Hypoparathyroidism and Pseudohypoparathyroidism

ABSTRACT

In primary hypoparathyroidism with hypocalcemia and hyperphosphatemia, deficient parathyroid hormone (PTH) secretion most commonly occurs from surgical excision of, or damage to, the parathyroid glands. The term idiopathic hypoparathyroidism describes isolated cases when a cause is not obvious, and there is no family history. However, hypoparathyroidism is also a feature common to a variety of hereditable syndromes that may present de novo. Familial isolated hypoparathyroidism may show autosomal dominant, autosomal recessive, or X-linked inheritance. Genes involved include PTH, SOX3, CASR, GNA11 and GCM2. Parathyroid hypoplasia is a frequent feature of 22q11.2 deletion syndrome with involvement of the TBX1 gene. The Hypoparathyroidism, Nerve Deafness, and Renal Dysplasia syndrome is due to haploinsufficiency of the GATA3 gene. Antibodies against parathyroid tissue are found in isolated hypoparathyroidism or combined with other endocrine deficiencies. Antibodies against the CASR occur in type 1 autoimmune polyglandular syndrome, due to mutations of the AIRE gene, or in acquired hypoparathyroidism. Disorders characterized by end-organ resistance to PTH are described collectively by the term pseudohypoparathyroidism (PHP), and PHP1A and PHP1B are caused by maternally-inherited changes at the imprinted GNAS complex gene that encodes the Gsα protein. Deleterious mutations of the PTH1R gene show resistance to PTH and PTHrP and present as Blomstrand lethal chondrodysplasia, Eiken syndrome, endochondromatosis, and primary failure of tooth eruption. Calcium and vitamin D are the standard therapy for the management of hypoparathyroidism, with hormone replacement [recombinant human PTH(1-84)] therapy recently becoming an option. Calcilytics, PTH analogs, and orally active small molecule PTH1R agonists may, in the future, join the treatment armamentarium.

PRIMARY HYPOPARATHYROIDISM

Primary hypoparathyroidism is caused by a group of heterogeneous conditions in which hypocalcemia and hyperphosphatemia occur as a result of deficient parathyroid hormone (PTH) secretion (1). This most commonly results from surgical excision of, or damage to, the parathyroid glands. However, autoimmune disease is also a significant factor in acquired cases, and genetic forms of hypoparathyroidism due to decreased PTH secretion are not rare (Table 1).

Table 1. Forms of Hypoparathyroidism having a Genetic Basis
Isolated 1) Autosomal dominant A) PTH mutation B) CASR activating mutation (ADH1) a) Bartter Syndrome Type V C) GCM2 mutation (dominant negative) D) GNA11 activating mutation (ADH2) 2) Autosomal recessive A) PTH mutation B) GCM2 mutation 3) X-linked Congenital multi-system syndromes* 1) DiGeorge 1 (22q11) & 2 (10p) 2) Barakat/HDR 3) Kenny-Caffey 1 & 2 and Sanjad-Sakati Metabolic disease 1) Mitochondrial neuromyopathies 2) Long-chain hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency 3) Heavy-metal storage disorders Autoimmune disease 1) Autoimmune polyendocrine syndrome type I (APS-1 / APECED) Parathyroid resistance syndromes 1) Pseudohypoparathyroidism 2) Blomstrand chondrodysplasia and related PTH receptor defects 3) Hypomagnesemia * Clarke et al. (2) list other potential syndromic associations with hypoparathyroidism, including: CHARGE (Coloboma, Heart defect, Atresia choanae, Retarded growth and development, Genital hypoplasia, Ear anomalies/deafness), Dubowitz, lymphedema, nephropathy & nerve deafness

Table 1. Forms of Hypoparathyroidism having a Genetic Basis

Isolated

1) Autosomal dominant

A) PTH mutation

B) CASR activating mutation (ADH1)

a) Bartter Syndrome Type V

C) GCM2 mutation (dominant negative)

D) GNA11 activating mutation (ADH2)

2) Autosomal recessive

A) PTH mutation

B) GCM2 mutation

3) X-linked

Congenital multi-system syndromes*

1) DiGeorge 1 (22q11) & 2 (10p)

2) Barakat/HDR

3) Kenny-Caffey 1 & 2 and Sanjad-Sakati

Metabolic disease

1) Mitochondrial neuromyopathies

2) Long-chain hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency

3) Heavy-metal storage disorders

Autoimmune disease

1) Autoimmune polyendocrine syndrome type I (APS-1 / APECED)

Parathyroid resistance syndromes

1) Pseudohypoparathyroidism

2) Blomstrand chondrodysplasia and related PTH receptor defects

3) Hypomagnesemia

* Clarke et al. (2) list other potential syndromic associations with hypoparathyroidism, including: CHARGE (Coloboma, Heart defect, Atresia choanae, Retarded growth and development, Genital hypoplasia, Ear anomalies/deafness), Dubowitz, lymphedema, nephropathy & nerve deafness

The signs and symptoms of hypoparathyroidism include evidence of latent or overt neuromuscular hyperexcitability due to hypocalcemia (Table 2). The effect may be aggravated by hyperkalemia or hypomagnesemia, but there is wide variation in the severity of symptoms. Patients may complain of circumoral numbness, paresthesias of the distal extremities, or muscle cramping, which can progress to carpopedal spasm or tetany. Laryngospasm or bronchospasm and seizures may also occur. Other less specific manifestations include fatigue, irritability, and personality disturbance. A comprehensive list of features associated with hypocalcemia can be found in the Endotext chapter, “Hypocalcemia: diagnosis and treatment” by Schafer & Shoback (3).

Severe hypocalcemia may be associated with a prolonged QT_c interval on electrocardiography, which reverses with treatment. More extensive cardiomyopathic changes may be seen. These include chest pain, elevated enzymes (CPK), left ventricular impairment, and T-wave inversion, suggestive of a myocardial infarction (4, 5) . Patients with chronic hypocalcemia may have calcification of the basal ganglia or more widespread intracranial calcification, detected by skull X-ray or CT scan. Also seen are extrapyramidal neurological symptoms (more often with intracranial calcification), subcapsular cataracts, band keratopathy, and abnormal dentition.

Table 2. Some Clinical Features of Hypocalcemia
· Neuromuscular irritability · Paresthesias · Laryngospasm · Bronchospasm · Tetany · Seizures · Chvostek sign · Trousseau sign · Prolonged QT interval on ECG

Table 2. Some Clinical Features of Hypocalcemia

· Neuromuscular irritability

· Paresthesias

· Laryngospasm

· Bronchospasm

· Tetany

· Seizures

· Chvostek sign

· Trousseau sign

· Prolonged QT interval on ECG

Increased neuromuscular irritability may be demonstrated by eliciting a Chvostek or Trousseau sign. A positive Chvostek sign is a prolonged reflex contraction of the facial muscle in response to a digital tap on the cheek just anterior to the ear. As with other hyperreflexias, up to 20% of normal individuals may demonstrate a slight positive reaction. A positive Trousseau sign is carpopedal spasm induced by inflation of a blood pressure cuff covering the upper arm to 20 mm Hg above systolic blood pressure for three minutes. This response reflects the heightened irritability of nerves undergoing pressure ischemia.

In hypoparathyroidism, serum calcium concentrations are decreased and serum phosphate levels are increased. Serum PTH is low or undetectable. (The important exception is PTH resistance, discussed further below.) Usually, serum 1,25-dihydroxyvitamin D (1,25(OH)₂D) is low, but alkaline phosphatase activity is normal. Despite an increase in fractional excretion of calcium, intestinal calcium absorption and bone resorption are both suppressed. The renal filtered load of calcium is decreased, and the 24-h urinary calcium excretion is reduced; nephrogenous cyclic AMP excretion is low and renal tubular reabsorption of phosphate is elevated.

The terms idiopathic or isolated hypoparathyroidism have been traditionally used to describe isolated cases of glandular hypofunction when a cause is not obvious and there is no family history. However, hypoparathyroidism is a feature common to a variety of heritable syndromes that may present de novo. Hypoparathyroidism can occur because of a congenital hypoplasia/aplasia with or without other congenital anomalies such as dysmorphic facies, immunodeficiency, lymphedema, nephropathy, nerve deafness or cardiac malformation. Thus, in patients with hypoparathyroidism of uncertain onset, a careful examination of craniofacial features and assessment of endocrine, cardiac and renal systems should be performed to exclude a syndromic cause. Similarly, autoimmune hypoparathyroidism can occur as an isolated endocrine condition or with other glandular deficiencies in a pluriglandular autoimmune syndrome, requiring attention to multi-organ endocrine dysfunction.

A significant number of patients with idiopathic hypoparathyroidism and hypercalciuria, but no other anomalies may be found to have de novo activating mutations of the CASR gene.

Because of the implications for treatment, CASR molecular screening of patients with this presentation is recommended (6, 7).

Familial Isolated Hypoparathyroidism

Familial isolated hypoparathyroidism (FIH) may show autosomal dominant, autosomal recessive, or X-linked inheritance.

In a few instances of autosomal dominant disease, a mutation in the PTH gene (MIM# 168450 (8) - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM) has been found. In one family, a missense mutation (C18R) in the signal sequence of the preproPTH precursor has been identified (9) and the mutant shown to be defective in vitro in processing preproPTH to proPTH, although, as patients had one normal gene copy, the autosomal dominant mode of inheritance remained unexplained. Then, further studies in transfected cells showed that the mutant was trapped in the endoplasmic reticulum (ER) promoting ER stress and apoptosis (10). In a family with autosomal recessive hypoparathyroidism, a different, homozygous, signal sequence mutation (S23P) segregates with affected status (11). This mutation may prevent proper cleavage of the signal peptide during processing of the nascent protein. In a girl with isolated hypoparathyroidism, a homozygous S23X signal sequence mutation was found predicting a truncated inactive PTH peptide (12). However, the circulating PTH level was not undetectable, suggesting some translational readthrough of the mutant preproPTH mRNA. A homozygous [Cys25]PTH(1-84) mutation that impairs PTHR1 activation was identified in an idiopathic hypoparathyroid family (13). Elevated circulating PTH levels were found in some (but not all) assays thus defining a novel form of hypoparathyroidism. In another family with autosomal recessive hypoparathyroidism, a donor splice site mutation at the exon 2/intron 2 junction of the PTH gene was identified (14). The mutation leads to exon skipping and loss of exon 2 containing the initiation codon and signal sequence of preproPTH mRNA. The SOX3 gene encodes a transcriptional factor likely involved in the embryonic development of the parathyroid gland (15). In two multigeneration families with X-linked recessive hypoparathyroidism exhibiting neonatal onset of hypocalcemia and parathyroid agenesis, the trait was mapped to a 906-kb region on distal Xq27 that contains three genes including SOX3 but no intragenic mutations were found (MIM# 307700). An interstitial deletion-insertion involving chromosomes 2p25.3 and Xq27.1 was found downstream of SOX3 and was speculated to exert a positional effect on SOX3 expression (16).

Gain-of-function mutations in the calcium-sensing receptor (CASR) gene (MIM#601199) have been identified in a number of families clinically diagnosed with autosomal dominant hypocalcemia type 1 (ADH1 – MIM#515361) (17, 18). In the parathyroid gland, the activated CASR suppresses PTH secretion, and in the kidney, it induces hypercalciuria that may contribute to the hypocalcemia. In many cases of ADH1, the family history is positive, but de novo mutations are quite common (19, 20). Mosaicism for de novo mutation in an otherwise healthy parent has been described (21), and may explain some cases of apparently recessive disease. Most importantly, there are implications for counseling parents about the risks of recurrence.

Almost all of the activating mutations are missense and appear almost equally divided between the amino-terminal third of the extracellular domain (ECD) and the transmembrane domain (TMD). Of special interest is the cluster of ECD mutations (A116T to C131W) that cause an increase in receptor sensitivity to extracellular calcium, suggesting that this region is critical for receptor activation. This cluster overlaps the two cysteine residues –cys-129 and cys-131– involved in the interface of the mature protein dimer (22). Further details can be found in the locus-specific database –http://data.mch.mcgill.ca/casrdb/ (23) and (24).

Although Bartter syndrome subtype V is represented by only a handful of cases with heterozygous severe activating mutations in the CASR (MIM#601199), it provides additional insight into the functioning of the CaSR in the thick ascending limb (TAL) of the nephron (25-27). Bartter syndrome encompasses a heterogeneous group of electrolyte homeostasis disorders, the common features of which are hypokalemic alkalosis, hyperreninemia, and hyperaldosteronism. Bartter syndrome subtypes I–IV are autosomal recessive disorders due to inactivating mutations in the following ion transporters or channels active in the TAL: type I, the sodium potassium-chloride cotransporter (NKCC2); type II, the outwardly rectifying potassium channel (ROMK); type III, the voltage-gated chloride channel (CLC-Kb); type IV, Barttin, a chloride channel beta-subunit that is required for trafficking of CLC-Ka and CLC-Kb. Patients with the autosomal dominant Bartter syndrome subtype V have, in addition to the classic features of the syndrome, hypocalcemia, and may exhibit neuromuscular manifestations, seizures, and basal ganglia calcifications. NKCC2 and ROMK in the apical membrane (luminal side) of the TAL have been proposed to generate a transepithelial electrochemical gradient that drives passive paracellular transport of Na⁺, Mg²⁺, and Ca²⁺ from the lumen to blood (28). The CASR is situated in the basolateral membrane (antiluminal side) and, when activated, increases 20-hydroxyeicosatetraenoic acid and decreases cAMP concentrations, both of which would inhibit ROMK and NKCC2 activities (28, 29). Thus, severe activating mutations of the CASR lead to the salt wasting of Bartter syndrome in addition to the hypercalciuric hypocalcemia of ADH1.

Heterozygous gain-of-function missense mutations of GNA11 have been identified in ADH patients without detectable CASR activating mutations (30-33). The GNA11 activating mutations increase the sensitivity of the parathyroid gland and renal tubule to extracellular calcium concentrations. Autosomal dominant hypocalcemia and hypoparathyroidism due to CASR and GNA11 mutations are now designated as ADH type 1 (MIM#601198) and type 2 (MIM#615361) respectively. The human Gα11 protein (a Gq family member – MIM#139313) has 359 amino acids with an α-helical domain in the NH₂-terminal region, a GTPase domain in the COOH-terminal region, and three switch regions (SR1-3) in the middle portion that change conformation based on whether GTP or GDP is bound (34). The R80C, R181Q, S211W, F341L, and V304M mutations found in hypocalcemic individuals are predicted by 3D modeling to alter the normal Gα11 protein structure. Moreover, cells stably expressing the CASR and transfected with the mutants exhibit increased sensitivity to changes in extracellular calcium (30-33).

Inactivating mutations in the CASR regulator, the adaptor protein 2 sigma subunit encoded by the AP2S1 gene, cause familial hypocalciuric hypercalcemia type 3 (35). The search for activating mutations in AP2S1 in familial and sporadic isolated hypoparathyroid patients negative for CASR or GNA11 mutations that would represent an additional genetic cause of ADH has thus far been negative (36, 37).

Recessively inherited FIH may occur with mutations of the glial cells missing-2 gene (GCM2; MIM#603716). The GCM2 gene localizes to chromosome 6p24.2 and encodes a transcription factor. It is expressed in the PTH-secreting cells of the developing parathyroid glands and is critical for their development in terrestrial vertebrates (38-40). A patient with neonatal hypoparathyroidism was found to be homozygous for a partial deletion acquired from both parents (41), and a pair of siblings with homozygous mutations has been reported (42). Additional studies have identified inactivating GCM2 mutations in cases with autosomal recessive FIH (43, 44). On the other hand, heterozygous mutations that cause dominant-negative GCM2 mutants have also been identified in patients with autosomal dominant hypoparathyroidism (43, 45, 46). Additional recessive and dominant GCM2 mutations have been noted in this gene that continues to be expressed in the adult parathyroid [see (47)]. Nevertheless, it appears that the prevalence of genetic defects affecting GCM2 function is not high in isolated hypoparathyroidism, as a recent study investigating 20 unrelated cases with this disorder (10 familial and 10 sporadic) failed to identify any GCM2 mutations segregating with the disease and/or leading to loss of function (48). Of further interest is that a genetic variant, Y282D that demonstrates significantly enhanced transcriptional activity relative to wild-type GCM2 associates with hyperparathyroidism in some cohorts of the sporadic primary disorder (49). Most recently, novel heterozygous active GCM2 variants that segregate with affected status in some kindreds with familial isolated hyperparathyroidism have been described (50). Thus, like CASR and GNA11, both gain-of-function and loss-of-function variants of GCM2 may contribute to calcemic disorders.

Hypoparathyroidism with Syndromic Features

Hypoparathyroidism due to parathyroid hypoplasia is a frequent feature of 22q11.2 microdeletions, the most common cause of DiGeorge syndrome 1 (DS1; MIM#188400) (51, 52) . This syndrome complex arises from a failure of the third and fourth pharyngeal pouches to develop, leading to agenesis or congenital hypoplasia of the parathyroid glands, thymus, and the anterior heart field. Patients with DS1 may typically present with neonatal hypocalcemic seizures due to hypoparathyroidism, severe infections due to thymic hypoplasia, and conotruncal heart defects (53). Because a microdeletion is involved, the identification of novel developmental genes in the 22q11 region has been keenly pursued. One of the genes is TBX1, encoding a DNA-binding transcription factor of the T-box family known to have important roles in vertebrate and invertebrate organogenesis and pattern formation (54, 55). Mouse models with Tbx1 haploinsufficiency established the essential contribution of this factor to conotruncal development (56), and placed it in developmental context during organogenesis (57, 58). However, while the Tbx1 null mutant mice had all the developmental anomalies of DS1 – thymic and parathyroid hypoplasia, abnormal facial structures and cleft palate, skeletal defects and cardiac outflow abnormalities – Tbx1 haploinsufficiency in mice was associated with only defects of the fourth pharyngeal pouch responsible for the cardiac outflow abnormalities (59). cDNA microarray analyses of mice lacking Tbx1 have identified Gcm2 as one of the downregulated genes in the pharyngeal region, indicating that Tbx1 is upstream of Gcm2 (60). Furthermore, as Tbx1 is regulated by sonic hedgehog (Shh) (61), a Shh-Tbx1-Gcm2 parathyroid developmental pathway is indicated.

The basis for the phenotypic differences between DGS1 patients who are heterogeneous for TBX1 loss and the Tbx1+/- mice is unclear but could reflect a species-specific gene dosage requirement together with roles of downstream genes regulated by Tbx1. Some patients may have late-onset DGS1 and develop symptomatic hypocalcemia in childhood or later with only subtle phenotypic abnormalities (62, 63). Of note is that the age of diagnosis in rare families with DGS1 patients having TBX1 inactivating (missense or frameshift) mutations ranged from 7 to 46 years in keeping with late-onset DGS1 (54).

The 22q11.2 deletion syndrome (22q11.2DS) encompasses a wider spectrum of clinical conditions that includes isolated congenital heart disease and velocardiofacial (VCF) syndrome (52). Associated craniofacial abnormalities include cleft palate, pharyngeal insufficiency and mildly dysmorphic facies. In the VCF syndrome, anatomical anomalies of the pharynx are prominent and hypernasal speech due to abnormal pharyngeal musculature with or without cleft palate is typical. In most patients, some degree of intellectual deficit is present and there is strong predisposition to psychiatric illness (schizophrenia or bipolar disorder) in adolescents and adults (64, 65). Further information, both clinical and educational, can be found at web sites specifically devoted to this condition [see (66)].

The 22q11.2DS is due to one of the most common microdeletions (1 in 4000 live births), and it may go clinically unrecognized in its milder or variant forms. Most cases with hypoparathyroidism (~50% of cases) are the result of de novo deletion through meiotic non-allelic homologous recombination, and driven by a unique cluster of low copy repeats designated LCR22 A-H [see (66, 67) ]. Most commonly (~85% of cases), a deletion of ~3 Mb is found, encompassing proximal repeats A to D. Many of the others (~10% of cases) involve atypical nested deletions including those spanning LCR22 A to B. Thus, LSR22 A to B, which includes the TBX1 gene, is the primary site contributing to parathyroid dysgenesis. Detailed characterization and long-term follow-up for the hypoparathyroid component of this disorder is ongoing.

Although most cases of DiGeorge syndrome are sporadic, as mentioned above autosomal dominant inheritance is not unknown. In utero influences may be important determinants of the clinical picture, since there are instances of monozygotic twins with discordant phenotypes (68-70). Phenocopies occur with diabetic embryopathy, fetal alcohol syndrome, and retinoid embryopathy. In rare instances, it has been shown that a phenotypically normal parent can transmit a microdeletion to an offspring. Such parents have been found to carry a duplication of the 22q11 on the second chromosome, and the combination of duplication and deletion alleles in a parent generates a balanced state, termed “gene dosage compensation” (71, 72).

Although the hypoparathyroidism affects about half of all carriers, it is usually not severe, and frequently treatment following neonatal hypocalcemia can be tapered or stopped in older children. However, the hypoparathyroidism may also remain asymptomatic until adolescence or emerge at times of stress, such as corrective cardiac surgery or severe infection, suggesting that continued surveillance of parathyroid gland reserve is important (73-75).

Traditionally, diagnosis of 22q11.2DS is established with specific cytogenetic studies -- usually with locus-specific fluorescence in-situ hybridization (FISH) testing. These tests will pick up many of the larger common deletions that involve regions of low-copy number repeats (LCRs). However, specific chromosomal array-based and MLPA analyses are now preferred, as they have been shown to have increased sensitivity for smaller deletions (66). Recently, the diagnostic power of next-generation sequencing has been harnessed to identify almost all of the microdeletions underlying sporadic and inherited forms of the disorder (52). Non-invasive prenatal screening and pre-implantation genetic diagnosis) are also clinically available (76). Because the clinical picture is so variable and the prevalence so high, testing for 22q11.2 microdeletion should be considered in the workup for any new hypoparathyroid case for which another cause is not found. Finally, distinct genetic defects can coexist with 22q11.2DS, as exemplified by the finding of concurrence of this syndrome in an adolescent with longstanding hypercalcemia who had familial hypocalciuric hypercalcemia type 3 due to an AP2S1 mutation (77).

Clinicians will also want to be aware that a small but significant minority (~10%) of patients will have associated autoimmune disease, driven in part, perhaps, by the thymus-based defect in T cell function (64,79). Among the more common (non-endocrine) conditions are arthritis, celiac disease, and autoimmune hematologic disease, particularly idiopathic thrombocytopenic purpura. Autoimmune thyroid disease, with either hypo- or hyperparathyroid states, has been reported (78, 79), and serum TSH assay should be measured regularly. It has been suggested that the later-onset hypoparathyroid disease may be partly autoimmune in origin, not developmental. A survey of 59 Norwegian patients showed discordance of adult onset disease with neonatal hypoparathyroidism, but a significant correlation with parathyroid autoantibodies and the presence of autoimmune disease (78).

The clinical features of DiGeorge syndrome, including hypoparathyroidism, also occur with other cytogenetic abnormalities, notably chromosome 10p haploinsufficiency (80, 81). Deletions of two non-overlapping regions of chromosome 10p contribute to DiGeorge syndrome 2; DS2 at 10p13-14 (82), and the Barakat or HDR (Hypoparathyroidism, Nerve Deafness, and Renal Dysplasia) syndrome (MIM#146255) (83, 84) at 10p14-10pter (85, 86). The latter is due to haploinsufficiency of GATA3 (MIM#131320), which encodes a dual zinc finger transcription factor (87) that is essential for normal embryonic development of the parathyroids, auditory system, and kidney. Since the original description, several additional GATA3 loss-of-function mutations have been described in HDR patients [e.g., (88-91)]. Heterozygous Gata3-deficient mice develop parathyroid abnormalities as revealed by challenge with a diet low in calcium and vitamin D that are due to dysregulation of the parathyroid-specific transcription factor, Gcm2. Gata3-/- embryos at E12.5 lack Gcm2 expression and have gross defects in the fourth pharyngeal pouches, including absent parathyroid/thymus primordia (92). GATA3 transactivates the GCM2 promoter and, with GCM2, forms part of a transcriptional cascade essential for the differentiation and survival of parathyroid progenitor cells.

In another congenital disorder, Kenny-Caffey syndrome, hypoparathyroidism is found variably associated with the typical picture of growth retardation, osteosclerosis, cortical thickening of the long bones, and delayed closure of the anterior fontanel (93-96). The original description of the syndrome was of the autosomal dominant form now identified as KCS-2 (MIM#127000) that is caused by heterozygous mutations in the FAM111A gene (97-99). The full functions of FAM111A and how mutations in it cause the disorder are unclear. FAM111A has some homology to peptidases, and is involved with chromatin structure during DNA replication (100). KS-2 is allelic to the lethal disorder, osteocraniostenosis (OCS, MIM#6023611). Hypocalcemia due to hypoparathyroidism was found in some OCS patients who survived the perinatal period (96).

A recessively inherited form of Kenny-Caffey syndrome (KCS-1, MIM#244460) was noted to be similar to the recessive Sanjad-Sakati syndrome (MIM#241410) characterized by congenital hypoparathyroidism, seizures, growth and developmental retardation and characteristic dysmorphic features, including deep set eyes, depressed nasal bridge with beaked nose, long philtrum, thin upper lip, micrognatia and large, floppy ear lobes. Radiographs showed medullary stenosis reminiscent of Kenny-Caffey syndrome (96, 101). Linkage studies localized the recessive KCS-1 and Sanjad-Sakati syndromes to 1q42-43, and causative mutations in the tubulin chaperone E, TBCE, gene were identified in what is now known as Hypoparathyroidism, Retardation and Dysmorphism (HRD) syndrome (96, 102, 103) . This highlighted the role of TBCE that binds microtubules and proteasomes and protects against misfolded stress (104) in parathyroid development (105).

Hypoparathyroidism due to Metabolic Disease

Hypoparathyroidism is also a variable component of the neuromyopathies caused by mitochondrial gene defects (106). Among these are the Kearns-Sayre syndrome (ophthalmoplegia, retinal degeneration, and cardiac-conduction defects) (MIM#530000), the Pearson marrow pancreas syndrome (lactic acidosis, neutropenia, sideroblastic anemia, and pancreatic exocrine dysfunction) (107) (MIM#557000) and mitochondrial encephalomyopathy (MIM#540000). The molecular defects range from large deletions and duplications of the mitochondrial genomes in a large number of tissues (108, 109) to single base-pair mutations in one of the transfer RNA genes found only in a restricted range of cell types (MIM#590050). The role of these mitochondrial mutations in the pathogenesis of hypoparathyroidism remains to be clarified. However, mutations in HADHB, that encodes the β-subunit of mitochondrial trifunctional protein, cause infantile onset hypoparathyroidism and peripheral polyneuropathy (110).

Long-chain hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency (MIM#600890)
is an inborn error of oxidative fatty acid metabolism that may be accompanied by hypoparathyroidism (111). Whether the parathyroid disease is directly related to the enzyme deficiency or secondary to the accompanying mitochondrial disease needs further study.

Parathyroid insufficiency and symptoms of hypocalcemia are occasionally seen in inherited metabolic disorders leading to excess storage of iron (thalassemia, Diamond-Blackfan anemia, hemochromatosis) or copper (Wilson disease)(112). In most instances, there is similar dysfunction in other endocrine glands, and the parathyroid disease is usually mild. Nonetheless, recognition of the hypoparathyroid state may help explain otherwise non-specific symptoms and aid in overall management of these multisystem diseases.

Autoimmune Hypoparathyroidism: Acquired and Inherited Disorders

Antibodies directed against parathyroid tissue have been detected in up to 38% of patients with isolated hypoparathyroid disease, and over 40% of patients having hypoparathyroidism combined with other endocrine deficiencies (113, 114). Subsequently, a survey of a parathyroid expression library led to the identification of one protein selectively associated with the autoimmune process, the NACHT leucine-rich-repeat protein 5 (NALP5). Elevated antibody titers occur in half the patients with autoimmune hypoparathyroidism, with or without another autoimmune disease, but uncommonly in other conditions without hypoparathyroidism (114, 115).

Antibodies against the extracellular domain of the parathyroid CASR were originally reported in more than half of patients with either type 1 autoimmune polyglandular syndrome (APS-1, also known as autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy or APECED), MIM# 240300, (116) or acquired hypoparathyroidism associated with autoimmune hypothyroidism (117). This finding was confirmed in a subsequent study of 51 cases of idiopathic hypoparathyroidism, but there was a 13% positive rate in controls (118). Other studies of APS-1 patients have also identified elevated CASR antibodies in some cases but at a lower frequency (119-121). Although some have suggested that CASR antibody assays are clinically indicated in acquired hypoparathyroidism (122), it remains to be seen whether the autoantibodies are of primary or secondary importance (114, 123). There is now good evidence that autoantibodies can be functional activators of CASR and thereby could induce hypoparathyroidism. While presently there may not be a convenient clinical test for this, patient sera have been demonstrated to activate the CASR transfected into HEK cells in vitro (124). In some hypoparathyroid patients, both autoimmune parathyroid destruction and suppression by CASR activation may co-exist (125).

In APS-1, the most common associated manifestations are hypoparathyroidism with mucocutaneous candidiasis and Addison's disease. Additional features include pernicious anemia, chronic active hepatitis, alopecia, keratitis, gonadal failure, thyroid disease, pancreatic insufficiency, and diabetes mellitus (116). The phenotype is highly variable and patients may not express all elements of the basic triad, leading to the suggestion that the criteria used for molecular screening be relaxed (125, 126). The disease usually presents in infancy with chronic oral thrush, followed by hypoparathyroidism in the first decade, and then adrenocortical failure in the third. Interestingly, there is nearly 100% penetrance of hypoparathyroidism in females, but less than 60% in males, even though the adrenal hypofunction affects both sexes equally (119). Moreover, patients who develop the adrenal hypofunction first are less likely to be male and may never develop hypoparathyroidism. The responsible gene, called the autoimmune regulator (AIRE), maps to chromosome 21q22 and encodes a transcriptional regulator (127-129) . In the absence of AIRE protein, tissue-specific self-antigens are not expressed in the thymus and multiorgan autoimmunity develops, because negative selection of the T cells bearing the autoantigens is disrupted (130). Many patients with APS-1 can be shown to have autosomal recessive inheritance of the AIRE defect. In families with autosomal recessive mutations of AIRE, obligate heterozygotes may also have common autoimmune disorders but APECED is not seen (131). A phenocopy leading to acquired APS-1 may occur when the AIRE gene is silenced by thymic neoplasia (132). APS-1 has been associated with more than 300 mutations of the AIRE gene, and updates can be found in the online mutation database (https://grenada.lumc.nl/LOVD2/mendelian_genes/home.php?select_db=AIRE).

PARATHYROID RESISTANCE SYNDROMES

Pseudohypoparathyroidism

Several clinical disorders characterized by end-organ resistance to PTH have been described collectively by the term pseudohypoparathyroidism (PHP). They are associated with hypocalcemia, hyperphosphatemia, and increased circulating PTH. Target tissue unresponsiveness to the hormone manifests as a lack of increased phosphate excretion and, in some cases, cAMP excretion in response to PTH administration (133). The biochemical characteristics of the different forms of PHP are contrasted with those of hypoparathyroidism in Table 3.

Table 3. Biochemical Characteristics of Hypoparathyroidism and Pseudohypoparathyroidism
Defects	Serum PO4	PTH	25(OH)D	1,25(OH)2D	UcAMP*	UPO4*	Multiple Endocrine Defects
Hypoparathyroidism	↑	↓	-	↓	-	-	Yes/No**
Pseudohypoparathyroidism
Type 1a	↑	↑	-	↓	↓	↓	Yes
Type 1b	↑	↑	-	↓	↓	↓	No/Yes#
Type 1c	↑	↑	-	↓	↓	↓	Yes
Type 2	↑	↑	-	↓	-	↓	No

↑, increased; ↓, decreased; -, normal;

*Response to PTH infusion

**, depending upon the etiology.

#, variable, mild defects of the thyroid axis due to TSH resistance may be seen.

Albright Hereditary Osteodystrophy

Fuller Albright first recognized that the likely cause of the hypoparathyroid state in PHP is a constitutive absence of target tissue responsiveness (134). In many patients, the end-organ resistance is accompanied by a specific pattern of physical findings, called Albright hereditary osteodystrophy (AHO; MIM#300800). Typically, patients have short stature, round facies, brachydactyly, obesity, and ectopic soft tissue or dermal ossification(s) (osteoma cutis) (Figure 1). In the calvaria, this may manifest as hyperostosis frontalis interna (135). Intracranial calcification(s), cataracts and band keratopathy, subcutaneous calcifications, and dental hypoplasia are also common but are likely the consequences of longstanding hypoparathyroid hypocalcemia (Table 4, see below Figure 1). The brachydactyly may be asymmetric or not, and may involve one or both hands or feet, but the pattern is quite distinctive (136, 137). The shortening tends to involve the first distal phalanx, with a thumbnail (or first toenail) that is wider than it is long. The fourth and fifth metacarpals (or metatarsals) are frequently shortened out of proportion to the others and the second metacarpal is often spared. Radiographic analysis of the hands (pattern profiling) may be helpful in assessment of the brachydactyly (Figure 1)(138).

Figure 1. Albright’s hereditary osteodystrophy (AHO) and characteristic skeletal abnormalities. A) A child with AHO exhibiting short stature, obesity, and round facies. B) The hand X-ray of a patient with AHO, displaying brachydactyly of the fourth and fifth metacarpal bones. C) Dimpling over the knuckles of a clenched fist (also known as Archibald sign), indicating the short metacarpals. D) Evidence of brachydactyly in the hand, reflecting the shortened fourth and fifth metacarpals and the distal phalanx of the thumb. Images are from: Levine, MA (139).

Table 4. Incidence of signs and symptoms in PHP with AHO^a
	Percentage
Short stature	80
Obesity	50
Craniofacial
Round face	92
^bLenticular opacities	44
Strabismus	10
^bDental hypoplasia	51
^bBasal ganglia calcification	50
Thickened calvaria	62
Mental deficit	75
Brachydactyly
Brachymetacarpia	68
Brachymetatarsia	43
Brachyphalangia	50
Other connective tissue features
Decreased bone density	15
Ectopic ossification	56
^bSubcutaneous calcification	55

a Taken from Drezner and Neelon (1995).

b Features common to other forms of chronic hypoparathyroid hypocalcemia.

Although affected patients are generally short as adults, their bone age as children may be advanced and growth accelerated (138). Patients with AHO may be predisposed to hypertension (140), conductive and sensorineural hearing loss (135, 141), cord compression due to spinal anomalies (142), and movement disorders due to basal ganglia calcification (143). The features of AHO may be subtle in infancy or early childhood; in a few, there is little to see even in adulthood. The round facies, short neck, and low, flat nasal bridge are often accompanied by central obesity (144). A study showed that the obesity phenotype occurs primarily in those patients who also have multiple hormone resistance, i.e., PHP1A (see below), and according to data from mice, a hypothalamic mechanism, rather than hypothyroidism, is the primary underlying cause (145, 146). Interestingly, a study showed that GNAS mutations are not uncommon in severe childhood-onset obesity in the absence of other typical PHP findings (147).

Patients with brachydactyly, mental retardation, and other features closely resembling AHO have been found to carry microdeletions of chromosome 2q37; brachydactyly-mental retardation, BDMR; MIM#600430 (148). Genes important for skeletal and neurological development lie within this region. Haploinsufficiency of HDAC4 (MIM#605314), encoding a histone deacetylase that regulates gene expression during the development of many tissues including the bone, is responsible for the brachydactyly and the mental retardation in those patients (149). Isolated brachydactyly type E (BDE, MIM#113300) has been associated in sporadic cases with mutations in HOX13 (MIM#168470) (150) and mutations in the PTHLH gene (MIM#168470) on 12p11.2 that encodes PTHrP have been implicated. In one family with autosomal BDE a cis-regulatory site downregulates PTHLH in translocation t(8;12)(q13;p11.2) and downregulates its targets ADAMTS-7 and ADAMTS-12 leading to impaired chondrogenic differentiation (151). Affected individuals of one large family with BDE, short stature, and learning difficulties had an ~900 bp microdeletion encompassing PTHLH (152). Additional individuals with BDE and short stature from other different kindreds were found to have PTHLH missense, nonstop, and nonsense mutations (152). Different translocations affecting chromosome 12p have also been identified in two families with BDE, leading to increased abundance of a long noncoding RNA on chromosome 12q, which regulates the expression of PTHLH in cis and of the SOX9 gene located on chromosome 17q in trans (153). BDE is associated with hypertension in some cases, in which the disease is inherited in an autosomal dominant manner (termed HTNB). Missense mutations in PDE3A, a gene encoding a cAMP/cGMP phosphodiesterase, have been recently found in several unrelated families with HTNB. These mutations cause increased cAMP hydrolytic activity and thus lead to diminished cAMP signaling (154). Some patients with AHO-like features have been described, who also showed platelet Gs hypofunction. Those patients were found to have IGF2 hypermethylation and SNURF hypomethylation, as well as imprinting defects within GNAS, the gene encoding the stimulatory G protein alpha-subunit (Gsα; see below) (155).

PHP1A

PHP1A patients are characterized by AHO, PTH resistance, and evidence of target organ resistance to other hormones. Patient-derived cells are found to have a reduction in the activity of the Gsα subunit, which is part of the membrane-associated heterotrimeric stimulatory G-protein complex - transducing signals between G-protein coupled receptors and adenylate cyclase (156-158). Adenylyl cyclase catalyzes the synthesis of the second messenger cAMP, and therefore, PHP1A patients tend to have a deficiency in cAMP generation, particularly in certain tissues. As explained above, this deficiency is clear when measuring cAMP excretion in response to PTH administration.

The GNAS gene (MIM#168470) encoding the Gsα protein maps to 20q13.2-13.3 and has at least 4 alternative transcriptional start sites (Figure 2) and an antisense transcript, GNAS-AS1 (159). The three upstream exons and the preceding promoter regions are genetically imprinted, i.e., methylated in an allele specific manner. The promoter of the Gsα transcript, which uses exon 1, is unmethylated. Unlike the other alternative GNAS products, Gsα expression is biallelic except in a small set of tissues, where Gsα is derived predominantly from the maternal allele (160-164) . This tissue-specific monoallelic Gsα expression affects the penetrance of the PHP phenotype. The maternal transmission of the hormone resistance in PHP1A (165) can be explained by the silencing of the paternal Gsα allele, which would otherwise allow expression of 50% of Gsα protein (166). Thus, the full expression of a coding GNAS mutation, which occurs in maternally transmitted cases, leads to AHO plus hormone resistance (PHP1A). On the other hand, if the same mutation is inherited paternally, it causes AHO alone. The latter is termed pseudopseudohypoparathyroidism (PPHP). Thus, PHP1A and PPHP can be found in the same families. Note that a systematic nomenclature and classification, “inactivating PTH/PTHrP signaling disorder” (iPPSD), has been suggested for PHP1A, PPHP, and related disorders arising from abnormal PTH and/or cAMP signaling, accounting for the underlying genetic/epigenetic abnormalities and associated phenotypes (167).

Despite clinical evidence supporting imprinting in portions of the kidney tubule, it has been difficult to confirm this experimentally in humans (168). The imprinting of GNAS is complex and involves multiple differentially methylated regions (DMR) (159). Moreover, it is tissue-specific and may vary with developmental stage, although key imprinting of the A/B (also referred to as 1A) DMR is thought to be a primary event that occurs during gametogenesis and is maintained thereafter (169). Ablation of the Gsα ortholog in mice (Gnas) has confirmed that maternal, but not paternal, transmission of the deleted allele results in PTH resistance. The homozygous deletion of Gnas is embryonic lethal (160). Comparison of Gsα expression in mice with maternally vs paternally disrupted Gsα expression also demonstrated that Gsα expression is predominantly maternal in the renal cortex, but not in renal medulla (160, 170) . PTH resistance is delayed until after infancy in most PHP1A patients, and a study using mice demonstrated that the silencing of the paternal Gsα allele develops postnatally (171).

Figure 2. Simplified view of the GNAS region and its transcripts. The normal allele-specific methylation and expression patterns of the four alternate first exons of GNAS which splice onto exon 2 to produce transcripts encoding NESP55, XLαs, 1A (referred to as A/B in humans), and Gsα (which uses exon 1). NESP55 and XLαs promoters are oppositely imprinted: NESP55 is expressed from the maternal allele and its promoter region is methylated on the paternal allele, whereas XLαs is expressed from the paternal allele and its promoter is methylated on the maternal allele. Gsα is paternally silenced in some tissues e.g., renal proximal tubule cells, indicated by the dashed arrow. NESP55 protein is unrelated to Gsα, and its entire coding region is located within its first exon. In contrast, XLαs and Gsα proteins have identical COOH-terminal domains (encoded by exons 2-13), while their unique NH2-terminal domains are encoded within their respective first exons. Exon A/B (1A) does not have a translational start site but is transcriptionally active. Loss of exon A/B imprinting (methylation) is associated with decreased Gsα expression in renal proximal tubules and some other hormone-responsive tissues and is the typical cause of PHP1B. (figure from Liu et al., 2000, with permission).

A variety of inactivating mutations in the Gsα-coding portion of the GNAS gene have been identified in PHP1A patients (172, 173). The spectrum includes missense mutations, point mutations impairing efficient and accurate splicing, and small insertion/deletion mutations. The 4-bp deletion in exon 7 (DGACT 188/190) has been observed in multiple unrelated cases, suggesting that this may be a hot spot (174, 175). Several other mutations have also been observed in more than one kindred, indicating that additional susceptibility regions may exist. The identification of de novo germline mosaicism (176) is consistent with the view that most sporadic cases harbor new mutations, but the separation of such sporadic cases from familial ones, in which there is suppression of phenotype due to imprinting, may be difficult without detailed molecular studies.

PHP1A cases have been described in which no mutations of the GNAS gene have been found by nucleotide sequence analysis of exons encoding Gsα. This may be because the mutation is in a regulatory region of the gene not yet examined, or it may be that a large deletion prevents amplification of the mutant allele for subsequent analyses. In cases without identified GNAS coding mutations, an assessment of Gsα bioactivity in erythrocytes is helpful in ruling out regulatory region mutations or large deletions. A 35-kb deletion spanning exons 1 through 5 has been identified by using comparative genome hybridization in a patient with PHP1A in whom coding mutations had been ruled out, but a marked reduction of erythrocyte Gsα activity demonstrated (177).

Typically, PHP1A is associated with multiple hormone resistance, including thyroid stimulating hormone (TSH) and gonadotropins, causing hypothyroidism and gonadal failure, respectively. Because the hypothyroidism may express before hypocalcemia is observed (178), early surveillance of thyroid function is warranted. However, thyroid replacement from birth does not appear to prevent the mental deficit typical of PHP1A. In women, the hypogonadism is partial (179), and thus, oral contraceptives may help regulate the menstrual cycle. Estrogen can antagonize bone resorption, leading to an exacerbation of hypocalcemia (180), but placental 1,25-dihydroxyvitamin D synthesis likely obviates this effect altogether in pregnancy so women are frequently normocalcemic at that time (181). Abnormalities of the somatotropin axis have also been reported, with documentation of subnormal growth hormone release following stimulation by L-arginine or growth hormone-releasing hormone (182, 183).

The tissue-specific silencing of the paternal Gsα allele also plays a key role in the development of the additional hormone resistance phenotypes, as monoallelic Gsα expression has been demonstrated in the thyroid, the ovaries, and the pituitary (161-164). Studies have revealed that obesity also develops primarily in patients who inherit the inactivating Gsα mutations from their mothers (184). Gsα is not imprinted in the white adipose tissue (185), but the investigations of mice in which Gsα is ablated conditionally in the brain showed that Gsα is also monoallelic in certain parts of the hypothalamus (145, 146), thus explaining the imprinted mode of inheritance of the obesity phenotype. This likely reflects impaired signaling downstream of the melanocortin receptor type-4 (MC4R), given that it signals via G proteins including Gsα and that inactivating MC4R mutations are causal for dominantly inherited morbid obesity (186, 187). Indeed, almost all GNAS mutations identified in a large cohort of children with severe obesity impaired MCR4 signaling in cell-based assays (147). In mice, ablation of the maternal but not paternal Gnas allele in the dorsomedial nucleus of the hypothalamus leads to obesity (145), similar to the findings in mice with the conditional MC4R deficiency in this part of the brain (188). Like obesity, it has been noted that cognitive impairment, a typical AHO feature, also develops primarily after maternal inheritance of the inactivating Gsα mutation (189), although the underlying mechanisms behind the parental-specific inheritance of this phenotype have yet to be defined.

PHP1B

PHP1B is typically not associated with AHO or a generalized reduction in Gsα expression (190-192). PHP1B patients show a defect in renal PTH signaling, but an apparently normal response to PTH in bone (193, 194). Affected individuals are therefore functionally hypoparathyroid but show normal skeletal architecture and development. Due to unimpaired PTH responsiveness in bone, however, signs of hyperparathyroid bone disease (osteitis fibrosa cystica) are occasionally observed, complicating the picture (195). Biochemical abnormalities suggestive of thyroid stimulating hormone resistance are also seen in some patients (164). In fact, sometimes, PHP1B cases can present first with hypothyroidism (196, 197). A study also demonstrated short stature and growth hormone deficiency in monozygotic twins with PHP1B (198). Abnormalities of renal uric acid handling have been documented (199, 200). However, clinically significant hormone resistance is restricted to PTH in most cases. Because the hormone resistance is mostly limited to PTH, it was thought at one time that these findings could be explained by a defect in the type-1 parathyroid hormone receptor (PTH1R, MIM#168468). Sequence analyses, however, found no mutations in protein-coding exons or gene promoter regions of the gene (201-203), and studies of PHP1B families show no linkage to PTHR1 (204, 205).

Most cases of PHP1B are sporadic, but a familial form of PHP1B with an apparent autosomal dominant mode of inheritance also exists (AD-PHP1B). In four AD-PHP1B kindreds, linkage to chromosome 20q13.3 was established, the same region which includes the GNAS locus (204). In these families, the pattern of transmission suggested paternal imprinting, and inheritance is therefore the same as for PHP1A. A further 13 PHP1B subjects were studied, some of whom had bone responsiveness to PTH (166). All lacked methylation of the alternate exon A/B, an epigenetic defect that is postulated to inhibit expression of the functional exon 1-containing Gsα transcript in renal tissues only (Figure 2). Thus, the loss of methylation of the maternal exon A/B allele leads to the silencing of the maternal as well as paternal Gsα allele, causing PTH resistance specifically in renal proximal tubule cells. A genetic analysis indicated that mutations in a regulatory region some distance from the GNAS coding exons were likely to account for the unique imprinting defect(s) associated with PHP1B (206). A search for the mutation revealed the presence of a 3 kb microdeletion that segregated with the disease in 12 kindreds with AD-PHP1B and also occurred in 4 sporadic cases (207). The deletion, flanked by direct repeats, removes 3 exons of the STX16 gene, which encodes syntaxin-16. Two other deletions within STX16 and larger deletions spanning both STX16 and its telomeric neighbor NPEPL1 have been identified in AD-PHP1B kindreds (208-211). In all these cases, maternal, but not paternal, inheritance of the STX16 deletion led to PTH resistance. Because STX16 is apparently not imprinted (208), loss of one copy of this gene is not predicted to underlie the PHP1B pathogenesis. Interestingly, two large deletions ablating NESP55 without any overlap with STX16 as the cause of PHP1B in families in whom affected individuals showed isolated loss of A/B methylation (211, 212). Note that the NESP55 region showed an apparent gain of methylation due to the deletion of the maternal allele.

In two other PHP1B kindreds, nearly identical deletions of the NESP55 DMR including exons 3 and 4 of the antisense transcript segregated with the disease (213). In this instance, however, the A/B DMR was not the only region to lose the differential methylation required to allow maternal expression of Gsα in the kidney. Maternal methylation was also lost in the regions of the XLαs and GNAS-AS1 promoters. Another kindred with these widespread epigenetic defects of GNAShas been described (214). The affected individuals in this kindred carried a maternally inherited deletion that removed antisense exons 3 and 4 with flanking intronic regions but not the NESP55 exon. Additional genomic deletions or rearrangements in the chromosomal regions comprising GNAS have also been identified and proposed to underlie the GNAS methylation abnormalities in some AD-PHP-Ib cases (215-219).

These PHP1B deletions point to two different imprinting control regions (ICRs) for the GNAS complex locus: one within the STX16 gene and the other at the NESP55 DMR. The ICR defined by the deletion at the neighboring STX16 gene seems to be in a different location in the mouse, because the targeted ablation of the region homologous to the 3-kb deletion caused neither Gnas methylation defects nor PTH resistance in mice (220). Recently, genome-wide methylation analysis of embryonic stem cells indicated that the A/B region is modestly hypomethylated compared to differentiated cells (221, 222), suggesting that this imprinted region differs from most other imprinted loci and is regulated critically in the early embryo. Subsequently, a study showed that deleting either the maternal STX16-ICR or the maternal NESP55-ICR results in significant further A/B hypomethylation in human embryonic stem cells (hESCs) (223). Moreover, while wild-type hESCs recovered their methylation following a transient inhibition of the maintenance DNA methyltransferase DNMT1 (mimicking the global demethylation process in the preimplantation embryo), the cells with maternally deleted STX16- or NESP55-ICR failed to regain methylation (223). This study also showed that the shortest region of overlap among the PHP1B-causing STX16 deletions was shown to harbor a pluripotent cell-specific enhancer element for the NESP55 promoter on the maternal allele (223). Taken together with a mouse study implicating NESP55 transcription in the regulation of maternal GNAS imprints (224), these findings strongly suggest that the GNAS exon A/B imprint is controlled, at least partly, in the early embryo by the NESP55 transcript that relies on the long-range enhancer within STX16. Thus, perturbation of this mechanism appears to be the underlying cause of the GNAS methylation defects observed in familial PHP1B cases.

Sporadic PHP1B cases also show broad GNAS epigenetic defects that involve A/B. In some of these cases, paternal uniparental disomy of different chromosome 20 segments have been reported as the likely cause of PHP1B in several such cases (225-229). The cause of the epigenetic defects and PTH resistance, however, remains unknown for most cases of sporadic PHP1B. GNAS methylation defects have been identified in some cases with hypomethylation at multiple maternally methylated imprinted regions (230-233). In fact, some of those cases show both PTH resistance and the clinical features resulting from the methylation changes of the other loci, such as Beckwith-Wiedemann Syndrome.

A recent study revealed that, in addition to the exon A/B DMR, methylation at a new GNAS region close to the GNAS-AS1 promoter (termed GNAS-AS2), is lost in patients who carry STX16 deletions (234). Note that this region is also affected in those cases that display broad GNAS methylation changes. Recently, two distinct subdomains with the GNAS-AS2 region have been identified, and a patient with partial loss of A/B methylation showed gain-of-methylation in one subdomain and no alteration in the other (235). The effect of methylation changes at GNAS-AS2 has yet to be determined at the level of gene expression, and their pathophysiologic significance is unclear. Two distinct PHP1B families have been recently described to carry maternal retrotransposon insertions in the large intron between exon XL and A/B of the maternal GNAS allele (236, 237). These cases had apparently normal levels of GNAS-AS2 methylation (235, 237), reflecting, perhaps, that the deleterious genetic alteration is located downstream of this DMR. The mechanism by which these retrotransposons cause A/B hypomethylation may entail perturbation of NESP55 transcription. The inserted sequence comprises multiple polyadenylation signals (AAUAAA), which may truncate the transcript prematurely, and one of the studies showed that the level of NESP55 transcript was reduced in patient-derived induced pluripotent cells (236).

A study compared the clinical phenotypes of PHP1B patients who show isolated A/B loss of methylation to those with broad GNAS methylation defects (238). No clinical differences could be established according to the pattern of GNASepigenetic defects, although serum PTH levels were significantly higher in females with broad GNAS methylation defects than females with isolated loss of 1A methylation. Another study also found an intrauterine growth advantage for both AD-PHP-1b and sporadic PHP-1b cases, but the results indicate that the sporadic cases are not as markedly growth accelerated as AD-PHP-1b cases at birth (239).

Contrary to the classical understanding that AHO features are unique to PHP1A, some studies have identified patients with PTH resistance and AHO features who show GNAS epigenetic defects rather than Gsα coding mutations (200, 240-242). Thus, there may be some overlap between the clinical and molecular features of PHP1A and PHP1B. It is possible that the AHO features observed in patients with GNAS epigenetic defects result from a genetic mechanism that is similar to the mechanism underlying the hormone resistance in PHP1A patients, i.e., due to monoallelic Gsα expression in additional tissues.

A PHP1B family with a novel Gsα mutation, deletion of isoleucine-382 in the carboxyl terminus has been described (243). In transfected cells this mutation led to uncoupling from the PTHR1 and isolated PTH resistance but not from other receptors, including TSH receptor. However, the same mutation showed uncoupling from multiple receptors, questioning the role of this mutation in the pathogenesis of PHP1B in this family. Such mutations within Gsα coding exons are likely to be a rare cause of PHP1B (166).

PHP1c and PHP2

Patients with PHP1c have multiple hormone resistance but normal Gsα activity. The defect may be in other components of the receptor-adenylate cyclase system, such as the catalytic unit, but some PHP1c cases have been reported to carry Gsα coding mutations (244). These mutations render the Gsα protein unable to mediate cAMP generation in response to receptor activation but do not affect basal adenylate cyclase stimulating activity or the ability to be activated by non-hydrolyzable GTP analogs (244-246). Thus, some forms of PHP1c appear to be an allelic variant of PHP1A. Finally, patients with PHP2 have a normal urinary cAMP response to PTH but an impaired phosphaturic response (247). The defect could be in the cAMP-dependent protein kinase (PKA), one of its substrates or targets, or in a component of the PTH-PKC signaling pathway.

Impaired PTH-induced phosphaturia with normal nephrogenous cAMP formation (i.e., PHP2) appears as the least common form of PHP. PHP2 is a sporadic disorder, but a familial form of PHP2 has been reported (248). In addition, a self-limited form of this disease in newborns has also been described, suggesting that it is transient in nature (249-251). The etiology and pathophysiological mechanisms behind this PHP variant remain unknown. Because patients show adequate nephrogenous cAMP generation in response to exogenous PTH, molecular defects downstream of cAMP production are implicated, such as protein kinase A (247). Accordingly, a study (252) has discovered a heterozygous mutation of the gene encoding the regulatory subunit of PKA (PRKAR1A) in three patients with multiple hormone resistance and acrodysostosis, a form of skeletal dysplasia that includes severe brachydactyly type-E and other skeletal findings that resemble AHO (also known as Maroteaux-Malamut syndrome (253, 254). Several other variants of PRKAR1A have also been identified in other patients with a similar phenotype (255-258). These mutations, including the recurrent mutation R368X leading to the truncation of the C-terminal 14 residues, impair cAMP binding to the regulatory subunit, thereby blocking the activation of PKA (252, 259-261). In addition to acrodysostosis, patients carrying this mutation display evidence for target organ resistance to PTH, thyrotropin, growth hormone-releasing hormone, and gonadotropins, but these findings are accompanied by elevated basal plasma and urinary cAMP levels and with an apparently normal cAMP response to exogenous PTH administration. In certain other patients with acrodysostosis, but mostly without hormone resistance, it has been shown that the disease is caused by missense mutations in PDE4D, which encodes a cAMP phosphodiesterase (258, 262, 263). Given that PDE4D is an enzyme that reduces the intracellular cAMP concentration, the PDE4D mutations are likely to be gain-of-function (264). The type of acrodysostosis caused by PRKAR1A mutations has been termed acrodysostosis-1 (MIM#101800), while the one caused by PDE4D mutations acrodysostosis-2 (MIM#614613). In addition, another subtype of cAMP phosphodiesterase, PDE3A, is affected in another disorder characterized by severe hypertension and brachydactyly type-E with short stature (154, 265), underscoring the importance of cAMP signaling in skeletal development and the regulation of vascular tone.

**Other Phenotypes Associated with GNAS Mutations**

In contrast to the PHP phenotype associated with inactivating GNAS mutations, a different form of sporadic bone disease, (polyostotic fibrous dysplasia) results from de novo GNAS mutations that cause constitutive Gsα activity (266). A more severe form of this disease (panostotic fibrous dysplasia) with hyperphosphatasia and hyperphosphaturic rickets has also been described (267, 268) . Patients carrying these activating mutations are mosaic for mutant and wild-type cells, indicating that the mutation is acquired during postzygotic development. These mutations affect the arginine residue at position 201 (exon 8) and, rarely, the glutamine at 227 (exon 9), and inhibit the intrinsic GTP hydrolase activity of Gsα, thereby leading to constitutive activity. Such constitutively activating mutations of GNAS are also found in a variety of endocrine and non-endocrine tumors, such as growth hormone-secreting adenomas (269) . A missense mutation in exon 13 (A366S) results in a Gsα protein that is unstable at 37°C, but constitutively active at lower temperatures (270, 271). Affected patients have PHP due to PTH resistance and precocious puberty (testotoxicosis) due to hormone-independent constitutive activation of luteinizing hormone receptors at lower ambient temperatures in the testes. Another Gsα mutant carrying Ala-Val-Asp-Thr amino acid repeats in the guanine-binding domain has been described in a patient with neonatal diarrhea and PTH resistance (272). In this instance, the mutant protein is unstable and localized to the cytoplasm rather than plasma membrane, which explains the hormone resistance. On the other hand, this mutation increases the rate of GDP-GTP exchange and, thus, confers overactivity. The increased activity of Gsα seems to be evident during the neonatal period in the gut, where the mutant localizes to the plasma membrane, thus explaining the diarrhea phenotype. Additional cases with missense Gsα mutations have been reported, presenting with clinical findings that likely reflect both gain and loss of Gsα function (273, 274).

Inactivating GNAS mutations have also been identified in patients with congenital osteoma cutis and progressive osseous heteroplasia (POH), suggesting that these connective tissue conditions are another variant in the phenotypic spectrum of GNAS-related disease (275-278). No genotype-phenotype correlation has been revealed regarding these disorders, as the same mutation can be associated with either typical AHO features or severe ossifications that involve deep connective tissues and skeletal muscle (279). Nonetheless, patients with POH inherit the GNAS mutation from their fathers or acquire this mutation de novo on the paternal GNAS allele. This parent-of-origin specific inheritance of POH was established by analyzing 18 unrelated kindreds with this disorder (280). In a single, three generation, kindred, the inheritance of the mutation from males led to POH, while the inheritance of the same mutation from females led to typical AHO. It thus appears likely that alterations in the activity of a paternally expressed GNAS product, such as XLαs, contribute to the pathogenesis of POH. However, POH-like features have also been seen in some patients with maternally inherited GNAS mutations (281). A study revealed that the distribution of POH lesions follows dermomyotomes and shows a tendency for one-sidedness, suggesting that post-zygotic second hits may contribute to the development of these lesions on top of the inherited heterozygous mutations of GNAS (282).

Differential Diagnosis and Genetic Counseling

Patients with dysmorphic features resembling AHO may require careful endocrinologic work-up to confirm and delineate the form of PHP that is present. Similar studies of family members may also be warranted, since the biochemical and clinical features vary within families. If PHP1A with AHO is established, genetic counseling may aid in understanding the multisystemic nature of the disorder, particularly in relation to the patient's growth and development, and later-onset connective tissue complications. For either PHP1A or PHP1B, extensive counseling may be required to adequately explain the various implications of paternal imprinting for the parent-specific recurrence risks in offspring. Germline mosaicism has been reported (176) , which is clearly important in assessing risks for recurrence in future sibs of a singleton family. Given the recently described complexities in the molecular, biochemical, and physical features of PHP1A and PHP1B, molecular testing is critical for achieving a clear diagnosis and validating the inheritance pattern in any given family.

THE PARATHYROID HORMONE RECEPTOR AND SKELETAL DYSPLASIAS

PTHR1 is a family B G protein-coupled receptor that signals through multiple different G proteins including Gsα (283). It responds to two ligands, PTH and the PTH-related peptide (PTHrP). It would thus be predicted that deleterious mutations might show resistance to PTH, as well as evidence for a defect of PTHrP action. Functional polymorphisms in the PTHR1 are associated with adult height and bone mineral density (284), emphasizing the role that the receptor and its ligands play in endochondral bone formation. Inactivating or loss-of-function mutations in the PTHR1 have been implicated in the molecular pathogenesis of Blomstrand lethal chondrodysplasia (BLC; MIM#215045), and other skeletal dysplasias and dental abnormalities (285). The rare, recessive BLC is characterized by short-limbed dwarfism with craniofacial malformations, hydrops, hypoplastic lungs and aortic coarctation (286-290). The bones show accelerated endochondral ossification and deficient remodeling. The Blomstrand disease has been subdivided into type I, which refers to the severe (classical) form, and type II, which refers to a relatively milder variant, and the difference between severity is attributed to complete or incomplete inactivation of the PTHR1, respectively (291, 292). A milder form of recessively inherited skeletal dysplasia, known as Eiken syndrome (MIM#600002), has also been linked to mutations of PTHR1 (293). Dominantly acting PTHR1 mutations have been identified in endochondromas of patients with enchondromatosis (Ollier's disease - MIM#166000), a familial disorder with evidence of autosomal dominance characterized by multiple benign cartilage tumors, and a predisposition to malignant osteocarcinomas (294, 295). As many patients with Ollier’s disease do not have PTHR1 mutations, it is likely that the condition is genetically heterogeneous (296). Dominantly inherited symmetrical enchondromatosis is associated with duplication of 12p11.23 to 12p11.22 that includes the PTHLH gene encoding PTHrP suggesting that abnormal PTHR1 signaling may underlie this unusual form of endochondromatosis (297). In addition, some cases of autosomal dominant nonsyndromic primary failure of tooth eruption (PFE) are due to loss-of-function mutations in the PTHR1 that are dominantly acting, leading to haploinsufficiency of the receptor (298-302) .

HYPOMAGNESEMIA

In humans, hypomagnesemia leads to a suppression of parathyroid hormone release and some degree of peripheral resistance. Although the exact molecular mechanism underlying the suppression of the parathyroid gland in hypomagnesemia is unknown, it is important to recognize that laboratory testing in cases of hypocalcemia with reduced PTH should include measurement of serum magnesium, particularly in newborns (303). Primary hypomagnesemia with secondary hypocalcemia (HSH) is an autosomal recessive disorder characterized by neuromuscular symptoms in infancy due to extremely low levels of serum magnesium and moderate to severe hypocalcemia. Homozygous mutations in the magnesium transporter gene transient receptor potential cation channel member 6 (TRPM6) cause the disease. HSH, a potentially lethal condition, can be misdiagnosed as primary hypoparathyroidism (304). Long-term prognosis after treatment with high dose of oral magnesium supplementation is good. Hypomagnesemia is also associated with long-term use of proton-pump inhibitors that decrease the luminal pH of the intestine by acting on the enterocyte apical TRPM6/7 channels (305, 306).

MANAGEMENT OF HYPOPARATHYROIDISM

Calcium and Vitamin D.

The goal of treatment in hypoparathyroid states is to raise the serum calcium sufficiently to alleviate acute symptoms of hypocalcemia and prevent the chronic complications (307, 308). The calcium concentration required for this purpose is generally in the low-normal range. It is equally important to ensure that treatment does not result in hypercalcemia, as even transient hypercalcemia could lead to nephrocalcinosis and renal failure. Acute or severe symptomatic hypocalcemia is best treated with intravenous calcium infusion. Initial doses of 2 to 5 millimoles of elemental calcium as the gluconate salt can be given over a 10 to 20 minute period, followed by 2 millimoles elemental calcium per hour as a maintenance dose, to be adjusted according to symptoms and biochemical response. Care must be taken to ensure that the infusion does not extravasate, as this can lead to severe tissue damage. Where possible treatment through central access is preferred. Ionized or total calcium levels should be monitored frequently. Doses in children 5 to 14 years of age need to be adjusted for body weight, while neonates and infants require age-specific dosing. If present, hyperphosphatemia, alkalosis and hypomagnesemia should be corrected concomitantly. Post-surgical hypocalcemia after thyroid or parathyroid surgery is now rarely severe and usually transient with appropriate management (309). However, the occasional patient can represent a significant problem, particularly if the indication for surgery is chronic hyperparathyroidism, and the post-operative hypoparathyroid state is permanent (310). The long-term effects of standard therapy, hypercalciuria, nephrolithiasis, nephrocalcinosis, ectopic tissue calcification and mood changes, remain a concern (311).

The mainstay of chronic treatment is oral calcium and activated vitamin D (calcitriol), which should be started as soon as possible to allow reduction and discontinuation of the intravenous calcium. Oral calcium comes in several forms, but calcium carbonate is generally the least expensive. A total of 20 to 80 millimoles elemental calcium daily (2 to 8 g calcium carbonate per day) is generally effective, but should be given in divided doses and adjusted on the basis of gastro-intestinal tolerance, relief of hypocalcemic symptoms, and appropriate biochemical response. Vitamin D is preferably administered as calcitriol (0.25 to 1.0 micrograms per day), but, with some conditions, pharmacological doses of cholecalciferol or ergocalciferol or calcidiol may be less expensive and equally efficacious (312). Cholecalciferol and ergocalciferol doses are more difficult to titrate, and given their long duration of action, any overdoses can result in sustained toxicity. It is, therefore, appropriate to institute a starting dose of 25,000 IU/day and titrate upwards (to 100,000 IU/daily) with an assessment of serum and urinary parameters afterward with follow-up at 6 and 12 months, even if the patient is relatively asymptomatic. However, the use of active vitamin D (calcitriol or alphacalcidol) is recommended given that the lack of PTH along with the accompanying hyperphosphatemia reduces renal conversion of 25-hydroxyvitamin D to active vitamin D (307, 308). Serum calcium and 24-hour urinary excretion should be carefully monitored when therapy is started and continued until the dosing is stabilized. Hypercalciuria that occurs as treatment is initiated, even prior to the normalization of the serum calcium, may warrant an assessment of nephrocalcinosis by renal ultrasound. Consequently, only a low-normal serum calcium concentration may be attainable, but many patients feel well enough that there is no need to entirely normalize the serum calcium. In this way, the risk of renal failure due to chronic hypercalciuria − especially problematic in patients with CASR activating mutations (6, 7) − is minimized. Even after normalization or near-normalization of serum calcium, a significant number of patients report problems with fatigue, exhaustion, and mood disturbances (e.g., depression, anxiety, hostility, and paranoid ideation) not in keeping with the degree of hypocalcemia, suggesting that there may be non-calcitropic effects of PTH not remedied by maintenance of normocalcemia alone (311). In an epidemiological and health-related quality of life study from Norway, postsurgical hypoparathyroid patients scored worse than those with nonsurgical hypoparathyroidism or pseudohypoparathyroidism (313), providing further support for the notion of direct effects of PTH on mood.

In pseudohypoparathyroidism, calcitriol (and not other forms of vitamin D) should be used for the treatment, because the PTH resistance in the proximal tubule does not allow for the efficient synthesis of 1,25(OH)₂D from 25-hydroxyvitamin D. In pseudohypoparathyroidism type 1A, there is also a degree of PTH resistance in the bone due to haploinsufficiency of Gsα. However, in pseudohypothyroidism type 1B, the bone is fully sensitive to PTH, so monitoring serum PTH levels during treatment is critical with the aim of normalizing or reducing PTH levels as much as possible (314, 315). Hypercalciuria as a result of the calcitriol and calcium treatment is a lesser concern in pseudohypoparathyroidism because PTH actions in the distal tubule are still functional, preventing excess loss of calcium in the urine and providing greater protection against nephrocalcinosis.

Hormone Replacement Therapy

Hormone replacement has been advocated as a potentially superior form of treatment for decades but only recently have preparations of recombinant human hormone –– teriparatide (PTH 1-34), full-length parathyroid hormone (PTH 1-84), and abaloparatide (PTHrP analog) — become available. In 2015, the U.S. Food and Drug Administration (FDA) approved recombinant human (rh) PTH (1-84) for the management of hypoparathyroidism (316). This provided an additional therapeutic option for the management of those patients who demonstrate poor control with the standard calcium and active vitamin D supplemental therapy. The FDA indication was for subjects with hypoparathyroidism of any etiology, except ADH, but including postsurgical cases. The FDA did not limit the duration of its use but approved rhPTH(1-84) with a “black box” warning because of the history of rat osteosarcoma and PTH use (317). However, no evidence for this in primates or in clinical use has been forthcoming (318), and the ‘black box’ warning has since been withdrawn (319).

The use of PTH in hypoparathyroidism was demonstrated initially with the amino-terminal fragment of PTH, teriparatide [PTH(1–34)] (320). Beneficial control in children and in adults occurred when teriparatide was administered daily, with better control when the peptide was administered in twice-daily dosing regimens (320-324). With a pump delivery system by which teriparatide could be administered continuously (325, 326), urinary calcium excretion fell, and markers of bone turnover normalized. A smaller daily dose was required with pump delivery vs multiple daily dosing regimens. An open-label trial of PTH(1–34) in adult subjects with postsurgical hypoparathyroidism showed improvement in quality of life (327). Beneficial effects on calcium homeostasis have also been demonstrated in specific ADH cases with activating CaSR mutations (328, 329).

The full-length PTH (1-84) mimics the secreted product of the parathyroid gland, and its longer biological half-life (than PTH(1-34) makes once-daily dosing feasible in the treatment of hypoparathyroidism (330-332). Studies by several groups have noted a substantial reduction in the requirement for calcium and active vitamin D (333-335); only transient reductions in urinary calcium excretion (331); a tendency for lumbar spine bone mineral density (BMD) to increase and that of the distal one-third radius to fall (334); a rapid increase in bone turnover, assessed by circulating markers and dynamic histomorphometric analyses of bone that achieves a new steady state that is higher than baseline values within 2–3 years (336); and improvements in quality of life in some studies (333, 337).

In a placebo-controlled 24-week clinical trial of rhPTH (1-84) in 130 hypoparathyroid patients the primary endpoints of a reduction by 50% in calcium supplements and in active vitamin D along with maintenance of the serum calcium were met in over half of the study subjects (338). There was a greater percentage of subjects in whom active vitamin D could be eliminated entirely while taking no more than 500 mg of oral calcium daily. The drug was titrated from 50 to 100 μg/d, with just over half of the subjects needing the highest dose. The rhPTH(1-84) reduced serum phosphate levels, improved the calcium-phosphate product, and maintained 1,25(OH)₂D and serum calcium levels in the normal range (339). In addition, therapy with a long-acting prodrug of PTH(1-34), TransCon PTH (palopegteriparatide), in hypoparathyroidism has been shown to improve scores in quality-of-life measures (340). However, despite these early positive results, the inconvenient route of administration, daily or twice daily subcutaneous injection, leads to most patients opting for conventional treatment with oral calcium and calcitriol.

The manufacturer of rhPTH(1-34) has recently decided to discontinue this product at the end of 2024 due to an unresolved supply issue (https://www.takeda.com/newsroom/statements/2022/discontinue-manufacturing-natpar-natpara/). In addition, the use of teriparatide or aboloparatide for hypoparathyroidism has not been approved by the FDA. Therefore, no available FDA-approved hormone replacement therapies currently exist for the management of this disorder.

Calcilytics

Calcilytics are small molecule allosteric modulators of the CASR that antagonize the calcium-sensing receptor and promote PTH secretion and are a promising alternative for disorders with intact but hypofunctioning parathyroid glands (341). Calcilytics inhibit the activation of the CASR in both the parathyroid and renal tubule, and thus, they not only promote PTH secretion but also increase renal calcium reabsorption and are, therefore, of potential interest for the treatment of ADH1. In contrast, clinical studies in patients with ADH1 treated with PTH(1-34) led to better control of blood calcium levels (324), but the effects of the activated CASR in the kidney led to continued increases in urinary calcium excretion, different from patients with postsurgical hypoparathyroidism (326, 329). Thus, while FDA approval was given for PTH treatment of hypoparathyroidism, ADH1 was excluded from the indication.

In cell culture experiments studying activating CASR mutants, calcilytics normalize the left-ward shift of the calcium response curve (342, 343). The utility of calcilytics was further demonstrated in studies of mice harboring activating Casrmutations. In one study, two knock-in mouse models of ADH1 with activating mutations in the Casr were generated. Daily oral administration of the calcilytic JTT-305/MK-3442 to these mice increased serum PTH and calcium levels and reduced urinary calcium excretion (310). Intraperitoneal injection of the calcilytic NPS2143 in the nuf mouse model of ADH1, transiently increased circulating PTH and calcium levels without increasing urinary calcium levels (342). In a preliminary clinical study, IV administration of the calcilytic NPSP795 to five patients with ADH1 increased their plasma PTH levels and decreased their fractional urinary calcium excretion (344). Calcilytics comprise two main classes of compounds; the amino alcohols (e.g., NPS2143, NPSP795, JTT-305/MK-5442) and the quinazolinones (e.g., ATF936 and AXT914) (341). While both classes of compounds corrected the gain-of-function properties of several of the ADH1 CASR mutations tested in vitro, a subset of mutations involving NPS2143 binding sites within the transmembrane domain of the CASR are not fully corrected with NPS2143 but are normalized with the quinazolinone drugs (ATF936 and AXT914) (345-347). Whether this is reflected in mouse model studies and clinical situations remains to be determined.

Cases of hypoparathyroidism presenting as ADH but without CASR mutations have been found to have activating mutations of the gene encoding Gα11, the alpha-subunit of the heterotrimeric G protein that couples the CASR to signaling pathways (348, 349). The syndrome has been designated ADH2. Even though Gα11 is downstream of the CASR, in vitro studies showed that the calcilytic NPS2143 rectifies the altered Ca²⁺ signaling of the overactive mutants (350). Knock-in mice harboring an ADH2 Gα11 activating mutation faithfully replicate ADH2 (351). Treatment with the calcilytic NPS2143 or a Gα11/q-specific inhibitor, YM-254890 (352), increased circulating PTH and calcium levels in the heterozygous mutant mice (351). Thus, calcilytics, by blocking the renal CASR, may have potential use for treating ADH1 and ADH2, as well as other forms of hypoparathyroidism.

Other Therapies

If the serum calcium attainable with oral calcium and calcitriol is below the normal range and the patient remains symptomatic, then a trial of a thiazide diuretic may be considered, with the aim of reducing the hypercalciuria to raise the serum calcium further. The argument for efficacy seems greatest for responsive forms of autosomal dominant hypocalcemia due to activating CaSR mutations, since the thiazide-sensitive transporter, SLC12A3 (MIM#600968), is a downstream target of and is suppressed by activated CaSR in the kidney. For reasons that are not clear, however, thiazides work well in only a subset of patients (353). It is critical to monitor serum potassium and magnesium levels, as thiazide use can increase renal losses of these cations with resulting hypokalemia and hypomagnesemia. Some authorities suggest thiazides should not be used in APS1 patients with adrenal insufficiency and in ADH1 patients with Bartter syndrome type V (307, 308).

As the serum calcium is normalized, elevated serum phosphate concentrations generally decline, but phosphate-binding gels such as aluminum hydroxide are occasionally helpful in reducing hyperphosphatemia at the beginning of therapy or in cases where there is persistent hyperphosphatemia. Patients who develop intracranial calcifications may experience seizures related to chronic neuropathic changes, and it may be necessary to add appropriate anti-epileptic medication(s). In all chronically hypocalcemic patients, ocular assessments should be performed periodically.

In cases with documented abnormalities of the somatotropin axis, the growth hormone replacement therapy is effective but has to be initiated as soon as possible (315, 354, 355).

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-- In memory of Dr. Geoffrey N. Hendy --

Dietary Advice for Individuals with Diabetes

ABSTRACT

The chapter summarizes the current information available from a variety of scientifically based guidelines and resources on dietary advice for those with diabetes. It is a practical overview for health care practitioners working in diabetes management. The chapter is divided into sections by content and includes sources for further reading. A primary message is that nutrition plans should meet the specific needs of the patient and take into consideration their ability to implement change. Often starting with small achievable changes is best, with larger changes discussed as rapport builds. Referral to medical nutrition therapy (MNT) provided by a Registered Dietitian Nutritionist (RDN) and a diabetes self- management education and support (DSMES) program is highlighted.

INTRODUCTION

This chapter will summarize current information available from a variety of evidence-based guidelines and resources on dietary advice for those with diabetes. The modern diet for those with diabetes is based on concepts from clinical research, portion control, and individualized lifestyle change. It requires open and honest communication between health care practitioner and patient and cannot be delivered by giving a person a diet sheet in a one-size-fits- all approach. The lifestyle modification guidance and support needed most often requires a team effort, ideally including a registered dietitian(RD) or registered dietitian nutritionist (RDN), or a referral to a diabetes self- management education and support (DSMES) program that includes dietary advice. Current (2024) recommendations of the American Diabetes Association (ADA) promote all health care professionals to refer people with diabetes for individualized medical nutrition therapy (MNT) provided by an RDN at diagnosis and as needed throughout the life span, in addition to DSMES (1). It is very important to note that dietary recommendations for those with diabetes are virtually the same recommendations for diabetes prevention and the health of the general population; however, it cannot be excluded that people with diabetes will require additional support to meet the recommendations.

Fang et al, reported that although there has been continued improvements in risk factor control and adherence to preventative practices over the past decades, half of U.S. adults with diabetes do not meet the recommended goals for diabetes care in 2015-2018 (2). This is a current and ongoing issue. Diet and lifestyle recommendations are cornerstones of advice to prevent and manage diabetes, however there are recognized barriers to heeding advice and implementing lifestyle change. First, there is a plethora of dietary information for diabetes management available from many sources, although not all is evidence-based or current. There are also social, cultural, and personal preferences unique to each individual that must be taken into consideration when making long-term dietary change. Many health care practitioners are not adequately trained to be confident in delivering dietary advice, and many food environments do not support healthy dietary intakes for all. There are also commercial determinants of health that influence dietary intakes, such as marketing advertising, and price discounting on certain foods. The following recommendations come from evidence-based guideline development processes and emphasize practical suggestions for implementing dietary advice for most individuals with diabetes.

GENERAL GOALS

Dietary advice for those with diabetes has evolved and have become more flexible and patient centered over time. Nutrition goals from the American Diabetes Association (ADA) 2024 include the following: (1)

To promote and support healthful eating patterns, emphasizing a variety of nutrient-dense foods in appropriate portion sizes, to improve overall health and:
- achieve and maintain body weight goals.
- attain individualized glycemic, blood pressure, and lipid goals.
- delay or prevent the complications of diabetes.
To address individual nutrition needs based on personal and cultural preferences, health literacy and numeracy, access to healthful foods, willingness and ability to make behavioral changes, and existing barriers to change.
To maintain the pleasure of eating by providing nonjudgmental messages about food choices while limiting food choices only when indicated by scientific evidence.
To provide an individual with diabetes the practical tools for developing healthy eating patterns rather than focusing on individual macronutrients, micronutrients, or single foods.

The American Association of Clinical Endocrinologists (AACE) guidelines have similar nutrition goals for people with type2 diabetes (3).

Putting Goals Into Practice

How should these goals best be put into practice? The following guidelines summarized from the ADA Standards of Care will address the above goals and provide guidance on nutrition therapy based on numerous scientific resources. The Diabetes Control and Complications Trial (DCCT) and other studies demonstrated the added value individualized consultation with a registered dietitian familiar with diabetes treatments, along with regular follow-up, has on long-term outcomes and is highly recommended to aid in lifestyle compliance (4). Medical nutrition therapy (MNT) implemented by a registered dietitian is associated with A1C reductions of 1.0–1.9% for people with type 1 diabetes and 0.3–2.0% for people with type 2 diabetes (1).

Target Guidelines For Macronutrients: The 3 Major Components Of Diet

Many studies have been completed to attempt to determine the optimal combination of macronutrients. Based on available data, the best mix of carbohydrate, protein, and fat depends on the individual metabolic goals and preferences of the person with diabetes. It’s most important to ensure that total energy intake is kept in mind for weight loss or maintenance (1).

CARBOHYDRATES

The primary goal in the management of diabetes is to achieve as near normal regulation of blood glucose as possible. Both the type and total amount of carbohydrate (CHO) consumed influences glycemia. Carbohydrate intake should emphasize nutrient-dense carbohydrate sources that are high in fiber (at least 14 g fiber per 1,000 kcal) and minimally processed (1). Dietary carbohydrate includes sugars, starch, and dietary fiber. Higher intakes of sugars are associated with weight gain and greater incidence of dental caries (5). Conversely, higher intakes of dietary fiber are associated with reduced non-communicable disease and premature mortality occurrence as well as improvements in body weight, cholesterol concentrations, and blood pressure (6, 7). These benefits with higher fiber intakes have been observed in the general population, for those with type 1, type 2, and pre diabetes, (8) and those with hypertension or heart disease (9).With this guidance in mind, eating plans should emphasize non-starchy vegetables, fruits, legumes, and whole grains, as well as dairy products with minimal added sugars (1, 10). There is less consistency of evidence for recommending an amount of overall CHO in the diet (1). This is in line with current World Health Organization for carbohydrate intakes for adults and children which stress the type of carbohydrate is important, with recommendations for fiber and vegetable and fruit intake, but no recommendations on CHO amount (7). Recent dietary guidelines for diabetes management from the European Association for the Study of Diabetes stress that a wide range of carbohydrate intakes can be appropriate, however both very high (>70%Total Energy (TE)) and low (<40%TE) intakes are associated with premature mortality (10). A recent comprehensive Cochrane systematic review of randomized controlled trials (RCTs) of adults with overweight or obesity with or without type 2 diabetes concluded that there is probably little to no difference in weight reduction and changes in cardiovascular risk factors up to two years' follow-up, when overweight and obese participants without and with T2DM are randomized to either low-carbohydrate or balanced-carbohydrate weight-reducing diets (11).Some of the reasons for these findings of a lack of effect with lower carbohydrate diets may be that: interventions do not consider the type of carbohydrate being consumed, with dietary fiber and sugar having differing physiological effects; the differing definitions of low CHO diets being applied; what CHO is replaced with; and that diets lower in CHO maybe difficult to maintain in the long term as they are not consistent with the socio, cultural, and personal preference of many. Current ADA recommendations relating to CHO are: (1)

Emphasize minimally processed, nutrient-dense, high-fiber sources of carbohydrate (at least 14 g fiber per 1,000 kcal).
People with diabetes and those at risk are advised to replace sugar-sweetened beverages (including fruit juices) with water or low-calorie or no-calorie beverages as much as possible to manage glycemia and reduce risk for cardiometabolic disease and minimize consumption of foods with added sugar that have the capacity to displace healthier, more nutrient-dense food choices.
Provide education on the glycemic impact of carbohydrate, fat, and protein tailored to an individual’s needs, insulin plan, and preferences to optimize mealtime insulin dosing.
When using fixed insulin doses, individuals should be provided with education about consistent patterns of carbohydrate intake with respect to time and amount while considering the insulin action time, as it can result in improved glycemia and reduce the risk for hypoglycemia.

Dietary Fiber

Current recommendations from the American Diabetes Association are that adults with diabetes should consume high fiber foods (at least 14g fiber per 1,000 kcal) (1). Current recommendations from the European Association for the Study of Diabetes are that adults with diabetes should consume at least 35g dietary fiber per day (or 16.7g per 1,000 kcal) (10).These two values are aligned, and higher than current World Health Organization recommendations for the general population of at least 25g dietary fiber per day, (7) although all three recommendations recognize a minimum intake level, with greater benefits observed with higher intakes. These values are appreciably higher than current dietary fiber intakes in the United States, which is approximately 16g per day. Our understanding of the importance of dietary fiber has changed in recent years. Dietary fiber is carbohydrate that is not digested by the stomach or absorbed in the GI tract. Instead, it is either degraded in the colon by the gut microbiota, or passes through the human body intact. Higher intakes of dietary fiber are associated with lower all-cause mortality, heart disease, T2 diabetes incidence, and certain cancers such colorectal cancer when compared with lower fiber intakes (6). The benefits for childhood intakes of dietary fiber and health outcomes later in life remain uncertain (12). There are several established physiological pathways that might explain these associations, such as reducing postprandial glycemia, competitive inhibition of saturated fat in the small intestine, and greater satiety leading to reduce subsequent intake. There are also more novel pathways proposed, such as modulation of the gut microbiota to increase branched and short chain fatty acids. Current recommendations by the World Health Organization are to obtain “naturally occurring dietary fiber as consumed in food” (7). Fiber supplements however are used frequently as additional dietary fiber sources, and may help individuals reach their fiber recommendations when sufficient amounts cannot be obtained from food alone. Fiber supplements can be extracted fiber (taken from a plant source) or synthetic. Few fiber supplements have been studied for physiological effectiveness to the same degree as inherent dietary fiber, so current best advice is to consume foods that are high in fiber (1, 7, 13).Recommended food sources of dietary fiber are minimally processed whole grains, vegetables, whole fruit and legumes (1, 7). An emphasis on minimally processed is made, as processing may reduce the benefits associated with intakes of these foods, (14-16) as well as introduce added nutrients such as saturated fats, sodium, and added sugars.

The website below contains links to a comprehensive table listing fiber content of foods, and a calculator to help select foods with higher fiber content to help reach daily fiber goals.

http://www.webmd.com/diet/healthtool-fiber-meter. In the Endotext chapter entitled “The Effect of Diet on Cardiovascular Disease and Lipid and Lipoprotein Levels” in the Lipid and Lipoprotein section provides several tables providing information on the fiber content of various foods.

Starch

Starch comprises most of the carbohydrates consumed globally, and is the storage carbohydrate found in refined cereals, potatoes, legumes, and bananas (16). Starch comprises two polymers: amylose (DP ~ 10³) and amylopectin (DP ~ 10⁴–10⁵). Most cereal starches comprise 15–30% amylose and 70–85% amylopectin. In their raw form, most starches are resistant to digestion by pancreatic amylase, but gelatinize in heat and water, permitting rapid digestion (16). Dietary starch intake is rarely directly reported, so the health effects of dietary starch intake are often assessed through key sources, such as refined grains and potatoes. For potatoes, meta-analyses of prospective observational studies have identified the health effects are largely determined by the cooking method (17). Fried and salted potatoes were associated with higher incidence of type 2 diabetes and hypertension. Boiled and roasted potatoes were not associated with increased or decreased risk to health (17). Some starches escape digestion, either naturally or due to food processing; these starches are called resistant starches.

Resistant Starches

Resistant starches are starch enclosed within intact cell walls. These include some legumes, starch granules in raw potato, retrograde amylose from plants modified to increase amylose content, or high-amylose containing foods, such as specially formulated cornstarch, which are not digested and absorbed as glucose. Resistant starches avoid digestion in the small intestine so do not contribute to postprandial glycemia and diabetes risk, and are instead fermented in the colon by the microbiota.

Sugars (Nutritive Sweeteners)

Sucrose, also known as “table sugar,” is a disaccharide composed of one glucose and one fructose molecule and provides 4 kcals per gram (16). Available evidence from clinical studies does not indicate that the overall amount of dietary sucrose is related to type 2 diabetes incidence, however it is related to body weight gain and increased dental caries (5). Given the association between excess body weight and type 2 diabetes occurrence, (18) there is rationale to promote a reduction of sugar intake related to diabetes occurrence, and replace sugar-sweetened beverages (including fruit juices) with water or no/low calorie beverages as much as possible (1).

Fructose is a naturally occurring monosaccharide found in fruits, some vegetables, and honey. High fructose corn syrup is used abundantly within the United States in processed foods as a less expensive alternative to sucrose. Fructose consumed in naturally occurring in foods such as fruit, (that also contain fiber) may result in better glycemic control compared with isocaloric intake of sucrose or fructose added to food, and is not likely to have detrimental effects on triglycerides as long as intake is not excessive (<12% energy).

A meta-analysis of 18 controlled feeding trials in people with diabetes compared the impact of fructose with other sources of carbohydrate on glycemic control. The analysis found that an isocaloric exchange of fructose for carbohydrates did not significantly affect fasting glucose or insulin and reduced glycated blood proteins in these trials of less than 12 weeks duration. The short duration is a potential limitation of the studies (19). Evidence exists that consuming high levels of fructose-containing beverages may have particularly adverse effects on selective deposition of ectopic and visceral fat, lipid metabolism, blood pressure, and insulin sensitivity compared with glucose-sweetened beverages (20). Thus, recommendations for dietary fructose tend to promote the reduction of fructose added to food, such as in fructose-containing beverages, while promoting whole fruit which can contain intrinsic fructose.

Non-Nutritive Sweeteners

Non-nutritive sweeteners provide insignificant amounts of energy and elicit a sweet sensation without increasing blood glucose or insulin concentrations. There are several FDA-approved sweeteners found to be safe when consumed within FDA acceptable daily intake amounts (ADI) (Table 1) (21).

Table 1. NON-NUTRITIVE SWEETENERS
Name	Main Source
Sucralose (Splenda®)	Sucralose is synthesized from regular sucrose, but altered such that it is not absorbed. Sucralose is 600 times sweeter than sucrose. It is heat stable and can be used in cooking. It was approved for use by the FDA in 1999.
Saccharine (Sugar Twin®, Sweet ‘N Low®)	Saccharine is 200 to 700 times sweeter than sugar. A cancer-related warning label was removed in 2000 after the FDA determined that it was generally safe.
Acesulfame K (Ace K, Sunette)	Acesulfame is 200 times sweeter than sucrose. It can be used in cooking. The bitter aftertaste of acesulfame can be greatly decreased or eliminated by combining acesulfame with another sweetener.
Neotame	Neotame is a derivative of the dipeptide phenylalanine and aspartic acid. It is 7,000-13,000 times sweeter than sucrose and does not have a significant effect on fasting glucose or insulin levels in persons with type 2 diabetes.
Aspartame (Equal®,NutraSweet®)	Aspartame is a methyl ester of aspartic acid and phenylalanine dipeptide. Although aspartame provides 4 kcal/g, the intensity of the sweet taste(200x sweeter than sucrose) means that very small amounts are required. The FDA requires any foods containing aspartame to have an informational label statement: “Phenylketonurics: contains phenylalanine.” Patients with phenylketonuria should avoid products containing Aspartame. Controversy has existed for many years around safety of this sweetener, but not from any major organizations.
Stevia (Truvia®)	Stevia derived from the plant stevia rebaudiana, is a non-caloric, natural sweetener. Stevia has been used as a sweetener and as a medicinal herb since ancient times and appears to be well-tolerated. It has an intensely sweet taste.
Luo han guo	Luo han guo is also known as monk fruit, or Swingle fruit extract. It is 150- 300 times sweeter than sucrose, and may have an aftertaste at high levels.

A review of 29 RCTs which included 741 people, 69 of which have type 2 diabetes, indicated that artificial sweeteners on their own do not raise blood glucose levels, but the content of the food or drink containing the artificial sweetener must be considered, especially for those with diabetes (22). This sentiment was echoed in recent WHO guidance on non-nutritive sweeteners for the general population (23) where their use was not recommended for weight loss, as the overall content of the processed food or drink was important.

Practical Tips For Carbohydrate Intake

Base meals and snacks around high fiber foods, such as whole grains, vegetables, whole fruit, and legumes.
Common whole grains include whole wheat, whole oats, brown rice, barley, and quinoa.
When purchasing wholegrain foods, check the label to make sure that the wholegrain is the first ingredient listed, and that energy from sugars is <10%.
Consume fruit, but chose whole fruit over dried, juiced, or further processed fruit.
Legumes are an excellent and cheap source of fiber and protein. Replace ground meat in meals such as casserole with lentils or legumes.
Strive to include a variety of vegetables in your meals each day, avoiding deep fried and heavily salted options.

FAT

Evidence is inconclusive for an ideal amount of total fat intake for people with diabetes; therefore, goals should be individualized.

In line with advice for the general public, people with diabetes should look to replace saturated and trans fats in the diet with mono and poly unsaturated fats (24). This is principally to lessen the increased risk of cardiovascular disease with high saturated and trans-fat intakes. Recent meta-analyses have found that decreasing the amount of saturated fatty acids and trans fatty acids, the principal dietary fatty acids linked to elevating LDL cholesterol, reduces the risk of CVD(25). The World Health Organization and American College of Cardiology currently recommend limiting the amount of dietary saturated and trans-fat intake (24, 26). Recommendations from the Institute of Medicine and the Academy of Nutrition and Dietetics for healthy individuals are that 20% to 35% of total energy should come from fat (27).Recommendations to reduce total fat intake are largely due to the high energy content of dietary fats, more so than protein or carbohydrate, and the risks associated with higher saturated fat intakes. Current recommendations for fat intakes from the American Diabetes Association focus on fat quality and its sources rather than quantity (1). They recommend:

Counsel people with diabetes to consider an eating plan emphasizing elements of a Mediterranean eating pattern, which is rich in monounsaturated and polyunsaturated fats and long-chain fatty acids such as fatty fish, nuts, and seeds, to reduce cardiovascular disease risk and improve glucose metabolism.

The American Heart Association has developed the Fat Facts to help individuals learn more about healthy vs. unhealthy fats. Among the campaign's top priorities is to encourage replacing high trans-fat partially hydrogenated vegetable oils,animal fats, and tropical oils with healthier oils and foods higher in unsaturated fats — monounsaturated and polyunsaturated.

See more at: https://www.heart.org/en/healthy-living/healthy-eating/eat-smart/fats/the-facts-on-fats

Monounsaturated Fatty Acids

Monounsaturated fats (MUFA) are in foods such as avocado, some fish, nuts, and nut butters. MUFA are also found in vegetable oils such as olive, peanut, avocado, and canola oil. Several large prospective observational studies have documented that diets rich in MUFA or PUFA and lower in saturated fat are associated with a reduced risk of CVD (28).Meta-analysis of RCTs comparing diets higher in MUFA vs CHO or PUFA demonstrated that high MUFA containing diets can improve metabolic parameters and reduce cardiovascular disease risk in people with T2D (29, 30).

Polyunsaturated Fatty Acids

Polyunsaturated fats (PUFAs) are found in foods such as walnuts, sunflower seeds, and some fish such as salmon, mackerel, herring, and trout. PUFA are also found in vegetable oils such as corn oil, safflower oil, and soybean oil. Both PUFA and MUFA are usually liquid at room temperature. A meta-analysis of feeding trials has indicated consistent positive effects when other macronutrients, such as saturated fats, are replaced with PUFA on glycemia, insulin resistance, and insulin secretion capacity (31). Substitution data from prospective observational studies also indicates that replacing saturated and trans fats with PUFA reduces all-cause mortality and coronary heart disease, (25) with a smaller body of evidence in those with diabetes indicating similar improvements in cardiovascular disease risk (30).

A few specific types of PUFA are referred to as Omega-3 fats. These are called eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and alpha-linolenic acid (ALA). These fats are particularly singled out and recommended to prevent or treat CVD; however, evidence does not support a beneficial role for the routine use of n-3 dietary supplements in diabetes management (1) or for the general population. EPA and DHA are found in fatty fish. ALA is found in nuts and seeds. Studies on the effect of omega-3 fatty acids (both from food and supplements) in persons with diabetes are limited and have been inconclusive (20). In addition to providing EPA and DHA, regular fish consumption may help reduce triglycerides by replacing other foods higher in saturated and trans fats from the diet, such as fatty meats and full-fat dairy products. Preparing fish without frying or adding cream-based sauces is recommended. Fish with high amounts of EPA and DHA include salmon, albacore tuna, mackerel, sardines, herring, and lake trout. Nuts and seeds high in ALA include walnuts, flax seeds, chia seeds and soybeans (16).

Saturated Fats

Saturated fats are usually solid or almost solid at room temperature. All land animal fats, such as those in meat, poultry, and dairy products, are predominantly saturated. Processed and fast foods also contain high amounts of saturated fats.Some vegetable oils also can be saturated, including palm, palm kernel, and coconut oils (16). Oil such as coconut and palm (sometimes referred to as tropical oils) are touted as healthful saturated fats since they are derived from plants, however this is not accurate (25). Current ADA recommendations are to limit all sources of saturated fats (1). The World Health Organization recommends limited consumption of saturated fats to less than 10% of total energy intake, (24)which is far less than the current average intake. When cooking with oil, choose non-tropical vegetable oils such as canola, corn, olive, peanut, safflower, soybean, and sunflower oils (16).

Few research studies have been undertaken to look at the difference between the amount of saturated fatty acids (SFA) in the diet and glycemic control and CVD risk in people with diabetes (30). The ADA recommends people with diabetes follow the guidelines for the general population (20).

In general, saturated fats are discouraged because they increase LDL-cholesterol and total cholesterol concentrations (24). Diets high in saturated fats have been implicated in an increased risk of cardiovascular disease. Three RCTs found that diets containing ≤7% SFA and ≤200 mg/day cholesterol reduced LDL cholesterol level from 9% to 12% compared tobaseline values or to a more standard Western-type diet (32). As saturated fats are progressively decreased in the diet,they should be replaced with unsaturated fats and high fiber carbohydrates, and not with trans fats or refined carbohydrates (25).

Trans Fats

Trans fatty acids (TFA) are also called hydrogenated fats, which are fats created when oils are "partially hydrogenated" (16). The process of hydrogenation changes the chemical structure of unsaturated fats by adding hydrogen atoms, or “saturating” the fat. Hydrogenation converts liquid oil into stick margarine or shortening. Manufacturers use hydrogenation to increase product stability and shelf-life. A large quantity of these fats can be produced at one time, saving manufacturing costs. Research trials indicate that TFA can increase LDL cholesterol and lower HDL cholesterol (33). Meta-analysis of prospective observational studies indicate higher intakes of trans fats are associated with higher cases of all-cause mortality, cardiovascular disease, and coronary heart disease (25). Although less prevalent by volume in the food supply, trans fats appear at least as harmful to health as saturated fats (25). Due to the observations from both RCTs and prospective observational studies, the World Health Organization currently recommends that the total intake of trans fats be less than 1% of total energy intake (24). With the mandatory TFA labeling in the United States in 2006, a big push has been made by food manufacturers to remove TFA from processed and baked goods. Although the TFA content in foods has decreased recently (through food reformulation), it is important to monitor the type of fat used to replace TFA, as it might be saturated fat. The FDA has determined that trans fats are no longer considered generally recognized as safe (GRAS).While manufacturers cannot add TFAs to foods anymore, they may still be produced during the food manufacturing, so consumers should still check the nutrition information panel on foods. The main sources of trans fats in the food supply today are highly processed foods such as cakes, cookies, potato chips, and animal products. They can also be produced in the home when frying foods in fat at high temperatures. While most trans fats in the diet now are created during food manufacturing, smaller amounts of trans fats are also found in ruminant animals (cows and sheep). At present there is insufficient evidence to indicate that the health effects differ between trans fats that are created in food manufacturing or ruminant derived, (25) so advice to reduce trans-fat intakes relates to total trans-fat (24).

Cholesterol

The body makes enough cholesterol for physiological functions, so it is not needed through foods. Older dietary guidelines formerly recommended avoiding or limiting consumption of foods high in cholesterol, in the idea that their intake would raise our own circulating cholesterol levels. Now however, it is understood that saturated fat intake has a stronger influence on human cholesterol levels, so recommendations focus on reducing saturated fat as the priority (24).

Table 2. DIETARY FATS
Type of Fat	Main Source
Monounsaturated	Canola, peanut, and olive oils; avocados; nuts such as almonds, hazelnuts, and pecans; and seeds such as pumpkin and sesame seeds.
Polyunsaturated	Sunflower, corn, soybean, and flaxseed oils, and also in foods such as walnuts, flax seeds, and fish.
Saturated	Whole milk, butter, cheese, and ice cream; red meat; chocolate; coconuts, coconut milk, coconut oil and palm oil.
Trans	Some margarines; vegetable shortening; partially hydrogenated vegetable oil; deep-fried foods; many fast foods; some commercial baked goods (check labels).

Stanols And Sterols

Plant sterols are naturally occurring cholesterol derivatives from vegetable oils, nuts, corn, woods, and beans. Hydrogenation of sterols produces stanols. The generic term to describe both sterols, stanols, and their esters is phytosterols. An important role of phytosterols is their ability to block absorption of dietary and biliary cholesterol from the gastrointestinal tract. The LDL lowering property of both sterols and stanols is considered equivalent in short term studies(34). The amounts of sterols and stanol esters found naturally in a normal diet are insufficient to have a therapeutic effect. Thus, many manufacturers add them to various foods for their LDL cholesterol lowering effects. You can find added phytosterols in margarine spreads, juices, yogurts, cereals, and even granola bars. Individuals with diabetes and dyslipidemia may be able to reduce total and LDL cholesterol by consuming at least 2 grams per day of plant stanols or sterols found in enriched foods (20). The evidence on long term use and in people with diabetes is less substantiated, as not many studies have been completed (35).

Practical Tips On Fat Intake

Fat intake should come primarily from good sources of mono and polyunsaturated fats: nuts and seeds, avocados, fish, and oils such as olive, canola, soybean, sunflower, and corn.
Limit intake of saturated fats by cutting back on processed and fast foods, red meat, and full-fat dairy foods. Try replacing red meat with beans, nuts, skinless poultry, and fish whenever possible, and switching from whole milk and other full-fat dairy foods to lower fat
In place of butter or margarine, use liquid vegetable oils rich in polyunsaturated and monounsaturated fats in cooking and at the table.
Keep trans-fat intakes as low as possible. Check food labels for trans fats, and limit fried

PROTEIN

Protein intake goals should be individualized based on an individual’s current eating patterns. The ADA Standards of Medical Care in Diabetes-2024 state that there is no evidence that adjusting the daily level of protein intake (typically1–1.5g/kg body weight/day or 10–20% total energy) will improve health in individuals without diabetic kidney disease, and research is inconclusive regarding the ideal amount of dietary protein to optimize either glycemic control or cardiovascular disease (CVD) risk (1). Some research has found successful management of weight and type 2 diabetes with meal plans including slightly higher levels of protein (23–32% total energy) for periods up to one year for those without kidney disease (10). Those with diabetic kidney disease (with albuminuria and/or reduced estimated glomerular filtration rate) should aim to maintain dietary protein at the recommended daily allowance of no more than 0.8g/kg desirable body weight/day (or 10-15% total energy) (10). The National Kidney Foundation recommends 0.8 g protein/kg desirable body weight for people with diabetes and stages 1–4 chronic kidney disease as a means of reducing albuminuria and stabilizing kidney function (36). Reducing the amount of dietary protein below 10% total energy is not recommended as it places people at risk of protein inadequacy (10).

The ADA recommends that in individuals with type 2 diabetes, ingested protein can increase insulin response without increasing plasma glucose concentrations. Therefore, carbohydrate sources high in protein should not be used to treat or prevent hypoglycemia (1). Further research is required to identify if the dietary source of protein (animal or plant) is important for health and diabetes. There is emerging evidence to suggest that plant sourced proteins may be superior for health, (1) however it is not yet known if this is due to the amino acid compositions of the proteins or unadjusted effects from the accompanying nutrients, such as saturated fats in meat sources and dietary fiber in plant sources of protein. Replacement of red meat in the diet with plant-based protein sources (such as beans and legumes) appears to produce both health and environmental co-benefits, as well as being cheaper (37-39).

Practical Tips For Protein Intake

Ideal plant protein sources include legumes, lentils, tofu, and tempeh (1/2c = 2 oz protein). Plant-based meat alternatives maybe also be used (i.e. Quorn), but be wary of meat alternatives that have high sodium and saturated fat
Nuts or seeds are another plant-based protein source to be encouraged, such as almonds, cashews, hazelnuts, filberts, Brazil nuts, macadamias, peanuts, pecans, walnuts, or sunflower, pumpkin seed or linseed. Nut butters are also a plant-based protein source but be mindful of added sodium and sugars.
Good sources of lean animal protein include: skinless poultry, lower fat cuts of beef or pork, fish or egg, and reduced fat dairy products (i.e. low fat or skim milk/yogurt, and cheese).
Protein sources should be a supplement to vegetables, fruits and whole grains for most meals, and not the entire meal.

TARGET GUIDELINES FOR MICRONUTRIENTS

There is no clear evidence that dietary supplementation with vitamins (such as Vitamin D), minerals (such as chromium), herbs, or spices (such as cinnamon or aloe vera) can improve outcomes in diabetes management where there are no underlying deficiencies. There is insufficient evidence for dietary supplements to be recommended for the purposes of improving glycemic control (1).

People with diabetes should be aware of the necessity for meeting vitamin and mineral needs from natural food sources through intake of a balanced diet. Specific populations, such as older adults, pregnant or lactating women, strict vegetarians or vegans, and individuals on very low energy diets may benefit from a multivitamin mineral supplement (1).Excessive doses of certain vitamin or mineral supplements when there is no deficiency has been shown to be of no benefit and may even be harmful. There is some evidence that those on metformin therapy are at higher risk of B12 deficiency and may need Vitamin B12 supplementation if tests indicate a deficiency (1, 40).

VITAMINS

Since type 2 diabetes is a state of increased oxidative stress, interest in recommending large doses of antioxidant vitamins has been high. Current studies demonstrate no benefit of carotene and Vitamins E and C in respect to improved glycemic control or treatment of complications. Routinely supplementing the diet with antioxidant supplements is not recommended due to lack of evidence showing benefit in large, placebo-controlled clinical trials and concerns regarding potential long-term safety (1, 40). There is also not adequate evidence to recommend routine Vitamin D supplementation without deficiency (1, 41).

MINERALS

Sodium

As for the general population, those with diabetes should limit sodium consumption to 2,300 mg/day (20, 42). Active steps to reduce current sodium intakes is necessary, as current intakes in the United States are around 3,400 mg/day, nearly 50% more than the recommended limit. The majority of sodium consumed is from processed foods. Food manufacturers and restaurants will need to provide additional reduced sodium alternatives to help accomplish consumption targets. For those with diabetes and hypertension, additional lifestyle modification beyond reducing sodium intake can be helpful, including: loss of excess body weight; increasing consumption of vegetables and fruit (8 –10 servings/day), and low-fat dairy products (2–3 servings/day); avoiding excessive alcohol consumption (no more than 2 servings/day in men and no more than 1 serving/day in women); and increasing physical activity levels. These nonpharmacological strategies may also positively affect glycemia and lipid control (20). The DASH (Dietary Approaches to Stop Hypertension) diet, which is high in vegetables and fruit, low-fat dairy products, and low in saturated and total fat; has been shown in large, randomized, controlled trials to significantly reduce blood pressure (43).

Magnesium

Studies in support of magnesium supplementation to improve glycemic control are unclear and complicated by differences in study designs as well as baseline characteristics. There is some evidence from observational data that higher dietary intake of magnesium may help prevent type 2 diabetes in both middle aged men and women at higher risk for developing the disease (44). Additional long-term studies are needed to determine the best way to assess magnesium status and how magnesium deficiency impacts diabetes management, however dietary sources of magnesium include nuts, whole grains, and green leafy vegetables can be encouraged as part of a healthy dietary pattern.

Chromium

Several studies have demonstrated a potential role for chromium supplementation in the management of insulin resistance and type 2 diabetes. According to the ADA position statement, the findings with more significant effects were mainly found in poorer quality studies, limiting transferability of the results. Routine supplementation of chromium is therefore not recommended for treating diabetes or obesity (45).

HERBAL SUPPLEMENTS

There has been interest in the past several years on the effect of cinnamon, curcumin, and other herbs and spices in individuals with diabetes. The most recent ADA Lifestyle Management recommendations conclude that after a review of the evidence, there is not enough clear data to substantiate recommending the use of herbs or spices as treatment for T2D (1). The ADA also states that the use of any herbal supplements, which are not regulated and vary in content, may provide more risk than benefit, in that herbs may interact with other medications that are taken to control diabetes (20).

PROBIOTICS

Probiotics (from pro and biota, meaning "for life"), are certain kinds of “good” bacteria found in fermented foods, such as yogurt, kefir, and kimchi and are available as supplements. They are naturally found in the gut and may be depleted due to poor diet, use of antibiotics, smoking, etc. Probiotics have been studied extensively to improve gut flora for use in treatment and possibly prevention of various disorders, including irritable bowel syndrome, diarrhea, constipation, and genitourinary infections, to name a few. Different strains and amounts may work better for some conditions over others, but the FDA does not oversee the supplements, so content and effectiveness are not regulated. They are generally considered safe, as they are found naturally in the digestive tract.

Some research has been done in people with gestational and type 2 diabetes using probiotic supplements and foods to determine if chronic inflammatory and glycemic markers can be improved (46). The premise is that the microbiota maybe connected to glucose metabolism by altering insulin sensitivity and inflammation. At present there is insufficient evidence to make recommendations for people with diabetes to take a probiotic for glycemic control.

ALCOHOL

Updated guidelines recommend there is no safe level of alcohol consumption (10). Adults with diabetes who chose to drink alcohol should do so in moderation (no more than one drink per day for adult women and no more than two drinks per day for adult men). Alcohol consumption may place people with diabetes at increased risk for hypoglycemia, especially if taking insulin or insulin secretagogues. Education and awareness regarding the recognition and management of delayed hypoglycemia due to alcohol with or without a meal are warranted. Risks of excessive alcohol intake include hypoglycemia (particularly for those using insulin or insulin secretagogue therapies), weight gain, and hyperglycemia (for those consuming excessive amounts). Hypoglycemia can occur through several mechanisms, including the inability of alcohol to be converted into glucose, the inhibitory effect of alcohol on gluconeogenesis, and its interference in normal counter regulatory hormonal responses to impending hypoglycemia. To decrease the risk of alcohol induced hypoglycemia, it is best to have the alcohol with food. Consuming alcohol in a fasting state may contribute to hypoglycemia in people with type 1 diabetes. Symptoms of hypoglycemia can be similar to drunkenness. When calculating the need for meal related boluses of insulin, one should account for the carbohydrate content of the alcohol if drinking sweet wines, liqueurs, or drinks made with regular juice or soda.

PUTTING IT ALL TOGETHER- FOR TYPE 1 DIABETES AND THOSE ON INSULIN

People taking insulin should be counseled on the importance of balancing food and beverage intake with timing and dosing of insulin. This is especially important for individuals with varied or hectic schedules such as shift workers, people that travel frequently, or anyone who has a schedule in which timing of meals and access to food is irregular (20).Numerous materials and resources are available that can be provided to people with diabetes to help them consider portion control, consistency in food intake and medication dosing, as well as planning to allow some flexibility in their daily self-care regimen (47). Ongoing support from a referral to medical nutrition therapy conducted by a registered dietitian (RD) or registered dietitian nutritionist (RDN), or a referral to a diabetes self- management education (DSMES) program that includes dietary advice is highly effective. The health care provider should provide individualized guidelines for a target blood glucose range, considering safety and health. For motivated people, teaching an insulin to CHO ratio and blood glucose correction factor may assist them with achieving blood glucose targets and achieving better glycemic control (1).

CARBOHYDRATE COUNTING

Carbohydrate counting is a tool that can be taught to the motivated, so that they can more easily estimate the amount (grams) of CHO in a particular food and adapt their insulin therapy accordingly (48). Furthermore, setting a target CHO intake for each meal allows those with diabetes to better match their CHO intake to the appropriate mealtime insulin dose. Potential advantages of CHO counting include improved glucose control, flexibility in food choices, a better understanding of how much insulin to take, and simplification of meal planning (49).

Carbohydrate (CHO) intake affects acute blood glucose levels. Monitoring carbohydrate, whether by carbohydrate counting, using the exchange method, or experienced- based estimation, remain an important strategy used in timing of medication administration and improving glycemic control (20). CHO counting methodology is based on the concept that each serving of CHO equals approximately 15 grams of CHO. Generally, blood glucose response to digestible carbohydrate is similar, however carbohydrate sources naturally high in fiber including whole grains, legumes, vegetables, and whole fruits should be encouraged over highly processed foods, fruit juices, and sweetened beverages. Insulin dosing also needs to be adjusted based on the protein and fat content of the mail as well, as high levels of either can slow down digestion and glucose uptake into circulation. On average woman require about 3-4 servings (45-60 grams), while men may need 4-5 servings (60-75 grams) of CHO at each meal (47). This number could vary depending on individual energy needs (i.e., pregnant/nursing, ill, etc.), medication, and level of physical activity.

A good online resource for basic carbohydrate counting can be found on the UCSF website:

https://dtc.ucsf.edu/living-with-diabetes/diet-and- nutrition/understanding-carbohydrates/counting- carbohydrates/

SPECIAL CONSIDERATIONS FOR THOSE WITH INTENSIVE INSULIN REGIMENS

The following guidelines are the starting point for the nutritional component of intensified insulin management regimens for those not on closed loop systems: (1, 50)

The initial diabetes meal plan should be based on the individual’s normal intake with respect to calories, food choices, and times of meals eaten.
Choose an insulin regimen that is compatible with their normal pattern of meals, sleep, and physical
Synchronize insulin with meal times based on the action time of the insulin(s) used.
Assess blood glucose levels prior to meals and snacks and at bedtime and adjust the insulin doses as needed based on intake.
Monitor A1C, weight, lipids, blood pressure, and other clinical parameters, modifying the initial meal plan as necessary to meet goals.
It is also important to educate those with diabetes on adjustment of prandial insulin considering premeal glucose levels, carbohydrate intake, and anticipated physical activity.
For those with diabetes who are overweight and on insulin, counseling on nutrition, weight management, and monitoring blood glucose continues to be important components of treatment. Medical nutrition therapy is recommended with continued emphasis on making lifestyle changes to achieve a weight loss of 5% or more to reduce the risk of chronic complications associated with diabetes, CVD, and other risk factors that contribute to early mortality.

CHILDREN AND ADOLESCENTS

While medical nutrition therapy provided by registered dietitians resulted in better glycemic control in children with newly diagnosed type 1 diabetes, a survey of 45 pediatric clinics revealed that only 25 clinics had an experienced pediatric/adolescent dietitian available for children with diabetes (51). Registered Dietitian Nutritionists who are trained and experienced with children and adolescent diabetes management should be involved in the multidisciplinary care team (52). The goals of nutrition therapy for children and adolescents with diabetes include the following: (1, 52)

Provide individualized nutrition therapy with guidance on appropriate energy and nutrient intake to ensure optimal growth and development.
Assess and consider changes in food preferences over time and incorporate changes into
Promote healthy lifestyle habits while considering and preserving social, cultural, and physiological well-
Achieve and maintain the best possible glycemic
Achieve and maintain appropriate body weight and promote regular physical activity.

Dietary Advice Should Start Gradually

- Emphasis should initially be on establishing supportive rapport with the child and family with simple instructions. More detailed guidelines should be administered later by the entire team, with focus on consistency in message, and should include dietary guidelines to avoid hypoglycemia. Instruction on carbohydrate counting should be provided as soon as possible after diagnosis (52).
- Nutritional advice needs to be given to all caregivers; babysitters, and extended family who care for the child.
- Nutrition guidelines should be based on dietary history of the family and child’s meal pattern and habits prior to the diagnosis of diabetes and focus on nutritional recommendations for reducing risk of associated complications and cardiovascular risk that are applicable to the entire family.
- Physical activity schedules need to be assessed, along with 24-hour recall, and 3-day food diary to determine energy Growth patterns and weight gain need to be assessed every 3-6 months and recommended dietary advice adjusted accordingly (51).
Dietary recommendations can be illustrated by use of the Plate method. There are numerous resources for visuals and educational materials using the plate method and some are specific to diabetes. Half the plate should consist of vegetables and fruit, while the other half is divided between whole grains and lean sources of protein. The dairy is represented by a glass of nonfat or 1% milk or other nonfat or low-fat dairy source. The general guidelines for macronutrients are similar to that of the adult population with diabetes (1, 10).

Figure 1. Choosemyplate.gov. Video and print materials can be found on the website.

PREVENTION OF HYPOGLYCEMIA

Hypoglycemia usually occurs when taking insulin, or when taking a sulfonylurea. To help prevent hypoglycemia, the following guidelines should be discussed:

Don't skip or delay meals or snacks. If taking insulin or sulfonylurea, be consistent about the amount eaten and the timing of meals and snacks.
Monitor blood glucose closely.
Measure medication carefully, and take it on time. Take medication as recommended by the physician coordinating diabetes care.
Adjust medication or eat additional snacks if physical activity The adjustment depends on the blood glucose test results and on the type and length of the activity.
Eat a meal or snack if choosing a drink with alcohol. Drinking alcohol on an empty stomach can contribute to hypoglycemia.
Record low glucose reactions. This can help the health care team identify patterns contributing to hypoglycemia and find ways to prevent them.
Carry some form of diabetes identification so that in an emergency others will know you have diabetes. Use a medical identification necklace or bracelet and wallet card.

SICK DAY MANAGEMENT

Eating and drinking can be a challenge when sick. The main rules for sick day management are:

Continue to take diabetes medication (insulin or oral agent).
Self-monitor blood
Test urine
Eat the usual amount of carbohydrate, divided into smaller meals and snacks if necessary.
Drink non-caloric, caffeine free fluids frequently.
Call the diabetes care team.

See more at: https://diabetes.org/getting-sick-with-diabetes/sick-days

PHYSICAL ACTIVITY

Regular physical activity has many health benefits. For individuals with diabetes, these benefits outweigh potential risks. Physical activity can improve glycemic control (1). People with diabetes should be encouraged to undertake regular physical activity to improve cardiovascular and overall fitness, weight control, and for improved psychological well-being and quality of life (20). Physical activity can be considered in terms of duration, intensity, modality, and regularity. The largest potential risks due to physical activity for people with diabetes relates most to the intensity of activity. Low intensity physical activity is safer than high intensity physical activity, and highly beneficial for those with diabetes. People with diabetes are encouraged to undertake at least 30 minutes physical activity each day, with modalities such as walking recommended. There is some evidence that walking is more beneficial to glycemia when undertaken within the two hours after meals, (53) as the skeletal muscles take glucose out of circulation to use as fuel. High intensity physical activity should be discussed first with the diabetes care team due to the potential risk of hypoglycemia and cardiovascular strain. To summarize, there are several factors that can affect the blood glucose response to physical activity: (54)

Individual response to physical activity
Duration, intensity, modality, and regularity of physical activity.
Timing and type of the previous
Timing and type of the insulin injection or other diabetes
Pre-physical activity blood glucose
Person’s fitness

In individuals taking insulin, blood glucose monitoring is necessary to adjust insulin dosing and carbohydrate intake to reduce hypoglycemia due to physical activity. To reduce the risk of hypoglycemia, when higher intensity physical activity is planned, it may be preferable to adjust the dose of insulin before the activity begins. On the other hand, if the physical activity is unplanned, blood glucose should be checked and a carbohydrate snack can be eaten as needed before the activity begins. If the blood glucose is less than 100mg/dL, a 15- to 30-g carbohydrate snack should be consumed, and glucose should be rechecked in 30 to 60 minutes. If glucose levels are less than 70 mg/dL, physical activity should be postponed. Depending on the blood glucose level at the start of physical activity, as well as duration and intensity of the activity, a snack may need to be consumed before, during and after the physical activity. Moderate intensity physical activity can increase glucose uptake significantly, which may require an additional 15 grams of carbohydrate for every 30-60 minutes of exercise above the normal routine (54).

Physical activity can increase the rate of absorption of insulin into limbs, especially when it is started immediately after the insulin injection. Inject insulin into a less-active area, such as the abdomen, to minimize the effect of physical activity on insulin absorption. Guidelines for glucose management with exercise exist for those with type 1 diabetes (55). The response to physical activity varies greatly in every individual, so adjustment in medication and food should be based on individual responses. Blood glucose monitoring is very important in understanding response patterns and tailoring a physical activity program (56).

TIMING OF INSULIN AND MEALS

The greatest risk for hypoglycemia results when the peak insulin action does not coincide with the peak postprandial glucose. For example, the longer duration of action of regular insulin may lead to increased risk of late postprandial hypoglycemia, compared with rapid-acting insulin analogs, which peak closer to meal consumption. In addition, when the pre-meal insulin dose is too large for a particular meal relative to its CHO content, hypoglycemia can result. Such a mismatch may occur due to errors in estimating CHO or food intake. Insulin calculations can be based on exchanges, carbohydrate counting, or predefined, set menus. If meals and the insulin regimen remain constant, then no problems will usually result. However, any changes in insulin or food intake require adjustment of one or the other, or both. Whatever regimen is employed, it must be individualized. Those taking rapid-acting insulin may choose to give their insulin dose after the meal, if unsure of amount of food to be consumed. This approach can be especially helpful in children or in nausea related to pregnancy or illness. If a smaller than normal meal is eaten, guidelines are available for reducing the insulin dose, or carbohydrate replacement in the form of fruit or fruit juice can be given, depending on the particular insulin regimen (57).

HYPOGLYCEMIA TREATMENT GUIDELINES

Hypoglycemia is defined as a low blood glucose level ≤70 mg/dL. Symptoms include anxiety, irritability, light- headedness and shakiness. Advanced symptoms include headache, blurred vision, lack of coordination, confusion, anger, and numbness in the mouth. Hypoglycemia must be treated immediately with glucose. Follow the 15/15 rule: take15 grams of simple carbohydrate which should increase blood glucose by 30-45 mg/dL within 15 minutes. When blood glucose dips below 70 mg/dL and oral carbohydrate can be administered, have one of the following "quick fix" foods right away to raise the glucose:

Glucose tablets (see instructions).
Gel tube (see instructions).
4 ounces (1/2 cup) of juice or regular soda (not diet).
1 tablespoon of sugar, honey, or corn
Hard candies, jellybeans, or gumdrops, see food label for how many to consume.

High-fat foods will delay peak of glucose levels from carbohydrate intake and should be avoided (e.g., treatment of hypoglycemia with chocolate bars). After 15 minutes, blood glucose should be checked again to make sure that it is increasing. If it is still too low, another serving is advised. Repeat these steps until blood glucose is at least 70 mg/dL. Then, a snack should be consumed if it will be an hour or more before the next meal.

Those who take insulin or a sulfonylurea should be advised to always carry one of the quick-fix foods with them, when driving, and also have available nearby when sleeping. Wearing a medical ID bracelet or necklace is also a good idea, as is having a glucagon emergency kit or nasal spray on hand and knowing how to administer, as well as training close contacts to administer as well.

WEIGHT LOSS FOR THOSE WHO WISH TO LOSE WEIGHT

While the general principles discussed so far apply to all people with diabetes, those with type 2 diabetes who are overweight or obese (BMI >25 kg/m² or >23 kg/m² for Asians) and wish to lose weight can require greater support to do so. Consistent evidence has indicated that intentional weight loss reduces blood glucose in people with type 2 diabetes, and improves most other major cardiometabolic risk factors (58, 59). Clinical guidelines state that weight loss through nutrition and physical activity are fundamental to type 2 diabetes management (60, 61). However, with so many weight loss “diets” available, confusion abounds and reinforces the absolute importance that health professionals provide consistent, evidence-based advice. It is also important to have realistic expectations about the speed at which weight is lost. Obesity does not occur overnight, and its treatment requires long term adjustments to energy intake and expenditure.

Many randomized, controlled trials and meta-analyses of trials have been undertaken and to ascertain which macronutrient combination leads to greater weight loss. A two-year head- to-head trial comparing four weight loss diets with differing macronutrient content concluded that all four reduced energy diets, regardless of macronutrient content, led to comparable modest weight loss with weight regain over time (62). This finding was reinforced by a recent comprehensive Cochrane systematic review of RCTs of adults with overweight or obesity with or without type 2 diabetes (11). This review concluded that there is probably little to no difference in weight reduction and changes in cardiovascular risk factors up to two years' follow-up, when overweight and obese participants without and with T2DM are randomized to either low-carbohydrate or balanced-carbohydrate weight-reducing diets. The understanding that focusing on reducing energy intake overall, rather than through a specific macronutrient, frees up weight loss advice so that it can be tailored to the individual’s personal, cultural, and social norms. In this context, understanding reasons for eating, portion size, the energy density of different foods, and factors that promote satiety such as high fiber intakes become essential for achieving and maintaining weight loss.

The most important variable in selecting a weight loss plan is the ability of the individual to follow it over the long term. Developing an individualized weight loss program together, preferably with a registered dietitian nutritionist familiar with diabetes management, along with regular follow-ups, will help promote and maintain weight loss. Initial physical activity recommendations alongside dietary changes should be moderate, gradually increasing the duration and frequency to at least 30 minutes a day of activities such as walking.

Current evidence indicates that ‘low’, and ‘very low’ energy diets using total or partial diet replacement formula diet products are highly effective for weight loss and reduction of other cardiometabolic risk factors when compared with food-based weight-loss diets (63-65). Furthermore, low-energy nutritionally complete formula diets with a ‘total diet replacement’ induction phase are the most effective dietary approach for achieving type 2 diabetes remission (65-67). Comparing ‘low’ with ‘very-low’ energy diets, many people find very-low-energy diets (420–550 kcal/day) difficult to sustain, and they do not generate greater weight loss than formula diets providing ~810 kcal/day (63, 65).

While fast weight loss is a highly desirable outcome, the ultimate health benefits from weight management are likely to depend on long-term weight loss maintenance (10). Long-term low-intensity structured programs, including support for changing food choice, eating pattern and physical activity, and psychological support for behavior change, can help to sustain new behaviors, relationships with foods and adherence to dietary advice, and thus improve weight-loss maintenance (68, 69). Given that dietary adherence can be socially and psychologically testing, skills and empathy from the health professional is needed, providing consistent, long-term, evidence-based support (70). Discussions with patients around weight loss should be entered into with their permission (71) and are important, given the prevalence of obesity (72) and its connection to diabetes incidence (18).

MEAL PLANNING APPROACHES

There is no one “diet” for diabetes. There are, however, many meal planning guidelines available for the people with diabetes. Listed in the information below are some of the meal planning approaches available.

Choose My Plate

Choose My Plate contains general, simple guidelines for healthy eating using a small plate to visually illustrate foods and portion control. Print materials and videos from the USDA are available at www.choosemyplate.gov. and The Joslin Diabetes Center https://www.joslin.org/info/diabetes-and-nutrition.html

Diabetes Place Mat

Figure 2. Nutrition Place Mat for Diabetes. A sturdy, heavily laminated, 11" by 17" place mat that can be easily used over and over to apply the meal plan.

One side of the Diabetes Place Mat lists food choices and individual portion sizes for each food category of the meal plan. This list replaces easily misplaced or damaged paper lists. When planning the meal, a wipe-off marker is used to write down the number of servings for each food category, as indicated on the plan. Then circle or tally the food choices in each category to track progress toward the plan’s targets. Carbohydrate categories - starch and bread, fruit, milk and other carbohydrates - which affect blood glucose and which can be exchanged for each other, are color coded in yellow for easy identification and proper selection. Other food categories - vegetables, meat, fat and free foods - are individually color-coded.

The other side of the Diabetes Place Mat illustrates the "Plate Method" of managing a diet for proper nutrition and control of blood glucose and weight. It shows the proportions of each food category that are appropriate for a healthy, balanced diet. The food groups shown on the top half of the Plate Method side are carbohydrates, which affect blood glucose the most - fruit, milk, and starch & bread. These are colored in yellow to distinguish them from the other food groups that don't significantly affect blood glucose (meat, vegetables, fat and free foods). The food categories are shown in proportion to how much of each might be eaten in a healthy, balanced diet. The plate method is a great plan for those who have poor math or reading skills or are non- English speaking.

Mediterranean-Style Eating

The Mediterranean-style eating pattern derived from the Mediterranean region of the world has been observed to improve glycemic control and cardiovascular disease risk factors. The Mediterranean eating pattern includes:

Vegetables, fruits, nuts, seeds, legumes, potatoes, whole grains, breads, herbs, spices, fish, seafood and extra virgin olive oil. Emphasis is placed on use of minimally processed foods, seasonal fresh and locally grown foods.
Olive oil is the primary fat, replacing other fats and oils (including butter and margarine).
Fresh fruit as daily dessert; sweets only
Low-to-moderate amounts of cheese and
Red meat limited to only 12 oz to 16 oz per month.

DASH Eating Plan

Dietary Approaches to Stop Hypertension (DASH) is a flexible and balanced eating plan that is based on research studies sponsored by the National Heart, Lung, and Blood Institute (NHLBI). The DASH diet emphasizes vegetables, fruit, fat-free or low-fat dairy, whole grains, nuts and legumes, and limit the intake of total and saturated fat, cholesterol, red and processed meats, sweets and added sugars, including sugar-sweetened beverages. Results from RCTs indicate reductions in glycemia, blood pressure, body weight, and -cholesterol concentrations (73). In prospective cohort studies the DASH diet is associated with reductions in the risk of CVD, CHD and stroke (73). DASH is considerably lower in sodium than the typical American diet.

Intermittent Fasting

The popularity of intermittent fasting has increased recently as a new way to lose weight and possibly lead to better control of Type 2 diabetes. There are many suggested types of intermittent fasts; some involve eating only on specific days, or not eating for a specified number of hours, alternated by day or hours in which food consumption is allowed. Others greatly restrict energy intake on some days but allow a more normalized diet on other days. There is no one specific intermittent fasting diet that has been proven to be beneficial. Since energy intake is restricted for certain periods of time, an individual with diabetes may lose weight over time if they maintain an overall energy deficit in relation to energy expenditure as is seen with any successful weight loss method.

For people with diabetes who are interested in intermittent fasting, current ADA guidance considers time-restricted eating or shortening the eating window adaptable to any eating pattern, and largely safe for adults with type 1 or type 2 diabetes (1, 22). However, anti-hyperglycemic medication use must be considered (74). For those on insulin or taking other anti-hyperglycemia medications, intermittent fasting may lead to hypoglycemic events that may become severe when medications are not adjusted down (75). Careful monitoring of blood glucose is required, and medication adjustment may be necessary. Overall, the simplicity of intermittent fasting and time-restricted eating may make these useful strategies for people with diabetes who are looking for practical eating management tools (1).

Gluten Free

Gluten is a protein commonly found in wheat, barley, rye, and other grains. A gluten free diet is essential to treat people with celiac disease. Celiac disease is an inflammatory condition in persons who are intolerant to gluten and suffer inflammatory and gastrointestinal side effects when gluten is consumed, leading to damage of the small intestine. It is noted that approximately 10% of people with type 1 diabetes also have celiac disease, which is significantly higher than the general population (1-2%). There seems to be no connection with Celiac disease and type 2 diabetes (76). There is no evidence of health benefits when avoiding gluten for those without celiac disease.

The gluten free diet has recently grown in popularity in persons who identify as gluten sensitive, but don’t have celiac disease. According to the ADA, people with T1D can follow a gluten free diet should they wish to, but it may provide additional challenges. Common CHO containing foods that do not contain gluten are: white and sweet potatoes, brown and wild rice, corn, buckwheat, soy, quinoa, sorghum, and legumes. These foods can be used in place of gluten containing grains.

MAYO Clinic Diet

Developed by the Mayo Clinic, a two-phase approach to lose and maintain body weight using the Mayo Clinic food pyramid. For more information see: https://diet.mayoclinic.org/diet/how-it-works.

Jenny Craig®

The plan emphasizes restricting energy, fat, and portions. Jenny's prepackaged meals and recipes do all three, plus emphasize healthy eating, an active lifestyle, and behavior modification. Personal consultants guide members through their journeys from day one. You'll gain support and motivation, and learn how much you should be eating, what a balanced meal looks like and how to use that knowledge once you graduate from the program. Jenny Craig offers two programs: its standard program and Jenny Craig for Type 2, which is designed for people with Type 2 diabetes by including a lower-carb menu, reinforcement of self-monitoring of blood glucose levels, consistent meals and snacks, and other self-management strategies for weight loss and support for diabetes control. Because you buy foods, this program can be more expensive, but convenient for some. For more information see: https://www.jennycraig.com/.

Vegan Diet

Veganism excludes all animal products from the diet – including dairy and eggs. Fruits, vegetables, leafy greens, wholegrains, nuts, seeds and legumes are the staples. It is restrictive, but beneficial for the cardiovascular system.

Weight Watchers®

The Weight Watchers assigns every food and beverage a point value, based on nutritional content and provides users with a maximum number of points they can consume per day. A backbone of the plan is multi-model access (via in-person meetings, online chat or phone) to support from people who lost weight using Weight Watchers, kept it off and have been trained in behavioral weight management techniques. For more information see: https://health.usnews.com/best-diet/weight-watchers-diet

Individualized Menus Provided by a RD/RDN

Many people with diabetes might like to have examples to follow when setting up meal plans. The menu describes in writing what foods and what quantities should be consumed over a period of days. A dietitian creates an individualized menu based on the specific nutritional counseling plan and incorporates the client’s unique preferences, schedule, etc. The client then has written examples to follow, and over time may learn how to independently create their own menus and substitutions to fit their individual lifestyle.

Month of Meals

These menus were created by committees of the Council on Nutritional Science and Metabolism of the American Diabetes Association, and staff of the American Diabetes Association National Service Center in response to frequent requests for menus from people with diabetes and their families. The menus are designed to follow the exchange groups and provide 45-50% of energy from CHO, 20% protein, and about 30% fat. The menus provide 1200 or 1800 calories, and instructions are provided on how to adjust caloric levels upward or downward. Each menu provides 28 days of breakfast, lunch, dinner and snacks with a different focus to help make planning meals easier.

Exchange List Approach

The Exchange Lists for Meal Planning were developed by the American Diabetes Association and the Academy of Nutrition and Dietetics, and have been in existence since 1950. The latest version of Choose Your Foods: Food Lists for Diabetes was released in 2019. The concept for this list is that foods are grouped according to similar nutritional value, and can be exchanged or substituted in the portion size listed within the same group. The exchange lists include:

Carbohydrate group – includes starches, fruit, milk and
Meat and Meat Substitutes group – four meat categories based on the amount of fat they
Fat group – contains three categories of fats based on the major source of fat contained: saturated, polyunsaturated or monounsaturated.

The exchange lists also give information on fiber and sodium content. They can be utilized for people with type 1 or 2diabetes. The emphasis for type 1 is on consistency of timing and amount of food eaten, while for type 2, the focus is on controlling the caloric values of food consumed. Use of the exchange list may be helpful for some people while others may benefit by learning from other carbohydrate counting resources available online and through numerous publications and resources.

Calorie Counting

These are meal planning methods that can be useful for people with type 2 diabetes who want to lose weight. Knowledge regarding the number of total calories in a given food (including pre-prepared and fast foods) and becoming adept at label reading, can help promote weight loss when incorporated into other lifestyle changes. One of the first studies designed to determine empirically if people can learn a calorie counting system and if estimated food intake improves with training demonstrated that use of the Health Management Resources Calorie System tool (HMRe, Boston, MA, USA) helped to teach people how to estimate food intake more accurately (77).

RESOURCES FOR DIABETES NUTRITION EDUCATION

Table 3. DIABETES NUTRITION EDUCATION RESOURCES
Choose My Plate	www.choosemyplate.gov
Eat Out, Eat Well	Your go-to resource for assembling healthy meals in just about any type of restaurant, from fast food to upscale dining and ethnic cuisines. Order from: The American Diabetes Assn., www.shopdiabetes.org, 1-800-232-6455
American Diabetes Associate: Standards of Care	Facilitating positive health behaviors and well-being to improve health outcomes: standards of Care in Diabetes. 2024 https://doi.org/10.2337/dc24-SINT
DNSG European Dietary Guidelines	The Diabetes Nutrition Study Group of the European Association for the Study of Diabetes. Diabetologia. 2023 https://doi.org/10.1007/s00125-023-05894-8
What Can I Eat? The Diabetes Guide to Healthy Food Choices2^nd Edition	A 28-page guide for planning meals and making the best food choices. Includes carb counting, glycemic index, plate method, eating out, meals/snack ideas, best food choices and more. Order from: The American Diabetes Assn., Inc. www.shopdiabetes.org, 1-800-232-6455
Eating Healthy with Diabetes, 5^th Edition	Picture cues for portion sizes and color codes for food types teach how to put together a healthy diet plan to manage diabetes Order from: The Academy of Nutrition and Dietetics. www.eatright.org or the American Diabetes Assn., Inc. www.shopdiabetes.org.
Diabetes Meal Planning Made Easy & Healthy Portions Meal Measure	Meet your health and nutrition goals with healthy diabetes meal plans, shopping strategies and our handy portion control guide. Order from: The American Diabetes Association, www.shopdiabetes.org, 800-232-6455
Diabetes Place Mat Kit for People with Diabetes	Order from: NCES Health & Nutrition Information Catalog- Available inSpanish https://www.ncescatalog.com/NCES- MyPlacemat-for-Diabetes_p_1103.html OR School Health Corporation https://www.schoolhealth.com/nutrition-place-mat-for-diabetes
The Complete Month of Meals Collection, 2017	Available from: Amazon.com or American Diabetes Association, 1-800-232-6455; www.shopdiabetes.org
Choose Your Foods: Food Lists for Diabetes	Order from: Academy of Nutrition and Dietetics OR American Diabetes Associations; www.eatright.org OR http://shopdiabetes.org or Amazon.com Available in Spanish
Diabetes Food Hub	www.diabetesfoodhub.org. A website available on the American DiabetesAssociation site that has meal planning, grocery lists, recipes, menus and healthy substitutions. Section in Spanish available.
The Complete Guide to Carb Counting	American Diabetes Association 4th edition. Has all the expert information you need to practice carb counting, whether you’re learning the basics or trying to master more advanced techniques. Order from American Diabetes Association, http://shopdiabetes.org or Amazon.com
Diabetes and CarbCounting for Dummies1^st Edition	By Sherri Shafer, RD, CDE. Provides essential information on how to strike a balance between carb intake, exercise, and diabetes medications while making healthy food choices. Available at Amazon.com

The resources listed above are a sampling of the many available, primarily from the American Academy of Nutrition and Dietetics and the American Diabetes Association. There are several other organizations and websites which have educational materials available:

Diabetes Care and Education (dce.org/public-resources/diabetes)
Joslin Diabetes Center (www.joslin.org)
National Diabetes Education Program (www.ndep.nih.gov; diabetes.niddk.nih.gov)

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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, β_A, β_B and β_C; the best characterized are activin A (β_Aβ_A) and activin B (β_Bβ_B). 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.

SECULAR TRENDS REGARDING PUBERTY

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 19^th century to 13 years of age in the late 20^th 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 G_sα 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 G_sα 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 6^th transmembrane segment and in the 3^rd 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
Hypergonadotropic Hypogonadism	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.
Hypogonadotropic Hypogonadism	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 (INSR_A or INSR_B) and the IGF1R of which IR_A-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. INSR_B-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 2.5^th 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.

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 dos