fat, 12–14% monounsaturated fatty acids, and 6–8% polyunsaturated fatty acids), 15% protein, and a minimum of 55% carbohydrates. The risk of an ASCVD event was reduced by approximately 25-30% in the Mediterranean diet group. Whether these diets differed in their effects on fasting lipid levels is unknown.

Finally, another secondary prevention trial of a Mediterranean diet has also demonstrated a reduction in cardiovascular events. The Lyon Diet Heart Study randomized 584 patients who had a MI within 6 months to a Mediterranean type diet vs usual diet (235,236). There was a marked reduction in events in the group of patients randomized to the Mediterranean diet (cardiac death and nonfatal MI rate was 4.07 per 100 patient years in the control diet vs. 1.24 in the Mediterranean diet; p<0.0001). Unfortunately, there is no indication of the number of patients with diabetes in the Lyon Diet Heart Study or whether patients with diabetes responded similar to the entire group. Lipid levels were similar in both groups in this trial (235).

The results of these three randomized trials indicate that we should be encouraging our patients to follow a Mediterranean type diet. It is likely that the beneficial effects of the Mediterranean diet on ASCVD is mediated by multiple mechanisms with alterations in lipid levels making only a minor contribution.

For additional information on the effect of diets on lipid levels and ASCVD see the chapter entitled “The Effect of Diet on Cardiovascular Disease and Lipid and Lipoprotein Levels” in the Lipids and Lipoproteins section of Endotext (229).

Bariatric surgery can have profound effects on weight and can result in marked improvements in lipid profiles with a decrease in TG and LDL-C and an increase in HDL-C (124,229) Additionally, observational studies have shown a decrease in cardiovascular events following bariatric surgery in patients with and without diabetes (237-241). For additional information see the chapter entitled “Obesity and Dyslipidemia” (124).

Ethanol and simple sugars, in particular fructose, increase serum TG levels in susceptible patients. In patients with hypertriglyceridemia efforts should be made to reduce the intake of ethanol, simple sugars, and fructose (229).

Lastly, in the past some "experts" advocated the addition of fish oil supplements to reduce cardiovascular events. However, both the Origin Trial and the ASCEND Trial did not demonstrate that fish oil supplements were beneficial in patients with T2DM or patients at high risk for the development of T2DM (207,208). It should be recognized that higher doses of fish oil are required to lower serum triglyceride levels (~ 3-4 grams of DHA/EPA per day) and are useful in treating patients with high TG levels (242). Most studies of fish oil supplements in patients with diabetes have demonstrated that this is a safe approach and that worsening of glycemic control does not occur in patients with diabetes treated with fish oil supplements (242). Additionally, in some patient's high dose fish oil increases LDL-C levels, particularly when serum TG levels are very high (242). For additional information on fish oil see the chapter on Triglyceride Lowering Drugs (209).

Drug Therapy

The effect of statins, fibrates, niacin, ezetimibe, omega-3-fatty acids, bile acid sequestrants, bempedoic acid, and PCSK9 inhibitors on lipid levels in patients with diabetes is virtually identical to that seen in non-diabetic patients (Table 12). Below we will highlight issues particularly relevant to the use of these drugs in patients with diabetes. For detailed information on lipid lowering drugs see the chapters on Triglyceride Lowering Drugs and Cholesterol Lowering Drugs (141,209).

STATINS

Statins are easy to use and generally well tolerated by patients with diabetes. However, statins can adversely affect glucose homeostasis. In patients without diabetes the risk of developing diabetes is increased by approximately 10% with higher doses of statin causing a greater risk than more moderate doses (243,244). The mechanism for this adverse effect is unknown but older, obese patients with higher baseline glucose levels are at greatest risk. In patients with diabetes, an analysis of 9 studies with over 9,000 patients with diabetes reported that the patients randomized to statin therapy had a 0.12% higher HbA1c than the placebo group indicating that statin therapy is associated with only a very small increase in HbA1c levels in patients with diabetes, which is unlikely to be clinically significant (245). Individual studies such as CARDS and the Heart Protection Study have also shown only a very modest effect of statins on HbA1c levels in patients with diabetes (163,166,246). Muscle symptoms occur in patients with diabetes similar to what is observed in patients without diabetes.

EZETIMIBE

Ezetimibe is easy to use and generally well tolerated by patients with diabetes. Ezetimibe does not appear to increase the risk of new onset diabetes (199,247,248).

FIBRATES

Fibrates are easy to use and generally well tolerated by patients with diabetes. When combining fibrates with statin therapy it is best to use fenofibrate as the risk of inducing myositis is much less than when statins are used in combination with gemfibrozil, which can inhibit statin metabolism (249). In the ACCORD-LIPID Trial the incidence of muscle disorders was not increased in the statin + fenofibrate group compared to statin alone (189). The dose of fenofibrate needs to be adjusted in patients with renal disease and fenofibrate itself can induce a reversible increase in serum creatinine levels. It should be noted that marked reductions in HDL-C levels can occur in some patients treated with both fenofibrate and a TZD (250).

Diabetic Retinopathy

Fenofibrate has been shown to have beneficial effects on diabetic eye disease. The FIELD study, described earlier, was a randomized trial of fenofibrate vs. placebo in patients with T2DM. Laser treatment for retinopathy was significantly lower in the fenofibrate group than in the placebo group (3.4% patients on fenofibrate vs 4.9% on placebo; p=0.0002) (191). Fenofibrate therapy reduced the need for laser therapy to a similar extent for maculopathy (31% decrease) and for proliferative retinopathy (30% decrease). In the ophthalmology sub-study (n=1012), the primary endpoint of 2-step progression of retinopathy grade did not differ significantly between the fenofibrate and control groups (9.6% patients on fenofibrate vs 12.3% on placebo; p=0.19). In patients without pre-existing retinopathy there was no difference in progression (11.4% vs 11.7%; p=0.87). However, in patients with pre-existing retinopathy, significantly fewer patients on fenofibrate had a 2-step progression than did those on placebo (3.1% patients vs 14.6%; p=0.004). A composite endpoint of 2-step progression of retinopathy grade, macular edema, or laser treatments was significantly reduced in the fenofibrate group (HR 0.66, 95% CI 0.47-0.94; p=0.022).

In the ACCORD Study a subgroup of participants was evaluated for the progression of diabetic retinopathy by 3 or more steps on the Early Treatment Diabetic Retinopathy Study Severity Scale or the development of diabetic retinopathy necessitating laser photocoagulation or vitrectomy over a four-year period (190). At 4 years, the rates of progression of diabetic retinopathy were 6.5% with fenofibrate therapy (n=806) vs. 10.2% with placebo (n=787) (adjusted odds ratio, 0.60; 95% CI, 0.42 to 0.87; P = 0.006). Of note, this reduction in the progression of diabetic retinopathy was of a similar magnitude as intensive glycemic treatment vs. standard therapy.

Taken together these results indicate that fibrates have beneficial effects on the progression of diabetic retinopathy. The mechanisms by which fibrates decrease diabetic retinopathy are unknown.

Diabetic Nephropathy

The Diabetes Atherosclerosis Intervention Study (DAIS) evaluated the effect of fenofibrate therapy (n= 155) vs. placebo (n=159) on changes in urinary albumin excretion in patients with T2DM (251). Fenofibrate significantly reduced the worsening of albumin excretion (fenofibrate 8% vs. placebo 18%; P < 0.05). This effect was primarily due to reduced progression from normal albumin excretion to microalbuminuria (fenofibrate 3% vs. 18% placebo; P < 0.001).

In the FIELD trial, fenofibrate reduced urine albumin/creatinine ratio by 24% vs 11% in placebo group (p < 0.001), with 14% less progression and 18% more albuminuria regression (p < 0.001) in the fenofibrate group than in participants on placebo (252). As expected, fenofibrate therapy acutely increased plasma creatinine levels and decreased eGFR but over the long term, the increase in plasma creatinine was decreased in the fenofibrate group compared to the placebo group (14% decrease; p=0.01). Similarly, there was a slower annual decrease in eGFR in the fenofibrate group (1.19 vs 2.03 mL/min/1.73m²annually, p < 0.001). The effect of fenofibrate on kidney function was greater in those with higher TG and lower HDL levels. End-stage renal disease, dialysis, renal transplant, and renal death were similar in the fenofibrate and placebo groups, but the incidence was low.

In the ACCORD-LIPID trial the post-randomization incidence of microalbuminuria was 38.2% in the fenofibrate group and 41.6% in the placebo group (p=0.01) and post-randomization incidence of macroalbumuria was 10.5% in the fibrate group and 12.3% in the placebo group (p=0.04) indicating a modest reduction in the development of proteinuria in patients treated with fenofibrate (189). There was no significant difference in the incidence of end-stage renal disease or need for dialysis between the fenofibrate group and the placebo group.

These studies suggest that fibrates may have a beneficial effect on diabetic kidney disease. One should recognize that reducing proteinuria is a surrogate marker and may not indicate a reduction in the development of end stage renal disease. The mechanisms accounting for decreased in proteinuria are unknown.

Amputations

In the FIELD study the risks of first amputation were decreased by 36% (p=0.02) and minor amputation events without known large-vessel disease by 47% (p=0.027) in the fenofibrate treated group (253). The reduction in amputations was independent of glucose control or dyslipidemia. No difference between the risks of major amputations was seen in the placebo and fenofibrate groups. The basis for this reduction in amputations is unknown.

Do Fibrates have an Independent Effect on Microvascular Disease?

Multiple studies cited above have now shown that fenofibrate decreases retinopathy, nephropathy, and amputation in the absence of large vessel disease. The effects are independent of blood glucose control. Given that there also was no effect of fenofibrate on cardiovascular (macrovascular) disease, these results may suggest that fenofibrate has an independent effect on microvascular disease. Further studies are warranted, but these results should be taken into account when considering treatment of marked hypertriglyceridemia in patients with diabetes.

BILE ACID SEQUESTRANTS

Bile acid sequestrants are relatively difficult to take due to GI toxicity (mainly constipation) (141). Diabetic subjects have an increased prevalence of constipation, which may be exacerbated by the use of bile acid sequestrants. On the other hand, in diabetic patients with diarrhea, the use of bile acid sequestrants may be advantageous. Bile acid sequestrants may also increase serum TG levels, which can be a problem in patients with diabetes who are already hypertriglyceridemic (141). An additional difficulty in using bile acid sequestrants is their potential for binding other drugs (141). Many drugs should be taken either two hours before or four hours after taking bile acid sequestrants to avoid the potential of decreased drug absorption. Patients with diabetes are frequently on multiple drugs for glycemic control, hypertension, etc., and it can sometimes be difficult to time the ingestion of bile resin sequestrants to avoid these other drugs. Colesevelam (Welchol) is a bile acid sequestrant that comes in pill, powder, or chewable bars and causes fewer side effects and has fewer interactions with other drugs than other preparations (254). The usual dose is3.75 grams per day and can be given as tablets (take 6 tablets once daily or 3 tablets twice daily), oral suspension (take one packet once daily), or chewable bars (take one bar once daily). Of particular note is that a number of studies have shown that colesevelam improves glycemic control in patients with diabetes resulting in an approximately 0.5% decrease in A1c levels (255).

NIACIN

Niacin is well known to cause skin flushing and itching and GI upset (256). Additionally, niacin reduces insulin sensitivity (i.e., causes insulin resistance), which can worsen glycemic control (256). Studies have shown that niacin is usually well tolerated in diabetic subjects who are in good glycemic control (257,258). In patients with poor glycemic control, niacin is more likely to adversely impact glucose levels. In the HPS2-Thrive trial, niacin therapy significantly worsened glycemic control in patients with diabetes and induced new onset diabetes in 1.3% of subjects that were non-diabetic (195). High doses of niacin are more likely to adversely affect glycemic control. Niacin can also increase serum uric acid levels and induce gout, both of which are already common in obese patients with T2DM (256). Additionally recent trials have reported an increased incidence of infection and bleeding with niacin therapy (256). However, niacin is the most effective drug in increasing HDL-C levels, which are frequently low in patients with diabetes.

OMEGA-3-FATTY ACIDS

A Cochrane review of fish oil in patients with diabetes have demonstrated that this is a safe approach and does not result in worsening of glycemic control in patients with diabetes (242). Fish oil effectively lowers TG levels but, in some patients, particularly those with significant hypertriglyceridemia, high dose fish oil increases LDL-C levels (242). It should be noted that fish oil products that contain just EPA (Vascepa) do not adversely affect LDL-C levels (259). When using fish oil to lower serum TG levels it is important to recognize that one is aiming to provide 3-4 grams of DHA/EPA per day. The quantity of these active omega-3-fatty acids can vary greatly from product to product. Prescription fish oil products contain large amounts of these active ingredients whereas the amount of DHA/EPA in food supplements can vary greatly and in some products levels are very low. Additionally, while prescription omega-3-fatty acid preparations have high levels of quality control, omega-3-fish oil food supplements may have contaminants and the dosage may not be precisely controlled.

PCSK9 INHIBITORS

The beneficial effects of PCSK9 inhibitors in patients with diabetes is similar to what is observed in non-diabetic patients. Additionally, except for local reactions at the injection sites PCSK9 inhibitors do not seem to cause major side effects. PCSK9 inhibitors do not appear to increase the risk of developing new-onset diabetes (260,261).

BEMPEDOIC ACID

The effect of bempedoic acid on LDL-C levels in patients with diabetes are similar to the decreases seen on non-diabetics. Patients with T2DM often have elevated uric acid levels and an increased risk of gouty attacks and a major side effect of bempedoic acid is elevating uric acid levels (141). In clinical trials, 26% of bempedoic acid-treated patients with normal baseline uric acid values experienced hyperuricemia one or more times versus 9.5% in the placebo group. Elevations in blood uric acid levels may lead to the development of gout and gout was reported in 1.5% of patients treated with bempedoic acid vs. 0.4% of patients treated with placebo. The risk for gout attacks were higher in patients with a prior history of gout (11.2% for bempedoic acid treatment vs. 1.7% in the placebo group). In patients with no prior history of gout only 1% of patients treated with bempedoic acid and 0.3% of the placebo group had a gouty attack.

In a meta-analysis, bempedoic acid therapy was associated with a decrease in the onset of diabetes and worsening of diabetes (RR 0.65, p = 0.03) (7/100 vs 6.4/100 patient years) (262). In a study focusing solely on the development of new onset diabetes it was reported that new-onset diabetes/hyperglycemia occurred less frequently with bempedoic acid vs placebo (263). In the bempedoic acid cardiovascular outcome trial (CLEAR Outcomes) the development of diabetes and worsening of pre-existing diabetes was similar in the bempedoic acid and placebo groups (187).

Table 12. Effect of Lipid Lowering Drugs
	LDL-C	HDL-C	TG

Statins	↓ 20-60%	↑ 5-15%	↓ 0-35%*
Bile acid sequestrants	↓ 10-30%	↑ 0-10%	↑ 0-10%**
Fibrates	↓ 0-15%***	↑ 5-15%	↓ 20-50%
Niacin	↓ 10-25%	↑ 10-30%	↓ 20-50%
Ezetimibe	↓ 15-25%	↑ 1-3%	↓ 10-20%
PCSK9 Inhibitors	↓ 50-60%	↑ 5-15%	↓ 5-20%
Bempedoic Acid	↓ 15-25%	↓ 5-6%	No change
High Dose Fish Oil	↑ 0- 50%***	↑ 4- 9%	↓ 20- 50%*

*Patients with elevated TG have largest decrease

** In patients with high TG may cause marked increase

*** In patients with high TG may increase LDL

Therapeutic Approach

FIRST PRIORITY- LDL-C

The first priority in treating lipid disorders in patients with diabetes is to lower the LDL-C levels to goal, unless TG are markedly elevated (> 500- 1000mg/dL), which increases the risk of pancreatitis. LDL-C is the first priority because the database linking lowering LDL-C with reducing ASCVD is extremely strong and we now have the ability to markedly decrease LDL-C levels. Dietary therapy is the initial step but, in almost all patients, will not be sufficient to achieve the LDL-C goals. If patients are willing and able to make major changes in their diet it is possible to achieve significant reductions in LDL-C levels but this seldom occurs in clinical practice (264).

Statins are the first-choice drugs to lower LDL-C levels and the vast majority of diabetic patients will require statin therapy. There are several statins currently available in the US and they are available as generic drugs and therefore relatively inexpensive. The particular statin used may be driven by price, ability to lower LDL-C levels, and potential drug interactions. Patients with ASCVD (secondary prevention patients) should be started on intensive statin therapy (atorvastatin 40-80mg per day or rosuvastatin 20-40mg per day). Given the extensive data showing that the lower the LDL-C the greater the reduction in ASCVD events most secondary prevention patients would benefit from the addition of ezetimibe to maximize LDL-C lowering. Ezetimibe is now a generic drug and therefore this strategy will not markedly increase costs. Similarly, primary prevention patients who are at high risk for cardiovascular events will also benefit from the use of high intensity statin therapy in combination with ezetimibe. Primary prevention patients at moderate risk can be started on moderate intensity statin therapy.

If a patient is unable to tolerate statins or statins as monotherapy are not sufficient to lower LDL-C to goal the second-choice drug is either ezetimibe or a PCSK9 inhibitor. Ezetimibe can be added to any statin. PCSK9 inhibitors can also be added to any statin and are the drug of choice if a large decrease in LDL-C is required to reach goal (PCSK9 inhibitors will lower LDL-C levels by 50-60% when added to a statin, whereas ezetimibe will only lower LDL-C by approximately 20%). Bile acid sequestrants and bempedoic acid are alternatives with the use of a bile acid sequestrant particularly useful if a reduction in A1c level is also needed. It should be noted that in statin intolerant patients with ASCVD or at high risk (approximately 45% with diabetes), bempedoic acid has been shown to reduce cardiovascular events by 13% (HR 0.87; 95% CI 0.79 to 0.96; P = 0.004) (187). Ezetimibe, PCSK9 inhibitors, bempedoic acid, and bile acid sequestrants additively lower LDL-C levels when used in combination with a statin, because these drugs increase hepatic LDL receptor levels by different mechanisms, thereby resulting in a reduction in serum LDL-C levels (141). Niacin and the fibrates also lower LDL-C levels but are not usually employed to lower LDL-C levels.

SECOND PRIORITY- NON-HDL-C

The second priority should be non-HDL-C (non-HDL-C = total cholesterol – HDL-C), which is particularly important in patients with elevated TG levels (>150mg/dL). Non-HDL-C is a measure of all the pro-atherogenic apolipoprotein B containing particles. Numerous studies have shown that non-HDL-C is a strong risk factor for the development of ASCVD (265). The non-HDL-C goals are approximately 30mg/dL greater than the LDL-C goals. For example, if the LDL goal is <100mg/dL then the non-HDL-C goal would be <130mg/dL. Drugs that reduce either LDL-C or TG levels will reduce non-HDL-C levels. To lower TG levels initial therapy should focus on glycemic control and lifestyle changes including weight loss if indicated and a decrease in simple sugars and ethanol intake. Additionally, if possible, discontinue medications that increase triglyceride levels. As discussed above, studies with the omega-3-fatty acid icosapent ethyl (EPA; Vascepa) added to statin therapy have reduced the risk of cardiovascular events. The National Lipid Association has recommended “that for patients aged ≥45 years with clinical ASCVD, or aged ≥50 years with diabetes mellitus requiring medication plus ≥1 additional risk factor, with fasting TGs 135 to 499 mg/dL on high-intensity or maximally tolerated statin therapy (±ezetimibe), treatment with icosapent ethyl is recommended for ASCVD risk reduction” (266). As noted earlier in this chapter there is controversy regarding the benefits of icosapent ethyl on cardiovascular outcomes with some experts interpreting the beneficial results of the REDUCE-IT trial as being due to the adverse effects of the mineral oil placebo (217).

VERY HIGH TG

Patients with very high TG levels (> 500-1000 mg/dL) are at risk of pancreatitis and therefore lifestyle interventions including diet, exercise, and weight loss if indicated should be initiated early. Treatment is a low-fat diet and glycemic control. Additionally, decreasing simple sugars and avoiding alcohol is beneficial. When the TG levels are very elevated (> 1000mg/dL) a very low-fat diet (5-20% of calories as fat) should be the primary treatment until the TG levels decrease to < 1000mg/dL. Treating secondary disorders that raise TG levels and when possible, stopping drugs that increase TG levels is essential. If the TG levels remain above 500mg/dL the addition of fenofibrate or omega-3-fatty acids is indicated.

LOW HDL-C

While there is strong epidemiologic data linking low HDL-C levels with ASCVD there is no clinical trials demonstrating that increasing HDL-C levels reduce ASCVD. Thus, the use of drugs such as niacin to raise HDL-C levels is not recommended.

CONCLUSION

Patients with diabetes, particularly T2DM, often have dyslipidemia. Modern therapy of patients with diabetes demands that we aggressively treat lipids to reduce the high risk of ASCVD in this susceptible population and in those with very high TG to reduce the risk of pancreatitis.

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Role Of Glucose And Lipids In The Atherosclerotic Cardiovascular Disease In Patients With Diabetes

ABSTRACT

Atherosclerotic cardiovascular disease (ASCVD) is a major cause of morbidity and mortality in both men and women with T1DM and T2DM. In patients with T1DM, intensive glycemic control results in a reduction in ASCVD. However, intensive glycemic control does not have a major impact in reducing ASCVD in patients with T2DM. Metformin, pioglitazone, SGLT2 inhibitors, and certain GLP-1 receptor agonists have been shown to decrease major cardiovascular events in patients with T2DM to a greater extent than other treatment modalities. In patients with T2DM other risk factors including, hypertension and dyslipidemia, play a major role in inducing ASCVD and control of these risk factors is paramount. In patients with T1DM in good glycemic control, the lipid profile is very similar to the general population. In contrast, in patients with T2DM, even with good glycemic control, there are frequently lipid abnormalities (elevated TG and non-HDL-C, decreased HDL-C, and an increase in small dense LDL). In both T1DM and T2DM, poor glycemic control increases TG levels and decreases HDL-C levels with modest effects on LDL-C levels. Extensive studies have demonstrated that statins decrease ASCVD in patients with diabetes. Treatment with high doses of potent statins reduces ASCVD events to a greater extent than low dose statin therapy. Adding fibrates or niacin to statin therapy has not been shown to further decrease ASCVD events. In contrast, studies have shown that the combination of a statin and ezetimibe or a statin and a PCSK9 inhibitor result in a greater decrease in ASCVD events than statins alone. Studies have suggested that EPA, an omega-3-fatty acid, when added to statins also reduces ASCVD events but this result is controversial. In statin intolerant patients with T2DM bempedoic acid decreases ASCVD events. Current recommendations state that most patients with diabetes should be on statin therapy. In certain patients with diabetes ezetimibe, PCSK9 inhibitors, and bempedoic acid can play a role in reducing ASCVD.

INTRODUCTION

ROLE OF GLYCEMIC CONTROL

Epidemiological studies have shown an association between the level of glycemic control and the development of ASCVD in both T1DM and T2DM (1,4,5,21,22). However, the association of glycemic control with ASCVD is considerably weaker than the association of glycemic control with the microvascular complications of diabetes, such as retinopathy and nephropathy (4). It must be recognized that epidemiological studies can only demonstrate associations and that confounding variables could account for the association between poor glycemic control and ASCVD. For example, patients with poor glycemic control may not undertake other preventive measures that could reduce ASCVD such as exercise, healthy diet, etc. Furthermore, the patients with poor glycemic control may have less compliance with therapies that reduce lipids and blood pressure. Therefore, randomized studies are essential in determining the role of glycemic control on ASCVD.

Early randomized studies, such as the University Group Diabetes Project (UGDP) and VA cooperative study, did not demonstrate a reduction in cardiovascular events in patients who were aggressively treated for glucose control (23-25). In fact, the data from these early studies suggested that improvements in glycemic control (VA cooperative study) or the use of certain drugs to treat diabetes (oral sulfonylureas in UGDP) may actually increase the risk of ASCVD.

Diabetes Control and Complications Trial (DCCT) and Kumamoto Studies

Latter studies, the DCCT in patients with T1DM and the Kumamoto study in patients with T2DM, while finding a decrease in ASCVD events (DCCT 41% decrease) in the subjects randomized to improved glycemic control did not have enough ASCVD events to demonstrate a statistically significant reduction (DCCT studied a population at low risk for ASCVD and the Kumamoto study had a very small number of subjects) (26-28). In contrast, both the DCCT and the Kumamoto study clearly demonstrated that improvements in glycemic control resulted in a reduction in microvascular disease (26-28). However, the long- term follow-up of the DCCT has demonstrated that those in the intensive glycemic control group had a decrease in ASCVD in subsequent years (29,30). The initial DCCT compared intensive vs. conventional therapy for a mean of 6.5 years. At the end of the study, a very large proportion of subjects agreed to participate in a follow-up observational study (Epidemiology of Diabetes Interventions and Complications- EDIC). During this follow-up period, glycemic control was relatively similar between the intensive therapy and conventional therapy group (glycosylated hemoglobin 7.9% vs. 7.8%) but during the actual trial there was a large difference in glycosylated hemoglobin levels (7.4% vs. 9.1%). After a mean 17 years of observation, the risk of any cardiovascular event was reduced by 42% and the risk of nonfatal myocardial infarction (MI), stroke, or death from ASCVD was reduced by 57% in the intensive control group. This study demonstrates that being in the intensive glycemic control group (for 6.5 of the 17 years of observation) is sufficient to have long-term beneficial effects on the risk of developing ASCVD in patients with T1DM. This beneficial effect was not entirely due to the prevention of microvascular complications as the differences between the intensive and conventional treatment groups for ASCVD persisted after adjusting for microalbuminuria and albuminuria. When an outcome of improved glycemic control is seen, or persists for years after the trial is over the phenomenon is called a “metabolic memory” effect.

UK Prospective Diabetes Study (UKPDS)

A similar finding has been reported with regard to T2DM. The UKPDS studied a large number of newly diagnosed patients with T2DM at risk for ASCVD. In this study improved glycemic control, with either insulin or sulfonylureas, reduced ASCVD by 16%, which just missed being statistically significant (p=0.052) (31). In the UKPDS, the improvement in glycemic control was modest (HbA1c reduced by approximately 0.9%) and the 16% reduction in ASCVD was in the range predicted based on epidemiological studies. The results of a 10-year follow-up of the UKPDS study have been reported (total duration of observation 25 years) (32). After termination of the study, glycosylated hemoglobin levels became very similar between the control and treatment groups. Nevertheless, risk reductions for MI became statistically significant for the insulin and the sulfonylurea group compared to controls (15% decrease, p=0.01).

DiGami Studies

Similarly, the DiGami study, which used insulin infusion during the peri-MI period to improve glycemic control followed by long-term glycemic control, demonstrated that survival post MI was significantly improved by good glycemic control (33). While this study focused on a highly-selected population and time period (patients undergoing a MI), the results are consistent with the hypothesis that improvements in glycemic control will reduce ASCVD. However, the DiGami 2 study did not confirm the benefits of tight glucose control beginning in the peri-MI period on outcomes (34). It must be noted though that the differences in glucose control achieved in DiGami 2 were much smaller than planned and the number of patients recruited was less than anticipated. Together these deficiencies could account for the failure to demonstrate significant differences in ASCVD events in this study.

ACCORD Study

Because of the need for more definitive data on the effect of glycemic control on ASCVD in T2DM, three large randomized trials, the ACCORD, ADVANCE, and VA Diabetes Trial, have been carried out. Much to everyone’s surprise and disappointment, improvement in glycemic control did not clearly result in a significant reduction in ASCVD in these trials.

The ACCORD study randomized 10,251 subjects with T2DM in the US and Canada with either a history of ASCVD or at increased risk for the development of ASCVD (35). Multiple different treatment protocols were used with the goal of achieving an A1c level < 6% in the intensive group and between 7-7.9% in the standard glycemic control group. During the trial the A1c levels were 6.4% in the intensive group and 7.5% in the standard group. As expected, the use of insulin therapy was much greater in the intensive group, as was the occurrence of hypoglycemia and weight gain. After a mean duration of 3.5 years this study was stopped early by the data safety monitoring board due to an increased all-cause mortality in the intensive treatment group (1.41% vs. 1.14% per year; hazard ratio 1.22 CI 1.01- 1.46). The primary outcome (MI, stroke, ASCVD death) was reduced by 10% in the intensive control group but this was not statistically significant (p=0.16). Of note, intensive glycemic control reduced the incidence of any MI (i.e. fatal or non-fatal) by 16%, nonfatal MI by 19%, coronary revascularization by 16%, and unstable angina by 19% (36).The explanation for the increased death rate in the intensive treatment arm remains unknown, but it has been speculated that the increased deaths might have been due to hypoglycemia, weight gain, too rapidly lowering A1c levels, or unrecognized drug toxicity. Long term follow-up of the ACCORD study did not reveal any beneficial effects on the primary outcome (nonfatal MI, nonfatal stroke, or cardiovascular death), death from any cause, and an expanded composite outcome that included all-cause death in the intensive glycemic control group (37).

ADVANCE Study

The ADVANCE study randomized 11,140 subjects with T2DM in Europe, Asia, Australia/New Zealand, and Canada who either had ASCVD or at least one other risk factor for ASCVD (38). In the intensive group the goal A1c was <6.5%. The achieved A1c levels during the trial were 6.3% in the intensive group and 7.3% in the standard treatment group. Of note is that compared to the ACCORD study, less insulin use was required to achieve these A1c levels. With regard to macrovascular disease (MI, stroke, and cardiovascular death), no significant differences were observed between the intensive and standard treatment groups (HR 0.94, CI 0.84-1.06, p=0.32). In contrast to the ACCORD trial, no increase in overall or cardiovascular mortality in the intensive treatment group was observed in the ADVANCE study. Long term follow-up did not demonstrate a decrease in the risk of death from any cause or major macrovascular events between the intensive-glucose-control group and the standard-glucose-control group (39).

VA Diabetes Trial

The VA Diabetes Trial randomized 1,791 subjects with poor glycemic control on maximal oral agent therapy or insulin (entry A1c 9.4%) (40). In the intensive group, the goal A1c was <6.0%. The achieved A1c levels during the trial were 6.9% in the intensive group and 8.5% in the standard treatment group. Similar to the other trials, a significant reduction in ASCVD was not observed in the intensive glycemic control group (HR 0.88, CI 0.74-1.05, p=0.12). Notably there were more ASCVD deaths and sudden deaths in the intensive treatment group, but this increase was not statistically significant. With long-term follow-up (approximately 10 years), the intensive-therapy group had a significantly lower risk of MI, stroke, congestive heart failure, amputation for ischemic gangrene, or cardiovascular-related death than did the standard-therapy group (hazard ratio, 0.83; P=0.04 (41). However, there was no reduction in cardiovascular or total mortality. Furthermore, with a longer period of follow-up (15 years) the risks of major cardiovascular events or death were not lower in the intensive-therapy group than in the standard-therapy group (42). In a careful analysis it was noted that that the risk of cardiovascular events was 17% lower in the intensive treatment group than in the standard control group during the approximate 10-year period when there was a separation of the glycated hemoglobin curves between the two groups, suggesting that glycemic control was reducing the risk of cardiovascular events (42).

Meta-analyses

In a meta-analysis of 6 randomized studies (UKPDS, Kumamoto, VA Feasibility study, ACCORD, ADVANCE, and VA Diabetes Trial) of intensive vs. conventional glycemic control in patients with T2DM (27,654 patients) there was no significant effect of tight blood glucose control on all-cause mortality (RR 1.03; 95% CI 0.90-1.17), cardiovascular mortality (RR 1.04; 95% CI 0.83-1.29), or nonfatal stroke (RR 1.02; 95% CI 0.88-1.17) but tight glucose control reduced the risk for nonfatal MI (RR 0.85; 95% CI 0.76-0.95) (43). In a meta-analysis of 4 studies (UKPDS, ACCORD, ADVANCE, and VA Diabetes Trial) the primary outcome was the composite of death from cardiovascular causes (including sudden death), non-fatal MI and non-fatal stroke, which was decreased by 9% (HR 0.91, 95% CI 0.84–0.99) in the intensive control group (44). Of note the risk of non-fatal/fatal MI was reduced by 15% (HR 0.85, 95% CI 0.76–0.94) in the intensive group without significant reductions in the risk of non-fatal/fatal stroke, fatal heart failure, all-cause mortality, or cardiovascular death.

Limitations of Cardiovascular Outcome Studies

Thus, while the epidemiological data strongly suggests that glycemic control would favorably impact ASCVD the recent randomized trials that were designed specifically to prove this hypothesis have failed to definitively demonstrate a clear link. There are several explanations for why these trials may not have worked as planned.

First, in the ACCORD, ADVANCE, and VA Diabetes Trial, other ASCVD risk factors were aggressively treated (lipid and BP lowering, ASA therapy). As a result of these treatments, the actual number of ASCVD events was considerably less than expected in these trials. The lower event rate may have reduced the ability to see a beneficial effect of glucose control. Additionally, the beneficial effects of glucose control maybe more robust if other risk factors are not aggressively controlled. In this regard, it is worth noting that in the earlier UKPDS, which showed that improved glycemic control reduced ASCVD events, both BP and lipids were not aggressively treated by current standards (systolic BP 135-140mm Hg, LDL-C 135-142mg/dL), which could be why this older trial demonstrated a benefit of improving glycemic control on ASCVD.

Second, these three recent trials were comparing relatively low A1c levels in both the intensive and usual control groups (A1c in intensive from ~6.4-6.9% and usual control group from ~7.0-8.4%). It is likely that both levels are on the “flatter” portion of the glycemic control-cardiovascular risk curve and that if one compared patients with intensive glycemic control with a control group with higher A1c values one would see more impressive results. If the difference in A1c levels were greater in the intensive and control groups the likelihood of seeing a reduction in cardiovascular events in the intensive group would be enhanced.

Third, all three trials were carried out by initiating tight control in patients with long standing diabetes who either had pre-existing ASCVD or were at high risk for ASCVD. It is possible that patients with a different clinical profile would be more likely to benefit from intensive glucose control. Subgroup analysis from these trials have suggested that patients with a shorter duration of diabetes, less severe diabetes, or the absence of pre-existing ASCVD actually benefited from intensive control. It may be that glycemic control is most important prior to the development of significant atherosclerosis. Clearly additional studies on different types of patients (i.e., newly diagnosed without evidence of ASCVD) will be necessary to definitively determine the role of glycemic control in different diabetic populations.

Fourth, the duration of these studies was relatively short and it is possible that a much longer duration of glycemic control is required to show benefits on ASCVD. In the UKPDS study the beneficial effects of intensive glucose control was not statistically significant at the end of the study but with an extended duration of follow-up (15-25 years) became statistically significant.

Fifth, it may be that glycemic control will be more important in patients with T1DM where abnormalities in glucose metabolism are a major reason for the increased risk of ASCVD. In contrast, patients with T2DM have multiple risk factors for ASCVD (dyslipidemia, hypertension, inflammation, insulin resistance, coagulation disorders, etc.) and glucose may play only a minor role in the increased risk. The differences in other cardiovascular risk factors could account for why intensive glycemic control produced a marked reduction in ASCVD in the DCCT (T1DM trial) and had only minimal effects in the trials carried out in patients with T2DM.

Finally, it is possible that certain treatments have side effects that mask the beneficial effects of glucose control. For example, hypoglycemia and weight gain could counterbalance the beneficial effects of improvements in glycemic control. It is possible that different treatment strategies could lead to more profound benefits (see below).

Summary

Thus, the currently available data do not definitively indicate that glycemic control will have major effects on reducing ASCVD in patients with T2DM. Furthermore, there are concerns that too tight control in patients with advanced disease could be harmful. In contrast, in patients with T1DM intensive glucose control appears to have a major beneficial effect on ASCVD based on the results of the DCCT.

THE EFFECT OF GLUCOSE LOWERING DRUGS ON ASCVD

Metformin

In the UKPDS, metformin, while producing a similar improvement in glycemic control as insulin or sulfonylureas, markedly reduced ASCVD by approximately 40% (45). In the ten-year follow-up the patients randomized to metformin in the UKPDS continued to show a reduction in MI and all-cause mortality (32). Two other randomized controlled trials have also demonstrated cardiovascular benefits with metformin therapy.

A study by Kooy et al compared the effect of adding metformin or placebo in overweight or obese patients already on insulin therapy (46). After a mean follow-up of 4.3 years this study observed a reduction in macrovascular events (HR 0.61 CI- 0.40-0.94, p=0.02), which was partially accounted for by metformin’s beneficial effects on weight. In this study the difference in A1c between the metformin and placebo group was only 0.3%.

Hong et al randomized non-obese patients with coronary artery disease to glipizide vs. metformin therapy for three years (47). A1c levels were similar, but there was a marked reduction in cardiovascular events in the metformin treated group (HR 0.54 CI 0.30- 0.90, p=0.026).

In contrast, long term follow-up (21 years) of individuals in the Diabetes Prevention Program with “pre-diabetes” did not demonstrate a reduction in cardiovascular event in individuals treated with metformin (48). A reduction in the use of metformin when the formal study ended after 3 years coupled with out-of-study metformin use over time may have diluted the potential effects of metformin therapy.

Support for the beneficial effects of metformin on atherosclerosis comes from long term follow-up of the Diabetes Prevention Program, which compared the effect of lifestyle changes or metformin in patients at high risk of developing diabetes (49). Coronary artery calcium scores were measured on average 13-14 years after randomization (49). There were no differences in coronary artery calcium scores between the lifestyle and placebo groups. However, in males, coronary artery calcium scores were significantly lower in the metformin group vs. the placebo group. In females treated with metformin coronary artery calcium scores were similar to the placebo group. The absence of a beneficial effect of metformin in women could be due to the lower baseline coronary artery calcium scores making it more difficult to demonstrate a beneficial effect. In HIV-infected patients with the metabolic syndrome metformin similarly reduced the progression of coronary artery calcium scores (50).

Thus, while there are no large cardiovascular outcome trials with metformin, together, the above results suggest that metformin may reduce ASCVD and that this effect is not due to improving glucose control. Metformin decreases weight or prevents weight gain and lowers lipid levels and these or other non-glucose effects may account for the beneficial effects on ASCVD.

Sulfonylureas

Based on the University Group Diabetes Project (UGDP) sulfonylureas carry a warning regarding an increased risk of ASCVD (24,25). However, the UKPDS studied a large number of newly diagnosed patients with T2DM at risk for ASCVD and in this study improved glycemic control with sulfonylureas reduced ASCVD by approximately 16%, which just missed being statistically significant (p=0.052) (31). In the UKPDS, A1c was reduced by approximately 0.9% and the 16% reduction in ASCVD was in the range predicted based on epidemiological studies. Thus, the reduction in cardiovascular events was likely due to improvements in glycemic control and not a direct benefit of sulfonylurea treatment. In support of this conjecture is that in the UKPDS, insulin treatment resulted in a similar decrease in A1c levels and reduction in cardiovascular events (31). Additionally, a large randomized cardiovascular outcome study (Carolina Study) reported that linagliptin, a DPP-4 inhibitor, and glimepiride, a sulfonylurea, had similar effects on cardiovascular events (hazard ratio 0.98) (51). Similarly, in the ADVANCE trial patients in the intensive therapy group were randomized to gliclazide and as noted above the occurrence of cardiovascular events was similar to the control group (38).Taken together these results suggest that sulfonylureas have a neutral effect on ASCVD.

Meglitinides

The Navigator study was a double-blind, randomized clinical trial in 9,306 individuals with impaired glucose tolerance and either ASCVD or cardiovascular risk factors who received nateglinide (up to 60 mg three times daily) or placebo (52). After 5 years, nateglinide administration did not alter the incidence of cardiovascular outcomes suggesting that meglitinides do not have an adverse or beneficial effect on cardiovascular events.

Thiazolidinediones

Studies with pioglitazone have suggested a beneficial effect on ASCVD. The PROactive study was a randomized controlled trial that examined the effect of pioglitazone vs. placebo over a 3-year period in T2DM with pre-existing macrovascular disease (53). With regard to the primary endpoint (a composite of all-cause mortality, non-fatal MI including silent MI, stroke, acute coronary syndrome, endovascular or surgical intervention in the coronary or leg arteries, and amputation above the ankle), there was a 10% reduction in events in the pioglitazone group but this difference was not statistically significant (p=0.095). It should be noted that both leg revascularization and leg amputations are not typical primary end points in ASCVD trials and these could be affected by pioglitazone induced edema. When one focuses on standard ASCVD endpoints, the pioglitazone treated group did demonstrate a 16% reduction in the main secondary endpoint (composite of all-cause mortality, non-fatal MI, and stroke) that was statistically significant (p=0.027). In the pioglitazone treated group, blood pressure, A1c, triglyceride, and HDL-C levels were all improved compared to the placebo group making it very likely that the mechanism by which pioglitazone decreased vascular events was multifactorial.

A multicenter, double-blind trial (IRIS Trial), randomly assigned 3,876 patients with insulin resistance (defined as score of more than 3.0 on the homeostasis model assessment of insulin resistance [HOMA-IR] index) but without diabetes and a recent ischemic stroke or TIA to treatment with either pioglitazone (target dose, 45 mg daily) or placebo (54). After 4.8 years, the primary outcome of fatal or nonfatal stroke or MI occurred in 9.0% of the pioglitazone group and 11.8% of the placebo group (hazard ratio 0.76; P=0.007). All components of the primary outcome were reduced in the pioglitazone treated group. Fasting glucose, fasting TG, and systolic and diastolic blood pressure were lower while HDL-C and LDL-C levels were higher in the pioglitazone group than in the placebo group. Although this study excluded patients with diabetes the results are consistent with and support the results of a protective effect of pioglitazone observed in the PROactive study.

In contrast, the TOSCA.IT study compared the effect of pioglitazone vs. sulfonylurea on ASCVD and did not observe a reduction in events with pioglitazone treatment (55). Patients with T2DM (n= 3028), inadequately controlled with metformin monotherapy (2-3 g per day), were randomized to pioglitazone or sulfonylurea and followed for a median of 57 months. Only 11% of the participants had a previous cardiovascular event. The primary outcome, was a composite of first occurrence of all-cause death, non-fatal MI, non-fatal stroke, or urgent coronary revascularization and occurred in 6.8% of the patients treated with pioglitazone and 7.2% of the patients treated with a sulfonylurea (HR 0.96; NS) (55). Limitations of this study are the small number of events due to the low-risk population studied and the relatively small number of participants. Additionally, 28% of the subjects randomized to pioglitazone prematurely discontinued the medication. Furthermore, it should be noted that when patients in this study were analyzed based on the risk of developing ASCVD those at high risk had a marked reduction in events when treated with pioglitazone compared to the sulfonylurea (56).Thus, the results of this study should be interpreted with caution.

Further support for the beneficial effects of pioglitazone on atherosclerosis is provided by studies that have examined the effect of pioglitazone on carotid intima-medial thickness. Both the Chicago and Pioneer studies demonstrated favorable effects on carotid intima-medial thickness in patients treated with pioglitazone compared to patients treated with sulfonylureas (57,58). Similarly, Periscope, a study that measured atheroma volume in the coronary arteries by intravascular ultrasonography, also demonstrated less atherosclerosis in the pioglitazone treated group compared to patients treated with sulfonylureas (59).

While the data from a variety of different types of studies strongly suggests that pioglitazone is anti-atherogenic, the results with rosiglitazone are different. Several meta-analyses of small and short-duration rosiglitazone trials suggested that rosiglitazone was associated with an increased risk of adverse cardiovascular outcomes (60,61). However, the final results of the RECORD study, a randomized trial that was specifically designed to compare the effect of rosiglitazone vs. either metformin or sulfonylurea therapy as a second oral drug in those receiving either metformin or a sulfonylurea on ASCVD events, have been published and did not reveal a difference in ASCVD death, MI, or stroke (62-64). Similarly, an analysis of patients on rosiglitazone in the BARI 2D trial also did not suggest an increase or decrease in ASCVD events in the patients treated with rosiglitazone (65). Thus, while the available data suggests that pioglitazone is anti-atherogenic, the data for rosiglitazone suggests a neutral effect. Whether these differences between pioglitazone and rosiglitazone are accounted for by their differential effects on lipid levels are unknown (see below for information on the effects of these drugs on lipid levels).

Numerous studies have shown that both pioglitazone and rosiglitazone increase the risk of heart failure (66).

DPP4 Inhibitors

Because of the importance of ASCVD in patients with diabetes the FDA is requiring manufacturers of new drugs to treat diabetes to carry out studies addressing ASCVD endpoints. The effect of the DPP4 inhibitors saxagliptin, alogliptin, sitagliptin, and linagliptin on ASCVD endpoints has been reported. In the saxagliptin study (SAVOR‐TIMI 53 trial), 16,492 patients with T2DM who had a history of cardiovascular events or who were at high risk were randomized to saxagliptin or placebo for 2.1 years (67). Saxagliptin did not increase or decrease cardiovascular death, MI, or ischemic stroke. Interestingly more patients treated with saxagliptin were admitted to the hospital for heart failure. The risk of heart failure with saxagliptin was greatest in patients at a high overall risk of heart failure (i.e., history of heart failure, impaired renal function, or elevated baseline levels of NT-proBNP) (68). Additionally, in the patients treated with saxagliptin the increase in heart failure was an early event with a 6-month rate of 1.1% vs. 0.6% in the placebo group (HR 1.80, p=0·001) and a 12-month rate of 1·9% vs. 1·3% (1.46; p=0.002) (68). In contrast, after 12 months no difference in the rate of heart failure was observed in the saxagliptin and placebo groups indicating that the development of heart failure is an early event (68).

In the alogliptin trial (EXAMINE), 5,380 patients with either an acute MI or unstable angina within the previous 15-90 days were randomized to alogliptin or placebo and followed for a median of 18 months (69). As seen in the saxagliptin study the rates of ASCVD events were similar in the alogliptin and placebo groups. The risk of hospitalization for heart failure was not statistically increased in the entire subset of patients treated with alogliptin (70). However, the hazard ratio for the subgroup of patients without heart failure at baseline was 1.76, p=0.026) (70).

In the sitagliptin trial (TECOS), 14,671 patients with established ASCVD were randomized to sitagliptin or placebo for 3 years (71). Sitagliptin did not decrease the risk of major adverse cardiovascular events or increase hospitalization for heart failure. Finally, in the linagliptin trial (CARMELINA), 6,979 patients at high risk for ASCVD were randomized to linagliptin or placebo for a median follow-up of 2.2 years (72). As in the other DPP4 inhibitor studies, linagliptin did not have a beneficial effect on ASCVD events. Additionally, linagliptin did not increase the risk of hospitalization for heart failure. Thus, these results indicate that DPP4 inhibitors do not increase or decrease ASCVD. The extent to which specific DPP4 inhibitors affect heart failure needs further investigation.

SGLT2 Inhibitors

EMPA-REG OUTCOME TRIAL

The effects of empagliflozin on cardiovascular morbidity and mortality in patients with T2DM has been reported (73). In this study, 7,020 patients at high risk for ASCVD were randomly assigned to receive 10 mg or 25 mg of empagliflozin or placebo once daily and were followed for 3.1 years. In the combined empagliflozin treated groups there was a statistically significant 14% reduction in the primary outcome (death from cardiovascular causes, nonfatal MI, or nonfatal stroke). As compared with placebo, empagliflozin treatment did not result in a significant difference in the occurrence of non-fatal MI or strokes. However, empagliflozin resulted in a significantly lower risk of death from cardiovascular causes (hazard ratio, 0.62), death from any cause (hazard ratio, 0.68), and hospitalization for heart failure (hazard ratio, 0.65). The beneficial effects of empagliflozin were noted to occur very rapidly and the beneficial effects on heart failure appeared to be the dominant effect compared to effects on ASCVD events. Decreases in cardiovascular outcomes and mortality with empagliflozin occurred across the range of cardiovascular risk (74). Additionally, the reduction in hospitalizations for heart failure and cardiovascular death were observed both in patients with and without heart failure at baseline (75).

CANVAS TRIAL

The effects of placebo vs. canagliflozin were determined in two combined trials involving a total of 10,142 participants with T2DM and high cardiovascular risk (76). The primary outcome was a composite of death from cardiovascular causes, nonfatal MI, or nonfatal stroke and the mean follow-up was 188 weeks. The primary outcome was reduced in the canagliflozin group (hazard ratio, 0.86; P=0.02). Death from any cause (hazard ratio 0.87; 95% CI 0.74-1.01) and death from ASCVD (hazard ratio 0.87; 95% CI 0.72-1.06) were reduced but were not statistically significant. Similarly, canagliflozin treatment did not result in a significant difference in non-fatal strokes or non-fatal MI (hazard ratio 0.90 for stroke and 0.85 for myocardial infarction). As seen with empagliflozin, hospitalization for heart failure was markedly reduced (hazard ratio 0.67; 95% CI 0.52-0.87) and this beneficial effect occurred rapidly. Notably, there was an increased risk of amputation (hazard ratio, 1.97; 95% CI, 1.41 to 2.75), which were primarily at the level of the toe or metatarsal. The basis for the increase in amputations is unknown. In other SGLT2 inhibitor studies an increase in amputations was not noted.

CREDENCE TRIAL

In a second canagliflozin trial that focused on kidney disease, a decrease in cardiovascular events was also observed (77). In this double-blind trial 4,401 patients with chronic kidney disease and T2DM were randomized to canagliflozin 100mg per day or placebo and followed for a median of 2.62 years. All the patients had an eGFR of 30 to <90 ml per minute per 1.73 m²and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000). In this trial hospitalization for heart failure was reduced by 39%. The relative benefits of canagliflozin for cardiovascular outcomes was similar in individuals across the spectrum of eGFR levels (78). In contrast to the CANVAS trial, an increased risk of amputations was not observed.

DECLARE–TIMI 58 TRIAL

The effect of dapagliflozin on cardiovascular events has also been reported (79). 17,160 patients, including 10,186 without ASCVD were randomized to dapagliflozin or placebo and followed for a median of 4.2 years. The primary outcome was a composite of major adverse cardiovascular events (MACE), defined as cardiovascular death, MI, or ischemic stroke. The primary efficacy outcomes were MACE and a composite of cardiovascular death or hospitalization for heart failure. Dapagliflozin did not result in a lower rate of major adverse cardiovascular events (8.8% in the dapagliflozin group and 9.4% in the placebo group; hazard ratio, 0.93; P=0.17) but did result in a lower rate of cardiovascular death or hospitalization for heart failure (4.9% vs. 5.8%; hazard ratio, 0.83; P=0.005), which reflected a lower rate of hospitalization for heart failure (hazard ratio, 0.73; 95% CI, 0.61 to 0.88). Interestingly, in the patients with a history of a previous MI, dapagliflozin reduced the risk of a MACE (HR 0.84; P=0.039), whereas there was no effect in patients without a previous MI (80). Additionally, there was no increase in lower extremities amputations in the dapagliflozin treated group.

VERTIS CV

Patients with ASCVD and T2DM were randomized to ertugliflozin 5mg (n=2752), 15mg (2747), or placebo (n=2747) and the primary composite outcome of cardiovascular death and non-fatal MI or stroke was determined after a mean duration of follow-up of 3.5 years (81). This trial did not demonstrate a significant difference in the primary endpoint (MACE) nor any components of the primary endpoint. However, heart failure hospitalizations were significantly reduced by 30% in the patients treated with ertugliflozin (HR 0.70; CI 0.54–0.90). The benefits on heart failure were observed in both patients with a history of heart failure (decreased 37%) and patients without a history of heart failure (decreased 21%) (82).

SUMMARY

Thus, all four SGLT2 inhibitor studies demonstrated a decrease in heart failure with SGLT2 inhibitor therapy without consistent effects on ASCVD events. For additional information on the beneficial effects of SGLT2 inhibitors and SGLT1/SGLT2 inhibitors on ASCVD and heart failure see the Endotext chapter entitled “Oral and Injectable (Non-Insulin) Pharmacological Agents for the Treatment of Type 2 Diabetes” (83).

GLP-1 Receptor Agonists

The effect of six GLP-1 receptor agonists on ASCVD has been reported.

ELIXA

In the ELIXA trial 6,068 patients with T2DM who recently had a MI or been hospitalized for unstable angina were randomized to placebo or lixisenatide and followed for a median of 25 months (84). The primary end point of cardiovascular death, MI, stroke, or hospitalization for unstable angina was similar in the placebo or lixisenatide groups.

LEADER TRIAL

In contrast, the LEADER trial has shown that liraglutide decreased cardiovascular events (85). In this trial 9,340 patients at high cardiovascular risk were randomly assigned to receive liraglutide or placebo. After a median time of 3.5 years, the primary outcome of death from cardiovascular causes, nonfatal MI, or nonfatal stroke occurred in significantly fewer patients in the liraglutide group (13.0%) than in the placebo group (14.9%) (hazard ratio, 0.87, P=0.01). Additionally, deaths from cardiovascular causes (hazard ratio 0.78, P=0.007) or any cause was lower in the liraglutide group than in the placebo group (hazard ratio, 0.85; P=0.02). Interestingly patients with established ASCVD or decreased renal function (eGFR < 60) appeared to derive the greatest benefit of liraglutide treatment (86,87). As expected, weight and blood pressure were decreased in the liraglutide treated group and A1c levels were also decreased by 0.4%.

SUSTAIN 6 TRIAL

In support of the beneficial effects of GLP1 receptor agonists to reduce cardiovascular events, semaglutide, a long acting GLP-1 receptor agonist, has been shown to also reduce cardiovascular events (88). In this trial, 3,297 patients with T2DM with established ASCVD, chronic heart failure, chronic kidney disease, or age >60 with at least one cardiovascular risk factor were randomized to receive once-weekly semaglutide (0.5 mg or 1.0 mg) or placebo for 104 weeks. The primary outcome of cardiovascular death, nonfatal MI, or nonfatal stroke occurred in 6.6% of the semaglutide group and 8.9% of the placebo group (hazard ratio, 0.74; P = 0.02). In this study, both body weight and A1c levels were decreased in the patients treated with semaglutide.

PIONEER 6

In the PIONEER 6 study 3,183 patients with T2DM at high cardiovascular risk (age ≥50 years with established cardiovascular or chronic kidney disease, or age ≥60 years with cardiovascular risk factors) were randomly assigned to receive oral semaglutide or placebo (89). After a median time of 15.9 months, major adverse cardiovascular events, the primary outcome, occurred in 3.8% of the subjects treated with oral semaglutide and 4.8% of the placebo group (HR 0.79; 95% CI 0.57 to 1.11). Deaths from cardiovascular causes were 0.9% in the oral semaglutide group and 1.9% in the placebo group (HR 0.49; 95% CI, 0.27 to 0.92) while death from any cause occurred in 1.4% in the oral semaglutide group and 2.8% in the placebo group (HR 0.51; 95% CI, 0.31 to 0.84). It should be noted that the primary outcome was not statistically decreased in this study, which may be due to the relatively small number of subjects studied and the short duration of the study that together resulted in a small number of events. Additionally, more patients in the placebo group received treatment with an SGLT2 inhibitor than in the oral semaglutide group and SGLT2 inhibitors are well recognized to reduce cardiovascular events, which could also have diminished the ability to observe a decrease in events in the oral semaglutide group. Because the direction of change in cardiovascular events in PIONEER 6 and glucose lowering, weight loss, and many other effects of oral semaglutide are very similar to injected semaglutide many experts consider the effects on cardiovascular to also be similar.

EXSCEL TRIAL

The effect of once weekly exenatide vs. placebo on cardiovascular outcomes was tested in 14,752 patients, 73% who had ASCVD (90). The primary outcome was the occurrence of death from cardiovascular causes, nonfatal MI, or nonfatal stroke. After a median follow-up of 3.2 years (duration of drug exposure 2.4 years) the primary outcome was reduced in the exenatide treated group but this difference just missed achieving statistical significance (hazard ratio 0.91; 95% CI 0.83-1.00; p=0.06). While not statistically significant these results are consistent with the results observed with liraglutide and semaglutide treatment. It should be recognized that a high percentage of patients discontinued exenatide therapy in this trial (>40%) and this could have adversely affected the ability of exenatide treatment to favorably effect ASCD outcomes.

HARMONY OUTCOMES TRIAL

The effect of once weekly albiglutide vs. placebo was tested in 9,463 patients with ASCVD (91). The primary outcome was first occurrence of cardiovascular death, MI, or stroke. After a median follow-up of 1.6 years a 22% decrease in the primary endpoint was observed in the albiglutide group (hazard ratio 0·78, p<0·0001). It should be noted that albiglutide is no longer available as it was removed from the market due to commercial considerations by Glaxo.

REWIND TRIAL

REWIND was a randomized study of weekly subcutaneous injection of dulaglutide (1.5 mg) or placebo in 9,901 patients with T2DM who had either a previous cardiovascular event or cardiovascular risk factors (approximately 70% of patients did not have prior ASCVD) (92). During a median follow-up of 5.4 years the primary outcome of non-fatal MI, non-fatal stroke, or death from cardiovascular causes was decreased by 12% in the dulaglutide treated group (HR 0.88, p=0.026). The decrease in events was similar in participants with and without previous ASCVD. In an analysis that focused on stroke it was noted that dulaglutide reduced ischemic stroke by 25% compared to placebo but had no effect on hemorrhagic stroke (93).

SUMMARY

Thus, four studies have clearly demonstrated that treatment with GLP-1 receptor agonists reduces cardiovascular events, two studies has provided data consistent with these results, and one study failed to demonstrate benefit (Table 1). In a meta-analysis of these seven trials it was observed that cardiovascular death, stroke, or MI was decreased by 12% (HR 0.88, p<0.0001), death from cardiovascular causes by 12% (HR 0.88, p=0.003), fatal or non-fatal stroke by 16% (HR 0.84, p<0.0001) and fatal and non-fatal MI by 9% (HR 0.91, p=0.043) (Table 1) (94). Why there are differences in results between these studies is unknown but could be due to differential effects of the GLP-1 receptor agonists, differences in the patient populations studied, or other unrecognized variables. For additional information on the beneficial effects of GLP-1 receptor agonists on ASCVD see the Endotext chapter entitled “Oral and Injectable (Non-Insulin) Pharmacological Agents for the Treatment of Type 2 Diabetes” (83).

Table 1. Summary of GLP-1 Receptor Agonist Cardiovascular Outcome Trials
	Number	Prior CVD	HbA1c	Mean Follow-up (years)	*Hazard Ratio (95% CI)**	P value
ELIXA Lixisenatide	6068	100%	7.7%	2.1	1.02 (0.89-1.17)	0.78
LEADER Liraglutide	9340	81%	8.7%	3.8	0.87 (0.78-0.97)	0.015
SUSTAIN 6 Semaglutide	3297	83%	8.7%	2.1	0.74 (0.58-0.95)	0.016
EXSCEL Exenatide	14,752	73%	8.0%	3.2	0.91 (0.83-1.00)	0.061
HARMONY Albiglutide	9463	100%	8.7%	1.6	0.78 (0.68-0.90)	<0.001
REWIND Dulaglutide	9901	31%	7.3%	5.4	0>88 (0.79-0.99)	0.026
PIONEER 6** Semaglutide oral	3183	85%	8.2%	1.3	0.79 (0.57-1.11)	0.17
Overall (94)					0.88 (0.82-0.94)	<0.001

*CVD death, MI, Stroke.

The mechanism accounting for this decrease in ASCVD is uncertain but could be related to reductions in HbA1c, body weight, systolic blood pressure, postprandial triglyceride levels, or the direct effect of activation of GLP-1 receptors on the atherosclerotic process such as improving endothelial function (95).

Tirzepatide

Tirzepatide activates both GLP-1 and GIP receptors. Long term cardiovascular trials with tirzepatide are underway. In a meta-analysis of seven randomized controlled trials with a duration of at least 26 weeks with 4,887 participants treated with tirzepatide and 2,328 control participants a 20% decrease in cardiovascular events was observed in the tirzepatide group (HR 0.80; 95% CI 0.57-1.11) suggesting that the effect of tirzepatide will be similar to the GLP-1 receptor agonists (96).

Acarbose

In the STOP-NIDDM trial 1,429 subjects with impaired glucose tolerance were randomized to placebo vs. acarbose and followed for 3.3 years (97). In the acarbose group a 49% relative risk reduction in the development of ASCVD events (hazard ratio 0.51; P =0.03) was observed. Among cardiovascular events, the major reduction was in the risk of MI (HR, 0.09; P =.02). In a smaller trial, 135 patients hospitalized for the acute coronary syndrome who were newly diagnosed with IGT were randomly assigned to acarbose or placebo (98). During a mean follow-up of 2.3 years the risk of recurrent major adverse cardiovascular event was decreased significantly in the acarbose group compared with that in control group (26.7% versus 46.9%, P < 0.05).

Despite these favorable observations a large trial failed to demonstrate a beneficial effect of acarbose in Chinese patients with impaired glucose tolerance (99). In a randomized trial acarbose vs. placebo was compared in 6,522 patients with coronary heart disease and impaired glucose tolerance. The primary outcome was cardiovascular death, non-fatal MI, non-fatal stroke, hospital admission for unstable angina, and hospital admission for heart failure and patients were followed for a median of 5 years. The primary outcome was similar in the acarbose and placebo groups (hazard ratio 0.98; 95% CI 0.86-1.11, p=0·73). No significant differences were seen for death from any cause, cardiovascular death, fatal or non-fatal MI, fatal or non-fatal stroke, hospital admission for unstable angina, hospital admission for heart failure, or impaired renal function.

Thus, whether acarbose favorably affects ASCVD in patients at high risk for developing diabetes is uncertain. Moreover, the effect of acarbose on ASCVD in patients with diabetes is unknown.

Cycloset

Cycloset is a quick-release bromocriptine formulation (bromocriptine-QR) that activates the D2 dopamine receptor and is approved for the treatment of diabetes. A 52 week, randomized, double-blind, multicenter trial evaluated cardiovascular safety in 3,095 patients with T2DM treated with bromocriptine-QR or placebo (100). The composite end point of first MI, stroke, coronary revascularization, or hospitalization for angina or congestive heart failure occurred in 1.8% of the bromocriptine-QR treated vs. 3.2% of the placebo-treated patients resulting in a 40% decrease in cardiovascular events (HR 0.60; CI 0.37– 0.96). Clearly further studies to confirm this finding and to elucidate the mechanism of this beneficial effect are required.

Bile Acid Sequestrants

Colesevelam is a non-absorbed, polymeric, LDL-C lowering and glucose lowering agent that is a high-capacity bile acid-binding molecule. This drug was developed primarily to lower LDL-C levels and was later noted to have favorable effects on blood glucose levels and was approved for improving glycemic control in patients with T2DM (101).

There have been no randomized studies that have examined the effect of bile acid sequestrants on cardiovascular end points in subjects with diabetes. In non-diabetic-subjects bile acid sequestrants have reduced cardiovascular events(102,103). Since bile acid sequestrants have a similar beneficial impact on LDL-C levels in diabetic and non-diabetic subjects one would anticipate that these drugs would also result in a reduction in events in the diabetic population.

Insulin

As described above in patients with T1DM the DCCT trial and in T2DM in the UKPDS trial demonstrated that insulin therapy reduced cardiovascular events by improving glycemic control (29-32). In the Origin Trial 12,537 people with cardiovascular risk factors plus impaired fasting glucose, impaired glucose tolerance, or T2DM were randomized to receive insulin glargine or standard care (104). The cardiovascular outcomes, which included nonfatal MI, nonfatal stroke, death from cardiovascular causes, revascularization, or hospitalization for heart failure, were similar in the glargine and placebo groups. Extended follow-up also did not demonstrate favorable effects on cardiovascular events in the glargine treated patients (105). Additionally, in patients with T2DM at high risk for cardiovascular events the occurrence of major cardiovascular events was similar in patients treated with degludec insulin or glargine insulin (106). These studies demonstrate that insulin does not accelerate atherosclerosis and by lowering glucose levels may decrease atherosclerosis, although the protective effects are mainly observed in patients with T1DM over a protracted period of time.

Other Studies

Finally, the Bari 2D study compared the effect of insulin sensitizers (metformin/TZD- mostly rosiglitazone) vs. insulin provision therapy (sulfonylureas/insulin) on cardiovascular outcomes in patients with T2DM and coronary artery disease (> 50% stenosis and positive stress test or > 70% stenosis and classic angina) (107,108). In this study, no differences in survival or cardiovascular endpoints were observed between metformin/TZD therapy vs. sulfonylurea/insulin therapy for the entire study. However, in the group with more severe coronary artery disease who were selected for coronary artery bypass surgery, the combination of coronary artery bypass and treatment with insulin sensitizers was associated with a lower rate of cardiovascular events. Why the metformin/TZD group only derived an enhanced benefit in the coronary artery bypass patients in this study is unknown. It should be noted that the vast majority of patients on TZD therapy were treated with rosiglitazone and, as discussed above, the effects of rosiglitazone on ASCVD do not appear to be as beneficial as pioglitazone.

Summary

These studies clearly demonstrate that the method by which one improves glycemic control may be very important with different drugs having effects in addition to glucose lowering that reduce cardiovascular events (table 2). While previous treatment algorithms have primarily focused on the effect of drugs on glycemic control, current treatment recommendations for patients with diabetes are using the results of these ASCVD trials to decide which drugs should be employed. For example, the ADA is recommending that in patients with high risk or established ASCVD an SGLT inhibitor or GLP1 receptor agonist with proven cardiovascular benefit should be part of the initial treatment regimen independent of A1c levels (109).

Table 2. Effect of Glucose Lowering Drugs on Atherosclerotic ASCVD
Metformin	Studies suggest benefit
Sulfonylureas	No effect
Meglitinides	No effect
Thiazolidinediones	Rosiglitazone no effect; Pioglitazone- studies suggest benefit
DPP4 Inhibitors	No effect on atherosclerosis.
SGLT2 Inhibitors	Marginal effect on ASCVD, Large effect on heart failure
GLP-1 Receptor Agonists	Decrease events
Tirzepatide	Study ongoing
Acarbose	No effect
Cycloset	Further studies required
Bile Acid Sequestrants	Decrease events, further studies required
Insulin	No effect

Thiazolidinediones clearly increase the risk of heart failure while saxagliptin and alogliptin may increase risk of heart failure. SGLT2 inhibitors decrease the risk of heart failure.

ROLE OF OTHER RISK FACTORS IN ASCVD

ROLE OF LIPIDS IN ASCVD

LIPID ABNORMALITIES IN PATIENTS WITH DIABETES

Table 3. Lipid Abnormalities in Patients with Diabetes
T1DM	Lipid profile is similar to controls if glycemic control is good
T2DM	Increased TG, VLDL, IDL, and non-HDL-C. Decreased HDL-C. Normal LDL-C but increase in small dense LDL, LDL particle number, and apolipoprotein B.
Poor glycemic control	Increased TG, VLDL, IDL, and non-HDL-C. Decreased HDL-C. Modest increase in LDL-C with increase in small dense LDL, LDL particle number, and apolipoprotein B.

EFFECT OF GLUCOSE LOWERING DRUGS ON LIPIDS

Table 4. Effect of Glucose Lowering Drugs on Lipid Levels
Metformin	Modestly decrease TG and LDL-C
Sulfonylureas	No effect
DPP4 inhibitors	Decrease postprandial TG
GLP1 analogues	Decrease fasting and postprandial TG, modestly decrease LDL-C
Tirzepatide	Decrease TG, modestly decrease LDL-C, increase HDL-C
Acarbose	Decrease postprandial TG
Pioglitazone Rosiglitazone	Decrease TG and increase HDL-C. Small increase LDL-C but a decrease in small dense LDL
SGLT2 inhibitors	Small increase in LDL-C and HDL-C
Colesevelam	Decrease LDL-C. May increase TG
Bromocriptine-QR	Decrease TG
Insulin	No effect

PATHOPHYSIOLOGY OF THE DYSLIPIDEMIA OF DIABETES

Figure 1. Pathophysiology of the Dyslipidemia of Diabetes

Increase in TG

There are a number of different abnormalities that contribute to the dyslipidemia seen in patients with T2DM and obesity (figure 1) (119-122,144-146).

OVERPRODUCTION OF VLDL BY THE LIVER

DECREASED DEGRADATION OF TRIGLYCERIDE RICH LIPOPROTEINS

Mechanism for the Increase in Small Dense LDL and Decrease in HDL

Role of Poor Glycemic Control

Role of Obesity and Inflammation

Role of Adipokines

EFFECT OF LIPID LOWERING ON ASCVD EVENTS IN PATIENTS WITH DIABETES

Monotherapy Studies

STATINS

Effect of Aggressive LDL-C Lowering with Statins

FIBRATES

The beneficial effect of monotherapy with fibrates (e.g., gemfibrozil, fenofibrate) on ASCVD in patients with diabetes is shown in Table 5. The results of these randomized trials suggest that monotherapy with this class of drug might reduce cardiovascular events in patients with diabetes, but the data is not very robust. The largest trial was the Field Trial (176). In this trial, 9,795 patients with T2DM between the ages of 50 and 75 not taking statin therapy were randomized to fenofibrate or placebo and followed for approximately 5 years. Fenofibrate therapy resulted in a 12% decrease in LDL-C, a 29% decrease in TG, and a 5% increase in HDL-C levels. The primary outcome was coronary events (coronary heart disease death and non-fatal MI), which were reduced by 11% in the fenofibrate group but did not reach statistical significance (p= 0.16). However, there was a 24% decrease in non-fatal MI in the fenofibrate treated group (p=0.01) and a non-significant increase in coronary heart disease mortality. Total ASCVD events (coronary events plus stroke and coronary or carotid revascularization) were reduced 11% (p=0.035). These beneficial effects of fenofibrate therapy on ASCVD were observed in patients without a previous history of ASCVD. In patients with a previous history of ASCVD no benefits were observed. Additionally, the beneficial effect of fenofibrate therapy was seen only in those subjects less than 65 years of age. The beneficial effects of fenofibrate in this study may have been muted by the increased use of statins in the placebo group, which reduced the differences in lipid levels between the placebo and fenofibrate groups. If one adjusted for the addition of lipid-lowering therapy, fenofibrate reduced the risk of coronary heart disease events by 19% (p=0.01) and of total ASCVD events by 15% (p=0.004). Thus, while the results of this large trial are intriguing they do not clearly show a benefit of fibrate therapy reducing ASCVD events. The number of patients with diabetes in the other fibrate trials are relatively small (table 4).

Table 5. Effect of Fibrate Monotherapy on Cardiovascular Outcomes
Study	Drug	#Diabetic subjects	%Decrease controls	% Decrease diabetics
Helsinki Heart Study (181)	Gemfibrozil	135	34	60*
VA-HIT (180)	Gemfibrozil	620	24	24
DIAS (182)	Fenofibrate	418	-	23*
Sendcap (183)	Bezafibrate	164	-	70
Field (176)	Fenofibrate	9795	-	11*

* Not statistically significant

NIACIN

EZETIMIBE

BEMPEDOIC ACID

OTHER DRUGS

Combination Therapy

STATINS + FIBRATES

STATIN + NIACIN

STATIN + EZETIMIBE

STATIN + PCSK9 INHIBITORS

STATINS + LOW DOSE OMEGA-3-FATTY ACIDS

STATINS + HIGH DOSE OMEGA-3-FATTY ACIDS

CURRENT GUIDELINES FOR SERUM LIPIDS

There are several different guidelines for treating lipids in patients with diabetes. While they all focus on lowering LDL-C there are differences between the various guidelines.

American Diabetes Association Guidelines

The 2023 American Diabetes Association (ADA) recommends that adult patients with diabetes have their lipid profile determined at the time of diabetes diagnosis and at least every 5 years thereafter or more frequently if indicated (219). This profile includes total cholesterol, HDL-C, TG, and calculated LDL-C. A lipid panel should be obtained immediately prior to initiating statin therapy. Once a patient is on statin therapy testing should be carried out 4-12 weeks after initiating therapy and annually thereafter to monitor adherence and efficacy. Lifestyle modifications including a reduction in saturated fat, trans fat, and cholesterol intake, weight loss if indicated, an increase in omega-3-fatty acids, viscous fiber, and plant stanols /sterol intake, and increased physical activity is indicated in all patients with diabetes. A focus on a Mediterranean style diet or Dietary Approaches to Stop Hypertension (DASH) diet should be encouraged. In patients with elevated TG levels glycemic control is beneficial and dietary changes and lifestyle changes including weight loss and abstinence from alcohol should be undertaken. Secondary disorders and medications that raise TG levels should be evaluated. Optimize glycemic control to improve TG and HDL-C levels. The recommendations for lipid lowering therapy are shown in table 6. If one follows these recommendations almost all patients with diabetes over the age of 40 will be on statin therapy and many under the age of 40 will also be treated with statins. The addition of ezetimibe should be considered to further lower LDL-C levels in high-risk primary prevention patients. In very high-risk patients with ASCVD if the LDL-C level on statin therapy is greater than 70mg/dL the use of ezetimibe or a PCSK9 inhibitor should be considered. The use of fibrates or niacin with statins were generally not recommended as there is no evidence of benefit. However, in patients with ASCVD or other cardiovascular risk factors on a statin with controlled LDL-C but elevated TG levels (135-499mg/dL) the addition of icosapent ethyl can be considered. Finally, in patients with fasting TG levels greater than 500mg/dL an evaluation for secondary causes of hypertriglyceridemia should be initiated and consideration of drug therapy to reduce the risk of pancreatitis.

Table 6. ADA Recommendations for Lipid Lowering Therapy
Primary Prevention
Age 20-39: With additional risk factors may be reasonable to initiate statin therapy
Age 40-75: Use moderate-intensity statin therapy* in addition to lifestyle therapy
Age 40-75: If at higher cardiovascular risk, including those with one or more ASCVD risk factors, it is recommended to use high intensity statin therapy to reduce LDL cholesterol by >50% and to target an LDL-C <70 mg/dL
Age 40-75: If at higher cardiovascular risk, especially those with multiple ASCVD risk factors and an LDL-C >70 mg/dL, it may be reasonable to add ezetimibe or a PCSK9 inhibitor to maximum tolerated statin therapy
Age > 75: Initiating moderate intensity statin therapy is reasonable after discussion and in patient already on statin therapy it is reasonable to continue statin therapy
Secondary Prevention
All ages: High intensity statin therapy**/maximally tolerated stain
For people with diabetes and ASCVD, treatment with high intensity statin therapy is recommended to target an LDL-C reduction of >50% and an LDL-C l goal of <55 mg/dL. Addition of ezetimibe or a PCSK9 inhibitor is recommended if this goal is not achieved

*Moderate intensity statin- atorvastatin 10-20mg, rosuvastatin 5-10mg, simvastatin 20-40mg, pravastatin 40-80mg, lovastatin 40mg, Fluvastatin XL 80mg, pitavastatin 3-4mg.

**High Intensity statin- atorvastatin 40-80mg, rosuvastatin 20-40mg.

American College of Cardiology and American Heart Association Guidelines

Table 7. Diabetes Specific Risk Enhancers That are Independent of Other Risk Factors in Diabetes
Long duration (≥10 years for type 2 diabetes mellitus or ≥20 years for type 1 diabetes mellitus Albuminuria ≥30 mcg of albumin/mg creatinine eGFR <60 mL/min/1.73 m2 Retinopathy Neuropathy ABI <0.9

Table 7. Diabetes Specific Risk Enhancers That are Independent of Other Risk Factors in Diabetes

Long duration (≥10 years for type 2 diabetes mellitus or ≥20 years for type 1 diabetes mellitus

Albuminuria ≥30 mcg of albumin/mg creatinine

eGFR <60 mL/min/1.73 m2

Retinopathy

Neuropathy

ABI <0.9

ABI indicates ankle-brachial index

American Association of Clinical Endocrinologists/American College of Endocrinology Guidelines

Table 8. ASCVD Risk Categories and Treatment Goals
Risk Category	Risk Factors/10-year risk	LDL-C mg/dL	Non-HDL-C mg/dL	Apo B mg/dL	TG mg/dL
Extreme Risk	Diabetes and clinical ASCVD	<55	<80	<70	<150
Very High Risk	Diabetes with one or more risk factors	<70	<100	<80	<150
High Risk	Diabetes and no other risk factors	<100	<130	<90	<150

European Society of Cardiology and European Atherosclerosis Society Guidelines

The European Society of Cardiology (ESC) and European Atherosclerosis Society (EAS) has 2019 guidelines for the treatment of lipids in patients with diabetes (223). These guidelines classify patients with diabetes as very high risk, high risk, or moderate risk (table 9). The recommended goals of therapy based on risk classification are shown in table 10. As with other guidelines intensification of statin therapy should be considered before the introduction of combination therapy. If the goal is not reached, statin combination with ezetimibe should be considered next.

Table 9. ESC/EAS Classification of Risk in Patients with Diabetes
Very High Risk- target organ damage, or at least three major risk factors, or early onset of T1DM of long duration (>20 years)
High Risk- without target organ damage, with DM duration >10 years or another additional risk factor
Moderate Risk- Young patients (T1DM <35 years; T2DM <50 years) with DM duration <10 years, without other risk factors. Calculated SCORE >1 % and <5% for 10-year risk of fatal CVD

Table 10. ESC/EAS Goals of Therapy in Patients with Diabetes
	LDL-C	Non-HDL-C	Apo B
Very High Risk	>50% reduction and <55mg/dL (<1.4mmol/L)	<85mg/d;	<65mg/dL
High Risk	>50% reduction and <70mg/dL (<1.8mmol/L)	<100mg/dL	<80mg/dL
Moderate Risk	<100mg/dL	<130mg/dL	<100mg/dL

European Society of Cardiology

Table 11. Cardiovascular Risk Categories and Goals in Patients with Type 2 Diabetes
Very high risk	Clinically established ASCVD or Severe target organ damage or 10-year risk of CVD> 20% using SCORE2-Diabetes	LDL-C < 55mg/dl Non-HDL-C <85mg/dL
High risk*	10-year risk of CVD 10% to < 20% using SCORE2-Diabetes	LDL-C < 70mg/dL Non-HDL-C <100mg/dL
Moderate risk*	10-year risk of CVD 5% to <10% using SCORE2-Diabetes	LDL-C < 100mg/dL
Low Risk*	10-year risk of CVD <5% using SCORE2-Diabetes	no recommendations

*Patients not meeting the very high-risk category.

My Guideline Recommendations

The approach I use is to combine these recommendations (Tables 12 and 13). In patients with diabetes who have pre-existing ASCVD I initiate intensive statin therapy. I prefer LDL or non-HDL-C goals over percent reduction goals. Given the extensive data showing that the lower the LDL-C the greater the reduction in cardiovascular events most secondary prevention patients would benefit from the addition of ezetimibe to maximize LDL-C lowering without markedly increasing costs (226). In patients with diabetes 40-75 years of age without pre-existing ASCVD I calculate the 10-year risk of developing ASCVD (http://www.cvriskcalculator.com/) and identify risk enhancing factors (Table 7) and other factors that increase risk that are not included in the calculator (for example family history, inflammatory disorders, etc.). I initiate intensive statin therapy if the 10-year risk is > 7.5% or if there are multiple risk factors/risk enhancers. I initiate moderate statin therapy if the risk is < 7.5% without multiple risk factors/enhancers. Four to twelve weeks after initiating statin therapy I obtain a lipid panel to determine if the LDL and non-HDL-C levels are at goal. In patients with pre-existing ASCVD or multiple risk factors/risk enhancers (i.e., very high-risk patients) my goal is an LDL-C < 55mg/dL and a non-HDL-C < 80mg/dL. In patients that are at high-risk the goal my goal is an LDL-C < 70mg/dL and a non-HDL-C < 100mg/dL. In patients with moderate risk an LDL-C goal of < 100mg/dL and a non-HDL c < 130mg/dL is appropriate. If the levels are not at goal, I first adjust the statin dose until the patient is taking the maximally tolerated statin dose and then consider adding additional medications. In patients with diabetes who are less than 40 years of age I initiate statin therapy if the patient has overt ASCVD, long standing diabetes, or risk factors/risk enhancers for ASCVD and the LDL and non-HDL-C levels are not at goal. In these younger patients I also calculate the life time risk of ASCVD events to start a discussion of beginning early therapy given the abundance of data indicating that initiating LDL-C lowering therapy early has great potential in markedly lowering ASCVD risk (226,227). In patients over 75 years of age with a reasonable life expectancy I begin moderate statin therapy and adjust based on response. When there is difficulty classifying a patient’s risk, I will obtain a coronary calcium score and use the score to help stratify the patient’s risk. In all cases the benefits and risks of lipid lowering therapy needs to be discussed with patients and the patient’s personnel preferences taken into account.

Table 12. ASCVD Risk Categories and Treatment Goals
Risk Category	Risk Factors/10-year risk	LDL-C mg/dL	Non-HDL-C mg/dL
Very High Risk	Diabetes and clinical ASCVD, multiple risk factors, 10-year risk > 20%	<55	<85
High Risk	Diabetes with one or more risk factors, 10-year risk >7.5% to <20%	<70	<100
Moderate Risk	Diabetes and no other risk factors. 10-year risk <7.5%	<100	<130

Table 13. Drug Therapy According to Risk Category that is Typically Required
Very High Risk	Intensive statin therapy + ezetimibe. Add PCSK9 if not close to goal
High Risk	Intensive statin therapy. Add ezetimibe if not at goal
Moderate Risk	Moderate statin therapy. Increase to intensive statin therapy if not at goal

TREATMENT OF LIPID ABNORMALITIES IN PATIENT WITH DIABETES

Life Style Changes and Weight Loss

Drug Therapy

The effect of statins, fibrates, niacin, ezetimibe, omega-3-fatty acids, bile acid sequestrants, bempedoic acid, and PCSK9 inhibitors on lipid levels in patients with diabetes is virtually identical to that seen in non-diabetic patients (Table 14). Below we will highlight issues particularly relevant to the use of these drugs in patients with diabetes. For detailed information on lipid lowering drugs see the chapters on Triglyceride Lowering Drugs and Cholesterol Lowering Drugs (141,209).

STATINS

EZETIMIBE

Ezetimibe is easy to use and generally well tolerated by patients with diabetes. Ezetimibe does not appear to increase the risk of new onset diabetes (199,247,248).

FIBRATES

Diabetic Retinopathy

Taken together these results indicate that fibrates have beneficial effects on the progression of diabetic retinopathy. The mechanisms by which fibrates decrease diabetic retinopathy are unknown.

Diabetic Nephropathy

Amputations

Do Fibrates have an Independent Effect on Microvascular Disease?

BILE ACID SEQUESTRANTS

Bile acid sequestrants are relatively difficult to take due to GI toxicity (mainly constipation) (141). Diabetic subjects have an increased prevalence of constipation, which may be exacerbated by the use of bile acid sequestrants. On the other hand, in diabetic patients with diarrhea, the use of bile acid sequestrants may be advantageous. Bile acid sequestrants may also increase serum TG levels, which can be a problem in patients with diabetes who are already hypertriglyceridemic (141). An additional difficulty in using bile acid sequestrants is their potential for binding other drugs (141). Many drugs should be taken either two hours before or four hours after taking bile acid sequestrants to avoid the potential of decreased drug absorption. Patients with diabetes are frequently on multiple drugs for glycemic control, hypertension, etc., and it can sometimes be difficult to time the ingestion of bile resin sequestrants to avoid these other drugs. Colesevelam (Welchol) is a bile acid sequestrant that comes in pill, powder, or chewable bars and causes fewer side effects and has fewer interactions with other drugs than other preparations (254). The usual dose is 3.75 grams per day and can be given as tablets (take 6 tablets once daily or 3 tablets twice daily), oral suspension (take one packet once daily), or chewable bars (take one bar once daily). Of particular note is that a number of studies have shown that colesevelam improves glycemic control in patients with diabetes resulting in an approximately 0.5% decrease in A1c levels (255).

NIACIN

OMEGA-3-FATTY ACIDS

PCSK9 INHIBITORS

BEMPEDOIC ACID

Table 14. Effect of Lipid Lowering Drugs
	LDL-C	HDL-C	TG

Statins	↓ 20-60%	↑ 5-15%	↓ 0-35%*
Bile acid sequestrants	↓ 10-30%	↑ 0-10%	↑ 0-10%**
Fibrates	↓ 0-15%***	↑ 5-15%	↓ 20-50%
Niacin	↓ 10-25%	↑ 10-30%	↓ 20-50%
Ezetimibe	↓ 15-25%	↑ 1-3%	↓ 10-20%
PCSK9 Inhibitors	↓ 50-60%	↑ 5-15%	↓ 5-20%
Bempedoic Acid	↓ 15-25%	↓ 5-6%	No change
High Dose Fish Oil	↑ 0- 50%***	↑ 4- 9%	↓ 20- 50%*

*Patients with elevated TG have largest decrease

** In patients with high TG may cause marked increase

*** In patients with high TG may increase LDL

Therapeutic Approach

FIRST PRIORITY- LDL-C

SECOND PRIORITY- NON-HDL-C

VERY HIGH TG

LOW HDL-C

CONCLUSION

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Idiopathic Short Stature and Growth Failure of Unknown Etiology

ABSTRACT

Idiopathic short stature (ISS) is defined by a height standard deviation score (SDS) ≤ -2.25 (≤1.2nd percentile) in pediatric patients for whom diagnostic evaluation has excluded other causes of short stature, and growth hormone levels are above 10 nanograms per milliliter in response to physiologic/pharmacologic stimulation. This definition, while comprehensive, is static and does not consider the importance of longitudinal follow up of growth, and consideration of evolving causes of growth hormone deficiency in these children. Using three illustrative cases, we highlight the importance of longitudinal evaluation of children with short stature to establish a growth pattern, identifying evolving causes of GH deficiency which may not be apparent on the initial work up, as well as exclusion of non-growth hormone related factors that affect growth including malabsorptive diseases, familial short stature, constitutional delay in puberty, Turner's syndrome, and SHOX Gene haploinsufficiency to accurately make a diagnosis of growth failure of unknown etiology (GFUE).

INTRODUCTION

In the 1980s, data on growth outcomes on patients who received growth hormone treatment were reported in an observational study (1); this included children who had a normal peak growth hormone response to stimulation, a group that was termed “constitutional short stature”. The term, idiopathic short stature (ISS), was later adopted to describe such patients (2). Guidelines for the use of growth hormone in children published by the Pediatric Endocrine Society defined ISS by a height standard deviation score (SDS) ≤-2.25 (≤1.2nd percentile) in pediatric patients for whom diagnostic evaluation excludes other causes of short stature, and with growth hormone levels above 10 nanograms per milliliter in response to physiologic/pharmacologic stimulation (3). The evaluation of children with short stature includes observation over a prolonged period to establish a growth pattern as well as the exclusion of non-growth hormone related factors that affect growth (4, 5). To monitor children's growth patterns in the United States, the Center for Disease Control (CDC) chart utilizes percentiles, with the 3^rd and 95th percentiles serving as the defined limits. Across the globe, the World Health Organization (WHO) charts are used to track a child's growth progression, and the limits are set at 2.3rd and 97.7th percentiles (5). There are wide variations in normal growth patterns and an even greater variety of conditions that manifest with growth abnormalities. Various factors contribute to the etiology of short stature, and this includes normal variants of growth such as familial short stature, constitutional delay in growth and puberty, as well as systemic conditions such as malabsorptive diseases, Turner's syndrome, and SHOX Gene haploinsufficiency, among others (Table 1). Diagnostic tests at initial presentation by pediatric endocrinologists focus on elucidating the underlying cause of poor growth (Table 2). Globally, malnutrition remains the main cause of poor growth (5).

In 2003, the Food and Drug Administration (FDA) approved the use of growth hormone (GH) for children with ISS. Two decades later, growth hormone therapy for idiopathic short stature remains controversial. ISS is not universally accepted as an indication for treatment with GH. This medication was approved for ISS in US, Canada, and Latin America, but not in the European Union or Japan (6). Along with other investigators, we have expressed concerns about the definition of ISS (7). The Pediatric Endocrine Society consensus statement did not specifically exclude patients with normal variants of growth such as constitutional growth delay and familial short stature (3). The definition focuses on stature, rather than growth and growth failure, therefore making GH treatment of such patients open to criticism for its use as only a height enhancer. In addition, as we have previously argued, a child whose height decelerates from the 75 to the 25th percentile without any known cause would also be of concern even though the height would not fit the ISS definition (7). If no cause is found after clinical, appropriate biochemical and radiologic evaluation, such a patient may have what we would prefer to term idiopathic growth failure (IGF). In as much as those initials are already in use for Insulin Like Growth Factor, we prefer the term GFUE, or growth failure of unknown etiology, thereby emphasizing the concern about growth failure, not just stature.

To illustrate the potential dilemmas in diagnosis, we present three patients with similar patterns of growth and a normal response to initial growth hormone stimulation testing with 2 provocative agents; each of these children may have been called ISS but demonstrate radically different outcomes.

Table 1. Common Causes of Short Stature and Growth Failure
Normal variants of growth
	Familial short stature
	Constitutional delay in growth and puberty (CDGP)
Systemic disorders
Endocrine	Growth hormone deficiency
	Hypothyroidism Cortisol excess (endogenous or exogenous)
Non-endocrine	Malnutrition
	Malabsorptive diseases
	Genetic syndromes: Turner's syndrome, Noonan’s syndrome, Achondroplasia, SHOX gene haploinsufficiency
	Chronic inflammatory conditions (e.g.: Inflammatory bowel diseases)
	Chronic medical conditions (e.g.: asthma on inhaled steroids)

Table 2. Diagnostic Tests at Initial Investigation of Idiopathic Short Stature and Growth Failure of Unknown Etiology
CBC, ESR
Urinalysis
Basic metabolic panel- BUN, creatinine, electrolytes
Celiac screen
Karyotype
TSH, Free T4
IGF1, IGFBP3
Bone age x ray

PATIENT 1

A boy aged 12 years and 5 months was evaluated for poor growth; both the father and paternal uncle were “late bloomers”. The height was at the 6th percentile, weight at 9th percentile, and Body Mass Index or BMI (weight in Kg divided by the square of height in meters) was at 32nd percentile, and his growth velocity was 3.8 cm per year. Clinical examination indicated Tanner stage 1 pubertal development with testes volume of 2-3mL bilaterally. Laboratory tests performed by the referring pediatrician before endocrine consultation showed normal thyroid function tests, sedimentation rate, C-Reactive protein, and no thyroid autoantibodies. A radiological bone age study confirmed delayed skeletal maturation, with a bone age of 10 years. No additional tests were recommended at the Pediatric endocrinology clinic, and he was observed clinically.

At 14 years of age, the patient showed no signs of puberty. Figure 1 shows his longitudinal growth during follow up. His height was the 2nd percentile, weight at the 6th percentile, BMI at the 31st percentile, and growth velocity remained at 3.8 cm per year. Delayed puberty in males is defined as the absence of testicular growth at an age that is 2 to 2.5 SD later than the reference population, which usually is around 14 years of age. (7) Laboratory results revealed low IGF-1 levels (-1.8 SDS for age and gender), while LH levels were close to pubertal levels, and testosterone levels remained prepubertal. A growth hormone (GH) stimulation test with the provocative agents, arginine and clonidine, was conducted due to the declining growth velocity and the low IGF-1 levels. Upon stimulation, peak GH level was 15 ng/ml. At 14 years and 6 months old, the patient's height was at the1st percentile and growth velocity had decreased to 1.9 cm per year. The laboratory tests at that visit showed that IGF-1 levels had increased to 226 ng/mL and testosterone was now 45.7 ng/dL, consistent with Tanner stage 2 pubic hair, and 4 ml testicular volume noted on examination. A bone age study confirmed delayed skeletal maturation, with a bone age of 10 years and 6 months. Since the patient had not progressed in puberty at the age of 14 years and 6 months, testosterone priming at a dose of 100 mg intramuscularly, once in 4 weeks for 3 months was initiated. At 15 years of age, his growth velocity had accelerated to 16.4 cm per year and his height was at the 5th percentile. Laboratory tests performed at 15.5 years showed that testosterone level was 301 ng/dL with a LH of 0.57 mIU/ml and IGF-1 levels had increased to 340 ng/ml. A bone age study showed the bone age to be consistent with 14 years and 6 months old. At 16 years and 11 months of age, the patient's height had reached the 36th percentile. Pubertal development had advanced with Tanner stage 3-4 for pubic hair development and testicular volume of 15mL bilaterally.

Figure 1. Patient 1 growth chart: Longitudinal growth chart in patient 1 before and after sex hormone priming.

DISCUSSION PATIENT 1

This patient’s height at presentation met the criteria for the diagnosis of Idiopathic Short Stature (ISS) since the patient's height was below -2.25 SD (8). However, with longitudinal follow-up, patient 1 ultimately proved to have constitutional growth delay (Figure 1). The diagnostic approach employed for this patient centered on monitoring. Once testosterone priming was employed, puberty progressed, and he reached his expected adult height. Through a targeted endocrine evaluation and longitudinal monitoring, the patient's condition was successfully managed without growth hormone treatment. This case underscores the importance of judicious use of testing.

The onset of puberty differs between boys and girls. In girls, 95% manifest at least one sign of puberty by 13 years of age whereas boys should begin signs of puberty by 14 years of age. In patients with delayed puberty and slow growth, a careful family history may be consistent with constitutional delay of growth and puberty. There often may be a history of a family member who also did not begin puberty until late into the teenage years, as in the case with the proband’s father and uncle. These patients can be difficult to diagnose because they may appear to be slowing in growth as they cross growth percentiles around the time of the anticipated pubertal growth spurt. Patients with constitutional growth delay fall behind their peers initially but have increased growth velocity later when puberty progresses and most reach adequate adult heights. Patients with constitutional growth delay have delayed bone ages and do not require treatment with growth hormone.

PATIENT 2

A boy presents at 12 years 9 months of age with growth failure and short stature. The patient's height had declined from 25th percentile to 10th percentile over 1.5 years, BMI was 40th percentile, growth velocity was 3.8 cm per year, and he was prepubertal. The parental target height was 176.7 cm (50th percentile). Laboratory tests showed that the testosterone level was 12 ng/dL, IGF-1 level was 125ng/mL (-2.06 SD), and bone age study was consistent with 12 years of age. Because of the low growth velocity and IGF1 level, the patient had a growth hormone (GH) stimulation test with provocative agents, arginine, and clonidine. The results showed a peak GH level of 16.9 ng/ml. At 13.5 years old, the patient’s height further declined to be at -2.25 SD and growth velocity was 2.84 cm per year. Figure 2 shows patient 2’s growth trajectory during follow-up. His testicular volume was 6mL bilaterally while tests showed IGF-1 to be114 ng/mL (-2.21 SD), thyroid function tests were normal, early morning cortisol level was 15.2 ug/dL, prolactin was 10 ng/ml, testosterone level 79 ng/dL, the luteinizing hormone was 3.2 mIU/mL, and the follicle-stimulating hormone was 2.1 mIU/mL, all consistent with pubertal progression. As puberty progressed, the growth velocity did not increase leading to a decline in height standard deviation. In the setting of this growth deceleration despite pubertal progression, a second growth hormone stimulation test with arginine and L-dopa was performed. The GH stimulation test revealed a peak GH level of 5.4 ng/ml. This low peak GH level is consistent with growth hormone deficiency (GHD). On pituitary magnetic resonance imaging, the pituitary gland appeared normal but small. The patient was treated with daily recombinant human growth hormone for 4.8 years. He reached an adult height at the 90^th percentile (1.3 SDS), slightly above mid parental height (Figure 2).

Figure 2. Patient 2 growth chart: Longitudinal growth chart in patient 2 before and after growth hormone therapy. GH denotes growth hormone. MPTH denotes mid-parental target height.

DISCUSSION PATIENT 2

This patient's referral to a pediatric endocrinologist stemmed from decline in height, subpar growth velocity considering stage of puberty, and low levels of insulin-like growth factor 1 (IGF-1). Based on his normal response to the first growth hormone (GH) stimulation test, the patient’s diagnosis was consistent with Idiopathic Short Stature (ISS). With continued monitoring, it was apparent that further growth deceleration occurred despite advent and progression of puberty. With an additional growth hormone (GH) stimulation test, the patient was ultimately diagnosed to have evolving growth hormone deficiency. GHD can be congenital or acquired, developing over time. The mechanism of idiopathic GHD might be due to a functional, transient decrease in somatomedin secretion insufficient to maintain growth as puberty sets in. Isolated GHD can be diagnosed using a combination of measuring growth factors, bone age X-rays, and growth hormone stimulation testing (9). Long-term monitoring as children progress into puberty is essential to uncover these instances of evolving GHD (9). It is imperative to emphasize the significance of prolonged follow-up and repeated testing to ensure the accurate diagnosis of GHD, rather than classifying the patient under the category of ISS or constitutional delay in growth and puberty (CDGP). In this case, physicians employed a meticulous approach of closely observing the decline in height and height velocity as puberty advanced, alongside screening tests such as IGF-1 measurement and multiple growth hormone stimulation tests. This comprehensive methodology allowed for the accurate diagnosis of GHD.

Despite the recognition of their many flaws, including poor reproducibility, non-physiologic assessment, and practical considerations, provocative tests remain part of the comprehensive evaluation of growth and are essential for the diagnosis of GHD (10). For GH stimulation testing, two agents that provoke GH secretion from the pituitary (L-dopa, clonidine, arginine, glucagon) are administered following an overnight fast. These provocative agents are not physiological and do not replicate normal secretory dynamics. In this patient with short stature, the clinical picture of low growth velocity as puberty progressed plus low IGF-1 levels are indicative of late, or evolving GHD.

PATIENT 3

A girl aged 13 years and 6 months was referred to a pediatric endocrinology clinic to evaluate her short stature. The patient had a medical history of ADHD diagnosed at the age of 7 years, and multiple surgeries for strabismus in both eyes. Her mid-parental height was at the 5^th percentile. Axillary hair developed at 11 years of age and breast development started around 10-11 years. At age 13.5 years, the height was at the 1st percentile (-2.27 SD), weight was at <1 percentile, and BMI was at the 6th percentile. Tanner stage of breast development was 3 and pubic hair had been shaved. The patient had small hands and feet, bilateral fifth finger clinodactyly, brachydactyly, and a slightly increased carrying angle. The bone age was12.5 years old with shortening and broadening of the metacarpals which raised concerns for hypochondroplasia. Chromosomal analysis showed a normal female karyotype, 46XX. SHOX gene analysis did not reveal mutations or deletions. This patient had a normal response to growth hormone stimulation test with provocative agents, arginine, and clonidine; peak growth hormone level was 18 ng/ml. The patient was referred to a medical geneticist to undergo further genetic testing to explain her short stature. Repeated chromosome analysis counting 50 cells, had normal female karyotype of 46XX excluding mosaic Turner Syndrome. The microarray results found gain in a 1.5 Mb copy at 10p15.3. The Fragile X test was negative, it did not reveal FMR1 CGG repeat expansions. A genetic testing panel for skeletal dysplasia investigated twenty-nine genes associated with skeletal dysplasia and the results showed no mutations or deletions. Whole exome sequencing did not reveal any variant in disease genes possibly associated with a short stature phenotype. In this patient with height -3 SDS and low mid parental height, treatment with growth hormone resulted in modest gains in height SDS. Figure 3 shows this patient’s growth before and after growth hormone therapy.

Figure 3. Patient 3 growth chart: Longitudinal growth chart in patient 2 before and after growth hormone therapy. GH denotes growth hormone.

DISCUSSION PATIENT 3

The cause of this patient’s short stature was not apparent despite extensive testing; thus, the diagnosis remains Growth Failure of Unknown Etiology, (GFUE) or Idiopathic short stature (ISS). After repeated genetic testing and thorough whole exome sequencing the etiology of the patient’s growth failure remained elusive. Case three presents the roadmap for a proper diagnosis of GFUE/ ISS using diagnoses of exclusion. The patient's height declined below >2.25 SD score below the mean height for a given age and sex, and through extensive genetic testing, there were no abnormalities found. Through a rigorous process of extensive evaluations and the systematic elimination of alternative causes for short stature, including malabsorptive diseases, familial short stature, constitutional delay in puberty, Turner syndrome, and SHOX Gene haploinsufficiency, the patient ultimately received a diagnosis of GFUE or idiopathic short stature.

ISS or GFUE may exist as a primary diagnosis alone or may be subcategorized to include ISS with familial short stature or constitutional delay of growth and puberty. Patients with ISS are often predicted to have an adult height below that expected based on mid parental height. It is thought that GH may increase the adult height of patients with ISS by 3.5 to 7.5 cm when compared with controls (11). GH-treated children with ISS have also been reported to achieve height gain similar to patients with isolated GHD (6). Additional considerations for treatment in conjunction with GH include gonadotropin- releasing hormone (GnRH) analogs and aromatase inhibitors. Both classes of medications seek to decrease bone age maturation while allowing the child to continue to grow. These agents are still considered experimental when used for improving growth and more studies are needed (12, 13). IGF-1 has received some attention recently as a potential treatment for ISS. Currently, IGF-1 is only approved for those patients with proven IGF-1 deficiency (IGF-1 levels –3 SD from the mean) with heights below –3 SD from the mean for age and normal stimulated GH levels. There is a lack of prospective studies that show clear benefit of IGF-1 for ISS. IGF-1 has also been associated with potential side effects, including hypoglycemia, headaches, lymphoid tissue hypertrophy, and coarsening of facial features (4). Further studies must be performed before IGF-1 can be considered a treatment option for GFUE/ISS.

The concept that short stature alone is a problem that must be treated often leads to patients seeking treatment for cosmetic, rather than medical reasons to attain an adult height the patient or family feel is appropriate. It is important to stress that short stature itself is not necessarily a medical problem. However, a pattern of growth consistent with growth failure (decreasing growth velocity, growth velocity consistently <50 percentile) regardless of height percentile should be evaluated further. If an underlying cause of growth failure cannot be found, we propose that it is appropriate to use a term such as “idiopathic growth failure” or “growth failure of unknown etiology” (GFUE) rather than “idiopathic short stature.” These terms take the focus off stature as the overriding problem, placing it into an element that all can agree needs investigation and, if persistent, needs intervention for its reversal. Height predictions always, but especially in the face of subnormal growth rates, are likely to be inaccurate and should be provided with a great deal of caution (5). We believe that familial short stature and constitutional delay of growth and puberty should be removed from the current overall category of “ISS” and placed under the category “normal variant.”

A child should be labeled as GFUE/ISS only after an in depth evaluation for other etiologies has been performed (7). As Next Generation Sequencing (NGS) and other diagnostic techniques become established, the number of patients with unidentified causes for growth failure is likely to diminish.

CONCLUSION

The three illustrative cases above help to show the significance of comprehensive testing, thorough evaluations, and rigorous longitudinal monitoring, to exclude currently known causes of short stature. Every effort should be made to avoid any bias in referral and evaluation of short stature based on gender, ethnic or racial differences (14). With these guidelines, two of the three patients initially presented with features aligned with the diagnostic criteria for growth failure of unknown etiology (GFUE)/ISS. Through subsequent testing and prolonged monitoring, alternative underlying diagnoses were identified: constitutional growth delay, and acquired, idiopathic, isolated growth hormone deficiency. In the third case, we present the roadmap for a proper diagnosis of GFUE/ ISS that is based on specific phenotypic characteristics. The diagnosis of ISS/GFUE should only be assigned after an exhaustive exploration of all diagnostic possibilities, ensuring that patients are provided with precise diagnoses and subsequently, if indicated, appropriate medication-based treatments to achieve height gain.

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Lipoprotein (a)

ABSTRACT

Lipoprotein(a) is an apo B-containing lipoprotein which has a unique plasminogen-like apolipoprotein(a) attached to apo B-100. The levels of lipoprotein(a) are under strong genetic regulation and vary by several hundred-fold in the general population. Although the exact function of lipoprotein(a) is still a mystery, recent studies have shown that lipoprotein(a) has a causal role in atherosclerosis and its level has been shown to be a risk factor for atherosclerotic cardiovascular disease (ASCVD) and calcific aortic valvular disease. Measurement of lipoprotein(a) level and comparison between different assays are challenging due to differences in reporting units and the absence of a reference method. Various guidelines recommend measurement of lipoprotein(a) levels in order to define cardiovascular risk. Lifestyle modifications have a minimal effect in reducing lipoprotein(a) levels. Currently available lipid-lowering therapies also result in only modest reductions in lipoprotein(a) levels. At present, there is no approved pharmacological treatment option for lowering lipoprotein(a) levels. More potent lipoprotein(a)-lowering medications are under active investigation to prove that lowering lipoprotein(a) level reduces ASCVD events. Lipoprotein(a), therefore, remains a viable and attractive therapeutic target in ASCVD risk reduction.

HISTORY

Lipoprotein(a) was first described by Kare Berg in 1963 (1) and later purified and characterized by Ehnholm and colleagues as part of human lipoproteins (2). Subsequent studies linked lipoprotein(a) to coronary artery disease (CAD) (3,4) and the threshold of 30 mg/dL was introduced (5). In 1987, amino acid sequences of apolipoprotein(a) [apo(a)], a major protein of lipoprotein(a), were identified and the LPA gene, encoding apo(a), was cloned (6,7). The sequence showed homology to plasminogen, a fibrinolytic proenzyme, but the protease domain was inactive (6). Apo(a) size polymorphism and its underlying molecular genetics were discovered and the inverse correlation between apo(a) isoform and lipoprotein(a) levels were demonstrated (8). Early observational and prospective studies showed higher levels of lipoprotein(a) in patients with CAD and myocardial infarction (MI) (4,5,9-12), but others did not (13,14). Lipoprotein(a) was later identified as a main carrier of oxidized phospholipids in the plasma (15). Results from large population-based studies along with Mendelian randomization studies in the past 2 decades have shown that lipoprotein(a) is a causal risk factor for atherosclerotic cardiovascular disease (ASCVD), especially MI and atherosclerotic stenosis (16-19). A surprise finding in 2013 also identified lipoprotein(a) as a key player in calcific aortic valvular disease (CAVD) (20). Currently, lipoprotein(a) is an active therapeutic target and results from ongoing phase 3 clinical studies will soon clarify the lipoprotein(a) hypothesis in ASCVD and CAVD.

STRUCTURE AND ASSEMBLY

Lipoprotein(a) is an apoB-containing lipoprotein resembling low-density lipoprotein (LDL), but apolipoprotein B-100 is covalently linked to a unique glycoprotein, called apolipoprotein(a) or apo(a). Apo(a), encoded by the LPA gene, is structurally similar to plasminogen, an important protein involved in fibrinolysis. In vitro, apo(a) has been shown to inhibit fibrinolysis (21)

Apo(a) is present only in a subset of primates, including humans, Old World monkeys, and orangutans, but not in New World monkeys or other primates. The exception is the occurrence of an apo(a)-like protein in the hedgehog (22). A characteristic feature of apo(a) is the presence of loop-like structure stabilized by 3 internal disulfide bonds, called “kringles”. These kringle domains are triple-loop structures found in other coagulation factors, including plasminogen. While plasminogen has 5 kringle domains (KI, KII, KIII, KIV and KV) and one protease domain at the end, apo(a) has only KIV, KV and an inactive protease domain (Figure 1).

Figure 1. Structure of lipoprotein(a) (from www.familyheart.org).

In apo(a), the number of the fourth kringle domain is highly variable due to expansion and differentiation of KIV into 10 different types of KIV domains, called KIV1-KIV10. While KIV1 and KIV3-KIV10 are present as single copies, kringle IV type 2 (KIV2) are further expanded, resulting in multiallelic copy number variation (1 to >40 copies). This expansion of KIV2 leads to a size polymorphism of apo(a), ranging from 300-800 kDa (23). Circulating levels of lipoprotein(a) are determined by the number of KIV2 copies. A low number of KIV2 copies results in small apo(a) isoforms, which lead to efficient secretion and contribute to higher levels of lipoprotein(a). On the other hand, the high number of KIV2 copies leads to large apo(a) isoforms and low levels of circulating lipoprotein(a).

Apo(a) is mainly synthesized in the liver. Newly formed apo(a) is then bound to apo B-100 of LDL to become lipoprotein(a). The assembly of lipoprotein(a) particles is a two-step process (24). The first step is the non-covalent docking of the lysine-binding site at the KIV7-KIV8 domains of apo(a) to the lysine residues at the N-terminus of apo B-100. The second step is the covalent binding through the disulfide bond between the 2 free cysteines in the KIV-9 of apo(a) and apo B-100 (25). The site of lipoprotein(a) assembly has been controversial whether it occurs intracellularly or extracellularly (23,25,26), but a recent study using a hepatocyte cell model suggests that the first non-covalent step occurs intracellularly, whereas the second covalent bond formation occurs extracellularly (27,28).

FUNCTION

The physiological function of lipoprotein(a) remains obscure (23). Earlier, it has been suggested that lipoprotein(a) may assist in wound healing (16,29). Lipoprotein(a) can interact with fibrin and other components of the extracellular matrix (30) via the lysine-binding sites in its kringle domains and it delivers cholesterol to the site of injury. Besides carrying cholesterol esters, free cholesterol, triglycerides, and phospholipids, lipoprotein(a) is the main carrier of oxidized phospholipids in the circulation (15). Lipoprotein(a) might also act as a carrier/scavenger for oxidized lipids since oxidized phospholipids and platelet-activating factor acetylhydrolase, an enzyme involved in hydrolyzing PAF, are found on lipoprotein(a) particles. In vitro, lipoprotein(a) can interfere with many steps of blood clotting and fibrinolysis (21), but the evidence for its thrombogenic role in vivo is less convincing (16).

In the Finnish population, loss-of-function variants in the LPA gene have been found (31). Although these subjects have very low levels of lipoprotein(a), no identifiable clinical abnormalities have been demonstrated (31). An increased risk of type 2 diabetes has been reported in those who have very low levels of lipoprotein(a) (32-36). Whether low levels of lipoprotein(a) are causally associated with type 2 diabetes is still unclear.

METABOLISM

Circulating levels of lipoprotein(a) are primarily determined by isoform size-dependent production rate and not catabolism (37). Fractional catabolic rates for lipoprotein(a) with short and long isoforms have been shown to be relatively indifferent (37). Liver is the main site of lipoprotein(a) catabolism, with a minor contribution from the kidney (26). Apo(a), apo B-100 and oxidized phospholipids carried on lipoprotein(a) may play a role in catabolism of lipoprotein(a) particles by acting as ligands for various receptors. A number of receptors have been shown to be associated with clearance of lipoprotein(a), including lipoprotein receptors, scavenger receptors, plasminogen receptors, Toll-like receptors (TLRs), and carbohydrate receptors or lectins (38). However, the relative contribution of these different receptors in lipoprotein(a) clearance remains to be explored. The lack of suitable animal models and the conflicting data on the role of certain receptors on the catabolism of lipoprotein(a) also make it difficult to conclude the definite role of these receptors. Following uptake into the cells, it has been shown that lipoprotein(a) dissociates into 2 components, with LDL being degraded in lysosomes and apo(a) being recycled to be re-secreted (39). It has been estimated that approximately 30% of apo(a) is recycled after lipoprotein(a) internalization into the cells (39), which could potentially contribute to the circulating level of lipoprotein(a).

GENETICS

Lipoprotein(a) levels are strongly determined by the genetic locus at the LPA gene on chromosome 6, which encodes apo(a). The LPA gene is closely related to the PLG gene, which encodes plasminogen. These 2 genes diverged about 40 million years ago with the loss of KI-KIII and the expansion and differentiation of KIV domain (23). The major genetic determinant is a copy number variation of the KIV2 repeat, which explains approximately 30-70% of the variability in lipoprotein(a) levels, depending on ethnicity (40). The apo(a) size is highly variable among individuals and depends on the number of KIV2 repeats in the LPA gene. Expression of a low number (10-22) of KIV2 repeats is characterized by small apo(a) isoforms and higher lipoprotein(a) levels, whereas a higher number (>23) of KIV2 repeats results in large apo(a) isoforms and lower lipoprotein(a) levels. Smaller apo(a) isoforms are associated with higher apo(a) production rates from the liver (41), whereas larger isoforms are more susceptible to proteosomal degradation in the endoplasmic reticulum (42). Subjects who carry a low number of KIV repeats have 4-5 times higher median lipoprotein(a) levels than those with a high number of KIV repeats (43).

Because every individual has 2 copies of this gene, 2 different isoforms may be present in plasma. In general, plasma levels of lipoprotein(a) are determined by the net production of apo(a) in each isoform. In Caucasians, with an increasing number of KIV repeats, and hence the larger apo(a) isoform, the frequency of the non-expressed allele is also increasing (44). As a result, the major isoform in circulation is mainly driven by the smaller ones. However, in about one-quarter of Caucasians, the larger allele is reported to be the dominant one (44) so the smaller allele is not always dominant (45). The frequency of the non-expressed alleles in Caucasians was highest in the mid-range whereas in African Americans, the non-expressed alleles were fairly distributed across apo(a) sizes (44).

It is of note that among those who carry the same number of KIV-2 repeats, there is a wide range of variability in the lipoprotein(a) levels. This could be explained by the presence of other genetic variants beyond the KIV repeat polymorphism that are also known to influence lipoprotein(a) levels (23,43,45) with a variable impact among various populations. A large genome-wide association meta-analysis has identified a number of independent single nucleotide polymorphisms (SNPs) around the LPA gene which are significantly associated with lipoprotein(a) levels (46). Certain SNPs are functionally lipoprotein(a)-increasing, such as rs1800769 and rs1853021, whereas other SNPS, such as rs10455872 and rs3798220, have no functional effect, but they are in linkage disequilibrium with other small apo(a) isoforms or other variants associated with elevated lipoprotein(a) levels (47).

On the contrary, other genetic variants of the LPA gene are associated with low levels of lipoprotein(a), such as the common splice variants, 4733G>A (48) and 4925G>A (49) in the KIV-2 repeat, and the missense variant rs41267813 (50). Other SNPs are null alleles which decrease lipoprotein(a) levels, such as rs41272114, rs41259144 and rs139145675. Individuals who carry these variants are found to be protected against the development of ASCVD.

Other genes, besides the LPA gene, have also been shown to influence lipoprotein(a) levels (45) although a meta-analysis has not confirmed the findings (51), suggesting that further studies with large sample sizes are required to replicate these findings. A recent genome-wide association study from the UK Biobank in almost 300,000 individuals has identified APOE, APOH, and CETP as additional loci that affect lipoprotein(a) levels (52).

Although lipoprotein(a) is under a strong genetic regulation, currently, there is no recommendation or advice to perform genetic testing for the LPA gene. Measuring lipoprotein(a) level in the circulation is considered sufficient since it reflects the overall genetic interaction of all variants.

LIPOPROTEIN(A) LEVEL

The distribution of lipoprotein(a) level in plasma is highly skewed with a tail toward higher levels as shown in Figure 2 (16,17,53). The range varies widely from <0.1 mg/dL to >300 mg/dL (<0.2 – 750 nmol/L). In people of European descent, 80% of the population have a serum level of lipoprotein(a) <40 mg/dL or 90 nmol/L (18). It is well known that plasma levels of lipoprotein(a) are different among various ethnicities, which are predominantly determined by lipoprotein(a) isoform size and other genetic variants in the LPA locus (45). Data from the UK Biobank showed that the mean lipoprotein(a) levels were lowest in Chinese individuals (16 nmol/L), followed by White (19 nmol/L), South Asian (31 nmol/L), and highest in Black individuals (75 nmol/L) (17). Data from subjects with various ethnicities have demonstrated that the level of lipoprotein(a) is slightly higher in women than in men (17,53-56), although other studies have shown a lack of difference between men and women (57,58).

Figure 2. Distribution of lipoprotein(a) in the Danish general population (from (16)).

Lipoprotein(a) level is fully expressed by the age of 2 years and the adult levels are usually achieved by 5 years (59). The level of lipoprotein(a) seems to be stable throughout life because it is under a strong genetic influence (60). Except for certain medical conditions, plasma lipoprotein(a) levels are relatively stable across the lifespan independent of lifestyle. A long-term study over a period of 15 years has shown that the overall absolute change in lipoprotein(a) levels is relatively modest but may be more pronounced in subjects with very high lipoprotein(a) levels (60). The level is also not affected by fasting (61).

Because lipoprotein(a) levels are predominantly determined by genetics, lifestyle modifications appear to have minimal effects (45). A low carbohydrate/high saturated fat diet was shown to result in a 15% decrease in lipoprotein(a) levels (62), whereas a decrease in saturated fat was associated with a 10-15% increase in lipoprotein(a) levels (63). Dietary changes may affect lipoprotein(a) levels in the opposite direction to LDL-C. Physical activity and exercise may affect lipoprotein(a) levels but the results are inconsistent and it may depend on the host, type, intensity, and duration (64).

As lipoprotein(a) is produced by the liver, a reduction in lipoprotein(a) levels is observed in liver disease (65,66). Liver transplantation has been shown to change apo(a) isoform to that of the donor with corresponding changes in lipoprotein(a) levels (67). In contrast, elevations of lipoprotein(a) levels are shown in nephrotic syndrome and chronic kidney diseases, either from impaired catabolism or increased hepatic production in response to protein loss in urine or in dialysis (68,69). A large increase is found among subjects who carry large apo(a) isoform sizes (70). Initiation of dialysis has no effect on lipoprotein(a) levels (71). Kidney transplantation can rapidly normalize lipoprotein(a) levels within several weeks (72-74).

Infection and inflammation also affect lipoprotein(a) levels. In acute and chronic inflammatory conditions, such as autoimmune diseases, lipoprotein(a) levels were increased (10,75), but in severe life-threatening conditions, such as sepsis and burns, lipoprotein(a) levels were decreased (76). Lipoprotein(a) levels increased during COVID infections, which might be responsible for increased thromboembolic events (77,78). The LPA gene contains interleukin-6 response elements (79) and elevated lipoprotein(a) levels could be decreased using interleukin-6 receptor blockade by tocilizumab (80). Following acute myocardial infarction, earlier results on the changes in lipoprotein(a) were conflicting, which could be due to differences in assay system (81,82).

Changes in endogenous sex hormones, including menopause and ovariectomy, have been found to have minimal effects on lipoprotein(a) levels (83,84), although a 27% increase after menopause has also been reported (85). Castration and orchidectomy have been shown to slightly increase lipoprotein(a) levels (86,87). Hormone replacement therapy in postmenopausal women reduces lipoprotein(a) levels by approximately 12-25% (85,88).

Longitudinal studies during pregnancy have shown that lipoprotein(a) levels are increased during the first trimester, peak at the middle of the second trimester and return to baseline after childbirth (89-91). Other studies, however, report no changes in lipoprotein(a) during pregnancy (92,93).

Hyperthyroidism is associated with a reduction in lipoprotein(a) level and treatment is found to increase it (94). Hypothyroidism is also associated with an elevation in lipoprotein(a) level and treatment with thyroxine can reduce it (94). Growth hormone (GH) therapy for GH-deficient adults can markedly increase lipoprotein(a) levels (95).

A decrease in lipoprotein(a) level has been reported in several genetic disorders of lipoprotein metabolism including abetalipoproteinemia, lecithin-cholesterol acyltransferase (LCAT) deficiency, and lipoprotein lipase deficiency (96). In contrast, an increase in lipoprotein(a) level is observed in familial hypercholesterolemia (FH) and familial defective apo B (96).

Conditions that have been reported to increase or decrease lipoprotein(a) levels are shown in Table 1.

Table 1. Conditions Associated with Changes in Lipoprotein(a) Levels
Increase	Decrease	No change
High carbohydrate/low fat diet (10-15%)	Low carbohydrate/high fat diet (10-15%)	Lifestyle intervention
Hypothyroidism (5-20%)	Hyperthyroidism (20-25%)	Alcohol consumption
Pregnancy (200%)	Hormone replacement therapy (25%)	Fasting
Castration/ovariectomy (small)	Severe acute-phase reactions (burn/sepsis)	Endogenous sex hormone
Growth hormone therapy (25-100%)	Tocilizumab (30-40%)	Menopause
Chronic kidney disease (200-400%)	Hepatitis/cirrhosis
Nephrotic syndrome (300-500%)
Severe inflammatory condition
Protease inhibitors/antiretroviral therapy

ISSUES IN LIPOPROTEIN(A) MEASUREMENT AND REPORTING

The wide variation in apo(a) isoform size among individuals poses challenges in measuring and reporting lipoprotein(a) levels. Most commercially available immunoassays use polyclonal antibodies which may cross-react with multiple KIV2 repeats (97). As a result, it may overestimate or underestimate lipoprotein(a) levels in subjects with large or small apo(a) isoforms, respectively. Currently, the latex-enhanced immunoturbidimetric method by Denka Seiken, Japan, has been shown to be less affected to apo(a) isoform size variability with high concordance of values when compared with the reference enzyme-linked immunosorbent assay (ELISA) method (98,99). Although the antibodies used in the Denka assay are still isoform dependent, the impact of apo(a) size variation is minimized by the use of five different calibrators (99). Nevertheless, results comparing six commercially available immunoassays, all of which use five-point calibrators, show a wide range of discrepancy among various assays (100). The reference ELISA method using monoclonal antibodies developed by a group at the University of Washington is considered the least apo(a) isoform size-sensitive immunoassay available (98,99). Recently, a newly developed LC-MS/MS method has been shown to be unaffected by the apo(a) isoform size polymorphism and proposed as a candidate reference method for standardization of lipoprotein(a) assay (101).

Besides the issues in measuring lipoprotein(a) levels, the reporting of lipoprotein(a) levels is also challenging. Currently, there are 2 types of commercially available assays to measure and report lipoprotein(a) levels. The first one reports the level in total lipoprotein(a) mass concentrations, which include the mass of apo(a), apo B-100, lipid, and carbohydrate components. The values are based on assay calibrators and are expressed in mg/dL. In the first method, there is no traceability of the various calibrators to the reference material (102). In the second method, the level is reported in lipoprotein(a) particle number and expressed in nmol/L of apo(a). The assay calibrators used in the second approach are traceable to the World Health Organization/International Federation of Clinical Chemistry and Laboratory Medicine (WHO/IFCCLM) secondary reference material (98) and the values are compared to the “gold standard” monoclonal antibody-based ELISA developed by Marcovina et al. (103). It is recommended that lipoprotein(a) levels should be measured with an assay which is least subjected to apo(a) isoform size and has been calibrated with the WHO/IFCCLM reference material. Current recommendations also encourage the reporting of lipoprotein(a) levels in molar units, but if it is not available, the units in which the assay is calibrated should be used for reporting (59,104,105).

It is known that different apo(a) isoform sizes give different molecular weights (106), therefore, direct conversion between molar and mass concentrations (i.e. nmol/L and mg/dL) using a single conversion factor could be misleading and is discouraged (59,99,106). Nevertheless, a factor of 2.0-2.5 is traditionally used to convert from a mass unit to a molar unit (97), although it may vary from 1.85 for a large apo(a) size to 2.85 for a small apo(a) size (99).

Since lipoprotein(a) and LDL have relatively similar size and density, cholesterol contained in lipoprotein(a) particles cannot be separated from that in LDL particles and is therefore collectively reported as LDL-C concentration. Previous experiments of isolated lipoprotein(a) particles showed that cholesterol accounted for approximately 30% of lipoprotein(a) mass concentration, therefore, lipoprotein(a)-cholesterol could be estimated by multiplying lipoprotein(a) mass (mg/dL) by 0.3 and used to correct LDL-C (lipoprotein(a)-cholesterol-corrected LDL-C) (59). However, a recent study directly measured lipoprotein(a)-cholesterol relative to lipoprotein(a) mass demonstrated that the percentage varied widely, ranging from 5.8% to 57.3% with a median of 17.3% (107), therefore, routine correction of LDL-C for lipoprotein(a)-cholesterol is currently not recommended in clinical practice (59). There are certain exceptions to this. First, in patients with clinically suspected familiar hypercholesterolemia (FH) and elevated lipoprotein(a) levels, measurement of lipoprotein(a)-cholesterol may be warranted since the corrected value may help exclude the diagnosis of FH and avoid unnecessary genetic testing (108). Second, correcting LDL-C for lipoprotein(a)-cholesterol may help explain the suboptimal response or resistance to statin therapy. High levels of lipoprotein(a) result in falsely elevated LDL-C. Since statins do not lower lipoprotein(a) levels, cholesterol in lipoprotein(a) is considered a statin-resistant fraction of LDL-C.

EFFECTS OF LIPOPROTEIN(A) ON VASCULAR DISEASE

Lipoprotein(a) exerts multiple effects that could be proatherogenic, proinflammatory, and prothrombotic.

Lipoprotein(a) could be atherogenic since lipoprotein(a) is small enough (<70 nm in diameter) to enter and become trapped in the vascular wall. Cholesterol carried on lipoprotein(a) particle could then be deposited in the arterial intima and aortic valvular leaflets. However, the number of circulating lipoprotein(a) particles is substantially lower than that of LDL particles. As a result, the amount of cholesterol deposited from lipoprotein(a) is expected to be much lower than that from LDL (109). Apo(a), the main protein of lipoprotein(a), also contains lysine-binding sites that could tightly bind to exposed surface of denuded endothelium of the vascular wall or the aortic valve leaflets. In an animal experiment of endothelial injury, it was found that lipoprotein(a) preferentially accumulated at injured sites, compared with LDL (110). These findings suggest that accumulation of lipoprotein(a) at the site of vascular injury could be the primary mechanism by which elevated lipoprotein(a) causes cardiovascular disease.

There is increasing evidence that oxidized phospholipids carried on lipoprotein(a) play a major role in atherogenesis and valvular calcification. In plasma, lipoprotein(a) is the main carrier of oxidized phospholipids (15). Interaction between oxidized phospholipids and apo(a) is mediated by the histidine residues in the KIV-10 domain of apo(a) (111,112). These oxidized phospholipids are both proatherogenic and proinflammatory (113). Oxidized phospholipids can induce inflammation by binding TLR2, TLR4, CD14 and CD36 on monocytes, macrophages, and endothelial cells (79,114). The result is an activation of multiple cytokines, chemokines, and adhesion molecules that mediate monocyte activation and migration into the vascular wall, endothelial dysfunction and proliferation, proliferation and migration of vascular smooth muscle cells into the atheromatous plaques, generation of reactive oxygen species, progression of vascular wall inflammation, and cell apoptosis that could lead to plaque rupture (115,116).

Structurally, apo(a) is similar to plasminogen, but the protease domain of apo(a) is catalytically inactive. Therefore, apo(a) may inhibit binding of plasminogen to endothelial cell surface receptor, compete with plasminogen for fibrin affinity sites, and interfere with fibrinolytic activity of plasminogen through a molecular mimicry mechanism (21). However, lowering lipoprotein(a) levels does not affect ex vivo fibrinolysis (117), suggesting that the role of lipoprotein(a) in thrombosis may involve other mechanisms beyond fibrinolysis (118). Lipoprotein(a) has also been shown to induce tissue factor expression, inhibit tissue factor pathway inhibitor, promote platelet activation and aggregation, and alter the structure of fibrin, which ultimately leads to thrombosis (118).

It is of note that these proatherogenic, proinflammatory, and prothrombotic effects of lipoprotein(a) have been demonstrated mainly from in vitro studies, and the clinical significance of these findings needs to be established from human studies.

LIPOPROTEIN(A) AND CARDIOVASCULAR DISEASE

Over the past two decades, data from epidemiological studies, meta-analyses, genome-wide association studies, and Mendelian randomization studies, have provided conclusive evidence that elevated lipoprotein(a) levels are associated with a higher risk of ASCVD (16,18,47,119-121). This is true in all ethnicities studied to date (17,55,122). The result of a genome-wide association study revealed that the most potent genetic association with coronary artery disease (CAD) was the LPA locus (121). A number of Mendelian randomization studies have shown that genetic variants associated with high lipoprotein(a) levels are more prevalent in subjects with ASCVD (18,47), whereas genetic variants associated with low lipoprotein(a) levels are protective against the development of ASCVD (31,49).

Several epidemiological studies, both in primary prevention and secondary prevention settings, have shown an association between lipoprotein(a) levels and ASCVD (17,19,55,120,123). The association is continuous and linear in different ethnicities without evidence of a threshold effect (17,120). Data from a large prospective cohort study from the UK Biobank showed a broadly linear relationship between lipoprotein(a) levels and ASCVD with an 11% increased risk of ASCVD with every 50 nmol/L increase in lipoprotein(a) levels (17). Another study also reported a relatively similar result, showing a 2.9% increased risk of CVD with every 18 nmol/L (10 mg/dL) increment in lipoprotein(a) levels (124).

Results from the clinical trials suggest that lipoprotein(a) might be a determinant of residual risk of ASCVD in patients who achieved low levels of LDL-C. For example, in the JUPITER trial using high-intensity rosuvastatin, on-statin lipoprotein(a) levels were associated with a residual risk of ASCVD even at low LDL-C levels (125). Similarly, findings from the FOURIER and ODYSSEY-OUTCOMES trials, using statins and PCSK9 inhibitors evolocumab and alirocumab, respectively, also demonstrated that patients with the higher baseline lipoprotein(a) levels were at an elevated risk of ASCVD irrespective of LDL-C levels (126,127). Lipoprotein(a) level is not associated with coronary artery calcium score, but both are independently associated with ASCVD risk (128). Elevated levels of lipoprotein(a) are associated with accelerated progression of atherosclerotic plaque (129).

The association between lipoprotein(a) levels and ASCVD is strongest for MI, atherosclerotic stenosis, and calcific aortic valvular disease (CAVD) (16). Additionally, very high levels of lipoprotein(a) levels are also associated with an increased risk of ischemic stroke (120,130) and heart failure (131), although the associations are weaker.

Not only that lipoprotein(a) is a determinant of ASCVD in patients with lower risk of ASCVD, but in subjects who are at higher risk of ASCVD, such as those with FH, elevated lipoprotein(a) levels have also been shown to be associated with a higher risk of ASCVD (132,133). Lipoprotein(a) is also an independent predictor of MI and mortality in patients with CKD (134).

Regarding prothrombotic effects of lipoprotein(a), although in vitro data demonstrated that lipoprotein(a) might impair fibrinolysis, a potential role for lipoprotein(a) in prothrombotic and antifibrinolytic activity in vivo remains unproven (21). Epidemiological studies and Mendelian randomization studies have failed to establish the major role of lipoprotein(a) in venous thrombosis in adults (135,136). In children, however, there is some evidence that elevated lipoprotein(a) level is a risk factor for arterial ischemic stroke and venous thromboembolism (137).

LIPOPROTEIN(A) AND CALCIFIC AORTIC VALVULAR DISEASE

Calcific aortic valvular disease (CAVD) is the disease of the aortic valve that includes early valvular sclerosis and advanced aortic valvular stenosis. CAVD is the leading cause of aortic valve replacement in developed countries, but there is currently no available medical treatment for CAVD. CAVD is characterized by thickening of the aortic valve leaflets with progressive stenosis of the aortic valve. Although the early stages of CAVD are asymptomatic, severe aortic stenosis may lead to significant left ventricular outflow obstruction and subsequent development of syncope, angina and heart failure.

Early cross-sectional studies have shown a strong association between elevated lipoprotein(a) and CAVD (138,139). Such association, however, cannot be used to indicate that lipoprotein(a) is causal for CAVD. In 2013, a genome-wide association study in subjects of European ancestry reported an association between genetic variants in the LPA locus and CAVD (20), which was also confirmed across multiple ethnic groups (20,140). Subsequent cohort and case-control studies have verified this finding and further linked lipoprotein(a) levels and oxidized phospholipids to CAVD (141-144). Lipoprotein(a) is now considered as a new causal risk factor for CAVD (16). Two recent meta-analyses show that plasma lipoprotein(a) levels ³50 mg/dL are associated with a 1.76-1.79-fold increased risk of CAVD (145,146). Elevated lipoprotein(a) levels have also been associated with the faster progression of CAVD and the increased risk for aortic valve replacement and cardiovascular death (143,146-149). In contrast, genetically lowered levels of lipoprotein(a) are associated with a 37% lower risk of aortic stenosis (150).

The normal aortic valve consists of endothelial cells and valvular interstitial cells. In the early stage of CAVD, increased permeability of valvular endothelial cells occurs and results in endothelial barrier dysfunction. Mechanical shear stress may cause further injury to the valvular endothelial cells, leading to endothelium denudation. As a result, lipid infiltration by LDL and lipoprotein(a) follows, similar to the development of atherosclerosis. Inflammatory responses ensue and induce the valvular interstitial cells to gain a myofibroblast-like phenotype and release collagen matrix along with other bone-related proteins. The later stage of CAVD is characterized by the osteogenic differentiation of the valvular interstitial cells, resulting in progressive calcification of the aortic valve. Apoptosis of the valvular interstitial cells can lead to formation of hydroxyapatite crystals and further calcification, creating the propagation of both inflammation and calcification. Progressive fibrosis and widespread calcification ultimately result in thickening and dysfunction of the aortic valvular leaflets.

The role of lipoprotein(a) in the molecular pathogenesis of CAVD is now accumulating. Apo(a) may increase the endothelial cell permeability through the Rho/RhoK signaling pathway, which is dependent on the lysine-binding site in the KIV10 domain. Since lipoprotein(a) is a major carrier of oxidized phospholipids in plasma, infiltration of lipoprotein(a) in the valvular tissue can deliver oxidized phospholipids to the valve. Oxidized phospholipids can directly attract monocytes and promote the transformation into macrophages. Oxidized phospholipids can then interact with various receptors on the macrophage, resulting in the release of several pro-inflammatory cytokines and subsequent formation of foam cells. Lipoprotein-associated phospholipase A2 (Lp-PLA2) from the macrophage carried on lipoprotein(a) can also act on oxidized phospholipids and generate lysophosphatidylcholine. Autotaxin, a lysophospholipase D enzyme secreted by the valvular interstitial cells and carried on lipoprotein(a) particles (151), can convert lysophosphatidylcholine into lysophosphatidic acid. Lysophosphatidic acid is both pro-inflammatory and pro-calcifying. By binding the lysophospholipid receptor on the valvular interstitial cells, it can activate the nuclear factor kappa B (NF-kB) and the Wnt-b-catenin pathways, leading to increased transcripts of IL-6 and various osteogenic genes, including runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP2). The result is the production of alkaline phosphatase, calcium deposition, inflammation, and calcification of the aortic valve (152).

These molecular pathways involved in the pathogenesis of CAVD provide several targets of interest, in addition to lipoprotein(a), that could be targeted and may lead to novel therapies for CAVD in the future.

GUIDELINE RECOMMENDATIONS FOR MEASUREMENT OF LIPOPROTEIN(A) LEVELS

Measurement of lipoprotein(a) levels could help refine the cardiovascular risk in certain conditions and various professional societies have recommended specific clinical conditions that warrant the measurement of lipoprotein(a) levels (59,105,153-156) as shown in Table 2.

Table 2. Guideline Recommendations for Lipoprotein(a) Measurement
Indications	AHA 2018	HEART UK 2019	NLA 2019	CCS 2021	BHS 2022	EAS 2022
1. Familial hypercholesterolemia		✓	✓	All Individuals	✓	All Adults
2. Family history (1^stdegree relatives) of premature ASCVD	✓	✓	✓		✓
3. Family history (1^stdegree relatives) of elevated lipoprotein(a)		✓	✓		✓
4. Personal history of premature ASCVD		✓	✓
5. Recurrent CVD despite optimal statin treatment			✓
6. Inadequate LDL-C reduction in response to statin			✓
7. Borderline or intermediate risk of ASCVD	✓	✓	✓		✓
8. Calcific valvular aortic stenosis		✓	✓		✓

It has been pointed out that different recommendations on measurement of lipoprotein(a) among various guidelines may be related to when the guidelines have been written (157). Most of the earlier guidelines list a number of conditions in which lipoprotein(a) should be measured, but the 2019 European Society of Cardiology/European Atherosclerosis Society (ESC/EAS) dyslipidemia guidelines have simplified this and recommended that lipoprotein(a) measurement should be considered at least once in each adult person’s lifetime (158). The rationale behind this recommendation is to identify individuals with very high levels of lipoprotein(a), i.e., >180 mg/dL or >430 nmol/L, who have a lifetime risk of ASCVD equivalent to that of heterozygous FH (158). Thus, measuring lipoprotein(a) levels in those with very high levels could make a significant contribution in the overall management to reduce cardiovascular risk. For the latest EAS lipoprotein(a) consensus statement published in 2022, it is recommended that lipoprotein(a) levels should be measured at least once in every adult (59). Incorporating lipoprotein(a) levels in the cardiovascular risk assessment could help improve risk stratification and provide comprehensive ASCVD risk evaluation. In addition, failure to consider an individual’s lipoprotein(a) level could lead to an underestimate of the absolute cardiovascular risk (59). For example, in a person with baseline risk of ASCVD events of 25%, a lipoprotein(a) level of 150 mg/dL will substantially increase the risk to 68% (59). A new risk calculator incorporating lipoprotein(a) levels and other traditional cardiovascular risk factors is now publicly available at http://www.lpaclinicalguidance.com.

Elevated lipoprotein(a) level is transmitted as a co-dominant trait. When an index subject is found to have high lipoprotein(a) level, screening for high lipoprotein(a) level in other family members is recommended and could help identify other affected family members (59). This approach is relatively similar to cascade screening in FH, except that genetic testing is not required. For children, there is a recommendation that children who have history of hemorrhagic or ischemic stroke, children of a parent with premature ASCVD and no other identifiable risk factors, and children of a parent with high levels of lipoprotein(a) should have lipoprotein(a) testing (59,154).

In general, the higher the lipoprotein(a) level, the greater risk for ASCVD. Therefore, the use of a specific threshold is biologically implausible and not appropriate. However, clinical practice guidelines prefer the use of a certain threshold for therapeutic decisions. An early study in patients who suffered acute MI proposed the lipoprotein(a) level of >30 mg/dL to reflect a higher risk of MI (5), and this cutoff values have been referred to in several guideline recommendations from various international societies (156,159,160). Another threshold level of lipoprotein(a) that has been used by other professional societies to confer an increased risk of ASCVD is 50 mg/dL or 100-125 nmol/L (153,154), which corresponds to the 80^th percentile in the European descent (45). A recent 2022 consensus statement from the EAS proposed a threshold of ³50 mg/dL (125 nmol/L) to rule in and <30 mg/dL (75 nmol/L) to rule out cardiovascular risk, whereas levels between 30-50 mg/dL (75-125 nmol/L) are grey zones (59). Different thresholds according to ethnicities have been proposed (161), although a large study from the UK Biobank has shown that the ASCVD risk among various ethnicities is similar when using a uniform threshold of ³150 nmol/L (17). At present, current management should remain the same regardless of ethnicity.

Interestingly, an inverse relationship between lipoprotein(a) levels and the risk of type 2 diabetes has been demonstrated from various studies and subjects with very low levels of lipoprotein(a) are at increased risk of diabetes (32-36,162). Some, but not all, genetic studies also suggest that low lipoprotein(a) level is causally associated with the risk of diabetes (33-35). The exact mechanism underlying this association is currently unclear. Whether potent specific lipoprotein(a)-lowering therapy might increase the risk of diabetes remains to be determined.

LIPOPROTEIN(A)-LOWERING TREATMENT

Currently, there is no approved medication for lowering lipoprotein(a) levels. Currently available lipid-lowering medications result in modest (10-30%) reduction in lipoprotein(a) levels (table 3) (163). Data from the Mendelian randomization studies have suggested that lowering lipoprotein(a) levels by approximately 100 mg/dL (215 nmol/L) would produce a similar benefit of reducing ASCVD risk by 22% as shown by lowering LDL-C levels by 38.6 mg/dL (1 mmol/L) using statin therapy (164). A separate analysis using data from primary prevention studies suggested that this number might be overestimated and lowering lipoprotein(a) levels by 65 mg/dL would be enough to give 22% ASCVD risk reduction (165,166). Similarly, data from the Copenhagen General Population Study demonstrated that lowering lipoprotein(a) by 50 mg/dL (105 nmol/L) within a 5-year period would produce a 20% reduction in major adverse cardiovascular events for secondary prevention (167). It is of note that lowering LDL-C levels of 38.6 mg/dL to give a reduction of an adverse cardiovascular event of 22% was used only as a benchmark and it is possible that lowering lipoprotein(a) levels by smaller amount may also be beneficial as previously shown for LDL-C lowering.

Although early studies report an increase in lipoprotein(a) levels after statin therapy, recent meta-analyses of statin therapy show that there is heterogeneity across studies. In addition, the changes in lipoprotein(a) levels are relatively small and may not be clinically meaningful (168,169). Currently, it is widely accepted that the benefits of statin in lowering LDL-C levels and decreasing ASCVD risk outweigh the potential risk associated with a small increase in lipoprotein(a) levels.

PCSK9 inhibitors, including evolocumab, alirocumab and inclisiran, can lower lipoprotein(a) levels by 10-30% and the absolute reduction is greatest in those with high lipoprotein(a) levels at baseline (126,127,170). A post-hoc analysis from the FOURIER trial using evolocumab reported a 23% decrease in adverse cardiovascular events in those with a baseline lipoprotein(a) level >37 nmol/L and a 7% reduction in those with <37 nmol/L (126). A significant relationship between a 15% lower risk per 25 nmol/L reduction of lipoprotein(a) levels was also observed. These data suggest that a small lowering in lipoprotein(a) level might have a clinical benefit. Results from the ODYSSEY Outcomes trial using alirocumab also showed a significant reduction in major cardiovascular events among those in the two highest quartiles of baseline lipoprotein(a) levels (127).

Interestingly, an exploratory analysis of the FOURIER trial further showed that lipoprotein(a) level was associated with future aortic stenosis (AS) events (new or worsening CAVD or aortic valve replacement) and that evolocumab therapy beyond one year might reduce these AS events (171). However, it should be noted that this post hoc analysis was performed in only a small number of subjects and the original trial was not designed to evaluate the impact of PCSK9 inhibitors on CAVD.

Other lipid-lowering medications, such as fibrates, ezetimibe, bempedoic acid and bile acid sequestrants, also have modest effects on lipoprotein(a) levels (163). Niacin can lower lipoprotein(a) levels by 20% due to decreased LPA mRNA and apo(a) production rate (172). Mipomersen and CETP inhibitors also decrease lipoprotein(a) levels by 20-30% (173). Currently, the use of these medications to lower lipoprotein(a) level is not advised.

Lipoprotein apheresis is an option to lower lipoprotein(a) levels in clinical practice, although levels of all apoB-containing lipoproteins are reduced after treatment (174). A median reduction in lipoprotein(a) level by 70% was observed (175) but the time-averaged reduction was around 30-35% (176). Notably, a reduction in CAD events has been reported from several retrospective studies (177-180). Lipoprotein apheresis could therefore be considered in those with very high levels of lipoprotein(a) and progressive cardiovascular disease despite optimal management of other risk factors (174). Worldwide, lipoprotein apheresis is not commonly performed except in Germany where it is approved for ASCVD patients who have elevated lipoprotein(a) levels (>120 nmol/L or >60 mg/dL) and recurrent ASCVD events, irrespective of LDL-C levels (175). Recently, it is also approved in the United States for patients with lipoprotein(a) levels >60 mg/dL and LDL-C levels >100 mg/dL with documented CAD or peripheral arterial disease (163).

Without specific lipoprotein(a)-lowering therapy yet available, in subjects with elevated lipoprotein(a) levels, intensive risk factor management, such as healthy diet and lifestyle behavior modifications, is recommended along with the intensification of statin therapy to lower the risk of ASCVD (59). Data from the EPIC-Norfolk population-based study showed that in subjects with elevated lipoprotein(a) above 50 mg/dL, those who modified their lifestyles to maintain ideal cardiovascular health had about one third of cardiovascular risk compared to those with poor cardiovascular health (181). These modifiable cardiovascular health scores included body mass index, healthy diet, physical activity, smoking status, high blood pressure, diabetes and cholesterol concentration (181). In those whose LDL-C target levels are not achieved, ezetimibe or PCSK9 inhibitors could be considered (154).

Aspirin has been shown to be associated with a cardiovascular risk reduction in subjects who carried the SNP rs3798220-C with elevated lipoprotein(a) levels from the Women’s Health Study and the Aspirin in Reducing Events in the Elderly (APREE) trial (182). However, this SNP was present in only a small percentage of Caucasian subjects (approximately 3-4%) in these 2 trials. Therefore, further studies with a larger number of subjects in broader populations are needed to confirm the benefits of aspirin.

NEW SPECIFIC LIPOPROTEIN(A)-LOWERING TREATMENT

Since apo(a) is exclusively produced by the liver, novel therapies targeting hepatic apo(a) production using antisense oligonucleotide (ASO) and small interfering RNA (siRNA) technologies are under active investigation.

Pelacarsen, formerly known as TQJ230, IONIS-APO(a)-LRx and AKCEA-APO(a)-LRx, is a second generation of apo(a) ASO, conjugated with N-acetylgalactosamine (GalNAc). Since GalNac is preferentially bound to asialoglycoprotein receptor on the cell surface of hepatocytes, the design of this GalNac-conjugated molecule will ensure the selective uptake by hepatocytes. Administered by subcutaneous injection every 4 weeks, it can bind apo(a) RNA in the hepatocytes, leading to an approximately 80% reduction in lipoprotein(a) levels in subjects with lipoprotein(a) level >150 nmol/L or approximately 60 mg/dL (183). Pelacarsen is generally well tolerated with the most frequently reported adverse event being injection site reactions. Currently, a phase 3 Lp(a)-HORIZON (NCT04023552) cardiovascular outcome study is underway to investigate the efficacy and safety of pelacarsen at the dose of 80 mg monthly for 4 years in subjects with ASCVD and lipoprotein(a) ³70 mg/dL.

Olpasiran, formerly known as AMG890 and ARO-LPA, is a GalNAc-conjugated siRNA that could be given every 3-6 months and result in a greater (>90%) reduction in lipoprotein(a) levels in subjects with lipoprotein(a) level >150 nmol/L or approximately 70 mg/dL (184). The most common adverse events were injection site reactions and hypersensitivity reactions. A cardiovascular outcome study (NCT05581303) is currently investigating the effects of olpasiran given every 12 weeks for 4 years in subjects with ASCVD and lipoprotein(a) levels ³200 nmol/L.

Zerlasiran, also known as SLN360, is another GalNAc-conjugated siRNA (185). A Phase 1 study investigated the safety and tolerability of zerlasiran after single ascending doses and multiple doses in healthy subjects with elevated lipoprotein(a) level ³150 nmol/L. A dose-dependent and sustained reduction in lipoprotein(a) level has been observed with a 98% reduction in the group receiving 600 mg of zerlasiran (185). A phase 2 ALPACAR-360 study (NCT05537571) to evaluate the Lipoprotein (a) lowering efficacy, safety and tolerability of zerlasiran in adult participants with elevated lipoprotein(a) (³125 nmol/L) at high risk of ASCVD is now underway.

Besides nucleic acid therapeutics to inhibit hepatic production of apo(a), small molecule inhibitors of lipoprotein(a) formation have been developed. Muvalaplin, an oral small molecule that blocks the initial noncovalent binding between apo(a) and apo B-100, thus disrupting lipoprotein(a) formation, has been reported to lower lipoprotein(a) level by 65% after 14 days of daily dosing in a phase 1 study (186). No serious adverse effects have been noted.

The effect of lipid-lowering medications on lipoprotein(a) levels is shown in Table 3.

Table 3. Effect of Lipid-Lowering Medications on Lipoprotein(a) Levels
Statins	No effect or slight increase
Ezetimibe	No effect or slight increase
Fibrates	No effect
Bempedoic acid	Minimal effect
Bile acid sequestrants	Minimal effect
Omega-3 fatty acids	No effect
Niacin	Decrease 15-25%
Lomitapide	Decrease 15-20%
Mipomersen*	Decrease 20-30%
CETP inhibitors*	Decrease 20-30%
Estrogen	Decrease 20-35%
PCSK9 inhibitors	Decrease 10-30%
Apo(a) ASO*	Decrease 80%
Apo(a) siRNA*	Decrease 90-98%
Apo(a) small molecule inhibitor*	Decrease 65%

*not currently available

The results of these ongoing and future clinical trials of lipoprotein(a) reduction are eagerly awaited and are expected to be the last piece of evidence supporting and confirming the causal relationship between lipoprotein(a) and ASCVD. Until a specific therapy for elevated lipoprotein(a) is available, intensive management of other modifiable cardiovascular risk factors is strongly recommended. Whether these lipoprotein(a)-lowering treatments would also be beneficial in CAVD remains to be further explored.

CONCLUSION

After 60 years of its discovery, several issues of lipoprotein(a) remain unresolved, including its function and metabolism. While the causality of lipoprotein(a) in ASCVD and CAVD has now been firmly established by epidemiological, genetic association, and Mendelian randomization studies, the next challenge is to prove that lowering lipoprotein(a) levels also leads to cardiovascular benefit in patients with elevated lipoprotein(a) levels.

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ABSTRACT

INTRODUCTION

CHARACTERISTICS OF TYPICAL MALE AND FEMALE EXTERNAL GENITALIA

GENERAL THOUGHTS ON AMBIGUOUS GENITALIA AT BIRTH

GENETICS OF SEX DETERMINATION AND DIFFERENTIATION

The Male Specific Pathway

The Female Specific Pathway

DIFFERENCES/DISORDERS OF SEX DEVELOPMENT (DSD)

The 46,XY Newborn With Ambiguous Genitalia

PARTIAL GONADAL DYSGENESIS

DEFICIENCY OF TESTOSTERONE BIOSYNTHESIS

5-ALPHA-REDUCTASE-2 DEFICIENCY (SRD5A2 VARIANTS)

GENETIC DEFECTS OF ANDROGEN ACTION

The 46,XX Newborn With Ambiguous Genitalia

ABNORMAL FETAL ANDROGEN PRODUCTION BY CONGENITAL ADRENAL HYPERPLASIA (CAH)

FETAL AND PLACENTAL AROMATASE DEFICIENCY

EXCESS MATERNAL ANDROGEN PRODUCTION

DRUGS ADMINISTERED TO THE MOTHER DURING GESTATION

Syndromes Associated with Ambiguous Genitalia

WORKUP OF NEWBORNS WITH AMBIGUOUS GENITALIA

Overall Assessment of the Child with Ambiguous Genitalia

Physical Examination of the External Genital Area

Basic Auxiliary Investigations in the First Days

KARYOTYPE

HORMONE STUDIES

IMAGING STUDIES

GENETIC STUDIES

DIFFERENTIAL DIAGNOSIS

46, XX DSD

46, XY DSD

Testing Early Postnatal Gonadal Function and Mini-Puberty

HCG Testing of Gonadal Function in a DSD Newborn

RISK OF MALIGNANCY OF THE DSD GONAD

SEX REGISTRATION OF A NEWBORN

FINAL REMARKS TO OVERALL MANAGEMENT

REFERENCES

ABSTRACT

INTRODUCTION

CLINICAL FEATURES

ASSESSMENT

DIAGNOSIS

1990 ACR Classification Criteria

2010 ACR Preliminary Diagnostic Criteria

2016 Revisions to the 2010/2011 Fibromyalgia Diagnostic Criteria

AAPT Diagnostic Criteria

EPIDEMIOLOGY

DIFFERENTIAL DIAGNOSIS

PATHOPHYSIOLOGICAL MECHANISMS

Abnormalities in Sensory Processing

Neurotransmitters

Cytokines

Hypothalamic-Pituitary-Adrenal Axis

Autonomic Nervous System

Psychological, Cognitive, and Behavioral Factors

Genetic Predisposition

SEROTONIN TRANSPORTER (5-HTT) GENE

D4 RECEPTOR GENE

CATECHOL-O-METHYL TRANSFERASE (COMT) GENE

OPIOID RECEPTOR μ 1 GENE (OPRM1)

ADRENERGIC RECEPTOR GENES

External Stressors

PERIPHERAL PAIN SYNDROME

INFECTIONS

PHYSICAL TRAUMA

PSYCHOLOGICAL DISTRESS

MANAGEMENT

Non-Pharmacological Management

PATIENT EDUCATION

EXERCISE

COGNITIVE-BEHAVIORAL APPROACHES

COMPLEMENTARY AND ALTERNATIVE APPROACHES

Acupuncture

Other

Pharmacologic Treatment

ANTIDEPRESSANTS

Tricyclic Antidepressants (TCAs)

Serotonin-Norepinephrine Reuptake Inhibitors (SNRIs)

Monoamine Oxidase Inhibitors

ANTICONVULSANTS

SEDATIVE HYPNOTIC AGENTS

D₄ RECEPTOR GENE

Epidemiology in Asia