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Normal and Abnormal Puberty

ABSTRACT

 

Puberty is a biological process that represents the development of secondary sexual characteristics and attainment of reproductive capacity, influenced by genetic, metabolic, environmental, ethnic, geographic, and economic factors. Pubertal onset is characterized by the increased kisspeptin and neurokinin B secretion leading to re-emergence of pulsatile gonadotropin releasing hormone signaling from the hypothalamus which stimulates increased pituitary secretion of luteinizing hormone and follicle stimulating hormone, which in turn stimulate gonadal sex hormone production. Precocious puberty refers to secondary sexual development occurring earlier than the lower end of normal age and delayed puberty refers to secondary sexual development occurring later than the upper end of normal age for the onset of puberty. These changes may represent a serious underlying condition or signify a common variation of normal for which treatment may not be necessary. Clinical evaluation should include a detailed history and physical examination, including anthropometric measurements, calculation of linear growth velocity, and evaluation of secondary sexual characteristics. This chapter summarizes the physiology of pubertal development, variations in pubertal development, and recent developments regarding human puberty.

 

INTRODUCTION

 

Puberty is the process through which reproductive competence is achieved (1). Physical characteristics associated with this process include the development of secondary sex characteristics, acceleration in linear growth velocity, and the occurrence of menarche in women and spermatogenesis in men. The sex chromosome karyotype of the embryo, XX or XY, determines the trajectory for differentiation of the gonads and development of the internal and external genital structures. This complex process, beginning in utero, depends on neuroendocrine signaling and gonadal components. Ultimately, integrated communication between the reproductive and metabolic systems is critical for timely pubertal development (2).

 

Pubertal development and neuroendocrine system maturation involve the ontogeny, activity, and interactions of the gonadotropin releasing hormone (GnRH) neurons. The onset of puberty is accompanied by increased kisspeptin and neurokinin B secretion causing the GnRH neurons to secrete GnRH in a pulsatile manner. Increased GnRH secretion stimulates pulsatile pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion (3). LH and FSH stimulate gonadal sex steroid secretion which promotes development of secondary sex characteristics and influences hypothalamic-pituitary function via negative feedback inhibition. This chapter summarizes the physiology of pubertal development, variations in pubertal development, and recent developments regarding human puberty.

 

CLINICAL FEATURES OF NORMAL PUBERTAL DEVELOPMENT

 

Children typically demonstrate a predictable sequence of physical changes during pubertal maturation. Within the chronologic age ranges for pubertal development, individual variations regarding age at onset and tempo of pubertal development are expected.

 

In humans, two physiological processes, gonadarche and adrenarche, govern pubertal transition. Gonadarche reflects the reactivation of the hypothalamic GnRH pulse generator which has been quiescent since late infancy. Increasing pulsatile GnRH secretion stimulates pulsatile gonadotropin secretion which, in turn, stimulates the growth and maturation of the gonads and gonadal sex steroid secretion. Increased estrogen secretion promotes breast development, cornification of the vaginal mucosa, and uterine growth in girls. Increased testosterone secretion promotes penile enlargement. The increased HPG axis activity culminates in folliculogenesis, ovulation, and menses in the female and spermatogenesis in the male.

 

In addition to gonadal sex steroid secretion, humans experience adrenarche signifying adrenal pubertal maturation. Adrenarche typically begins prior to the first visible physical manifestation of gonadarche, breast development, or testicular enlargement. Pubarche, the physical manifestation of adrenarche, is characterized by the development of pubic hair, axillary hair, apocrine odor, and acne. Adrenarche indicates increased adrenal cortical zona reticularis activity and is accompanied by increased secretion of dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), androstenedione, and 11-hydroxyandrostenedione (4, 5). These so-called “adrenal androgens” are C19 steroids which do not bind directly to the androgen receptor and can be peripherally converted to more potent androgens. Circulating concentrations of two adrenal 11-oxyandrogens, 11-hydroxyandrostenedione and 11-ketotestosterone increase with adrenarche. Whereas 11-hydroxyandrostenedione has minimal androgenic activity, 11-ketotestosterone is almost as potent as testosterone. During adrenarche, 11-ketotestosterone appears to be the major bioactive adrenal C19 steroid and may be responsible for the physical changes associated with pubarche (6).

 

Gonadarche and adrenarche are dissociated such that the absence of adrenarche does not prevent gonadarche or the attainment of fertility (7). Curiously, the phenomenon of adrenarche appears to be limited to humans and a few species of non-human primates (8, 9). The factors that drive the dynamic changes within a strictly defined zona reticularis within the adrenal cortex, are still poorly defined. How adrenarche and increased adrenal C19 steroids impact brain development during human adolescence is indeterminate (10). Urinary steroid hormone profiling suggest that adrenarche may be a gradual process that likely begins earlier than previously believed (11).

 

STAGING OF PUBERTY

 

Tanner and colleagues followed the pubertal development of children living in an orphanage in the UK. Their five-stage classification system continues to be commonly utilized for clinical assessments (12, 13, 14). For girls, Tanner staging is used to describe breast and pubic hair development (See Figure 1). For boys, Tanner staging is used to describe testicular volume, penile development, and pubic hair development (See Figure 2). Tanner and his colleagues also described that the tempo of puberty varies between individuals.

 

Figure 1. Tanner Staging for pubertal development in girls. In girls, breast development is rated from 1 (preadolescent) to 5 (mature), and stage 2 (appearance of the breast bud) marks the onset of pubertal development. Pubic hair stages are rated from 1 (preadolescent, no pubic hair) to 5 (adult), and stage 2. Although pubic hair and genital or breast development are represented as synchronous in the illustration, they do not necessarily track together and should be scored separately. Figure 1 Adapted with permission from Carel JC, Léger J. Clinical practice. Precocious puberty. N Engl J Med. 2008;358(22):2367 and Klein DA, Emerick JE, Sylvester JE, Vogt KS. Disorders of Puberty: An Approach to Diagnosis and Management. Am Fam Physician. 2017 Nov 1;96(9):590-599. PMID: 29094880

Figure 2. Tanner Staging for pubertal development in boys. In boys, genital development is rated from 1 (preadolescent) to 5 (adult); stage 2 marks the onset of pubertal development and is characterized by an enlargement of the scrotum and testis and by a change in the texture and a reddening of the scrotal skin. Pubic hair stages are rated from 1 (preadolescent, no pubic hair) to 5 (adult), and stage 2 marks the onset of pubic hair development. Although pubic hair and genital or breast development are represented as synchronous in the illustration, they do not necessarily track together and should be scored separately. In normal boys, stage 2 pubic hair develops at an average of 12 to 20 months after stage 2 genital development. Figure 2 Adapted with permission from Carel JC, Léger J. Clinical practice. Precocious puberty. N Engl J Med. 2008;358(22):2367 and Klein DA, Emerick JE, Sylvester JE, Vogt KS. Disorders of Puberty: An Approach to Diagnosis and Management. Am Fam Physician. 2017 Nov 1;96(9):590-599. PMID: 29094880

 

Girls

 

The typical first clinical sign of puberty in girls is the appearance of breast tissue with elevation of the breast and papilla; this is considered to be Tanner Stage 2 (Figure 1). Initially, breast development (thelarche) may be unilateral. Many girls complain of mild breast tenderness or discomfort during this stage that subsequently resolves. Tanner stage 3 breast development is considered to be additional enlargement of the breast and areola. During Tanner stage 4, the papilla forms a secondary mound above the breast; this stage is often very rapid. Tanner stage 5 represents mature breast development due to recession of the areola to the contour of the breast. Palpation of the breast is obligatory to differentiate breast tissue from lipomastia. In children with obesity without breast development, a palpable depression beneath the nipple in the center of the areola when examined in the supine position gives the impression of a donut and is referred to as the ‘donut’ sign.  Breast ultrasound correlates reasonably well with Tanner staging by palpation and can detect breast development slightly earlier than physical exam (15). In most instances, breast development is evident before pubic hair development. Typically, the pubertal growth spurt in girls occurs concurrently with the onset of breast development with only 4-6 cm of linear growth occurring after menarche, however this may be variable.

 

The appearance of sexual hair including pubic hair (pubarche) signifies the onset of adrenarche. In girls, Tanner stage 2 pubic hair is characterized by sparse, coarse, lightly pigmented hairs along the labia majora. For Tanner stage 3, pubic hair becomes progressively darker, coarser, and spreads over the mons pubis. For Tanner stage 4, pubic hair continues to extend to become an inverse triangle, with spread to the medial aspects of the thighs being considered Tanner stage 5.

 

With the onset of ovarian estrogen secretion, the vaginal mucosa changes from shiny bright red to pale pink appearance due to cornification of the vaginal mucosa. Increased estrogen secretion promotes uterine growth and causes physiologic leukorrhea, a thin, white, non-foul-smelling vaginal discharge that typically begins 6 to 12 months before menarche. Menarche occurs, on average, 2 to 2.5 years after the onset of breast development (See figure 3A). During the first-year post-menarche, menses are usually irregular and anovulatory. These early years are characterized by inconsistent ovulation and varying lengths of follicular and luteal phases. Ultimately, coordinated maturation of the hypothalamic, pituitary, and ovarian components occurs culminating in cyclic monthly ovulation (16). Although full HPG axis maturation generally occurs over several years, by three years post-menarche, most cycles are between 21-35 days.

 

Figure 3A and 3B. Average ages and sequence of pubertal development. Panel A: girls; Panel B: boys.

 

Boys

 

For boys, increased testicular volume is the first physical finding indicating onset of gonadarche (See Figure 2 and Figure 3B). Palpation of the testes and use of a Prader orchidometer is essential for accurate assessment. A Prader orchidometer is a collection of 3-D ellipsoids ranging in volume from 1 to 25 mL (See Figure 4). During gonadarche, testicular volume increases, and the penis increases in length and diameter. Flaccid penile length can be measured using a straight edge on the dorsal surface from the pubic ramus to the tip of the glans while compressing the suprapubic fat pad and applying gentle traction to stretch to penis.

 

Figure 4. Prader Orchidometer.

 

Increased testicular volume represents Sertoli cell proliferation, differentiation, and eventually, the initiation of spermatogenesis. The onset of puberty is defined as a testicular volume > 4 ml and a testicular length > 2.5 cm. The volume of mature human testis is approximately 20-25 ml. Spermatozoa (spermaturia) can be found in early morning urine samples beginning during genital stage 3 (16). Nocturnal sperm emissions may also begin around this time.

 

For boys, Tanner stage 2 pubic hair consists of downy hairs at the base of the penis. During pubic hair stage 3, the hair becomes longer, darker, and extends over the junction of the pubic bones. For pubic hair stage 4, the extent of hair has increased, but has not yet achieved the adult male escutcheon with spread to the medial aspects of the thighs that would be considered Tanner stage 5. Additional features include axillary hair, increased size of the larynx, voice break with deepening of the voice, increased bone mass, and increased muscle strength. Terminal hair develops in androgen-dependent regions on the face and trunk approximately three years after appearance of pubic hair. The distribution and density of beard, chest, abdominal, and back hair varies among individuals.

 

Peak height velocity is both age and sex-dependent. It occurs earlier in girls, between Tanner breast stages 2 and 3, and later in boys, between Tanner testis stages 3 and 4.

 

Approximately 50% of boys experience pubertal gynecomastia (17). Typically, pubertal gynecomastia is transient and most prominent in mid-puberty when the ratio of circulating estradiol to testosterone concentrations is relatively higher.

 

DISCOVERY OF THE HYPOTHALAMIC-PITUITARY-GONADAL (HPG) AXIS

 

Since ancient times, it was known that castration of animals and humans interfered with development of secondary sex characteristics and fertility (14). In 1935, Ernst Laquer and colleagues isolated testosterone from several tons of steer testes (18). Later that year, Adolf Butenandt, Gunter Hanisch, Leopold Ruzicka, and A. Wettstein published the chemical synthesis of testosterone (19, 20). After showing that follicular fluid obtained from a sow ovary was able to induce cornification of vaginal mucosa, Edgar Allen and Edward Doisy isolated the active substance, estrone (21). Donald MacCorquodale, Stanley Thayer, and Edward Doisy isolated estradiol from 8000 pounds of sow ovaries in 1935 (22). Philip Smith, Bernhard Zondek, Hermann Zondek, H.L. Fevold and colleagues, and Geoffrey Harris established the functional relationships involved in HPG axis function (23, 24, 25, 26). Roger Guillemin and Andrew Schally engaged in a vigorous competition to identify hypothalamic releasing hormones including GnRH (27, 28, 29). Ernst Knobil and his colleagues identified that pulsatile GnRH secretion was essential for sustained pituitary gonadotropin secretion (28, 30). Fred Karsch and Ernst Knobil independently developed the concept of the “GnRH pulse generator” (31). In the 1970s, Melvin Grumbach and colleagues measured circulating gonadotropin concentrations in the human fetus (32). Around the same time, Charles Faiman and Jeremy Winter also reported gonadotropin concentrations in normal and agonadal children (33). Their collective findings led to recognition of early postnatal HPG axis activity followed by quiescence of the HPG axis during childhood until resumption of GnRH pulse generator activity at the onset of puberty.

 

Ontogeny of GnRH Neurons

 

Reproductive competence depends on the meticulous development of the GnRH neuron system. In the human fetus, GnRH neurons initially develop in the olfactory placode outside the central nervous system. The olfactory placodes invaginate at approximately 39 days of gestation in the human. Based on the appearance of immunoreactive GnRH protein, the GnRH neuron specification occurs between 39-44 days of gestation (34). The developing GnRH neurons are associated with the embryonic vomeronasal organ. Available data suggest that the GnRH neuron precursors are distinct from those giving rise to the vomeronasal neurons (35).

 

Subsequently, the GnRH neurons migrate accompanied by olfactory-derived axons, olfactory epithelial sheath cells, and blood vessels towards the cribriform plate (36). Migration of the GnRH neurons seems to pause at the nasal/forebrain junction prior to crossing the cribriform plate (37). During this “pause” phase, multiple tissues, chemokines, growth factors, and neurotransmitters appear to form gradients influencing movement of GnRH neurons. Upon reaching the hypothalamus, the GnRH neurons disperse to their final locations sending projections to the median eminence to release GnRH into the hypophyseal portal vasculature.

 

The precise origin and particular factors responsible for the specification and differentiation of GnRH neuron precursors remain enigmatic. Inaccessibility of developing human GnRH neurons has led to development of alternative approaches to elucidate the history of GnRH neurons. One approach has involved a protocol to generate GnRH neurons from human pluripotent stem cells (38). With this approach, Yellapragada et al. demonstrated that dose- and time-dependent FGF8 signaling via FGFR1 is indispensable for human GnRH neuron ontogeny (39). Using a differentiation trajectory analysis approach, DLX family of transcription factors have been reported to promote in vitro human GnRH neuron differentiation (40).

 

Components of the HPG Axis

 

Gonadotropin-releasing hormone is a decapeptide (pGlu-His-Trp-Ser-Trp-Gly-Leu-Arg-Pro-Gly-NH2) derived from a 92-amino acid precursor, preproGnRH, that was characterized in 1984 (41). LH and FSH are synthesized in the same gonadotroph cell located in the anterior pituitary. LH and FSH are glycoproteins consisting of two subunits. The alpha subunits are identical whereas the beta subunits confer hormone specificity. Each GnRH pulse stimulates an LH pulse.

 

During human gestation, human chorionic gonadotropin (hCG) drives fetal testicular testosterone secretion in the developing male fetus early during gestation. The pituitary gland begins to secrete gonadotropins with LH and FSH becoming detectable in fetal blood after 14 weeks of gestation (42, 43). Initially, pituitary gonadotropin secretion appears to be GnRH-independent with progressive transition to kisspeptin-GnRH regulation of pituitary gonadotropin secretion during the third trimester (44). Peak gonadotropin concentrations occur around the midpoint of gestation followed by a progressive decline attributed to suppression by placental estrogens (45). In the male fetus, testicular testosterone secretion is essential for normal development of internal and external male genital structures. Comparatively, the fetal ovary is quiescent.

 

As noted above, GnRH stimulates pituitary LH and FSH secretion. LH and FSH signal through their cognate receptors which are G-protein coupled receptors (46).

 

GONADS

 

The gonads synthesize sex steroids from cholesterol. In the testis, LH acting through the LH receptor stimulates conversion of cholesterol to testosterone in the Leydig cell. In specific target tissues such as external genital skin and the prostate, testosterone is converted to dihydrotestosterone by the enzyme, 5α-reductase type 2 encoded by the SRD5A2gene. Testosterone influences pituitary LH secretion through negative feedback either via direct actions or indirectly after conversion to estradiol. FSH acting through the FSH receptor promotes growth of seminiferous tubules and supports sperm development. Growth of the seminiferous tubules and increasing numbers of germ cells accounts for increasing testicular volume during puberty.

 

In females, the two cell-two gonadotropin model applies to ovarian steroidogenesis. LH stimulates the theca cell to synthesize androstenedione which diffuses to the granulosa cell where FSH-stimulated aromatase activity stimulates estradiol synthesis. Estradiol has both negative feedback and positive feedback. Estradiol mediated positive feedback is required to elicit the LH surge responsible for ovulation.

 

Activin and inhibin are heterodimeric glycoproteins secreted by the gonads. Inhibins consist of an alpha subunit and one of two homologous yet distinct beta subunits, βA or βB. Inhibin B is composed of an alpha subunit and a βB subunit whereas inhibin A consists of an alpha subunit and a βA subunit. Inhibins are secreted by Sertoli cells in the testes and granulosa cells in the ovary. Inhibin B influences pituitary FSH secretion by negative feedback. In prepubertal boys, inhibin B concentrations reflect Sertoli cell mass and function. After puberty, inhibin B concentrations reflect spermatogenesis (47). Inhibin B correlates inversely with FSH levels in adult men. Activins are dimers of inhibin β subunits, βA, βB and βC; the best characterized are activin A (βAβA) and activin B (βBβB). Activin A stimulates pituitary FSH secretion(48, 49). Follistatin is a monomeric protein that modulates activin activity and can irreversibly inhibit activin activity.

 

Leydig cells secrete insulin-like peptide 3 (INSL3), a small peptide that, in utero, acts through the relaxin-like family peptide receptor 2 (RXFP2) to promote trans-abdominal testicular descent. INSL3 concentrations increase in boys during puberty (50).

 

HYPOTHALAMUS

 

The hypothalamus serves as the rheostat for many physiological functions especially reproduction and growth. The adult human hypothalamus contains approximately 2000 GnRH neurons with cell bodies diffusely distributed in a rostro-caudal continuum (34). The GnRH neurons send projections to the median eminence that terminate in close association with the capillaries of the primary plexus of the hypophyseal portal circulation. Synchronized activity of the GnRH neurons leads to episodic GnRH release into the median eminence with consequent pulsatile pituitary gonadotropin secretion.

 

An extrinsic hypothalamic neuronal network, known as the GnRH pulse generator, governs GnRH neuron function. This network is located within the infundibular nucleus (known as the arcuate nucleus in non-human species). In the human, the GnRH pulse generator is responsible for tonic gonadotropin secretion; pulsatile LH and FSH secretion regulate testicular function in men and modulate ovarian function, especially folliculogenesis in women. In women, the developing follicle secretes increasing amounts of estradiol ultimately triggering an LH surge followed by ovulation. In adult men, pulse frequency is relatively constant at approximately one pulse every 90-120 minutes. Among women, pulse frequency varies across the menstrual cycle from approximately one pulse per hour during the follicular phase and one pulse every 180 minutes during the luteal phase.

 

Among GnRH deficient women, pulsatile GnRH administered at a frequency simulating the follicular phase led to ovulatory menstrual cycles (51). In a preclinical model, administration of pulsatile GnRH to prepubertal rhesus female monkeys initiated pubertal development including ovulatory menstrual cycles (52). Thus, puberty in girls and boys is entirely dependent on resumption of pulsatile GnRH release.

 

Although the GnRH pulse generator was conceptualized by Fred Karsch and Ernst Knobil, the anatomic location of the pulse generator was indeterminant. Identification of loss of function variants in the kisspeptin receptor (KISS1R) gene in patients with congenital hypogonadotropic hypogonadism launched the investigations establishing kisspeptin, neurokinin B, dynorphin, and their cognate receptors as major components of the pulse generator (53, 54). Kisspeptin signals through its receptor, KISS1R, expressed on GnRH cells. Neurokinin B is a decapeptide encoded by the TAC3 (Tac2 in rodents) gene and its cognate receptor encoded by NK3R gene. Both the kisspeptin and neurokinin B receptors are G-protein coupled receptors. Dynorphin is an opioid peptide that signals through a kappa-opioid receptor which is also a G-protein coupled receptor.

 

Due to the inaccessibility of human brain, especially the pubertal brain, the contemporary model of the GnRH pulse generator has been delineated by preclinical studies performed in rodents, sheep, and non-human primates (55). This model predicts that reciprocal interactions within a network of kisspeptin neurons in the infundibular nucleus leads to synchronous intermittent activation transmitted to GnRH neurons by kisspeptin fibers that project to the median eminence. These kisspeptin fibers are closely associated with GnRH projections targeting the portal capillaries (56).

 

Based on the detection of kisspeptin, neurokinin B, and dynorphin in the arcuate kisspeptin neurons of mice and sheep, these neurons have been labeled as KNDy neurons (57). Preclinical data suggest that KNDy neurons serve as the intrinsic GnRH pulse generator (58). Kisspeptin and neurokinin B stimulate GnRH release whereas dynorphin appears to be inhibitory. Coordinated interactions of these neuropeptides within the arcuate kisspeptin neuronal network are ostensibly central to the neurobiology of the GnRH pulse generator resulting in pulsatile kisspeptin output. However, the applicability of these findings to human biology remains to be confirmed.

 

In humans, the HPG axis is active during gestation and the early neonatal period followed by the quiescent years of childhood until the onset of puberty occurs. This pattern suggests that diverse mechanisms integrate the hierarchical activation and deactivation of various stimulatory and inhibitory neuronal pathways ultimately regulating pubertal onset and progression towards reproductive maturity. Thus, a central inhibition of the axis occurs during childhood. For puberty to occur, increased expression of the key factors, KISS1, NKB3, and GnRH, must begin along with decreased expression of the various inhibitory factors. In other words, during the pubertal transition, the balance between inhibitory and stimulatory factors shifts to favor the re-activation of the HPG axis, onset of pubertal changes, and reproductive competence.

 

Identifying the proximate factors and specific interactions responsible for the on-off-on pattern of HPG axis activity in humans has been a longstanding enigma. Starting with clinical findings, the availability of more sophisticated tools and preclinical models have begun to identify pieces of the puzzle to elucidate the fine details of HPG axis functioning. One factor involved in the suppression of puberty was identified in families with paternally inherited GnRH-dependent/central precocious puberty (CPP). Exome sequencing analyses in multiple families with CPP identified loss of function variants in the makorin 3 (MKRN3) gene (59). This gene, mapped to the Prader Willi region at chromosome 15q11.2, is exclusively expressed from the paternal allele. Consistent with the hypothesis that MKRN3 suppresses the GnRH pulse generator, circulating MKRN3 concentrations decline during puberty (60, 61, 62). 

 

The MKRN3 protein is an E3 ubiquitin ligase consisting of 507 amino acids. It is expressed in KNDy neurons. The protein has five zinc finger domains. Regarding its function, the protein can ubiquitinate substrates and can undergo auto-ubiquitination (63). MKRN3 ubiquitinates methyl-CpG-DNA binding protein 3 (MBP3) interfering with GnRH1transcription (64). Available preclinical data suggest that MKRN3 functions as a brake on neuronal GnRH release (65). One potential factor influencing MKRN3 expression is microRNA (miRNA) miR-30. Using a rat model, hypothalamic miR-30 expression increased while Mkrn3 expression decreased during puberty. In addition, treatment with agents that interfered with the binding of miR-30 to Mkrn3 were associated with delayed puberty in female rats (66). Using proteomics, MKRN3 targets include insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) and several members of the polyadenylate-binding protein family (67). The decline of hypothalamic Mkrn3 expression in mice and serum MKRN3 protein levels in females prior to the onset of puberty support the hypothesis that MKRN3 suppresses pubertal initiation possibly through effects on prepubertal hypothalamic development and plasticity (61, 67)

 

Preclinical studies have provided persuasive evidence regarding the regulatory role of epigenetic modifications in pubertal maturation. Epigenetics refers to changes in gene expression and/or activity independent of changes in the primary nucleotide sequence (68). Epigenetic changes include DNA modifications such as methylation/demethylation and histone post-translational modifications such as acetylation/deacetylation. Other post-translational protein modifications such as ubiquitination may also influence protein function. Ubiquitination involves the transfer of ubiquitin to a protein altering its function typically by interfering with protein actions or by promoting protein degradation. As noted above, the MKRN3 protein can function as a ubiquitin ligase. Noncoding RNAs such as miRNAs provide yet another regulatory mechanism.

 

Another example of epigenetic regulation of pubertal maturation involves two mutually antagonistic histone methylating complexes, the Poly-comb and Trithorax groups. The Poly-comb group represses gene transcription while the Trithorax group appears to function as a gene activator. Preclinical studies performed in rats showed that the Poly-comb group effectively silenced Kiss1 expression until the onset of puberty when increased methylation of the Eed and Cbx7 genes occurred leading to decreased Eed and Cbx7 expression and increased Kiss1 expression (69). Recruitment of the Trithorax activity group further enhanced.  Kiss1 expression (70, 71). Genome wide association studies have implicated zinc finger (ZNF) genes. In nonhuman primates, expression of two ZNFs, GATAD1 and ZNF573, decreases upon pubertal reactivation of the GnRH pulse generator (71).

 

Clinically, it has long been recognized that extremes of body energy status such as chronic malnutrition or severe obesity influence the HPG axis especially in girls and women. The hypothalamic kisspeptin neurons integrate various peripheral and central metabolic signals reflecting energy intake, energy expenditure, and environmental circumstances. Signal coordination between reproductive and metabolic neurons can be direct or indirect. For example, leptin does not directly regulate kisspeptin neurons yet acts as a permissive factor for the onset of puberty (72). Cellular energy and metabolic sensors include mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), and sirtuin 1 (SIRT1) (73). Depending on energy status, mTOR and AMPK promote or repress puberty, respectively, by activating or inhibiting Kiss 1 neurons in the arcuate nucleus. Other factors such as melanocortin and agouti-related peptides also interact with kisspeptin pathway (74). In the hypothalamus, neuronal nitric oxide (NO) appears to act on GnRH neurons to integrate metabolic and gonadal information (75, 76). Detailed reviews regarding the neurobiology of the GnRH pulse generator are beyond the scope of this chapter and are available elsewhere (77, 78, 79, 80, 81, 82).

 

MINI-PUBERTY

 

Facilitated by the availability of more sensitive hormone assays, Forest and her colleagues described a transient period of increased HPG axis activity in early infancy (83, 84). Following the low gonadotropin concentrations at birth, gonadotropin concentrations were found to rise in both boys and girls within weeks of birth (85). This period of transient gonadotropin secretion has been designated as “minipuberty”. Gonadotropin concentrations in the immediate neonatal period are likely low due to in-utero suppression by placental estrogen. With removal of the placental estrogen suppression, the HPG axis is functional. Relevantly, physical findings typical of pubertal sex steroid secretion are absent with the rare exception of vaginal bleeding attributed to decreased exposure to placental estrogen.

 

Over the first few years of life, sexual dimorphism in gonadotropin concentrations occurs (86) Boys have higher LH concentrations which peak between 2-10 weeks of age and decline by 4-6 months of age. Girls have higher FSH concentrations which may remain elevated until 2-4 years of age.

 

In boys, LH stimulates testicular testosterone secretion with testosterone concentrations typically peaking around 1 month of age followed by a decline to prepubertal concentrations by 7-12 months of age. During this phase, the number of germ cells and Sertoli cells increase and penile size increases (87, 88). The proliferation of Sertoli cells leads to a transient increase in testicular volume (89). Sertoli cells secrete Anti-Mullerian Hormone (AMH) and inhibin B. Since Sertoli cells do not express androgen receptors during this stage, spermatogenesis does not occur and AMH secretion remains high (90, 91). A temporary increase in the number of Leydig cells also occurs, but subsequent fetal Leydig cell apoptosis reduces fetal Leydig cell number (92). Longitudinal data obtained from healthy boys suggests a temporal dissociation of Leydig and Sertoli cell activity during minipuberty (93). These data suggest that single blood sample may be insufficient to assess HPG axis activity during early infancy and that obtaining several consecutive samples may be more informative. Curiously, gonadotropin and testosterone concentrations are higher among preterm boys. In addition, increases in testicular volume and penile length are greater in preterm boys compared to full term boys essentially enabling catch-up for testicular volume and penile length (94). Some small studies have documented an exaggerated physiologic hormonal response in extremely premature infants (95).

 

In girls, the gonadotropins promote granulosa cell proliferation and ovarian estrogen and AMH secretion (96). As would be anticipated, AMH concentrations remain much lower in girls compared to boys (97). A longitudinal study involving healthy full-term infant girls demonstrated two gonadotropin peaks in early infancy with one peak occurring around days 15 to 27 and a later peak occurring at days 164-165 (98). Again, collecting several consecutive samples may be more informative than a single blood sample to assess for minipuberty in infancy.

 

This transient time period of an active HPG axis, provides an opportunity to diagnose individuals with differences/variants of sex development (DSD/VSD). In a series including both healthy infants and infants with DSD, testosterone measured by LC-MS/MS, AMH concentration, and LH/FSH ratio provided the best discrimination between sexes. The cut-point for LH/FSH ratio was 0.32. Inhibin B and AMH levels were higher in boys with minimal overlap in girls (99). Infants with Turner Syndrome usually have elevated FSH concentrations. Surprisingly, gonadotropin concentrations are typically not elevated in patients with complete androgen insensitivity.

 

This brief interval of HPG axis activity can also help diagnose congenital hypogonadotropic hypogonadism in boys who present with micropenis accompanied by low gonadotropin and testosterone concentrations (100). Testosterone, LH, FSH, AMH, and inhibin B concentrations may provide information regarding the functionality of testicular tissue in infant boys (101).

 

As noted above, the human HPG axis displays an “on-off-on” pattern. The biological basis and rationale for transient post-natal HPG axis activity during the first few months of life are enigmatic. At birth, the brain is still plastic with ongoing development. Most axon and synapse formations are completed during the first year of life. Does this transient HPG axis activity imprint specific areas in the brain? Does minipuberty influence future patterns for female and male reproductive function with cyclic gonadotropin patterns in females and not in males? Are gonadal hormones during infancy able to affect future fertility, gender identity, sexual orientation, behaviors, and risk for autism spectrum dysfunction? Data are accruing regarding patterns of hormone secretion during the first six months of life. However, the factors that initiate and terminate this transient period of HPG axis activity and maintain the quiescence of the HPG axis until the onset of puberty are still unknown.

 

 

Over the past few decades, several studies have observed that puberty is beginning at a younger age. Clinical studies examining ages of the onset of puberty depend on the criteria used to denote puberty. Onset of breast development and age at menarche are the conventional indicators of puberty in girls. Prospective observations and retrospective questioning of parents and young girls through in-person questioning has been used to record age at menarche; shorter recall intervals provide the greatest accuracy regarding the details of menarche (102, 103). For boys, age at voice change has been used as a surrogate marker because accurate ascertainment of pubertal onset in boys requires testicular exams using an orchidometer, thus, effectively excluding large-scale epidemiologic clinical studies (104).

 

During medieval times, available evidence suggests that puberty began around 10-12 years of age. However, the tempo of puberty was slow with menarche occurring closer to 15 years in rural areas and 17 years in London (105). Presumably, undernutrition, increased infections, and greater physical exertion impacted both the timing and tempo of puberty during medieval times (106). The age of menarche declined from 16 to 17 years in the early 19th century to 13 years of age in the late 20th century in Europe and North America. Similarly, the age at menarche has declined in the Yunnan Province in China (107). This decline has been attributed to the improvement in socioeconomic conditions. Currently, the dialogue continues as to whether the trend towards earlier puberty is persisting and, if so, what are the factors driving this process. 

 

Data regarding pubertal milestones in American girls were obtained through the cross-sectional Third National Health and Nutrition Examination Survey (NHANES III) between 1988 and 1994.  Among these American girls, mean ages in years for breast development, pubic hair development, and menarche were 9.5, 9.5, and 12.1 for non-Hispanic black girls; 9.8, 10.3, and 12.2 for Mexican-American girls; and 10.3, 10.5, and 12.7 years for non-Hispanic white girls, respectively (108). In 1997, the Pediatric Research in Office Settings (PROS) study reported earlier onset of thelarche with the caveat that breast palpation was not performed (109). The Copenhagen Puberty Study reported that mean age at breast development was lower in the 2006 cohort compared to the 1991 cohort whereas mean age at menarche was similar in both cohorts. Independent of BMI, gonadotropin concentrations were comparable between these cohorts while estradiol concentrations were lower in the 2006 cohort (110).

 

Beginning in 2004, the Breast Cancer and Environment Research Program (BCERP) prospectively recruited three cohorts of girls aged 6-8 years. This program recruited non-Hispanic white, Hispanic, non-Hispanic black girls, and Asian girls living in New York, Ohio, and California. The overall median age at menarche was 12.25 years with ethnic background median ages as follows: Hispanic girls 11.6 years, black girls at 11.8 years, white girls at 12.5 years, and Asian girls at 12.0 years (111). This cohort differed from the NHANES III study because Hispanic girls experienced menarche earlier than the black girls. These studies, all performed in the United States, report race and ethnicity-related differences in onset of pubertal milestones. Detailed assessment of the potential impact of socio-economic factors was not performed. Notably, differences noted in pubertal timing are smaller than the overall variation among individuals in the population. Most importantly, clinical decision-making should reflect an individual patient's characteristics and family history with less dependence on racial or ethnic backgrounds.

 

Comparable studies from Spain and Greece have also reported earlier onset of breast development and slower pubertal tempo (112, 113). Thus, available data including a systemic review of international studies largely confirm the ongoing trend for earlier breast development with minimal decline in age at menarche (114).

 

Several questions regarding this earlier onset of puberty, predominantly earlier thelarche, need to be considered. Does this earlier breast development reflect earlier resumption of GnRH pulse generator activity, extragonadal estrogen production, or environmental exposures? What, if any, is the relationship of BMI to puberty? Another consideration is that race/ethnicity are socio-political constructs and are not fully representative of biology. While genetic ancestry likely influences the onset of puberty, nutritional factors and environmental exposures play important roles. Hence, should cut-off points based primarily on race/ethnicity continue to be utilized?

 

Based on single unstimulated gonadotropin concentrations, data from the Copenhagen puberty in girls study suggest that gonadotropin concentrations are not obviously increased in girls with early thelarche. Thus, the phenomenon of early thelarche appears to be independent of gonadotropin secretion and may not signify early resumption of GnRH pulse generator activity (115).

 

The possibility that exposure to endocrine-disrupting chemicals (EDCs) can induce early thelarche has been questioned. EDCs are defined as exogenous chemicals that interfere with hormone action. EDCs include phthalates, phenols, phytoestrogens, organochlorine pesticides, polybrominated flame retardants, diphenyl ethers, heavy metals, and perfluorochemicals. In addition to pesticides, these chemicals can be found in common household products such as hair products, soaps, toothpaste, perfumes, plastics, essential oils, and cleaning products (116). Valid assessment of the consequences of EDCs on puberty is problematic because exposure may occur in utero and generally involves a mixture of assorted EDCs with differing half-lives and activities. Differences in the duration and route of the exposure(s), methodology to detect EDCs, and potential sample contamination further confound analyses. One potential example regarding EDCs involved transient past exposure to organochlorine pesticides among internationally adopted girls in Belgium who subsequently developed precocious puberty (117). Animal models suggest that EDCs can affect puberty through epigenetic mechanisms (118). Nevertheless, most data available regarding the consequences of EDCs on human puberty are inconclusive (119).

 

Relationship with BMI

 

Observational data has shown a relationship between BMI and age at puberty in girls (120, 121). The BCERP study found that girls who were overweight or obese at baseline experienced menarche 0.3 years earlier with age at thelarche being inversely correlated with BMI. The BCERP also concluded that BMI had a greater effect than ethnic background on age at menarche (111). Limited data exist regarding the relationship of BMI to pubertal onset in boys. The Puberty Cohort of the Danish National Birth Cohort reported that increased BMI was associated with earlier onset of puberty in boys and girls (122). Among boys, pubertal milestones, testicular enlargement, voice break, and testosterone concentrations showed inverse correlation with BMI (104). Hence, available evidence strongly indicates an inverse relationship between BMI and the onset of puberty in both boys and girls.  

 

Yet, investigating the relationship between puberty and BMI is confounded by potential hormonal and genetic influences (123). Obesity may be associated with hyperinsulinemia and lower sex hormone binding globulin concentrations with consequent higher free sex steroid concentrations. In addition, some genes influence both BMI and pubertal timing (124, 125). The pro-opiomelanocortin (POMC) and central melanocortin systems provide one example of the intricate interrelationships between nutrient signaling and reproductive function. Neurons expressing POMC, producing α-MSH (melanocyte-stimulating hormone), have been suggested to stimulate puberty onset and gonadotropin secretion via modulation of arcuate Kiss1 neurons (126, 127).

 

Genetic Factors

 

Genetic  factors influence pubertal timing as evidenced by twin studies demonstrating > 50% hereditability for menarche (128). Skeletal maturation, age at pubertal growth spurt, and Tanner staging also show greater concordance between monozygotic twins compared to dizygotic twins emphasizing the relevance of genetic variation in the timing of puberty. Thus, 50-80% of variation in the timing of puberty onset may reflect genetic variation (129). Parental self-reports regarding pubertal timing are associated with timing of specific pubertal milestones in offspring of the concordant sex (130, 131). Genome-wide association studies (GWAS) have detected loci associated with age at menarche (132). Some loci appear to be common and independent of ancestry. A large-scale trans-ethnic GWAS, involving 38,546 women of diverse and predominantly non-European ancestry or ethnicity, identified a novel locus in chromosome 10p15 that is associated with early menarche. This region maps to intron 7 of the aldo-keto reductase Family 1, member C4 (AKR1C4) gene, a member of family of enzymes involved in steroid metabolism and action (133).

 

To summarize, the secular trends suggesting an earlier onset of puberty appear to be persistent although the age at menarche appears to be relatively static. Likely contributing factors include the rising prevalence of obesity, exposure to potential EDCs, specific dietary influences, and decreased physical activity.

 

VARIATIONS IN PUBERTAL DEVELOPMENT

 

Timing of the onset of puberty reflects complex interactions between hormonal and neuronal signals with genetic, metabolic, and environmental factors. These interactions presumably begin early in development and ultimately lead to the re-activation of the HPG axis concomitant with the onset of puberty. Multiple factors, both known and unknown, influence the reactivation of the GnRH pulse generator modulating pubertal onset. As noted above, familial patterns of pubertal development and twin studies highlight the role of genetic factors. Studies of families with either delayed or precocious puberty led to discovery of genes relevant to pubertal onset. In addition, genetic factors including single nucleotide polymorphisms (SNPs) have been associated with pubertal timing in both sexes and across ethnic groups. Epigenetic mechanisms have been suggested to affect the development and function of the GnRH neuronal network ultimately influencing HPG axis function. How confounders such as socioeconomic, environmental, and nutritional status influence pubertal development is unclear. These factors can influence puberty timing, HPG axis function, and fertility.

 

Precocious puberty is defined as the development of puberty prior to age 8 in girls, and age 9 in boys (134, 135). In girls, delayed puberty is defined as the absence of breast development by age 13 years, absence of menarche by age 15 or lack of menses after 3 years since breast development. In boys, delayed puberty is defined as absence of pubertal development by age 14 (136). Evaluation of a child with abnormal timing of puberty entails thorough knowledge of normal pubertal development, typical variations of normal pubertal development, and causes of abnormal pubertal development. The next section focuses on the evaluation of a patient presenting with a variation in pubertal development.

 

PRECOCIOUS PUBERTY  

 

Traditionally, the diagnosis of precocious puberty is considered when signs of puberty develop prior to 8 years of age in girls and 9 years in boys (137). These ages are based on Tanner’s original observations on English children regarding typical ages at specific pubertal stages. However, these age criteria should be used as guidelines to complement the evaluation of individual patients. Precocious puberty can be categorized as central or gonadotropin-dependent precocious puberty (CPP) or non-gonadotropin-dependent or peripheral precocious puberty (PPP). Additionally precocious puberty can be further classified as familial or sporadic and syndromic or non-syndromic. The specific etiologies and management differ between the two broad categories of CPP or PPP. Potential consequences of early puberty and menarche in girls include increased risks for breast cancer and diabetes as adults (138, 139).

 

Central Precocious Puberty or Gonadotrophin Dependent Precocious Puberty

 

Central precocious puberty (CPP) is associated with early maturation of the HPG with premature reactivation of the GnRH pulse generator and sequential maturation of breasts and pubic hair in females. In males, sequential maturation of testicular volume, penile enlargement, and pubic hair is observed. Typically, the pubertal characteristics are appropriate for the child's sex (isosexual). Despite the earlier onset of puberty, the sequence of pubertal events is usually normal. CPP is due to organic lesions in approximately 40-100 percent of boys whereas idiopathic precocious puberty is the most common diagnosis in girls (69-98%) (140). These children have accelerated linear growth for age, advanced bone age, and pubertal levels of LH and FSH. A Spanish observational report described an annual incidence of CPP ranging between 0.02 and 1.07 new cases per 100,000 (141) while a Korean study reported an incidence of 15.3 per 100,000 girls, and 0.6 per 100,000 boys (142). Distinguishing among CPP, isolated premature thelarche, and premature adrenarche is important because the pathophysiology and therapeutic interventions differ.

 

CNS LESIONS/INSULTS

 

CPP can be associated with central nervous system lesions. Hamartomas of the tuber cinereum are congenital benign lesions comprised of heterotopic gray matter, neurons, and glial cells. The prevalence is approximately 1 in 200,000 children (143). Hamartomas are the most commonly recognized CNS lesions associated with CPP in very young children. Hamartomas can be categorized as para-hypothalamic, attached or suspended from the floor of the third ventricle, or as intrahypothalamic, in which the mass is enveloped by the hypothalamus and distorts the third ventricle. The lesions do not grow over time, do not metastasize, and do not produce β-human chorionic gonadotropin-(β-hCG). In some instances, hamartomas are associated with gelastic (laughing or crying) seizures. Yet, most patients with hypothalamic hamartomas do not display neurological symptoms (144, 145). Most hypothalamic hamartomas are sporadic and appear to be idiopathic. Hypothalamic hamartomas can also occur in Pallister-Hall Syndrome (PHS) and oral-facial-digital syndrome (OFD) types I and VI (146). Genetic variants in the sonic hedgehog pathway have been associated with hypothalamic hamartoma (147, 148). The mechanism(s) through which hypothalamic hamartomas lead to CPP is unknown. Hamartoma located close to the infundibulum or tuber cinereum are often associated with CPP whereas those functionally connected to the mammillary bodies and limbic circuit are typically associated with epilepsy without CPP (149, 150). As discussed below, medical treatment is usually indicated for hypothalamic hamartomas associated with CPP. Surgical treatment should be limited to large hamartomas complicated by severe refractory drug-resistant epilepsy (151).

 

CNS tumors such as astrocytomas, ependymomas, and pinealomas have rarely been associated with CPP. Among girls, factors associated with CNS lesions include: (1) age younger than 6 years; (2) absence of pubic hair; and (3) estradiol concentrations greater than 30 pg/ml (110 pmol/L) (152, 153). As noted above, suspicion for CNS lesions is higher for boys than for girls.

 

Neurofibromatosis type 1 (NF1) is an autosomal dominant multi-system neurocutaneous disorder due to loss-of-function variants in the neurofibromin-1 (NF1) gene located at chromosome 17q11.2. NF1 is often associated with CPP typically due to optic glioma. The glioma is usually a benign pilocytic astrocytoma that can occur anywhere along the optic tract; the most common locations are within the optic nerve or chiasm. CPP has also been described in NF1 in the absence of optic glioma (154). Children with meningomyelocele and spina bifida also have an increased incidence of CPP. Although the precise mechanism responsible for CPP in these children is unclear, associated factors may include increased perinatal intracranial pressure and brainstem malformations such as Chiari II malformations (155). The mechanistic link between CPP and Rathke cleft cysts, Chiari malformation, and pineal and arachnoid cysts is unclear.

 

Septo-optic dysplasia (SOD) is a heterogeneous congenital condition characterized by presence of at least two features of the classic triad which include optic nerve hypoplasia, anterior pituitary hormone deficiencies, and midline brain anomalies. SOD is associated with genetic variants in HESX1, SOX2, SOX3, and OTX2 genes. Although SOD is typically associated with delayed puberty, CPP can occur (156, 157).

 

CNS tumors may be treated with CNS irradiation (158). In some instances, CNS irradiation is associated with acquired CPP (159). In this situation, concurrent growth hormone (GH) deficiency may be present. The linear growth spurt of CPP may mask the decreased linear growth velocity due to GH deficiency. Hence, in this setting, consideration should be given to evaluating the GH axis by provocative GH testing. If testing shows GH deficiency, the patient may benefit from treatment with GH combined with GnRH agonist therapy. Rarely, CPP occurs following head trauma and can develop many years after the injury(160, 161).

 

SECONDARY CPP

 

Some children exposed to elevated circulating high sex steroid concentrations occurring in other disorders such as McCune-Albright syndrome, congenital adrenal hyperplasia, and virilizing adrenocortical tumors may develop a secondary CPP (163). These individuals typically have accelerated bone age maturation. The precise mechanism responsible for development of the secondary CPP is unclear. The secondary CPP may represent a priming effect of sex steroids on the hypothalamus or potentially as the consequence of the acute decrease in sex steroid concentrations with treatment of the underlying etiology (164) (165).

 

NON-SYNDROMIC CPP

 

Specific genetic variants have been associated with non-syndromic CPP (See Table 1) (166). Loss of function MKRN3variants are the most reported genetic cause of familial CPP. Paternally inherited loss-of-function MKRN3 variants have been reported in up to 33-46 percent of familial cases of CPP and nearly 0-20% percent of sporadic cases (167) . To date, at least 70 deleterious MKRN3 variants have been identified in patients with CPP. These variants lead to diminished inhibition of puberty results in early onset of puberty. Differing ubiquitination patterns suggests that MKRN3 has multiple molecular mechanisms associated with CPP (168). Curiously, a GWAS study investigating parental effects on pubertal development reported that the paternal allele of a specific SNP (rs12148769, G>A) in MKRN3 was associated with age at menarche in healthy girls suggesting that variants in this region affect pubertal timing within the normal range (132). Although circulating MKRN3 concentrations decrease with onset of puberty, peripheral blood MKRN3 concentrations are not adequately sensitive to distinguish CPP (169).

 

TABLE 1. Genes Associated with Central Precocious Puberty (175, 461)

 

Gene

(Reference/s)

Protein encoded

Genetic locus

Comments

MKRN3

(59, 63, 167, 462)

Makorin ring finger protein 3

15q11-q13

Loss-of-function mutation

KISS1R (previously named GPR54)

(463, 464, 465)

Kisspeptin receptor

19p13.3

Gain-of-function mutation

KISS1

(465)

 

Kisspeptin

1q32

Gain-of-function mutation

DLK1

(466, 467, 468)

Delta-like homolog 1

14q32

-Loss-of-function mutation

-Metabolic abnormalities (obesity, type 2 diabetes, hyperlipidemia)

ESR1

(469, 470)

Estrogen receptor 1

6q25.1-q25.2

Mutations/polymorphisms, epigenetic change

CYP19A1

(471)

 

Aromatase

15q21

(TTTA)n polymorphism, epigenetic change

 

Evaluation of another family with CPP led to identification of a loss of function variant in the delta-like 1 homologue (DLK1) gene. DLK1, also known as preadipocyte factor 1, plays a role in the Notch signaling pathway. DLK1 is a paternally expressed gene located at chromosome 14q32.2. Two differentially methylated regions influence the DLK1 imprinting pattern. DLK is located within the genetic locus associated with Temple syndrome. Temple syndrome is characterized by prenatal growth retardation, hypotonia in infancy, motor delay, small hands, CPP, and short stature. In addition to DLK1 loss, two other genes from the paternally inherited chromosome, RTL1 and DIO3, results in Temple Syndrome. Genetic findings associated with Temple syndrome include maternal uniparental disomy, paternal deletion, or loss of differential methylation at the DLK1/MEG3 region on chromosome 14 (170). Women with DLK1 variants also have a metabolic phenotype characterized by overweight/obesity and insulin resistance (171).

 

Gain-of-function variants in the kisspeptin 1 gene (KISS1) and its cognate receptor, KISS1R, gene have been identified in children with CPP. A heterozygous variant in the KISS1 gene, p.Pro74Ser, was identified in a boy who developed CPP at one year of age; in vitro studies suggested that this variant was more stable than the normal protein leading to a prolonged duration of action (172). A girl with precocious puberty was found to have a variant in the KISS1R gene; in vitro studies of this p.Arg386Pro variant showed prolonged activation of the intracellular signaling pathways following kisspeptin stimulation (173, 174).

 

Among a series of 586 children with familial CPP, both maternal and paternal inheritance patterns were found. Variants in MKRN3 were the most common cause in paternally inherited CPP. Among the maternally inherited cases, genetic analysis detected rare variants of unknown significance (175).

 

SYNDROMIC CPP

 

In addition to genetic and idiopathic CPP, CPP can occur as a feature in specific syndromes. Pallister-Hall and Temple Syndrome are described above. Other syndromes associated with CPP include Cowden and Cowden-like cancer predisposition syndromes associated with PTEN, SDHB-D and KLLN gene variants. These disorders are characterized by multiple multisystemic hamartomas which may be associated with CPP when the skull base, infundibulum, or hypothalamus are affected. Although Prader-Willi syndrome is typically associated with delayed puberty, CPP has also been reported (176). Other genetic syndromes associated with CPP include tuberous sclerosis and Williams-Beuren (See Table 2). Williams-Beuren is associated with genetic variant at chromosome 7q11.23 (177). Rare cases of precocious puberty have also been reported in Russell Silver syndrome (178).

 

Table 2. Syndromic Causes of Central Precocious Puberty Without CNS Lesions (CPP)

Gene (Reference/s)

Genetic locus

Comments

MECP2

methyl-CpG-binding protein 2

(472)

 

Xq28

Rare forms of Rett syndrome

X-linked dead-box helicase 3

(461)

Xp11.4

Neurodevelopmental delay

Xp22.33 deletion, SHOX region

(473)

Xp22.33

Body disproportion, short stature, Madelung deformity

Xp11.23-p.11.22 duplication

(474)

Xp11.23-p11.22

Intellectual disability, speech delay, electroencephalogram abnormalities, excessive weight, skeletal anomalies

Temple syndrome

-DLK1

Maternal uniparental disomy or paternal deletion

(170, 473)

14q32.2

Imprinting defect, act via DLK1,

Prenatal and postnatal growth failure, hypotonia, small hands and/or feet, obesity, motor delay

Prader-Willi syndrome

- MKRN3

Paternal deletion or maternal uniparental disomy of chromosome 15q11-q13

(475)

15q11-q13

Changes to the imprinted MKRN3 and/or MAGEL2genes

Hypotonia, obesity, growth failure, cognitive disabilities, hypogonadism

Silver-Russell syndrome

Hypomethylation of chromosome 11p15 or maternal uniparental disomy of chromosome 7       

(476)

 

11p15.5

Possible imprinted or recessive factors, not well elucidated,

Prenatal and postnatal growth retardation, relative macrocephaly, prominent forehead, body asymmetry, feeding difficulties

Williams-Beuren

(177, 477, 478)

7q11.23

Distinct face, cardiovascular disease, short stature, intellectual disability, hyper-sociability

Kabuki syndrome

(479)

12q13.12

Downregulation of estrogen receptor activation

Neurodevelopmental phenotypes, typical distinct face, short stature

Mucopolysaccharidosis type IIIA or Sanfilippo disease

(480)

17q25.3

Severe neurologic deterioration, visceromegaly, skeletal abnormalities

 

NONPROGRESSIVE PRECOCIOUS GONADARCHE

 

Some children experience a nonprogressive (or slowly progressing) CPP (179). Typically, basal gonadotropin concentrations are prepubertal. In general, children with nonprogressive CPP show no or minimal pubertal responsiveness to GnRH stimulation. Height potential is generally unaffected. Typically, these individuals do not usually benefit from GnRH-Ra therapy. Physical findings alone cannot distinguish between progressive and nonprogressive CPP. Presumably this early pubertal development reflects a transient premature activation of the GnRH pulse generator. Longitudinal follow-up to assure that puberty is not progressive is the most appropriate management.

 

GONADOTROPH ADENOMA

 

The anterior pituitary gland consists of highly differentiated ectoderm-derived cells expressing specific hormones such as LH, FSH, GH, prolactin, and ACTH. LH and FSH are secreted by gonadotrophs which are derived from the steroidogenesis factor 1(SF-1) lineage. Gonadotroph adenomas, a type of pituitary adenoma, account for approximately 40% of pituitary adenomas  (180, 181) in adults. In children, gonadotroph adenomas can very rarely cause central precocious puberty (182). Though, most gonadotroph adenomas are nonfunctional and benign, rare cases of functional adenomas have been reported. Hormone profiles of functioning adenomas most commonly show elevated FSH concentrations with or without increase in LH concentrations. Elevated TSH secretion resulting in hyperthyroidism may occur concurrently (180, 181).

 

GUT MICROBIOME AND PUBERTY

 

Microbiota interact with a variety of metabolic and endocrine pathways of the host through genetic expression of more than 100 times the human genome. The gut microbiome variety, composition and impact on health depend on a vast number of variables, both internal, such as age, genetic factors, gender, and endocrine and immune systems, as well as external factors, such as diet, environment, drugs, and pathogens. The relationship between sex hormones and gut microbiome is complex. Sex steroids may directly or indirectly influence the sex-specific gut microbiome that develops during puberty (183). One study reported several gut microbiome alterations in girls with CPP including Ruminococcus bromii, Ruminococcus callidus, Roseburia inulinivorans, Coprococcus eutactus, Clostridium sporosphaeroides, Clostridium lactatifermentans, Alistipes, Klebsiella and Sutterella (176). Although the evidence of the interaction between microbiota and sex hormones remains limited, evidence of diversity of the gut microbiota at different pubertal stages and that alterations may occur in girls with CPP represents an area for potential future development in the prediction and prevention of precocious puberty (184).

 

Treatment of central precocious puberty

 

GONADOTROPIN-RELEASING HORMONE ANALOGS

 

Long-acting Gonadotropin-releasing hormone analogs (GnRHa) have been the standard treatment of CPP since the mid-1980s (185, 186). The GnRHa are super-agonists that bind to the pituitary GnRH receptor downregulating the endogenous pituitary GnRH receptor resulting in decreased gonadotropin and sex steroid secretion. These medications are modified preparations of the native GnRH decapeptide engineered to increase potency and duration of action by substituting a D-isomer amino acid for the naturally occurring L-glycine at position 6. In some analogs, the tenth amino acid is deleted with modification of the naturally occurring L-proline at position 9 (14).

 

Several distinct GnRHa preparations are available differing in route of administration and duration of action (See Table 3) (28). The choice of a specific GnRHa depends on patient, caregiver, and physician preference and on insurance coverage/payment/authorization. Treatment with GnRHa leads to regression or stabilization of pubertal symptoms, deceleration of linear growth velocity, and slowing of skeletal maturation. Some girls experience estrogen withdrawal bleeding about 2-3 weeks following the first injection. Parents and the patient should be counseled to expect this episode of vaginal bleeding (187).  

 

 Table 3. Currently Available GnRHa Therapeutic Options

GnRHa Preparations                         

Dose  

Frequency       

Route

Goserelin

3.6mg

Once a month

intramuscular

Leuprolide

7.5mg

Once a month

intramuscular

 

11.25mg

Once a month

intramuscular

 

15mg

Once a month

intramuscular

 

11.25mg

Every 3 months

intramuscular

 

30mg

Every 3 months

intramuscular

 

45mg

Every 6 months

intramuscular

Leuprolide

45mg

Every 6 months

subcutaneous

Triptorelin

22.5mg

Every 6 months

intramuscular

Nafarelin

800mcg

Twice daily

intranasal

Histrelin

50mg

Annually *

Subdermal implant

*May be used up to 2 years (481).

 

Adverse Effects

 

In general, GnRHas are safe and effective. Adverse events include injection site reactions and sterile abscesses at the site of the injection or implant (188, 189, 190) which may result in loss of efficacy. Minor reported side effects include headaches, hot flashes, vaginal withdrawal bleeding, and mood swings (191). Extremely rare side effects include hypersensitivity reactions, seizures, slipped capital femoral epiphysis, idiopathic intracranial hypertension, and anaphylaxis. One concern regarding the histrelin implant is possible device fracture during extraction; ultrasound-guided removal of the remaining fragments may be necessary (192).

 

GnRHas, specifically only leuprolide and degarelix, have been associated with prolonged QT interval. A prolonged QT interval increases the risk of developing torsades de pointes (TdP) which is a ventricular arrhythmia associated with sudden cardiac death. Low serum potassium or magnesium may exacerbate the risk for prolonged QT interval. Individuals also taking anti-psychotics (typical and atypical), anxiolytics, and anti-depressants may have an increased risk for prolonged QT intervals when taking leuprolide. Hence, providers should inquire regarding other medications, history of congenital heart disease, and family history of Long QT Syndrome or sudden death. If positive, the provider should obtain screening and follow-up EKGs.

 

Studies conflict regarding how GnRHa treatment impacts weight gain and BMI. Some studies have reported weight gain during treatment (193, 194, 195, 196) whereas others have not found any significant change in weight or BMI (197, 198).As with all patients, counseling patients regarding the pre-treatment weight trajectory and healthy lifestyle is beneficial. Women with a history of CPP have been reported to have similar adult weight to the general population (199).

 

Bone mineral density is typically elevated at diagnosis with deceleration in bone mineral accrual during treatment. However, follow-up several years after treatment shows normal bone mineral density compared to population norms (200). Available outcome data suggest that fertility is not compromised for women or men with histories of CPP (192, 201, 202, 203, 204).

 

Despite suggestions that CPP is associated with subsequent development of PCOS, available data are inconsistent. Prospective longitudinal studies are needed to adequately address this concern (205, 206).

 

Who to Treat?

 

For patients less than 7 years of age with a confirmed diagnosis of CPP, the benefit of GnRHa treatment is generally unequivocal. However, the value of GnRHa treatment may be unclear for the peripubertal child (typically a girl) with onset of puberty between 7-9 years of age especially when treatment is unlikely to improve the predicted adult height (PAH) (207). Some girls and their families are comfortable with early pubertal onset and early menarche. In contrast, some girls and their families are distraught when even contemplating early puberty and premature menarche. Consistent evidence-based data regarding negative psychosocial consequences in children with CPP are lacking (208). Further, it may be challenging to justify the medical benefits of GnRHa therapy for early puberty due to the accompanying burdens of increased physician office visits and financial impact. Shared decision-making involving the patient, parents, and medical staff is indispensable to address the benefits and risks of GnRHa in the individual patient (209) . 

 

Goals of Treatment

 

Goals of GnRHa treatment include prevention of pubertal progression and height preservation (210). Growth velocity can significantly decline in some children during GnRHa treatment particularly in those with a markedly advanced bone age (211). The use of other height augmenting medications including recombinant human growth hormone (GH) (212, 213, 214, 215), stanozolol (216, 217), and oxandrolone (218) have been explored but none are recommended for sole use or as an adjunct to GnRHa therapy (219, 220). 

 

Increasing adult height must be judged considering the financial and psychological burdens of this intensive treatment regimen (221). Several recent studies have recommended treatment beyond a bone age of 12 years, however more rigorous studies are needed before such treatment is endorsed (222, 223).

 

Another goal of CPP treatment is to mitigate psychosocial distress and prevent adverse mental health outcomes. One epidemiological study of over 7000 women showed that adolescents with early age of menarche had higher rates of depression and antisocial behavior, which persisted into adulthood (224). Adverse psychosocial experiences reported in girls with early age at menarche include increased likelihood of teenage pregnancy and childbearing, sexual and physical assault, and reduced likelihood of high school graduation (225). However, studies thus far do not show that GnRHa therapy can mitigate these effects. One small study of 36 girls with CPP treated with GnRHas evaluated behavioral health diagnosis and health-related quality of life and found no abnormalities in psychological functioning (226). In a small study of 15 girls with CPP treated with GnRHa and 15 age-matched controls, comprehensive test batteries revealed similar scores in cognitive performance, behavioral, and psychosocial problems (227). A review of 15 studies evaluating the psychosocial impact of CPP showed an increased psychosocial and health-related quality of life burdens with CPP compared with controls (228). The same study showed qualitative data demonstrating emotional lability in patients with CPP and that physical differences associated with sexual precocity could increase feelings of shame and embarrassment which further increase isolation and social withdrawal (228). Again, larger studies are needed to better establish if and how GnRHas influences the psychosocial issues associated with CPP.

 

Monitoring of Treatment

 

Treatment efficacy can be monitored by repeat clinical exams assessing pubertal progression, ultrasensitive LH, FSH and sex hormone concentrations (estradiol in girls, testosterone in boys), rate of progression of bone maturation, estimates of PAH and change in PAH, and patient satisfaction. No uniform consensus exists regarding the optimal strategy for monitoring treatment efficacy in children with CPP. Progression of breast or testicular development may indicate poor adherence, treatment failure, or incorrect diagnosis (188).

 

Random basal LH concentrations to confirm treatment efficacy may be unhelpful because random LH levels often fail to revert to a prepubertal range even when the HPG axis is fully suppressed (229, 230). Therefore, random LH concentrations cannot be used to indicate treatment failure. To confirm gonadotropin suppression, a GnRH stimulation test with short-acting GnRH or, alternatively, a single LH sample 30–120 min after long-acting GnRH analog administration may be performed (231, 232) and different protocols exist regarding the specific timing and number of LH and FSH measurements (233). Some clinicians prefer to utilize clinical indices particularly in areas where hormone determinations are costly.

 

During treatment, breast tissue usually becomes softer with variable changes in size. The rate of bone maturation typically slows with adequate treatment resulting in a decline in BA/CA or a change in BA divided by time. Recent data show that the decline in BA/CA is non-linear and that larger declines are seen in the first 18 months of treatment (222). Thereafter, a slower rate of decrease suggests maintenance of suppression rather than treatment failure.

 

Height velocity is typically rapid prior to treatment and decreases on treatment. The height deceleration is most apparent during the first 18 months of treatment, similar to the deceleration in skeletal maturation. Subsequently, a prepubertal growth rate is often evident (222). Ideally, the rate of bone maturation decelerates resulting in a net gain in height potential. Therefore, calculating PAH during treatment helps assess efficacy. It is also important to understand that mid-parental height (MPH) influences height outcome. GnRHa treatment for CPP may restore genetic potential but rarely causes PAH to surpass genetic potential. Therefore, treatment efficacy by PAH assessment is always in comparison to MPH.

 

Discontinuation of Therapy

 

The decision to discontinue GnRHa treatment needs to be tailored to meet the patient’s specific needs. Factors influencing the decision-making process include synchronizing pubertal progression with peers, patient readiness for resumption of puberty, recent linear growth velocity, bone age X-ray results, and adult height prediction (234). Specific considerations for the developmentally delayed child may be reviewed with the caregivers (137, 234, 235). Pubertal manifestations generally reappear within months of discontinuation of GnRHa treatment; the mean time to menarche is approximately 16 months (217, 218). Several studies have reported that ovulatory function and menstrual cycles are normal once they resume (137, 236).

 

Eripheral Precocious Puberty or Gonadotropin-Independent Precocious Puberty

 

Peripheral precocious puberty (PPP) is due to either excessive endogenous gonadal or adrenal sex steroid secretion (estrogens or androgens) or from exogenous exposure to sex steroids. Ectopic gonadotropin secretion typically from a germ-cell tumor often located in the CNS can also lead to PPP. PPP may be appropriate for the child's sex (isosexual) or inappropriate, with virilization of females and feminization of males (heterosexual). In most instances, pubertal development is incomplete, and fertility is not attained. Etiologies of PPP include:

 

MCCUNE-ALBRIGHT SYNDROME

 

McCune-Albright syndrome (MAS) is an uncommon disorder characterized by the triad of gonadotropin-independent precocious puberty, irregular café-au-lait skin pigmentation and fibrous dysplasia of bone (237, 238).  It has been recognized more recently that MAS may exist as a “form fruste” with only one or two features (239). MAS affects both boys and girls. Importantly, precocious puberty is not observed in all affected individuals and tends to be more common among girls.

 

MAS is due to a somatic cell (post-zygotic) variant arising early during embryogenesis in the GNAS1 gene which is located at chromosome 20q13.3. This gene encodes the Gsα protein coupled to the G-protein membrane receptors for glycoprotein hormones. Vertical transmission has not been reported suggesting that germline variants are embryonic lethal. Variability in post-zygotic expression of the deleterious variant results in a mosaic pattern of tissue expression and inconsistent clinical manifestations between affected individuals (237).

 

Two missense variants, Arg201His and Arg201Cys, are the most frequently identified variants. These variants lead to loss of the α-subunit’s intrinsic GTPase activity resulting in inappropriate cyclic AMP production and constitutive receptor activation. The net result is autonomous ligand-independent signaling by LH, FSH, TSH, GHRH, and ACTH receptors leading to the associated hyperfunctioning endocrinopathies(237).

 

The café-au-lait lesions are generally large with irregular “coast of Maine” borders and typically do not cross the midline. The café-au-lait lesions result from increased tyrosinase gene expression and melanin production in affected melanocytes (240).

 

Bone manifestations are characterized by dysplastic lesions with abnormal bone turnover and inadequate mineralization. These lesions can be associated with pain, malformations, fractures, or nerve compression. The somatic cell gain-of-function variants alter the differentiation of multi-potent skeletal stem cells resulting in the replacement of normal bone and marrow with immature woven bone and fibrotic stroma. The dysplastic tissue is characterized by abundant osteoclast-like cells. Although the somatic Gsα skeletal variants arise during embryogenesis, bone development appears to be normal in utero.

 

Bony lesions become apparent during early childhood typically reaching the maximal burden in young adulthood. The variability in the somatic cell expression accounts for the variability in the location and extent of the fibrous dysplasia.  To date, an accurate ascertainment of risk to develop bone disease is unavailable. However, younger age and higher skeletal burden score derived from scintigraphic bone scans appear to predict longitudinal progression of bone disease. Importantly, evolution of bony lesions is not associated with the extent of endocrine manifestations (241).

 

Overproduction of fibroblast growth factor 23 (FGF23) by skeletal cells bearing the GNAS1 variant can lead to increased urinary phosphate excretion and decreased renal 1-α-hydroxylase activity (242). Although overt hypophosphatemic rickets is uncommon due to compensatory mechanisms, affected individuals often manifest increased serum FGF23 levels and renal phosphate wasting (243).

 

In the gonads, these variants induce ligand independent activation of gonadotropin receptors resulting in subsequent autonomous ovarian estrogen and testicular testosterone secretion in affected prepubertal girls and boys, respectively.

 

Girls may develop recurrent estrogen-secreting cysts accompanied by breast development and linear growth acceleration. Spontaneous resolution of a cyst decreases the estrogen concentration resulting in withdrawal vaginal bleeding. The sequence of pubertal development may be atypical with vaginal bleeding preceding breast development. Hence, MAS should be considered in females with recurrent ovarian cysts and vaginal withdrawal bleeding. Ovarian torsion rarely occurs. Bloodwork may reveal elevated estradiol concentrations with suppressed gonadotropin concentrations. Pelvic ultrasound typically shows one or more ovarian cysts and uterine enlargement. Nevertheless, serum estradiol concentrations and pelvic ultrasound results may be unremarkable following spontaneous involution of an ovarian cyst. Estrogen exposure may lead to accelerated skeletal maturation with adverse consequences on final adult height. In some instances, a secondary gonadotropin-dependent precocious puberty develops. In adult women, the persistent autonomous ovarian activity can lead to abnormal uterine bleeding, menometrorrhagia, which may be so severe as to require blood transfusion. Spontaneous pregnancies can occur, but relative infertility is common (244).

 

Among boys, autonomous GNAS1 activation in the testes leads to Leydig and Sertoli cell hyperplasia which can be associated with macro-orchidism. Scrotal ultrasound may show focal masses, diffuse heterogeneity, and microlithiasis. Differing from typical pubertal progression, testicular volume in boys with MAS does not accurately indicate pubertal status. Substantial autonomous testosterone production is uncommon. Approximately 15% of boys manifest clinical signs of excessive androgen secretion (239). Leydig cell hyperplasia, the most common histologic finding of the testes, carries a low risk of malignant transformation. Thus, conservative management with periodic scrotal ultrasound imaging is appropriate for follow-up of testicular masses detected in boys with MAS (245).

 

Other features associated with MAS include thyrotoxicosis, growth hormone excess (gigantism or acromegaly), and Cushing syndrome. Hypercortisolism is uncommon, typically occurs during the first year of life, and is associated with higher mortality attributed to secondary infections (246). Specific laboratory evaluation and treatment for associated endocrine features should be obtained. Genetic variants can be found in other nonendocrine organs (liver, intestines, and heart) resulting in cholestasis and/or hepatitis, intestinal polyps, and cardiac arrhythmias, respectively (247, 248). Since GNAS1 variants are considered to be weak oncogenes, the risk for malignant transformation is slightly higher than for the general population (239). In addition, women with MAS have an increased risk for breast cancer attributed to earlier estrogen exposure (249).

 

The diagnosis of MAS is usually based on the characteristic clinical features. Due to GNAS1 variant mosaicism, only 20-30% of peripheral blood lymphocytes are positive for the variant using traditional PCR-based testing. However, variant detection is greater than 80% in the affected tissues (250). Newer circulating cell free DNA testing offers a potential methodology to assess for MAS variants (251) . Importantly, negative testing, especially of peripheral blood lymphocytes, does not exclude the diagnosis of MAS.

 

Therapeutic goals focus on treating specific clinical manifestations. For manifestations related to puberty, current medications either inhibit sex steroid biosynthesis or block their actions at the level of end organs. Minimal evidence-based data are available because of the low prevalence of MAS.  Ketoconazole, an anti-fungal medication, has been used because it inhibits the steroidogenic cytochrome P450 enzymes decreasing adrenal and gonadal steroidogenesis (252). However, ketoconazole may interfere with cortisol synthesis; patients need to be monitored for possible adrenal insufficiency and may benefit from use of stress dose hydrocortisone treatment. Rarely hepatic toxicity can occur. 

 

Aromatase inhibitors prevent conversion of androgens to estrogens. Initial reports for testolactone, fadrozole, and anastrozole were disheartening because no enduring beneficial effects on skeletal growth and bone maturation were observed. Letrozole has been used and showed sustained beneficial effects on skeletal maturation and predicted final height in one small series (253).

 

Selective estrogen receptor modulators such as tamoxifen and fulvestrant have been used. Tamoxifen has both agonist and antagonist activity at the estrogen receptor. Despite reports regarding the efficacy of tamoxifen to reduce vaginal bleeding accompanied by positive effects on bone, this medication has been reported to increase risk of endometrial disease in adult women (254). In view of potential risks for endometrial cancer, tamoxifen should be used with great caution in women with MAS (255).

 

Fulvestrant is a pure estrogen receptor blocker administered by intramuscular injections at monthly intervals. In one small series, vaginal bleeding was reduced with complete cessation of vaginal bleeding in only 8/25 girls. The rate of skeletal maturation decreased without any significant change in linear growth velocity or predicted adult height. Fulvestrant was reported to be well tolerated; additional studies are needed to supplement these initial findings (256).

 

In the past, surgery cystectomy or oophorectomy had been performed in girls with MAS (257). Since cyst recurrence is common, cystectomy should be avoided if possible. Women with MAS have the potential for fertility and spontaneous pregnancy; hence, oophorectomy should be avoided (258).

 

For boys with MAS associated precocious puberty, therapeutic interventions include androgen receptor blockers, aromatase inhibitors, and ketoconazole to interfere with testosterone synthesis (258). Combination therapy with bicalutamide and anastrozole was successfully utilized in one boy with PPP due to MAS (259). Bicalutamide is a potent nonsteroidal antiandrogen that binds to and inhibits the androgen receptor and increases the receptor’s degradation. Surgical intervention should only be considered for rapidly enlarging palpable testicular masses due to the risk of malignancy (245).

 

PREMATURE MENARCHE AND OVARIAN CYSTS  

 

Functioning ovarian follicular cysts can secrete estradiol resulting in isolated premature vaginal bleeding or peripheral precocious puberty (260, 261). Additional signs of puberty may be absent in girls with isolated premature menarche. Although some girls may present with slight breast development followed by vaginal bleeding. The bleeding typically lasts only a few days and is usually attributed to spontaneous resolution/regression of an estrogen-secreting ovarian cyst. By the time a pelvic ultrasound can be obtained, the cyst has resolved, and the ultrasound shows no abnormalities. Isolated premature menarche may be limited to a single episode or may be recurrent. In most instances, linear growth velocity, onset of cyclic menstrual cycles, and final adult height are unaltered.

 

Differential diagnosis includes sexual abuse, vaginal foreign body, vaginal infections, MAS, or primary hypothyroidism(262). Due to the intermittent nature of these cysts, conservative medical management is usually appropriate (263). Large cysts may predispose to ovarian torsion (264, 265, 266, 267). Patients with ovarian torsion usually present with short duration of pain and systemic symptoms such as vomiting. Given the low frequency of malignancy in such an ovarian, detorsion with or without cystectomy is generally preferred (268). Gonadectomy should be avoided to preserve fertility. Rarely, rhabdomyosarcoma or sclerosing stromal tumors can present with vaginal bleeding.

 

OVARIAN TUMORS  

 

Estrogen-secreting ovarian tumors are a rare cause of peripheral precocious puberty. Specific types of tumors include granulosa cell, gonadal stromal cell, ovarian sex cord stromal, and theca cell tumors.

 

Juvenile granulosa cell tumors (JGCT) are the most common ovarian tumors. Typically, these tumors present with rapidly progressive isosexual precocity (269). Most JGCT are large enough to be palpated during an examination and are typically limited to the ovary at the time of diagnosis. Circulating estradiol concentrations may be extremely elevated with suppressed gonadotropin concentrations. Circulating tumor markers including α-fetoprotein (AFP), lactate dehydrogenase (LDH), β-human chorionic gonadotropin (β-hCG), cancer antigen 125 (CA-125), and inhibin B can be identified. Genetic somatic variants have been identified in juvenile granulosa cell tumors. Over 60% of JGCT carry in frame duplications in the AKT1 gene; this gene codes for a kinase involved in ovarian mitogenic signaling (270). Other identified variants include KMT2C-truncating and the ribonuclease III domain of DICER1 variants.  In contrast to adult granulosa cell tumors of the ovary, variants in the FOXL2 gene are generally not found in JGCT. Ollier and Maffucci syndromes, rare disorders associated with benign cartilaginous enchondroma, have been associated with JGCT (271). Surgical excision with peritoneal cytology for staging is the primary treatment.

 

Rarely, other tumors including gonadoblastoma, lipid tumors, cystadenomas, and ovarian carcinomas can secrete sex steroids. Finding elevated serum inhibin and AMH concentrations suggest that the tumor cells are derived from granulosa or Sertoli cells.

 

Sex cord tumors with annular tubules can occur in patients with Peutz-Jeghers Syndrome. Peutz-Jeghers Syndrome is an autosomal dominant disorder associated with mucocutaneous pigmentation, gastrointestinal polyposis, and genetic variants in the STK11 gene located at chromosome 19p13.3. (272) The gonadal tumors can be multi-focal, bilateral, and can differentiate into granulosa cell or large cell calcifying Sertoli cell tumors with the potential to secrete estrogen. Thus, girls may present with precocious puberty whereas boys may present with gynecomastia.

 

Sertoli-Leydig cell tumors are rare ovarian tumors often associated with somatic or germline DICER1 variants (273). Most are unilateral, but bilateral tumors have been described.  These tumors contain testicular structures, Sertoli and Leydig cells, and can rarely secrete androgens. Hence, girls can virilize with pubic hair development (274, 275). Girls known to carry germline DICER1 variants should undergo regular pelvic ultrasounds to screen for ovarian tumors (276).

 

LEYDIG CELL TUMORS  

 

Leydig cell tumors are a subtype of testicular stromal tumors that arise from testosterone producing Leydig cells. In prepubertal boys, presenting features include penile enlargement, acne, development of pubic and axillary hair, and accelerated linear growth velocity. Examination of the testes typically show asymmetric testicular volume due to a unilateral testicular tumor. Leydig cell tumors are usually benign. Bloodwork shows elevated circulating testosterone concentrations and suppressed gonadotropin concentrations. Ultrasound is useful to assess testicular volume and morphology.

 

Treatment involves surgical removal of the tumor. When possible, testis-sparing enucleation is preferred to radical orchiectomy to preserve testicular function and fertility. The surgical approach is dictated by the intraoperative assessment of tumor size, location, and the amount of remaining normal testicular parenchyma (277, 278).

 

HUMAN CHORIONIC GONADOTROPIN SECRETING GERM CELL TUMORS  

 

During early gestation, primordial germ cells migrate from the hindgut to the gonads. In some instances, the germ cells can migrate to locations outside of the gonad, fail to undergo apoptosis, proliferate in these atypical locations, and ultimately become hCG-secreting germ cell tumors (279). Common locations for germ cell tumors include the CNS, lung, or liver (280). Boys and men with Klinefelter syndrome are at higher risk to develop extra-gonadal GCTs particularly in the mediastinum (281). In addition, hepatoblastoma secreting hCG and α-fetoprotein can also present with precocious puberty (282). Due to the similarity between hCG and LH which have identical α-subunits and related β-subunits, tumor-derived hCG stimulates testicular LH receptors resulting in testosterone secretion. In the prepubertal boy, the aberrant hormone exposure can result in precocious puberty (27, 28). Testicular volume may not increase since seminiferous tubule growth does not occur in the absence of FSH stimulation. Bloodwork shows elevated hCG and testosterone concentrations with suppressed/variable LH and FSH concentrations.

 

Prepubertal girls generally do not develop isosexual precocious puberty with hCG-secreting germ cell tumors because in the absence of FSH, the granulosa cells do not express aromatase and are unable to synthesize estradiol.

 

GERM CELL TUMORS

 

Chromosomal aneuploidy or genetic variants can interfere with gonadal development resulting in dysgenetic gonads. In this situation, the appropriate microenvironment for normal germ cell maturation is absent, thereby disrupting the normal maturational progression for germ cells. This situation may result in the development of gonadal germ cell tumors. Precursor lesions of germ cell tumors include germ cell neoplasia in situ (GCNIS, formerly termed carcinoma in situ – CIS) and gonadoblastoma (283). Subsequently, dysgerminoma, seminoma, or non-seminoma may develop. Usually, such germ cell tumors do not secrete significant amounts of sex steroids.

 

FAMILIAL MALE LIMITED PRECOCIOUS PUBERTY (FMPP)

 

Familial male-limited precocious puberty (also known as testotoxicosis) is due to an autosomal dominant activating germline variant in the LH/choriogonadotropin receptor (LHCGR) gene located at chromosome 2p21. The LH/CG receptor is a G-protein coupled receptor (284). The variant is associated with autonomous ligand-independent receptor signaling leading to Leydig cell hyperplasia and premature testosterone secretion in prepubertal boys. Pathogenic missense variants associated with FMPP tend to congregate in an apparent hot spot located in the 6th transmembrane segment and in the 3rd intracellular loop (285).

 

Affected males typically present between two to six years of age with penile enlargement, linear growth acceleration, advanced skeletal maturation, acne, and pubarche. The testes are usually symmetrically enlarged due to the Leydig cell hyperplasia, but the size is disproportionately smaller compared to the testosterone levels (286, 287). A large portion of the testicular volume is formed by Sertoli cells which are not stimulated in this condition (286, 287). Circulating testosterone concentrations are elevated with suppressed gonadotropin concentrations. Although adult height is generally compromised, fertility has been reported (288). Longitudinal follow-up with testicular self-examination and scrotal ultrasound is recommended because malignant testicular germ cell tumors have been described in a few individuals (289).

 

Therapeutic goals include decreasing autonomous testicular testosterone secretion and slowing epiphyseal maturation. To date, several medications including ketoconazole, spironolactone, bicalutamide, and aromatase inhibitors have been used with varying efficacy (286). To date, the most effective therapy is combination treatment with an anti-androgen and an aromatase inhibitor (290, 291). If secondary GnRH-dependent precocious puberty develops, GnRH agonist therapy can be added to the therapeutic regimen. Abiraterone, a selective CYP17A1 inhibitor, was utilized in a young boy with bilateral Leydig cell tumors and resistance to the usual combination regimen of an anti-androgen and aromatase inhibitor. He required glucocorticoid replacement therapy and monitoring for possible excessive mineralocorticoid action because of abiraterone treatment-associated iatrogenic 17-hydroxylase/17,20-lyase deficiency (292).

 

Although inherited as an autosomal dominant disorder, girls do not develop precocious puberty (293). Since only the LHCGR gene is affected, the presumably minimally increased theca cell androgens cannot be aromatized to estradiol in the absence of FSH stimulation. Importantly, asymptomatic women can transmit the affected allele to their sons.   

 

PRIMARY HYPOTHYROIDISM

 

Children with profound chronic primary hypothyroidism may present with precocious puberty. Van Wyk and Grumbach described this association in 1960 (294). Clinical features in girls include early breast development, vaginal bleeding, and galactorrhea. Boys present with testicular enlargement. Pubic and axillary hair are absent. Typical features associated with hypothyroidism such as short stature, impaired linear growth, puffy face, dry skin, constipation, and delayed skeletal maturation (despite pubertal changes) are usually evident. Pituitary imaging shows an enlarged pituitary gland. Abdominal ultrasound may show ovarian enlargement with or without ovarian cysts. Labs show mild elevation in FSH levels but LH levels usually remain prepubertal.

 

Levothyroxine therapy induces regression of pubertal symptoms, stops vaginal bleeding, and decreases pituitary volume. However, final height may often be compromised due to accelerated skeletal maturation upon initiation of thyroxine treatment. One potential mechanism for the precocious puberty is cross-reactivity of TSH at the ovarian FSH receptor. TSH and FSH share a common α-subunit with hormone specificity due to the differing β-subunits (295). This mechanism was tested using recombinant human TSH in an in vitro bioassay which, at high concentrations, was able to stimulate human FSH receptors (296, 297, 298, 299).

 

VIRILIIZING CONGENITAL ADRENAL HYPERPLASIAS

 

The virilizing congenital adrenal hyperplasias (CAHs) are autosomal recessive disorders associated with impaired adrenal steroidogenesis due to genetic variants in steroidogenic enzyme genes. The most common is 21-hydroxylase deficiency due to variants in the 21-hydroxylase gene (CYP21A2) located at chromosome 6p21.33. Clinically, congenital adrenal hyperplasias reflect a phenotypic spectrum ranging from presentation in neonatal period with classic salt-losing CAH to presentation during infancy/todder age with classic simple virilizing CAH to later presentations with non-classic CAH. Milder or non-classic forms have been described for 11-β-hydroxylase deficiency and 3β-hydroxysteroid dehydrogenase deficiency (300).

 

Children with non-classic CAH typically present with premature pubarche characterized by pubic/axillary hair development, acne, accelerated linear growth velocity, and advanced skeletal maturation. Girls may have clitoromegaly whereas boys may have phallic enlargement with prepubertal testicular volume. Adult women with non-classic CAH usually present with irregular menses, hirsutism, and infertility.

 

The diagnostic test for 21-hydroxylase deficiency is an elevated 17-hydroxyprogesterone (17-OHP) concentration. Early morning basal 17-OHP values have been suggested as an effective screening test with reports of 100% sensitivity and 99% specificity with a threshold value of 200 ng/dl (6 nmol/L) to diagnose NCAH in children who present with premature pubarche (301). If the diagnosis is highly suspected despite relatively normal 17-OHP concentrations, an ACTH stimulation test may be indicated to exclude the diagnosis of 21-hydroxylase deficiency. For an ACTH stimulation test, following collection of a basal blood sample, 0.25 mg synthetic ACTH (Cortrosyn) is administered by intravenous or intramuscular routes; a second blood sample is collected at 30 and/or 60 minutes. Physician preference governs the timing of the ACTH-stimulated 17-OHP concentration. In the future, 21-deoxycortisol and 11-oxyandrogens may be increasingly utilized in the diagnosis and management of 21-hydroxylase deficiency (302, 303). The reader is referred to more extensive discussion of the virilizing CAH (304, 305).

 

ADRENOCORTICAL TUMORS

 

Androgen-secreting adrenocortical tumors are extremely rare causes of PPP accounting for less than 1% of all childhood malignancies. Most tumors occur in children younger than 4 years of age with a second smaller peak in adolescents. Pediatric adrenocortical tumors are categorized as adenomas or carcinomas based on histological features. However, histopathologic differentiation may be challenging, and biologic behavior of the tumor may help with this categorization (306).

 

Pediatric adrenocortical carcinoma is more common in girls than boys and has a bimodal pattern with peaks under age 5 and over 10 years of age (307). Adrenocortical tumors are associated with several genetic syndromes such as Li-Fraumeni syndrome and Beckwith-Wiedemann syndrome (BWS). Li-Fraumeni Syndrome is an autosomal dominant familial cancer syndrome associated with germline p53 gene variants. The p53 gene (or TP53 gene) is a tumor suppressor gene located at chromosome 17p13.1, and codes for the protein p53. Malignancies associated with Li-Fraumeni syndrome include adrenocortical carcinomas, breast cancer, brain tumors, and sarcoma. The incidence of adrenocortical tumors is 10-15 times higher in southern Brazil; this has been attributed to the higher prevalence of the R337H variant of the TP53 gene (308).

 

Beckwith-Wiedemann syndrome is characterized by macroglossia, macrosomia, organomegaly, neonatal hypoglycemia due to hyperinsulinism, and abdominal wall defects. This disorder is associated with uniparental disomy in the 11p15 chromosomal region leading to IGF2 growth factor overexpression. Although only 1% of children with Beckwith-Wiedemann Syndrome will develop adrenocortical carcinomas, these adrenal tumors account for approximately 20% of the neoplasms in children with this disorder (309). Other disorders associated with adrenal tumors include Multiple Endocrine Neoplasia Syndrome Type 1 (MEN1) and Carney complex (310).

 

The next section reviews variants of puberty associated with early pubertal changes and are important differentials to consider in the evaluation of CPP.

.

PREMATURE THELARCHE

 

Premature thelarche is the premature development of glandular breast development. The breast development may be unilateral or bilateral. Typically, premature thelarche develops in otherwise healthy girls between 12-24 months of age and is self-limited. No other pubertal changes are evident; linear growth velocity is normal and pubic/axillary hair are absent. On physical examination, the areolae and vaginal mucosa are prepubertal. The diagnosis can usually be made on a clinical basis without bloodwork or bone age X-rays (311). Pelvic ultrasound showed increased prevalence of ovarian microcysts in girls with premature thelarche compared to age-matched controls; no correlation between ovarian cysts, gonadotropin concentrations, and estradiol concentrations has been found (312). Longitudinal follow-up is appropriate to confirm the diagnosis and assess for the unlikely possibility of progression to CPP.

 

PREMATURE ADRENARCHE

 

Pubarche refers to the appearance of pubic/axillary hair, increased apocrine odor, and acne due to the onset of adrenarche. Adrenarche refers to the pubertal maturation of the adrenal zona reticularis. Adrenarche which normally occurs in children between 6-8 years of age and is characterized by increased secretion of the adrenal androgen precursors DHEA, DHEAS, and androstenedione.

 

Premature adrenarche is characterized by premature pubarche, which is defined as the development of pubic or axillary hair before 8 years in girls or 9 years in boys. There is no breast development in females and no testicular enlargement in males. Bone age is usually not advanced. Premature adrenarche is a diagnosis of exclusion. Thus, exclusion of other disorders such as CAH, androgen-secreting tumors, exogenous androgen exposures, and other rare genetic disorders such as apparent cortisone reductase and PAPS synthase 2 (PAPSS2) deficiencies is essential (313).

 

Children with premature adrenarche and early androgen excess may be at a higher risk to develop the metabolic syndrome. Waist circumference (WC), waist/hip ratio, and total and truncal fat mass increase are detected in premature adrenarche. Increases in systolic and diastolic blood pressure (BP), total cholesterol (TC), very low-density lipoprotein (VLDL), TC/high density lipoprotein (HDL), low density lipoprotein (LDL)/HDL ratio, and atherogenic index (AI) have been reported. Increased insulin concentrations starting from prepubertal ages may occur suggesting that premature adrenarche may be one of the first symptom of insulin resistance (IR) in childhood (314, 315). T2DM may occur in a subset of these cases. Ovarian hyperandrogenism, hirsutism, ovulatory dysfunction, and polycystic ovaries may be more frequent in girls with premature adrenarche during post pubertal ages than normal population. Although, early retrospective data in a homogenous population suggests an association between premature adrenarche and adolescent hyperandrogenism (316), more recent longitudinal data suggests that premature adrenarche was not associated with adolescent ovarian dysfunction and was only associated with lower SHBG concentrations (317).

 

EXPOSURE TO EXOGENOUS SEX STERIODS  

 

Feminization, including gynecomastia in males, has been attributed to excess estrogen exposure from creams, ointments, and sprays. Other possible sources of estrogen exposure include contamination of food with hormones, phytoestrogens (e.g., in soy), and over-the-counter remedies such as lavender oil and tea tree oil (318, 319, 320, 321). Similarly, virilization of young children has been described following inadvertent exposure to androgen-containing creams/gels (322).

 

Endocrine-Disrupting Chemicals 

 

Various endocrine-disrupting chemicals (EDCs) are found in the environment  (323, 324, 325, 326).  Most EDCs have chemical structures similar to those of endogenous sex steroids. These chemicals can disrupt steroid hormone receptor binding and hormone metabolism altering hormone concentrations or changing hormone synthesis/degradation (327). EDCs can act beyond steroid hormone receptors by affecting transcriptional modulators and direct effects on genes. Some EDCs have mixed activities, and most EDCs include several different chemicals. The patient’s age and duration of exposure modulate the consequences of EDC exposure. In addition, EDCs can be classified as persistent (long-lasting) or non-persistent (short half-lives). Environmental EDC exposures can be transgenerational such that future generations could be affected (328). Mechanisms for EDC exposures include ingestion, topical use, inhalation, and transfer across the placenta (329).

 

The consequences of mixed “cocktail” EDC exposures on pubertal development are indeterminate. A systematic review with a stringent meta-analysis found no consistent association between xenobiotic EDCs and pubertal timing apart from an insinuation that, in girls, postnatal exposure to phthalates could be associated with earlier thelarche and later pubarche, consistent with their anti-androgenic properties. Methodological heterogeneity, limited number of studies, and variability in statistical analyses constrained the conclusions of this systematic review. Hence, future longitudinal epidemiologic studies to clarify the specific EDCs, age at exposure, and duration of exposure will be valuable (327, 330).

 

 

DELAYED PUBERTY

 

Gonadarche associated with the reactivation of the GnRH pulse generator, is signified by breast development in girls and testicular enlargement in boys. Delayed puberty is defined as absence or delayed onset of gonadarche at a chronologic age >2 standard deviations later than the population mean. In girls, delayed puberty is defined as absence of breast development by age 13 years or lack of menarche by age 15 years (331) or 3 years from onset of thelarche (332). In boys, delayed puberty is defined as the lack of testicular enlargement to a volume >= 4 ml by age 14 years (333). Delayed puberty is more common in boys than in girls.

 

Four main categories of delayed puberty have been described (See Table 4).

  • transient hypogonadotropic hypogonadism associated with delayed maturation of the HPG axis also known as constitutional delay of growth and puberty (CDGP)
  • hypergonadotropic hypogonadism characterized by primary gonadal dysfunction with consequent elevated LH and FSH concentrations.
  • hypogonadotropic hypogonadism with low LH and FSH concentrations due to congenital (CHH) or acquired causes
  • functional hypogonadotropic hypogonadism (FHH), as seen in chronic health disorders such as cystic fibrosis, renal failure, inflammatory bowel disease, restrictive eating disorders etc.

 

Table 4. Etiologies of Delayed Puberty (458)

Condition

Etiology

Constitutional Delay of Growth and Puberty

Genetic basis has infrequently been described in HS6ST1, FTO, IGSF10, EAP1 genes

Hypergonadotropic Hypogonadism

Congenital:

-Klinefelter’s syndrome

-Turner syndrome

-Gonadal dysgenesis

-Anorchia

-Primary ovarian insufficiency

-Testicular regression syndrome

-Genetic causes: FMR1, STAG3, NR0B1, NR5A1, FOXL2, WT1 and others

-Galactosemia

Acquired:

-Infectious (mumps)

-Autoimmune (polyglandular syndromes)

-Surgery (torsion, trauma)

-Chemotherapy (alkylating agents)

-Radiation

-Gonadal tumor

Hypogonadotropic Hypogonadism

Congenital:

-Isolated HH: Over 50 genes have been identified; notable are ANOS1, FGFR1, FGF8, PROK2, CHD7, KISS1, KISS1R, GNRH, GNRHR and others

-Prader Willi

-CHARGE syndrome

-Noonan

-Bardet-Biedl

- Panhypopituitarism associated with genetic variants in PROP1, HESX1, LHX, LHB, FSHB and others.

Acquired:

-Central nervous system tumors (e.g., craniopharyngiomas, germinomas), cysts,

-Cranial surgeries,

-Cranial radiation therapy greater than

 30 Gy

-Other inflammatory, autoimmune (hypophysitis), and infiltrative (Langerhans cell histiocytosis) diseases of the pituitary gland

Functional Hypogonadotropic Hypogonadism

-intense physical stress (competitive gymnastics, ballerina syndrome)

-emotional stress (elevated glucocorticoids)

-caloric deficit (anorexia nervosa)

-chronic systemic illness (celiac, inflammatory bowel disease, CF, renal disease)

-endocrinopathies (hypothyroidism, excess glucocorticoids, hyperprolactinemia)

-medication adverse effects

-pituitary iron deposits in chronic transfusion dependent children

 

Transient Hypogonadotropic Hypogonadism Associated with Delayed Maturation of the HPG Axis/ Constitutional Delay of Growth and Puberty (CDGP)

 

Transient hypogonadotropic hypogonadism also known as constitutional delay in growth and puberty (CDGP) is the most common etiology of delayed puberty occurring in 70% of boys and 32% of girls with delayed puberty (334). In both sexes, CDGP is self-limited and is considered to represent a variant of normal pubertal timing. CDGP has a strong genetic component, with a positive family history of delayed puberty reported in 50% to 75% of cases (335).

 

Distinguishing between CDGP and congenital hypogonadotropic hypogonadism (CHH) may be challenging because these conditions share clinical features, hormone levels, and radiological findings. Inhibin B and LH levels tend to be lower in boys with CDGP, but the overlap in values precludes the use of these hormones to distinguish between CDGP and CHH.

 

For boys, 3-4 months of steroid priming with testosterone followed by 3-4 months of observation is commonly used to discriminate CHH from CDGP (336). It has been suggested that this sex steroid exposure stimulates resumption of the HPG axis activity leading to secondary sex characteristics typical of male puberty (337). Individuals who show no pubertal progression during the observation period should be evaluated for CHH or another disorder affecting the HPG axis. Estradiol priming has been used similarly in girls to distinguish CHH from CDGP (338). Due to the differences in the long-term outcomes, accurate diagnosis is essential, with CDGP being largely a diagnosis of exclusion (339).

 

CDGP occurs more commonly in family members of individuals with CHH compared to the general population (340). Individuals with CDGP appear to have higher prevalence of pathogenic variants compared to unaffected family members or controls (341). Some genetic variants have been detected in both individuals with CDGP and CHH; these genes include HS6ST1, PROKR2, TAC3, TAC3R, and IL17RD (342). Genetic variants associated primarily with CDGP include IGFS10, EAP1, and FTO (338).

 

Hypergonadotropic Hypogonadism

 

Pubertal delay associated with hypergonadotropic hypogonadism is usually associated with disorders affecting gonadal function, specifically gonadal steroidogenesis. With the onset of gonadarche and increased GnRH and gonadotropin secretion, inadequate gonadal steroid secretion and lack of negative feedback leads to increasing gonadotropin secretion. These conditions may be present at birth or acquired.

 

TURNER SYNDROME  

 

Turner Syndrome refers to deletions or structural rearrangements of the X chromosome. The

reported incidence is around 1 in 2500 liveborn female births (343). The initial in utero process of ovarian differentiation proceeds normally with migration of the primordial germ cells into the developing ovary during the fourth week of gestation. By 18 weeks of gestation, premature degeneration of ovarian follicles has begun. The ovarian follicles are typically replaced by connective tissue resulting in the characteristic streak gonad. This accelerated follicular atresia usually leads to premature ovarian insufficiency. Girls with Turner syndrome have gonadal dysgenesis or “streak gonads” in 85% of cases at birth. However, because adrenal androgen secretion is not impaired, the onset of pubarche usually occurs at a normal time. Typical clinical features of girls with Turner syndrome include short stature, short/webbed neck, shield shaped chest with the appearance of widely spaced nipples, cubitus valgus, and Madelung deformity of the forearm and wrist, shortened fourth metacarpals/metatarsals, horseshoe kidneys, coarctation of the aorta, increased risk for autoimmune conditions, and aberrant development of the lymphatic system. Many girls with Turner syndrome may remain undiagnosed until later in childhood or adolescence when they present with short stature and/or delayed puberty. With increased utilization of noninvasive prenatal screening (NIPS), many girls with Turner Syndrome are detected prenatally. The reader is referred to other publications for more extensive discussion regarding the features and approach to multidisciplinary health care management (344, 345, 346).

 

KLINEFELTER SYNDROME

 

Klinefelter Syndrome is a chromosomal aneuploidy characterized by 47, XXY karyotype and premature testicular insufficiency. Increased NIPS utilization has led to detection of many boys in utero and is estimated to occur in 1 in 667 males based on prenatal cytogenetic analysis (347). However, many men remain underdiagnosed, with less than 10% of patients being diagnosed prior to puberty. Men with Klinefelter syndrome typically present with tall stature, incomplete puberty, or gynecomastia. Generally, the onset of puberty is not delayed. Klinefelter syndrome is associated with small firm testes, Sertoli cell dysgenesis, impaired spermatogenesis, and variable degrees of testosterone deficiency (348). Learning disabilities, language and visuospatial processing defects, and neuropsychiatric conditions such as attention-deficit/hyperactivity disorder and depression are common (349). If a tumor is found in the anterior mediastinum, a karyotype should be performed to evaluate for Klinefelter syndrome because of its association with mediastinal germinoma (350). Despite normal BMI, the body fat percentage, and the ratio between android fat percentage and gynoid fat percentage are significantly higher than normal. They may also have an impaired bone metabolism starting during childhood and adolescence. Systematic studies are needed to evaluate whether testosterone replacement therapy during puberty will improve these parameters (351). The reader is referred to other publications for more extensive discussion regarding the features and approach to multidisciplinary health care management (352, 353, 354, 355, 356).

 

DIFFERENCES OF SEX DEVELOPMENT  

 

Differences of sex development (DSDs) are a group of conditions where external genital development is atypical. These disorders are associated with chromosomal anomalies, genetic variants, and environmental influences (357). Gonadal function is impaired in some types of DSDs resulting in primary gonadal failure and hypergonadotropic hypogonadism. Detailed review of DSDs is beyond the scope of this chapter. The interested reader is referred to other Endotext chapters for more extensive review of DSDs.

 

GENETIC CAUSES OF PREMATURE OVARIAN INSUFFICIENCY (POI)

 

POI can present with primary or secondary amenorrhea.  Fragile X-associated premature ovarian insufficiency is among a family of disorders caused by the expansion of a CGG trinucleotide repeat sequence located in the 5′ untranslated region (UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene on the X chromosome. One etiology is premutation of the FMR1 gene associated with 55-200 CGG repeats without abnormal methylation of the neighboring CpG island and promoter, responsible for both fragile X associated premature ovarian insufficiency in females and fragile X associated tremor ataxia syndrome in males and females where patients may present with mild to moderate intellectual disability, intentional tremor and cerebellar ataxia, peripheral neuropathy, Parkinsonism, and urinary and bowel incontinence.

 

The X chromosome carries many genes that govern follicular maturation and overall ovarian function, and numerical and structural changes in this chromosome, as in Turner syndrome or triple X syndrome, are associated with POI.

 

Multiple genes are involved in ovarian differentiation, oocyte development, and, ultimately, folliculogenesis and variants in these genes may be associated with premature ovarian insufficiency (358, 359). The clinical phenotype ranges from delayed puberty to secondary amenorrhea (360). 

 

GALACTOSEMIA

 

Galactosemia is a rare cause of delayed puberty. Classic galactosemia is a rare inborn error of galactose metabolism due to a defect in the gene encoding the galactose-1-phospate uridyltransferase enzyme (GALT). The prevalence is approximately 1/30,000–60,000 (361). Early manifestations include lactose intolerance, jaundice, failure to thrive, lethargy, hepatocellular damage, renal tubular disease, and cataracts. A galactose-free diet can reverse the neonatal symptoms. However, some long-term complications such as developmental delay, intellectual disability, epilepsy, osteoporosis, and premature ovarian insufficiency may still develop. In females, hypergonadotropic hypogonadism resulting in delayed puberty, primary or secondary amenorrhea, and infertility may occur (362, 363). Available data from patients with classic galactosemia suggest that the primary ovarian insufficiency is due to dysregulation of pathways essential for folliculogenesis culminating in premature ovarian insufficiency (364). Several previous cohort studies in males showed delayed puberty and below-target final height (365, 366, 367), however a recent study with 47 males showed that puberty and fertility were normal and in contrast to earlier reports, AMH, testosterone and Inhibin B levels were normal (361).

 

Hypogonadotropic Hypogonadism

 

Pubertal delay associated with hypogonadotropic hypogonadism is usually associated with disorders affecting the neurons that secrete GnRH or the pituitary gonadotrophs that secrete the FSH and LH. These conditions may be present at birth or acquired as described below.

 

CONGENITAL HYPOGONADOTROPIC HYPOGONADISM

 

The initiation and maintenance of reproductive capacity in humans depends on pulsatile GnRH secretion. Congenital hypogonadotropic hypogonadism (CHH) results from the absence of the normal pulsatile GnRH secretion or deficient pituitary gonadotropin secretion leading to delayed puberty and infertility. The number of genetic loci associated with CHH continues to expand (Table 4). CHH may be associated with variants in genes involved in the development or migration of GnRH neurons as well as genes involved in the secretion or action of GnRH (368). Autosomal recessive, autosomal dominant, X-linked, and oligogenic inheritance have been described (369, 370). Additional genetic influences include epigenetic factors (371). Clinical heterogeneity has been described between and within families (372).

 

Given the developmental origins of GnRH neurons in the olfactory placode, CHH can be associated with anosmia or hyposmia. The association of CHH and anosmia is known as Kallmann syndrome. Classic Kallmann syndrome is associated with variants in the ANOS1 gene which is mapped to the X chromosome. Other features of Kallmann syndrome due to ANOS1 variants include unilateral renal agenesis, sensorineural hearing loss, dental agenesis, synkinesia (alternating mirror movements), and cleft lip/palate (373).

 

In syndromic CHH, associated clinical features may help identify the possible gene(s). For example, clinical features associated with FGFR1 variants include anosmia/hyposmia, cleft lip/cleft palate, dental agenesis, and skeletal anomalies. CHH can also occur in the CHARGE syndrome, which is characterized by coloboma, congenital heart disease, choanal atresia, genital anomalies, ear anomalies, and development delay. CHH can occur with impaired pituitary development associated with PROP1, HESX1, or LHX variants. CHH is also associated with variants in the GnRH, GnRHR, LHB, and FSHB genes (See Table 5). Although some genetic loci are common to both CDGP and CHH, the genetic architectures of these two conditions are largely distinct (374).

 

Table 5. Genes Associated with Delayed Puberty (338, 482, 483)

Gene

(Reference/s)

Protein encoded

Genetic locus

Associated features/syndromes

                                         SYNDROMIC CAUSES

FGFR1/FGF8

 

(484, 485)

Fibroblast Growth Factor Receptor 1/fibroblast growth factor 8

8p11.23

Hartsfield syndrome

LEPR/LEP

(486, 487, 488)

Leptin receptor and Leptin

1p31.3

Severe obesity syndromes

PCSK1

(489)

Prohormone convertase 1 gene

5q15

Obesity, ACTH deficiency, diabetes

DMXL2

(490)

DmX-like protein 2

15q21

Polyendocrinopathy, Polyneuropathy syndrome

RNF216/

OTUD4

(491)

Ring finger protein 216/ OTU domain-containing protein 4

4q31.21

Gordon Holmes

PNPLA6

(492, 493)

Patatin-like phospholipase domain-containing protein 6

19p13.2

Gordon Holmes, Oliver McFarlane,

Lawrence Moon, Boucher-Neuhauser syndrome

SOX10

(494)

Sex determining region Y-Box transcription factor 10

22q13.1  

Wardenburg syndrome

SOX2

(495)

Sex determining region Y-Box transcription factor 2

3q26.33

Optic nerve hypoplasia, CNS abnormalities

SOX3

(496)

Sex determining region Y-Box transcription factor 3

Xq27.1

Intellectual disability, craniofacial abnormalities, multiple pituitary hormone deficiencies

IGSF1

(497, 498)

Immunoglobulin superfamily member 1

Xq26

Associated with X-linked central hypothyroidism, macro-orchidism

HESX1

(499)

HESX homeobox 1

3p14.3

Hypopituitarism, septo-optic dysplasia

CHD7

(500, 501, 502)

Chromodomain helicase DNA binding protein 7

8q12.2

CHARGE syndrome

POLR3A/

POLR3B

(503, 504, 505)

 

RNA polymerase III

12q23.3

Hypomyelination, hypodontia

NROB1

(DAX-1)

 

(506, 507)

Nuclear Receptor Subfamily 0 Group B Member 1/ dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1

Xp21

Adrenal hypoplasia

REV3L/

PLXND1

(508)

 

Catalytic subunit of DNA polymerase zeta

6q21

Möbius syndrome

PWS

(509, 510)

 

15q11.2

Prader-Willi syndrome

BBS1, BBS2, ARL6, BBS4, BBS5, MKKS, BBS7, TTC8, BBS9, BBS10,

TRIM32, BBS12

(511, 512)

Encoded protein may play a role in eye, limb, cardiac and reproductive system development

11q13.2, 20p12, 16q21, 15q22.3‐23, 14q32.1

(multiple loci)

Bardet-Biedl syndrome

PHF6

(513)

Plant homeodomain (PHD)-like finger protein 6 

Xp26.2

Borjeson‐Forssman‐Lehmann syndrome

SMCHD1

 

(514)

Structural maintenance of chromosomes flexible hinge domain containing 1

18p11.32

Bosma arhinia microphthalmia syndrome

TBC1D20/

RAB18

(459, 515)

TBC1 Domain Family Member 20, GTPase activator proteins of Rab-like small GTPases

 

20p13

Warburg micro syndrome

HDAC8

(516)

 

Histone deacetylase 8

Xq13.1

Cornelia de Lange syndrome

                                          NON-SYNDROMIC CAUSES

FGF17

(517)

Fibroblast Growth Factor 17

8p21.3

 

ANOS1 (KAL1)

(518, 519)

Kallmann syndrome protein, which is now known as Anosmin 1

Xp22.31 

involved in fibroblast growth factor (FGF) signaling

GNRHR/

GNRH1

(520, 521, 522)

 

Gonadotropin-releasing hormone receptor/ gonadotropin-releasing hormone 1

4q13.2

 

KISS1R/

KISS1

(54, 523)

Kisspeptin-1 receptor/ kisspeptin-1

19p13.3

 

 KLB

(524)

Klotho Beta

4p14

Metabolic defects

TAC3/TACR3

(342, 525)

Tachykinin 3, Tachykinin 3 receptor

Encodes neurokinin b

4q24

 

IL17RD

(517)

 

Interleukin 17 Receptor D

3p14.3

 

DUSP6

(517)

 

Dual specificity phosphatase 6

12q22–q23

 

SEMA3A/

SEMA3E/

SEMA7A

(526)

Semaphorin 3A

7q21.11

 

SPRY4

(517)

Sprouty homolog 2

5q31.3

 

FLRT3

(517)

Fibronectin leucine rich transmembrane protein 3

20p11

 

PROKR2/

PROK2

(527, 528)

Prokineticin-2 and Prokineticin receptor 2

3p13

 

WDR11

(529)

 

WD repeat domain 11

10p26.12

 

 

CCDC141

(530)

Coiled-Coil Domain Containing 141

2q31.2

 

FEZF1

(531)

FEZ family zinc finger 1

7q31.32

 

LHB

(532)

Luteinizing hormone

19q13.33

 

FSHB

(533, 534)

Follicle-stimulating hormone

11p14.1

 

AXL

(458)

 

AXL receptor tyrosine kinase

19q13.2

 

EAP1

(535)

Enhanced at puberty 1

14q24

Trans-activates the GnRH promoter

LGR4

(536)

Receptor for R-spondins which, once activated, potentiates the canonical Wnt signaling pathway

11p14.1

 

TUBB3

(483)

Microtubule protein β-III-tubulin

16q24.3

Congenital fibrosis of the extraocular muscles

WDR11, PROP1, PROK2, PROKR2

(529)

Bromodomain and WD repeat-containing protein 2, Homeobox protein 

prophet of PIT-1, prokinectin 2

10q26.12,

5q35.3,

3p13

Combined pituitary hormone deficiency

FTO

(482, 537)

Fat mass and obesity-associated protein

16q12.2

Mice lacking FTO had significantly delay in pubertal onset

 

ACQUIRED HYPOGONADOTROPIC HYPOGONADISM

 

Several conditions are associated with primary gonadal insufficiency. These conditions include auto-immune disorders, trauma, neoplasia, vascular events, and infection. Autoimmune disorders can be associated with premature ovarian and testicular insufficiency. Biallelic mutations in the autoimmune regulator (AIRE) gene are associated with autoimmune polyendocrine syndrome type 1 which is also known as autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. Associated features include mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency (375). The detection of autoantibodies directed against tissue-specific antigens suggests an autoimmune diagnosis.   

 

Antineoplastic chemotherapy with alkylating agents, as well as localized ionizing radiation, may permanently damage germ cells leading to infertility. In males, Sertoli cells are more susceptible to such toxicity than Leydig cells such that testosterone production may remain intact despite Sertoli and germ cell injury. Mumps orchitis should be considered, especially in unvaccinated males. Decreased blood flow to the gonads from surgical injury (e.g., orchiopexy in boys), torsion, or trauma can lead to ischemia and atrophy, with resultant primary testicular insufficiency.

 

The presence of otherwise normal male external genitalia associated with nonpalpable gonads indicates that the testes were present and functioning at least early in gestation. The “vanishing testes syndrome” also known as testicular regression is associated with atrophy or regression of testicular tissue initially formed during early embryonic development. Potential etiologies of the testicular regression include in utero vascular disruption or testicular torsion. Pathogenic variants of the DEAH-box RNA helicase DHX37 (DHX37) gene have been identified in boys with testicular regression and in association with gonadal dysgenesis (376).

 

In addition to autoimmune etiologies, premature ovarian insufficiency can be associated with ovarian/pelvic tumors, chemotherapy, especially alkylating agents, and radiation therapy. The location of the pelvic tumor and treatments influence the ovarian reserve and risk for premature ovarian insufficiency. Low or declining serum AMH levels provide an indirect measure of ovarian reserve. However, due to much variability and lack of diagnostic thresholds, measuring AMH values does not accurately predict ovarian insufficiency in cancer survivors (377).

 

Acquired hypogonadotropic hypogonadism (HH) can be due to central nervous system tumors such as craniopharyngiomas and germ cell tumors. Such tumors can disrupt the hypothalamic-pituitary stalk or can impact pituitary function producing decreased gonadotropin production. Hyperprolactinemia due to prolactin-secreting adenomas can cause acquired HH (378). Other central nervous system disorders associated with acquired HH include hypophysitis, histiocytosis, and hemochromatosis. Intracranial surgeries and/or cranial radiation therapy greater than 30 Gy are known risk factors for HH. Moderate to severe trauma to the brain is associated with injuries to the hypothalamus, stalk (infundibulum), or pituitary gland itself; the consequences of traumatic brain injury may not manifest for many years. Chronic steroid treatment can be associated with acquired HH in boys with Duchenne muscular dystrophy (379, 380). Inflammatory and infiltrative diseases of the pituitary gland are other rare causes of acquired HH.

 

FUNCTIONAL HYPOGONADOTROPIC HYPOGONADISM

 

Functional HH is the hypothalamic response to intense physical or emotional stress, caloric deficit, or chronic systemic illness (381). In this situation, the otherwise normal HPG axis fails to function due to the concomitant stress. Puberty can be delayed or stalled until the underlying condition has been adequately addressed. The finding of hypercortisolemia in women with functional HH associated with restrictive eating disorders highlights the relevance of HPA axis function in FHH (382). Importantly, functional HH can have long lasting adverse consequences on bone health (383).

 

The hypothalamus receives numerous inputs regarding body energy status and subsequently modulates reproductive status based on this information. Hence, nutritional status and energy output influence HPG axis activity in part via leptin signaling which regulates the sensitivity of the pituitary to GnRH (384). Energy deficits may occur due to weight loss, excessive energy expenditure (rigorous physical activity, renal disease, cystic fibrosis, congenital heart disease), decreased caloric intake or malabsorption (disordered eating behaviors, bowel disorders such as celiac, Crohn’s, and ulcerative colitis) are associated with delayed or stalled puberty and  functional hypothalamic amenorrhea (385, 386, 387). Elevated circulating levels of cytokines (as seen in some acute or inflammatory conditions) may also inhibit the HPG axis. Elevated prolactin levels, due to prolactinoma or severe primary hypothyroidism may inhibit gonadotropin release.

 

Some boys with obesity have low gonadotropin and testosterone levels and manifest delayed puberty (388). It is important to recognize that certain medications such as antipsychotics (typical and atypical), certain antidepressants, and opioids can alter menses (364). 

 

Treatment of Delayed Puberty

 

A variety of therapeutic regimens for pubertal induction have been described for both boys and girls. However, large, randomized trials providing evidence-based data regarding the optimum regimen are lacking (389). Sex steroid replacement therapy remains a mainstay of treatment. The type and route of administration of the sex steroids is dependent on patient preference, insurance coverage, and health care provider practices. Importantly, the specific treatment regimen depends on the underlying etiology of the pubertal delay. Future novel therapies could include kisspeptin and neurokinin B analogs (390).

 

BOYS

 

Pulsatile GnRH therapy is the most physiological method and can induce adult secondary sex characteristics, achieving normal adult testosterone concentrations, and spermatogenesis (370) in boys with HH. However, the inconvenience of wearing a mini-pump and conflicting outcome data limits its usefulness. Other approaches include hCG, FSH, hMG, and/or GnRH treatments. Despite much heterogeneity, a systematic study reported that treatment with hCG and FSH induced greater increase in testicular volume and rate of spermatogenesis compared to hCG alone (391)370). Importantly, available limited data suggest that testosterone administration prior to gonadotropin treatments does not interfere with the beneficial effects on testicular growth and spermatogenesis. Based on the physiologic roles of LH and FSH, pubertal induction should begin with FSH to promote testicular maturation followed by combined FSH and hCG treatment. The subsequent hCG treatment will promote testicular testosterone secretion leading to virilization, growth spurt, and psychosocial development.

 

Still, at the present time, testosterone is the most established treatment for pubertal induction in boys with delayed puberty. Traditionally, IM testosterone esters, primarily testosterone enanthate or testosterone cypionate, have been used. A subcutaneous testosterone enanthate auto-injector has recently been approved, but this approach requires more weekly injections and is more expensive. However, no evidence-based guidelines exist for testosterone-induced pubertal initiation. Potential adverse consequences of testosterone therapy include erythrocytosis, premature epiphyseal closure especially with excessive doses which may result in aggressive behavior, mood swings, and priapism.

 

Other testosterone formulations include testosterone gels, pills, and pellets. Limitations of testosterone gels include difficulties in accurately titrating low doses, potential testosterone exposure to household members, and the cost. Oral methyltestosterone and its 17α-derivatives have been associated with hepatic dysfunction and should be avoided. Oral testosterone undecanoate was approved by the FDA in 2019 to treat hypogonadal adult men. However, due to its short half-life, multiple daily doses are necessary, and no data are available regarding use for puberty induction. Testosterone pellets require surgical placement every 3-4 months, are expensive, and often spontaneously extrude (392).

 

For the younger adolescent boy with a strong family history of CDGP, reassurance and continued clinical monitoring may be adequate. However, discerning CDGP from CHH is essential because the treatment, genetics, and psychosocial implications differ. Hence, low dose testosterone for 3-4 months followed by a similar period of observation may be helpful to distinguish CDGP from CHH. Individuals with CHH will show persistently low gonadotropin and sex steroid hormone levels after the 3–4-month period of observation whereas individuals with CDGP will usually show spontaneous pubertal progression. Curiously, testosterone exposure apparently activates GnRH production and secretion leading to “reversal” with onset of HPG axis activity in some boys with CHH. This reversal is associated with specific genetic variants and may be transient (393, 394).

 

GIRLS

 

Timely induction of pubertal development is fundamental. Two major goals of estrogen therapy are mimicking typical pubertal progression with breast development and promoting adequate uterine growth (395). Although pulsatile GnRH treatment can be used, this approach has no advantage over estrogen for pubertal induction in girls. Though all therapeutic approaches utilize estrogens, details regarding specific formulations and methods of administration vary. Transdermal estradiol is preferred for replacement therapy because this approach avoids the first pass through the liver and the potential for adverse effects on clotting factors.

 

Typically, low transdermal estradiol doses are used for the initial phase of pubertal induction. Transdermal estradiol doses of 3-7 mcg/day can be achieved by cutting matrix patches (0.014-0.025 mg/24 h) into quarters or eighths. Subsequently, the dose can be increased approximately every six months until adult replacement dosage is achieved taking about 24-36 months to do so. High initial estrogen doses should be avoided due to increased likelihood for atypical breast development characterized by prominent nipples with little supporting breast tissue. High estrogen doses should also be avoided as premature epiphyseal fusion could impair additional linear growth.

 

Oral micronized 17β-estradiol can be used for those with severe skin irritation or aversion to the use of a patch. Oral preparations containing conjugated equine estrogens or ethinyl estradiol should be avoided for both pubertal induction and maintenance therapy. Most combined oral contraceptives contain ethinyl estradiol at doses higher than appropriate for induction of puberty. Approximately 18-24 months after initiation of unopposed estrogen therapy, progestogens can be added to induce withdrawal bleeding and to reduce the risk for endometrial hyperplasia. Progestogens can be introduced earlier if breakthrough vaginal bleeding occurs. Pelvic ultrasounds before and during pubertal induction can be planned to assess uterine size and shape as well as to evaluate endometrial thickness to ascertain optimal timing to introduce progestins. Progestins vary in potency and can be administered by transdermal, oral, or uterine routes. Although increased potency may have beneficial effects on withdrawal bleeding, greater progestogenic side effects may develop.

 

No evidence-based data exist, and no single regimen has been demonstrated to be superior. Pubertal induction therapy should be individualized based on clinical response and other auxologic parameters.

 

Oral contraceptive pills may be used for convenience but should be limited to after completion of pubertal development. Since some girls may experience sporadic ovulation, contraception should be utilized by those at risk of undesired pregnancies.

 

EVALUATION OF A CHILD WITH A VARIATION IN PUBERTAL DEVELOPMENT

 

The diagnostic tools are comparable for the evaluation of either precocious or delayed puberty. Detailed medical history and physical examination provide the preliminary information to guide the differential diagnosis for a child with a variation in pubertal development (165, 396) (see Figures 5-8). Laboratory, imaging, and genetic studies are subsequently utilized to ascertain the specific diagnosis. The tools for evaluation of a child with a variation in pubertal development are described below. The tools are comparable, but the interpretation of test results differs for precocious and delayed puberty.

 

Figure 5. Algorithm to evaluate a girl presenting with precocious puberty. *follow clinical progression every 3-6 months. FSH: Follicle Stimulating Hormone; LH: Luteinizing Hormone; CAH: Congenital Adrenal Hyperplasia; ACTH: Adrenocorticotrophic Hormone; GnRH: Gonadotropin Releasing Hormone; DHEAS: Dehydroepiandrosterone Sulfate; 17OHP: 17-hydroxy progesterone; NF-1: Neurofibromatosis-1.

Figure 6. Algorithm to evaluate a boy presenting with precocious puberty. FSH: Follicle Stimulating Hormone; LH: Luteinizing Hormone; MRI: Magnetic Resonance Imaging.

Figure 7. Algorithm to evaluate a girl presenting with delayed puberty. FSH: Follicle Stimulating Hormone; LH: Luteinizing Hormone; CDGP: Constitutional Delay in Growth and Puberty; CHH: Congenital Hypogonadotropic Hypogonadism; MRI: Magnetic Resonance Imaging.

Figure 8. Algorithm to evaluate a boy presenting with delayed puberty. FSH: Follicle Stimulating Hormone; LH: Luteinizing Hormone; CDGP: Constitutional Delay in Growth and Puberty; CHH: Congenital Hypogonadotropic Hypogonadism; MRI: Magnetic Resonance Imaging.

 

History and Physical Examination

 

The medical history focuses on the timing and sequence of the pubertal changes in the patient as well as parents, grandparents, and siblings. Review of past medical history and medications (including chemotherapy and nutritional supplements) is essential. Inquiry regarding exposures (tea tree/lavender oils, sex steroids, radiation) may help to identify potential environmental factors (162). Obtaining birth history, length and weight, history of SGA (397), prematurity, or CNS insult at birth or later provide relevant information (162).

 

A history of gelastic seizures may point to a hypothalamic hamartoma. Inquiry regarding use of transdermal testosterone by a family member may identify the cause of premature virilization. Pubertal delay associated with micropenis, anosmia, cryptorchidism, deafness, choanal atresia, hearing loss, and/or digital abnormalities suggests congenital hypogonadotropic hypogonadism (CHH). A family history of anosmia, subfertility, and deafness should be sought for those with pubertal delay. Multiple syndromes are associated with CHH (see Table 3); suggestive features include absent/reduced sense of smell, choanal atresia, hearing loss, morbid obesity, visual impairment. Family history of precocious or delayed puberty in close relatives may be discovered (398). Behavioral difficulties or learning disabilities may be associated with specific syndromes such as Turner or Klinefelter syndromes.

 

A complete physical examination including height, weight, arm span, and sitting height is essential. Review of the child’s growth curves provides valuable information regarding changes in linear growth and weight gain. Acceleration in linear growth and upward crossing of centiles may be seen in precocious puberty. A gradual downward crossing of centiles may be noted in constitutional delay in growth and puberty (CDGP) as linear growth slows compared to peers who are entering puberty (399). Pubic hair development (pubarche) may also be delayed in CDGP as opposed to CHH where adrenarche occurs at the normal age for population (372).

 

Physical exam includes ascertainment of the sexual maturity rating for breast, pubic hair, and testicular volume based on the scoring system derived by Tanner and colleagues (Figure 1). Due to challenges in discriminating lipomastia from true glandular breast development, palpation of the breasts is important. Firm glandular tissue under the areolae is indicative of thelarche. Accurate measurement of testicular volume using an orchidometer is essential (see Figure 2). A testicular volume of ≤ 1.1 mL has a reported sensitivity and specificity of 100% and 91%, respectively, for CHH (400).

 

The physical examination should assess for midline defects, dysmorphic features, visual field abnormalities, and features characteristic for specific syndrome. For example, short stature, cubitus valgus, low hair line, widely spaced nipples, and delayed puberty suggest Turner Syndrome. The physical examination needs to include palpation of the thyroid gland, skin examination for acne or café-au-lait macules (which would suggest neurofibromatosis or McCune-Albright syndrome) and a visual field exam. Melanocytic macules typical of Peutz-Jeghers syndrome could point to the presence of a sex cord tumor causing gonadotropin independent (peripheral) sexual precocity.

 

Laboratory Evaluation

 

Laboratory evaluation assists the diagnostic process to identify the etiology of “off-time puberty.” Circulating gonadotropin and sex steroid concentrations reflect HPG axis status (187, 236). Most current gonadotropin assays are sandwich assays specific to the β-subunit. Ultrasensitive FSH and LH assays should be used when available. For LH, samples should preferably be obtained in the morning. The lower limit of detection for most ultrasensitive immunochemiluminescent assays (ICMA) is ≤0.1 mIU/mL (230, 401, 402).

 

When the clinical concern is precocious puberty, LH concentrations greater than 0.3-0.5 mIU/mL suggest central precocious puberty (CPP) with higher cut-points increasing the sensitivity and specificity of the LH determination (403). Elevated basal LH levels show high sensitivity and specificity for boys when high quality immunochemiluminometric assays (ICMA) is used (404). Different cut-points need to be used to interpret LH concentrations in girls under two years of age because LH concentrations may be elevated at this age leading to misdiagnosis of CPP followed by inappropriate treatment during this phase of development (405). For the child with physical signs of premature puberty, LH concentrations in the prepubertal range are consistent with either peripheral precocity or a benign pubertal variant such as premature thelarche. Typically, LH and FSH concentrations are suppressed in children with peripheral precocious puberty (406).

 

In the evaluation for delayed puberty, low gonadotropin concentrations suggest a central etiology such as CDGP or hypogonadotropic hypogonadism while elevated gonadotropin concentrations suggest primary gonadal insufficiency. Random gonadotropin concentrations may provide only limited information because gonadotropin secretion is pulsatile. Distinguishing hypogonadotropic hypogonadism from CDGP is often challenging because LH, FSH and sex hormone reference intervals vary widely even in healthy adolescents (407). Similarly, due to significant overlap in hormone reference intervals, GnRH agonist and human chorionic gonadotropin (hCG) stimulated gonadotropin (408) and sex steroid concentrations fail to distinguish youth with CHH from those with CDGP (407, 409).

 

Due to the small structural differences between steroid molecules, immunoassays are confounded by cross-reactivity issues. Assay issues are amplified in children because commercial immunoassays for estradiol and testosterone are usually designed to measure hormone concentrations within the normal adult reference interval. Hence, most estradiol immunoassays have low sensitivity and specificity to quantify the low concentrations (< 30 pg/ml) typically found in prepubertal children and individuals with hypogonadism. Similar issues occur with testosterone immunoassays. Hence, steroid hormone concentrations should be measured by liquid chromatographic separation followed by mass spectrometry (LC-MS/MS). Serum testosterone is best measured using LC-MS/MS technology to limit cross-reactivity and increase sensitivity and specificity especially when low hormone concentrations might be anticipated. LC-MS/MS is also the optimal technique to measure circulating concentrations of other steroids including 17-hydroxyprogesterone, DHEA, androstenedione, and the 11-oxy androgens. It offers greater sensitivity and specificity and allows simultaneous measurement of multiple hormone concentrations (410).

 

Sex steroids such as estradiol and testosterone circulate bound to sex hormone binding globulin (SHBG). Tissue availability of the free hormone, presumed to be the active moiety, is regulated by SHBG. Direct free testosterone concentrations should be avoided because direct immunoassays have poor reproducibility and reliability. When free testosterone concentrations need to be determined, equilibrium dialysis should be performed despite known potential limitations including increased expense, reliance on total testosterone accuracy, temperature control, and sample dilution (411).

 

Another confounding factor is biotin (vitamin B7) which is an over-the counter supplement by itself or as an addition to many preparations used to strengthen nails and hair. Biotin interferes with the technical aspects of immunoassays and can lead to either falsely elevated or falsely low result when streptavidin binding is utilized in the assay detection system. When immunoassay results seem incongruous, use of biotin-containing products should be queried. Biotin does not interfere with LC-MS/MS assays (412).

 

GnRH or GnRH AGONIST STIMULATION TEST

 

Historically, the established gold standard to diagnosis CPP was the LH and FSH response to a standard bolus of native GnRH. With decreased availability of native GnRH, most stimulation tests are now performed with the GnRH agonist (GnRHa) leuprolide acetate, a synthetic nonapeptide with much greater potency. The timing and peak values of FSH and LH levels differ between GnRH and leuprolide acetate. Following native GnRH administration, LH levels peak after 20–40 minutes, followed by a decline. With leuprolide acetate, peak LH occurs between 0.5 - 4 hours followed by sustained LH elevation.

 

The optimal cutoff value of peak stimulated LH for identifying children with CPP is unclear due to assay variability. For most LH assays, a value of 3.3 to 5 mIU/mL defines the upper limit of normal for stimulated LH values in prepubertal children. Stimulated LH concentrations above this range suggest CPP (232). Children with progressive CPP tend to have a high stimulated LH:FSH ratios compared with those with non- or intermittently progressive precocious puberty. Measuring the appropriate sex steroid 24 hours following GnRHa administration can help confirm a CPP diagnosis (413). However, obtaining this second sample may burden the family because of the need for a second venipuncture, expense of another hormone determination, and missed school and work.

 

As noted above regarding basal LH levels, care must be taken in interpreting the results of GnRH stimulation test in females under the age of two years, as both basal and stimulated LH levels can be elevated as part of the normal hormonal changes associated with mini-puberty (405).

 

To assess GnRH production by the hypothalamus, kisspeptin-stimulated LH response has been proposed to identify individuals with GnRH deficiency and thus CHH. Kisspeptin stimulates GnRH secretion, thus promoting LH, and to a lesser extent FSH, secretion.

 

One study found that maximal LH rise after kisspeptin administration was more accurate for diagnosis of men with GnRH deficiency than GnRH stimulation testing (414). A similar study in adolescents with pubertal delay (3 females and 13 males), peak LH post kisspeptin stimulation was demonstrated to be superior to GnRH stimulation testing for predicting capacity to progress through puberty (noting that the LH cut off values were different and an ideal cutoff value still needs to be determined) (346). Further research is required to better define the parameters of using kisspeptin stimulation in clinical practice (404).

 

In children with precocious pubarche, measurement of adrenal steroids may be necessary to help distinguish between peripheral precocity and benign premature adrenarche. Children with premature adrenarche can have mild elevation in adrenal hormones (415). Since premature adrenarche is a diagnosis of exclusion, further investigation for congenital adrenal hyperplasia and virilizing adrenal tumors may be indicated. In children, an early-morning 17-hydroxyprogesterone (17-OHP) value >200 ng/dL (6 nmol/L) has a high sensitivity and specificity for non-classic congenital adrenal hyperplasia secondary to 21-hydroxylase deficiency. An adrenocorticotropic hormone (ACTH) stimulation test is needed to confirm the diagnosis (313, 416). An ACTH stimulation test involves administration of 0.25 mg synthetic ACTH (1-24) or 15 mcg/kg for children up to 2 years of age with blood samples obtained at baseline and either 30 or 60 minutes after synthetic ACTH administration. Although 21-hydroxylase deficiency is the most common virilizing form of CAH, 17-hydroxypregnenolone, DHEA, and 11-deoxycortisol determinations may be necessary to assess for 3β-hydroxysteroid dehydrogenase or 11β-hydroxylase deficiencies.

 

Boys with hypogonadotropic hypogonadism tend to have lower inhibin B values compared to boys with CDGP. However, a validated cut-point for inhibin B concentrations remains to be established (417, 418, 419). FSH stimulated inhibin B concentrations < 116 pmol/L have been demonstrated in a study of adolescents with delayed puberty to have more accurate diagnostic discrimination and a promising test for prediction of onset of puberty (414).

 

Human chorionic gonadotropin concentrations can be measured in males to evaluate for the possibility of an hCG-secreting tumor leading to peripheral precocity (280). A thyroid-stimulating hormone (TSH) concentration should be measured if chronic primary hypothyroidism is suspected as the underlying cause for the sexual precocity, known as the Van-Wyk-Grumbach syndrome (296, 298) .

 

Targeted diagnostic tests are warranted in some cases to investigate for specific causes of apparent functional hypogonadotropic hypogonadism, such as anti-transglutaminase IgA for celiac disease. Despite promising data, measurement of AMH and INSL3 in addition to testosterone, as endocrine markers to guide the differential diagnosis (418, 420), need additional studies  

 

The testosterone response to long-term hCG stimulation and peak serum FSH response to GnRH were found to be significantly different in CHH patients (421). However, there are potential long-term drawbacks to prolonged hCG therapy in males who are FSH-naïve regarding premature stimulation of Sertoli and germ cell differentiation prior to FSH exposure (338). 

 

Imaging Studies  

 

BONE AGE

 

Assessing the skeletal maturation based on a radiograph of the left hand and wrist is an important diagnostic tool in pubertal evaluation. For the commonly utilized Greulich and Pyle method, the patient’s bone age radiograph is compared with an atlas of radiographs from children of known ages (422). For the Tanner-Whitehouse 2 method, 20 different hand and wrist bones are scored. Bone age standards are largely based on hand and wrist radiographs obtained from children of European ancestry between the 1930s to the 60s (423). Despite this limitation, the bone age radiograph is a valuable indicator regarding sex steroid exposure and epiphysial (growth plate) maturation. Additional factors such as other hormones, obesity, genetics, nutritional status, various disease states, and certain medications can influence the rate of epiphyseal maturation (424) (425).

 

Bone age has been used to predict adult height using the tables of Bayley and Pinneau (426), but reliability is low with a tendency toward overestimation (427). The use of automated measurement systems with artificial intelligence has increased, mitigating previous limitations due to intra- and inter-observer variability (428, 429). Bone age readings within two standard deviations of the chronologic age are considered to be within normal limits. A delayed bone age is usually observed in patients with delayed puberty and an advanced bone age is observed with precocious puberty. One exception is patients with precocious puberty associated with hypothyroidism (Van Wyk Grumbach syndrome) where the bone age is delayed despite pubertal changes. In some instances, monitoring the predicted adult height (PAH) during the course of treatment of pubertal disorders helps to assess treatment efficacy.

 

ULTRASOUND IMAGING

 

In females, pelvic ultrasound is a rapid, non-invasive, and relatively low-cost method to ascertain the anatomy of the ovaries and uterus, ovarian volume, and uterine development. This imaging is generally readily accessible and does not require sedation, radiation, or use of contrast material. However, the quality of the device and operator experience influence the analysis.

 

During puberty, increased gonadotropin secretion promotes ovarian growth, increased estradiol secretion, and increased uterine volume (430). Girls with CPP have increased uterine size and ovarian volumes compared to prepubertal girls or those with premature thelarche. However, the overlap between prepubertal and early pubertal girls for ovarian volume and uterine size confounds interpretation of the ultrasound findings (431) (432). In a prepubertal patient with isolated vaginal bleeding, a normal pelvic ultrasound does not exclude the diagnosis of a functional ovarian cyst because the cyst may have regressed prior to imaging. Pelvic ultrasounds should be obtained in girls with primary amenorrhea who fail progesterone withdrawal to assess for Mullerian duct and renal anomalies. For patients with rapid development of secondary sex characteristics, pelvic ultrasound studies may be needed to assess for gonadal tumors.

 

The use of Doppler ultrasound to assess utero-ovarian blood flow may also provide helpful information. With increased estradiol secretion and stimulation of the estrogen receptors, vascular resistance of the uterine arteries is reduced. The pulsatility index (PI) is defined as the difference between peak systolic flow and end-diastolic flow divided by the mean flow velocity; the PI reflects impedance to blood flow distal to the sampling point. A review showed that PI is lower among pubertal girls. However, definitive cut-points for PI values have not been established. In addition, testing is operator dependent (433, 434, 435, 436).

 

Ultrasound examination of the testes, especially if asymmetric in size, should be performed in males with peripheral precocity to evaluate for the possibility of a Leydig cell tumor (437, 438). Testicular ultrasound imaging should be performed regularly to assess for testicular rest tissue in boys with congenital adrenal hyperplasia (439).

 

MR AND CT IMAGING

 

Brain MR or CT imaging is performed to define brain and pituitary anatomy. Brain and pituitary MR is helpful to assess for intracranial pathology among those with CNS symptoms. Most studies recommend a contrast-enhanced brain MRI for girls with onset of secondary sexual characteristics before six years of age because of higher rates of CNS abnormalities in these patients (137). In a 2018 meta-analysis (440), the prevalence of intracranial lesions was 3 percent among girls presenting with CPP after six years of age, compared with 25 percent among those presenting before six years. Thus, girls with pubertal onset between six and eight years of age may not need the MRI in the absence of clinical evidence of CNS pathology (441, 442, 443). MRI should be limited to high-risk individuals (younger age, neurologic symptoms) (444). Current guidelines recommend that in otherwise asymptomatic girls with CPP, a discussion occur with the parents regarding the pros and cons of brain imaging and assist in informed decision making (137, 445, 446). While contrast-enhanced brain MRIs are recommended for all boys presenting with CPP (412), one study found that these rates may be overestimated. The prevalence of intracranial lesions among boys who were healthy, did not have neurological symptoms, and were diagnosed with CPP was lower than that previously reported and none of the identified lesions necessitated treatment, suggesting the need to globally reevaluate the prevalence of pathological brain lesions among boys with CPP (447).

 

For children with delayed puberty, MR imaging of the pituitary gland and olfactory structures can assess for features of CHH such as absence of the olfactory bulbs (448, 449, 450).

 

Pelvic MRI is helpful to characterize and stage pediatric ovarian masses due to excellent soft tissue contrast. In addition, MR imaging does not involve the use of ionizing radiation and allows better assessment of the abdomen and kidneys. Disadvantages of MRI include that it is time-consuming, expensive, and may require sedation.

 

In both girls and boys, adrenal tumors can cause peripheral precocious puberty, progressive virilization, and/or markedly elevated serum adrenal androgens (e.g., DHEAS). If diagnoses such as congenital adrenal hyperplasia and exogenous androgen or testosterone exposure have been excluded, such patients should have an imaging study of the adrenal glands (451, 452, 453). CT may be preferable for evaluation, staging, surgical planning for adrenal tumors (454). Despite radiation exposure, CT can be readily performed in emergent situations.

 

Genetic Testing  

 

A karyotype can help with a diagnosis of Turner or Klinefelter syndrome (455).Newer sequencing technologies along with increased knowledge regarding genes involved in puberty has advanced the usefulness of genetic testing (456). The known genetic causes of CPP and HH have increased exponentially over the past five years. Genetic testing could therefore precede brain MRI, at least in familial CPP cases (167, 457).

 

Patients with delayed puberty associated with phenotypic features such as anosmia/hyposmia, synkinesia, or hearing loss, the probability of detecting a pathogenic variant on genetic testing for HH is increased (458) (459, 460). Consideration should be given to using genetic testing early in the diagnostic process while recognizing the limitations of genetic testing. Challenges in using genetic testing as a discriminatory test between CHH and CDGP remain, and more research is needed in this area.

 

SUMMARY

 

Pubertal development and maturation of the neuroendocrine system involve the ontogeny, activity, and interactions of the GnRH neurons. Pubertal onset is accompanied by an increase in kisspeptin and neurokinin B secretion regulating the pulsatile GnRH secretion that stimulates pulsatile pituitary LH and FSH secretion. LH and FSH stimulate gonadal sex steroid secretion promoting development of secondary sex characteristics and influencing hypothalamic-pituitary function via negative feedback inhibition.

 

Alterations of gut microbiome at different pubertal stages may present an area for future development in the prediction and prevention of precocious puberty. Use of genetic testing including targeted next generation sequencing and whole exome sequencing may have increasing utility as diagnostic tools early on in the evaluation of pubertal disorders.

 

Discovery regarding the details of normal reproductive physiology followed by identification of the genetic basis for disorders of pubertal timing established our current knowledge base for the evaluation and management of children with disorders affecting the timing of puberty. Despite the vast expansion of our knowledge, much remains to be learned about the physiology and regulation of the HPG axis from the fetus to the young adult.

 

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Pediatric implications of normal insulin-GH-IGF-axis physiology

ABSTRACT

 

Understanding the involvement of the insulin-GH-IGF-axis in the different phases of human growth, development, and metabolism is the key to understanding human pathophysiology. The normal physiological actions of the axis optimize human growth and metabolism to impact adult height by approximately one third. IGF binding proteins modulate access of circulating IGF-I to the tissues and modulate IGF-I and -II access to the type 1 IGF receptor (IGF1R) at the cellular level. Complete lack of IGF1R signaling is most likely not compatible with a viable human fetus, while allelic haploinsufficiency impairs brain development and causes severe short stature. Lack of insulin receptor signaling in Leprechaunism may result in the rare event of an alive but severely small for gestational age baby that will only survive if treated with recombinant-IGF-1 to substitute inulin receptor signaling with IGF1R signaling via their common intracellular pathways. IGF-I gene defects result in mental retardation and severe fetal and postnatal growth failure with GH hypersecretion and marked insulin resistance. Likewise, IGF2 gene defects or imprinting defects cause severe fetal growth failure but somewhat less adverse effects on postnatal growth, more variable effects on brain development, and an absence of marked metabolic effects. GH fine-tunes insulin and IGF-I signaling with no impact on IGF-II expression and has a minor impact on fetal development and growth. GH effects on lipolysis are established in the newborn and ensure gluconeogenesis and prevents hypoglycemia after birth. The complete absence of GH expression such as in GHRHR or GH1 gene defects or absence of GH signaling in GHR or STAT5B gene defect leads to an adult height of 120-130cm if untreated, and has severe metabolic consequences. Even excess of insulin, GH, IGF-I and IGF-II signaling are associated with severe metabolic disease and excess growth and/or obesity. Malnutrition or malabsorption causes decreased insulin signaling which reduces GHR expression and blocks the GH signaling pathway leading to IGF-I expression (GHR uncoupling), while GH’s metabolic actions on lipolysis and gluconeogenesis are unaffected. GH signaling attenuate insulin actions on glucose metabolism which causes insulin resistance and hyperinsulinemia or may precipitate diabetes. However, insulin signaling pathways that enhances GHR function or suppress IGFBP-1 or SHBG production are still intact and promote anabolism, optimize growth, enhance androgen actions and play a mechanistic role in premature adrenarche and PCOS. Long-term nutritional deprivation compromises growth, while from a developmental perspective, decreased insulin signaling (leading to GHR uncoupling) prolongs life (at least in some experimental animal models) which ensures that fertile age is reached, and survival of the species is ensured. For the health of the general population, the subtle changes in insulin, GH and IGF-I signaling associated with gene polymorphisms or epigenetic changes programmed during fetal and early postnatal life and affecting gene expression are important. They determine growth and pubertal development in childhood and predispose the individual for developing the metabolic diseases and malignancies in adult life, as predicted by the Barker hypothesis. As the roles of the insulin-GH-IGF-axis in growth and metabolism, often discussed separately, are intimately linked they will be described jointly here.

 

EARLY WORK DEFINING THE INSULIN-GH-IGF-AXIS

 

Daughaday realized that the mitogenic effect of GH in the growth plate was not direct but mediated by Insulin-like Growth Factor-I (IGF-I), at that time named sulphation factor or somatomedin C (1). Another effect of IGF-I was insulin-like and not inhibited by insulin specific antibodies (2,3,4) and therefore it was named non-suppressible Insulin-like activity (NSILA). Hall and Van Wyk purified IGF-I from human muscle extracts (5,6) and realized that these biological activities originated from the same molecule. They also identified significant quantities in blood (7). The primary structure of IGF-I and Insulin-like Growth Factor-II (IGF-II) was discovered by Froesch and coworkers as a result of their persistent work to characterize the metabolic activity of NSILA (8,9). Soon after, the mitogenic activity of the sulphation factor or somatomedin C as well as somatomedin A was shown to be identical to IGF-I (10). Rechler and Nissley demonstrated that IGF-II was identical to multiplication stimulating activity, a factor known to stimulate DNA synthesis in chick embryo fibroblasts (11).

 

The concept that binding proteins existed for peptide hormones like the IGFs, similar to those for steroid and thyroid hormones, were suggested by studies from Zapf and Froesch (12) and by Hintz (13), demonstrating that NSILA was present in high molecular weight complexes in serum. The binding was exclusive to IGFs and did not apply to insulin or proinsulin despite their structural similarities. High molecular weight IGF-I complexes with IGFBPs were GH dependent (14) and formed a ternary complex composed of IGFBP-3 (15), the Acid Labile Subunit (ALS) (16) and IGF-I or IGF-II. Low molecular weight complexes contained IGF-I or IGF-II bound to an insulin regulated liver derived protein IGFBP-1 (17, 18), at first called the 28 kDa binding protein or PP12 (19). The existence of other IGF binding proteins, six in total, became clear when Hossenloop (20) developed Western ligand blotting as a technique to quantify these proteins. The components of the IGF-IGFBP-system are outlined in Figure 1.

 

Figure 1. The primary structures of IGF-I, IGF-II and insulin are similar. IGFs are produced by many differentiated cell types, and their bioactivity in the extracellular fluids or in the circulation are coordinated by six IGF binding proteins (IGFBP-1 through -6). IGFBP-3, the major binding protein in serum is stimulated by GH and it forms a large 150 kDa ternary complex with IGF-I or -II and the GH regulated acid labile subunit (ALS). IGFBP-5, an important supporter of bone tissue formation, also forms ternary complexes with IGF-I or -II and ALS. IGFBP-1, suppressed by insulin, is one of several binding proteins in the smaller 50 kDa binary complexes with IGF-I or –II. IGFBP-2 has inverse association with insulin under many physiological conditions. In contrast, IGFBP-4, -5 and -6 do not appear to be directly regulated by GH or insulin and are important local regulators of IGF activity in bone and the CNS. The type 1 IGF receptor (IGF1R) is the mediator of the mitogenic, anti-apoptotic, differentiating and metabolic effects of both IGF-I and -II. The structural similarity of the IGF1R with the insulin receptor (IR) explains the formation of hybrid receptors in cells that expresses both receptors such as myocytes and pre-adipocytes. Cross reactivity among the ligands and the receptors have been demonstrated, although it has minor importance under physiological conditions but may cause non-islet-cell tumor hypoglycemia due to unprocessed pro-IGFs with markedly decreased binding affinity to IGFBPs. A second receptor, exclusively binding IGF-II, work as a scavenger receptor and is identical to the mannose-6-phosphate receptor, known to direct proteins for degradation in the lysosomes. A second level of control of IGF bioactivity is exerted by IGFBP proteases which release IGF-I activity after fragmentation of IGFBPs. Specific production and regulation of IGFBP proteases at the tissue level controls processes such as ovulation and atherosclerosis. Furthermore, interaction of IGFBPs and IGFBP proteases with the extracellular matrix modify the binding affinity for the IGFs and are involved in prolonging the actions of IGFs at the tissue level. Extracellular matrix also signals though integrin receptors on the cell surface and modifies IGF-1R signaling. This figure also shows the existence of IGFBP-related proteins with markedly lower affinity for the IGFs and with physiological roles not related to their IGF binding.

 

ANIMAL EXPERIMENTS ESSENTIAL FOR THE UNDERSTANDING OF HUMAN INSULIN-GH-IGF AXIS PHYSIOLOGY

 

Insulin, IGF-I, and IGF-II and Their Receptors

 

Efstratiadis’ series of knock-outs of the insulin-GH-IGF-axis in mice in the early 1990s clearly confirmed its importance in fetal and postnatal growth and metabolism (21). It also predicted the phenotype of experiment of nature in humans with gene defects in the axis yet to be discovered. The studies opened new insights, not least the equal importance of IGF-I and IGF-II in fetal growth, reducing birth weight by about 60 % in both Igf1 knock-out (Igf1ko) and Igf2ko animals and demonstrating that the previous perceived concept that there was a fetal (IGF-II) and a postnatal (IGF-I) form of IGF was incorrect. IGF-I and -II had actions through the type 1 IGF receptor (IGF1R) demonstrated by Igf1rko animals with 45% of wild type birth weight and no further effect when crossed with Igf1ko animals. While Igf1ko animals were viable depending on genetic background and were non-fertile, the Igf1rko animals died from respiratory failure but with an absence of apparent malformations. Interestingly, crossing Igf2ko with Igf1rko resulted in further growth retardation indicating that IGF-II had actions through an additional receptor. Another new insight came from knock-out of the ‘mysterious type 2 IGF receptor’, identical to the mannose-6 phosphate receptor (M6P-R), specifically binding IGF-II and involved in internalization of proteins for lysosomal degradation. Knock-out of the Igf2r/M6pr resulted in increased serum and tissue levels of IGF-II and fetal overgrowth (140% of wild-type birth weight) (22). This receptor works to clear IGF-II and its presence in endothelial cells may, at least partly, explain the lack of endocrine actions of IGF-II due to its proteolytic lysosomal degradation (23). Thus, IGF-II effects on fetal growth are paracrine/autocrine actions mediated by the IGF1R. Knock-out of the Igf2r/M6pr gene combined with Igf2ko/Igf1rko could partly rescue growth retardation, a finding that was explained by IGF-II actions via the insulin receptor (INSR). The formation of heterodimers, more commonly named hybrid receptors, between type A or B isoforms of the insulin receptors (INSRA or INSRB) and the IGF1R of which IRA-IGF1R are highly expressed in the fetus (and in malignant cells) and activated by IGF-II, may further point to the importance of IGF-II during the fetal period. INSRB-IGF1R hybrids comprises up to 30 % of INSR and IGF-I receptors in muscle due to high expression of both and this hybrid predominantly responds to IGF-I (less to insulin) and explains the important role of IGF-I in growth and metabolism in skeletal muscle.

 

Postnatally, the Igf1ko mice continued to grow poorly, resulting in an adult weight 30% of wild-type and with poor gonadal function and delayed bone development. Knock-out of GH or its receptor (GHR), both expressed in the mouse fetus, did not affect birth size, indicating that the Igf1 gene is not under GH control during the fetal period. The actions of GH and its receptor on growth in mice were obvious from postnatal day 15 and largely slowed growth resulting in a 50 % reduction of wild-type adult weight. On the other hand, double Ghrko/Igf1ko resulted in further postnatal growth retardation relative to Igf1ko mice completely obstructing further weight gain after postnatal day 15 and supporting previous studies suggesting that progenitor cells in the growth plate require direct GH actions (24).

 

IGFBPs and IGFBP Proteases  

 

Like the above attempts to pinpoint the role of important ligands and receptors in the axis, steps to assess the role of IGF binding proteins involved in modulating IGF-I and IGF-II bioactivity were taken (reviewed by Pintar (25)). In contrast to the pronounced phenotypes caused by mutations in receptors and their ligands, the growth phenotypes of the various IGFBP knock-out animals were far less pronounced as were the metabolic changes observed (26,27,28). It was argued that there is a large degree of redundancy among the functions of the IGFBPs which to some extend contradicts their specialized functions in various tissues (29). However, this idea was to some extent supported by the finding of somewhat more pronounced phenotypes in double and triple knock-out animals (30). This is largely in accordance with the absence of reports of IGFBP gene defects causing growth retardation in humans. The most affected phenotype identified was that of Igfbp4ko mice who were growth retarded at birth and displayed poor postnatal growth (30). No such mutation has been identified in humans. IGFBP-4 is specifically degraded by the metalloproteinase PAPP-A (Pregnancy Associated Plasma Protease -A) produced by the placenta as well as bone and ovary. In Pappa knock-out animals a 20-30% reduction in body weight was reported (31). Interestingly, the growth restriction phenotype of mice null for Pappacould be rescued by disruption of IGF-II imprinting during embryonic development (32).

 

Endocrine Versus Paracrine Autocrine IGF-I

 

One of the controversies of the area has been the relative contribution to linear growth of circulating endocrine IGF-I largely produced by the liver versus peripherally produced IGF-I with major paracrine/autocrine actions on local tissues. The major importance of paracrine/autocrine IGF-I was demonstrated by liver specific Igf1ko mice (Ligf1ko) with largely unaffected longitudinal growth (33). Circulating levels were 20% of wild-type with compensatory elevation of GH, insulin resistance and hyperinsulinemia. With age the animals developed type 2 diabetes, underlining the metabolic consequences of largely elevated GH combined with circulating IGF-I deficiency (34). Somewhat unexpectedly, this animal model closely resembles children and adolescents with type 1 diabetes, as further elaborated on below.

 

Another model to assess the relative importance of endocrine versus paracrine/autocrine IGF-I is the liver-specific Ghrko mouse. It produces a similar phenotype but with more specific hepatic consequences of absent GH signaling (35).

 

INSULIN-GH-IGF AXIS PHYSIOLOGY, A PEDIATRIC PERSPECTIVE

 

Insulin-GH-IGF-Axis and Human Fetal Growth

 

IGF-I controls the pace of the cell cycle from early on in human embryogenesis. INSR and IGF1R is expressed in human pre-implantation blastocysts already from the 8-cell stage, while IGF-II is expressed already in the oocyte (36). After implantation, IGF-I is produced in the human embryo (37), but until then the source of IGF-I is thought to be the female reproductive tract, and it is known that the availability of the IGF-I ligand is important for blastocyst growth in human in vitro fertilization - IVF (38). IGF-I production is controlled by nutritional factors in the early embryo and even later during human fetal development (39). Circulating endocrine IGF-I increases with gestational age (40) and is strongly correlated with fetal growth in the second part of gestation (41,42). However, little has been reported concerning the regulatory control of the IGF1 gene in the human fetus. IGFBPs can be identified in the human fetal circulation (40), and recently the role of IGFBP-5 in regulating fetal growth was suggested by fetal growth retardation in the absence of a specific IGFBP-5 protease, PPAP-A2 (43). Insulin continues to be permissive for IGF-I production even after GH is established as the major pituitary stimuli controlling endocrine as well as paracrine/autocrine IGF-I, as described below.

 

Fetal Growth Restriction and Programming of the Insulin-GH-IGF-Axis Setpoint

 

Insulin resistance has been developmentally advantageous for mankind until very recent decades of excess food and sedentary life style. It was proposed by Barker et al (44) in his ‘fetal and infant origin of adult disease’ hypothesis that intrauterine restriction of growth compensated by excessive postnatal catch-up growth results in an increased risk of developing disease entities of the metabolic syndrome later in life. In his early epidemiological studies, he demonstrated that there is a U-shaped relationship between birth weight and risk of obesity, insulin resistance, type 1 diabetes, hypertension, dyslipidemia and ischemic heart disease, with lower birth weight (within the normal range) imposing a risk. Notably, at higher birth weights this risk rises again which may represent genetic risks of obesity and type 2 diabetes. The concept was that poor fetal nutrition would lower fetal IGF-I and program the child to a low IGF-I setpoint and slower postnatal growth, an epigenetic phenomenon that could be preserved over a few generations (45). At the same time, small for gestational age (SGA) babies becomes insulin resistant (46) and this trait is enforced by a low endocrine IGF-I setpoint (47), creating the best physiological circumstances for the storage of fat during short times of food availability in a world with limited access to food. However, in a world of plenty, this advantage would turn into a disadvantage and give fast increases in body weight, hyperinsulinemia, and the development of metabolic syndrome problems early on (reviewed by Dunger et al (48). New information even suggests that the parental nutritional state can impose epigenetic metabolic changes in the fetus (49).

 

Figure 2. In the fetus (insert) IGF-I increases with gestational age toward birth. Endocrine circulating IGF-I is strongly nutritionally dependent and correlated with birth size. Pituitary GH control of IGF-I production is not fully established during the first year of human life. The ability of serum IGF-I levels to increase during childhood is dependent on the shift from binary complexes of IGF-I with short half-life to a complete dominance of IGF-I bound in a stable ternary complex with the GH dependent proteins IGFBP-3 and ALS. Both these proteins increase, when pituitary GH control of the axis is established. During pubertal development, sex steroids change the set-point of negative IGF-I feedback and allow a peak of IGF-I in mid-puberty. Total IGF-I levels decline to low levels in senescence. Serum IGF-I reference values based on Juul (50).

 

Postnatal Establishment of Pituitary GH Control of IGF-I Production

 

In humans, full GH control of the IGFI gene, as well as the IGFBP3 and Acid Labile Subunit (ALS) genes, is developmentally regulated and established not until the first year of life. IGF-I and IGFBP-3 levels increase slowly from birth until a more rapid increase and peak during puberty, which is followed by a decline toward low levels in senescence (50, 51) (Figure 2). The late establishment of pituitary GH control of the axis is strongly supported by animal data from GHR KO mice reviewed above, and by a new model of Laron syndrome (GHR defect) in pigs (52). In accordance, newborns with mutations in the GH Releasing Hormone Receptor (GHRHR) gene resulting in an isolated GH deficiency (GHD) phenotype was associated with normal birth weight in one cohort (53) and slightly subnormal birth weight in another (54). In a subgroup of 12 children with congenital isolated GHD, birth weight (1·1 ± 0·8 SD) and length (0·5 ± 1·3 SD) was not affected (Mehta A). GH1 mutations appear to be slightly more affected with mean birth weights of −1.0 ± 0.9 (54). Studies of common polymorphisms in GH1 demonstrate dose effects of 150 and 100 grams in term newborns of normal and low birth weight, respectively (55).  Somewhat contradictory to the observations in animals, Savage et al (56) reported 27 prepubertal children with severe GH insensitivity syndrome (GHIS or Severe Primary IGF Deficiency (SPIGFD)) to have a median (range) birth weight SDS of -0.72 (1.75 - (-3.29)) and birth length SDS of -1.59 (0.63 - (-3.63)). SPIGFD in these patients were defined by phenotypic and biochemical characteristics and they were treated with recombinant human (rh)IGF-1 in one of the clinical trials leading to approval of this therapy, as described later. There was no complete genetic characterization of these patients and 7 patients had a normal serum GH Binding Protein (GHBP) suggesting that the extracellular part of GHR was not affected. In a monograph, Professor Zvi Laron (57), who gave his name to this syndrome, reported that birth weight is unaffected while birth length is slightly on the shorter side. In summary, human fetal growth is only marginally affected by GH. GH is detectable and the GHR is expressed in the human fetus and the metabolic effects of GH on lipolysis are essential to maintain normal levels of glucose in the newborn.

 

Given this critical role of GH in adjusting metabolism to the fasting condition, it is plausible that the metabolic effects of GH are required for optimal linear growth of the human fetus and that this explains marginal effects on linear growth of the human fetus. However, strict GH control of IGF-I in the human fetus would predict severe growth retardation of the above-mentioned genetic defects comparable with the birth size observed in defects in the IGF-I gene. And that is not observed: Children with IGF1 defects suffer from far more severe fetal growth retardation with birth weight SDS around -4 and birth length SDS of -5 to -6 (reviewed in (58). In the study by Mehta et al (Mehta A), children with congenital isolated GH deficiency demonstrated growth retardation as already at 6 months of age (−2·6 ± 1·0 SD and −2·2 ± 1·3 SD in weight and length, respectively) prior to starting treatment. This suggests that the developmental GH control of IGF-I production is established early after birth. It is in accordance with data from Jensen RB and Juul A et al (Jensen RB) suggesting that low IGF-I is a marker of GH deficiency early in life. Children with combined pituitary hormone deficiencies were even more growth retarded at 6 months (Metha A).

 

GHR Signaling Pathway to IGF1 Gene Transcription

 

The important cell signaling steps associated with GH stimulated IGF1 gene activation, transcription and IGF-I production are detailed in the Endotext chapter ‘Normal Physiology of Growth Hormone in Adults´. Briefly, GH binding to preformed dimeric GHR - JAK2 complexes introduces structural changes in the receptor complex that separates JAK2 inhibitory and kinase active sites and enables trans-phosphorylation of the two JAK2 molecules (reviewed in (59). The GHR belongs to the class 1 cytokine receptors which uses STAT as one of its principal secondary messengers, and the subsequent phosphorylation of two STAT5b molecules results in a phospo-STAT5b homodimer which translocate to the cell nucleus, binds to STAT5b recognition sites on the IGF1 gene promoter, and initiates transcription (Figure 3).

 

Primary and Secondary IGF-I Deficiency

 

IGF-I deficiency may be the result of low or inadequate production of GH. This condition is known as GH deficiency (GHD) but in analogy with other pituitary deficiencies leading to peripheral hormone deficiencies the term secondary IGF-I deficiency was proposed (Rosenfeld et al). GHD is severe in GH1 or GHRH-R gene defects and in some children with congenital GHD as well as after treatment of brain tumors with radiation therapy. However, these conditions are rare and most children treated with rhGH has less severe GHD or an indication not associated with GHD. Disorders of GH in childhood is outlined in the Endotext chapter Disorders of Growth Hormone in Childhood by Murray and Clayton (132)

 

Primary IGF-I deficiency or GH insensitivity is severe in homozygous genetic defects in genes including the GH receptor (Laron syndrome) gene, STAT-5b gene and IGF-I gene. Less severe growth retardation is reported in children with homozygous genetic defects in the ALS gene. Treatment with rhGH do not improve growth in these cases while rhIGF-1 is efficient in SPIGFD and approved by FDA and EMA. In many patients with severe primary IGF-I deficiency (SPIGFD) defined by low IGF-I (less than – 3 SDS or the 2.5th percentile), severe short stature (Height SDS less than – 3), normal GH secretion and absence of acquired insensitivity to GH (discussed below) genetic defects may be absent. Still treatment with rhIGF-1 may be as efficient as in patients with confirmed homozygous GHR defects (131).

 

Insulin Enhancement of GHR Signaling to IGF1 Gene Transcription

 

Insulin signaling enhances the GH signaling pathway to enable IGF-I production in the fed state and promotes linear growth and other anabolic responses (60, 61, 62). Moreover, GH signaling to elicit IGF1 gene transcription is blocked in the absence of appropriate insulin signaling, a phenomenon also known as un-coupling, and resulting in growth arrest (60, 61, 63, 64) (Figure 3). This is partly a result of insulin’s effects on hepatic GHR expression, and partly a post-receptor signaling effect as unraveled by extensive animal studies (reviewed in (60). In obese individuals with hyperinsulinemia, hepatic GHR expression is enhanced as indicated by elevated GHBP levels reflecting proteolytic cleavage of highly expressed surface GHR and release of the extracellular part to the circulation (65). This allows obese individuals to maintain normal serum IGF-I levels despite markedly diminished GH secretion (66, 67). Consequently, obese individuals have attenuated GH responses to GH secretagogues (68).

 

Figure 3. Multiple, partly identical, pathways have been described to be activated by the GHR, the INSR and many other hormone kinase receptors not shown on the slide. Limiting this cartoon to the GHR and INSR, still the complexity is very high and the potential candidate hubs for crosstalk are numerous. The crosstalk that, following activation of the GHR, leads to resistance to specific signaling events from the INSR (related to glucose metabolism) is more well established and describe in detail in the Endotext chapter ‘Normal Physiology of Growth Hormone in Adults´. In the current review the focus is on the crosstalk that is executed by activation of the INSR and results in enhanced signaling from the GHR leading to gene activation of IGF1 and other GH dependent genes such as IGFBP3 and ALS. There are basically no studies addressing this crosstalk on the cellular level despite the strong evidence for INSR signaling being permissive for IGF1 transcription. Given that mTORC1 and mTORC2, downstream the INSR, are essential hubs for substrate and energy sensing and thus controlling the switch between cell anabolism and catabolism, they appear to be strong candidates to determine whether IGF1 should be on or off. A further argument for their candidate role is the so far limited evidence of mTORC1 and mTORC2 involvement in branched chain amino acid sensing directly enhancing IGF1 transcription. The unique role of the Jak2, STAT5b pathway in connecting the GHR with IGF1 gene activation has not been challenged and is thus the major candidate pathway to be affected by INSR signaling crosstalk. It is less clear which of the signaling pathways from the GHR that results in enhanced lipolysis although the STAT5b pathways has been implicated. This is particularly interesting given that GHR induced lipolysis does not require INSR signaling crosstalk. The reader is encouraged to seek specific information regarding other GHR and INSR pathways depicted in the cartoon but not further discussed in this review.

 

While short-term fasting decreases serum IGF-I but does not affect GHBP (69), the triad of IGF-I deficiency, poor growth and pubertal delay/arrest in long term nutritional deficiency such as in anorexia nervosa is associated with low GHBP that is partly restored with weight gain (63). Also, circulating IGF-I deficiency due to hepatic under-insulinization in type 1 diabetes is associated with low GHBP levels. Normal circulating IGF-I and GHBP are fully restored only after experimental intra-peritoneal (70) or intraportal (71) insulin delivery.

 

Functional/Acquired IGF-I Deficiency - Uncoupling of GHR Signaling to IGF1 Gene Transcription and Maintained GHR Metabolic Signaling Due to Insulin Deficiency

 

Increased GHR signaling in obese children does not generally result in elevated IGF-I, due to negative feedback inhibition of GH. In contrast, impairment of GH signaling due to insulin deficiency cannot generally be compensated by GH hypersecretion. This is true in fasting children (63) and adults (62). The exact mechanisms by which insulin and GH signaling crosstalk on the post-receptor level is not yet understood (Figure 3). More recent data suggesting that FGF21 plays a role in mediating these events needs further confirmation (72). Interestingly, GH signaling leading to activation of lipolysis in adipose tissue and increased hepatic glucose production via both glycogenolysis and gluconeogenesis in the liver, is not affected by the absence of insulin crosstalk (reviewed in (72)). This has important implications in securing substrate mobilization and gluconeogenesis during fasting and explains the cardinal hypoglycemic symptoms in GHD and GHIS newborns in the absence of intrauterine growth retardation (IUGR). GH signaling leading to lipolysis is thought to involve STAT5b. Most information comes from animal models and involves GH signaling in the liver, but in mouse adipose tissue GHR KO downregulates beta-3 adrenergic receptor expression and inhibits lipolysis (73). GH effects are lost if STAT5b signaling is blocked (74), gene transcript profiles of GHR KO and STAT5b KO animals overlap largely, and STAT5b controls key regulator enzymes involved in lipid metabolism (75). However, if STAT5b mediates both metabolic signaling and IGF-I production it still needs to be understood where the two pathways diverge, and why GH metabolic signaling is not blocked in the absence of insulin crosstalk. In humans, recent studies have identified new GH signaling responses involving GH downregulation of fat-specific protein (FSP27), a negative regulator of lipolysis. MEK/ERK activation and inhibition of peroxisome proliferator-activated receptor-γ (PPARγ) are involved, and this offers an alternative signaling pathway from the GHR (76).

 

Interactions Among Endocrine Axes

 

The activity of the insulin-GH-IGF-axis is dependent on the other endocrine axes which have permissive actions on GH stimulated IGF-I expression and affect IGFBPs and proteases (Figure 4). For example, thyroxine is needed to enhance GH effects on endocrine IGF-I expression and a normal GH-IGF-IGFBP-axis is needed for optimal thyroid hormone production (77). Sex steroids further enhance the function of the GH-IGF-axis, most likely by attenuating pituitary and hypothalamic sensitivity to IGF-I negative feedback (78). The pivotal role of sex steroids on the setpoint of the axis is reflected by the peak circulating levels of IGF-I and IGFBP-3 reached in mid-puberty (50,51). On the other hand, GH via its stimulation of local IGF-I is important for testicular production of testosterone and spermatogenesis (79), and the local IGF-IGFBP-axis is involved in selection and growth of the primary follicle in the ovary, estradiol production and ovulation (80, 81). Finally, cortisol has impact on the actions of the GH-IGF-axis on growth by blocking IGF1R signaling leading to apoptosis (82) despite normal endocrine IGF-I levels (83).

 

Figure 4. Hypothalamic GH releasing hormone and somatostatin establish the pulsatile pituitary GH secretion that is established as the main regulator of endocrine and paracrine/autocrine IGF-I production during the first year of life in humans. Insulin is permissive for this regulation by modulating GHR signaling, and normal beta-cell release of insulin is required for normal liver derived endocrine IGF-I levels measured in serum (blue insert) that in most cases is a good marker of the local production and actions of IGF-I. During fasting the GH regulation of IGF-I is uncoupled, resulting in decreased IGF-I (and catabolism) and elevated GH secretion and maintained lipolytic signals securing gluconeogenesis and preventing hypoglycemia. Apart from insulin, the endocrine thyroid axis is important for normal GH induced IGF-I production and during pubertal development sex steroids from the gonads enhance the performance of the GH-IGF-axis presumable by relaxation of the negative IGF-I feedback on GH secretion allowing a higher set-point of the axis. Whether this is an estradiol effect is not fully elucidated but it is suggested by the fact that non-aromatizable androgens such as oxandrolone do not affect IGF-I levels. The actions of the adrenal axis are most likely local and involve actions on IGF1R signaling leading to apoptosis of stem cells in the growth plate and thus irreversible loss of height. Cortisol excess leaves endocrine IGF-I and GH levels largely unaffected.

 

Discordance Between Endocrine and Paracrine/Autocrine IGF-I

 

An important example of metabolic and mitogenic consequences of an unbalanced endocrine versus autocrine/paracrine insulin-GH-IGF-axis comes from observations in children and adolescents with type 1 diabetes (Figure 5). They suffer specifically from insulin deficiency in the hepatic portal circulation as a result of the subcutaneous delivery of insulin (reviewed by Dunger (64)). This attenuates their endocrine production of circulating IGF-I despite excessive GH secretion (84). Circulating IGF-I deficiency and GH hypersecretion induce insulin resistance which is further augmented by insufficient suppression of hepatic glucose output. To overcome this, higher subcutaneous insulin doses are needed to maintain glycemic control, and this results in aggravated systemic hyperinsulinemia. The importance of local tissue hyperinsulinemia and GH hypersecretion in generating high paracrine/autocrine IGF-I production and promoting mitogenic vascular events leading to diabetic long-term complications should not be underestimated. Based on this insight, a promising new drug targeting the alphaVbeta3 integrin affecting IGF-I signaling in smooth muscle cells has been found to inhibit the development of atherosclerotic lesions in diabetic pigs (85). Another consequence of a compensatory increased in local IGF-I activity is the finding of normal childhood and pubertal linear growth despite endocrine IGF-I deficiency in type 1 diabetes (86). It is interesting that the endocrine and paracrine/autocrine changes in the insulin-GH-IGF-axis observed in children with type 1 diabetes closely resembles those observed in liver IGF-I KO mice which eventually leads to diabetes in the KO mice. Given that portal delivery of insulin, which has the potential to completely restore IGF-I levels in type 1 diabetes (70, 71, 87), remains an experimental treatment, rhIGF-1 treatment to restore circulating IGF-I and suppress GH and decrease insulin needs appears to be the most feasible approach to take (88). In a 6-month clinical trial of a single daily injection of rhIGF-1 improved glycemic control in adolescents with type 1 diabetes were found (89). Long-term studies on diabetic vascular complications have yet to be performed.

 

If paracrine/autocrine IGF-I production is lost in addition to liver-derived IGF-I, the metabolic consequences become obvious. This situation was first reported in a boy with a deletion of exon 4 of the IGF-I gene (90) resulting in severe fetal and post-natal growth arrest, poor brain development and extreme insulin resistance with compensatory hyperinsulinemia and acanthosis nigricans. A short trial of treatment with rhIGF-1 resulted in normalization of circulating IGF-I, suppression of GH hypersecretion and a markedly decreased insulin response to a meal tolerance test (91). In this example and in type 1 diabetes, it has been discussed whether the normalization of glucose metabolism following rhIGF-I therapy is most importantly associated with insulin-like effects of IGF-I on glucose uptake in muscle or suppression of GH hypersecretion? Although most studies support the importance of GH suppression, prolonged actions of IGF-I similar to that of long-acting insulin analogs in type 1 diabetic patients are important. IGF-I is equipotent with insulin in stimulating glucose uptake in human muscle but has less effects in fat and liver (92). Reports that newborns with a complete lack of insulin effects due to inactivating defects in the INSR gene can now survive for extended time into adolescence when treated with recombinant IGF-I, that stimulate glucose uptake via the IGF1R sharing common signaling pathways with the INSR, support an important direct role of IGF-1 signaling on metabolism (93).

 

Figure 5. Changes in liver derived endocrine IGF-I measured in the circulation and paracrine/autocrine IGF-I are in most cases concordant. In the absence of practical and validated methods to measure IGF-I at the tissue site of action, paracrine/autocrine IGF-I activity is assessed by determining known physiological actions of IGF-I such as growth or glucose metabolism. Type 1 diabetes is a condition with discordant changes in endocrine vs. paracrine/autocrine changes in IGF-I that in many ways resembles those reported in a mouse model of conditional knock-out of IGF-1 expression in the liver. In type 1 diabetes, insulin deficiency in the liver, caused by a systemic rather than a portal insulin replacement therapy, results in a functional GHR signaling defect to IGF-I transcription (uncoupling). Low endocrine IGF-I production decreases circulating IGF-I and results in decreased negative pituitary feedback and GH hypersecretion. The lack of direct IGF-I effects on glucose uptake in muscle and the diabetogenic effects of GH (including maintained signaling to lipolysis) decreases insulin actions on glucose metabolism (known as insulin resistance). The portal insulin deficiency also fails to suppress hepatic glucose production. In other to maintain glycemic control, the increased insulin requirement can only be met by more subcutaneous insulin leading to systemic hyperinsulinemia. There is no direct information about local paracrine/autocrine IGF-I activity, but there are several indications that tissue hyperinsulinemia and GH hypersecretion results in a compensatory increase of tissue IGF-I activity. Firstly, linear growth is not impaired in children and adolescents with Type 1 Diabetes despite of their low endocrine IGF-I (comparable to levels in short stature children), indicating a compensatory upregulation of local IGF-I activity (IGF-I being the most important stimulator of longitudinal growth). Secondly, it is plausible that increased local IGF-I activity contributes to diabetes complications known to be tightly associated with increased rather than decreased IGF-I activity. While type 1 diabetes is not generally associated with increased risk of cancer, the increase in local production of IGF-I in obesity and type 2 diabetes may contribute.

 

Liver Disease and Endocrine Versus Paracrine/Autocrine IGF-1 Production

 

In children with severe liver disease, there may be similar discrepancies between circulating endocrine levels of IGF-I and IGF-I activity in peripheral levels contributed by paracrine/autocrine secretion of IGF-I (94). However, less is known about peripheral IGF-I activity and it is possible that there are more secondary metabolic and nutritional issues that could lower local IGF-I production and impact on linear growth. Particularly in liver cirrhosis associated with thalassemia major, which concomitantly can impair pituitary GH secretion, there is no secondary upregulation of local IGF-I and linear growth failure is common (95). Recently, increased IGF-I expression was reported in obese children with non-alcoholic fatty liver disease (NAFLD) and it was combined with upregulation of IGF1R (96), not expressed in the normal liver but involved in liver repair, such as after liver resection in a mouse model (97).

 

GH and Cytokine Crosstalk

 

STAT5b phosphorylation is also mediated by activation of other members of the cytokine receptor family and has an impact on immunological function: this is evident from the finding of immune deficient symptoms in children with STAT5b genetic defects (98) but these are not found in GHD and GHIS children.

 

Negative Control of GHR Signaling

 

Control of the GH signaling cascade is also under inhibitory control, principally by two mechanisms. Firstly, tyrosine phosphatases including PTP dephosphorylate GHR associated molecules. In Noonan’s syndrome genetic defects in the PTPN11 gene may affect this pathway and has been implicated in poor growth and poor response to GH therapy, although reports are conflicting (99, 100). In addition, the SOCS gene is activated by GH signaling and works as a short intracellular negative feedback loop which rapidly down-regulate GHR activity by internalization and receptor ubiquitinoylation resulting in lysosomal and proteasomal degradation (101).

 

Other Nutritional Signals to the GH-IGF-Axis

 

Nutritional supplementation increasing dietary protein intake from cow’s milk increases endocrine IGF-I, while an equal intake of animal protein from meat does not (102). It is possible that the amino-acid composition that differs depending on the dietary source may contribute, and there are other constituents in skimmed milk not present in meat. More likely, however, it is explained by a higher carbohydrate intake in the milk group (while fat content was higher with meat supplementation) and the finding that fasting insulin levels doubled (103), in accordance with the essential regulatory role of insulin on GHR signaling discussed above. There is, however, direct evidence for a regulatory role of amino-acids on IGF-I production that is independent of insulin. Branched chained amino-acids (BCAA) are known to stimulate cell growth by the activation of mTORC2, a protein complex that controls protein synthesis in cells by sensing nutrient and energy availability and is also one of the main signaling pathways of IGF-I and insulin (Figure 3). The role of BCAA has been studied in rats given a restricted diet containing high levels of BCAA, compared to a group given low levels of BCAA (104).

 

The availability of nutrients in the circulation might also have a direct effect on the production of IGF-I. Human breast adipocytes cultured in high glucose levels have been found to produce more IGF-I compared to adipocytes cultured in low glucose levels (105).

 

IGF-II

 

IGF-II is a paracrine/autocrine hormone which is as essential for fetal growth as IGF-I (21). IGF-II may also contribute considerable to postnatal growth although the absence of endocrine effects makes it difficult to study in humans. Less is known about the metabolic actions of IGF-II. Growth promoting actions of IGF-II is via the IGF1R and IGF1R-INSR hybrid receptors which has preference for IGF-II actions if the A isoform of the INSR receptor – expressed in the fetus and in malignantly transformed cells - pairs with the IGF1R in the hybrid (106). The human IGF2 gene is an imprinted gene (107) exclusively expressed from the paternal allele in certain tissues (reviewed by Rossignol (108). The imprinted promoter region is found in a complex configuration with the H19 gene on chromosome 11 and shares two important imprinting regions with this gene. Methylation of the imprinting region ICR1 results in expression of the paternal IGF2allele, while H19 gene expression is suppressed. Correct imprinting should lead to expression of the paternal allele only and sufficient expression of IGF-II for normal growth. Loss of methylation of the ICR1 on the paternal allele results in the phenotype of Silver Russell syndrome (SRS) which may also arise from other genetic aberrations that have not yet been linked to the IGF-II production or signaling cascade including maternal uniparental dyssomnia of chromosome 7.

 

SRS is characterized by proportional IUGR with severe SGA at birth, relative sparing of the brain with close to normal head circumference at birth, severe feeding difficulties in infancy and childhood (which in contrast to Prader-Willi syndrome does not rebound into feeding obsession later in life), postnatal growth retardation and body asymmetry. As indicated by the SRS phenotype, IGF2 gene transcription of some organs are not controlled by imprinting. The relative normal development of the brain and the relative macrocephaly of SRS is explained by the lack of imprinting control of IGF-II expression in the brain. Children with genetic mutations in the expressed paternal allele of the IGF2 gene, were reported to have an SRS phenotype. They had somewhat more pronounced psychomotor developmental problems compared with the SRS phenotype, which has increased risk of autism spectrum defects including attention deficit hyperactivity disorder. In SRS, there is a normal postnatal expression of the IGF2 gene in the liver leading to normal levels of circulating IGF-II. Interestingly, the normal endocrine levels of IGF-II do not overcome the postnatal growth restriction. A similar lack of endocrine IGF-II effects on growth and metabolism was reported in the IGF-I deficient child mentioned previously with a loss of exon 4 of the IGF1 gene. He had compensatory increased GH, IGFBP-3, ALS as a result of lack of negative IGF-I feedback and secondary to the increased IGF binding capacity, increased IGF-II (see also the chapter on IGF-binding proteins below). Another observation in favor of this view is the lack of a correlation between newborn cord levels of IGF-II and birth size (41). This contrasts with a strong positive correlation between cord blood IGF-I concentrations and birth size. The role of IGF-II should be viewed in the light of Efstradiadis series of knock-out experiments in mice (21) where the Igf2ko mice had the same degree of fetal growth retardation as the igf1ko which demonstrates that IGF-II is a paracrine/autocrine and not an endocrine hormone.

 

It is possible that circulating IGF-II after release from the ternary complex is cleared from the circulation by binding to the IGF2R – identical to the mannose-6-phosphate receptor – which is associated with lysosomes and results in degradation of IGF-II in endothelial cells (23). In the elegant mouse KO experiments by Efstradiadis et al (21), KO of the IGF2R resulted in fetal overgrowth. However, the largely elevated IGF-II serum levels in that model are more likely a secondary finding, while the lack of clearance of paracrine/autocrine IGF-II is the explanation for the excessive growth.

 

IGF Binding Proteins

 

Six IGFBPs bind IGF-I and IGF-II inside and outside the circulation and has impact on IGF bioactivity (reviewed by Clemmons (109). The IGFBP-related proteins share some structural similarities with the six IGFBPs but have no relevant impact on IGF bioactivity. GF-I passes the endothelium intact primarily via IGF1R mediated transcytosis and this process is essential for endocrine actions of liver derived IGF-I (110). Limited experimental evidence from animal and tissue cultures suggest that IGF-I complexed with IGFBP-1 and -2 may leave the circulation, although the extent and importance is unclear (Bar 1990).  After endothelial passage IGF-I redistributes to soluble IGFBPs in the extravascular fluids or IGFBPs bound on extracellular matrix or cell surfaces (111). The concentration of unbound IGF-I in the circulation is likely to be proportional to unbound IGF-I concentrations in the tissues, but they are not equal and may have different relationship in different target tissues with differentially-expressed IGFBPs.

 

As pointed out earlier in this review, transgenic animal studies disrupting one or more IGFBPs have not suggested that a marked growth phenotype should be expected in children and no IGFBP mutations causing growth retardation in children had been reported to the best of my knowledge. Interestingly, as predicted by the Igfbp4-ko and the pappa-ko animal models described previously, a human PAPP-A2 gene defect with growth phenotype was recently reported as detailed below (43).

 

Free IGF-I Assays

 

Assays claiming to measure free circulating IGF-I have been developed, but it is unclear to what extent different techniques are influenced by redistribution of IGF-I among IGFBPs associated with the assay procedure (112). Anyway, the fact that IGF-I redistribute among extravascular IGFBPs after passing the endothelium is likely to affect the local tissue bioavailability even more. Moreover, the fact that most data in the literature originate from one assay technique established in one single laboratory has resulted in a lack of confirmatory reports. In a few cases, measurements with different free IGF-I assays have been reported from the same study/experiment with large differences in results (113). The bottom line is that measurements of free IGF-I have not been demonstrated to better predict different physiological or pathophysiological conditions in humans and do therefore not provide any clinically important contributions (114, 112). Techniques to assess IGF-I at the tissue site of action pose practical and methodological challenges. Attempts to establish and validate a method to determine local tissue levels by microdialysis have been reported in adolescents with type 1 diabetes, where endocrine levels are a poor marker of local IGF-I activity (115).

 

Ternary Complex Formation

 

The developmental establishment of GH control over the IGF1, IGFBP3 and IGFALS genes in early childhood initiates the dominance of the ternary complex formed by IGF-I or IGF-II and IGFBP-3 (or IGFBP-5) and ALS as the quantitatively most important circulating form of IGF-I and IGF-II (reviewed by Baxter (116). In the human fetus and newborn, serum IGFBP-3 and ALS concentrations are low and ternary complex formation is absent (117). Although IGF2 gene expression is not under GH control, the circulating levels of IGF-II are largely influenced by GH status since IGF-II (as well as IGF-I and IGFBP-3) is rapidly cleared from the circulation if not bound in the ternary complex. This can be observed in children with SPIGFD, who are deficient in IGF-I as well as IGFBP-3 and ALS, and in whom sc injected rhIGF-I displays a very fast serum clearance rate (118). As mentioned, formation of the ternary complex also governs the circulating levels of IGFBP-3 which under physiological conditions is present in a 1:1 molar relationship with IGF-I plus IGF-II. ALS is a large glucoprotein that under physiological conditions are present in a two-fold molar excess (16).

 

Immunometric IGFBP-3 assays have been claimed to be more predictive of GH status in very young children; however, the support for that is weak. It is rather a misconception related to problems of commercial IGF-I assays at the lower end of IGF-I detection. Moreover, IGFBP-3 has been claimed to provide information about IGF-I bioavailability from calculating the molar ratio of total IGF-I to IGFBP-3. Given that both IGF-I and IGFBP-3 are rapidly cleared from the circulation if unbound, using the IGF-I/IGFBP-3 ratio and disregarding IGF-II concentrations (that are 2-3-fold those of IGF-I on a molar basis) does not make any sense. During puberty, for example, IGF-I bioactivity is increased (114). This is dependent on the 3-4-fold increase in total IGF-I (50), which consequently results in an increase in unbound IGF-I, even if the increase is matched with the same absolute molar increase in IGFBP-3 (and complexed with ALS in a ternary complex). A common view is that increased IGF-I bioactivity depends on a higher IGF-I/IGFBP-3 molar ratio during puberty. However, the increase in molar ratio is entirely explained by the fact that IGF-I and IGFBP-3 increase with the same number of moles per liter, but with a larger relative increase in IGF-I than IGFBP-3 and with IGF-II molar concentrations being unchanged (112).

 

IGFBP Proteolysis and Physiological Consequences

 

The fact that proteolytic cleavage of IGFBP-3 is common, and may result in falsely elevated IGFBP-3 immunoactivity, is the most likely reason for observing a low IGF-I/ IGFBP-3 ratio. Under certain physiological conditions first described in pregnancy (119, 120), specific proteases cleave IGFBP-3 into several proteolytic fragments of which each may retain immunoactivity and thus give rise to signals in an immunometric assay (121). This will lead to overestimations of the IGFBP-3 immunoreactivity in pregnancy, which is already truly increased due to increased placental GH tonus. It may also lead to the erroneous conclusion that IGF-I bioactivity is decreased. On the contrary, IGF-I bioactivity is increased in the maternal circulation resulting from increased total serum IGF-I and decreased binding affinity of fragmented IGFBP-3 (122). There is strong experimental evidence that IGFBP proteolysis results in lower IGF binding affinity. The finding that partial IGFBP-3 proteolysis, such as in pregnancy, does not disrupt the ternary complex, has questioned its significance. However, evidence for increased IGF-I bioactivity in a ternary complex with fragmented IGFBP-3 exists (123). IGFBP-3 proteolysis has also been described in insulin resistant states such as fasting, obesity and type 1 and 2 diabetes (62, 124, 125). While several known proteolytic enzymes such as those involved in blood clotting (126) and cancer metastasis (127, 128) have been identified as IGFBP-3 proteases, the identity of the pregnancy protease is still not resolved.

 

Recently, a human gene mutation of PAPP-A2, a circulating and tissue protease with IGFBP-5 (and to some extent IGFBP-3) as its primary substrate (Gaudamauskes et al), was demonstrated to have a marked growth phenotype involving fetal and post-natal growth retardation in children in a consanguineous family (43). Largely elevated levels of circulating IGF-I but as a result of absence of proteolysis of IGFBP-5 and IGFBP-3, necessary for disruption of ternary complex formation, IGF-I bioactivity in serum is low and presumably tissue bioactivity of IGF-I (and IGF-II) is low. Functionally, this is a state of severe primary IGF-I deficiency (despite of elevated total serum IGF-I) and pharmacokinetic studies suggested that sc. Injections of rhIGF-1 resulted in a fraction of unbound IGF-I in serum despite the impaired proteolysis of IGFBP-5 and IGFBP-3 (133). Attempts to improve linear growth by rhIGF-I treatment has been reported to result in some improvements in a few affected children but not all (134, 135)

 

It is beyond the scope of this chapter to review the overwhelming evidence from cell biology experiments demonstrating the important role of IGFBPs in modulating IGF bioactivity and the role of IGFBP proteases and their actions at the cellular level. Furthermore, IGFBPs other than IGFBP-3 may play a role in the access of IGF-I to various tissues (129).

 

SUMMARY

 

In the present review the pivotal role of nutrition and insulin in determining the regulation and actions of the GH-IGF-axis is reviewed. For the pediatrician, caring for patients in a phase of rapid growth and development, it is important to refer to normality and understand the requirements for a normal insulin-GH-IGF-axis in order to succeed in this task. In the complex work-up, treatment and management of growth disorders a thorough understanding of the normal physiology of the axis is essential in taking the right actions (130, 131). From the normal physiology of this axis, it is possible to understand the consequences of various genetic defects and disorders that affect its regulation and function. The most severe conditions associated with defects in the axis may cause a loss of adult height of approximately 1/3 and may cause severe developmental and neurological deficits and compromise pubertal maturation and fertility. Minor changes in the setpoint of the axis caused by programming of the fetus exposed to intra-uterine growth retardation may predispose the individual for poor linear growth and later metabolic disease, insights that the pediatrician should be aware of and consider in order to improve health and prevent later disease.

 

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Evaluation and Treatment of Dyslipidemia in the Elderly

ABSTRACT

 

The definition of elderly is arbitrary. In this chapter we will define elderly as greater than 75 years of age because both the US and European lipids guidelines use this age to differentiate therapy recommendations. Atherosclerotic cardiovascular disease (ASCVD) is a major cause of morbidity and mortality in the elderly. Age is a key risk factor for ASCVD and with identical risk factors the 10-year risk of an ASCVD event markedly increases with age. In fact, an older individual with excellent risk factors can still have a high risk for having an ASCVD event. ASCVD begins early in life and progresses until it leads to clinical events later in life. The age that one develops clinical manifestations of ASCVD is dependent on the severity of individual risk factors, the number of risk factors, and the duration of exposure to the risk factors. Elderly individuals have a long exposure to risk factors so even when the risk factors are relatively modest the cumulative effects can be sufficient to result in clinical ASCVD events. This explains why age is such a key variable in determining the risk of developing an ASCVD event. Cardiovascular outcome studies have demonstrated that lowering LDL-C levels with statins, ezetimibe, or PCSK 9 monoclonal antibodies will reduce ASCVD events in elderly patients with pre-existing cardiovascular disease (secondary prevention). In elderly patients without cardiovascular disease (primary prevention) the available data does not definitively demonstrate a decrease in ASCVD events with statin or ezetimibe therapy but is suggestive of a benefit (note there are no primary prevention trials with PCSK9 inhibitors). Additional data is required to determine if bempedoic acid and icosapent ethyl reduce ASCVD events in patients ≥ 75 years of age. Studies are currently underway to provide definitive information on whether statin therapy is beneficial as primary prevention in the elderly. In deciding whether to treat an elderly patient with lipid lowering drugs one needs to consider the following factors; the higher the LDL-C level the greater the benefit of lowering LDL-C, the greater the decrease in LDL-C the greater the benefit, the higher the absolute risk of ASCVD the greater the benefit of lowering LDL-C, life expectancy, competing non-cardiovascular disorders, risk of drug side effects, potential for drug interactions, and patient preferences. In elderly patients without pre-existing ASCVD one should estimate the patient’s risk of developing ASCVD events and in conjunction with the general principles described above discuss with the patient a treatment plan. Determining the coronary calcium score can be helpful if there is uncertainty regarding the appropriate decision. If the decision is to treat our goal in primary prevention patients is often an LDL-C < 100mg/dL but in high-risk patients our goal may be an LDL-C < 70mg/dL. Elderly patients with ASCVD should be treated with lipid lowering drugs to reduce ASCVD unless there are contraindications. At a minimum our goal is an LDL-C < 70mg/dL but we would prefer an LDL-C < 55mg/dL if they can be achieved with a statin + ezetimibe. In very high-risk patients our goal is an LDL-C < 55mg/dL and adding a PCSK9 inhibitor may be required to achieve these levels in some patients. Age per se should not be used to withhold therapy with lipid lowering drugs that can reduce the risk of ASCVD events.

 

INTRODUCTION

 

Due to a decreasing birth rate and a longer life expectancy the population is getting older. According to the US census in 2020 there were approximately 50 million people between 65 and 84 years of age (14.9% of the total population) and approximately 6 million between 85 and 99 years of age (1.89% of the total population). The number of Americans ages 65 and older is projected to increase to 82 million by 2050 (23% of the total population). World-wide there are 703 million people aged 65 or older, which is projected to reach 1.5 billion by 2050 (1 in 6 people). It is well recognized that atherosclerotic cardiovascular disease (ASCVD) increases with age and is a major cause of morbidity and mortality in the elderly. In addition to an increased risk of coronary artery disease there is more than a doubling of the prevalence of peripheral arterial disease, cerebrovascular disease, and abdominal aortic aneurism with each decade of life (1). Unfortunately, the elderly (≥ 75 years of age) have not been well represented in lipid lowering cardiovascular outcome trials.

 

The definition of elderly is arbitrary. In this chapter we will define elderly as greater than 75 years of age because both the US and European guidelines use this age to differentiate therapy recommendations. In both the “Guideline on the Management of Blood Cholesterol: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines” and the “2019 ESC/EAS Guidelines for the Management of Dyslipidemias: Lipid Modification to Reduce Cardiovascular Risk” the recommendations for those over 75 years of age differ from recommendations for younger individuals (2,3). Thus, where possible we will focus on studies in individuals greater than 75 years of age.

 

LIPID LEVELS IN THE ELDERLY

 

Lipid levels in US adults from the National Health and Nutrition Examination Survey (NHANES) 2003-2004 are shown in Table 1 (4). Compared to 30–69-year-olds there is a slight decrease in LDL-C, non-HDL-C, and triglycerides with similar HDL-C levels in individuals 70-79 years of age. Other cross-sectional studies have reported similar results (5-11). Prospective studies with longitudinal follow-up have also observed small decreases in total cholesterol, LDL-C, and HDL-C levels in men and women as they become elderly (6,7,9,12-14). It should be noted that the changes in lipid levels reported with aging are relatively small and vary somewhat from study to study. The clinical significance of these small changes is uncertain.

 

Table 1. Lipid Levels in U.S. Adults (NHANES 2003-2004)

Age

LDL-C (mg/dL)

Non-HDL-C (mg/dL)

HDL-C (mg/dL)

Triglycerides (mg/dL)

20-29

104

126

54

105

30-39

120

146

54

118

40-49

124

152

53

144

50-59

123

154

55

141

60-69

126

157

54

145

70-79

119

148

56

133

 

Studies have demonstrated that older individuals have an exaggerated postprandial lipemia compared with younger individuals (15,16). While elevated postprandial triglycerides is associated with an increased risk of ASCVD whether this plays a causal role in increasing ASCVD is uncertain.

 

It is well recognized that with increasing age the likelihood of other medical disorders increases and this can affect lipid levels. For example, inflammation and infections can decrease LDL-C and HDL-C levels (17). Additionally, poor nutrition due to illness or social-economic factors could decrease lipid levels in the elderly. Finally, frailty is a syndrome associated with aging and increases with age. It is usually associated with a lowering of total, LDL-C, and non-HDL cholesterol levels (18-20).

 

AGE IS AN IMPORTANT RISK FOR ATHEROSCLEROTIC CARDIOVASCULAR DISEASE

 

The clearest way to illustrate the importance of age as a key risk factor for atherosclerotic cardiovascular disease (ASCVD) is to compare the 10-year risk at different ages using an updated version of the AHA/ACC pooled cohort equation. Shown in table 2 are four examples of the effect of age on 10-year risk in different clinical situations that demonstrate the marked effect of age on ASCVD risk. Similarly, using the SCORE risk calculator for determining the 10-year risk of fatal cardiovascular disease also demonstrates the very large impact of age on risk (figure 1). It is obvious that age is a major determinant of ASCVD risk.

 

Table 2. Ten Year Risk of Developing ASCVD

 

Age 55

Age 65

Age 75

Male, white, BP 130, TC 200, HDL-C 45, non-smoker, no diabetes

6.3%

13.9%

26.2%

Female, African American, BP120, TC 180, HDL-C 50, non-smoker, no diabetes

2.8%

6.3%

13.2%

Male, African American, BP140, TC 200, HDL-C 50, smoker, no diabetes

10.1%

14.9%

20.5%

Female, white, BP 140, TC 180, HDL-C 50, non-smoker, diabetes

4.4%

12.2%

32.9%

https://tools.acc.org/ascvd-risk-estimator-plus/#!/calculate/estimate/.

BP= systolic BP mm Hg, TC= total cholesterol mg/dL.

 

Figure 1. Systematic Coronary Risk Estimation chart for European populations at high cardiovascular disease risk (from 2019 ESC/EAS Guidelines for the management of dyslipidaemias (3)).

 

In fact, an older individual with excellent risk factors can have a high risk for having an ASCVD event. For example, using the AHA/ACC pooled cohort equation a 75-year-old white male with a total cholesterol of 180mg/dL, an HDL-C of 50mg/dL, a blood pressure of 120/80 mmHg, who is not diabetic, doesn’t smoke, and is on no medications still has a 10-year risk for an ASCVD event of 21.7%. A 75-year-old white female with the same risk factors also has a relatively high 10-year risk (14.1%). Using the SCORE estimator (figure 1) for a fatal CVD event it is also apparent that many older individuals, particularly males, are at high risk even when they are non-smokers with an excellent total cholesterol and blood pressure. For example, a 70-year-old male, non-smoker with a total cholesterol of 160mg/dL and systolic BP of 120 mmHg still has a 13% 10-year risk of death from CVD.

 

WHY ARE OLDER INDIVIDUALS AT HIGHER RISK FOR ASCVD?

 

It is widely recognized that atherosclerosis begins early in life and slowly progresses ultimately resulting in clinical manifestations later in life (21). Numerous studies have demonstrated the presence of atherosclerosis in young individuals (22-27). In the Bogalusa Heart Study autopsies were performed on 204 young people 2 to 39 years of age (22,28). In the coronary arteries fatty streaks were very common (50 percent at 2 to 15 years of age and 85 percent at 21 to 39 years of age). More advanced raised fibrous-plaque lesions in the coronary arteries were present in 8 percent of individuals 2 to 15 years of age and 69 percent of individuals 26 to 39 years of age. The extent of the atherosclerotic lesions correlated positively with BMI, systolic and diastolic BP, total cholesterol, LDL-C, and triglyceride levels and negatively with HDL-C levels. The extent of the atherosclerotic lesions was greatest in individuals who had multiple risk factors. The Pathobiological Determinants of Atherosclerosis in Youth [PDAY] study examined the effect of risk factors for atherosclerosis in 1079 men and 364 women 15 through 34 years of age who died due to accidents, homicide, or suicide (23,29). Atherosclerosis of the aorta and right coronary artery was measured and increased with age, LDL-C levels, glycohemoglobin levels, BMI, and smoking while HDL-C levels were negatively associated with the extent of fatty streaks and raised lesions in the aorta and right coronary artery. Finally, in a study of US service members (mean age 25.9 years; range 18-59 years; 98.3% male) who died of combat or unintentional injuries (n= 3832) the effect of risk factors on coronary atherosclerosis was determined (27). Atherosclerosis prevalence was increased by age, dyslipidemia, hypertension, and obesity. Taken together these studies clearly demonstrate that atherosclerosis begins early in life with the prevalence increasing with age and the extent and onset of lesions is influenced by risk factors, including dyslipidemia.

 

Moreover, the presence of risk factors early in life is associated with an increase in atherosclerosis later in life (30-32). A meta-analysis that included 4380 participants from 4 prospective studies that collected cardiovascular risk factor data during childhood (age 3 to 18 years) and measured carotid intima-media thickness (CIMT) in adulthood (age 20 to 45 years) reported that total cholesterol, triglycerides, BP, and BMI measured in childhood were predictive of elevated CIMT in adults (33). Additionally, increased LDL-C and/or decreased HDL-C during adolescence predict an increase in CIMT later in life (34). Importantly, an increased total cholesterol or BP early in life also predicted an increased risk of developing cardiovascular disease later in life (35-38).

 

Genetic studies have further illustrated the key role of risk factors and duration of exposure to the risk factor as key variables determining the time when clinical manifestations of ASCVD occur. In patients with homozygous familial hypercholesterolemia (FH) LDL-C are markedly elevated and cardiovascular events can occur early in life. Greater than 50% of untreated patients with homozygous FH develop clinically significant ASCVD by the age of 30 and cardiovascular events can occur before age 10 in some patients (39). In patients with heterozygous FH LDL-C levels are elevated but not to the levels seen with homozygous FH and cardiovascular events occur later in life but still at a relatively younger age. Untreated males with heterozygous FH have a 50% risk for a fatal or non-fatal myocardial infarction by 50 years of age whereas untreated females have a 30% chance by age 60 (39). Conversely, individuals with genetic variants in PCSK9, HMG-CoA reductase, LDL receptor, NPC1L1, or ATP citrate lyase that lead to a decrease in LDL-C levels have a reduced risk of developing cardiovascular events (40,41). The relationship between genetic disorders that alter LDL-C levels and the time to develop clinical cardiovascular events is illustrated in figure 2. The figure clearly illustrates that the age when one clinically manifests ASCVD depends on the level of LDL-C. With very high LDL-C levels clinical events occur early in life and with low LDL-C levels events will occur at an older age leading to the concept of LDL years.

 

Figure 2. Relationship between cumulative LDL-C exposure and age of developing cardiovascular disease. (from (41)).

 

The degree and duration of other risk factors also seems to play a role in when the clinical manifestations of ASCVD are expressed. For example, for cigarette smoking, cigarettes/day, smoking duration, and pack-years all increase the risk of cardiovascular disease (42). Interestingly smoking fewer cigarettes/day for a longer duration was more deleterious than smoking more cigarettes/day for a shorter duration (42,43). Additionally, while smoking cessation lowers the risk of ASCVD events an increased risk persists for decades after smoking cessation (44). These observations suggest that the effect of smoking is related to the number of cigarettes smoked and the duration of the smoking (i.e., pack years). Similarly, in patients with diabetes glycemic control and duration of diabetes influences the development of ASCVD complications (45-48). At any given age, a 10-year longer diabetes duration was associated with a 1.1-1.5-fold increased risk of stroke and 1.5-2.0-fold increased risk of MI (45).

 

Thus, ASCVD begins early in life and progresses until it leads to clinical events such as a myocardial infarction or stroke later in life. The age that one develops clinical manifestations of ASCVD is dependent on the severity of individual risk factors, the number of risk factors, and the duration of exposure to the risk factors. Elderly individuals have a long exposure to risk factors so even when the risk factors are relatively modest the cumulative effects can be sufficient to result in clinical ASCVD events. This explains why age is such a key variable in determining the risk of developing an ASCVD event.

 

DOES LIPID LOWERING REDUCE EVENTS IN THE ELDERLY

 

Below we discuss lipid lowering drug studies that report the effect on cardiovascular outcomes that are relevant to clinical decisions in elderly individuals. For additional and more detailed information on lipid lowering cardiovascular outcome studies see the Endotext chapters on “Cholesterol Lowering Drugs” and “Triglyceride Lowering Drugs” (49,50).

 

Statins

 

Few statin studies have focused on lowering LDL-C in elderly patients, which we define as individuals greater than 75 years of age. The Prosper Trial determined the effect of pravastatin 40mg/day (n= 2891) vs. placebo (n= 2913) on cardiovascular events in older subjects (70-82 years of age) with pre-existing vascular disease or who were at high risk for vascular disease (51). The average age in this trial was 75 years of age and approximately 45% had cardiovascular disease. As expected, pravastatin treatment lowered LDL-C by 34% compared to the placebo group. The primary end point was coronary death, non-fatal myocardial infarction, and fatal or non-fatal stroke, which was reduced by 15% (HR 0.85, 95% CI 0.74-0.97, p=0.014). However, in the individuals without pre-existing cardiovascular disease pravastatin did not significantly reduce ASCVD events (HR 0.94; CI 0.77–1.15). In contrast, in individuals with cardiovascular disease pravastatin therapy significantly reduced ASCVD events (HR 0.78, CI 0.66–0.93). Thus, this study demonstrated benefits of statin therapy in the elderly with cardiovascular disease but failed to demonstrate benefit in the elderly without cardiovascular disease.

 

A meta-analysis by the Cholesterol Treatment Trialists of 28 trials with 14,483 of 186,854 found a reduction in LDL-C levels with statin therapy that was similar in the participants ≥75 years of age compared to younger individuals. Moreover, statin therapy resulted in a decrease in cardiovascular events in all age groups including participants ≥75 years of age (Figure 3) (52). In the participants ≥75 there was a 13% reduction in ASCVD events per 39mg/dL decrease in LDL-C (RR 0.87; 95% CI 0.77–0.99). This analysis included four trials done exclusively among people with heart failure or receiving renal dialysis, for whom statin therapy shows little or no benefit (50). A second analysis was performed after elimination of these four trials and there was an 18% reduction in ASCVD events per 39mg/dL decrease in LDL-C (RR 0.82; 95%CI 0.70-0.95). Similar to the Prosper Trial a decrease in ASCVD events was clearly demonstrated in individuals with pre-existing cardiovascular disease (secondary prevention) but in individuals without pre-existing cardiovascular disease (primary prevention) the decrease in ASCVD events was not statistically significant (Figure 4- analysis included all studies). After excluding the trials in patients with heart failure or receiving renal dialysis, statin therapy reduced major ASCVD events by 26% per 39mg/dL decrease in LDL-C (RR 0.74; 95% CI0.60 − 0.91) in patients with pre-existing cardiovascular disease but only by 8% in patients without pre-existing cardiovascular disease (RR 0.92; 95%CI 0.72 − 1.16).

 

Figure 3. Effect of Statin Treatment on Major Vascular Events. Modified from (44).

Figure 4. Effect of Statin Treatment on Major Vascular Events in Individuals With and Without Pre-Existing Cardiovascular Disease. Modified from (44).

 

A statin trial not included in the Cholesterol Treatment Trialists meta-analysis was carried out in patients with an ischemic stroke or a transient ischemic attack who were treated with statins and/or ezetimibe with a target LDL-C level < than 70mg/dL (n= 1430) or an LDL-C 90mg/dL to 110mg/dL (n= 1430) (53). The primary end point was ischemic stroke, myocardial infarction, new symptoms leading to urgent coronary or carotid revascularization, or death from cardiovascular causes. The mean LDL-C level was 65mg/dL in the lower-target group and 96 mg/dL in the higher-target group. After median of 3.5 years the primary end point occurred in 8.5% of the patients in the lower-target group and 10.9% of the patients in the higher target group (HR 0.78; 95% CI 0.61 to 0.98; P=0.04). In patients < 65 years of age only a 7% decrease in the primary end point was observed (HR 0.93; 95%CI 0.63–1.36) whereas more impressive decreases in the primary endpoint were observed in patients 65-75 years of age (37% decrease; HR 0.63 95% CI 0.42–0.95) and > 75 years of age (23% decrease; HR 0.77; 95%CI 0.49–1.22). These results are consistent with the Cholesterol Treatment Trialists meta-analysis demonstrating that elderly patients with pre-existing cardiovascular disease lowering LDL-C levels reduces ASCVD events.

 

There are observational studies demonstrating that statin treatment for the primary prevention of ASCVD is effective in older patients (54-59). For example, in US veterans ≥75 years of age and free of ASCVD at baseline, new statin use was significantly associated with a lower risk of ASCVD events (HR 0.92; 95% CI 0.91-0.94) and cardiovascular mortality (HR 0.80; 95% CI 0.78-0.81) when compared to statin nonusers (55). Similarly, in a Danish nationwide cohort initiation of statin therapy in patients > 70 years of age without pre-existing cardiovascular disease there was a 23% lower risk of major vascular events per 39mg/dL decrease in LDL-C (HR 0.77; 95% CI 0.71-0.83), which was similar to what was observed in younger individuals (54). Finally, in nursing home residents without ASCVD statin use reduced all-cause mortality in individuals with and without dementia (59). These observational studies while suggestive of a benefit of statin therapy for primary prevention in older individuals cannot provide definitive proof as there is always the possibility of residual confounding. Nevertheless, they provide additional support that statin therapy provides benefits in elderly patients without pre-existing cardiovascular disease.

 

Thus, in older patients with cardiovascular disease lowering LDL-C levels with statins clearly reduces cardiovascular events but in older patients without cardiovascular disease the data demonstrating that statins reduce cardiovascular events is less robust but suggests a reduction in ASCVD events.

 

Ezetimibe

 

IMPROVE-IT TRIAL

 

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 (60). The IMPROVE-IT Trial was a large trial with over 18,000 patients randomized to simvastatin 40mg vs. simvastatin 40mg + ezetimibe 10mg per day. On treatment LDL-C levels were 70mg/dL in the statin alone group vs. 54mg/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 rehospitalization, coronary revascularization, or stroke) in the statin + ezetimibe group (HR 0.936; 95% CI 0.887-0.988; p=0.016). Cardiovascular death, non-fatal MI, or non-fatal stroke were reduced by 10% (HR 0.90; 95% CI 0.84-0.97; p=0.003). The effect of age on the benefits of statin + ezetimibe therapy is shown in figure 5. In elderly individuals (≥ 75 years of age) the combination of ezetimibe and simvastatin reduced ASCVD events.

 

Figure 5. Primary endpoint in the IMPROVE-IT trial in different age groups. Modified from (60).

 

EWTOPIA 75 TRIAL

 

EWTOPIA 75 was a multicenter, randomized trial in Japan that examined the preventive efficacy of ezetimibe for patients aged ≥75 years (mean age 80.6 years), with elevated LDL-C (≥140 mg/dL) without a history of coronary artery disease (primary prevention) who were not taking lipid lowering drugs (61). Patients were randomized to ezetimibe 10mg (n=1,716) or usual care (n=1,695) and followed for 4.1 years. The primary outcome was a composite of sudden cardiac death, myocardial infarction, 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. 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 in usual care group). 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 suggests that lowering LDL-C in elderly individuals without cardiovascular disease can reduce ASCVD events.

 

RACING TRIAL

 

The RACING trial was a randomized, open-label trial in patients with ASCVD carried out in South Korea (62). Patients were randomly assigned to either rosuvastatin 10 mg with ezetimibe 10 mg (n= 1894) or rosuvastatin 20 mg (n= 1886). The primary endpoint was cardiovascular death, major cardiovascular events, or non-fatal stroke. The median LDL-C level during the study was 58mg/dL in the combination therapy group and 66mg/dL in the statin monotherapy group (p<0.0001). The primary endpoint occurred in 9.1% of the patients in the combination therapy group and 9.9% of the patients in the high-intensity statin monotherapy group (non-inferior). Non-inferiority was observed in patients with baseline LDL-C levels < 100mg/dL and >100mg/dL (63).

 

In the RACING trial 574 participants (15.2%) were aged ≥75 years and there was no difference in the primary endpoint between the combination therapy group and the high-intensity statin monotherapy group in these elderly participants (64). However, in participants ≥75 years of age moderate-intensity statin with ezetimibe combination therapy was associated with lower rates of drug related intolerance with drug discontinuation or dose reduction (2.3% vs 7.2%; P = 0.010).

 

This study demonstrates that moderate intensity statin plus ezetimibe was non-inferior to high-intensity statin therapy with regards to cardiovascular death, major cardiovascular events, or non-fatal stroke. The lower prevalence of discontinuation or dose reduction caused by intolerance to the study drug was seen with combination therapy indicating that using a moderate intensity dose of a statin plus ezetimibe is a useful strategy in patients that do not tolerate high intensity statin therapy or where there are concerns about statin toxicity with high doses.

 

PCSK9 Inhibitors

 

FOURIER TRIAL

 

The FOURIER trial was a randomized, double-blind, placebo-controlled trial of evolocumab vs. placebo in 27,564 patients with ASCVD and an LDL-C level of 70 mg/dL or higher who were on statin therapy (65). The primary end point was cardiovascular death, myocardial infarction, stroke, hospitalization for unstable angina, or coronary revascularization and the key secondary end point was cardiovascular death, myocardial infarction, 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 levels (LDL-C level on treatment approximately 30mg/dL). In this trial 6233 of the participants were > 69 years of age and the decrease in LDL was similar in participants > 69 years of age and younger individuals (66). A 14% reduction in the primary endpoint (HR 0.86; 95% CI 0.74–0.99) and a 18% reduction in the secondary endpoint (HR 0.82; 95% CI 0.69–0.98) was observed in the participants > 69 years of age, which was similar to the decreases seen in younger individuals (66). The effect of treatment with evolocumab on the primary and secondary endpoint in specific age groups is shown in table 3 (66). These results demonstrate that lowering LDL-C with a PCSK9 inhibitor decreases ASCVD events in elderly patients.

 

Table 3. Effect of Evolocumab Treatment on Cardiovascular Outcomes in Different Age Groups

 

< 65

65-75

>75

Primary Endpoint

HR 0.86; 95%CI 0.78–0.94

HR 0.86; 95%CI 0.76–0.97

HR 0.78; 95%CI 0.60–1.02

Secondary Endpoint

HR 0.79; 95%CI 0.69–0.90

HR 0.82; 95%CI 0.70–0.95

HR 0.78 95%CI 0.58–1.04

For the primary endpoint the P interaction for the three age groups = 0.84

For the secondary endpoint the P interaction for the three age groups = 0.94.  

 

ODYSSEY TRIAL

 

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 apolipoprotein B level of at least 80 mg/dL while on high intensity statin therapy or the maximum tolerated statin dose (67). 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 myocardial infarction, fatal or nonfatal ischemic stroke, or unstable angina requiring hospitalization. In this trial 5084 (26.9%) individuals were ≥ 65 years of age, 1007 (5.3%) ≥ 75 years of age, and 42 (0.2%) ≥ 85 years of age (68). The baseline and decrease in LDL-C levels were similar in participants ≥65 years of age and those <65 years of age (LDL-C at baseline approximately 94mg/dL and after 4 months of treatment approximately 40mg/dL) (67,68). In the individuals ≥ 65 years of age there was a 22% decrease in the primary endpoint (HR 0.78; 95% CI 0.68–0.91) and in those < 65 years of age a 11% decrease (HR 0.89; 95% CI 0.80–1.00) (68). The secondary endpoint of all-cause death, myocardial infarction, or ischemic stroke was also reduced in the ≥ 65 participants (HR 0.78; 95% CI 0.68–0.90) and < 65 participants (HR 0.91; 0.82–1.02) (68). In participants ≥ 75 years of age the primary endpoint was reduced by 15% (HR 0.85; 95% CI 0.64–1.13) (68). When plotted as a continuous variable the relative benefit of alirocumab over placebo on the primary endpoint was consistent across the entire age range (figure 6).

 

Figure 6. Relative benefit of alirocumab at various ages. Modified from reference (68).

These two studies demonstrate that lowering LDL-C with a PCSK9 inhibitor decreases ASCVD events in elderly patients with pre-existing cardiovascular disease.

 

Bempedoic Acid

 

The CLEAR Outcome trial was a double-blind, randomized, placebo-controlled trial involving patients with cardiovascular disease or at high risk of cardiovascular disease who were unable or unwilling to take statins ("statin-intolerant" patients) (69). The patients were randomized to bempedoic acid 180 mg (n= 6992) or placebo (n= 6978) and the median duration of follow-up was 40.6 months. In this trial 44% of the participants were between ≥65 to < 75 years of age and 15% were ≥ 75 years of age. As expected, LDL-C levels were decreased by 21% in the bempedoic group compared to placebo (29mg/dL difference). The primary endpoint, death from cardiovascular causes, nonfatal myocardial infarction, nonfatal stroke, or coronary revascularization, was reduced by 13% in the bempedoic acid group (HR 0.87; 95% CI 0.79- 0.96; P = 0.004). The effect of age on the primary endpoint is shown in table 4.

 

Table 4. Effect of Bempedoic on Cardiovascular Outcomes in Different Age Groups

< 65

HR 0.87; 95% CI 0.74-1.02

≥65-<75

HR 0.83, 95% CI 0.72-0.96

≥ 75

HR 0.95, 95% CI 0.77-1.16

Interaction P value = 0.60

 

Niacin and Fibrates

 

Because of the robust effect of statins in lowering LDL-C levels and cardiovascular events recent trials of both niacin and fibrates have focused on the addition of these lipid lowering drugs to statin therapy. The AIM-HIGH trial was designed to determine if the addition of Niaspan, an extended-release form of niacin, to aggressive statin therapy would result in a further reduction in ASCVD events in patients with pre-existing cardiovascular disease (70) while the HPS 2 Thrive trial determined the effect of adding extended-release niacin (2000mg/day) combined with laropiprant, a prostaglandin D2 receptor antagonist, to statin therapy on ASCVD events in  patients with pre-existing vascular disease (71). Unfortunately, both of these trials failed to demonstrate a decrease in ASCVD events with the addition of niacin to statin therapy. The absence of benefit and increased side effects from niacin therapy has markedly reduced enthusiasm for treating patients with niacin to reduce ASCVD event. For additional details on these two studies and other niacin cardiovascular outcome studies see the Endotext chapter “Triglyceride Lowering Drugs” (49).

 

The ACCORD-LIPID Trial was designed to determine if the addition of fenofibrate to aggressive statin therapy in patients with pre-existing cardiovascular disease or at high risk for developing cardiovascular disease would result in a further reduction in cardiovascular disease in patients with Type 2 diabetes (72). The PROMINENT trial determined whether pemafibrate, a new selective PPAR-alpha activator, in patients on statin therapy with diabetes and pre-existing cardiovascular disease or at high risk for developing cardiovascular disease would reduce cardiovascular events (73). Disappointingly, neither trial demonstrated benefits from adding a fibrate to statin therapy. For additional details on these two studies and other fibrate ASCVD outcome studies see the Endotext chapter “Triglyceride Lowering Drugs” (49).

 

Thus, there is currently little enthusiasm for adding either niacin or a fibrate to statin therapy to reduce ASCVD events. One should recognize that like all studies these trials have limitations, that are discussed in detail in reference (49), and it is possible that future trials could resurrect the use of niacin and/or fibrates for decreasing ASCVD.

 

Omega-3-Fatty Acids (Fish Oil)

 

Numerous studies have determined the effect of low dose fish oil (< 1 gram per day) on ASCVD and found that they do not consistently reduce the risk of cardiovascular disease (49). Described below are ASCVD outcome studies that have used higher doses.

 

JAPAN EPA LIPID INTERVENTION STUDY (JELIS)

 

JELIS was an open label study without a placebo in patients with total cholesterol levels > 254mg/dL with cardiovascular disease (n= 3,664) or without cardiovascular disease (n=14,981) who were randomly assigned to be treated with 1800 mg of EPA (Vascepa) + statin (n=9,326) or statin alone (n= 9,319) with a 5-year follow-up (74). The primary endpoint was any major coronary event, including sudden cardiac death, fatal and non-fatal myocardial infarction, and other non-fatal events including unstable angina pectoris, angioplasty, stenting, or coronary artery bypass grafting. Total cholesterol, LDL-C, and HDL-C levels were similar in the two groups but plasma TGs were modestly decreased in the EPA treated group (5% decrease in EPA group compared to controls; p = 0.0001). In the EPA plus 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). In participants < 61 years of age the primary endpoint was reduced by 24% (HR 0.76; 95%CI 0.57–1.00) while in individuals ≥ 61 years of age the primary endpoint was reduced by 16% (HR 0.84; 95% CI 0.68–1.02; p interaction 0.57). Unstable angina and non-fatal coronary events were significantly reduced in the EPA plus statin 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 myocardial infarction, stroke, or cardiovascular death. A subjective endpoint has the potential to be an unreliable endpoint in an open label study and is a limitation of the JELIS Study. Unfortunately, we do not have information on elderly patients (≥ 75 years).

 

REDUCE-IT

 

REDUCE-IT was a randomized, double-blind trial of 2 grams twice per day of EPA ethyl ester (icosapent ethyl) (Vascepa) vs. mineral oil placebo in 8,179 patients with hypertriglyceridemia (135mg/dL to 499mg/dL) and established cardiovascular disease or high cardiovascular disease risk (diabetes plus one risk factor) who were on stable statin therapy (75). The primary end point was a composite of cardiovascular death, nonfatal myocardial infarction, 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; P<0.001), indicating a 25% decrease in events. In participants <65 years of age the primary end point was reduced by 35% (HR 0.65; 95% CI 0.54–0.78) while in participants ≥ 65 years of age the primary end point was reduced by 18% (HR 0.82; 95%CI 0.70–0.97; P Value for Interaction 0.06). The cardiovascular benefits of EPA were similar across baseline levels of TGs (<150, ≥150 to <200, and ≥200 mg per deciliter). 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 ASCVD risk reduction was not associated with attainment of a normal TG level. Unfortunately, information on an elderly subgroup (≥ 75 years) is not available. 

 

It should be noted that in this trial mineral oil was used as the placebo. In the placebo group the LDL-C, non-HDL-C, and hsCRP levels were increased compared to the EPA group during the trial (LDL-C 96mg/dL vs 85mg/dL; non-HDL-C 130mg/dL vs. 113mg/dL; hsCRP 2.8mg/L vs. 1.8mg/L). The impact of these adverse changes on clinical outcomes is uncertain and whether they contributed to the apparent beneficial effects observed in the individuals treated with EPA is unknown.

 

STRENGTH TRIAL

 

The STRENGTH Trial was a double-blind, randomized, trial comparing 4 grams per day of a carboxylic acid formulation of omega-3 fatty acids (EPA and DHA; Epanova) (n = 6,539)) vs. corn oil placebo (n = 6539) in statin-treated participants with high cardiovascular risk, hypertriglyceridemia, and low levels of HDL-C (76). Approximately 55% of patients had established cardiovascular disease and approximately 70% had diabetes. Median LDL-C level was 75.0 mg/dL, median TG level was 240 mg/dL, and median HDL-C level was 36 mg/dL. There were minimal differences in the change in LDL-C and HDL-C levels between the treated and placebo groups after treatment for 12 months but as expected there was a greater reduction in TG levels in the group treated with omega-3-fatty acids (−19.0% vs −0.9%). The primary endpoint was a composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, coronary revascularization, or unstable angina requiring hospitalization which occurred in 12.0% of individuals treated with omega-3 CA vs. 12.2% treated with corn oil (HR 0.99; P = .84). There were no significant differences between the treatment groups with regard to the risk of the individual components of the primary end point over the 3-4 years of the study. Thus, in contrast to the JELIS and REDUCE-IT trials the STRENGTH trial did not demonstrate a benefit of treatment with a mixture of omega-3-fatty acids (EPA + DHA).

 

OMEMI TRIAL

 

The OMEMI trial was a randomized trial of 1.8 grams per day of omega-3-fatty acids (930 mg EPA and 660 mg DHA) (n= 505) vs. corn oil placebo (n= 509) in patients aged 70 to 82 years with a recent myocardial infarction (2-8 weeks) (77). Baseline LDL-C was approximately 76mg/dL, HDL-C was 49mg/dL, and TGs 110mg/dL. The primary endpoint was a composite of nonfatal myocardial infarction, unscheduled revascularization, stroke, all-cause death, and heart failure hospitalization after 2 years of follow-up. The primary endpoint occurred in 21.4% of patients on omega-3-fatty acids vs. 20.0% on placebo (HR 1.08; P=0.60). TGs levels decreased 8.1% in the omega-3-fatty acid group and increased 5.1% in the placebo group (between group difference 13.2%; P<0.001) while changes in LDL-C were minimal in both groups. Thus, similar to the STRENGTH trial no benefits on cardiovascular disease were observed with EPA + DHA treatment.

 

SUMMARY

 

  • High dose EPA (JELIS and REDUCE-IT) reduced ASCVD outcomes while high dose EPA+DHA (STENGTH and OMEMI) did not decrease ASCVD outcomes.
  • The decrease in TG levels is not a major contributor to the beneficial effect of high dose EPA as the combination of high dose EPA+DHA lowers TG levels to the same degree as EPA alone without benefit. Additionally, the JELIS trial only lowered TG levels by 5% but nevertheless reduced ASCVD events. It is likely that the beneficial effects of EPA seen in the JELIS and REDUCE-IT trials are multifactorial with TG lowering making only a small contribution to the decrease in cardiovascular disease. 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 ASCVD events (78).
  • Whether EPA has special properties that resulted in the reduction in ASCVD events in the REDUCE-IT trial or there were flaws in the trial design (i.e., 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% in the mineral oil placebo group (75). Additionally, Apo B levels were increased by 7% (6mg/dL) by mineral oil (75). 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 (75,79). 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 (76). 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 (80,81). Ideally, another large randomized ASCVD trial with EPA ethyl ester (icosapent ethyl) (Vascepa) using a placebo other than mineral oil would help resolve this controversy. In the meantime, clinicians will need to use their clinical judgement on whether to treat patients with modest elevations in TG levels with EPA (icosapent ethyl; Vascepa) balancing the potential benefits of treatment vs. the potential side effects.

 

Summary of Lipid Lowering Drug Studies

 

The above results clearly demonstrate that lowering LDL-C levels with statins, ezetimibe, or PCSK 9 monoclonal antibodies will reduce ASCVD events in elderly patients (≥ 75 years of age) with pre-existing cardiovascular disease. In elderly patients without cardiovascular disease (primary prevention) the available data does not definitively demonstrate a decrease in ASCVD events with statin or ezetimibe therapy but is suggestive of a benefit (note there are no primary prevention trials with PCSK9 inhibitors). Additional data is required to determine if bempedoic acid and icosapent ethyl reduce ASCVD events in patients ≥ 75 years of age.

 

Studies in Progress

 

Studies are currently underway to provide definitive information on whether statin therapy is beneficial as primary prevention in the elderly. STAREE (NCT02099123) is a multicenter randomized trial in Australia of atorvastatin 40mg vs. placebo in adults ≥ 70 years of age without cardiovascular disease and PREVENTABLE (NCT04262206) is a multicenter randomized trial in the USA of atorvastatin vs. placebo in adults ≥ 75 years of age without cardiovascular disease (82,83). Other trials in the elderly that are in progress include SCOPE (NCT03770312) which is a multicenter randomized trial in Korea of low intensity vs. high intensity statin therapy in adults 76-85 years of age without CVD and SITE (Statins In The Elderly) (NCT02547883) which is a trial in France of patients ≥ 75 years of age on statin therapy who will be randomized to continue statin therapy or stop statin therapy.

 

SIDE EFFECTS OF LIPID LOWERING DRUGS

 

In this section we will describe the potential side effects of lipid lowering drugs with an emphasis on side effects likely to be seen in the elderly. Elderly patients may be more susceptible to side effects due to decreased renal function, decreased drug metabolism by the liver, polypharmacy leading to drug interactions, and co-morbidities. For a detailed discussion of the side effects of lipid lowering drugs see the Endotext chapters entitled “Cholesterol Lowering Drugs” and “Triglyceride Lowering Drugs” (49,50).

 

Statins

 

An umbrella review of meta-analyses of observational studies and randomized controlled trials examined 278 unique non-CVD outcomes from 112 meta-analyses of observational studies and 144 meta-analyses of RCTs and found that the only adverse effects associated with statin therapy were the development of diabetes and muscle disorders (84). For a detailed discussion of the side effects of statin therapy a scientific statement from the American Heart Association provides a comprehensive review (85).

 

DIABETES

 

After many years of statin use it was recognized that statins increase the risk of developing diabetes. In a meta-analysis of 13 trials with over 90,000 subjects, there was a 9% increase in the incidence of diabetes during follow-up among subjects receiving statin therapy (86). All statins appear to increase the risk of developing diabetes. In comparisons of intensive vs. moderate statin therapy, Preiss et al observed that patients treated with intensive statin therapy had a 12% greater risk of developing diabetes compared to subjects treated with moderate dose statin therapy (87). Older subjects, obese subjects, and subjects with high glucose levels were at a higher risk of developing diabetes while on statin therapy. In the Prosper trial in elderly subjects (70-82 years of age; average age 75), diabetes developed in 6.6% of patients treated with pravastatin vs. 5.1% of patients in the placebo group (51). Thus, statins may be unmasking and accelerating the development of diabetes that would have occurred naturally in these subjects at some point in time. In patients without risk factors for developing diabetes, treatment with statins does not appear to greatly increase the risk of developing diabetes.

 

In balancing the benefits and risks of statin therapy it is important to recognize that an increase in plasma glucose levels is a surrogate marker for an increased risk of developing micro and macrovascular disease (i.e., an increase in plasma glucose per se is not an event but rather increases the risk of future events). In contrast, statin therapy is preventing actual clinical events that cause morbidity and mortality. Furthermore, it may take many years for an elevated blood glucose to induce diabetic complications while the reduction in cardiovascular events with statin therapy occurs relatively quickly (after one-year benefits are seen). Finally, the number of patients needed to treat with statins to avoid one cardiovascular event is much lower (10-20 depending on the type of patient) than the number of patients needed to treat to cause one patient to develop diabetes (100–200 for one extra case of diabetes) (88). Patients on statin therapy, particularly those with risk factors for the development of diabetes, should be periodically screened for the development of diabetes with measurement of fasting glucose and/or HbA1c levels.

 

COGNITIVE DYSFUNCTION

 

Several randomized clinical trials have examined the effect of statin therapy on cognitive function and have not indicated any increased risk (for review see (89-92)). The Prosper Trial was designed to determine whether statin therapy will reduce cardiovascular disease in older subjects (age 70-82) (51). In this trial cognitive function was assessed repeatedly and no difference in cognitive decline was found in subjects treated with pravastatin compared to placebo (51,93). In the Heart Protection Study over 20,000 patients were randomized to simvastatin 40mg or placebo and again no significant differences in cognitive function was observed between the statin vs. placebo group (94). Additionally, a Cochrane review examined the effect of statin therapy in patients with established dementia and identified 4 studies with 1154 participants (95). In this analysis no benefit or harm of statin therapy on cognitive function could be demonstrated in this high-risk group of patients. Thus, randomized clinical trials do not indicate a significant association between statins and cognitive function.

 

MUSCLE

 

The most common side effect of statin therapy is muscle symptoms and many patients will discontinue the use of statins due to muscle symptoms. These can range from life threatening rhabdomyolysis, which is very rare, to myalgias, which are a common complaint (96). The risk of serious muscle disorders due to statin therapy is very small, particularly if one is aware of the potential drug interactions that increase the risk. In a case-control study with a cohort of 252,460 new users of lipid-lowering medications in U.S. health plans 21 cases of rhabdomyolysis were compared to 200 controls without rhabdomyolysis (97). Statin users >65 years of age had four times the risk of hospitalization for rhabdomyolysis than those under age 65.

The Cholesterol Treatment Trialists analyzed individual participant data on the development of muscle symptoms from 19 double-blind trials of statin versus placebo with 123,940 participants and four double-blind trials of a more intensive vs. a less intensive statin regimen with 30,724 participants (98). After a median follow-up of 4.3 years 27.1% of the individuals taking a statin vs. 26.6% on placebo reported muscle pain or weakness representing a 3% increase greater than placebo (risk ratio- 1.03; 95% CI 1.01-1.06) (Table 5). The specific muscle symptoms caused by statin therapy, myalgia, muscle cramps or spasm, limb pain, other musculoskeletal pain, or muscle fatigue or weakness were similar to those caused by placebo. The slight increase in muscle symptoms in the statin treated individuals was manifest in the first year of therapy but in the later years muscle symptoms were similar in the statin treated and placebo groups. The relative risk of statin induced muscle symptoms was greater in women than men. Intensive statin treatment with 40-80 mg atorvastatin or 20-40 mg rosuvastatin resulted in a higher risk of muscle symptoms than less intensive or moderate-intensity regimens but different statins at equivalent LDL-C lowering doses had similar effects on muscle symptoms. As shown in figure 7 muscle pain or weakness was slightly increased in patients > 65 years of age and similar in patients > 65 and ≤ 75 and those > 75 years of age. It should be noted that in individuals > 75 years of age the occurrence of muscle pain or weakness occurred in 39.6% of the individuals on the placebo, demonstrating the very high occurrence of muscle symptoms in this age group.

 

This study demonstrates that there is a small increase in muscle symptoms that primarily manifests in the first year of therapy. Statin therapy caused approximately 11 additional complaints of muscle pain or weakness per 1000 patients during the first year, but little excess in later years. Of particularly note is that 26.6% of patients taking a placebo had muscle symptoms demonstrating a very high frequency of this clinical complaint (even higher in patients > 75). Given the high prevalence of muscle complaints and the small increase attributed to statins it is very difficult to determine if a muscle complaint is actually due to the statin, which presents great clinical difficulties in patient management.

 

Table 5. Effect of Statin vs. Placebo on Muscle Symptoms

Symptom

Statin Events

Placebo Events

RR (95% CI)

Myalgia

12.0%

11.7%

1·03 (0·99–1·08)

Other musculoskeletal pain

13.3

13.0

1·03 (0·99–1·08)

Any muscle pain

26.9%

26.3%

1·03 (1·01–1·06)

Any muscle pain or weakness

27.1%

26.6%

1·03 (1·01–1·06)

 

Figure 7. Occurrence of muscle pain or weakness in different age groups in the Cholesterol Treatment Trialists meta-analysis.

 

While the results of the randomized trials suggest that muscle symptoms are not frequently induced by statin therapy, in typical clinical settings a significant percentage of patients are unable to tolerate statins due to muscle symptoms (in many studies as high as 5-25% of patients) (99-101). Additionally, when patients know that they are taking a statin they are more likely to have muscle symptoms (i.e. the nocebo effect) (102). Clinically differentiating statin induced myalgias from placebo induced myalgias is difficult, as there are no specific symptoms, signs, or biomarkers that clearly distinguish between the two. Thus, while statin induced myalgias are a real entity careful studies have shown that in the majority of patients with “statin induced muscle symptoms” the symptoms are not actually due to statin therapy. In the clinic it is difficult to be certain whether the muscle symptoms are actually due to true statin intolerance or to other factors.

 

A detailed discussion of statin induced muscle symptoms and a clinical approach to this problem is presented in the Endotext chapter entitled “Cholesterol Lowering Drugs” (50). In the section “Patients with Statin Intolerance” in this chapter we discuss the clinical approach to treating these patients.

 

Ezetimibe

 

Ezetimibe has not demonstrated significant side effects.

 

PCSK9 Monoclonal Antibodies

 

In a subgroup of patients from the FOURIER trial a prospective study of cognitive function (EBBINGHAUS Study) was carried out and no significant differences in cognitive function was observed over a median of 19 months in the PCSK9 treated vs. placebo group (103).

 

An issue of concern is whether lowering LDL-C to very low levels has the potential to cause toxicity. In a number of the PCSK9 studies a significant number of patients had LDL-C levels < 25mg/dL. For example, in the Odyssey long term study 37% of patients on alirocumab had two consecutive LDL-C levels below 25mg/dL and in the Osler long term study in patients treated with evolocumab 13% had values below 25mg/dL (104,105). In these short term PCSK9 studies, toxicity from very low LDL-C levels has not been observed. Additionally, in patients with Familial Hypobetalipoproteinemia LDL levels can be very low and these patients do not have any major disorders other than hepatic steatosis, which is not mechanistically due to low LDL-C levels (106). Similarly, there are rare individuals who are homozygous for loss of function mutations in the PCSK9 gene and they also do not appear to have major medical issues (107). Finally, in a number of statin trials and the IMPROVE-IT trial (statin + ezetimibe) there have been patients with very low LDL-C levels and an increased risk of side effects has not been consistently observed in those patients (108-111). Thus, with the limited data available there does not appear to be a major risk from markedly lowering LDL-C levels.

 

Bempedoic Acid

 

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 (package insert). In the CLEAR Outcomes trial elevated uric acid levels occurred in 10.9% of the patients on bempedoic acid compared to 5.6% taking the placebo (69). Elevations in blood uric acid levels may lead to the development of gout. 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) (package insert). 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 (package insert). In the CLEAR Outcomes trial gout was increased in the bempedoic acid group (3.1% vs. 2.1%) (69).

 

In clinical trials tendon rupture occurred in 0.5% of patients treated with bempedoic acid vs. 0% of placebo treated patients and involved the rotator cuff (the shoulder), biceps tendon, or Achilles tendon (package insert). Tendon rupture occurred within weeks to months of starting bempedoic acid and occurred more frequently in patients over 60 years of age, in those taking corticosteroid or fluoroquinolone drugs, in patients with renal failure, and in patients with previous tendon disorders. In the CLEAR Outcomes trial tendon rupture was similar in the bempedoic acid and placebo group (bempedoic acid 1.2% and placebo 0.9%) (69).

 

Bempedoic acid treatment resulted in a mean increase in serum creatinine of 0.05 mg/dL compared to baseline. Approximately 3.8% of patients treated with bempedoic acid had BUN levels that doubled vs. 1.5% in the placebo group and about 2.2% of patients treated with bempedoic acid had creatinine values that increased by 0.5 mg/dL vs. 1.1% in the placebo group (package insert). Renal function returned to baseline when bempedoic acid was discontinued. In the CLEAR Outcomes trial renal impairment was increased in the bempedoic acid group (11.5% vs.8.6%) as was the change from baseline creatinine (0.05±0.2 mg/dL vs. 0.01±0.2 mg/dL) (69).

 

Omega-3-Fatty Acids

 

 At very high doses, omega-3-fatty acids can inhibit platelets and prolong bleeding time. However, at the recommended doses this has not been a major clinical problem but nevertheless when patients are on anti-platelet drugs one should be alert for the possibility of bleeding problems (Package Inserts for Lovaza, Vascepa, and Epanova). In the REDUCE-IT trial serious bleeding events occurred in 2.7% of the patients in the icosapent ethyl group and in 2.1% in the placebo group (P=0.06) (75). 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 than in the placebo group. In the STRENGHT trial any bleeding events and major bleeding events were similar in the omega-3 fatty acid group and placebo group (76).  A recent review found no evidence for discontinuing the use of omega-3 fatty acid treatment before invasive procedures or when given in combination with other agents that affect bleeding (112).

 

An increase in new-onset atrial fibrillation was observed in the REDUCE-IT trial in the patients treated with icosapent ethyl 4 grams/day (5.3% vs. 3.9%) and in the STRENGTH trial in the patients treated with omega-3-fatty acids (2.2% vs 1.3%)

 

CURRENT GUIDELINES AND LDL-C GOALS

 

This section discusses guidelines as they pertain to elderly patients.

 

2018 AHA/ACC/Multi-Society Report

 

The following summarizes the 2018 AHA/ACC guidelines (2).

 

PRIMARY PREVENTION

 

  • For individuals >75 years of age, randomized controlled trials of statin therapy do not provide strong evidence for benefit, so clinical assessment of risk status in a clinician–patient risk discussion is needed for deciding whether to continue or initiate statin treatment.
  • In individuals ≥ 75 years of age with an LDL-C level of 70 to 189mg/dL, initiating a moderate-intensity statin may be reasonable. Goal is to reduce LDL-C by 30-49% (note these guidelines recommend percent reduction rather than absolute LDL-C goals).
  • In individuals ≥ 75 years of age it may be reasonable to stop statin therapy when functional decline (physical or cognitive), multimorbidity, frailty, or reduced life-expectancy limits the potential benefits of statin therapy.
  • A shared decision-making process between clinicians and patients that individualizes decisions is indicated, with regular periodic reassessment.
  • Determining coronary artery calcium (CAC) score will help in determining which patients will benefit the most. For older adults with CAC scores of zero, the likelihood of benefits from statin therapy does not outweigh the risks. Limiting statin therapy to those with CAC scores greater than zero, combined with clinical judgment and patient preference, could provide a valuable awareness with which to inform shared decision-making.

 

SECONDARY PREVENTION

 

  • In patients ≥75 years of age with clinical ASCVD, it is reasonable to initiate moderate- or high-intensity statin therapy after evaluation of the potential for ASCVD risk reduction, adverse effects, and drug–drug interactions, as well as patient frailty and patient preferences. The goal of moderate statin therapy is to reduce LDL-C by 30-49% and the goal of high-intensity statin therapy is to reduce LDL-C by ≥ 50%. In very high-risk patients, a goal of an LDL-C < 70mg/dL and non-HDL-C < 100mg/dL is reasonable.
  • In patients ≥75 years of age who are tolerating high-intensity statin therapy, it is reasonable to continue high-intensity statin therapy after evaluation of the potential for ASCVD risk reduction, adverse effects, and drug-drug interactions, as well as patient frailty and patient preferences.

 

PATIENTS WITH DIABETES

 

  • In patients ≥ 75 years of age with diabetes mellitus and who are already on statin therapy, it is reasonable to continue statin therapy.
  • In patients ≥ 75 years of age with diabetes mellitus without cardiovascular disease it may be reasonable to start moderate statin therapy after a clinician-patient discussion of the potential benefits and risks of therapy. The goal is to decrease LDL-C by 30-49%.

 

2019 ESC/EAS Guidelines

 

The following summarizes the 2019 ESC/EAS guidelines (3).

 

  • Treatment with statins is recommended for older people with ASCVD in the same way as for younger patients.
  • Treatment with statins is recommended for primary prevention, according to the level of risk, in older people aged ≤ 75 years.
  • Initiation of statin treatment for primary prevention in older people aged >75 years may be considered, if at high-risk or above.
  • It is recommended that the statin is started at a low dose if there is significant renal impairment and/or the potential for drug interactions, and then titrated upwards to achieve LDL-C treatment goals.
  • In patients at very-high risk in primary or secondary prevention the goal is a 50% reduction in LDL-C and an LDL-C < 55mg/dL.
  • In patients at high risk in primary or secondary prevention the goal is a 50% reduction in LDL-C and an LDL-C < 70mg/dL.

The ESC/EAS criteria for very high risk and high risk are shown in table 6.

 

Table 6. ESC/EAS Criteria for Very-High Risk and High Risk for ASCVD Events

Very High Risk

Documented ASCVD or unequivocal on imaging

DM with target organ damage or at least three major risk factors, or early onset of T1DM of long duration (>20 years)

Severe CKD (eGFR <30 mL/min/1.73 m2)

A calculated SCORE ≥ 10% for 10-year risk of fatal CVD

Familial Hypercholesterolemia with ASCVD or with another major risk factor

High Risk

Markedly elevated single risk factors, in particular LDL-C >190 mg/dL or BP ≥ 180/110 mmHg

Patients with Familial Hypercholesterolemia without other major risk factors

Patients with DM without target organ damage, with DM duration ≥ 10 years, or another additional risk factor

Moderate CKD (eGFR 30-59 mL/min/1.73 m2)

A calculated SCORE ≥ 5% and <10% for 10-year risk of fatal CVD.

 

Our Approach

 

Our approach is based on concepts taken from both the ACC/AHA and ESC/EAS guidelines (i.e., we try to utilize the best ideas from each guideline). There are several general principles regarding lipid lowering therapy that should be considered in deciding who to treat (113).

 

  • The higher the LDL-C level the greater the benefit of lowering LDL-C.
  • The greater the decrease in LDL-C the greater the benefit.
  • The higher the absolute risk of ASCVD the greater the benefit of lowering LDL-C.

 

Additional factors that need to be considered, particularly in elderly patients, include

 

  • Life expectancy.
  • Competing non-cardiovascular disorders.
  • Risk of drug side effects.
  • Potential for drug interactions.
  • Patient preferences.

 

PRIMARY PREVENTION

 

Given the absence of definitive outcome trials demonstrating the benefit of decreasing LDL-C levels in patients ≥ 75 years of age without cardiovascular disease one must use clinical judgement in deciding who to treat. It should be recognized, as discussed in detail earlier, that the available evidence suggests that decreasing LDL-C will reduce ASCVD events in the elderly. Our approach is to determine ASCVD risk and then balance the risk with competing factors such as life expectancy, non-cardiovascular disorders, potential for drug interactions, and patient preferences. We use the approach described below to determine risk.

 

Step 1- Calculate the 10-year risk of an ASCVD event using the AHA/ACC pooled cohort equation. In Europe one can use the SCORE OP risk prediction algorithms (114). This will provide an estimate of the risk of the patient having an ASCVD event/death.

Step 2- To gain further insight on the risk of ASCVD one can determine if patient has any risk enhancing factors (tables 7 and 8). This can help further stratify the patient’s risk.

Step 3- If after discussion with the patient, you and/or the patient is uncertain on the level of risk and the appropriate treatment plan obtaining a coronary calcium score (CAC) can be helpful. A CAC score of zero indicates low risk for ASCVD and allows one to not start statin therapy (2). Note that a CAC score of zero in cigarette smokers, patients with diabetes mellitus, those with a strong family history of ASCVD, and possibly chronic inflammatory conditions such as HIV, may still be associated with a substantial 10-year risk (2).

 

Following these steps, we can estimate the risk for ASCVD events and in conjunction with the general principles described above discuss with the patient a treatment plan. If the decision is to treat our goal is often an LDL-C < 100mg/dL and non-HDL-C < 130mg/dL but in high-risk patients our goal may be an LDL-C < 70mg/dL and non-HDL-C < 100mg/dL.

 

Table 7. Risk-Enhancing Factors

 Family history of premature ASCVD (males, age <55 y; females, age <65 y)

 Primary hypercholesterolemia (LDL-C ≥160mg/dL; non-HDL-C ≥190mg/dL

 Metabolic syndrome (increased waist circumference, elevated triglycerides [>175 mg/dL], elevated blood pressure, elevated glucose, and low HDL-C [<40 mg/dL in men; <50 in women mg/dL] are factors; tally of 3 makes the diagnosis)

 Chronic kidney disease (eGFR 15–59 mL/min/1.73 m2 with or without albuminuria; not treated with dialysis or kidney transplantation)

 Chronic inflammatory conditions such as psoriasis, RA, or HIV/AIDS

 History of premature menopause (before age 40 y) and history of pregnancy-associated conditions that increase later ASCVD risk such as preeclampsia

 High-risk race/ethnicities (e.g., South Asian ancestry)

 Lipid/biomarkers: Associated with increased ASCVD risk

  Persistently* elevated, primary hypertriglyceridemia (≥175 mg/dL);

  If measured:

  1. Elevated high-sensitivity C-reactive protein (≥2.0 mg/L)

  2. Elevated Lp(a) ≥50 mg/dL or ≥125 nmol/L

  3. Elevated apoB ≥130 mg/dL

  4. ABI <0.9

 ABI= ankle-brachial index, RA= rheumatoid arthritis.

Modified from reference (2).

 

Table 8. Factors Modifying Systematic Coronary Risk Estimation Risks

Social deprivation: the origin of many of the causes of CVD.

Obesity and central obesity as measured by the body mass index and waist circumference, respectively.

Physical inactivity.

Psychosocial stress

Family history of premature CVD (men: <55 years and women: <60 years).

Chronic immune-mediated inflammatory disorder.

Major psychiatric disorders.

Treatment for HIV infection.

Atrial fibrillation.

Left ventricular hypertrophy.

Chronic kidney disease.

Obstructive sleep apnea syndrome.

Metabolic associated fatty liver disease.

Modified from reference (3).

 

PATIENTS WITH DIABETES

 

In patients ≥ 75 with diabetes without pre-existing cardiovascular our approach is very similar to that described for primary prevention. In addition to the risk enhancers listed in tables 7 and 8 there are specific diabetes risk enhancers that clinicians should factor in their decisions (table 9). Also, as noted above, in the presence of diabetes a zero CAC score is not as strong an indicator of low risk for ASCVD as in non-diabetics.

 

In patients with diabetes because they usually have multiple risk factors and are at high risk for ASCVD events our typical LDL-C goal is < 70mg/dL and non-HDL-C < 100mg/dL. In the rare situation where there are minimal other risk factors an LDL-C goal < 100mg/dL and non-HDL-C < 130mg/dL is reasonable. 

 

Table 9. Diabetes-Specific Risk Enhancers That Are Independent of Other Risk Factors

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

Albuminuria ≥30 mcg of albumin/mg creatinine

eGFR <60 mL/min/1.73 m

Retinopathy

Neuropathy

ABI <0.9

ABI= ankle-brachial index.

Modified from reference (2).

 

SECONDARY PREVENTION

 

Studies have shown that lowering LDL-C levels with statins, ezetimibe, and PCSK9 monoclonal antibodies reduces ASCVD events in older adults with ASCVD. Thus, unless there are contraindications older patients with ASCVD should be treated with lipid lowering drugs to reduce ASCVD events. In elderly patients we will often employ a modest statin dose (for example atorvastatin 10-20mg or rosuvastatin 5-10mg) in combination with ezetimibe 10mg and then increase the statin dose, if necessary, based on lipid levels and the patient tolerating the treatment regimen. At a minimum our goal is an LDL-C < 70mg/dL and a non-HDL-C level < 100mg/dL but we would prefer lower values (ideally LDL-C < 55mg/dL and non-HDL-C < 85mg/dL) if they can be achieved with a statin + ezetimibe. In very high-risk patients (table 10) our goal is an LDL-C < 55mg/dL and non-HDL-C < 85mg/dL and adding a PCSK9 inhibitor may be required to achieve these levels in some patients.

 

Table 10. Criteria for Very High Risk

Very high-risk includes a history of multiple major ASCVD events or one major ASCVD event and multiple high-risk conditions.

 

Major ASCVD Events

 Recent ACS (within the past 12 months)

 History of MI (other than recent ACS event)

 History of ischemic stroke

 Symptomatic peripheral arterial disease (history of claudication with ABI <0.85, or previous revascularization or amputation)

 

High-Risk Conditions

 Age ≥65 y

 Heterozygous familial hypercholesterolemia

 History of prior coronary artery bypass surgery or percutaneous coronary intervention outside of the major ASCVD event(s)

 Diabetes mellitus

 Hypertension

 CKD (eGFR 15-59 mL/min/1.73 m2)

 Current smoking

 Persistently elevated LDL-C (LDL-C ≥100 mg/dL [≥2.6 mmol/L]) despite maximally tolerated statin therapy and ezetimibe

 History of congestive HF

ABI= ankle-brachial index; ACS= acute coronary syndrome.

Based on reference (2).

 

TREATMENT

 

Lifestyle

 

The lifestyle changes described below are recommended for all adults and are not specific for elderly individuals or for individuals with cardiovascular disease. The lifestyle changes recommended will lower lipid levels and are likely to reduce the risk of ASCVD.

 

EXERCISE

 

There is little debate that exercise is beneficial and that all individuals should be physically active. It is recommended that individuals participate in at least 150 minutes of moderate-intensity aerobic physical activity (for example 30 minutes 5 times per week) or 75 minutes per week of vigorous-intensity physical activity (115,116). Additionally, it is recommended that individuals participate in 2 days per week of muscle-strengthening activity (116). Because of the loss of muscle mass with aging it is very important to incorporate resistance training into the exercise program of elderly individuals.

 

A meta-analysis of exercise in the older individuals (>60 years of age) found that aerobic exercise decreased triglyceride and LDL-C levels and increased HDL-C levels while resistance exercise decreased LDL-C levels (117). Exercise also increases fitness and helps with weight loss. It should be noted that many elderly individuals may have substantial medical and social barriers to participating in exercise programs. Comorbidities, such as osteoarthritis, may limit exercise tolerance and make exercise challenging. Older individuals should be encouraged to be as active as possible.

 

DIET

 

For a detailed discussion of the effect of diet on lipids, lipoproteins and ASCVD see the Endotext chapter entitled “The Effect of Diet on Cardiovascular Disease and Lipid and Lipoprotein Levels (118). There is general agreement on what constitutes a healthy diet and a brief summary of the Guidelines for Americans 2020-2025 is shown in table 11 and the guidelines from the American College of Cardiology/American Heart Association are shown in table 12.

 

Table 11. Guidelines for Americans 2020-2025

Recommend

Limit

Vegetables of all types—dark green; red and orange; beans, peas, and lentils; starchy; and other vegetables

Added sugars—Less than 10 percent of calories per day

Fruits, especially whole fruit

Saturated fat—Less than 10 percent of calories per day

Grains, at least half of which are whole grain

Sodium—Less than 2,300 milligrams per day

Dairy, including fat-free or low-fat milk, yogurt, and cheese, and/or lactose-free versions and fortified soy beverages and yogurt as alternatives

Alcoholic beverages—Adults can

choose not to drink or to drink in moderation by limiting intake to 2 drinks or less in a day for men and 1 drink or less in a day for women

Protein foods, including lean meats, poultry, and eggs; seafood; beans, peas, and lentils; and nuts, seeds, and soy products

 

Oils, including vegetable oils and oils in food, such as seafood and nuts

 

Full guideline is available at DietaryGuidelines.gov

 

Table 12. ACC/AHA Dietary Recommendations to Reduce Risk of ASCVD (115)

1. A diet emphasizing intake of vegetables, fruits, legumes, nuts, whole grains, and fish is recommended

2. Replacement of saturated fat with dietary monounsaturated and polyunsaturated fats can be beneficial

3. A diet containing reduced amounts of cholesterol and sodium can be beneficial

4. As a part of a healthy diet, it is reasonable to minimize the intake of processed meats, refined carbohydrates, and sweetened beverages

5. As a part of a healthy diet, the intake of trans fats should be avoided

 

A summary of the effect of individual dietary constituents on lipid and lipoprotein levels is shown in table 13 (118). This table summarizes the results of numerous randomized trials examining the effect of dietary manipulations on lipid and lipoprotein levels.

 

Table 13. Summary of the Effect of Dietary Constituents on Lipid and Lipoproteins

SFA

Increase LDL-C and modest increase HDL-C

MUFA and PUFA

Decrease LDL-C

TFA

Increase LDL-C and decrease HDL-C

Cholesterol

Increase LDL-C

Carbohydrates

Increase TGs, increase greater with simple sugars particularly fructose

Fiber

Decrease LDL-C

Phytosterols

Decrease LDL-C

SFA= saturated fatty acids, MUFA= monounsaturated fatty acids, PUFA= polyunsaturated fatty acids, TFA= trans fatty acids.

 

There is a huge literature describing the effect of diet on the risk of ASCVD and this literature is often conflicting and controversial (118). Several well recognized investigators have discussed the limitations of the information linking various diets and dietary constituents and the risk of disease (119,120). The major problem is that almost all of the information is based on observational studies and well conducted randomized trials measuring important ASCVD outcomes are very rare. Observational studies can demonstrate associations but do not definitively indicate that there is a cause-and-effect relationship. Unrecognized confounding variables can result in false associations.

 

Some of the more recent randomized dietary trials that have examined the effect of diet on ASCVD events are described below. For a discussion of other studies see reference (118). The PREDIMED trial employing a Mediterranean diet (increased monounsaturated fats) reduced the incidence of major ASCVD events (121). In this multicenter 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. The average age of participants in this trial was 67. 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 < 70 and ≥ 70 years of age. 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 (122). The CORDIOPREV study and the Lyon Diet Heart Study were randomized trials that demonstrated that a Mediterranean diet reduces ASCVD events in patients with cardiovascular disease (secondary prevention) (123,124). Unfortunately, these studies did not have a sufficient number of patients > 70 years of age for analysis of the effect of the diet in older patients with pre-existing cardiovascular disease.

 

The results of these three randomized trials indicate that following a Mediterranean type diet reduces ASCVD. 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.

 

LIPID LOWERING DRUGS

 

Current guidelines and lipid lowering goals are discussed in the guidelines section above. In this section we will focus on clinical decisions regarding the use of lipid lowering drugs. To maximize benefits of lipid lowering therapy we think it is important to achieve LDL-C goals.

 

Elderly Patients on Lipid Lowering Therapy

 

In elderly patients on lipid lowering therapy, we usually continue therapy if they are tolerating the medications without side effects. We will periodically check a lipid panel to make sure that they are achieving the goals of therapy. If not, we will adjust the lipid lowering medications to achieve the desired LDL-C goals. We will make changes in therapy if circumstances change. For example, if a patient develops metastatic cancer and is transferred to palliative or Hospice care we will stop the lipid lowering therapy. Similarly, if a new drug is required the current lipid lowering drugs may need to be changed to avoid drug interactions. Thus, in most patients continuing lipid lowering therapy is appropriate.

 

Primary Prevention in Elderly Patients

 

In elderly patients we usually initiate therapy using moderate-intensity statin therapy if therapy is indicated as discussed above. We typically use either atorvastatin 10-20mg or rosuvastatin 5-10mg. Our reason for using these statins is that if one needs to lower LDL-C further we can just increase the dose of the statin and not need to start a new statin. In certain circumstances we might use another statin to avoid drug interactions (for example in a patient living with HIV we might use pitavastatin). After 6-12 weeks on statin therapy, we check a lipid panel and if the patient is having any medication side effects. If the LDL-C is not at goal we will either increase the statin dose or if we feel that the patient is at risk for statin toxicity add ezetimibe 10mg instead. In a healthy elderly patient at high risk for ASCVD (for example high LDL-C, diabetes, and hypertension) we do not hesitate to use high-intensity statin therapy (atorvastatin 40-80mg and rosuvastatin 20-40mg) plus ezetimibe 10mg to achieve the LDL-C goal.

 

Secondary Prevention in Elderly Patients

 

Unless than is a contraindication we frequently start these patients on high-intensity statin therapy. After 6-12 weeks on statin therapy, we check a lipid panel and if the patient is having any medication side effects. We will often add ezetimibe as studies have shown that the greater the lowering of LDL-C the greater the reductions in ASCVD events. Additionally, ezetimibe is generic (i.e. inexpensive) and doesn’t typically cause side effects. We will use PCSK9 inhibitors following the principle that the higher the LDL-C and the greater the risk of ASCVD events the greater the cost effectiveness of using expensive PCSK9 inhibitors.

 

Mixed Hyperlipidemia

 

In patients with mixed hyperlipidemia (elevated LDL-C and triglyceride levels) Initial drug therapy should also be a statin unless triglyceride levels are greater than 500-1000mg/dL. If triglycerides are > 500-1000mg/dL initial therapy is directed at lowering triglyceride levels (49,125). In addition to lowering LDL-C levels, statins are also effective in lowering triglyceride levels particularly when the triglycerides are elevated. If LDL-C is not lowered sufficiently ezetimibe is a reasonable next step. The approach to the patient whose LDL-C levels are at goal but the triglycerides and non-HDL-C are still elevated is not clearly defined. As discussed above studies have failed to demonstrate that adding a fibrate or niacin reduces ASCVD events. The REDUCE-IT trial has demonstrated that icosapent ethyl (Vascepa) decreases ASCVD events in this patient population but as discussed in detail above the results of this study are debated because the mineral oil placebo increased LDL-C. non-HDL-C, hsCRP, and other biomarkers associated with an increased risk of ASCVD events. It is debated by various experts whether the beneficial effect seen in this study was due to the positive effects of icosapent ethyl or to negative effects of the placebo. Clinicians will need to use their clinical judgement on whether to treat patients with elevations in TG and non-HDL-C levels with icosapent ethyl balancing the potential benefits of treatment vs. the potential side effects. In making this decision in our elderly patients it is worth noting that in participants <65 years of age the primary end point was reduced by 35% (HR 0.65; 95% CI 0.54–0.78) while in participants ≥ 65 years of age the primary end point was reduced by 18% (HR 0.82; 95%CI 0.70–0.97; P Value for Interaction 0.06). Information on patients ≥ 75 years of age is not available.  

 

Patients with Statin Intolerance

 

Statin intolerance is frequently due to myalgias but on occasion can be due other issues, such as increased liver or muscle enzymes, cognitive dysfunction, or other neurological disorders. The percentage of patients who are “statin intolerant” varies greatly but in clinical practice a significant number of patients have difficulty taking statins.

 

It can be difficult to determine if the muscle symptoms that occur when a patient is taking a statin are actually due to the statin or are unrelated to statin use. The first step in a “statin intolerant patient” is to take a careful history of the nature and location of the muscle symptoms and the timing of onset in relation to statin use to determine whether the presentation fits the typical picture for statin induced myalgias. The characteristic findings with a statin induced myalgia are shown in table 14 and findings that are not typical for statin induced myalgia are shown in table 15. The disappearance of symptoms within a few weeks of stopping statins and the reappearance after restarting statins is very suggestive of the symptoms being due to true statin intolerance. An on-line tool (htpp://tools.acc.org/statinintolerance/#!/) and an app produced by the ACC/AHA are available. This tool characterizes patients based on 8 criteria into possible vs. unlikely to have statin induced muscle symptoms (table 16).

 

Table 14. Characteristic Findings with Statin Induced Myalgia

Symmetric

Proximal muscles

Muscle pain, tenderness, weakness, cramps

Symptom onset < 4 weeks after starting statin or dose increase

Improves within 2-4 weeks of stopping statin

Cramping is unilateral and involves small muscles of hands and feet

Same symptoms occur with re-challenge within 4 weeks

 

Table 15. Symptoms Atypical in Statin Induced Myalgia

Unilateral

Asymmetric

Small muscles

Joint or tendon pain

Shooting pain, muscle twitching or tingling

Symptom onset > 12 weeks

No improvement after discontinuing statin

 

Table 16. Diagnosis of Statin Associated Muscle Symptoms

Symptom timing

Symptom type

Symptom location

Sex

Age

Race/ethnicity

CK elevation > 5 times the upper limit of normal

Known risk factors for statin induced muscle symptoms and non-statin causes of muscle symptoms

 

One should also check a CK level but this is almost always in the normal range. If the CK is not elevated and the symptoms do not suggest a statin induced myalgia one can often reassure the patient and continue statin therapy. This is often successful and studies have shown that many patients that stop taking statins due to “statin induced myalgia” can be successfully treated with a statin (50). If the CK is elevated it should be repeated after instructing the patient to avoid exercise for 48 hours. Also, the CK levels should be compared to CK levels prior to starting therapy if available. If the CK remains elevated (3x upper limit of normal) the statin should be discontinued. Similarly, if the CK is normal but the symptoms are suggestive of a statin induced myalgia the statin should also be discontinued. The next step is to determine if one can identify reversible factors that could be increasing statin toxicity (hypothyroidism, drug interactions).  If none are identified the next step after the myalgias have resolved is to try a low dose of a different statin that is metabolized by a different pathway (for example instead of atorvastatin, which is metabolized by the CYP3A4 pathway, rosuvastatin, which has a different pathway of metabolism). Because statin side effects are dose related, a low dose of a statin may often be tolerated. One can also try several different statins as sometimes a patient may tolerate one statin and not others. A meta-analysis has shown that every other day administration of statins is as effective as daily administration in lowering lipid levels and therefore is a very reasonable strategy (126). In some instances, using a long-acting statin (rosuvastatin or atorvastatin) 1-3 times per week can work (we usually start with once per week and then slowly increase frequency as tolerated) (127). In these circumstances (low doses or 1-3 times per week) the reduction in LDL-C may not be sufficient but one can use combination therapy with other drugs such as ezetimibe, bempedoic acid, or PCSK9 inhibitors to achieve LDL-C target goals.

 

If after trying various approaches a patient still has myalgias and is unable to tolerate statin therapy one needs to utilize other approaches to lower LDL-C levels. We typically use the ezetimibe and bempedoic acid combination pill (Nexlizet), which can lower the LDL-C level by approximately 40%, which is often sufficient (128). If needed one could add a PCSK9 inhibitor to further decrease LDL-C.

 

CONCLUSION

 

ASCVD is a major cause of morbidity and mortality in elderly patients. In elderly patients with pre-existing ASCVD randomized clinical trials have shown that lipid lowering drug therapy with statins, ezetimibe, and PCSK9 inhibitors reduce ASCVD events. Thus, most elderly patients with ASCVD should be treated with lipid lowering drugs unless there are contraindications such as limited life expectancy, competing non-cardiovascular disorders, high risk of drug interactions or drug side effects. In elderly patients without ASCVD if they are already taking lipid lowering drugs and if they are tolerating the medications without side effects continuing therapy is usually reasonable as long as the clinical circumstances have not changed. In elderly patients not on lipid lowering therapy and without cardiovascular disease studies have suggested that lipid lowering therapy is beneficial but further studies are required to definitively demonstrate benefit. In these patients one needs to determine the patient’s risk for ASCVD events and then discuss the potential benefits and side effects with the patient to make a shared decision on whether to initiate therapy. Age per se should not be used to withhold therapy with lipid lowering drugs that can reduce the risk of ASCVD events.

 

ACKNOWLEDGEMENTS

 

This work was supported by grants from the Northern California Institute for Research and Education. The authors would like to thank Dan Streja, the original author of this chapter, who provided the framework for this updated chapter.

 

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The Effect of Diet on Cardiovascular Disease and Lipid and Lipoprotein Levels

ABSTRACT

 

The role of lipids and lipoproteins as causal factors for cardiovascular disease (CVD) is well established. Dietary saturated fatty acids (SFA), which are in milk, butter, cheese, beef, lamb, pork, poultry, palm oil, and coconut oil increase LDL-C and HDL-C. The increase in LDL-C is due to a decrease in hepatic LDL clearance and an increase in LDL production secondary to a decrease in hepatic LDL receptors. Monounsaturated fatty acids (MUFA) are in olive, canola, peanut, safflower, and sesame oil, and avocados, peanut butter, and many nuts and seeds and polyunsaturated fatty acids (PUFA) are in soybean, corn, and sunflower oil, and some nuts and seeds, tofu, and soybeans. Both MUFA and PUFA lower LDL-C by increasing hepatic LDL receptor activity. Dietary cholesterol is found in egg yolks, shrimp, beef, pork, poultry, cheese, and butter and increase LDL-C but the effect is modest and varies with approximately 15-25% of individuals being hyper-responders with more robust increases. Dietary cholesterol reduces hepatic LDL receptor activity, decreasing the clearance and increasing the production of LDL. Trans fatty acids (TFA) occur naturally in meat and dairy products and are formed during the partial hydrogenation of vegetable fat. TFA increase LDL-C and decrease HDL-C. Carbohydrates (CHO) can be divided into high-quality, for example fruits, legumes, vegetables, and whole grains, or low-quality, which include refined grains, starches, and added sugars. CHO increase TG with low quality CHO, particularly added sugars, having a more robust effect. Dietary CHO, particularly fructose, promotes hepatic de novo fatty acid synthesis leading to increased VLDL secretion. Fiber is found mostly in fruits, vegetables, whole and unrefined grains, nuts, seeds, beans, and legumes and phytosterols are naturally occurring constituents of plants and are found in vegetable oils, cereals, nuts, fruit and vegetables. Both dietary fiber and phytosterols decrease LDL-C by decreasing intestinal cholesterol absorption.

Summary of the Effect of Dietary Constituents on Lipid and Lipoproteins

SFA

Increase LDL-C and modest increase HDL-C

MUFA and PUFA

Decrease LDL-C

TFA

Increase LDL-C and decrease HDL-C

Cholesterol

Increase LDL-C

CHO

Increase TGs particularly simple sugars

Fiber

Decrease LDL-C

Phytosterols

Decrease LDL-C

With regards to CVD there are very few well conducted randomized controlled trials and most of the information is derived from observational studies that demonstrate associations. These observational studies have found that fruits, vegetables, beans/legumes, nuts/seeds, whole grains, fish, yogurt, fiber, seafood omega-3 fatty acids, and polyunsaturated fats were associated with a decreased risk of CVD while unprocessed red meats, processed meats, sugar-sweetened beverages, high glycemic load CHO, and trans-fats were associated with an increased risk of CVD. Randomized trials have shown that a Mediterranean diet reduces CVD. Based on this information current guidelines for the general population recommend 1. A diet emphasizing intake of vegetables, fruits, legumes, nuts, whole grains, and fish 2. Replacement of SFA with MUFA and PUFA 3. A reduced amount of dietary cholesterol 4. Minimizing intake of processed meats, refined CHO, and sweetened beverages and 5. Avoidance of TFA. For individuals with a high LDL-C limiting dietary SFA, TFA, and cholesterol and increasing fiber and phytosterols will help lower LDL-C while in individuals with high TG limiting low quality CHO, particularly simple sugars, and ethanol with weight loss, if indicated, will help lower TG.

 

INTRODUCTION

 

There is a huge literature describing the effect of diet on the risk of cardiovascular disease (CVD) and this literature is often conflicting and controversial. Several well recognized investigators have discussed the limitations of the information linking various diets and dietary constituents and the risk of disease (1,2). The major problem is that almost all of the information is based on observational studies and well conducted randomized trials measuring important cardiovascular outcomes are very rare. Observational studies can demonstrate associations but do not necessarily indicate that there is a cause-and-effect relationship. Unrecognized confounding variables can result in false associations. In several instances a robust association was observed in observation trials but randomized trials failed to confirm these observations (3). For example, several observational studies showed that higher vitamin E intake from dietary sources or supplements was associated with a lower risk of CVD (4-8), but randomized controlled trials failed to demonstrate a reduction in cardiovascular events with vitamin E supplementation (9-12). Observational studies have also reported that vitamin B6, B12, or folic acid intake reduced the risk of CVD (13-15), but again randomized controlled trials failed to demonstrate a benefit of increased vitamin intake on CVD (16-19). These results point to potential deficiencies in observational studies and the need to recognize that the associations demonstrated in observational studies may not always be causal. Therefore, in this chapter, where possible, we will focus on randomized controlled trials.

 

Moreover, even the interpretation of the results of observational trials is often debated. For example, a 2019 meta-analysis and systematic review published in the Annals of Internal Medicine reached the conclusion that “the magnitude of association between red and processed meat consumption and all-cause mortality and adverse cardiometabolic outcomes is very small, and the evidence is of low certainty” (20). This conclusion is contrary to the recommendations of almost all dietary guidelines and as would be expected this resulted in a critique challenging this conclusion (21). There are numerous other instances where there are conflicting results and interpretations in the literature linking diet with CVD making it difficult to sort out fact from fiction.

 

The information pertaining to the effect of dietary manipulations on lipid and lipoprotein levels are frequently based on randomized controlled trials rather than observational studies and therefore tend to be more consistent. However, even in these studies the results are sometimes conflicting. There are many factors that could account for this variability including the heterogeneity in study settings, type of individuals studied, study designs, differences in baseline diets, adherence to the study diet, differences in types of diet or dietary composition, methods and accuracy of the methods used to measure lipid and lipoprotein levels, and many other factors.

 

Additionally, the clinician should recognize that the lipid response of an individual patient to dietary manipulations can vary greatly, is very modest on average (in the range of 10% reductions, typically), and in most cases will not prevent the need for lipid lowering medications. The importance of genetic differences on these responses is often under recognized by patients and providers. For example, individuals with an apo E4 allele have a more robust decrease in LDL-C in response to a decrease in dietary fat and cholesterol than subjects carrying the apo E3 or apo E2 alleles (22). Polymorphisms in other genes have also been shown to modulate the lipid and lipoprotein response to dietary manipulations (22,23). Clinical conditions can also affect the response to diet. For example, the expected lipid and lipoprotein response to a low cholesterol, low saturated fatty acids (SFA) diet is blunted in obese individuals (24). Therefore, the effect of a specific diet can vary from individual to individual and the clinician will have to monitor a patient’s response.

 

It should be recognized that when one increases or decreases a particular macronutrient in the diet (lipids, carbohydrates (CHO), or protein) there needs to be a reciprocal change in another macronutrient to maintain caloric balance. It can therefore be difficult to know whether the increase in a particular nutrient or a decrease in another nutrient is accounting for the observed effect (for example decreasing SFA and increasing CHO). Where possible I will try to specify which nutrient was decreased and which was increased in the studies described.

 

Finally, it is important to look at the effect of diet on lipids independent of weight loss. Weight loss per se can affect lipid levels resulting in a decrease in triglycerides and LDL-C and an increase in HDL-C levels (25). For a detailed discussion of the effect of weight loss on lipid levels see the chapter on obesity and dyslipidemia (25).

 

In this chapter we will first discuss the effect of various macronutrients, then specific foods, and finally specific diets on lipids and lipoprotein levels.  

 

DIETARY SATURATED FATTY ACIDS

 

Major sources of saturated fatty acids (SFA) in the diet are milk, butter, cheese, other dairy products, beef, lamb, pork, poultry particularly the skin, palm oil, palm kernel oil, and coconut oil (tables 1 and 3).

 

Table 1. Fatty Acid Composition of Foods High in Saturated Fat

 

Total Fat

grams/100 grams

SFA

grams/100 grams

MUFA

grams/100 grams

PUFA

grams/100 grams

Hamburger

15.0

5.89

6.66

0.49

Pork loin

13.3

5.23

6.19

1.20

Chicken

12.6

3.50

4.93

2.74

Lamb

15.1

6.90

7.00

1.20

Whole milk*

3.9

2.5

1.0

0.1

Gouda cheese**

30.6

20.3

7.4

0.9

Butter***

82.2

52.1

20.9

2.8

*TFA = 0.1g/100g; **TFA = 1.1g/100g; TFA = 2.9g/100g.

TFA= trans fatty acids, MUFA= monounsaturated fatty acids, PUFA= polyunsaturated fatty acids.

 

Effect of Dietary Saturated Fatty Acids on Cardiovascular Disease

 

OBSERVATIONAL STUDIES

 

Dietary guidelines uniformly recommend reducing the intake of SFA. There are a large number of observational trials that have shown an association between dietary SFA intake and CVD (26-31). However, there are meta-analyses that have not found an association between dietary SFA intake and CVD (32-36). A possible explanation for this discordance is whether the SFA in the diet is replaced by polyunsaturated fatty acids (PUFA) vs. replaced by CHO. When SFA is replaced by PUFA there is a reduction in CVD whereas replacement with CHO has no benefit on CVD (27-29,37-39). However, replacement of SFA with high quality CHO may be beneficial (27,37,38). Additionally, in one study SFA from meat was associated with an increased risk of CVD while SFA from dairy products was associated with a decrease in CVD (40). Thus, the source of SFA may be important. 

 

As noted above, observational studies can demonstrate an association but are not able to definitively demonstrate a causal relationship. It is therefore essential to review the results of randomized controlled trials on the effect of decreasing dietary SFA on preventing cardiovascular events.

 

RANDOMIZED CONTROLLED OUTCOME TRIALS

 

This section will review the major randomized trials analyzing the effect of decreasing SFA intake on preventing CVD. Studies with very few participants, few cardiovascular events, or very short-term studies will not be included. It is important to note that many of these studies were carried out in the 1950’s and 1960’s when the diagnosis and treatment of CVD was very primitive compared to current standards. Also, typical diets were much different (higher in SFA) and mean plasma cholesterol levels were higher. Lastly, the methodology of these studies was not up to the current standards by which randomized controlled trials are performed (small number of patients, often not blinded, inadequate statistical power, non-specific endpoints, etc.). Thus, the accuracy of these trials and the relevancy of these older studies to current times is uncertain.

 

In a study from England initiated in 1957, 252 men under the age of sixty-five who recently

had a myocardial infarction were assigned to a low-fat diet or usual diet (41). The low-fat diet was limited to 40 grams per day of fat with decreases in butter and meat. The intake of fat during the trial was approximately 100-120 grams per day in the usual diet group and slightly greater than 40 grams per day in the low-fat diet group. At the time of the study the typical diet was high in SFA so a decrease in total fat would have resulted in a significant decrease in SFA. During the trial serum cholesterol levels were in the 240mg/dL range in the usual diet group and 220mg/dL in the low-fat diet group. There were no differences between the two groups in cardiovascular events during the 5 years of the trial. To see a reduction in cardiovascular events with the modest reduction in serum cholesterol levels this study would have required a much larger number of participants.

 

The Oslo Diet-Heart Study randomized men under 65 years of age with a history of a myocardial infarction to a diet low in SFA and cholesterol, and high in PUFA (n=206) or their usual diet (n=206) (42). Cholesterol levels were approximately 295mg/dL and decreased to approximately 240mg/dL in the patients on the low SFA diet with minimal changes in the control group. After 5 years major cardiovascular events and cardiovascular mortality were reduced in the group on the low SFA diet (Events- 61 low SFA group vs. 81 control group; Mortality- 38 low SFA group vs. 52 control group).

 

The Medical Research Council soya-bean trial randomized men under 60 years of age with a recent myocardial infarction to continue their usual diet (n=194) or a diet low in SFA and containing 85 grams of soya-bean oil daily (PUFA) (n=199) (43). The low SFA diet lowered cholesterol from 272 to 213mg/dL (22% decrease) while in the controls, cholesterol decreased from 273 to 259mg/dL (6% decrease). The primary outcome was first relapse (myocardial infarction, angina, sudden death). After 4 years, 62 of 199 in the soybean oil group had a recurrent coronary event compared with 74 of 194 in the usual diet group; the difference, −18% (95% CI, −38 to 7), was not statistically significant but given the small number of participants was suggestive of benefit.

 

The Los Angeles Veterans Administration Center study randomized 422 men to the conventional control diet and 424 to the experimental diet low in SFA and cholesterol and enriched in PUFA (44,45). 30% of the men had CVD. The baseline plasma cholesterol was 233mg/dL and on treatment there was a 13% decrease in the experimental diet compared to controls. Over 8 years the primary endpoint of myocardial infarction and sudden death from ischemia were reduced in the experimental diet group (control 67 vs experimental diet 45). The difference in the primary end point of the study-sudden death or myocardial infarction was not statistically significant but when these data were pooled with those for cerebral infarction and other secondary end points, the totals were 96 in the control group and 66 in the experimental group; P=0.01. Fatal atherosclerotic events occurred in 70 patients in the control group and 48 in the experimental group (P<0.05). For all primary and secondary end points the incidence rates were 47.7% and 31.3% for the control and experimental groups respectively (P= 0.02).

 

The Finnish Mental Hospital Study was carried out in two mental hospitals. One hospital was switched to a diet low in SFA and cholesterol and relatively high in PUFA, while the other hospital continued the usual hospital diet (46-48). After 6 years the type of diet was reversed in each hospital. The individuals in this study were hospitalized men between 34 to 64 years of age and women age 44 to 64 years. During the study individuals were removed from the study and others added to the study cohort. The serum cholesterol level on the usual diet was 268mg/dL while on the low SFA diet the serum cholesterol level was 226mg/dL. The incidence of CVD was consistently much lower during the low SFA diet periods than during the normal-diet periods but detailed comparisons are difficult due to the lack of randomization of individuals and the adding and removal of individuals during the study leading to only 36% of the men and 20.6% of the women completing both periods of the study. Nevertheless, this study provides evidence of the benefit of a diet low in SFA and cholesterol and enriched in PUFA.

 

The Sydney Diet Heart Study was a randomized controlled trial conducted from 1966 to 1973 that evaluated the effects of increasing linoleic acid from safflower oil (PUFA ~ 15% of calories) in place of SFA (<10% of calories) in men aged 30-59 years with a history of coronary artery disease (49). Participants were randomized to the dietary intervention group (n=221) or a control group with no specific dietary instruction (n=237). Baseline cholesterol levels were ~280mg/dL and decreased to 267mg/dL in the control group and 244mg/dL in the diet intervention group. Compared with the control group, the intervention group had an increased risk of all-cause mortality (17.6% v 11.8%; P=0.051), cardiovascular mortality (17.2% v 11.0%; P=0.037), and mortality from coronary heart disease (16.3% v 10.1%; P=0.036) over the 5 years of the trial. The reason for the increase in mortality is not clear but the safflower oil margarine substitute for animal fats may have contained trans fatty acids, which could have increased CVD.

 

The DART trial was a multicenter trial in men less than 70 years of age with a diagnosis of an acute myocardial infarction (50). There were several different dietary approaches used in this trial but the one of interest reduced fat intake to 30% of total energy and increased the PUFA/SFA ratio to 1.0 (n=1018) vs. no advice (n=1015). The fat advice group reduced SFA from 15% to 11% of total calories, increased PUFA from 7% to 9%, and increased carbohydrate intake from 44% to 46%. Cholesterol levels were reduced by 3.6% (baseline 252mg/dL) in the diet advice group. During the 2-year trial the number of cardiovascular events were similar in the diet group vs. no advice group.

 

The Minnesota Coronary Survey was a 4.5-year, randomized trial that was conducted in six Minnesota state mental hospitals and one nursing home and included 4,393 men and 4,664 women (51). The trial compared the effects of the usual diet (18% SFA, 5% PUFA, 16% monounsaturated fatty acid (MUFA), 446 mg dietary cholesterol per day) versus a low SFA and cholesterol treatment diet (9% SFA, 15% PUFA, 14% MUFA, 166 mg dietary cholesterol per day). The mean duration of time on the diets was 384 days, with 1,568 subjects consuming the diet for over 2 years. The baseline serum cholesterol level was 207 mg/dL, falling to 175 mg/dL in the treatment group and 203 mg/dL in the control group. No differences between the treatment and control groups were observed for cardiovascular events, cardiovascular deaths, or total mortality, perhaps due to the relatively short duration of this study.

 

The Women’s Health Initiative trial randomized 19,541 postmenopausal women 50-79 years of age to the diet intervention group and 29,294 women to usual dietary advice (52). The goal in the diet intervention group was to reduce total fat intake to 20% of calories and increase intake of vegetables/fruits to 5 servings/day and grains to at least 6 servings/day. Fat intake decreased by 8.2% of energy intake in the intervention vs the comparison group, with small decreases in SFA (2.9%), MUFA (3.3%), and PUFA (1.5%) with increased consumption of vegetables, fruits, and grains. LDL-C levels were reduced by 3.55 mg/dL in the intervention group while levels of HDL-C and TGs were not significantly different in the intervention vs comparison groups. The dietary intervention did not significantly decrease CVD. In fact, in the women with pre-existing CVD there was an increase in cardiovascular events with diet therapy.

 

Summary of Dietary Randomized Controlled Trials 

 

In reviewing these randomized controlled trials, it appears that the dietary studies that produce a long-term decrease in plasma cholesterol levels resulted in a reduction in cardiovascular events (Oslo Diet-Heart Study, soya-bean trial, Los Angeles Veterans Administration Center, Finnish Mental Hospital Study) while the dietary studies that did not produce a long-term decrease in plasma cholesterol levels failed to demonstrate a reduction in CVD. The baseline plasma cholesterol levels in the positive studies tended to be high and allowed for a robust cholesterol lowering with dietary manipulation. Additionally, as will presented in the next section the greater the reduction in SFA in the diet the greater the decrease in TC and LDL-C levels and many of the positive studies were carried out in an era when the content of SFA in the diet was high. Additionally, studies in non-human primates have also demonstrated that reducing SFA intake reduces atherosclerosis (53,54).

 

These results correspond very nicely with the large number of trials demonstrating that using a variety of different pharmacologic agents that lower plasma cholesterol levels results in a decrease in cardiovascular events (55). In an analysis comparing cholesterol lowering with diet vs. drug therapy it was observed that a similar decrease in cardiovascular events occurred adjusting for the magnitude of cholesterol lowering (56). Thus, it would appear that diets that decrease dietary SFA and thereby lead to a significant decrease in plasma cholesterol levels for an extended period of time have benefits on CVD with the caveat that there is not an increase in other nutrients that will adversely affect other parameters thereby negating the beneficial effects of decreasing SFA. For example, an increase in dietary simple sugars for SFA could lead to an increase in TG levels with negative effects.

 

REVERSAL OF ATHEROSCLEROSIS TRIALS

 

Two studies have examined the effect of decreasing dietary SFA on atherosclerotic lesions.

 

The St Thomas’ Atherosclerosis Regression Study (STARS) determined the effect of decreasing dietary saturated fat in the diet (n=26) vs. usual diet (n=24) in men less than 66 years of age with a plasma cholesterol greater than 234mg/dL referred for coronary angiography to investigate angina pectoris or other findings suggestive of coronary heart disease (57). In the diet group total fat intake was reduced to 27% of dietary energy, saturated fatty acid content to 8-10% of dietary energy, and dietary cholesterol to 100 mg/1000 kcal; omega-6 and omega-3 polyunsaturated fatty acids were increased to 8% of dietary energy, and plant-derived soluble fiber intake was increased to the equivalent of 3-6 g polygalacturonate/1000 kcal. During the trial LDL-C levels were 163mg/dL in the diet intervention group vs.182mg/dL in the usual diet group. Additionally, TGs decreased in the diet intervention group (206mg/dL to 165mg/dl) with no change in TG levels in the usual diet group. After approximately 3 years coronary angiography revealed that the percentage of patients who showed progression of coronary narrowing was significantly reduced by the dietary intervention (usual diet 46% vs, dietary intervention 15%), whereas the percentage who showed an increase in luminal diameter rose significantly (usual diet 4% vs. dietary intervention 38%). While the number of cardiovascular events was small, they were significantly reduced in the dietary intervention group (usual diet 36% vs dietary intervention 11%; p< 0.05). Finally, the improvement in angiographic appearance correlated with LDL-C levels.

 

The Lifestyle Heart Trial was a one year randomized, controlled trial to determine whether lifestyle changes affect coronary atherosclerosis in patients with angiographically documented coronary artery disease (58). Patients were assigned to the lifestyle group (low-fat vegetarian diet, stopping smoking, stress management training, and moderate exercise) (n= 22) or a usual-care control group (n=19). The lifestyle diet contained approximately 10% of calories as fat PUFA/SFA ratio greater than 1), 15-20% protein, and 70-75% predominantly complex carbohydrates. Cholesterol intake was limited to 5 mg/day or less. In the lifestyle group LDL-C decreased from 153mg/dL to 96mg/dl (37% decrease) whereas in the usual care group LDL-C decreased from 168mg/dL to 159mg/dL. Patients in the lifestyle group reported a 91% decrease in the frequency of angina, a 42% decrease in the duration of angina, and a 28% decrease in the severity of angina. In contrast, patients in the usual care group reported a 165% increase in the frequency of angina, a 95% increase in the duration of angina, and a 39% increase in the severity of angina. In the lifestyle group regression of coronary atherosclerosis occurred in 18 of the 22 patients (82%) whereas in the usual care group progression of coronary atherosclerosis occurred in 10 of 19 patients (53%).

 

These two regression trials provide strong support for the results observed in the randomized cardiovascular outcome studies described above i.e., that lowering LDL-C levels by decreasing dietary SFA can reduce atherosclerosis and cardiovascular events.

 

Effect of Dietary Saturated Fatty Acids on Lipid Levels          

 

It should be recognized that when one increases or decreases a particular macronutrient in the diet there needs to be a reciprocal change in another macronutrient to maintain caloric balance.

The effect of substituting PUFA, MUFA, or carbohydrates (CHO) for SFA is shown in table 1. Note that this table shows the effect of replacing 5% of energy from SFA for the indicated dietary component. Thus, going from a diet where 15% of the calories is from SFA to a diet where 10% of the calories is from SFA is estimated to lower LDL-C levels from 6 to 9mg/dL depending on which dietary component replaces the SFA. To keep this decrease in LDL-C in perspective it is estimated that a 40mg/dL decrease in LDL-C induced by statin therapy will result in an approximate 20% decrease in cardiovascular events over a 5 year period of time but the lifetime benefits of a 10 mg/dL decrease in LDL-C due to genetic variants will result in a 16–18% decrease in cardiovascular events (59). The effect on TGs is dependent on the dietary component replacing SFA with CHO resulting in a large increase in TG levels. One should note that there is also a decrease in HDL-C with replacement of SFA (table 2).

 

Table 2. Effect of Decreasing Dietary Saturated Fatty Acids on Lipid Levels

Dietary Component

LDL-C (mg/dL)

TGs (mg/dL)

HDL-C (mg/dL)

PUFA

−9.0    

-2.0

-1.0

MUFA

-6.5

+1.0

-6.0

CHO

-6.0

+9.5

-2.0

PUFA- polyunsaturated fatty acids; MUFA- monounsaturated fatty acids; CHO- carbohydrates.

Effects on lipoprotein lipids of replacing 5% of energy from SFA with the 5% of energy from the specified dietary component. Table adapted from references (26,60).

 

SFA in the diet predominantly increases LDL-C levels, predominantly larger, cholesterol-enriched LDL, with modest increases in HDL-C (60,61). As expected, Apo B and apo AI levels also increase (60). These effects are observed in both men and women (60). The effect of a decrease or increase in SFA intake on lipids and lipoproteins is linear with a consistent effect on serum lipids and lipoproteins across a wide range of SFA intakes (60). Of note the effects of decreasing SFA intake was observed even when the SFA intake was already less than 10% of the daily energy intake. Most studies have suggested that replacement of SFA with carbohydrate or unsaturated fat modestly increases Lp(a) but the results have varied from study to study with replacement of SFA with unsaturated fat from particular food sources such as nuts showing no increase in Lp(a) (62).

 

Individual SFA have diverse biological and cholesterol-raising effects with chain length of SFA playing an important role in determining the effect on lipid and lipoprotein levels. The most commonly consumed SFA are palmitic acid (16:0; major source: vegetable oil, dairy, and meat), stearic acid (18:0; meat, dairy, and chocolate), myristic acid (14:0; dairy and tropical oil, particularly coconut oil) and lauric acid (12:0; dairy and tropical oil). A meta-analysis of 60 controlled trials by Mensink et al. reported an increase in LDL-C and HDL-C concentrations by isocaloric replacement of carbohydrates with palmitic, myristic, and lauric acids (63). As expected, apolipoprotein B and A-I also increase (60,64). Myristic and palmitic acids increased LDL-C and HDL-C levels to a similar extent, whereas lauric acid had the largest LDL-C- and HDL-C-raising effect (63,65). Stearic acid did not increase LDL-C levels (63,65).The lack of an association between stearic acid and changes in LDL-C levels has been linked to a slower and/or less efficient absorption as well as desaturation of stearic acid to oleic acid (66). Compared with carbohydrates, an increased intake of lauric, myristic, palmitic or stearic acid lowered TG levels (63,65). For a specific individual many factors including lifestyle factors such as overall dietary composition and physical activity, clinical conditions such as obesity, insulin resistance and hypertriglyceridemia, as well as genetic factors may modify these responses.

 

MECHANISM FOR THE INCREASE IN LDL-C

 

Dietary SFA have been shown to decrease hepatic LDL receptor activity, protein, and mRNA levels and this results in a decrease in the clearance of circulating LDL leading to increased LDL-C levels (67,68). Additionally, the decrease in LDL receptors could result in an increase in the conversion of intermediate density lipoproteins to LDL rather than clearance by the liver (i.e., LDL production is enhanced).

 

SFA have been shown to decrease the formation of cholesterol esters, a reaction catalyzed by the enzyme acyl CoA:cholesterol acyltransferase (ACAT) (68). Free cholesterol in the endoplasmic reticulum is the primary regulator of the activation of sterol receptor binding protein (SREBP), which translocates to the nucleus and enhances the transcription of the LDL receptor (69). Elevated levels of cholesterol in the endoplasmic reticulum prevents the activation of SREBP (69). When free cholesterol is esterified into cholesterol esters it no longer prevents the activation of SREBP and the up-regulation LDL receptor expression. Thus, SFA by decreasing the formation of cholesterol esters and increasing free cholesterol may lead to the down-regulation of LDL receptor expression (68).

 

DIETARY MONOUNSATURATED AND POLYUNSATURATED FATTY ACIDS

 

Olive oil, canola oil, peanut oil, safflower oil, sesame oil, avocados, peanut butter, and many nuts and seeds are major sources of MUFA (table 3). Soybean oil, corn oil, sunflower oil, some nuts and seeds such as walnuts and sunflower seeds, tofu, and soybeans are major sources of PUFA (table 3). Omega-3-fatty acids, eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6), are mostly found in fish and other seafood, while another omega-3 fatty acid, alpha-linolenic acid (ALA, 18:3) is found mostly in nuts and seeds such as walnuts, flaxseed, and some vegetable oils such as soybean and canola oils. The body is capable of converting ALA into EPA and DHA but the conversion rates are low.

 

Table 3. Fat Composition of Oils, Lard, Butter, and Margarine

Type of Oil

SFA (%)

MUFA (%)

PUFA (%)

Corn oil

13.6

28.97

57.43

Safflower oil (linoleic)

6.51

15.1

78.4

Canola oil

7.46

64.1

28.49

Almond oil

8.59

73.19

18.22

Olive oil

14.19

74.99

10.82

Soybean oil

16.27

23.69

60.0

Sesame oil

14.85

41.53

43.62

Sunflower oil (linoleic)

10.79

20.42

68.8

Avocado oil

12.1

73.8

14.11

Peanut oil

17.77

48.58

33.65

Palm oil

51.57

38.7

9.73

Coconut oil

91.92

6.16

1.91

Lard

41.1

47.23

11.73

Butter

68.1

27.87

4.0

Margarine (soft)

20

47

33

Margarine (hard)

80

14

6

U.S. Department of Agriculture

 

Effect of Dietary Monounsaturated and Polyunsaturated Fatty Acids on Cardiovascular Disease

 

MONOUNSATURATED FATTY ACIDS

 

Many meta-analyses, but not all, have failed to demonstrate that MUFA intake reduces cardiovascular events (29,33,35,70). However, one meta-analysis and the Nurses’ Health Study and Health Professionals Follow-Up Study, two very large observational studies, found that MUFA when delivered from plant sources was protective but MUFA from other sources was not protective from developing cardiovascular events (71,72).

 

The PREDIMED a randomized controlled outcome trial employing a Mediterranean diet (increased MUFA) reduced the incidence of major CVD (73-75). In this multicenter trial, carried out in Spain, over 7,000 individuals at high risk for developing CVD 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. In the patients assigned to the Mediterranean diets there was 29% decrease in the primary composite end point (myocardial infarction, stroke, and death from CVD), which was primarily due to a decrease in strokes. The Mediterranean diet resulted in a small but significant increase in HDL-C levels and a small decrease in both LDL-C and TG levels (76). The changes in lipids were unlikely to account for the beneficial effects of the Mediterranean diet on CVD.

 

The Lyon Diet Heart Study randomized 584 patients who had a myocardial infarction within 6 months to a Mediterranean type diet vs usual diet (77,78). The oils recommended for salads and food preparation were rapeseed and olive oils exclusively. Additionally, they were also supplied with a rapeseed (canola) oil-based margarine. There was a marked reduction in events in the group of patients randomized to the Mediterranean diet (cardiac death and nonfatal myocardial infarction rate was 4.07 per 100 patient years in the control diet vs.1.24 in the Mediterranean diet; p<0.0001). Lipid levels were similar in both groups in this trial (77).

 

The CORDIOPREV study was a single center randomized trial that compared a Mediterranean diet to a low-fat diet in 1,002 patients with cardiovascular disease (79). 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 has not been reported.

 

The results of these three randomized trials indicate that a Mediterranean diet enriched in plant MUFA reduce the risk of CVD. It is likely that the beneficial effects of the Mediterranean diet on CVD is mediated by multiple mechanisms with alterations in lipid levels making only a minor contribution. It should be noted that in addition to an increase in MUFA the diet also includes low to moderate red wine consumption, high consumption of whole grains and cereals, low consumption of meat and meat products, increased consumption of fish, and moderate consumption of milk and dairy products. As in many dietary studies it is difficult to change a single variable and therefore the interpretation of which factor or factors account for the benefits is difficult to untangle.

 

POLYUNSATURATED FATTY ACIDS

 

Recent meta-analyses of the effect of PUFA on cardiovascular events in observational studies have demonstrated either no effect or a modestly lower risk of CVD and mortality (80-84). Randomized trials are described in the section on saturated fats and CVD and describe the results of replacing SFA with PUFA. It appears that dietary PUFA has a neutral effect on CVD except in the circumstances where it replaces SFA and results in a sustained decrease in plasma cholesterol levels leading to a decrease in cardiovascular events.

 

OMEGA-3-FATTY ACIDS

 

As discussed in detail in the chapter entitled “Triglyceride  Lowering Drugs” numerous randomized controlled trials of the effect of low dose omega-3-fatty acids (approximately ≤1 gram/day) on CVD have been published and the bulk of the evidence indicates no benefit (85). The effect of pharmacologic doses of omega-3-fatty acids (≥1.8 grams/day) on cardiovascular outcomes is discussed in the chapter entitled “Triglyceride  Lowering Drugs” (85).

 

Effect of Dietary Monounsaturated and Polyunsaturated Fatty Acids on Lipid Levels

 

Table 4 shows the effect of substituting PUFA or MUFA for carbohydrates on LDL-C, HDL-C, and TG levels. Both PUFA and MUFA decrease LDL-C and TGs but PUFA induces a greater decrease (60). Both PUFA and MUFA increase HDL-C levels (60).

 

Table 4. Effect of Decreasing Dietary Carbohydrate on Lipid Levels

Dietary Component

LDL-C (mg/dL)

TGs (mg/dL)

HDL-C (mg/dL)

PUFA

-4.3

-9.2

1.2

MUFA

-1.8

-6.6

1.6

PUFA- polyunsaturated fatty acids; MUFA- monounsaturated fatty acids;

Effects on lipoprotein lipids of replacing 5% of energy from carbohydrates with the 5% of energy from the specified dietary component. Table adapted from reference (60).

 

In a meta-analysis of 14 studies no significant differences in TC, LDL-C, or HDL-C  levels were observed when diets high in MUFA or PUFA were compared directly (86). TG levels were modestly but consistently lower on the diets high in PUFA (P = .05) (86).

 

While high dose omega-3-fatty acids (3-4 grams/day) lower TG levels, lower doses (≤1 gram/day) have minimal effects on lipid levels (85).

 

MECHANISM FOR THE DECREASE IN LDL-C

 

Unsaturated fatty acids increase hepatic LDL receptor activity, protein, and mRNA abundance, which will increase the clearance of LDL from the circulation (67,68). Unsaturated fatty acids are a preferred substrate for ACAT and thereby result in an increase in cholesterol ester formation and a decrease in free cholesterol in the liver (68). A decrease in hepatic free cholesterol will result in the up-regulation of LDL receptor expression leading to a decrease in LDL-C levels. PUFA also increase membrane fluidity leading to an increase in the ability of LDL receptors to bind LDL (67). Additionally, the increase in LDL receptors could result in a decrease in the conversion of intermediate density lipoproteins (IDL) to LDL due to increased uptake of IDL by the liver (i.e., LDL production is decreased).

 

DIETARY TRANS FATTY ACIDS

 

The two major sources of dietary trans fatty acids (TFA) are those that occur naturally in meat and dairy products as a result of anaerobic bacterial fermentation in ruminant animals and those formed during the partial hydrogenation of vegetable fat (the fatty acids in vegetable oils have cis double bonds) (87). Partial hydrogenation and the formation of TFA converts the liquid vegetable oil into a solid form at room temperature allowing for ease of use in food products and increased shelf life (87,88). TFA acids were widely used in baked products, packaged snack foods, margarines, and crackers (88). With the recognition of the adverse effects of TFA the use of partial hydrogenated oils in food products has markedly diminished World-wide and in the US is no longer allowed.

 

Effect of Trans Fatty Acids on Cardiovascular Disease

 

A meta-analysis by de Souza and colleagues of 5 studies with 70,864 participants found that the relative risk of coronary heart disease mortality disease was increased with dietary TFA (1.28; p=0.003) (34). Similarly, the relative risk of coronary heart disease was also increased (1.21; p<0,001) (34). Another meta-analysis by Chowdhury and colleagues of 5 studies with 155,270 participants found that the relative risk of coronary events was increased with higher intake of TFA (RR 1.16; CI 1.06-1.27) (33). It has been estimated that a 2 percent increase in energy intake from TFA was associated with a 23 percent increase in the incidence of coronary heart disease (88). Thus, observational studies have consistently demonstrated that an increase in dietary TFA increase the risk of CVD. Clearly it would not be ethical to carry out randomized trials of the effect of TFA acids on CVD.

 

Effect of Trans Fatty Acids on Lipid Levels

 

The effect of replacing SFA, MUFA or PUFA with TFA acids is shown in table 5. TFA increase LDL-C levels and decrease HDL-C levels. Of note TFA increase LDL-C even when substituting for SFA. There appears to be a nearly linear relationship between TFA intake and LDL-C concentration, but this relationship does not seem to exist between TFA intake and HDL-C (89). HDL-C seems to be lowered significantly by TFA only when intake is >2% to 4% of the total energy intake (89). TFA also increases TG and Lp(a) levels (88). Additionally, dietary TFA increases small dense LDL and the increase correlates with the quantity of TFA in the diet (90). 

 

Table 5. Effect on Lipids of Replacing Various Fatty Acids with Trans Fatty Acids

Dietary Component

LDL-C (mg/dL)

HDL-C (mg/dL)

SFA

2.0

-2.0

PUFA

11.5

-1.3

MUFA

9.5

-1.5

SFA- saturated fatty acids; PUFA- polyunsaturated fatty acids; MUFA- monounsaturated fatty acids. All results are statistically significant (P<0.05) except the increase in LDL-C with SFA replacement. Effects on lipoprotein lipids of replacing 5% of energy from various fatty acids with 5% of the energy from TFA. Table adapted from reference (88).

 

Replacing carbohydrates with TFA results in an increase in LDL-C and apo B and no change in HDL-C, apo AI, or TG levels (63).

 

RUMINANT TRANS FATTY ACID

 

A key question now that TFA derived from partial hydrogenation of vegetable fat in the diet have been markedly reduced is whether ruminant derived TFA which are present in milk, butter, cheese, and beef have harmful effects similar to industrial created TFA. It is important to note that ruminant derived TFA have a different composition with ruminant TFA being enriched in vaccenic acid, which is the predominant TFA, and conjugated linoleic acid (89,91). Also the quantities of ruminant TFA ingested is much lower than the quantities of industrial TFA ingested (89). In an analysis of a large number of studies of the effect of ruminant and industrial TFA on lipid levels it was observed that the effect of ruminant TFA on LDL-C and HDL-C was similar but slightly less than that of industrial TFA (the difference was not significant) (91). Whether the low quantities of ruminant TFA in the diet will influence the risk of CVD is unknown (89) but a meta-analysis of 4 observational trials did not find a link between ruminant-TFA intake (increments ranging from 0.5 to 1.9 g/day) and the risk of CHD (RR=0.92; CI 0.76-1.11; P=0.36) (92). Another meta-analysis also did not find a link between ruminant TFA and CVD (34). 

 

MECHANISM FOR THE LIPID EFFECTS OF TRANS FATTY ACIDS

 

The mechanism for the increase in LDL-C levels by dietary TFA is thought to be due to decreased LDL-Apo B catabolism without a change in LDL-Apo B production (87,93). The decrease in HDL-C induced by TFA has been attributed to an increase in HDL Apo A-I catabolism without a significant change in HDL apoA-1 production rate (87,93). Additionally, TFA increases CETP activity which could increase the transfer of cholesterol esters from HDL to LDL thereby contributing to the decreased HDL-C levels and increased LDL-C levels (94).

 

DIETARY CHOLESTEROL

 

The primary food sources of dietary cholesterol are egg yolks, shrimp, beef, pork, poultry, cheese, and butter with the top five food sources being eggs and mixed egg dishes, chicken, beef, burgers, and cheese (table 6) (95). In the US the typical cholesterol intake varies from 50 to 400mg per day with a mean of 293 mg/day (348 mg/day for men and 242 mg/day for women) (96).

 

Table 6. Cholesterol Content of Food

Food

mg per 100 grams

Egg

373

Butter

215

Shrimp

125

Cheese

108

Beef

90

Chicken

88

Pork

80

Ice Cream

47

 

Effect of Dietary Cholesterol on Cardiovascular Disease

 

In reviews of prospective observational studies an association between dietary cholesterol and CVD has not been clearly demonstrated with some studies reporting an association and others no association (97,98). Most of these studies did not adjust for the amount and types of fatty acids consumed, which could influence the results as foods containing large amounts of cholesterol are also rich in SFA. Dietary cholesterol was not associated with cardiovascular risk among >80,000 nurses and 43,000 male health care professionals after adjusting for energy intake, PUFA, trans fatty acid, and SFA intake (99,100).

 

Most foods that contain cholesterol also contain significant amounts of SFA. An exception are eggs which contain significant amounts of cholesterol and only small amounts of SFA (95). It is therefore of interest to examine the effect of egg consumption on CVD. In an analysis of 7 cohort studies no association between egg intake and coronary heart disease was observed and egg intake may be associated with a reduced risk of stroke (101). A recent meta-analysis of 23 prospective studies with 1,415,839 individuals and a median follow-up of 12.28 years also found that increased consumption of eggs was not associated with increased risk of CVD (102). Other meta-analyses and reviews have also not demonstrated a consistent link between eggs and CVD (98,103-105). However, a recent very large meta-analysis with 3,601,401 participants with 255,479 events showed that the consumption of 1 additional 50-g egg daily was associated with a very small increase in CVD risk (pooled relative risk, 1.04; 95% CI 1.00-1.08) (106). Thus, eggs have either no effect or a very small effect on CVD that can be seen only in very large studies.

 

There appears to be no randomized studies of the effect of decreasing cholesterol intake on CVD. Do recognize that the studies of decreasing dietary SFA intake described earlier also result in a decrease in cholesterol intake. Thus, at this time there is very limited data linking dietary cholesterol intake with an increased risk of CVD. 

 

Effect of Dietary Cholesterol on Lipid Levels   

 

In a meta-analysis of fifty-five studies with 2,652 subjects the predicted change in LDL-C levels for an increase of 100 mg dietary cholesterol per day adjusted for dietary fatty acids ranged from 1.90mg/dL to 4.58 mg/dL depending upon the model employed (107). An increase of 200mg dietary cholesterol per day increased LDL-C levels from 3.80mg/dL to 6.96mg/dL. It should be noted that the effect of dietary cholesterol levels is greater the higher the LDL-C level (107). For a baseline LDL-C level of 100, 125, 150, and 175 mg/dL the predicted increase in LDL-C for a change in dietary cholesterol of 100mg is 2.7, 3.6, 4.6, and 5.5 mg/dL respectively (107). While the absolute increase is greater if the LDL-C level is higher the percentage increase is similar. Moreover, cholesterol feeding does not alter number of LDL particles – instead it increases the cholesterol content of the LDL particles leading to the formation of large buoyant LDL (108).

 

The effect of dietary cholesterol on HDL-C levels differs in males and females. In men an increase of 100mg of dietary cholesterol results in a 0.30 to 1.44mg/dL decrease in HDL-C levels while in women this results in a 0.50 to 1.61 increase in HDL-C levels (107). Dietary cholesterol does not impact TG or VLDL cholesterol levels (97). 

 

Approximately 15-25% of the population have an increased response to dietary cholesterol with greater increases in LDL-C levels (i.e., sensitive or hyper-responders), while the majority respond minimally (i.e., non-sensitive or hypo-responders) (109). An intake of 100 mg/day dietary cholesterol leads to a 3-4-fold difference in LDL-C concentration between hyper- and hypo-responders (an increase of 2.84 mg/dL vs. 0.76 mg/dL (110). The mechanism for the increase in cholesterol absorption in hyper-responders is unknown. On average 50% (typical range 40-60%) of dietary cholesterol is absorbed but this varies from person to person (111). A high-cholesterol diet leads to significant increases in non-HDL-C levels in insulin-sensitive individuals but not in lean or obese insulin-resistant subjects whereas HDL-C levels increased in all 3 groups (112). The above observations demonstrate the variable response of lipid and lipoprotein levels that can occur in response to dietary manipulations and emphasize how the response of an individual can be variable.

 

MECHANISM FOR THE INCREASE IN LDL-C

 

The increase in LDL-C levels by dietary cholesterol is due to a decrease in hepatic LDL receptors (111). Cholesterol absorbed by the small intestine is packaged into chylomicrons which deliver dietary cholesterol to the liver (111). This increases hepatic cholesterol levels which down-regulates the expression of LDL receptors leading to a decrease in the clearance of LDL from the circulation (111). Additionally, the decrease in LDL receptors could result in an increase in the conversion of intermediate density lipoproteins to LDL rather than clearance by the liver (i.e., LDL production is enhanced).

 

DIETARY CARBOHYDRATES

 

Carbohydrates (CHO) can be divided into high-quality CHO, for example fruits, legumes, vegetables, and whole grains, or low-quality CHO, which include refined grains (such as white bread, white rice, cereal, crackers, and bakery desserts), starches (potatoes), and added sugars (sugar-sweetened beverages, candy). The high-quality CHO are typically enriched in fiber and have a low glycemic index/glycemic load (i.e., are slowly absorbed and thus do not rapidly increase plasma glucose levels). The low-quality CHO have a high glycemic index and load and rapidly increase plasma glucose levels.

 

Effect of Dietary Carbohydrates on Cardiovascular Disease

 

OBSERVATIONAL STUDIES

 

When SFA is replaced by CHO there is no reduction in CVD whereas replacement of SFA with high quality CHO may be beneficial (27,37,38). A study by Jakobsen and colleagues found that replacing SFA with CHO with a low-glycemic index value is associated with a lower risk of myocardial infarction whereas replacing SFA with CHO with a high-glycemic index values is associated with a higher risk of myocardial infarction (113). Meta-analyses and reviews of the association of glycemic index with CVD have varied with some showing an association of low glycemic index with CVD and others reporting no link (114,115). Two very large studies found that a diet with a high glycemic index was associated with an increased risk of cardiovascular disease (116,117). It should be noted that in the largest study the relative risk for CVD was relatively modest (RR 1.15; 95% CI 1.11-1.19) (117). An increase in cardiovascular morbidity and mortality was associated with an increase in added sugar intake (118-121). Hazard ratios were 1.30 (95% CI- 1.09-1.55) and 2.75 (95% CI-1.40-5.42), respectively, comparing participants who consumed 10.0% to 24.9% or 25.0% or more calories from added sugar with those who consumed less than 10.0% of calories from added sugar (118). Additionally, in the Health Professionals Follow-up Study participants in the top quartile of sugar-sweetened beverage intake had a 20% higher relative risk of coronary heart disease than those in the bottom quartile (RR=1.20; 95% CI- 1.09-1.33) after adjustment for multiple risk factors (122).

 

RANDOMIZED CONTROLLED TRIALS

 

Three of the randomized trials described above in the SFA and CVD section provide information on the role of CHO on CVD. The British Medical Research Council studied 252 men after a myocardial infarction aiming to reduce total fat from 41% to 22% of calories and maintaining total fat at 41% in the control group (41). The type of fat was similar in the high- and low-fat groups, mainly saturated fat from dairy products and meat. It is likely that the decrease in fat calories was substituted by an increase in CHO calories. The type of CHO that replaced the SFA was not specified but the authors indicated that there was a marked increase in sugar intake in the low- fat diet group. There was no difference between the two groups in cardiovascular events during the 5 years of the trial. The DART study decreased SFA which were substituted with PUFA and CHO (50). During the 2-year trial cardiovascular events were similar in the decreased SFA vs. PUFA and CHO group. Finally, the Women’s Health Initiative trial randomized 19,541 postmenopausal women 50-79 years of age to the diet intervention group and 29,294 women to usual dietary advice (52). The goal in the diet intervention group was to reduce total fat intake to 20% of calories and increase intake of vegetables/fruits to 5 servings/day and grains to at least 6 servings/day (i.e., CHO}. Fat intake decreased by 8.2% of energy intake in the intervention vs the comparison group, with small decreases in SFA (2.9%), MUFA (3.3%), and PUFA (1.5%) fat with increased consumption of CHO. The dietary intervention did not significantly decrease CVD even though the CHO recommended was high quality CHO. These randomized studies do not provide support for a benefit of substituting CHO for fat in reducing CVD. Of particular note is the Women’s Health Initiative which decreased fat intake and increased high quality CHO and observed no cardiovascular benefits in contrast to the results of observational studies.

 

Effect of Dietary Carbohydrates on Lipids

 

Replacing SFA, MUFA, or PUFA with CHO results in an increase in TGs and a decrease in HDL-C levels (60,63). Replacing SFA with CHO results in a decrease in LDL-C while replacing MUFA or PUFA with CHO results in an increase in LDL-C (see tables 2 and 4) (60,63). In addition, dietary CHO increases the quantity of small dense LDL particles (123). The consumption of moderate amounts of fructose or sucrose (40-80 grams/day) in healthy young men was sufficient to increase small dense LDL levels (124). The effect of increasing dietary CHO on Lp(a) levels has been variable (62).

 

Conversely, decreasing CHO in the diet and adding fat results in an increase in LDL-C and HDL-C levels and a decrease in TG levels (125). In a meta-analysis of eleven randomized controlled trials with 1,369 participants comparing low fat/high CHO diet to high fat/low CHO diet it was found that the high fat/low CHO led to an increase in LDL-cholesterol (6.24mg/dL; 95 % CI 0.12- 12.9) and HDL-C (5.46mg/dL; 95% CI 3.51- 7.41) compared with subjects on the low fat/high CHO diets (126). The high fat/low CHO decreased TG levels (-22.9mg/dL; 95 % CI -13.4- -32.6 (126). Another meta-analysis of 23 randomized controlled trials also found that a high fat/low CHO diet increased LDL-C and HDL-C levels and decreased TG levels (127). These studies nicely demonstrates that a high fat diet will increase LDL-C and HDL-C levels while a high CHO diet will increase TG levels and decrease HDL-C levels.

 

COMPARISON OF DIFFERENT CARBOHYDRATES ON LIPIDS

 

A meta-analysis of twenty-eight randomized controlled trials comparing low- with high glycemic index diets (1,272 participants) reported that low glycemic index diets significantly decreased LDL-C levels by 6.2mg/dL; P < 0.0001) with no effect on HDL-C or TGs (128). The decrease in LDL-C was related to the amount of fiber and/or phytosterols in the low glycemic diet (see Fiber and Plant Sterols/Stanols section below).

 

High fructose corn syrup (HFCS) has become a major source of fructose intake (HFCS made for beverages contains 55% fructose and 45% glucose). Because sucrose and HFCS are major contributors to total CHO intake there has been interest in the effect of fructose, glucose, and sucrose on lipid levels. In a comparison of isocalorically substituting starch for glucose, fructose, or sucrose there were no difference in TG levels but there was a decrease in LDL-C (approximately 7.8mg/dL) (129).

 

A meta-analysis by Te Morenga and colleagues examined the effect of the addition of sugar on lipid levels. In studies where energy intake was isocaloric, sugar intake increased TG levels by 11.7mg/dL, LDL-C by 6.6mg/dL, and HDL-C by 0.8mg/dL (130). In a similar meta-analysis by Fattore and colleagues an isocaloric substitution of free sugars for complex CHO increased TGs by 8.3mg/dL, LDL-C by 7.1mg/dL, and HDL-C by 1.3mg/dL (131). The increase in TG and LDL-C levels were larger in the trials where greater amounts of free sugar were employed.

 

In a meta-analysis of adding fructose to the diet there was no significant effect on fasting TG levels at dietary fructose < 100 grams per day but at higher amounts fructose increased fasting TG levels (132). Fructose is more likely to have adverse effects on lipids when intake is high and/or when caloric excess is present. For example, in young healthy individuals, a 2-week intervention with 25% of energy requirements as HFCS or fructose sweetened beverages resulted in significant increases in fasting LDL-C, small dense LDL particles, non-HDL-C, apo B, and HDL-C and postprandial TGs (133). High quantities of glucose did not affect LDL-C, non-HDL-C, Apo B, HDL-C, or postprandial TG levels but did increase fasting TG levels (133).

 

Thus, the effect of CHO on lipids can vary depending upon the particular type of CHO studied (table 7). In the case of glycemic index (complex CHO) and starch vs sugar some of the difference in lipid response could be due to other dietary constituents (i.e., fiber, phytosterols).

 

Table 7. Summary of the Effect of Different Carbohydrates on Lipid and Lipoproteins

Comparisons

Effect on Lipids and Lipoproteins

Low GI vs. High GI

High GI increases LDL-C

Sugar vs. Starch

Sugar increases LDL-C

Sugar vs. Complex CHO

Sugar increases LDL-C and TGs

Fructose vs. Glucose

Fructose increases LDL-C and HDL-C and postprandial TGs

Sugar- sucrose, glucose, or fructose

 

MECHANISM OF THE EFFECTS OF CARBOHYDRATES ON LIPIDS

 

Dietary CHO promote hepatic de novo fatty acid synthesis by providing substrate for fatty acid synthesis (Figure 1). This is particularly the case when there is caloric excess. Additionally, the glucose provided by dietary CHO stimulates insulin secretion which also increases hepatic fatty acid synthesis. The increase in fatty acid synthesis in the liver enhances TG synthesis which promotes VLDL formation and secretion leading to an increase in plasma TG levels. 

 

Figure 1. Carbohydrates stimulate VLDL production by stimulating de novo fatty acid synthesis.

 

Fructose is more potent at increasing de novo fatty acid synthesis than glucose. Small quantities of fructose in the diet are metabolized in the small intestine to glucose and organic acids and do not affect systemic metabolism while high quantities of fructose can escape intestinal metabolism and are delivered to the liver (134). In the liver fructose but not glucose activates SREBP1c and ChREBP leading to the increased expression of the genes that synthesize fatty acids stimulating hepatic lipogenesis (134,135). Additionally, fructose metabolism in the liver is not inhibited providing an unlimited supply of fructose carbons for lipogenesis. In contrast, the first steps in glucose metabolism can be inhibited and thus the utilization of glucose for lipogenesis is regulated (134). In addition, fructose inhibits fatty acid oxidation whereas glucose does not (135). These differences in the metabolism of fructose and glucose in the liver explain the increased ability of fructose to stimulate hepatic lipogenesis and the enhanced formation and secretion of VLDL. In the addition to increased VLDL production fructose does not stimulate the secretion of insulin, which plays a key role in stimulating lipoprotein lipase activity and the clearance of TG rich lipoproteins. The failure of dietary fructose to induce an increase in lipoprotein lipase activity may lead to a decrease in the clearance of TG rich lipoproteins compared to dietary glucose, which stimulates insulin secretion.

 

The elevation in TG rich lipoproteins in turn may have effects on other lipoproteins (25) (Figure 2). Specifically, cholesterol ester transfer protein (CETP) mediates the equimolar exchange of TGs from TG rich VLDL and chylomicrons for cholesterol from LDL and HDL (25). The increase in TG rich lipoproteins per se leads to an increase in CETP mediated exchange, increasing the TG content and decreasing the cholesterol content of both LDL and HDL particles. This CETP-mediated exchange underlies the commonly observed reciprocal relationship of low HDL-C levels when TG levels are high and the increase in HDL-C when TG levels decrease. The TG on LDL and HDL are then hydrolyzed by hepatic lipase and lipoprotein lipase leading to the production of small dense LDL and small HDL particles.

 

Figure 2. The effect of hypertriglyceridemia on LDL and HDL.

 

DIETARY PROTEIN

 

Effect of Dietary Protein on Cardiovascular Disease

 

In a meta-analysis of 10 studies with 425 ,781 participants intake of plant protein was associated with a decrease in cardiovascular mortality (136). Other meta-analyses have also found that intake of plant proteins was associated with a lower risk of cardiovascular mortality (137-139). In some but not all studies animal protein intake increased the risk of cardiovascular mortality (136-139). The differences in outcomes observed between plant and animal proteins could be due to increased intake of SFA with animal proteins and increased fiber and phytosterol intake with plant proteins. 

 

Effect of Dietary Protein on Lipids

 

Because a high protein diet is often associated with an increase in SFA intake it is important to control for this variable in determining the effect of dietary protein on lipid levels. In a meta-analysis of a high vs. low protein diets in individuals on a low-fat diet no difference in LDL-C, HDL-C, or TG levels were observed (140). In another meta-analysis of 24 trials with 1,063 participants that compared isocaloric diets matched for fat intake but with differences in protein and CHO  intakes no differences in LDL-C and HDL-C levels were observed but TG levels were decreased in the high protein diet group (-20.2mg/dL) (141). Greater weight loss and decreased CHO intake in the high protein diet group likely contributed to the decrease in TGs. In a meta-analysis where fat intake was not controlled the high protein diet was associated with an increase in HDL-C levels and a decrease in TG levels (142). It is obviously difficult to determine the effect of dietary protein on lipid levels as other dietary constituents are changing (SFA, CHO) and secondary effects induced by changes in protein intake (weight loss) could influence lipid levels.

 

DIETARY FIBER

 

Dietary fiber are non-digestible carbohydrates including non-starch polysaccharides, cellulose, pectins, hydrocolloids, fructo-oligosaccharides and lignin. Fiber is found mostly in fruits, vegetables, whole grains, nuts, seeds, psyllium seeds, beans, and legumes. There are two main types of dietary fiber; soluble and insoluble. The main sources of soluble fiber are fruits and vegetables and insoluble fiber are cereals and whole-grain products. Most high fiber foods contain both soluble and insoluble fiber. A summary of the fiber content of some foods is shown in tables 8-11.

 

Table 8. Fiber Content of Selected Vegetables

Vegetables

 

Serving

Size

Total Fiber/ Serving (g)

Soluble Fiber/ Serving (g)

Insoluble Fiber/ Serving (g)

Cooked vegetables

Turnip

½ cup

4.8

1.7

3.1

Peas, green, frozen

½ cup

4.3

1.3

3.0

Okra, frozen

½ cup

4.1

1.0

3.1

Potato, sweet, flesh

½ cup

4.0

1.8

2.2

Brussels sprouts

½ cup

3.8

2.0

1.8

Asparagus

½ cup

2.8

1.7

1.1

Kale

½ cup

2.5

0.7

1.8

Broccoli

½ cup

2.4

1.2

1.2

Carrots, sliced

½ cup

2.0

1.1

0.9

Green beans, canned

½ cup

2.0

0.5

1.5

Beets, flesh only

½ cup

1.8

0.8

1.0

Tomato sauce

½ cup

1.7

0.8

0.9

Corn, whole, canned

½ cup

1.6

0.2

1.4

Spinach

½ cup

1.6

0.5

1.1

Cauliflower

½ cup

1.0

0.4

0.6

Turnip

½ cup

4.8

1.7

3.1

Raw vegetables

Carrots, fresh

1, 7 ½ in. long

2.3

1.1

1.2

Celery, fresh

1 cup chopped

1.7

0.7

1.0

Onion, fresh

½ cup chopped

1.7

0.9

0.8

Pepper, green, fresh

1 cup chopped

1.7

0.7

1.0

Cabbage, red

1 cup

1.5

0.6

0.9

Tomato, fresh

1 medium

1.0

0.1

0.9

Mushrooms, fresh

1 cup pieces

0.8

0.1

0.7

Cucumber, fresh

1 cup

0.5

0.2

0.3

Lettuce, iceberg

1 cup

0.5

0.1

0.4

Adapted from Anderson JW. Plant Fiber in Foods. 2nd ed. HCF Nutrition Research Foundation Inc, PO Box 22124, Lexington, KY 40522, 1990.

 

Table 9. Fiber Content of Selected Legumes

Legumes (cooked)

Serving Size

Total Fiber/ Serving (g)

Soluble Fiber/ Serving (g)

Insoluble Fiber/ Serving (g)

Kidney beans, light red

½ cup

7.9

2

5.9

Navy beans

½ cup

6.5

2.2

4.3

Black beans

½ cup

6.1

2.4

3.7

Pinto beans

½ cup

6.1

1.4

4.7

Lentils

½ cup

5.2

0.6

4.6

Black-eyed peas

½ cup

4.7

0.5

4.2

Chick peas, dried

½ cup

4.3

1.3

3

Lima beans

½ cup

4.3

1.1

3.2

Adapted from Anderson JW. Plant Fiber in Foods. 2nd ed. HCF Nutrition Research Foundation Inc, PO Box 22124, Lexington, KY 40522, 1990.

 

Table 10. Fiber Content of Selected Fruits

Fruits

Serving

Size

Total Fiber/ Serving (g)

Soluble Fiber/ Serving (g)

Insoluble Fiber/ Serving (g)

Apricots, fresh w/skin

4

3.5

1.8

1.7

Raspberries, fresh

1 cup

3.3

0.9

2.4

Figs, dried

1 ½

3

1.4

1.6

Mango, fresh

½ small

2.9

1.7

1.2

Orange, fresh

1 small

2.9

1.8

1.1

Pear, fresh, w/skin

½ large

2.9

1.1

1.8

Apple, red, fresh w/skin

1 small

2.8

1

1.8

Strawberries, fresh

1 ¼ cup

2.8

1.1

1.7

Plum, red, fresh

2 medium

2.4

1.1

1.3

Applesauce, canned

½ cup

2

0.7

1.3

Apricots, dried

7 halves

2

1.1

0.9

Peach, fresh, w/skin

1 medium

2

1

1

Kiwifruit, fresh

1 large

1.7

0.7

1

Prunes, dried

3 medium

1.7

1

0.7

Grapefruit, fresh

½ medium

1.6

1.1

0.5

Blueberries, fresh

¾ cup

1.4

0.3

1.1

Cherries, black, fresh

12 large

1.3

0.6

0.7

Banana, fresh

½ small

1.1

0.3

0.8

Melon, cantaloupe

1 cup cubed

1.1

0.3

0.8

Watermelon

1 ¼ cup cubed

0.6

0.4

0.2

Grapes, fresh w/skin

15 small

0.5

0.2

0.3

Raisins, dried

2 tbsp

0.4

0.2

0.2

Adapted from Anderson JW. Plant Fiber in Foods. 2nd ed. HCF Nutrition Research Foundation Inc, PO Box 22124, Lexington, KY 40522, 1990.

 

Table 11. Fiber Content of Grains

Food

Serving

Size

Total Fiber/ Serving (g)

Soluble Fiber/ Serving (g)

Insoluble Fiber/ Serving (g)

Wheat bran

½ cup

12.3

1.0

2.7

Barley, pearled, cooked

½ cup

3.0

0.8

2.2

Oatmeal, dry

⅓ cup

2.7

1.4

11.3

Bread, pumpernickel

1 slice

2.7

1.2

1.5

Wheat flakes

¾ cup

2.3

0.4

1.9

Bread, rye

1 slice

1.8

0.8

1.0

Bread, whole wheat

1 slice

1.5

0.3

1.2

Rice, white, cooked

½ cup

0.8

trace

0.8

Bread, white

1 slice

0.6

0.3

0.3

Adapted from Anderson JW. Plant Fiber in Foods. 2nd ed. HCF Nutrition Research Foundation Inc, PO Box 22124, Lexington, KY 40522, 1990.

 

Effect of Dietary Fiber on Cardiovascular Disease

 

Several meta-analyses have demonstrated that an increase in total fiber, soluble fiber, and insoluble fiber are associated with a decrease in cardiovascular events (143-148). The greater the intake of fiber the greater the reduction in risk of cardiovascular events.  

 

Effect of Dietary Fiber on Lipids

 

In a meta-analysis of randomized controlled trials the effect of fiber on lipid levels was evaluated (149). Increased dietary fiber decreased total cholesterol (TC) (−7.8mg/dL; 95% CI −13.3 to −2.3), LDL-C (−5.5mg/dL; 95% CI −8.6 to −2.3), and HDL-C levels ( −1.17mg/dL; 95% CI −2.34 to −0.39) (149,150), There was no change in TG levels. A meta-analysis of randomized controlled studies of whole-grain foods vs non-whole-grain foods found that the whole-grain diet lowered LDL-C (-3.51mg/dL; P < 0.01) and TC levels (-4.68mg/dL; P < 0.001) compared with the non-whole grain foods (151). HDL-C and TG levels were not significantly altered by the whole grain diet. Moreover, 3.4 g of psyllium (Metamucil), a soluble fiber, decreased LDL-C with no significant effects on HDL-C or TGs (152,153). In a meta-analysis of 28 randomized trials psyllium lowered LDL by 12.9mg/dL (P < 0.00001) (154). A mean reduction in LDL-C concentrations of about 1.1 mg/dL can be expected for each g of water-soluble fiber in the diet (155,156).

 

MECHANISM OF EFFECT OF FIBER ON LDL-C

 

Fiber is thought to decrease cholesterol absorption by the small intestine (157,158). This leads to a decrease in cholesterol content of chylomicrons and a reduction in the delivery of cholesterol to the liver. The decrease in cholesterol in the liver upregulates LDL receptors resulting in a decrease in plasma LDL-C levels. Fiber may also decrease small intestinal absorption of bile acids which will lead to the increased utilization of hepatic cholesterol for the synthesis of bile acids (159). This will also decrease hepatic cholesterol levels inducing an increase in the expression of LDL receptors lowering plasma LDL-C levels. Finally, colonic fermentation of dietary fiber with production of short-chain fatty acids, such as acetate, propionate, and butyrate, is postulated to inhibit hepatic cholesterol synthesis contributing to a decrease in LDL-C levels (159). 

 

PLANT STEROLS AND STANOLS (PHYTOSTEROLS)

 

Plant sterols and plant stanols (phytosterols) are naturally occurring constituents of plants and are found in vegetable oils, such as corn oil, soybean oil, and rapeseed oil and cereals, nuts, fruits, and vegetables. The intake of plant sterols and stanols is about 200–400 mg/day. The most commonly occurring phytosterols in the human diet are β-sitosterol, campesterol, and stigmasterol. Higher intakes can be achieved by consuming a vegetable-based diets such as a vegetarian diet (400-800mg/day) or by consuming food products enriched with plant sterols or stanols (for example margarines or yogurt). If using foods enriched in phytosterols it is best to take them with main meals to enhance their effectiveness. High doses of phytosterols can affect the absorption of fat-soluble vitamins. The plant sterol and stanol content of different foods is shown in table 12.

 

Table 12. Plant Sterol and Stanol Contents in Different Foods

Food item

Plant Sterols

(mg/100 g)

Plant Stanols

(mg/100 g)

Vegetable oils

Corn oil

686-952

23-33

Rapeseed oil (canola oil)

250-767

2-12

Soybean oil

221-328

7

Sunflower oil

263-376

4

Olive oil

144-193

0.3-4

Palm oil

60-78

Traces

Cereals

Corn

66-178

-

Rye

71-113

12-22

Wheat

45-83

17

Barley

80

2

Millet

77

-

Rice

72

3

Oats

35-61

1

Vegetables

Broccoli

39

2

Cauliflower

18-40

Traces

Carrot

12-16

Traces

Lettuce

9-17

0.5

Potato

7

0.6

Tomato

7

1

Fruits and berries

Avocado

75

0.5

Passion fruit

44

Not detected

Raspberry

27

0.2

Orange

24

Not detected

Apple

12-18

0.8

Banana

12-16

Not detected

Adapted from Piironen V and Lampi AM (160)

 

Effect of Phytosterols on Cardiovascular Disease

 

There is minimal data on the effect of phytosterols on cardiovascular events. From the effect on LDL-C levels one would anticipate that phytosterols would reduce CVD.

 

Effect of Phytosterols on Lipids

 

Plant sterols or plant stanols at a dose of 3 grams per day lowers LDL-C by approximately 12% (161). Higher doses do not dramatically further lower LDL-C levels and lower doses have less effect on LDL-C (for example 2 grams/day lowers LDL-C by 8%) (161).  HDL-C levels are not affected by plant sterols or stanols but TG levels decrease modestly (~6%) with a greater absolute reduction in individuals with high TG level (percent change is the same) (162). To achieve these high doses consuming food products enriched is phytosterols is necessary.

 

MECHANISM OF EFFECT OF PHYTOSTEROLS ON LDL-C

 

Plant sterols or plant stanols reduce LDL-C levels by competing with cholesterol for incorporation into micelles in the gastrointestinal tract, resulting in decreased cholesterol absorption (163). This leads to the decreased delivery of cholesterol to the liver and the up-regulation of LDL-receptor expression lowering LDL-C levels.

 

SUMMARY OF THE EFFECT OF DIETARY CONSTITUENTS ON LIPID LEVELS

 

A summary of the major effects of dietary constituents on lipid levels is shown in table 13, typically under isocaloric feeding conditions in short-term feeding studies. Dietary SFA, TFA, and cholesterol increase LDL-C levels whereas CHO increases TG levels. MUFA, PUFA, fiber and phytosterols decrease LDL-C and TFA decrease HDL-C levels.

 

Table 13. Summary of the Effect of Dietary Constituents on Lipid and Lipoproteins

SFA

Increase LDL-C and modest increase HDL-C

MUFA and PUFA

Decrease LDL-C

TFA

Increase LDL-C and decrease HDL-C

Cholesterol

Increase LDL-C

CHO

Increase TGs, increase greater with simple sugars particularly fructose

Fiber

Decrease LDL-C

Phytosterols

Decrease LDL-C

 

EFFECT OF SPECIFIC FOODS ON CARDIOVASCULAR DISEASE

 

There are a large number of observational trials linking various foods with either an increased or decreased risk of CVD. A large meta-analysis by Micha et al reported that fruits, vegetables, beans/legumes, nuts/seeds, whole grains, fish, yogurt, fiber, seafood omega-3 fatty acids, polyunsaturated fats, and potassium were associated with a decreased risk of CVD while unprocessed red meats, processed meats, sugar-sweetened beverages, and sodium were associated with an increased risk of CVD (164). A similar meta-analysis by Bechthold et al found that whole grains, vegetables and fruits, nuts, and fish consumption were associated with a decrease in CVD while red meat, processed meat, and sugar sweetened beverage consumption was associated with an increase in CVD (165). Note, as discussed in the introduction, observational studies have limitations and cannot be assumed to indicate cause and effect. Additional one can find other meta-analyses that reach different conclusions than the results described above. For example, a meta-analysis by Zeraatkar et al and a meta-analysis by Vernooij et al reached the conclusion that meat and processed meat were not associated with a significant increase in CVD (20,166). Thus, one needs recognize that while these studies can suggest beneficial and harmful effects of eating certain foods more definitive studies are required to be certain. For a detailed analysis of the limitations of observational dietary studies see articles by Ioannidis and Nissen (1,2).

 

Only a single randomized trial has examined the effect of specific foods on CVD events. The DART trial randomized men with an acute myocardial infarction to at least two weekly portions (200-400 g) of fatty fish (mackerel, herring, kipper, pilchard, sardine, salmon, or trout) (n=1015) or no dietary advice (n=1018) (50). After approximately 2 years total mortality was significantly lower (RR 0.71; CI 0.54-0.93) in the fish advice group than in the no fish advice group, due to a reduction in ischemic heart disease deaths. There were no significant differences in ischemic heart disease events (RR 0.84; CI 0.66-1.07). In a separate portion of the DART trial there was also a group of men with an acute myocardial infarction randomized to increased intake of cereal fiber (18 grams/day) (n=1017) vs. no dietary advice (n=1016). No reduction in cardiovascular events was seen in the cereal fiber group.

 

Clearly addition randomized trials are required to determine the true benefits of specific foods on cardiovascular events.

 

EFFECT OF SPECIFIC FOODS ON LIPID LEVELS

 

In contrast to the paucity of randomized controlled trials on the effect of specific foods on cardiovascular disease there are an abundance of studies on the effect of specific foods on lipid and lipoprotein levels. Given the large number of studies in many instances I will cite the results of meta-analyses to provide the reader with the typical effects that are observed. It should be noted that the effect of specific foods on lipid and lipoprotein levels tend to be small and therefore the results can be inconsistent from study to study.

 

Nuts and Seeds

 

The most consumed edible tree nuts are almonds, hazelnuts, walnuts, pistachios, pine nuts, cashews, pecans, macadamias, and Brazil nuts. Peanuts are botanically groundnuts or legumes, and are widely considered to be part of the nut food group. Nuts are generally consumed as snacks (fresh or roasted), in spreads (peanut butter, almond paste), or as oils or baked goods. Seeds come in all different sizes, shapes and colors. Popular seeds include flax, pumpkin, sunflower, chia, sesame, and mustard seeds.

 

Nuts and seeds are rich in MUFAs, such as oleic acid and in PUFAs, such as linoleic acid and alpha-linolenic acid (ALA). They also contain small amounts of SFA. Almonds, cashews, hazelnuts, pistachios and macadamian nuts have a high MUFA content (>50%) content when compared with other nuts. For other nuts (e.g., Brazil nuts, pine nuts, and walnuts) the PUFA content is high (>50%), while peanuts and pecans have been found to contain relatively high levels of both MUFA and PUFA (table 14). Nuts are a good source of dietary fiber, ranging from 4-11 g/100 g and phytosterols.

 

Table 14. Nutrient Composition of Nuts

Nuts

 

PUFA

(g/100 g)

MUFA

(g/100 g)

SFA

(g/100 g)

Fiber

(g/100 g)

Walnuts

47.2

8.9

6.1

6.7

Peanuts

15.6

24.4

6.3

8.8

Pistachios

13.7

23.8

5.6

10.3

Almonds

12.3

31.6

3.8

12.5

Hazelnuts

7.9

45.7

4.5

9.7

Cashews

7.8

23.8

7.8

3.3

Pecans

21.6

40.8

6.2

9.6

Macadamias

1.5

58.9

12.1

8.6

 

Consumption of nuts and seeds lower TC and LDL-C levels in healthy subjects or patients with moderate hypercholesterolemia (167-172). Nuts had no significant or minimal effect on increasing HDL-C. The benefits of nuts and seeds vary depending on the type, nutrient composition, and quantity of nuts and seeds consumed. Studies have noted that the estimated cholesterol lowering effect of nuts was greater in individuals with higher initial values of LDL-C and in those with a lower baseline BMI (169).

 

Walnuts: A meta-analysis on the effect of walnuts on lipid levels that included 365 participants showed a decrease in LDL-C (9.2 mg/dL), while HDL-C or TG were not significantly affected (173). In another meta-analysis that analyzed 1,059 participants with a walnut enriched diet LDL-C was lowered by 5.5 mg/dL (174).

 

Almonds: A meta-analysis of 15 studies with 534 participants found that almonds decreased LDL cholesterol (5.8 mg/dL; 95% CI: -9.91, -1.75 mg/dL) and apo B (6.67 mg/dL; 95% CI: -12.63, -0.72 mg/dL) (175). Triglycerides, apo A1, and lipoprotein (a) showed no differences.

 

Pistachio nuts: A meta-analysis of twelve randomized studies reported that pistachio nuts decreased LDL-C -3.82 mg/dL (95% CI, -5.49 to -2.16) and TG -11.19 mg/dL (95% CI, -14.21 to -8.17) levels without effecting HDL-C levels (176).

 

A meta-analysis by Houston et al analyzed the effect of a variety of different nuts on lipid levels (table 15) (177). They found that in general nuts lowered LDL-C and minimally lowered TG levels but had no effect on HDL-C levels. A meta-analysis found that whole flaxseed reduced TC and LDL-C by 6 and 8 mg/dL, respectively (178). Thus, both nuts and seeds lower LDL-C levels.

 

Table 15. Effect of Nuts on Lipid Levels

 

Number of analyses

Number of participants

Effect estimate (mmol/L)

95% CI

LDL Cholesterol

Almond

32

2439

-0.15 [-0.22, -0.08]

Brazil nut

4

307

-0.30 [-0.70, 0.11]

Cashew nut

3

432

 0.02 [-0.12, 0.16]

Hazelnut

6

374

-0.01 [-0.15, 0.12]

Macadamia

6

410

-0.11 [-0.27, 0.04]

Mixed nuts

10

791

 0.04 [-0.06, 0.14]

Peanut

10

1021

 0.08 [-0.04, 0.20]

Pecan

6

295

-0.23 [-0.46, 0.00]

Pistachio

12

736

-0.15 [-0.30, 0.00]

Walnut

35

2582

-0.12 [-0.18, -0.06]

Triglycerides

Almond

32

2439

-0.02 [-0.05, 0.02]

Brazil nut

4

307

 0.04 [-0.54, 0.63]

Cashew nut

3

432

-0.02 [-0.11, 0.07]

Hazelnut

5

313

 0.11 [-0.02, 0.25]

Macadamia

5

342

-0.10 [-0.21, 0.00]

Mixed nuts

11

888

-0.01 [-0.07, 0.06]

Peanut

10

1021

-0.09 [-0.16, -0.02]

Pecan

6

295

-0.11 [-0.24, 0.03]

Pistachio

9

498

-0.12 [-0.21, -0.03]

Walnut

35

3109

-0.09 [-0.12, -0.06]

Table based on data from a meta-analysis by Houston et al (177). To convert mmol/L cholesterol to mg/dL multiply by 39 and to convert mmol/L triglycerides to mg/dL multiply by 88.

 

Whole Grains

 

Whole grains include barley, brown rice, buckwheat, bulgur (cracked wheat), millet, oatmeal, and wild rice. Whole grains contain ~80% more dietary fiber than refined grains, as the latter are milled, a process that removes bran and germ. Refined grains include white flour, white rice, white bread, and corn flower. Health benefits ascribed to whole grains are mainly due to the presence of fiber and bran. A meta-analysis of fifty-five trials with 3900 participants comparing various grains found that oat bran was the most effective intervention strategy for lowering LDL-C (- 12.5mg/dL; 95% CI – 17.2 to – 7.4mg/dL) compared with control (179). Oats also reduced LDC (- 6.6mg/dL; 95% CI – 10.9 to 2.73mg/dL). Barley, brown rice, wheat and wheat bran were not effective in improving blood lipid levels compared with controls. Another meta-analysis also found that whole-grain oats decreased LDL-C levels (–16.7 mg/dL; P < 0.0001) (180).

 

Soy Protein

 

Soybeans and soy products as well as supplements contain soy proteins. In a meta-analysis of 43 randomized studies with 2,607 participants the decrease in LDL-C levels reductions for soy protein ranged between −4.2 and −6.7 mg/dL (P<0.006) (181). Numerous other meta-analyses have reported similar decreases in LDL-C (182-187).  In addition, soy protein also decreases TG levels (~2-10mg/dL) and increases HDL-C levels (~1-2mg/dL). Soy protein does not affect Lp(a) levels (188). The amount of soy protein that is recommended for lipid lowering is 25–50 grams per day (189).

 

The decrease in LDL-C is due to the indirect effect of soy protein decreasing the intake of animal protein (SFA and cholesterol) and the intrinsic effects of bioactive compounds in soy protein (190). The intrinsic effect of soy protein might be mediated by phyto-estrogens that could increase levels of HDL-C and TG and decrease levels of LDL-C (189).   

 

Garlic

 

Garlic supplements are available in several different forms, including garlic powder, allicin, aged garlic extract, and garlic oil. Several meta-analyses have shown that garlic lowers TC levels with variable effects on LDL-C, HDL-C, and TG (191-198). Some studies find a decrease in LDL-C and others a decrease in TG levels. The longer the duration of treatment and the higher the baseline TC the greater the effect. In one meta-analysis TC was reduced by 17 ± 6 mg/dL and low-density lipoprotein cholesterol by 9 ± 6 mg/dL in individuals with elevated TC levels (>200 mg/dL) if treated for longer than 2 months (191). In another meta-analysis garlic powder and aged garlic extract were more effective in reducing TC levels, while garlic oil was more effective in lowering serum TG levels (192). In a meta-analysis of garlic administration to patients with diabetes TC decreased 16.9mg/dL, LDL decreased 9.7mg/dL, TG decreased 12.4mg/dL, and HDL-C increased 3.19mg/dL (all p=0.001) (199). Lp(a) levels are not altered by garlic (198).The mechanism by which garlic alters lipid levels is unknown.

 

Tea

 

Green tea contains many catechins (e.g., epigallocatechin-3-gallate) that influence lipid metabolism in animal models and have been shown to upregulate LDL receptors in liver and suppress PCSK9 production (200,201). Epigallocatechin gallate may also interfere with the intestinal absorption of lipids (202). Most but not all meta-analyses have shown that drinking green tea or black tea decreases TC and LDL-C levels with no significant effect on HDL-C or TG levels (203-214). The reduction in LDL-C is approximately 5-10mg/dL.

 

Coffee

 

Coffee contains cholesterol-increasing compounds; diterpenes such as cafestol and kahweol (215,216). The amount of these cholesterol increasing compounds in coffee depends on how the coffee is prepared (215,216). Boiling coffee beans extracts diterpenes due to the prolonged contact with hot water resulting in high concentrations in the coffee whereas brewed filtered coffee because of the short contact with hot water and retention of diterpenes by the filter paper has lower concentrations of diterpenes. Instant coffee has very low levels of diterpenes (216). The concentration of the cholesterol-raising compound cafestol is negligible in drip-filtered, instant, and percolator coffee but high in unfiltered coffee such as French press, Turkish, or Scandinavian boiled coffee. Levels of cafestol are intermediate in espresso and coffee made in a Moka pot.

 

A meta-analysis of 18 trials found that the consumption of unfiltered, boiled coffee dose-dependently increased TC and LDL-C concentrations (23 mg/dL and 14 mg/dL, respectively), while consumption of filtered coffee resulted in only small changes (TC increased by 3 mg/dL and no effect on LDL-C concentration) (217). Additionally, decaffeinated coffee had a smaller effect and the increase in cholesterol levels was greatest in individuals with hypercholesterolemia. Thus coffee, depending upon how it is prepared, can increase TC and LDL-C levels.

 

Chocolate and Cocoa

 

Cocoa is the non-fat component of finely ground cocoa beans that is used to produce chocolate. Cocoa is rich in flavanols which are low‐molecular‐weight monomeric compounds, such as epicatechin or complex higher‐molecular‐weight oligomeric and polymeric compounds (218). The flavanol content in cocoa products can vary greatly and is dependent on the crop type, post‐harvest handling practices, and manufacturer processing techniques. The flavanol content of milk and white chocolate is low or even absent (218).

 

In a meta-analysis of 21 studies with 986 participants very small effects on LDL-C and HDL-C levels were observed (LDL-C 2.7mg/dL decrease; HDL-C 1.2mg/dL increase) with no change in TG levels with chocolate and/or cocoa intake (219). In another meta-analysis there was a decrease in TG levels (-8.8mg/dL), an increase in HDL-C (2.3mg/dL), and a non-significant decrease in LDL-C (-10.1mg/dL) (220). In studies where the epicatechin dose was greater than 100mg per day the decrease in LDL-C levels was greater (5.5mg/dL) (219). Another meta-analysis of 19 studies found that LDL decreased by 3.3mg/dL and HDL-C increased by 1.8mg/dL with cocoa intake (221). A meta-analysis of 10 clinical trials with 320 participants that focused on dark chocolate found a 6.23mg/dl decrease in LDL-C with no significant changes in HDL-C and TG (222). Thus chocolate/cocoa causes a small decrease in LDL-C levels. 

 

Alcohol

 

It is recommended that females consume no more than 1 drink per day of alcohol (equivalent to 15 grams per day) and that males consume no more than 2 drinks per day (equivalent to 30 grams per day). Alcohol has a relatively high caloric level (7 calories/gram).

 

EFFECT OF ALCOHOL ON LIPID LEVELS

 

In a meta-analysis of 25 studies with an average consumption of 40.9 grams of alcohol per day HDL-C concentrations increased by 5.1 mg/dL (223). HDL-C levels increased by 0.122- 0.133 mg/dL per gram of alcohol per day. Consuming 30 grams of alcohol a day would therefore increase HDL-C concentrations by approximately 3.99 mg/dL compared with an individual who abstains (an 8.3% increase from pretreatment values). The increase in HDL-C was observed regardless of sex, duration of study, median age, or beverage type but the increase was greater in individuals with baseline HDL-C < 40mg/dL and who were sedentary. As expected apo A1 levels also increased. In a meta-analysis of 35 studies TG concentrations increased by 0.19 mg/dL per gram of alcohol consumed a day (P=0.001) and 5.69 mg/dL per 30 g consumed per day (5.9% increase over baseline) (223). The increase in TG levels was seen regardless of beverage type and appeared to be greater in males than females.

 

In a more recent meta-analysis of 33 studies with 796 participants HDL-C levels were increased by 3.67mg/dL by alcohol intake (224). Apo A1 levels were also increased but there were no significant differences in TC, LDL-C, TG, or Lp(a) with alcohol intake. The greater the consumption of alcohol the greater the increase in HDL-C levels. When the consumption of alcohol was greater than 60 grams per day (4 drinks) TG levels were also increased (24.4mg/dL).

 

In a meta-analysis of 14 studies, comparing 548 beer drinkers and 532 controls TC levels were significantly higher in the beer drinkers compared to controls (difference 3.52 mg/dL; p<0.001) (225). In a meta-analysis of 18 studies, comparing 626 beer drinkers and 635 controls HDL-C levels were higher in the beer drinkers compared to controls (difference 3.63 mg/dL: p<0.001) (225). This increase in HDL-C levels in beer drinkers were seen in both males and females. LDL-C and TG levels were not significantly different between beer drinkers and controls (LDL-C difference -2.85 mg/dL; p = 0.070; TG difference 0.40 mg/dL; p = 0.089) (225).

 

Genetic factors play a role in the HDL response to alcohol (226). Individuals with an apoE2 allele have greater HDL-C increase and those with an apoE4 allele have a blunted increase in HDL-C with alcohol intake (226).In addition to an increase in HDL-C levels studies have suggested that the ability of HDL to facilitate the efflux of cholesterol from cells is enhanced by alcohol intake (226,227).

 

One should note in the meta-analyses described above alcohol doesn’t appear to have a major impact on TG levels. However, it must be recognized that the amount of alcohol consumed is a key variable (228,229). At low to moderate amounts alcohol has either no effect or might even decrease TG levels (228). However, at high amounts of alcohol intake increases in TG levels are observed (228,229). As noted above one meta-analysis noted that the consumption of 60 grams per day of alcohol increased TG levels (224). Moreover, alcohol consumed with a meal increases and prolongs the postprandial increase in TG levels (228,229). Additionally, genetic factors and the presence of other abnormalities play a role in the TG response to alcohol intake (229). For example, the increase in TG levels after red wine was -4%, 17%, and 33% in individuals with a BMI 19.60-24.45, 24.46- 26.29, and 26.30-30.44, respectively (P = .001) demonstrating that the increase in TG was strongly influenced by BMI (230). Finally, in patients with pre-existing hypertriglyceridemia moderate alcohol intake increased TG levels (338 ± 71 mg/dL to 498 ± 117 mg/dL; P < 0.05) (231).  

 

MECHANISM FOR THE EFFECT OF ALCOHOL ON HDL

 

 The mechanism for the increase in HDL-C levels is likely due to an increased production of apo A1 and A2 (232). Additionally, alcohol inhibits cholesteryl ester transfer protein (CETP) activity, which will also increase HDL-C levels (229).

 

MECHANISM FOR THE EFFECT OF ALCOHOL ON TRIGLYCERIDES

 

Alcohol increases VLDL secretion by the liver (229). The increased production and secretion of VLDL is due to a number of factors including a) alcohol increases lipolysis in adipose tissue and increases the delivery of fatty acids to the liver b) alcohol increases hepatic fatty acid transporters increasing the uptake of circulating fatty acids c) alcohol increases hepatic de novo fatty acid synthesis d) alcohol decreases the beta oxidation of fatty acids in the liver (229,233,234). Together these effects lead to an increased supply of fatty acids in the liver facilitating TG synthesis and the formation and secretion of VLDL.

 

While moderate alcohol intake increases lipoprotein lipase (LPL) activity acute alcohol intake inhibits LPL activity, which could explain the observation that alcohol consumed with a meal increases postprandial TG levels ((228,229).

 

Summary of the Effect of Specific Foods on Lipid Levels

 

Table 16. Major Effects of Specific Foods on Lipid Levels

Nuts and Seeds

Decrease TC and LDL-C

Whole Grains

Decrease LDL-C

Garlic

Decrease TC, LDL-C, TG

Green and Black Tea

Decrease TC and LDL-C

Coffee (depends on method of preparation)

Increase TC, LDL-C, TG

Cocoa and Chocolate

Decrease LDL-C

 

SPECIFIC DIETS

 

The effect of several dietary strategies on lipid levels is discussed below. Randomized controlled trials on the effect of specific diets on cardiovascular outcomes were discussed in earlier sections (saturated fatty acids section and monounsaturated fatty acids section).  

 

Mediterranean Diet

 

Mediterranean diets have an abundance of plant foods, including vegetables, legumes, nuts, fruits, and grains, fish, and low to moderate red wine consumption. Low consumption of meat and meat products and moderate consumption of milk and dairy products is encouraged. In the PREDIMED trial the Mediterranean diet resulted in a small but significant increase in HDL-C levels and a small decrease in both LDL-C and TG levels (76). In the Lyon Diet Heart Study lipid levels were similar in the Mediterranean and usual diet groups (77). The cardiovascular outcome benefits of both of these randomized outcome trials are discussed in the effect of MUFA on CVD section. In a meta-analysis of the effect of a Mediterranean diet on lipid levels little or no change in LDL-C, HDL-C, and TGs was observed (235). Another meta-analysis reported a 4.6mg/dL decrease in LDL-C and a 0.61mg/dL increase in HDL-C (236).

 

Dietary Approach to Stop Hypertension (DASH) Diet

 

The DASH diet promotes the consumption of fruits, vegetables, low-fat dairy products, whole grains, poultry, fish, and nuts and a decrease in the intake of red meat, sweets, sugar-containing beverages, total fat, saturated fat, and cholesterol. In the initial DASH trial total fat and SFA intake was reduced in the DASH diet group (total fat 27% vs. 39% of calories; SFA 6.2% vs. 15% of calories). MUFA and PUFA intake were similar but cholesterol intake was decreased (194mg/day vs 324mg/day). As expected, CHO and fiber intake were increase (CHO 59% vs. 49% of calories; fiber 35grams/day vs. 17grams/day). The DASH diet lowered TC (15.6 to 19.5mg/dL), LDL-C (11.7 to 15.5mg/dL), and HDL-C (3.12 to 3.90mg/dL) (237). TG levels were not significantly affected. In a meta-analysis of twenty studies of the DASH diet reporting data for 1917 participants TC was decreased (7.8mg/dL; P=0.001) and LDL was decreased (3.9mg/dL; p<0.03) (238). HDL-C and TG levels were not significantly altered (238). Similar results were seen in other meta-analyses (239,240).

 

Portfolio Diet

 

The portfolio dietary pattern is a plant-based dietary pattern that includes four cholesterol-lowering foods; a) tree nuts or peanuts, b) plant protein from soy products, beans, peas, chickpeas, or lentils, c) viscous soluble fiber from oats, barley, psyllium, eggplant, okra, apples, oranges, or berries, and d) plant sterols initially provided in a plant sterol-enriched margarine. In a meta-analysis of 5 studies with 439 participants LDL-C was lowered by 17% (28.5mg/dL; p< 0.001) and TGs by 16% (24.6mg/dL; p< 0.001) with no change in HDL-C or weight (241).

 

Nordic Diet

 

The Nordic diet is based on the consumption of different healthy foods such as whole grains, fruits (such as berries, apples, and pears), vegetables, legumes (such as oats, barley, and almonds), rapeseed oil, fatty fish (such as salmon, herring and mackerel), shellfish, seaweed, low-fat choices of meat (such as poultry and game), low-fat dairy, and decreased intake of salt and sugar-sweetened products. In a meta-analysis of 5 studies LDL-C was decreased by 11.7mg/dL (p= 0.013) with no changes in TG or HDL-C levels (242). In another meta-analysis of 6 studies LDL-C was decreased by 10.1mg/dL with no changes in TG or HDL-C levels (243).

 

Ketogenic Diet

 

Low CHO diets can contain variable amounts of CHO. When the CHO levels are very low, they stimulate the formation of ketones. In a typical ketogenic diet CHO contribute <10% of calories (< 50 grams/day), protein approximately 30% of calories, and fat approximately 60% of calories with no restrictions on the type of fat or cholesterol levels. These diets can be high in beef, poultry, fish, oils, various nuts/seeds, and peanut butter, with moderate amounts of vegetables, salads with low-carbohydrate dressing, cheese, and eggs. Fruits and fruit juices, most dairy products with the exception of hard cheeses and heavy cream, breads, cereals, beans, rice, desserts/sweets, or any other foods containing substantial amounts of CHO are avoided.

 

It is well recognized that a ketogenic diet results in an increase in LDL-C levels, which varies depending upon the type of fat ingested, the degree of carbohydrate restriction, the presence of other medical conditions, weight loss on the diet, and genetic background (244). This increase in LDL-C levels is best illustrated in children treated with a ketogenic diet for epilepsy and in healthy individuals on a ketogenic diet (245-250). In some studies HDL-C is also increased (246-249). A meta-analysis of randomized studies in normal-weight adults found that a ketogenic diet increased LDL-C by 42mg/dL and HDL-C by 13.7mg/dL with no significant changes in TG levels (251). It should be noted that the increase in LDL-C is often not observed or is modest in patients with obesity or the metabolic syndrome (252,253).

 

While the typical increases in LDL-C levels observed with a ketogenic diet are relatively modest, recently a series of reports have described marked elevations in LDL-C levels in some patients on a ketogenic diet (253-255). For example, Goldberg et al reported 5 patients with marked increases in LDL-C levels on a ketogenic diet (256). Three patients had LDL-C levels greater than 500mg/dL. Similarly, Schaffer et al described 3 patients in which a very low carbohydrate diet induced LDL-C levels greater then 400mg/dL (257). Finally, Schmidt et al reported 17 patients with LDL-C levels greater than 200mg/dL on a ketogenic diet (258). In these patients there was an average increase in their LDL-C level of 187 mg/dL (258). The elevations in LDL-C levels decrease towards normal with cessation of the ketogenic diet (256-258). It should be noted that most of the patients with marked elevations in LDL-C in response to a ketogenic diet had normal LDL-C levels prior to the dietary change (255).

 

Many of the individuals who develop marked increases in LDL-C on a very low carbohydrate ketogenic diet have low TG levels, elevated HDL-C levels, and are thin (253,255). This phenotype has been called the lean mass hyper-responder (LMHR) phenotype (253,255). LMHR individuals have been defined as having TG <70mg/dL, HDL-C > 80mg/dL, and LDL-C > 200mg/dL (253,255). The mechanism for the marked increase in LDL-C levels is unknown. It may be due to a genetic predisposition in certain individuals (Apo E2/E2 genotype or high polygenic risk score for hypercholesterolemia) (256). Therefore, it is important for clinicians to monitor lipid levels in patients electing to follow a very low CHO/high fat diet.    

 

Comparison of Low Fat vs. Low Carbohydrate Weight Loss Diets

 

Numerous randomized studies have compared the effect of low fat vs. low CHO weight loss diets on lipid levels. In a study by Foster et al 154 obese individuals were randomized to a low-fat diet and 153 obese individuals to a low CHO diet (259). In the low CHO diet during the first 12 weeks of treatment participants were instructed to limit CHO intake to 20 grams/day in the form of low–glycemic index vegetables after which the diet was gradually liberalized. In the low-fat diet participants were instructed to limit energy intake with approximately 55% of calories from CHO, 30% from fat, and 15% from protein. Participants were instructed to limit calorie intake, with a focus on decreasing fat intake. After 6 months weight loss was similar in both diet groups. The effect on lipid levels at 6 months is shown in table 17. As one would expect the low CHO was very effective at lowering TG levels and increasing HDL-C levels while the low-fat diet was very effective at lowering LDL-C levels. The large weight loss in this trial may have contributed to the large reduction in lipid levels. A review of a large number of meta-analyses comparing a low CHO diet vs. low fat weight loss diet similarly described that the low CHO diet lowered TG levels and increased HDL-C and LDL-C levels compared to the low-fat diet (244). Note the increase in LDL-C with the low-CHO diet was blunted in patients with diabetes or pre-diabetes (244). Also, the increase in LDL-C levels is likely to be greater in low CHO diets that are enriched in SFA (244).

 

Table 17. Comparison of Low Fat vs. Low Carbohydrate Weight Loss Diet on Lipid Levels

 

Low Fat Diet

Low Carbohydrate Diet

 

Weight

-11.3kg

-12.2kg

NS

TGs

-24mg/dL

-49mg/dL

P<0.001

LDL-C

-9.5mg/dL

0.5mg/dL

P<0.001

HDL-C

1.0mg/dL

6.2mg/dL

P<0.001

 

Comparison of Vegetarian and Omnivore Diet on Lipid Levels

 

Vegetarian diets exclude all animal flesh. A meta-analysis of 19 studies comparing a vegetarian vs. omnivore diet found that consumption of vegetarian diets resulted in a 12.2mg/dL decrease in LDL-C (p < 0.001) and 3.4mg/dL decrease in HDL-C (p < 0.001) and a nonsignificant increase in TG levels (5.8 mg/dL; P = 0.090) compared with consumption of an omnivorous diet (260). Vegan diets, which exclude all animal products, were associated with larger LDL-C reductions than lacto-ovo vegetarian diets. A meta-analysis of 11 clinical trials comparing a vegetarian vs. omnivore diet observed similar results (LDL‐C decreased 13.3mg/dL ; P<0.001; HDL decreased 3.9mg/dL; P<0.001) (261). It is likely that a decrease in dietary SFA and cholesterol and an increase in dietary fiber and phytosterols account for the differences in a vegetarian and omnivore diets.

 

Comparison of 14 Different Diets on Lipid Levels

 

In a network meta-analysis of 121 eligible trials with 21, 942 overweight or obese patients Ge and colleagues compared the effect of 14 different diets on LDL-C and HDL-C levels (236). The diets could be grouped into low CHO diets (Atkins, South Beach, Zone), moderate macronutrients diets (Biggest Loser, DASH, Jenny Craig, Mediterranean, Portfolio, Slimming World, Volumetrics, Weight Watchers), and low-fat diets (Ornish, Rosemary Conley). The effect of these different diets on LDL-C and HDL-C levels are shown in table 18. It should be noted that despite considerable weight loss the effect of these diets on LDL-C and HDL-C levels was very modest except for the LDL-C lowering seen with the Portfolio diet. Unfortunately, a comparison of the effect of these diets on TG levels was not reported.

 

Table 18. Effect of Different Diets in Comparison with Usual Diet

Diet vs. Usual Diet

Decrease in Weight (Kg)

Change in LDL-C (mg/dL)

Change in HDL-C (mg/dL)

Atkins

5.46

+2.75

-3.41

Zone

4.07

+2.89

+0.33

Dash

3.63

-3.93

+1.90

Mediterranean

2.87

-4.59

+0.61

Paleolithic

5.31

-7.27

+2.52

Low Fat

4.87

-1.92

+2.13

Jenny Craig

7.77

-0.21

+2.85

Volumetrics

5.95

-7.13

+0.13

Weight Watchers

3.90

-7.13

+0.88

Rosemary Conley

3.76

-7.15

+2.04

Ornish

3.64

-4.71

+4.87

Portfolio

3.64

-21.29

+3.26

Biggest Loser

2.88

-3.90

+0.01

Slimming World

2.15

N/A

N/A

South Beach

9.86

+0.64

-3.60

Dietary Advice

0.31

+2.01

+1.71

 Summary of results of popular named diets network meta-analysis for outcomes at six months

 

In a study carried out in a single center the Atkins, Zone, Weight Watchers, and Ornish diets were compared and the effect on TG levels was also reported (262). Table 19 shows the results of this study at 2 months, a period at which dietary compliance was still high. The magnitude of weight loss was similar but the decrease in LDL-C that occurs with weight loss was blunted with a diet that was high in fat (Atkins diet). In contrast HDL-C levels increased with a high fat diet, particularly SFA (Atkins diet) and decreased with a very low-fat diet (Ornish diet). The weight loss induced decrease in TG levels was blunted by a high CHO intake (Ornish diet). These observations confirm and extend the results described above.

 

Table 19. Effect of Different Diets on Lipid Levels

 

Weight (kg)

LDL-C (mg/dL)

HDL-C (mg/dL)

TG (mg/dL)

Atkins

-3.6

1.3

3.2

-32

Zone

-3.8

-9.7

1.8

-54

Weight Watchers

-3.5

-12.1

-0.2

-9.2

Ornish

-3.6

-16.5

-3.6

-0.4

 

Summary

 

While diets can significantly affect lipid levels it should be recognized that the effect is typically relatively modest compared to drug therapy. Whether these modest effects on lipid levels can reduce the risk of CVD has not been tested in randomized controlled trials and given the difficulty of carrying out such long-term diet studies is likely not to be attempted. However, diet therapy can be initiated early in life and has the potential to result in long-term decreases in lipid levels. Given that studies have shown that long-term modest reductions in LDL-C levels can have major effects on the risk of CVD (a 10mg/dL life-long decrease in LDL-C due to polymorphisms in ATP citrate lyase, HMGCoA reductase, LDL receptor, PCSK9, and NPC1L1 resulted in a 16%-18% decrease in cardiovascular events (263)) it is likely that a similar long-term decrease induced by dietary changes would also be effective in decreasing CVD. A life-long 70mg/dL decrease in TG levels due to polymorphisms in the lipoprotein lipase gene resulted in a 23% decrease in coronary heart disease suggesting that long-term decreases in TG levels due to dietary changes would also be beneficial (264). Thus, long-term reductions in lipid levels induced by diet therapy may reduce the lifetime risk of developing CVD.

 

CURRENT DIETARY GUIDELINES

 

Most dietary guidelines recommended to the general population to prevent disease are very similar so I will only present the recommendations of two organizations. A brief summary of the Guidelines for Americans 2020-2025 is shown in table 20 and the guidelines from the American College of Cardiology/American Heart Association are shown in table 21.

 

Table 20. Guidelines for Americans 2020-2025

Recommend

Limit

Vegetables of all types—dark green; red and orange; beans, peas, and lentils; starchy; and other vegetables

Added sugars—Less than 10 percent of calories per day

Fruits, especially whole fruit

Saturated fat—Less than 10 percent of calories per day

Grains, at least half of which are whole grain

Sodium—Less than 2,300 milligrams per day

Dairy, including fat-free or low-fat milk, yogurt, and cheese, and/or lactose-free versions and fortified soy beverages and yogurt as alternatives

Alcoholic beverages—Adults can

choose not to drink or to drink in moderation by limiting intake to 2 drinks or less in a day for men and 1 drink or less in a day for women

Protein foods, including lean meats, poultry, and eggs; seafood; beans, peas, and lentils; and nuts, seeds, and soy products

 

Oils, including vegetable oils and oils in food, such as seafood and nuts

 

Full guideline is available at DietaryGuidelines.gov

 

Table 21. ACC/AHA Dietary Recommendations to Reduce Risk of ASCVD (265)

1. A diet emphasizing intake of vegetables, fruits, legumes, nuts, whole grains, and fish is recommended

2. Replacement of saturated fat with dietary monounsaturated and polyunsaturated fats can be beneficial

3. A diet containing reduced amounts of cholesterol and sodium can be beneficial

4. As a part of a healthy diet, it is reasonable to minimize the intake of processed meats, refined carbohydrates, and sweetened beverages

5. As a part of a healthy diet, the intake of trans fats should be avoided

ASCVD- Atherosclerotic CVD

 

DIETARY RECOMMENDATIONS FOR PATIENTS WITH LIPID DISORDERS

 

Elevated LDL-C

 

The dietary approach to reduce LDL-C levels is to avoid TFA and decrease SFA and cholesterol intake while increasing intake of fiber and phytosterols (266). Additionally, weight loss if appropriate can be helpful in lowering LDL-C levels (266). Certain foods are effective in lowering LDL-C levels such as tree nuts or peanuts, plant protein from soy products, beans, peas, chickpeas, or lentils, and viscous soluble fiber from oats, barley, psyllium, eggplant, okra, apples, oranges, or berries and if possible, can be added to the individual’s diet (236,241). If one combines multiple nutritional changes one can have significant reductions in LDL-C levels (table 22).

 

Table 22. Effect of Multiple LDL-C Lowering Changes on LDL-C Levels

Nutritional Intervention

Estimated LDL-C Decrease

Replace 5% of energy from SFA with MUFA or PUFA

5% to 10%

7.5 grams/day viscous fiber

6% to 9%

2 grams/day plant sterols/stanols

5% to 8%

Replace 30 grams animal protein or CHO with plant protein

3% to 5%

Loss 5% body weight if excess adiposity

3% to 5%

Total Effect

22% to 37%

Table adapted from (267).

 

While diet alone usually does not reduce LDL-C sufficiently it adds to the beneficial effect of cholesterol lowering drugs. In a comparison of LDL-C lowering a low-fat diet alone lowered LDL-C by 5%, a statin alone by 27%, and the combination of low-fat diet plus statin by 32% demonstrating an independent and additive effect of combining diet and lipid lowering medications (268). 

 

Modestly Elevated Triglycerides

 

The dietary approach to reduce TG levels is to reduce CHO intake particularly simple and refined sugars and to avoid or minimize alcohol intake (266). Weight loss if appropriate can be very helpful in lowering TG levels (25,266).

 

Markedly Elevated Triglycerides

 

In patients with marked elevations in TGs due to the Familial Chylomicronemia Syndrome a diet very low in fat is often necessary to prevent episodes of pancreatitis (<10% of calories from fat) (269). In patients with this disorder medium chain TGs may be helpful. In patients with the Multifactorial Chylomicronemia Syndrome who present with markedly elevated TGs (>1000mg/dL) initial dietary treatment should be a very low-fat diet until the TGs decrease. Once the TGs decrease one can initiate the diet described above for individuals with modestly elevated TGs.

 

Elevated Lipoprotein (a)

 

There is no evidence that healthy dietary changes significantly lower Lp(a) levels (62,270) . In fact, it should be noted that reducing SFA intake while decreasing LDL-C levels increases Lp(a) levels (271). In certain patients with high Lp(a) levels one may need to balance the benefits of decreasing LDL-C levels with the risks of increasing Lp(a) levels (271).

 

Effect of Dietary Advice on Lipid and Lipoprotein Levels

 

In a meta-analysis of 44 randomized studies with 18,175 healthy adult participants comparing dietary advise vs. no or minimal advice found that dietary advice reduced total serum cholesterol by 5.9mg/dL (95% CI 2.3 to 9.0) and LDL-C by 6.2mg/dL (95% CI 3.1 to 9.4) with no change in HDL-C and TG levels (272). In a meta-analysis of 7 studies with 1081 participants that compared consultation with a dietician vs. usual care there was no difference in the absolute change in TC, LDL-C, or HDL-C levels but TG levels were decreased by 19.4mg/dL (95%CI -37.8 to -1.8; p=0.03) (273). Similarly, in a meta-analysis of 5 randomized trials in 912 patients with type 2 diabetes found that dietary advice from a dietician vs. usual care resulted in a small decrease in LDL-C (6.6mg/dL) in the group receiving advice from the dietician (274). Finally, as discussed earlier the Women’s Health Initiative trial randomized 19,541 postmenopausal women 50-79 years of age to the diet intervention group and 29,294 women to usual dietary advice (52). The goal in the diet intervention group was to reduce total fat intake to 20% of calories and increase intake of vegetables/fruits to 5 servings/day and grains to at least 6 servings/day. Fat intake decreased by 8.2% of energy intake in the intervention vs the comparison group, with small decreases in SFA (2.9%), MUFA (3.3%), and PUFA (1.5%) with increased consumption of vegetables, fruits, and grains. LDL-C levels were reduced by 3.55 mg/dL in the intervention group while levels of HDL-C and TGs were not significantly different in the intervention vs comparison groups. Taken together these studies illustrate that diet therapy under many circumstances has only modest effects on lipid and lipoprotein levels. Of course, there are studies and individual patients where major reductions in lipid levels occur. For example, in a life style modification study including a vegetarian diet by Ornish and colleagues a marked decrease in LDL-C was observed (153mg/dL decreasing to 96mg/dL) (58). One is most likely to see dramatic effects the greater the change in diet (for example going from a typical Western diet to a vegetarian low-fat diet) and the higher the baseline lipid levels. Patient ability to follow the dietary advise is crucial for success.

 

ACKNOWLEDGEMENTS

 

This work was supported by grants from the Northern California Institute for Research and Education.

 

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Osteoporosis in Men

ABSTRACT

 

While progress has been made, osteoporosis in men is still under-diagnosed and under-treated.  In general, men fracture about 10 years later than women, with large increases in fracture risk after about age 75, although a small number of men may present with vertebral fractures in middle age. There is overlap between secondary causes of osteoporosis and risk factors for primary osteoporosis, but men with fragility fractures or low bone density require evaluation by history and physical examination as well as a short list of laboratory tests. Bone mineral density by dual-energy x-ray absorptiometry remains the best test for diagnosing osteoporosis in men, although opportunistic bone density measurements from CT scans are promising. Clinicians should recommend a comprehensive program of treatment with fall risk reduction, attention to diet and vitamin D status, and pharmacologic treatment. In general, medications that work in women should lead to fewer fractures in men, although there are few studies in men with fracture risk reduction as the primary outcome. Most men with osteoporosis should be treated with oral or intravenous bisphosphonates, but men at very high fracture risk should be considered for initial anabolic treatment. Compared to women, men are more likely to die after hip fracture. The long-term management of men with osteoporosis is based solely on a few studies in women.

 

INTRODUCTION

 

Despite new information and even some attention in popular publications, osteoporosis in men remains under-appreciated, under-diagnosed, and under-treated. While the evidence base for evaluation and management of male osteoporosis will always be less than that of female osteoporosis, there is enough information available to identify those men at highest risk, evaluate them thoroughly, and treat them with a program that will reduce osteoporotic fractures.  Nonetheless, there are many impediments to quality care at all stages: recognition, diagnosis, assessment, and management (both short- and long-term). In this chapter, the challenges for the primary care and specialty clinician will be addressed with the purpose of providing an approach to reducing osteoporotic fracture in men.

 

DEFINITION, CLASSIFICATION, AND EPIDEMIOLOGY OF MALE OSTEOPOROSIS

 

Definitions of Osteoporosis in Men

 

In an older adult, regardless of gender, a fall from a standing position should not result in a fracture. Hence, one definition of osteoporosis is just such a fracture. By consensus, some fractures are considered osteoporotic; and others may or may not be, even if they occur with minimal trauma. For the most commonly used fracture risk calculator FRAX (see below), low trauma fractures of the spine, hip, forearm (radius and ulna), and humerus are considered osteoporotic. Pelvic, rib, and sternal fractures may also be osteoporotic. Most authorities do not count skull or digital fractures, and ankle fractures are the most controversial. Interestingly, in a study (1) of older men (MrOS, see below), any fracture after age 50 increased the risk of osteoporotic hip fracture, when combined with bone mineral density (BMD) measured by dual energy x-ray absorptiometry (DXA). The above is compatible with the standard definition of osteoporosis as compromised bone strength leading to increased risk of low trauma fracture (2).  A more operational diagnosis relies on DXA measurements, with a BMD T-score of -2.5 or worse in the spine or hip serving as the diagnosis of osteoporosis (3). This means that the patient’s BMD is at least 2.5 standard deviations below the normal young mean. As the BMD decreases, the fracture risk rises markedly. In men there has been great controversy about the normative database that should be used for the calculation of T-scores. Based on the fact that men and women fracture at similar (overlapping but not quite identical) absolute bone density measurements (in g/cm2), several major osteoporosis organizations, including the International Society for Clinical Densitometry (ISCD), recommend use of the young, white female normative database for all T-score calculations (4). The reader is directed to a discussion of this subject (5), and more details about DXA are discussed below. While the man with a T-score of -2.5 or less is clearly at the highest risk for fracture, more fractures occur in men with T-scores between -1 and -2.5, what is called osteopenia or low bone mass. The reason for this is that there are many more men in this category. For example, baseline DXA testing was done in the Rotterdam study (6), a large, long-term observational study. In men, 29% of hip fractures were in those with osteoporosis by DXA, 64% had osteopenia, and 7% had normal bone density. DXA measures bone quantity, and fracture risk is also determined by bone quality, which is impossible to measure definitively with current clinical tools. Thus, fracture risk calculators have been established, based on epidemiological data, to reflect bone quality and add to the predictive power of DXA. The most commonly used fracture risk calculator is FRAX (7), available online as www.sheffield.ac.uk/FRAX/. FRAX calculates the 10-year risk of hip fracture and of major osteoporotic fracture (MOF) based on the femoral neck BMD in g/cm2 plus a series of risk factors: age, sex, previous fracture, parental hip fracture, current smoking, having more than 3 alcoholic drinks daily, rheumatoid arthritis, exposure to systemic glucocorticoid drugs, and secondary osteoporosis. It also can be calculated using the body mass index (BMI) as a surrogate for femoral neck BMD.  While some studies (e.g. 8) suggest that FRAX works better in women than men, the calculator has been adopted internationally. There are other risk calculators, such as the Garvan nomogram (9), which unlike FRAX includes falls as a risk factor for determining fracture risk. It is interesting to note that at age 50, a man has a risk of experiencing an osteoporotic fracture of 13 to 25%, depending on the population studied. A much smaller percentage of men over age 50 have T-scores of -2.5 or worse, although the proportion increases with age. In a study of NHANES data, osteoporosis was defined from FRAX calculations: a 10-year hip fracture risk of > 3% or MOF of > 20% (10,11,12). Using this definition 16% of American men at age 50 and 46% at age 80 met criteria for osteoporosis, much more similar to actual incidences of osteoporotic fracture (12). There is some evidence (e.g. 13) that treating women who meet this fracture risk criterion respond to current osteoporosis treatment. There are, to my knowledge, no studies in men that show that diagnosing osteoporosis in a man by this method and treating him with standard medication leads to fewer fractures. Indeed Ensrud (14) has reported that men with osteoporosis by DXA have the best response to osteoporosis treatment, compared to those with better BMD.  However, as will be described below, studies of osteoporosis medications in men have almost always used the more liberal male normative database for the calculation of the T-score and accepted men with osteopenia plus a history of an osteoporotic fracture for inclusion. In these studies, such men responded to the treatment regimen with improvements in the standard surrogates for fracture. It is also interesting that the Rotterdam study (6) mentioned above also used sex-specific normative databases for the DXA diagnosis of osteoporosis. Had they used the female database for all participants, the group with osteoporosis by DXA at baseline would have accounted for an even smaller percentage of the hip fractures observed. A practical approach to the diagnosis is provided below.

 

There are other potential tools for determining fracture risk. For example, FRAX Plus (15) will be released soon. It will add falls, diabetes mellitus, and other risk factors to the fracture risk prediction. Trabecular Bone Score (16) can be derived from DXA of the spine. It reflects bone architecture and can be added to FRAX calculations. It is thought to be a reflection of bone quality (17). The reader is directed to the chapters on osteoporosis in women, which will include other methods to better quantify fracture risk.

 

Epidemiology of Osteoporosis in Men

 

Fractures in men occur about 10 years later in life than in women (18). Men, with generally bigger bones, have more to lose over time. In addition, men do not undergo the rapid increase of bone turnover that occurs with menopause and the marked drop in estradiol secretion.  Instead, it is well-accepted that the loss of sex steroids in men is a much more gradual process (19), and it is interesting to note that, with aging, BMD is more closely associated with serum bioavailable estradiol levels than with any serum measure of testosterone (20). Nonetheless, in middle-aged men presenting usually with vertebral fractures or low spine BMD by DXA, one of the causes of osteoporosis earlier in life is hypogonadism. This type of osteoporosis is analogous to what Riggs and Melton labelled postmenopausal osteoporosis in a seminal paper (21) many years ago. They described osteoporosis in women soon after menopause as loss of mostly trabecular bone (and thus vertebrae were particularly at risk) and associated with the dramatic drop in ovarian estrogen production. Men with organic causes of hypogonadism (for example, pituitary tumors) may also present with very low serum testosterone levels and osteoporosis. There are other causes of this earlier type of osteoporosis in men, including hypercalciuria (22) and secondary causes, which may not be very apparent clinically.  An example of the latter is celiac disease, which may not bring the patient to clinical attention but can lead to early fracture risk. (See below for other secondary causes of osteoporosis in men).  Finally, there have been reports of genetic disorders leading to so-called idiopathic osteoporosis in men, such as low levels of IGF-I without abnormalities in growth hormone (23) and low serum bioavailable estradiol levels (24). It is much more likely for a man to experience an osteoporotic fracture after age 75 than at middle age, but the clinician needs to know that early osteoporosis occurs and that it should lead to evaluation and treatment.

 

The majority of fractures in men occur later in life. The Rotterdam Study (6) assessed only nonvertebral fractures because the date of vertebral fractures was much more difficult to ascertain. In men the incidence of nonvertebral fracture accelerates after about age 75. The incidence of nonvertebral fractures in men at ages 80 to 84 is about the same as the incidence in women ages 70 to 74. This observation is the basis for stating that fractures occur about 10 years later in men than women, and it may explain why men come to fracture with more co-morbidities than women, a possible explanation for why men do relatively poorly after hip fracture in particular. As used in FRAX, risk factors for fracture, presumably reflecting bone quality, magnify the impact of bone quantity (DXA) on fracture risk. In the FRAX calculation age, prior fracture, and history of parental fracture are the most important variables. Not well known is a report (25) from Leslie and colleagues proposing that the age at which a parent has fractured a hip is important. If the parent has fractured before age 80, this adds greatly to the patient’s risk of fracture, whereas if the parental hip fracture occurred late in life, the impact on fracture risk is much less. The analogy with familial heart disease is striking: early heart disease, particularly in a patient’s mother, makes the patient at much increased risk for cardiac events. 

 

Risk Factors and Secondary Causes of Osteoporosis

 

Table 1 summarizes potential risk factors and secondary causes of osteoporosis, most of which pertain to men as well as to women. Aspects specific to men are discussed below.

 

Table 1. Conditions, Diseases and Medications that Cause or Contribute to Osteoporosis and Fractures

Lifestyle Factors

Low Calcium Intake

Vitamin D Insufficiency

Excess Vitamin A

High Caffeine Intake

High Salt Intake

Aluminum (in antacids)

Inadequate Physical Activity

Immobilization

Smoking

Falling

Thinness

Alcoholism

Genetic Factors

Cystic Fibrosis

Homocystinuria

Osteogenesis Imperfecta

Ehlers-Danlos Syndrome

Hypophosphatasia

Gaucher’s Disease

Idiopathic Hypercalciuria

Porphyria

Glycogen storage diseases

Marfan Syndrome

Riley-Day Syndrome

Hemochromatosis

Menkes Steely Hair Syndrome

Parental History of Hip Fracture

Androgen Insensitivity

Turner’s & Klinefelter’s Syndromes

Endocrine Disorders

Adrenal Insufficiency

Diabetes Mellitus

Hyperthyroidism

Cushing’s Syndrome

Hyperparathyroidism

Hypogonadal States

Panhypopituitarism

Athletic Amenorrhea

Anorexia Nervosa and Bulimia

Hyperprolactinemia

Premature Ovarian Failure

 

Gastrointestinal disorders

Celiac Disease

Inflammatory Bowel Disease

Primary Biliary Cirrhosis

Gastric Bypass

Malabsorption

GI Surgery

Pancreatic Disease

 

Hematologic Disorders

Hemophilia

Multiple Myeloma

Systemic Mastocytosis

Leukemia

Lymphoma

Sickle Cell Disease

Thalassemia

 

Rheumatic and Autoimmune Diseases

Ankylosing Spondylitis

Lupus

Rheumatoid Arthritis

 

Miscellaneous Conditions and Diseases

Chronic Obstructive Pulmonary Disease

Muscular Dystrophy

Amyloidosis

End Stage Renal Disease

Parenteral Nutrition

Chronic Metabolic Acidosis

Epilepsy

Post-Transplant Bone Disease

Congestive Heart Failure

Idiopathic Scoliosis

Prior Fracture as an Adult

Depression

Multiple Sclerosis

Sarcoidosis

HIV/AIDS

 

Medications

Anticoagulants (heparin)

Cancer Chemotherapeutic Drugs

Gonadotropin Releasing Hormone Agonists

Anticonvulsants

Lithium

Aromatase Inhibitors

Depo-medroxyprogesterone

Barbiturates

Glucocorticoids (> 5mg of prednisone or equivalent for > 3 months)

Cyclosporine A

Tacrolimus

 

Table from the Endotext chapter entitled “Osteoporosis: Clinical Evaluation” by E. Michael Lewiecki.

 

The Osteoporotic Fractures in Men Study (MrOS) has provided a great deal of information. This long-term US observational study included about 6000 men for more than 15 years (26). Of the many important findings from the study, one is of particular interest. What are the characteristics of men, in addition to DXA, that predict hip fracture?  In this excellent report (1), several surprising factors were discovered and others were expected. Of the latter group, age >75, current smoking, Parkinson’s disease, hyperthyroidism, hyperparathyroidism, and decreased cognitive function were risk factors that greatly increased the fracture risk prediction, when added to BMD. More interestingly, several other risk factors were found: low dietary protein, any fracture after age 50, divorce, tricyclic anti-depressants or hypoglycemic agents, tall stature, and the inability to do chair stands. Having 4 of these risk factors increased hip fracture risk 5-fold in men with osteoporosis by DXA.

 

Secondary causes of osteoporosis are thought to be particularly important in men, but there is overlap between what might be called a secondary cause of osteoporosis and in another context a risk factor for primary osteoporosis. In addition, while treatment of a secondary cause may be adequate to lower fracture risk, a man will possibly be at risk for primary osteoporosis as he ages – and need osteoporosis specific treatment. Hyperthyroidism, hyperparathyroidism, and hypercalciuria are well-characterized secondary causes of osteoporosis in men.  A particularly important cause is glucocorticoid excess, usually due to treatment of an inflammatory disorder with systemic glucocorticoids. Glucocorticoid-induced osteoporosis (GIOP) is considered the most important medication-related type of osteoporosis and is of particular concern because fracture risk is increased (27) after 3 months of prednisone equivalent doses of 5 to 7.5 mg daily – and maybe earlier (28) and maybe even lower doses.  There is evidence that men are less likely to be evaluated and treated for GIOP (29), perhaps because clinicians again do not think that osteoporosis happens in men. While endogenous Cushing’s syndrome leads to GIOP, most cases are due to exogenous glucocorticoids, and about 1% of the adult population may be taking such medications at any particular time. 

 

Multiple myeloma may present with osteoporosis-like vertebral fractures; hence, this diagnosis must be in the differential diagnosis of the new patient presenting this way. Malabsorption, particularly celiac disease, is another potential secondary cause of osteoporosis. While type 2 diabetes mellitus is clearly associated with increased fracture risk (30), bone density is usually not decreased, whereas in type 1 diabetes mellitus, BMD is variable. Celiac disease is associated with type 1 diabetes mellitus, and thus it should be considered in men with type 1 diabetes mellitus and a fracture. Mastocytosis is associated with osteoporosis, although the mechanism is not fully understood. Hemochromatosis, presumably via some of its consequences is also on the list of secondary causes. Immobilization leads to loss of bone.  Spinal cord injury is much more common in men than women, and bone is lost distal to the cord lesion and may be worse than immobilization per se because of comorbidities (31). The fracture risk is high in men with spinal cord injury, and other types of decreased mobility should be considered when assessing men: stroke, Parkinson’s disease, multiple sclerosis, etc. 

 

Men with HIV now have life expectancies close to those without HIV, but the risk of osteoporosis and fracture is greater. Fractures appear about 10 years earlier than in men without HIV.  In a systematic review of large numbers of people living with HIV (mostly men), the relative risk of a low trauma fracture was 1.51 and the relative risk of a hip fracture was 4.09 (32). In a meta-analysis of bone mineral density testing (33), low bone mass (osteopenia) and osteoporosis by dual energy x-ray absorptiometry (DXA) was more prevalent in persons (again mostly men) with HIV compared to non-infected controls.  Interestingly, initiation of antiretroviral therapy (ART) appeared to be associated with lower bone density, confirmed by a subsequent randomized trial (34). ART with tenofovir alafenamide appears to have better renal safety than tenofovir disoproxil fumarate (35) and may have less impact on bone. Fortunately, men with HIV appear to response well to bisphosphonate therapy for osteoporosis 36).    

 

Osteoporosis and Hypogonadism

 

As mentioned above, men with organic causes of hypogonadism are at risk for fracture and may present at middle age or later with fracture or low BMD. More common and more controversial is the parallel decrease in BMD and serum testosterone levels with aging. While not proven, it is reasonable to assume that as testosterone and muscle mass diminish with age, falls will increase, leading to fractures. The relationship between serum testosterone and BMD is less clear. As previously mentioned, Khosla and colleagues reported (20) that BMD was more strongly related to bioavailable estradiol levels than any measure of testosterone. Of course, in men the major source of estradiol is aromatization of testosterone. The importance of estrogen is illustrated by the impact of iatrogenic hypogonadism. Prostate cancer often responds to androgen withdrawal, and for men with rising PSA levels or other evidence of recurrence or spread, androgen withdrawal may be accomplished by orchiectomy (not done very often today) or by use of analogs of gonadotropin releasing hormone (GnRH).  Some GnRH analogs acutely increase gonadotropins LH and FSH such that an androgen receptor blocker such as bicalutamide or nilutamide is given on a short-term basis until the pituitary is down regulated.  GnRH analog treatment results in serum testosterone levels that are essentially zero and in very low levels of estradiol (the remaining estrogens are presumably from conversion of adrenal androgens). Some men are treated with an androgen blocker alone. In most studies (37) bone loss is much more profound in the men treated with GnRH analogs compared to men treated with only androgen receptor blockers, who have normal estradiol levels. Abiraterone (38) blocks conversion of precursors to androgens and may be used in concert with GnRH analogs.  Prednisone is needed to prevent mineralocorticoid excess due to the enzyme blockage caused by abiraterone. The dose is 5 mg twice daily, a little more than a replacement dose. This potentially adds to the risk that men treated with this combined androgen deprivation therapy (ADT) will have particularly increased fracture risk. However, the most widely cited study (39) of fracture in men on ADT is several years old, done before abiraterone was approved. The important finding from this study was that while ADT given to a man who has a rising PSA level after primary treatment of prostate cancer leads to a 10-year survival rate of 80 to 90%, the 5-year fracture rate was almost 20% in Caucasian men and 2/3 or ¾ of that rate in African-Americans. Thus, the profound hypogonadism of ADT is clearly a major risk for fracture. 

 

This still leaves unanswered whether testosterone given to men with decreased serum testosterone levels associated with aging would benefit from testosterone replacement. There are no studies large enough to show a fracture benefit of such treatment. In a careful study (40) of older men with low serum testosterone levels, testosterone gel or placebo gel was used for one year. At the end of the year, there was a modest increase in BMD by DXA and also by quantitative computed tomography (qCT). More importantly, there was an increase in bone strength by finite element analysis of the qCT data. The Endocrine Society Male Osteoporosis Guideline (41) states that older men at risk for fracture should be treated with osteoporosis-specific medications but those who also have symptomatic hypogonadism can be considered for testosterone replacement. The likely impact of testosterone deficiency on muscle and the bone strength response to testosterone replacement make it plausible that testosterone replacement will lead to fewer fractures. The TRAVERSE study (42) is a large study of testosterone replacement on cardiovascular safety in older hypogonadal men. There was no increase in cardiovascular events in the men treated with testosterone gel (43), nor was there evidence of increased prostate cancer risk or urinary retention (44). Interestingly, there were more fractures in the men receiving testosterone replacement (45). However, the fractures occurred soon after starting replacement, and the majority were ankle and risk fractures (45, 46). This suggested to Grossmann and Anawalt (46) that testosterone-induced changes in behavior may have been the etiology of the fracture increase.

 

SCREENING AND DIAGNOSTIC EVALUATION IN MEN

 

DXA Testing Men

 

From this extensive review of pathogenesis and epidemiology of osteoporosis in men, it is possible to postulate which men should be screened for osteoporosis and how they should be evaluated.  Age is a major risk factor for fracture.  At what age should a man undergo DXA testing and does such testing lead to fewer fractures? The Endocrine Society Guideline (41) suggests DXA testing in most men at age 70 or above. The United States Preventive Services Task Force (47) states that there is insufficient evidence to recommend DXA testing in men, although it supports DXA testing in women by age 65. There are few studies demonstrating that DXA screening in women leads to fewer fractures. The recent SCOOP study (48) from the UK revealed that a two-stage method of choosing women for testing by first calculating FRAX using BMI as a surrogate for femoral neck BMD resulted in fewer hip fractures. In this study, women at low risk for fracture by FRAX were not screened further. Those at high risk were treated, and those in the middle had a DXA. Based on DXA results and recalculation of FRAX with femoral neck DXA results, women at risk were placed on therapy and had fewer fracture than those not screened for osteoporosis. There are no similar prospective studies in men, but Colon-Emeric and colleagues (49) used the Department of Veterans Affairs and Medicare databases to determine the impact of screening men with DXA. Overall, screening did not lead to fewer fractures. However, strategic screening did.  Men aged 80 or older, men on systemic glucocorticoids or ADT, and men with FRAX calculated with BMI (somewhat like the SCOOP study women) had fewer fractures if they were screened by DXA. In addition, men over age 65 with several other risk factors (including rheumatoid arthritis, alcohol or tobacco abuse, chronic obstructive pulmonary disease, chronic liver disease, stroke, Parkinson’s disease, gastrectomy, hyperthyroidism, hyperparathyroidism, or traditional anti-seizure drug use) were also likely to benefit, should they have a DXA done. This study was observational and done with the Department of Veterans Affairs population, which tends to be sicker than the general population and from the population of the prospective study, MrOS. Nonetheless, the findings are compatible with the epidemiology of fractures in men and can serve as a basis for clinical care.  It is unrealistic to expect that a study like SCOOP will be done in men. The SCOOP population was about 12,500 women; a male version would likely need approximately 40,000 participants.  Based on the Colon-Emeric observational study (49) and studies from MrOS (1), Table 2 suggests which older men that should be screened for osteoporosis by DXA.

 

Table 2.  Which Men Should Be Screened (by DXA) for Osteoporosis?

Men > 50 Years Old

After a fragility fracture (usually vertebral in younger group)

On chronic glucocorticoids

Organic causes of hypogonadism

Hypercalciuria

Men > 65 Years Old

All of the above plus:

On androgen deprivation therapy for prostate cancer

High risk for fracture based on FRAX using BMI

Current smoking/COPD

Alcohol abuse/chronic liver disease

Rheumatoid arthritis

Parkinson’s disease or other mobility disorder

Gastrectomy/bariatric surgery

Hyperthyroidism

Hyperparathyroidism

On enzyme-inducing anti-seizure medications for > 2 years

Men > 80 Years Old

If not already screened, all men over 80 should have a DXA (unless there is a contraindication).

    

In the United States, reimbursement for DXA testing is limited. This may be one reason that so few men are assessed for fracture risk. One potential method to identify men at risk for fracture is to assess bone density from CT scans done for other reasons. There are several methods of so-called opportunistic bone density evaluation that have been used (e. g. 50), including a study done in male veterans (51). It is likely that artificial intelligence can be harnessed to make this process even more efficient. Whether finding men at risk this way will lead to more clinical evaluation and treatment and fewer fractures remains to be determined.

 

Beyond DXA: Laboratory Evaluation of Osteoporosis in Men

 

If a man has osteoporosis by DXA or meets other criteria for osteoporosis or has low bone mass (osteopenia) but may be at higher risk for fracture, what other tests should be done? Spine x-rays or vertebral fracture analysis (images of the spine by DXA machines) may reveal vertebral fractures that increase subsequent fracture risk. There are no specific blood tests for osteoporosis, and the evidence base for the tests that follow may be weak. Nonetheless, it makes clinical sense to do a few laboratory tests to look for secondary causes/risk factors for osteoporosis and to ensure the safety of treatment, should it be indicated. Many patients will have had some of these tests as part of their general medical care, so the actual addition to routine testing may be small. For all patients, assessments of serum calcium and phosphate and renal function are necessary to look for hypercalcemia (which might signal hyperparathyroidism) and to determine if some osteoporosis treatments can be safely given.  Avoiding controversies about ideal levels of serum 25-OH vitamin D in the general population, there is consensus that for the patient with osteoporosis, the target level should be 30 ng/ml (52). All of those tests mentioned may help to identify the unusual patient with osteomalacia.  Serum alkaline phosphatase reflects bone formation and turnover, among other things. It is interesting that low serum alkaline phosphatase may be a sign of hypophosphatasia (53), a disorder of variable severity that may present as osteoporosis. Such patients should not be treated with anti-resorptive agents. An automated complete blood count should be done, particularly if there is any suspicion of multiple myeloma because about 75% of such patients will have anemia. All of the above tests, with the exception of 25-OH vitamin D, may be done as routine screening tests in many people visiting primary care clinicians, although measurement of 25-OH vitamin D has become very common as well. Once the 25-OH vitamin D level is at goal, a 24-hour urine for calcium and creatinine (and possibly sodium) may help to signal hypercalciuria, or in in the case of low urinary calcium excretion, may reflect malabsorption. For a patient suspected of hyperparathyroidism or hyperthyroidism, appropriate testing for parathyroid hormone (PTH) or thyroid hormones/TSH should be done. Similarly, for patients in whom there is a suggestion of another secondary cause of osteoporosis, specific tests such as serum protein electrophoresis, celiac antibodies, cortisol, tryptase, etc. can be done.  More controversial is whether serum testosterone should be measured.  Most symptoms of hypogonadism are non-specific, such as fatigue. Decreased libido is considered the most specific symptom, but decreased muscle mass and decreased beard growth might be present.  For the symptomatic man, measurement of early morning testosterone is reasonable. Many experts may suggest measurement of free and bioavailable testosterone as well as gonadotropins. The diagnosis of hypogonadism requires two early morning (preferably fasting) testosterone measurements (54). We would also measure PSA and review the hematocrit and hemoglobin before considering testosterone replacement. In addition, measurement of testosterone should only be done if the clinician would consider testosterone replacement, likely in addition to an osteoporosis-specific treatment. In the Veterans Affairs population, routine laboratory testing was found to reveal new secondary causes and/or osteoporosis risk factors (55). In contrast, in the healthier MrOS cohort, routine testing was found to be less helpful (56).

 

Table 3.  Practical Approach to the Man with Osteoporosis

History and Physical Exam

           Evidence of secondary causes of osteoporosis, risk factors

           Family history

           Height versus maximum attained height

           Gait

           Kyphosis

           General condition of teeth

           Evidence of significant visual abnormalities

           Ability to rise from chair without using hands

           Tenderness to percussion of spine

Standard Laboratory Tests

            Serum Chemistries: Calcium, Phosphate, Alkaline Phosphatase, Albumin

            Measure of Renal Function (e.g. serum creatinine, eGFR)

            Complete blood count

            Serum 25-OH vitamin D

            When 25-OH vitamin D is at goal: 24-hour urine calcium, creatinine, and maybe sodium

Laboratory Tests in Specific Cases (triggered by history and physical exam)

            Thyroid function tests (TSH, Free T4, maybe Total T3)

            Parathyroid hormone (PTH)

            Ionized Calcium

            Total, Free, and Bioavailable Testosterone

            LH, FSH, Prolactin

            CTX or other marker of bone resorption

            Bone Specific Alkaline Phosphatase (or other marker of bone formation)

            Celiac antibodies

            Serum/Urine Protein Electrophoresis

            Magnesium

            Tryptase

            Tests for cortisol excess (e.g. urinary free cortisol, dexamethasone suppression test, midnight salivary cortisol)

Images          

            X-rays of thoracic and lumbar spine

            X-rays of fractured bone

            Pituitary imaging (usually MRI)

 

MANAGEMENT OF OSTEOPOROSIS IN MEN

 

Non-Pharmacologic Management of Osteoporosis

 

One criticism heard about current osteoporosis treatment is that it focuses only on pharmacologic methods. A more comprehensive approach to osteoporosis treatment is preferred. Indeed, there are ways to reduce fracture that do not involve prescription of drugs, and they should be an important part of the therapeutic regimen. While there has been controversy about the role of calcium and vitamin D on fracture risk and on potential side effects, such as cardiovascular events, discussion of these controversies can be found in other chapters. One recent meta-analysis (57) concluded that daily calcium and vitamin D are likely to be salutary for osteoporosis. The widely-cited Institute of Medicine report (58) suggested 1000 to 1200 mg of elemental calcium in the diet and vitamin D intake of 400 to 800 units per day. As stated above, most experts would suggest that a target vitamin D level of 30 ng/ml is reasonable for patients with osteoporosis. From MrOS (1) we learned that the protein content of the diet is also important. A liquid protein supplement might be a good source of calcium and protein for some older men. In my own experience, older men who live alone may have poor diets, and such protein supplements may be an easy way to augment their diet.

 

Fall risk reduction is also very important. In most cases, patients fall first, fracture second.  Thus, attention to eyesight, avoidance of drugs that affect standing blood pressure or cause sleepiness or confusion, and home safety are very important parts of a comprehensive osteoporosis treatment program. Treatment of cataracts, for example, leads to fewer fractures (59). In MrOS (1) use of hypoglycemic agents was associated with increased hip fracture risk.  People with seizure disorders fall; thus, control of epilepsy is important. Avoidance of alcohol, opiates, benzodiazepines, and psychiatric drugs is suggested, but of course some patients may require medications that can cause drowsiness or imbalance. Anti-hypertensive medications need to be titrated such that postural hypotension does not occur. Convincing a man to use a walking aid may be challenging. Night lights, elimination of loose throw rugs and extension cords, and care with pets are also important to avoid falls. Consultation with Occupational Therapy and/or Physical Therapy should be considered in many cases. Exercise prescriptions should aim to improve muscle strength as well as balance. Risk factors for falls are listed in table 4.

 

Table 4. Risk Factors for Falls Adapted From Guidelines of the National Osteoporosis Foundation

Environmental Risk Factors

Lack of assistive devices in bathrooms, loose throw rugs, low level lighting, obstacles in the walking path, slippery outdoor conditions

Medical Risk Factors

Age, anxiety and agitation, arrhythmias, dehydration, depression, female gender, impaired transfer and mobility, malnutrition, orthostatic hypotension, poor vison and use of bifocals, previous fall, reduced mental acuity and diminished cognitive skills, urgent urinary incontinence, Vitamin D insufficiency (serum 25-OH-D < 30ng/ml (75nmol/l)), medications causing over-sedation (narcotic analgesics, anticonvulsants, psychotropics), diabetes

Neurological and Musculoskeletal Risk Factors

Kyphosis, poor balance, reduced proprioception, weak muscles

Other Risk Factors; Fear of falling

The presence of any of these risk factors should trigger consideration of further evaluation and treatment to reduce the risk of falls and fall-related injuries.

Table from the Endotext chapter entitled “Osteoporosis: Clinical Evaluation” by E. Michael Lewiecki.

 

Medications for Osteoporosis in Men

 

The pharmacologic treatment of osteoporosis in men is by and large the same as treatment in women. Alendronate, risedronate, zoledronic acid, denosumab, teriparatide, and abaloparatide are all FDA approved for men with osteoporosis. Most men have been treated with bisphosphonates, similar to women. Alendronate was the first modern bisphosphonate approved by regulatory agencies in the mid-1990’s; it was shown to change surrogates of fracture (BMD and bone turnover markers) in men similarly to women (60). Although fracture risk reduction was not the primary outcome of the study, there were fewer morphometric vertebral fractures in the men randomized to alendronate compared those on placebo.  Similarly, risedronate and zoledronic acid have been shown to increase bone density in men to a similar degree as in women (61, 62). A criticism by some is that current surrogates for fracture may not be adequate, and that raising BMD or suppressing bone turnover markers in men is not enough evidence to conclude that fracture risk will be lowered by bisphosphonates. In a two-year study (63) with morphometric vertebral fractures as the primary outcome, Boonen et al demonstrated that zoledronic acid not only increased BMD in men, compared to placebo infusions, but it also led to fewer vertebral fractures. Specifically, at 2 years there was a 67% relative risk reduction and 3.3% absolute risk reduction in morphometric vertebral fractures. Thus, the clinician can be confident that if the patient is compliant and adherent to bisphosphonate treatment, fracture risk should be decreased.

 

All of the cited studies in men used a male normative database for calculation of the T-score. Men were eligible for the studies if they had osteoporosis by this criterion or had osteopenia (usually a T-score of -2) plus history of a low trauma facture. In women treated with bisphosphonates, vertebral fractures are decreased by about half and hip fractures by a third.  In the zoledronic acid registration trial in women (64), at 2 years the relative risk reduction of morphometric vertebral fractures was 71% and the absolute risk reduction was 5%. Compared to the study in men, at baseline the women were older, were more likely to have had a previous fracture, and had lower BMD.  It is impossible to compare results between the two gender-specific studies in any meaningful way, other than to conclude that zoledronic acid works similarly in men and women. 

 

The usual starting treatment for osteoporosis is oral alendronate 70 mg by mouth once weekly.  As in women, oral alendronate (or most preparations of risedronate) has to be taken on an empty stomach with just a glass of water, and the patient is instructed to take nothing else by mouth for at least 30 minutes. In general, this is not a problem, but for men also taking levothyroxine and/or proton pump inhibitors, timing may be difficult. In patients on levothyroxine, one strategy is to have them take the levothyroxine in the middle of the night, when older men are likely to need to urinate. This does not work for bisphosphonates because lying down after taking the bisphosphonate may lead to esophageal irritation. For the man with gastro-esophageal reflux disease (GERD), avoidance of oral bisphosphonates is indicated if the GERD is not under good control. For such men and for those unable or unwilling to adhere to the correct oral regimen, intravenous zoledronic acid, 5 mg given over 15 minutes or more, is a reasonable choice. The FDA-approved interval for zoledronic acid is one year. In our experience (65, 66), increasing the interval to 1.5 years or so allows all bisphosphonate patients to have a 5-year initial treatment period. Long-term management of osteoporosis in men is discussed below.  An alternative oral treatment is risedronate given as a monthly 150 mg tablet.  For some men, particularly those with a high pill burden, this may be an attractive regimen.

 

As an alternative to bisphosphonates, another anti-resorptive or anti-bone turnover medication is denosumab, an antibody against RANK Ligand. Among the earliest uses of this medication was a study of a high-risk group, men on ADT for prostate cancer. In this important study, Smith and colleagues (67) randomized men receiving GnRH analogs to profoundly suppress testosterone secretion to denosumab or placebo. There were fewer morphometric vertebral fractures in the men who were given denosumab as a subcutaneous injection every 6 months compared to men receiving placebo injections. After a study (68) showing that denosumab altered surrogates for fracture in men similarly to the effect in women, the drug was approved for osteoporosis in men, regardless of etiology. Interestingly, denosumab increases forearm bone density, something not found with bisphosphonate treatment (60). In long term studies of bisphosphonates in women (69, 70) BMD rises and then plateaus after a few years of treatment.  In contrast, studies in women have shown continued increases in BMD for at least 10 years with continued denosumab treatment (71). The consequences of this plus the impact of withdrawal of osteoporosis treatment will be discussed below.

 

As men age, there is thinning of trabeculae, whereas in women there is loss of trabecular number and the spacing between trabeculae increases (72). Thus, while the changes in vertebral fracture risk appear very similar in men and women, the impact on fracture could be different. DXA does not capture all of the changes with time. More recent studies (73) with high resolution peripheral quantitative computed tomography (HR-pQCT) also show sex-specific changes, but the studies are small. To my knowledge, there are no bone biopsy or HR-pQCT studies that demonstrate sex-dependent differences in response to therapy.

 

In women anabolic agents increase trabecular thickness and connectivity and increase cortical bone thickness. Of late, increased use of such agents as the initial treatment has been advocated for those patients at highest risk for fracture based on recent studies in women (74) demonstrating benefits to starting with anabolic treatment. In the United States, only teriparatide and abaloparatide are FDA approved for osteoporosis in men. There is another anabolic agent, romosozumab, that is approved for women, but there is no reason to believe that it would not work in men. There is one published report of improved fracture surrogates in men given romosozumab (75). Abaloparatide works similarly in men and women (76, 77). The use of anabolic agents, regardless of the patient’s sex, is limited by inconvenience of treatment (both teriparatide and abaloparatide are administered as a daily subcutaneous injection) and cost.  While romosozumab is given as a monthly injection in a clinician’s office, its cost in the United States is similar to that of abaloparatide, which is somewhat cheaper than teriparatide. In Japanese women, a higher dose of teriparatide given weekly or semi-weekly has been found to be effective (78), but there have been no studies of similar preparations in Europe or the United States. Based on studies in women, anabolic agents should be considered, including off-label use, for men at the highest risk of fracture. In a 3-year study (79) of men and women with glucocorticoid-induced osteoporosis, teriparatide was shown to result in fewer spine fractures than alendronate. More recently, a study in women (80) showed that anabolic treatment led to fewer fractures than anti-resorptive treatment with risedronate. Until there are better surrogates for fracture, there will never be a study comparing fracture risk in men treated with anabolic agents compared to anti-resorptives. The data from studies in women are convincing, and there is no physiological reason to question whether men would respond differently. A recent systematic review and meta-analysis of randomized controlled trials (81) led to the conclusion that osteoporosis drugs work the same in men and women.

 

In summary, initial treatment of osteoporosis in men should be comprehensive, with attention to diet, exercise, vitamin D, fall risk reduction, and home safety. After vitamin D is satisfactory, and possibly after dental work is completed, most men can be treated with bisphosphonates, usually oral alendronate. For those who cannot take an oral preparation, intravenous zoledronic acid is the drug of choice. For men at the highest risk for fracture, based on fracture history, DXA, risk factors, and risk calculators, a 1 to 2-year course of anabolic treatment should be considered, although teriparatide and abaloparatide can be prescribed for more than 2 years, if needed. For very high-risk patients, the anabolic therapy can be followed by 2 years of denosumab treatment, followed by consolidation with a bisphosphonate. For those men with CKD 4, denosumab is a good choice but must be continued indefinitely. Denosumab is also appealing for men on ADT who receive long-acting GnRH analogs every 6 months because they can receive denosumab at the same visit. However, there are rapid bone loss and potential vertebral fractures in women who have recently withdrawn from denosumab (82, 83). In one observational study in men (84) zoledronic acid prevented the loss of bone after men had discontinuation of denosumab.

 

Long-Term Management of Osteoporosis in Men

 

There are no long-term studies of osteoporosis treatment in men. Hence, all suggestions for management must be made from the few studies in women. The FLEX trial (69) showed that 10 years of alendronate in women led to fewer clinical vertebral fractures than 5 years of alendronate followed by 5 years of placebo tablets. The HORIZON extension trial (70) showed that a plan of 6 annual infusions of zoledronic acid was associated with fewer morphometric vertebral fractures than 3 annual infusions of zoledronic acid followed by 3 placebo infusions.  Based on these studies plus some other information, a task force of the American Society for Bone and Mineral Research recommended an approach to long-term osteoporosis management (85). While the approach was aimed mostly at postmenopausal women, the task force recommended that it be applied to men as well. In this approach, the initial treatment period is 5 years for oral bisphosphonates and 3 years for zoledronic acid. At the end of the initial treatment period, the patient is re-assessed by history, physical examination, and repeat BMD. Those patients remaining at elevated fracture risk should continue treatment and be re-assessed again in 2 to 3 years. Those patients whose fracture risk has been demonstrably decreased by treatment can interrupt therapy and be re-assessed at 2 to 3 years. Beyond 10 years of treatment there are no studies, and so clinical judgement will be necessary to manage such patients. I have proposed, based on studies in women (86, 87) and men (65), that the interval between zoledronic acid infusions can be lengthened such that each patient would receive 3 infusions of zoledronic acid over 5 years. This creates a 5-year initial treatment plan for the majority of people with osteoporosis: all but those at highest risk for fracture. For the latter group, initial therapy should be anabolic for the first 1 to 2 years, and then the patient would be placed on anti-resorptive agents. While this approach to long-term osteoporosis management makes sense, it will likely never be supported by large randomized trials.

 

A summary of a practical approach to the evaluation of osteoporosis in men is shown in Table 5.

 

Table 5.  Approach to Osteoporosis Treatment in Men

For All Men: Conservative Treatment

Fall risk reduction/home safety

Adequate calcium, vitamin D, dietary protein

Weight bearing exercise/balance training

Smoking cessation/minimization of alcohol intake

Treat Secondary Osteoporosis with Specific Therapy

Men with Borderline Fracture Risk

Conservative treatment

Repeat DXA in 2 to 3 years

Use FRAX to demonstrate low risk

Osteoporosis by DXA, Osteopenia + Fracture, High Risk by FRAX

Oral alendronate or risedronate or intravenous zoledronic acid

Clinical reassessment every year

Repeat DXA at 2 to 3 years

Change Rx if response inadequate

Repeat DXA at 5 years to consider drug holiday versus continued Rx

Very High Risk by DXA, FRAX, Clinical Findings

Anabolic Rx for 1 to 2 years

Then denosumab for 2 years

Then 1 year of alendronate or 1 infusion of zoledronic acid

DXA at 2 to 3 and 5 years

Drug holiday versus continued treatment based on fracture risk after treatment

.

CONCLUSIONS

 

Despite the overall paucity of evidence underpinning osteoporosis evaluation and treatment in men, it is important to identify men at risk for fracture, evaluate them efficiently, and treat them.  As more men live long enough to fracture, the burden of male osteoporosis will increase. In addition, because men with hip fracture are more likely to die after fracture (88), compared to women of the same age, improving diagnosis and treatment is likely to save lives, decrease suffering, and lead to lower costs.     

 

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Cushing Syndrome/Disease in Children and Adolescents

ABSTRACT

 

Endogenous Cushing syndrome (CS) is a rare pediatric endocrine condition commonly caused by pituitary corticotroph tumors or less often by adrenal or ectopic sources. The typical presentation of the child with CS includes weight gain with height deceleration, characteristic skin findings, and hormonal and biochemical findings indicative of excessive glucocorticoid production. The diagnostic evaluation of the patient with suspected hypercortisolemia initially involves the confirmation of cortisol excess in blood and/or urine, and then the identification of source. The first line of management usually requires surgical treatment of a pituitary or adrenal lesion. In persistent or recurrent disease, re-operation, medical treatment, or radiation should be considered.

 

INTRODUCTION

 

Cushing syndrome (CS) describes the exposure of the body to supraphysiologic levels of glucocorticoids. Although exogenous (iatrogenic) CS is common, endogenous pediatric CS, is a rare pediatric endocrine condition. Population studies of the incidence of the disease have shown that endogenous CS occurs in about 3-50 cases per million people per year, depending on the population studied; pediatric patients in these studies represent 6-7% of all cases (1-3).  

 

ETIOLOGY

 

Endogenous CS can be classified as ACTH-dependent (pituitary or ectopic) or ACTH-independent CS (adrenal-related, Table 1) (4). The etiology of pediatric CS differs based on the age group of the patient (5). In patients younger than 5 years of age, ACTH-independent CS is more common compared to older children and adolescents who usually present with ACTH-dependent CS. Ectopic CS (ECS) is rare at any age group (5).

 

Table 1. Causes of Cushing Syndrome

Type

Source

 Mechanism

Exogenous

 

Iatrogenic

 Exogenous administration of supraphysiologic doses of glucocorticoids or ACTH

Endogenous

 

ACTH-dependent

Pituitary

Corticotroph pituitary neuroendocrine tumor

Pituitary blastoma

Ectopic

 Neuroendocrine tumors secreting ACTH and/or CRH

 

 

 ACTH-independent

Unilateral adrenal (except in metastatic disease)

 Cortisol-secreting adrenocortical adenomas and carcinomas

Bilateral adrenals

Bilateral micronodular adrenocortical disease

-       Primary pigmented nodular adrenocortical disease (PPNAD), isolated or in the context of Carney complex

-       Isolated micronodular adrenocortical disease (iMAD)

Bilateral macronodular adrenocortical disease

 

ACTH-Dependent Cushing Syndrome

 

ACTH-dependent CS is most commonly due to a corticotroph pituitary neuroendocrine tumor (PitNET, also called pituitary adenoma or Cushing disease, (CD). These are monoclonal lesions that continue to express some of the characteristics of the normal corticotroph cell which can be useful in the diagnostic evaluation of patients (6, 7). Corticotroph-secreting PitNETs are usually microadenomas with median diameter of 5mm and do not often show signs of invasion to the cavernous sinus or other parasellar structures (8). Rare cases of aggressive PitNETs have been reported in the pediatric population with either resistance to treatment or distant metastasis (metastatic PitNETs) (9). These are associated with specific histologic subtypes, such as Crooke cell adenomas (9).

 

Infantile onset of ACTH-dependent CS with a pituitary lesion is often due to a pituitary blastoma. In 2014 de Kock et al, collected tissues from several infants who had been diagnosed with very young onset CD and reported that the tumors were consistent with pituitary blastomas as they had histologic findings of undifferentiated epithelium Rathke-like cells, mixed with hormone producing cells (10). They were able to identify germline and/or somatic DICER1 gene defects in these patients, suggesting that pituitary blastoma is a rare but almost pathognomonic presentation of DICER1 syndrome (10).

 

ECS is due to neuroendocrine tumors secreting ACTH and/or CRH outside the hypothalamic-pituitary axis. In older children and adolescents, the most common source of ECS are bronchial carcinoids, thymic carcinoids, and gastro-entero-pancreatic NETs (11-13). By contrast, in children younger than 5-10 years of age, ECS often presents in the context of pediatric specific tumors such as Wilm’s tumors, neuroblastomas, and others (13, 14).

 

ACTH-Independent Cushing Syndrome

 

ACTH-independent CS is commonly caused by unilateral adrenocortical tumors, cortisol-producing adenomas or carcinomas (5). Cortisol-producing adenomas are benign lesions with isolated cortisol secretion, while adrenocortical carcinomas are aggressive tumors and may commonly co-secrete cortisol and androgens in up to 80% of all cases (15, 16).

 

Bilateral adrenocortical disorders account for <2% of all cases of CS but some subtypes may be more prevalent in children compared to adults given their association with germline genetic defects (17). Micronodular adrenocortical disease is the most common type of bilateral adrenocortical disorder in pediatric patients. This category may be further divided in primary pigmented micronodular adrenocortical disease (PPNAD) where the adrenals present with multiple dark brown pigmented micronodules (due to lipofuscin deposition with most with diameter of <1cm) with internodular cortical atrophy, or the absence of these findings referred to as isolated micronodular adrenocortical disease (i-MAD) (18). PPNAD may be identified in the context of Carney complex (CNC) and less often as isolated PPNAD (19). Bilateral macronodular adrenocortical disease presenting with bilateral macronodules (most with diameter of ≥1cm) is rare in the pediatric population.

 

GENETICS

 

Genetic causes are found in less than half of the patients with pediatric CD and more commonly in adrenal-related CS. For patients presenting with pediatric onset CS, it is recommended to obtain genetic testing directed to the source of hypercortisolemia, i.e. adrenal vs. pituitary causes.  Although the yield in CD may be low, in cases of pituitary blastomas or bilateral micronodular disease genetic testing has higher chance of identifying the genetic cause and lead to screening for other related manifestations that may be important, such as cardiac myxomas in patients with CNC.

 

ACTH-Dependent Cushing Syndrome

 

Germline mutations are identified in less than 10% of patients with pediatric CD (8). Of the most common causes are MEN1 (causing multiple endocrine neoplasia type 1 syndrome, MEN1), CDKN1B (causing MEN4), and CABLES1 gene defects (20). Genes associated with familiar isolated pituitary adenoma (FIPA) syndrome, such as AIP, SDHx, and MAX, or syndromes associated with pituitary tumors amongst other manifestations, such as CNC due to PRKAR1A gene defects, do not commonly cause corticotropinomas and have only been reported in few case reports (21).

 

As mentioned above, young children (<2 years old) presenting with pituitary blastomas should be screened for DICER1 gene defects (10). DICER1 codes for an endoribonuclease that processes miRNAs (22). Patients with DICER1 or pleuropulmonary syndrome present with multiple tumors in lungs, kidneys, multinodular goiter, and other manifestations. Pituitary blastomas are present in less than 10% of all patients and always within the first years of life (23).

 

Somatic mutations are more likely to be identified in corticotropinomas. USP8 mutations in the 14-3-3 binding motif hotspot region of the gene have been reported as the cause of 40-60% of adults with CD (24, 25). Pediatric data suggest that USP8 mutations are less common and identified in up to 30% of cases (26). USP8 is a deubiquitinase involved in recycling of the epidermal growth factor receptor (EGFR) and mutations in the hotspot region led to increased catalytic activity, activation of the EGF pathway, and increased POMC expression. In children, USP8 mutant tumors presented with larger size and higher risk for persistent disease after surgery or recurrence after initial remission (26). Data in adult patients did not confirm this finding, and the prognostic value of identifying a USP8 mutation is still unclear (27). Other somatic mutations identified in corticotropinomas include USP48, TP53, and BRAF, but the incidence in pediatric patients is unknown (28). Finally, in a subset of patients with pediatric corticotropinomas large genomic chromosomal deletions/gains are identified and are associated with larger tumor and higher risk of invasion of the cavernous sinus (29).

 

ECS may present in various neuroendocrine tumors and the genetic background is associated with the primary tumor. MEN1, MEN2 (RET gene mutations), and some gene fusions have been described according to the tissue involved in ectopic ACTH secretion (30, 31).

 

ACTH-Independent Cushing Syndrome

 

Pediatric cortisol producing adrenocortical carcinomas may present in the context of TP53 mutations (32). In the Brazilian South and Southeast population, high prevalence of a germline founder TP53 mutation (p.R337H) is associated with high incidence of pediatric adrenocortical carcinomas (33, 34). Germline TP53 mutations may also present as Li-Fraumeni syndrome where patients have high risk for breast, central nervous system, bone, and other tumors (35). Cortisol-producing adrenocortical adenomas may be associated with gene defects leading to activation of the cyclic AMP (cAMP) protein kinase A (PKA) pathway, such as somatic mutations in PRKACA, PRKAR1A, and PRKACB genes (36, 37). Finally, somatic gene defects in the Wnt signaling pathway have also been identified in adrenocortical tumors (38).

 

ACTH-independent CS due to PPNAD presents commonly in the context of CNC (39). CNC is an autosomal dominant multiple neoplasia syndrome caused by inactivating mutations of the gene PRKAR1A, coding for the regulatory subunit 1 alpha of PKA, or less often linked to a second locus at chromosome 2p16 (40-42). Inactivating mutations in PRKAR1Alead to constitutive activation of PKA and downstream pathways (18). Patients with CNC present with several manifestations including PPNAD, pituitary abnormalities most often presenting as growth hormone dysregulation or acromegaly, thyroid nodules or carcinomas, testicular tumors, cardiac and skin myxomas, characteristic skin lesions, breast myxomatosis or adenomas, osteo-chondro-myxomas and psammomatous melanotic schwannomas (40). PPNAD in CNC is often diagnosed in the third decade of life but patients as young as in the first decade of life have been reported (43). Additional information about CNC can be found in the chapter entitled “Carney Complex” of Endotext (40).

 

Additional genetic defects associated with bilateral adrenocortical disease include PRKACA genomic gains, PDE11A, and PDE8A gene defects identified in patients with macronodular adrenocortical disease or isolated micronodular disease (44-46). PRKACA codes for the catalytic subunit of PKA, and chromosomal gains lead to increased PKA signaling (47). PDE11A and PDE8A codes for phosphor-diesterases that catalyze and decrease cAMP levels. Inactivating mutations in these genes lead to increased circulation of cAMP and increased PKA activity (44, 48). Macronodular adrenocortical disease due to ARMC5 gene defects often seen in adults is rare in the pediatric population (49).

 

Neonatal ACTH-independent CS may be seen in the context of McCune-Albright syndrome (MAS) (50). In these cases, CS presents within the first year of life and may have detrimental and rapidly developing symptoms which may even lead to death. However, if managed medically, neonatal CS in MAS may resolve on its own (51). Rare cases of neonatal onset adrenocortical disease have also been reported in the context of Beckwith-Wiedemann syndrome (52, 53).

 

PRESENTATION

 

The presentation of pediatric CS has similarities and differences from that in adults (Table 2).

 

Table 2. Presenting Findings in Pediatric Patients with Cushing Syndrome

Clinical findings

Anthropometric

Height deceleration

Weight gain

Cardiovascular

Hypertension

Musculoskeletal

Fractures

Proximal muscle weakness

Skin

Striae

Facial Plethora

Easy bruising

Acne

Hirsutism

Acanthosis nigricans

Abnormal fat deposition

Neuropsychiatric

Behavioral changes (compulsive behavior, overachievement tendency, irritability)

Psychiatric disorders (depression, anxiety)

Changes in cognitive function

Sleep disturbance (difficulty falling asleep)

Memory problems

Reproductive system

Delayed puberty

Irregular menses

Immunologic

Increased risk for infections

Laboratory and imaging findings

Complete blood count

Elevated total white blood cell, neutrophil and monocyte counts

Decreased lymphocyte and eosinophil count

Elevated neutrophil-to-lymphocyte ratio

Biochemistry

Hypokalemia

Hypercalciuria

Elevated ALT

Hyperlipidemia

Hyperglycemia with elevated insulin levels

Coagulation factors

Increased coagulation factors

Decreased aPTT

Echocardiogram

Cardiac hypertrophy

DXA

Decreased bone mineral density

 

The hallmark of pediatric CS is weight gain with concomitant height deceleration (Figure 1) (54). This finding can help discriminate patients with CS from with simple obesity who often have preserved height percentile (55). Fat deposition in pediatric patients may not be as prominently centripetal as noted in adults, and may present often as generalized obesity similar to other causes (56). Although height deceleration is seen in most cases of growing children, patients may not be short at presentation, may have completed growth by the time hypercortisolemia occurred, or may be exposed to episodic hypercortisolemia which may have more limited effect on their height (5, 8, 57). Bone age is often within the expected range for the chronologic age or advanced in pediatric patients with endogenous CS, and is correlated with the levels of adrenal androgens which are often increased in ACTH-dependent CS (58).

 

Figure 1. Typical growth chart of a pediatric patient with Cushing syndrome (A) compared to a child with obesity (B).

 

Dermatologic findings present in CS include striae (which are present in 60-80% of patients and may not have the characteristic appearance of deep purple color and thickness as in adults), facial plethora, acne (more common in ACTH-dependent CS possibly due to stimulation of adrenal zona reticularis by ACTH), hirsutism in women, hypertrichosis, acanthosis nigricans, and easy bruising (8, 56, 59).

 

Patients with CS often present with delayed puberty in males and females and/or irregular menses and secondary amenorrhea in females (8).

 

As in patients with iatrogenic CS, pediatric patients with endogenous CS present with decreased bone mineral density, with lower scores in the spinal measurements (60-62).  Proximal muscle weakness although reported is less frequent than adult patients (54).

 

Pediatric patients with endogenous CS, especially younger in age, often present with behavioral and neurocognitive changes. They may report behavioral changes including compulsive behaviors with overachievement goals, described as excellent students, along with increased anxiety and irritability (63). They may also report mood changes, depressed mood, sleep problems, and memory issues similar to adults. Headaches are common in pediatric patients and can be noted in up to 80% of them (8).

 

Hypercortisolemia and its related obesity lead to metabolic syndrome (64). Patients often present with insulin resistance and up to 30% of patients may have impaired glucose metabolism (56). Hyperlipidemia and elevated ALT as a surrogate marker of metabolic associated fatty liver disease (MAFLD) are also present in almost half of the patients (8, 56). Hypertension is present in almost 50% of patients with endogenous CS and cases of posterior reversible encephalopathy syndrome (PRES) due to hypertensive emergency have been reported as the initial manifestation of CS in pediatric patients (8, 65).

 

Similar to adults, pediatric patients with CS present with a hypercoagulable state associated with abnormal levels of procoagulants, antifibrinolytics, and anticoagulant factors, such as factor VIII, antithrombin III, protein C and S, and prolonged partial thromboplastin time (PTT) (66). Although in adult patients with CS the risk of venous thromboembolism is more studied, the exact incidence, risk, and thromboprophylaxis protocols in children are not as well delineated and depend on clinical judgement (67).

 

Additional findings in pediatric CS include characteristic abnormalities in CBC due to glucocorticoids effects including increased WBC count, neutrophil count, low normal lymphocyte count, and increased neutrophil-to-lymphocyte ratio (NLR) (68). Although immunosuppression may lead to severe infections in patients with significantly elevated cortisol levels, in most pediatric cases infections are limited to less clinically significant areas such as skin infections etc. (69). However, in very young patients, especially in neonatal CS, or patients with severe hypercortisolemia, such as in ECS, opportunistic infections may lead to significant morbidity and even death and prophylaxis should be initiated (14, 70).

 

Electrolyte abnormalities seen in endogenous CS include hypokalemia, uncommon overall but seen more frequently in ECS, and hypercalciuria which may lead to nephrolithiasis (8, 71).

 

Patients with hypercortisolemia also present with other hormonal defects including abnormal thyroid function test with a pattern of central hypothyroidism, abnormal GH secretion with IGF-1 levels usually preserved within the reference range, and suppressed gonadotropins (72-75). Tumor stalk compression effects may lead to hyperprolactinemia, although this is uncommon due to the small size of most corticotropinomas. Androgen levels are commonly elevated in ACTH-dependent CS due to adrenal zona reticularis stimulation from ACTH, or in adrenocortical carcinomas where co-secretion of cortisol and DHEAS may be seen.

 

DIAGNOSIS

 

The diagnostic evaluation of pediatric CS follows the guidelines of the endocrine and pituitary society adjusted for the pediatric population (7, 76, 77). Screening for hypercortisolemia is preferably done with at least two of the following tests: 24-hour urinary free cortisol (UFC, measured on 2-3 days), midnight (or late night) cortisol measured on 2-3 days, and suppression of cortisol to low dose dexamethasone (76). Specific details on these tests can be found in the chapter entitled “Endocrine Testing Protocols: Hypothalamic Pituitary Adrenal Axis” of Endotext (78).

 

Confirming the Diagnosis of Cushing Syndrome

 

The loss of the diurnal rhythm of ACTH/cortisol secretion is the first abnormality noted in patients with CS (79, 80). In clinical practice, salivary cortisol has been used to measure midnight or late night cortisol levels as it is convenient and can be collected at home (78, 81). If this is not available, then serum midnight cortisol is an alternative accurate screening test (77). A serum cortisol level of ≥4.4mcg/dL was able to distinguish almost all pediatric patients with CS with a sensitivity of 99% and a specificity of 100% (7). Serum cortisol needs to be measured from an indwelling catheter that has been placed at least 2 hours prior to sampling. We instruct patients to turn off all screens by 10pm and blood should be collected without awakening the patient (82).

 

The 24-hour urine collection should be performed on two or three days, to ensure optimal urine collection and account for the known day-to-day variability in urinary cortisol in patients with CS (83, 84). It is generally recommended to collect urine on days of routine physical activities and avoid days when increased stressful activities are expected, like competitive sports games etc. (85). Additionally, patients are advised to consume normal amount of fluids as excessive fluid intake and urine output may lead to false positive results (86). The urine samples should be measured for urine creatinine to ensure normal kidney function, but we do not routinely correct UFC levels for the urine creatinine level as this may lead to inaccurate results (87).

 

The low dose or 1mg overnight dexamethasone suppression test is performed similar to adults (78). Dose adjustment has been used in several studies, though no study has been done to specifically investigate the appropriate dose in children with CS. Various protocols recommend the use of 15mcg/kg, 25mcg/kg, or 0.3mg/m2 (max 1mg) once at 11pm for the overnight test or 1200mcg/kg/day (max 2mg/day) divided Q6 hours for two days (88, 89). Measurement of a serum dexamethasone level at the same time as cortisol is important to ensure the desired dexamethasone level has been reached.

 

If screening labs suggest cortisol excess, it is important to rule out physiologic/non-neoplastic hypercortisolism (previously known as pseudo-Cushing syndrome) (90). If suspicion is high, additional testing should be considered, including dexamethasone-CRH test (if available) or DDAVP stimulation test. If results remain inconsistent, close monitoring with repeat physical examination and labs within 3 months should be offered to monitor clinical and biochemical findings while at the same treating causes that may contribute to activation of the hypothalamic-pituitary-adrenal axis (90).

 

Identifying the Source of Hypercortisolemia

 

Once endogenous CS is confirmed, the next step is to identify the source of hypercortisolemia. ACTH levels are used to guide next steps. Elevated ACTH levels of >20-29pg/mL suggest ACTH-dependent CS while suppressed ACTH is consistent with ACTH-independent CS (7). Intermediate values may need further evaluation for both ACTH-dependent and ACTH-independent causes, but most often a non-suppressed ACTH level suggests ACTH-dependent CS, except in the case of mild subclinical hypercortisolemia or cyclical CS.

 

In cases of ACTH-dependent CS, additional biochemical and imaging studies include pituitary MRI (with and without contrast, pituitary protocol), CRH stimulation test (if available), DDAVP stimulation test and/or high dose dexamethasone suppression test. Corticotroph PitNETs are often shown as hypo-enhancing microadenomas in pituitary MRI (Figure 2), but a normal/negative MRI may be seen in up to 30% of patients (91). In cases of normal MRI or biochemical testing inconsistent with pituitary source, bilateral inferior petrosal sinus sampling (BIPSS) is the gold standard in diagnosing or ruling out CD. Non-invasive strategies are described if BIPSS is not feasible and/or not available (92). In our pediatric patients, all patients who showed suppression to high dose dexamethasone administration and stimulation to CRH/DDAVP consistent with a pituitary source, had CD irrespective of imaging findings (8).

 

For patients suspected to have ECS, further evaluation should include imaging of the neck, chest, abdomen, and pelvis with thin cuts as carcinoids can be small in diameter. Chest imaging is preferably done with CT due to higher accuracy in the lung parenchyma, but some centers use MRI for abdominal/pelvic imaging to reduce radiation. Nuclear imaging, preferably with Ga-68 DOTATATE or, if not available or negative, with 18F-FDG PET, may identify some of these ectopic sources (11, 13, 93). If a lesion found on imaging studies is suspicious but not convincing, one may attempt venous sampling close to the possible lesion for measurement of CRH and/or ACTH and compare the levels to a peripheral source (11). If a gradient is reported then this may further support the diagnosis of ectopic tumor (11). Other markers of potential interest in these cases include chromogranin A and CRH, which may be helpful in the follow-up of patients. Patients with ECS may present with pituitary hyperplasia if CRH is co-secreted, which should be considered when interpreting the imaging and biochemical results.

 

When ACTH-independent CS is suspected, imaging of the adrenals is the best next step. Imaging can be preferably with CT since it has good accuracy for lesions <1cm and less artifacts due to motion, but MRI may be an alternative to avoid radiation exposure. Ultrasound however is not accurate in identifying adrenal lesions other than large adrenocortical tumors (14). In ACTH-independent CS, it is important to review the anatomy of both adrenals; noting a unilateral lesion with atrophy of the contralateral adrenal supports the diagnosis of unilateral disease, whereas bilateral symmetrical adrenal enlargement or bilateral normal appearing adrenals suggests bilateral disease (Figure 2). In case of bilateral micronodular adrenocortical disease, adrenal anatomy is often read as normal or sometimes asymmetric appearance of the contour of the adrenals described as “beads on a string” may be apparent (94).

 

When bilateral adrenocortical disease is suspected, confirmation of the diagnosis prior to proceeding with surgical intervention involves the performance of Liddle’s test (95). The paradoxical increase of urinary free cortisol or 17-hydroxy steroids with increasing doses of dexamethasone is pathognomonic for PPNAD (95).

 

Figure 2. Typical imaging findings in a patient with Cushing disease (A-B), a cortisol-producing adrenal adenoma (C) and bilateral micronodular adrenocortical disease (E). Postcontrast sagittal (A) and coronal (B) MRI images of the pituitary showing a microadenoma (tip of arrows) as hypoechoic lesion. (C) Axial adrenal CT of a patient with a left adrenal adenoma (yellow asterisk) and atrophic contralateral adrenal (blue outline). (E) Axial adrenal CT of a patient with bilateral micronodular adrenocortical disease showing normal appearing adrenals (blue outline) with bilaterally symmetric thickness of the limbs of the adrenals.

 

TREATMENT

 

Surgical intervention is the first line of treatment in all types of CS whenever the source is identified. In patients with CD, transsphenoidal resection of the pituitary tumor is the preferred approach. Endoscopic or microscopic approaches have been attempted. A recent meta-analysis has not showed significant differences in the remission rate between the two approaches overall, but endoscopic approach may be preferrable in macroadenomas (96, 97). In very young patients, pneumatization of the sphenoid sinus may be incomplete and the surgical approach more be more difficult but transsphenoidal access is still possible (98). In rare cases of very young children with pituitary lesions or in giant complex pituitary tumors, the transcranial approach may be considered (99). Remission is defined as postoperative nadir cortisol levels of <2-5mcg/dl and early postoperative hypocortisolemia is a sensitive marker of durable remission (76, 100). In cases of non-remission patients may be managed with immediate reoperation and partial hypophysectomy (101). In the pediatric cohorts the remission rate after surgery ranges from 62 to 98% depending on the cohort and the criteria used (8, 102-104).

 

In cases of ACTH-independent CS, bilateral or unilateral adrenalectomy is recommended depending on the underlying cause (105). Although unilateral adrenalectomy has been suggested in cases of bilateral macronodular adrenocortical disease, data on unilateral adrenalectomy in micronodular adrenocortical disease are not clear (105).

 

ECS should primarily be managed with surgical resection.

 

In cases of persistent CD after surgery, medical therapy or radiation should be considered. At this time, no medical therapy for CS in the pediatric population has been approved by the FDA in the US and all treatments are considered as off-label use, but ketoconazole is approved by the European Medical Association for children >12 years of age. Medical therapies are divided in those directed to adrenal steroidogenesis, to pituitary tumor function, or to peripheral glucocorticoid action. Most commonly, steroidogenesis inhibitors are considered first line as they are more potent and faster acting. Of these, ketoconazole, metyrapone, osilodrostat, levo-ketoconazole and others have been used (106). Radiation therapy could be considered as an alternative second-line treatment but requires medical management and close monitoring until the radiation effect is apparent (107, 108). Finally, bilateral adrenalectomy is reserved for cases of severe CS persistent despite surgical or medical intervention. This is followed by lifelong adrenal insufficiency and patients should be monitored for the risk of Nelson syndrome (109).

 

POSTOPERATIVE MANAGEMENT

 

After successful surgical management, patients experience adrenal insufficiency. In CD the median duration of adrenal insufficiency is almost 12 months (110). Additionally, management of patients after remission of CS should also target symptoms of glucocorticoid withdrawal which may require supraphysiologic doses of glucocorticoids for a period of time and slow tapering to physiologic levels (111).

 

After recovery of the axis, regular screening for possible recurrence should be offered. Long term recurrence has been reported in 8-20% of pediatric patients with CD after initial postoperative remission (8). Screening for recurrence should be done preferably as in adults with two midnight or late-night salivary cortisol levels or with overnight dexamethasone suppression test annually (76).

 

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Familial Isolated Pituitary Adenoma

ABSTRACT

 

Familial Isolated Pituitary Adenoma (FIPA) is a term used to identify a genetic condition with pituitary tumors without other endocrine or other associated abnormalities. FIPA families contribute around 2% to the overall incidence of pituitary tumors. FIPA is a heterogeneous disease both in terms of the clinical phenotype as well as from the genetic background point of view. Some FIPA families have been identified to have germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene leading to incomplete penetrance of young-onset, mostly growth hormone, mixed growth hormone/prolactin-secreting, or prolactin-secreting pituitary adenomas. Due to the low penetrance, almost half of the AIPmutation-positive patients do not have a positive family history. Duplication of the orphan G protein coupled receptor GPR101 gene, located on Xq26.3, leads to high penetrance pituitary hyperplasia or adenoma resulting in infant-onset GH excess, usually with concomitant hyperprolactinemia, named X-linked acrogigantism (XLAG). The majority of the FIPA families, however, have no known genetic mutation. Their clinical picture includes various types of pituitary adenomas, either homogeneous (all affected family members have the same adenoma type) or heterogeneous (different adenoma types within the same family), presenting with low penetrance and an age of onset not significantly different from patients with sporadic pituitary adenomas. Here we review the clinical features, genetics and screening aspects of FIPA.

 

INTRODUCTION

 

Familial Isolated Pituitary Adenoma (FIPA) is a relatively new term. Introduced by Professor Beckers in 1999, FIPA describes families with pituitary adenoma and no other associated symptoms (1, 2). As opposed to occurring in isolation, familial pituitary adenomas have been recognized in several syndromic diseases, such as the classical MEN1 syndrome or Carney complex or the most recently described, such as hereditary paraganglioma syndromes (3-5), MEN4, and DICER1 syndrome (6) (Figure 1).  For additional information we refer the reader to other chapters within ENDOTEXT on syndromic familial pituitary adenomas.

 

Figure 1. Germline or Mosaic Mutations Causing Pituitary Tumors. Details for the syndromic forms can be found, among others, in the following sections https://www.endotext.org/chapter/multiple-endocrine-neoplasia-type-i/, https://www.endotext.org/chapter/carney-complex/, https://www.endotext.org/chapter/pituitary-adenomas-in-childhood/ and in these references (6-10).

 

Descriptions of familial pituitary adenoma families have been around for several hundreds of years, but only over the last decade has the clinical phenotype and, in some cases, the genetic abnormality been described. Interestingly, some of the patients with germline mutations present as simplex patients without any known family history, either due to low penetrance or due to de novo mutations.

 

Figure 2. Family Trees Demonstrating Examples of the Various Types of FIPA Families. In some AIP mutation-negative FIPA families unaffected obligate carriers can be identified by their position in the family tree, while in other family’s possible carriers of the unidentified gene cannot be identified. AIP mutation-positive kindreds can be ‘families’ or simplex cases. Most XLAG kindreds are simplex cases with females having de novo germline mutations while males have somatic mosaic mutations.

 

Previous data suggest that FIPA families contribute around 2% of the overall incidence of pituitary tumors, but this number may increase with increasing recognition of this clinical entity.

 

Around 10-20% of all FIPA families and 50% of familial isolated GH-producing Tumor families (11, 12) have been identified to have mutations within the aryl hydrocarbon receptor interacting protein (AIP) gene, located at 11q13. Germline mutations in AIP have also been identified in patients with young-onset pituitary adenomas, mostly GH-secreting or prolactin-secreting or silent GH/prolactin-producing adenomas with no apparent family history. These are called ‘simplex’ cases. Until recently, no somatic mutations had been described in the AIP gene in pituitary or other tumors (1). Duplication of the orphan G protein-coupled receptor GPR101 causes X-linked acrogigantism (XLAG) (13).While most of the XLAG cases are due to de novo mutations (germline or somatic mosaicism (14, 15)), to date three families have also been described. The causative gene for the rest and therefore the vast majority (90% only considering kindreds with 2 or more affected subjects) of FIPA families is currently unknown (16). Recently, a microdeletion upstream the GHRH gene, on chromosome 20, has been identified as another possible cause of severe infant-onset gigantism (17). New candidate genes are under active investigation in somatic and familial cases of pituitary adenomas (18), but some need further validation. Representative examples of FIPA family trees are shown in Figure 2.

 

CLINICAL FEATURES OF FIPA

 

Families with AIP mutations usually have a characteristic phenotype, which is usually substantially different from that ofAIP mutation-negative phenotype. In this section, we compare characteristics of AIP-mutated and non-AIP-mutated FIPA. Germline chromosomal defects leading to gigantism, including XLAG and a recently described microdeletion in chromosome 20 that leads to GHRH overexpression, have a drastically different phenotype and are discussed separately below.

 

Tumor Types

 

FIPA families can be homologous (i.e. all affected family members have the same type of tumor) or heterologous (i.e. family members can have different type of tumor) (Figure 2). Therefore, pure acromegaly, pure prolactinoma, and pure non-functioning pituitary adenoma (NFPA) families have been identified, while also mixed families such as acromegaly-prolactinoma, acromegaly-NFPA, prolactinoma-NFPA, prolactin-corticotrophinoma or even acromegaly-prolactinoma-NFPA families have been described. Somato-mammotrophinomas occur commonly, but are not consistently reported, probably as a result of variations in the reporting of tumor histology type. Figure 3a, b and c demonstrate the distribution of histological tumor types in FIPA families.

 

Figure 3a. Proportion of histological tumor types in the AIP positive FIPA population in the International FIPA Consortium cohort (n=911) (19).

Figure 3b. Proportion of tumor types in AIP mutation-positive FIPA families (12).

Figure 3c. Proportion of tumor types in AIP mutation-negative FIPA families (12).

 

In a study including familial as well as simplex (apparently sporadic) patients with germline AIP mutations, 78% of 96 patients developed GH-secreting adenomas (20) (half of the GH-secreting adenomas were somato-mammotrophinomas), 13.5% of patients developed prolactinomas, 7% developed non-functioning pituitary adenomas (NFPAs), and 1 patient developed a TSH-secreting adenoma. In another study, comprising 171 patients carrying AIPmutations, based on clinical diagnosis 70% had somatotrophinomas, 11% mixed GH/PRLomas, 12% had prolactinomas, and 8% had clinically non-functioning tumors (12). On histological testing some tumors show plurihormonal profile (Figure 3b). It is important to note that some non-functioning tumors are found to be somatotroph/lactotroph upon histological examination (21) – these are therefore ‘silent adenomas’. The distribution of tumors amongst 318 non-AIPmutated FIPA families (1310 patients) is represented in Figure 3c (12). Somatotrophinomas are the most common tumor type in both AIP mutation-positive and negative FIPA families (12, 19).

 

Gender Distribution

 

While higher numbers of males are identified with AIP mutations both in familial and simplex setting (12, 20), ascertainment bias due to physiological later puberty of boys and their normally taller stature cannot be ruled out (19), as in a carefully-studied large AIP mutation family equal number of affected males and females are present (22). There is a greater prevalence of females within AIP mutation-negative families, probably due to a higher number of prolactinomas (19).

 

Age of Onset

 

AIP gene mutation-positive FIPA patients have an earlier age of onset of diagnosis compared to those with AIP mutation-negative familial (23) or sporadic (20) pituitary adenomas. The age of onset of pituitary adenoma symptoms is 8 years earlier in the AIP mutation-positive group (mean age 19 years, SD ± 9.5, p<0.001), with diagnosis being made 6 years earlier (mean age of diagnosis 24.3, SD ± 11.9 vs 30, SD ± 13.5, p<0.001) than in the AIP mutation-negative population (12). In our international FIPA cohort, the familial cohort with AIP mutation-positive tumors had a peak age of onset during the 2nd and 3rd decades of life, with 65% of these patients’ developing symptoms aged ≤18 years (28.8% in the AIP mutation-negative group) and 87% by the age of 30 years (12). Previous work has shown that those families with AIP mutation-negative tumors demonstrate a more even spread of occurrence between the ages of 20 and 50, with a peak incidence around the age of 30 years old (19); the latest data suggests that the modal age group (42%) is 20-29 years (12). 

 

Young (<30 years) onset simplex patients, the AIP mutation-positive group, also developed tumors at a younger age than the mutation-negative group, with median ages of 16 years (IQR 14.8-22.3) and 22 years (IQR 16-26) respectively (19).

 

In the Bart’s international cohort, over 80% of the families with AIP mutations have at least one affected patient with gigantism or disease onset before the age of 18 years, while only 3 out of 46 AIP mutation-negative families have an onset of pituitary adenoma before the age of 18 years (23). Interestingly, probably due to earlier recognition of symptoms in affected FIPA families, the age of tumor onset appeared to be earlier in the second generation than in the first (mean age 29 ±10.2 years vs. 50.5± 14.2 years p<0.0001) (24).

 

Disease Penetrance

 

Disease penetrance in FIPA is incomplete. As there is a clear natural bias of affected patient referral and the clinical and genetic data in the individual families are incomplete, the calculation of disease penetrance is difficult. Additionally, it is important that penetrance always be considered in the context of the subject’s age.

 

In AIP positive mutation families, current data suggests 12.5-30% penetrance, but ranges between 10-90%, also depending on available data (19, 20, 23). It seems that the nature of the AIP mutation (truncating or non-truncating) does not have any effect on penetrance (19).

In AIP mutation-negative families, penetrance calculations are even more difficult as carrier unaffected family members (other than obligate carriers) cannot be distinguished from non-carrier unaffected subjects. The current calculation based on affected subjects, obligate carriers and 50% of potential carriers suggest 38±16% (23), but this is obviously a very significant overestimate.

 

Another way to compare penetrance between AIP positive and negative families is to count the known affected subjects within families. Penetrance in AIP mutation-negative families is probably lower than in AIP mutation-positive families, as the mean number of patients with disease in AIP mutation-positive families is 3.2±1.8 and in AIP mutation-negative families 2.2±0.5, P<0.001 (23).

 

De novo AIP mutation has been described in two cases so far: in a child with prolactinoma (c.721A>T; p.Lys241*) where the AIP mutation was not found in the parents (paternity confirmed) or his sister (19, 25). A second case was with identical twin gir