Puberty: Gonadarche and Adrenarche

Pubertal Staging

For both sexes, the genital and pubic hair changes that unfold at puberty are classified into five stages: stage 1 is prepubertal and stage 5 is adult ( Fig. 18.1 ; Table 18.1 ). These physical changes reflect the changing hormonal milieu due to gonadarche (as in the case of breast or testicular enlargement) or adrenarche (as in the case of pubic hair development). Although the physical sequelae of gonadarche and adrenarche generally occur concomitantly, discordance between the two processes may also occur in normal development.

During puberty, increased ovarian estrogen secretion promotes breast development in girls. The development of breast buds with increased areolar diameter is considered to be stage 2; greater enlargement of the breasts occurs in stage 3, accompanied by increased pigmentation of the areolae and nipples. During stage 4, the areolae are mounded above the breast tissue. Recession of the areola to the general breast contour represents breast stage 5. Palpation of the breast is necessary to better differentiate between the breast tissue and lipomastia. Additional effects of estrogen at this stage of development include cornification of the vaginal mucosa, uterine growth, and morphogenesis of an adult female body habitus.

Menarche follows an anovulatory cycle and generally occurs 2 to 3 years after the onset of breast development. Menstrual cycles during the first year after menarche are typically irregular and anovulatory, with most ranging in duration from 21 to 45 days. By three years postmenarche, over 90% of adolescent females have 10 or more menstrual cycles peryear with an average menstrual interval of 36.5 days. Nevertheless, cycles can remain irregular until the fifth year postmenarche.

Although primordial and preantral follicles predominate during the prepubertal years, small antral follicles can develop during this phase of maturation. These small follicles are gonadotropin-independent. Ovarian volume increases with the onset of puberty, achieves maximum volume soon after (between menarche and age 16 years), and remains stable or decreases slightly thereafter. Ovarian volume assessed by three-dimensional MRI in healthy adolescents and young adults between the ages of 13 and 25 years was 10.8 ± 3.7 mL. Polycystic ovary morphology (PCOM) is commonly detected in healthy adolescent girls and is generally not associated with decreased ovulatory rate, hyperandrogenism, or metabolic abnormalities in this age group. During the early postmenarcheal period, ovarian morphology on transabdominal ultrasound shows multicystic ovaries and increased ovarian volume that differs from ovarian morphology observed in older women. ^, Healthy adolescents and young adults were recently found to have 15.2 ± 4.0 follicles per ovary, which exceeds the 12 follicles per ovary cutoff of the Rotterdam Criteria for PCOM.

In girls, increased adrenal C19 steroid (androgen) secretion is considered to be responsible for the development of darker hairs along the labia, which is classified as pubic hair stage 2. The hair becomes darker and coarser during pubic hair stage 3, spreading over the pubic symphysis with gradual progression to a full female escutcheon. Apocrine odor may precede or accompany the development of pubic hair. Associated findings include axillary hair, acne, and oiliness of skin and hair.

For boys, increasing testicular and adrenal androgen secretion contributes to the secondary sex characteristics. Genital stage 2 is characterized by an increase in testicular volume and enlargement of the scrotum. At stage 2, the testes are approximately 4 to 8 mL in volume, with the longest axis being approximately 2.5 cm. The volume of the mature human testis is approximately 20 to 30 mL and primarily reflects increased growth of the seminiferous tubule due to Sertoli cell proliferation and differentiation as well as initiation of spermatogenesis. At genital stage 3, further growth of the testes has occurred, and the length and diameter of the penis have increased. At genital stage 4, penile size has increased and the scrotal skin has become darkened. Palpation and use of an orchidometer are preferable to inspection. Male pubic hair stage 2 consists of downy hair at the base of the penis. For pubic hair stage 3, the hair is longer and 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. Other secondary sexual characteristics in boys include axillary hair, increased size of the larynx, deepening of the voice, increased bone mass, and increased muscle strength. Approximately three years after the appearance of pubic hair, terminal hair appears in androgen-dependent regions on the face and trunk where it may develop for several years thereafter. There is considerable variation in the distribution and density of beard, chest, abdominal, and back hair, presumably reflecting genetic differences. The appearance of spermatozoa in early morning urine specimens (spermaturia) occurs during genital stage 3. Gynecomastia is observed in 50% of boys during pubertal development. This is typically the most prominent in midpuberty when the ratio of circulating concentrations of estradiol to testosterone is relatively high. In most instances, gynecomastia resolves spontaneously by 16 years of age.

The pubertal growth spurt in girls occurs concurrently with the onset of breast development and reaches its peak in Tanner Stage 3 to 4 prior to menarche. Usually, only 4 to 6 cm of growth occurs after menarche. The pubertal growth spurt in boys, with an average height velocity of 9.5 cm per year, occurs around the genital end of Tanner stage 3 and the beginning of Tanner stage 4. In general, the age at peak height velocity shows an inverse relationship with the magnitude of the growth spurt. ^, Linear growth is approximately 99% complete for girls at a bone age of 15 years and for boys at a bone age of 17 years.

Breast development in girls and testicular enlargement in boys generally precede pubic hair development. Yet, the tempo for pubic hair development is faster such that synchrony between genital and pubic hair development occurs during the later stages of puberty. Schemata for the temporal development of the secondary sexual characteristic and their relationship to growth velocity are shown for girls and boys in Figs. 18.2 and 18.3 , respectively.

A mixed cross-sectional and longitudinal study of 730 healthy Danish boys showed that voice break and peak height velocity occur during later male puberty. Voice break had a mean age of 13.6 years and was moderately correlated with other male pubertal milestones gonadarche (11.5 years), (testicular enlargement (11.6 years), axillary odor (11.8 years), pubarche (12 years), axillary hair (13.1 years) and peak height velocity (13.7 years)). The authors found that at voice break, testosterone was 10.9 nmol/L, LH was 2.5 mIU/mL and testicular volume was about 12 cc. The longitudinal portion of the study showed that voice break can occur over a broad range of testosterone levels.

Secular Trends and Racial and Ethnic Differences in the Onset and Tempo of Puberty

Reports of secular changes in the onset of puberty have focused on girls and have typically used age at menarche as the biomarker for puberty. Gluckman and Hanson have suggested that menarche occurred between 7 and 13 years in Paleolithic and Neolithic times. ^, Based on an analysis of 994 medieval adolescent skeletons (10–25 years) for the pubertal stage, it appears that adolescents likely began puberty around 10 to 12 years, with menarche occurring closer to 15 years in rural areas and 17 years in London. Boys experienced protracted pubertal development during the 10th to 17th centuries. Potential factors that contributed to later puberty during medieval times included poor diet, increased infections, and greater physical exertion. ^,

Available historical data indicate that this was followed by a decline in the age of menarche in Europe and North America from the early 19th century (16–17 years of age) to the latter half of the 20th century (13 years of age). This trend has been attributed to the improving socioeconomic conditions during this epoch. Although recent data from North America, several European countries, and other regions of the industrialized world suggest that the trend of earlier menarche has been reduced or halted, breast and pubic hair development are occurring earlier than they would have 50 years ago in both North America and Europe. ^,

The biology underlying this continued earlier secular trend in sexual development in girls, which in some populations is loosely associated with a similar trend in growth, is unclear, and may or may not involve an earlier onset of gonadarche or adrenarche. This earlier onset of breast development is not associated with increased gonadotropin or estradiol concentrations, suggesting that it represents a gonadotropin-independent event. ^, Earlier breast development assessed by palpation was reported in NHW girls, which was likely related to the increased BMI of this group. Analogous studies of boys are limited, but no striking sex differences in secular trends in puberty and growth are apparent. Using both genital staging and the orchidometer, the Copenhagen Puberty Study reported pubertal onset occurring three months earlier in Danish boys and reported that obesity advanced the onset of testicular enlargement ; however, other studies suggest that obesity delays the onset of puberty in boys. More recently, the Copenhagen Puberty Study used two-sample Mendelian randomization (MR) to determine the causal effect of genetically determined BMI on genetically determined pubertal timing. The study used GWAS of 97 BMI-determining variants from the GIANT consortium and GWAS for voice-breaking from 55,871 male research participants in the 23andMe study. They found significant associations between higher BMI and earlier voice break and a linear association between BMI and the timing of male pubertal milestones. In this study, all studied male pubertal events other than pubarche occurred earlier in obese boys compared to nonobese boys.

The age at onset of puberty varies between ethnic groups. Among American girls, mean ages for breast development, pubic hair development, and menarche were 9.5, 9.5, and 12.1, respectively, for NHB girls; 9.8, 10.3, and 12.2, respectively, for MXAM girls; and 10.3, 10.5, and 12.7 years, respectively, for NHW girls. ^, Data obtained through the cross-sectional Third National Health and Nutrition Examination Survey (NHANES III) between 1988 and 1994 showed that NHB girls enter puberty first, followed by MXAM and NHW girls. ^, Based on the NHANES III study, luteinizing hormone (LH) and inhibin B concentrations associated with onset of breast development were evaluated. As would be anticipated, LH and inhibin B concentrations increased with pubertal progression. Cut-points for Tanner 2 breast development were LH 1.04 mIU/mL (95% confidence interval [CI]: 0.71–1.37) and inhibin B 17.89 pg/mL (95% CI: 11.63–24.15). The respective median ages at hormonal onset based on LH concentrations were 10.7, 10.6, and 10.1 years for NHW, MXAM, and NHB girls, respectively. Girls with low birthweight and greater postnatal weight gain had relatively earlier onset of puberty based on LH concentrations and a similar pattern regarding pubertal onset was noted based solely on postnatal weight gain.

For boys in NHANES III, median estimated ages for genital and pubic hair stage 2 were 9.3 and 11.1, respectively, for NHB boys; 10.4 and 12.3, respectively, for MXAM boys; and 10.1 and 12.0, respectively, for NHW youths. For genital and pubic hair stage 5, median ages were 14.9 and 15.2, respectively, for NHB boys; 15.8 and 15.7, respectively, for MXAM boys; and 16.0 and 15.6, respectively, for NHW boys. Using the NHANES III data, LH, testosterone, and inhibin B concentrations increased as puberty progressed. Likely reflecting individual and diurnal variation, no single or combination hormone cut-point was found to be predictive of physical pubertal status, either genital or pubic hair stage 2, in this population. ^,

Ethnic differences affecting the rate of skeletal maturation have been described in the Generation R study in the Netherlands; using the Greulich and Pyle standards, skeletal age was most advanced in Asians, intermediate in Africans, and least advanced in white Europeans. Skeletal age was more advanced in girls compared to boys in all three ancestral groupings. These ethnic differences in skeletal age persisted after adjustment for age, height, and BMI. Similar findings of advanced skeletal age were found in a longitudinal cohort of American children categorized as African or non-African descent; skeletal age was more advanced in girls. ^, Normative data in the Greulich and Pyle standard are largely derived from hand and wrist radiographs obtained from a non-Hispanic European white population from the 1930s to the 1960s. Nevertheless, available data suggest that genetic and environmental factors influence rates of skeletal maturation. Hence, clinicians need to be cognizant of these limitations when using the Greulich and Pyle standards to assess skeletal maturation in children from other ethnic/racial backgrounds. ^,

The age at puberty reflects interactions between genetic, prenatal, and environmental factors. Twin studies indicate that heredity is responsible for approximately 50% of the variation in age at menarche. ^, Indeed, pubertal timing of both parents has been demonstrated to influence the timing of pubertal onset of both boys and girls independent of sex. Investigation of genetic variants associated with onset of puberty identified a specific variant, −29G>A, in the promoter region of the follicle-stimulating hormone (FSH) receptor ( FSHR ) gene; breast development occurred 7.4 months later among homozygous carriers of the −29G>A variant compared to the −29GG+GA carriers.

Steroidogenesis

The biosynthetic pathways for gonadal and adrenal steroids are considered together because of their similarities and their importance in understanding the physiology and pathophysiology of puberty ( Fig. 18.4 ; see Chapter 4 ). Synthesis of steroids from cholesterol requires the expression of specific enzymes, receptors, cofactors, and other proteins in the adrenal cortex and the gonads. Steroidogenesis is regulated by specific trophic hormones, ACTH, LH, and FSH.

The adrenal cortex consists of three zones, the zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR). The ZG synthesizes aldosterone, a mineralocorticoid, and is primarily regulated by potassium concentrations and renin-angiotensin. The ZF synthesizes cortisol. Steroidogenesis in the ZF is primarily governed by ACTH. The ZR synthesizes C-19 steroids, such as DHEA, DHEAS, androstenedione, androstenediol, and 11β-hydroxyandrostenedione.

Recent studies have confirmed that the human adrenals secrete 11-oxygenated 19-carbon (C ₁₉ ) steroids (11-oxygenated androgens, 11OAs). The 11-OAs include 11β-hydroxyandrostenedione (11OHA4), 11β-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), and 11-ketodihydrotestosterone (11KDHT). The androgenic activities of 11KT and 11KDHT are comparable to testosterone (T) and dihydrotestosterone (DHT), respectively. In a study of predominantly Caribbean Hispanic prepubertal children with premature adrenarche (n = 13) and controls (n = 11), it was shown that 11OAs were elevated in premature adrenarche compared to controls and a high correlation of T and A4 with 11-ketosterone and 11β-hydroxyandrostenedione. In fact, 11KT values were 4-fold higher than those for T, indicating that 11KT is the major active androgen in the circulation in PA. 11KT was elevated even in children who presented with PA but did not have elevated DHEAS. It was proposed that serum 11OA measurement may be a more accurate way to screen and classify children with PA than DHEAS.

ACTH is a 39 amino acid peptide derived following proteolytic cleavages of proopiomelanocortin (POMC). Its actions are mediated by the ACTH receptor, a seven-transmembrane G protein-coupled receptor encoded by MC2R. This pathway utilizes cyclic adenosine monophosphate (cAMP)-dependent protein kinase A. The acute effects of ACTH include uptake of plasma low-density lipoproteins, stimulation of cholesterol esterase activity, enhanced synthesis and phosphorylation of steroidogenic acute regulatory protein (StAR), cholesterol transfer across the inner mitochondrial membrane, and increased cortisol secretion. The chronic effects of ACTH involve stimulation of transcription and translation of steroidogenic enzyme genes.

In the gonads, LH and FSH modulate steroid biosynthesis. LH promotes ovarian theca cell and testicular Leydig cell steroidogenesis; its actions are mediated by its cognate receptor, LHCGR. Acting through the FSHR, FSH stimulates aromatase expression to promote estrogen biosynthesis in the ovary and Sertoli cell growth in the testis. The LH and FSH receptors are both G protein-coupled receptors and contain leucine-rich repeats in their large ectodomains.

Most enzymes involved in steroidogenesis are cytochrome P450s (CYPs) or hydroxysteroid dehydrogenases (HSDs). The rate-limiting step of steroidogenesis is the transport of cholesterol into mitochondria mediated by StAR. Within the mitochondria, cholesterol desmolase (also known as side-chain cleavage or P450scc) converts cholesterol into pregnenolone. One enzyme, 17α-hydroxylase/17,20-lyase (P450c17), encoded by the CYP17A1 gene, is the qualitative regulator of adrenal and gonadal steroidogenesis. This enzyme mediates 17α-hydroxylation to convert pregnenolone into 17α-hydroxypregnenolone. In the ZR, ovarian theca, and Leydig cells and this same enzyme catalyzes scission of the C17–20 bond to produce DHEA. Although this one protein is capable of two distinct enzymatic reactions, these enzyme activities are differentially regulated. Factors known to modulate 17,20-lyase activity include: (1) the amount of P450 oxidoreductase (POR); (2) the expression of cytochrome b ₅ (CYB5A); (3) the phosphorylation of serine/threonine residues on P450c17; and (4) the phosphorylation of noncanonical P450c17 residues. POR is a protein that transfers electrons from nicotinamide adenine dinucleotide phosphate to microsomal cytochrome P450 enzymes, such as P450c17, P450c21, and aromatase (P450aro). CYB5A modulates adrenal androgen secretion by increasing the 17,20-lyase activity of (P450c17).

The Δ ⁵ -steroids are converted to the Δ ⁴ -steroids by 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2), the adrenal and gonadal specific isoform. This enzyme converts pregnenolone to progesterone in the ZG, 17-hydroxypregnenolone to 17-hydroxyprogesterone in the ZF, and DHEA to androstenedione in the ZR. In the ZF, 17-hydroxyprogesterone (17-OHP) is converted to 11-deoxycortisol by 21-hydroxylase (P450c21) and, subsequently, to cortisol by 11β-hydroxylase (P450c11β).

The adrenals, ovaries, and testes synthesize sex steroids. The ZR of the adrenal cortex produces DHEA, DHEAS, androstenedione, androstenediol, and 11β-hydroxyandrostenedione. DHEA sulfotransferase (SULT2A1) converts DHEA to DHEAS and this enzyme is also expressed in the liver. Sulfation of steroids by SULT2A1 requires a sulfate donor, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), and the enzyme PAPS synthase. In the ovary, androstenedione is synthesized in the theca cell and diffuses into the granulosa cell, where it is aromatized by aromatase (P450aro) to estrone and converted to estradiol by 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1). Estradiol binds the estrogen receptor. In Leydig cells, androstenedione is converted to testosterone by HSD17B3. In androgen target cells, such as those in the external genitalia and prostate, testosterone is converted to DHT by 5α-reductase type 2. In some androgen-sensitive tissues, such as bone and adipose, testosterone is converted to estradiol by aromatase. Peripheral tissues, including the kidney, adipose, and prostate and genital skin, can convert the adrenal-derived 11-OAS, 11ß-hydroxyandrosterone (11OHA4), and 11ß-hydroxytestosterone (11OHT) into the more potent androgens, 11-ketoandrostenedione (11KA4) and 11-ketosterone (11KT). Androgenic actions are mediated by the androgen receptor. ^,

The 17β-hydroxysteroid dehydrogenase enzymes comprise a large family of enzymes involved in steroid biosynthesis and metabolism. The differences in tissue distribution, substrate preferences, subcellular localization, and mechanisms of regulation influence the cellular steroid microenvironment. The type 1 isozyme, 17βHSD1, is expressed in the ovaries, placenta, endometrium, and liver, where it favors conversion of estrone to estradiol. The type 3 isozyme, 17βHSD3, is expressed in the testis, where it preferentially converts androstenedione to testosterone. The type 5 enzyme, 17βHSD5, is an aldo-keto-reductase (AKR) enzyme (AKR1C3) that is expressed in steroidogenic and peripheral tissues; it can convert androstendione to testosterone.

Through investigations of the tammar wallaby and patients with disordered steroidogenesis, the presence of another pathway leading to dihydrotestosterone (DHT) synthesis was elucidated. In this “alternative backdoor pathway,” 17-OHP undergoes 3α- and 5α-reduction followed by 17,20-lyase, 17β-hydroxysteroid dehydrogenase, and 3α-oxidation steps to generate DHT in the absence of the “classic” intermediates, DHEA, androstenedione, and testosterone. In humans, since 17-OHP is not a favorable substrate for the 17,20-lyase reaction, this pathway acquires functional importance in disorders of steroidogenesis associated with increased 17-OHP concentrations, such as congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency and oxidoreductase deficiency.

Once secreted, sex steroids circulate bound to sex hormone-binding globulin (SHBG) and to albumin. The unbound or free hormone is the bioavailable form that diffuses passively into target cells and interacts with nuclear steroid receptors. Steroid hormone receptors are ligand-dependent transcription factors comprised of three functional domains: the N-terminal domain serves to modulate function, the DNA-binding domain mediates binding of the receptor to DNA, and the ligand-binding domain binds to steroid. Steroid receptor activity is modulated by various tissue-specific cofactors; both coactivators and corepressors can influence receptor function.

Steroids also act through nongenomic mechanisms. For example, testosterone can activate phospholipase C, leading to calcium influx into Sertoli cells, and can activate the mitogen-activated protein kinase as well as other intracellular mediators. Sex steroids can be metabolized to inactive forms by a variety of enzymes. Glucuronidation decreases the biological activity of steroid hormones and increases solubility to facilitate renal excretion. This process, catalyzed by UDP-glucuronyltransferase (UGT) enzymes, involves the transfer of glucuronic acid from uridine diphosphoglucuronic acid to steroid hormones. In humans, the UGT2B isoforms show greater specificity for C19 androgens. A second mechanism is sulfoconjugation, in which DHEA sulfotransferase catalyzes conversion of DHEA to DHEAS, and estrogen sulfotransferase converts estrogens to estrone sulfate. The inactive sulfated steroids can be hydrolyzed to active forms by steroid sulfatase.

Activation of Gonadarche

Gonadarche reflects reactivation of the hypothalamic-pituitary-gonadal (HPG) axis. The increased pituitary secretion of LH and FSH stimulates gonadal steroidogenesis, development of the physical manifestations of puberty, completion of gametogenesis, and maintenance of fertility. LH and FSH are heterodimeric proteins consisting of a common α-subunit and a unique β-subunit. Both LH and FSH are glycosylated peptides. Glycosylation appears to modulate hormone stability, protein folding, cellular trafficking, circulating serum half-life, and receptor signaling. The temporal increments in circulating LH and FSH concentrations at the time of puberty, and their relationships to those of the gonadal steroids, testosterone and estradiol, respectively, at this stage of development, have been well documented in both boys and girls. ^, The actions of LH and FSH are mediated by their cognate seven-transmembrane domain G protein-coupled receptors: the LH receptor (LHCGR) and FSHR, respectively.

In the female, LH stimulates androgen production by the theca cells of the ovarian follicles and progesterone secretion from luteinized granulosa cells of the corpus luteum. FSH is critical for the process of follicular recruitment and selection. In the granulosa cells of the developing follicle, FSH induces expression of aromatase, which is responsible for the aromatization of the theca cell-derived androgens into estrogens. FSH also induces LHCGR expression in granulosa cells of the dominant follicle, which selectively amplifies the effect of declining FSH concentrations on the dominant follicle. ^, In the male, LH regulates the secretion of testosterone from Leydig cells.

FSH, together with testosterone, is responsible for initiating and maintaining spermatogenesis. The action of FSH in this regard is indirect and exerted on the somatic Sertoli cell of the seminiferous tubule. While the actions of testosterone are also indirect, several somatic cell types in the testis (Sertoli, Leydig, and peritubular) express AR and are considered to be involved in the control of spermatogenesis.

The pubertal drive to the pituitary-gonadal axis is generated by a diffusely distributed network of hypothalamic neurons expressing GnRH-1, known as the “hypothalamic GnRH pulse generator.” As the name implies, the hypothalamic GnRH pulse generator produces intermittent discharges of GnRH into the hypophysial portal circulation, which is obligatory for gonadotropin synthesis and secretion by the pituitary gonadotrophs. LH and FSH secretion is stimulated by GnRH acting through its receptor, GnRH-R1, located on gonadotropin-secreting cells (gonadotrophs) in the pituitary gland. A unique feature of the human GnRHR, a 7-transmembrane domain G protein-coupled receptor, is its lack of a C-terminal cytoplasmic domain. Other factors studied during puberty include inhibins, activins, antimüllerian hormone (AMH), insulin-like factor-3 (INSL3), and osteocalcin. Activins and inhibins are members of the transforming growth factor-β (TGF-β) superfamily composed of a common α-subunit and two β-subunits (β _A and β _B ). The activins are dimers consisting of only the β-subunits; activin A is a dimer of β _A subunits and activin B is a dimer of β _B subunits. The activins are synthesized in the gonadotropes and influence FSH secretion. Mature inhibins are dimers composed of a common α-subunit covalently linked with one of two β-subunits (β _A and β _B ). The α/β _A and α/β _B dimers are known as inhibin A and inhibin B, respectively. The gonadal inhibins, like the gonadal steroids, play both an endocrine role in the regulation of gonadotropin secretion and a paracrine role within the gonads. Inhibin B, which is synthesized in part by the Sertoli cell, is the principal inhibin secreted by the testis. Inhibin B concentrations are low in prepubertal boys and increase with the onset of puberty. ^, The pubertal increase in inhibin B may be attributed to Sertoli cell proliferation and to the initiation of spermatogenesis, both of which reflect the increased gonadotropin drive to the testis at the time of gonadarche.

In girls, circulating levels of inhibin A and B are low or undetectable prior to puberty. Inhibin B begins to rise with the onset of puberty, as does inhibin A in breast stages 3 and 4. Adult levels are attained at approximately 14 to 15 years of age. During the menstrual cycle, inhibin A levels are elevated in the luteal phase, while inhibin B predominates in the circulation of the follicular phase. However, the role of ovarian inhibins in regulating gonadotropin secretion in pubertal and premenopausal women remains to be fully elucidated.

Osteocalcin is secreted by osteoblasts and is a marker of bone formation. Most available data regarding the potential actions of osteocalcin on puberty are derived from mouse studies. Studies of mice have shown that osteocalcin facilitates testicular testosterone secretion and pancreatic β-cell proliferation. The testicular effects of osteocalcin appear to be independent of the HPG axis as the osteocalcin receptor is not expressed in the hypothalamus or pituitary of mice. ^, Posttranslationally, osteocalcin is carboxylated on three glutamic acid residues. The carboxylated form of the molecule is considered to be biologically inactive, whereas the undercarboxylated form is biologically active. Osteocalcin signals through its cognate receptor, GPRC6A, which is expressed by Leydig cells. Interestingly, increased osteocalcin concentrations are associated with the pubertal rise in testosterone concentrations in boys. Osteocalcin shows sexual dimorphism because it modulates Leydig cell testosterone production, but not ovarian estrogen production. The discovery of a missense mutation in the GPRC6A gene in two men with primary testicular failure, oligospermia, and glucose intolerance encourages speculation that osteocalcin influences testicular function in humans.

Antimüllerian hormone (AMH), also known as müllerian-inhibiting hormone (MIH), is another member of the TGF-β superfamily. AMH signals by binding to a specific type-II receptor (AMHR2); this receptor heterodimerizes with one of several type-I receptors (ALK2, ALK3, and ALK6), leading to recruitment of Smad proteins that are translocated to the nucleus to regulate target gene expression. AMH is secreted by the Sertoli cells of the developing testis and stimulates regression of the müllerian ducts during male fetal development. Postnatally, AMH is secreted by the Sertoli cells of the testis. In boys, AMH concentrations decline at puberty. Although FSH stimulates AMH production, testosterone inhibits Sertoli cell AMH secretion. The decline in AMH concentrations appears to be closely associated with rising inhibin B concentrations at the onset of puberty in boys, the latter presumably reflecting androgen-induced differentiation of the Sertoli cell. Both AMH and inhibin B concentrations are low in boys with bilateral anorchia or complete hypogonadotropic hypogonadism (HH).

Immunohistochemical studies of human ovaries showed no AMH staining in primordial follicles. AMH expression is highest in growing preantral and small antral follicles and disappears in larger follicles (>8 mm). In the ovaries, AMH is primarily secreted by the granulosa cells of the preantral and antral follicles. Among females, AMH concentrations begin to increase in infancy to achieve a plateau during adolescence until age 25 years. Subsequently, AMH concentrations decline and correlate inversely with age. ^, AMH plays a role as a gatekeeper of follicular development.

Data obtained from studies in rodents have demonstrated the presence of the AMH receptor, AMHRII, in gonadotropes. One report using rats showed that AMH stimulated FSH secretion in immature female rats. Subsets of GnRH neurons express the AMH receptor. In vitro and in vivo studies have demonstrated that AMH increases GnRH-dependent LH pulsatility and secretion. Thus AMH likely influences the migration of GnRH neurons during gestation and contributes to HPG axis function during prepubertal, pubertal, and adult years.

INSL3, a peptide hormone secreted by Leydig cells, plays a major role in directing the transabdominal phase of testicular descent during gestation. INSL3 signaling is mediated by the G protein-coupled relaxin family peptide receptor 2 (RXFP2). After birth, fetal Leydig cells involute followed by quiescence until puberty. Adult-type Leydig cells are derived from a different population of precursors and are dependent on LH stimulation. INSL3 concentrations are low during infancy, rise during puberty, and reflect Leydig cell function during adulthood. Longitudinal data demonstrated that INSL3 concentrations rise with onset of puberty and increasing testicular volume.

INSL3 is also secreted by ovarian theca cells, predominantly by those surrounding medium and large growing follicles. INSL3 and its receptor may play a role in autoregulatory feedback to maintain theca cell androgen production. Despite many variations, INSL3 concentrations tend to rise during late puberty in girls; the variation likely reflects that INSL3 is secreted largely by growing follicles and is a marker of theca cell activity.

In the pubertal and postpubertal individual, the ovaries and testes are governed by feedback control systems. GnRH and the gonadotropins comprise the feed-forward components from hypothalamus to pituitary and from pituitary to gonad, respectively. In turn, steroid and protein hormones from the gonads provide the feedback signals that regulate the secretion of LH and FSH. The feedback actions of these gonadal hormones, which involve both negative and stimulatory (positive) actions, may be exerted directly at the level of the pituitary gonadotrophs to modulate expression of the genes encoding LHβ and FSHβ ( LHB and FSHB , respectively). Feedback may also be exerted indirectly at the level of the hypothalamus to regulate the release of GnRH.

In the male, a negative feedback action of testosterone and inhibin B are the major regulators of LH and FSH secretion, respectively. The action of testosterone is predominantly exerted at the hypothalamic level, while that of inhibin appears to occur directly at the pituitary. The role of aromatization of testosterone to estradiol in mediating the negative feedback action of this androgen on LH secretion continues to be an area of active investigation. The feedback control of LH and FSH throughout the menstrual cycle is complicated and involves both negative and positive feedback actions of ovarian steroids at both the hypothalamic and pituitary levels (see also Chapter 9 ). The maintenance of normal ratios of circulating LH and FSH concentrations is important for gonadal function, particularly for folliculogenesis and ovulation.

In conditions under which pulsatile GnRH release is compromised, such as occurs in anorexia nervosa and during periods of strenuous physical training especially in young women, gonadotropin secretion is attenuated and pubertal development is arrested. Thus the pituitary-gonadal axis in both males and females may be viewed as being a slave to the hypothalamic GnRH pulse generator, and this analogy should be held in mind when considering the mechanisms triggering the onset of gonadarche.

Hypothalamic Gonadotropin-Releasing Hormone Pulse Generator

The human hypothalamus contains approximately 2000 diffusely distributed GnRH neurons, many of which send their projections to the median eminence, where they intermittently discharge their peptide into the primary plexus of the hypophysial portal circulation, thereby providing the pituitary gonadotrophs with the pulsatile stimulation essential for maintaining gonadotropin secretion. The mammalian GnRH neuron displays unique characteristics in that the distal portion of the neuron functions as an autonomous regulatory region gathering synaptic input from other neurons; this region lies upstream of the short distal axons responsible for secreting GnRH into the portal circulation. ^, At the median eminences, these fibers are intertwined, encased by tanycytes (specialized ependymal cells of the third ventricle); they project to multiple blood vessels and receive numerous synaptic inputs. Tanycytes influence GnRH output by modulating access of GnRH neurons to the fenestrated vessels of the median eminence. ^,

Compelling evidence indicates the fundamental role of KNDy neurons in the arcuate nucleus (also called the infundibular nucleus) as a major component of the neurobiological mechanism that underlies GnRH pulse generation. ^, These hypothalamic neurons, named because they coexpress kisspeptin, NKB, and dynorphin, project their axons to the median eminence, where they mingle intimately with GnRH fibers en route to the portal vessels.

GnRH pulse generation is achieved by reciprocal stimulatory (NKB) and inhibitory (dynorphin) connections within the arcuate nucleus; the net output of the pulse generator is relayed to GnRH fibers by an intermittent kisspeptin signal. Kisspeptin, an extremely potent GnRH secretagogue, signals through its cognate receptor, KISS1R, which is expressed in GnRH neurons. Using immunohistochemistry, kisspeptin was detected in the anterior and intermediate lobes of the pituitary in monkeys but was not apparently colocalized with gonadotrophs, somatotrophs, or lactotrophs. ^, Loss-of-function mutations in the genes encoding for kisspeptin ( KISS1 ), the kisspeptin receptor ( KISS1R ), ^, NKB ( TAC3 ), or the NKB receptor ( TACR3 ) in humans are associated with hypogonadotropic hypogonadism. ^,

Detailed reviews of the neurobiology of the GnRH pulse generator and its applicability to humans are beyond the scope of this chapter but are readily available.

Fetal Development of the Gonadotropin-Releasing Hormone Pulse Generator

Using three-dimensional (3D) imaging and transparent human fetal brains, Casoni et al have suggested that approximately 2000 GnRH neurons reside in the hypothalamus and approximately 8000 are widely distributed in other areas of the brain. Meticulously orchestrated development of the GnRH neurons and olfactory neurons in conjunction with precise spatiotemporal expression of multiple factors is essential for normal HPG axis function ( Fig. 18.5 ). Specific mutations in families with disorders of puberty and studies of transgenic mice have established some of the factors involved in GnRH neuron migration. Elements essential to GnRH neuron development, migration, and function include cytoskeletal proteins, adhesion molecules, neurotransmitters, growth factors, receptors, and transcription factors. Some factors function in multiple regions and may have diverging effects depending on the context of the molecular environment. Elucidation of the neurobiology and ontogeny of the GnRH neurons will improve understanding of the pathophysiology of HH and, perhaps, lead to novel therapies.

The GnRH neurons are born in the olfactory placode and migrate during early fetal development from the nose through the forebrain to the hypothalamus. The fetal ontogeny of the GnRH neurons can be classified into several stages, each with distinct regulatory mechanisms: (1) differentiation of GnRH neurons; (2) migration with axons of the vomeronasal nerve across the cribriform plate and into the forebrain; (3) localization in the hypothalamus and development of processes to the median eminence; and (4) attainment of final location and functionality.

Most vertebrates have two distinct olfactory systems: the main olfactory system responsible for recognition of volatile odorants and the vomeronasal system responsible for detecting pheromones. During the fifth week of gestation in humans, the olfactory placodes develop as thickenings of the ectoderm on the ventrolateral sides of the head. GnRH neuronal differentiation occurs between 39 and 44 days of gestation. Despite the recognition that GnRH progenitor stem cells are derived from this embryonic olfactory placode, the precise lineage of the GnRH neuron remains unclear.

Between weeks 5 and 6 of human gestation, the migratory mass of neural crest-derived migratory cells and olfactory neurons contains a small number of GnRH neurons. Subsequently, around the sixth week of gestation, the GnRH neurons begin their migration along vomeronasal nerves through the cribriform plate and eventually find their way to the hypothalamus. The GnRH neurons migrate into the brain along two distinct migratory pathways: a ventral pathway directed towards presumptive hypothalamic regions and a dorsal pathway directed toward pallial and subpallial telencephalic regions. These GnRH neurons, accompanied by olfactory ensheathing cells (OECs), travel along with the terminal, vomeronasal, and olfactory nerves into the brain. OECs are glial cells that guide GnRH and olfactory nerves to the forebrain. OECs express several factors important for GnRH migration, such as semaphorin 4D, signaling and neuronal migration factor (NSMF), and stromal-derived growth factor 1 (SDF-1). Available data indicate that SOX10 promotes development of OECs.

Two proteins, chromodomain helicase DNA-binding protein 7 (CHD7) and SOX10, influence neural crest cell development and eventual migration. CHD7 is a large protein that participates in chromatin remodeling and transcription; it interacts with other proteins and may regulate genes involved in neural crest cell guidance. In mice heterozygous for Chd7 mutations, Fgfr1 expression in the olfactory placode, GnRH1 and Otx2 expression in the hypothalamus, and GnRHR expression in the pituitary were decreased, supporting a role for CHD7 upstream of Fgf8 and Fgfr1 in the development and maintenance of GnRH neurons.

Other factors involved in the differentiation of GnRH neurons include fibroblast growth factor-8 (FGF8), fibroblast growth factor receptor-1 (FGFR1), heparan sulfate 6- O -sulfotransferase 1 (HS6ST1), and AMH. FGF8 influences craniofacial development, neuroendocrine cell proliferation, cell fate specification, and cell survival. ^, In the developing GnRH neuron, FGFR1 is the preferred receptor for FGF8. The FGFR1 is a tyrosine kinase receptor composed of three extracellular immunoglobulin domains, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. Upon ligand binding, FGFR1 and its coreceptor dimerize, leading to autophosphorylation and protein kinase activity. ^, The extracellular domain of FGFR1 interacts with heparan sulfate proteoaminoglycan, its coreceptor. Heparan sulfates are cell membrane and matrix-associated proteoglycans involved in neural development. These polysaccharides undergo nonrandom modifications of the sugar moieties to facilitate cell-to-cell communication. HS6ST1 introduces a sulfate at the 6- O position within heparan sulfate. This action appears to be necessary for FGFR1 function.

AMH is reported to play roles in both the migration of GnRH neurons during fetal life and the postnatal function of the GnRH neuron. Immunohistochemical studies have identified AMH and AMHR in human fetal GnRH neurons and adult hypothalami. ^,

Correct targeting and movement of GnRH neurons depend on multiple cues provided by several factors. These signals may act directly or indirectly through the scaffold of olfactory neurons. Chemokine gradients likely influence GnRH neuronal movement. Such factors include SDF-1 and gamma-aminobutyric acid (GABA). Curiously, SDF-1 and GABA appear to exert divergent effects to accelerate or retard, respectively, neuronal migration. SDF-1 acts through its receptor, CXCR4, via a G protein-activated inward rectifier potassium channel. SDF-1 has been observed in the nasal mesenchyme (NM), whereas its receptor, CXCR4, has been localized in migrating GnRH neurons and olfactory/vomeronasal nerve axons. Further, CXCR4-deficient mice exhibit a loss of GnRH neurons and impaired migration, suggesting the importance of SDF-1/CXCR4 signaling in the development of this system.

CCDC141 encodes a coiled-coil domain containing protein that is expressed in GnRH neurons and olfactory fibers. Knockdown of Ccdc141 did not change olfactory axon outgrowth but was associated with decreased GnRH cell migration out of the nasal pit. In mice, Ccdc141 expression, correlated with migration in nasal regions and decreased when GnRH neurons entered the forebrain, appears to affect cellular motility through its interactions with myosin II.

Additional adhesion and guidance molecules include ANOS1, ephrins, and prokineticin 2 (PROK2). The gene encoding ANOS1 (previously known as KAL1 ) is located at Xp22.3 in the pseudoautosomal region of the X chromosome. Anosmin-1 is an extracellular matrix glycoprotein that contains a whey acidic protein-like protease inhibitor domain and four fibronectin type III domains. It promotes the formation of the lateral olfactory tract and neurite development. Anosmin may also serve as (1) an adhesion molecule to guide migrating GnRH neurons and (2) as a chemoattractant for olfactory axon pathfinding. Also, it may interact with FGFR1. Ephrins are cell surface molecules that play a major role in axon guidance and signal through their cognate membrane tyrosine kinase receptors. PROK2 signals through the prokineticin receptor 2 (PROKR2), a member of the rhodopsin G protein-coupled receptor family. PROK2 and its receptor ( PROKR2 ), a G protein-coupled receptor, appear to play major roles in olfactory bulb neurogenesis and GnRH neuron migration. However, neither protein is expressed in GnRH neurons. Based on the findings in mice with targeted PROK2 mutations, the GnRH neurons appear to be trapped with olfactory neurons with arrested migration just after crossing the cribriform plate.

Semaphorins comprise a large and diverse family of secreted and membrane-associated proteins that influence the navigation of growing axons and play a role in neural network formation. Four class 3 semaphorins, Sema3A, Sema3B, Sema3C, and Sema3F, are expressed around the developing olfactory/vomeronasal region. Semaphorin-3A ( SEMA3A ) is a secreted protein with repulsive effects on primary olfactory axons expressing the coreceptor neuropilin-1 (Nrp1), which may influence the migration of GnRH neurons. Semaphorin 3A is also expressed by OECs. Semaphorin-3E ( SEMA3E ) on the other hand protects maturing GnRH neurons from cell death. Semaphorin 4D is a membrane-bound semaphorin that can also be proteolytically released into the extracellular space in an active form. It can act as a proangiogenic factor through the coupling of its cognate receptor, PlexinB1, with the hepatocyte growth factor (HGF) receptor Met tyrosine kinase (MET). Both semaphorin 4D and PlexinB1 are highly expressed in the developing olfactory placode and the developing NM.

\Semaphorin 7A (Sema 7A) appears to play a role in GnRH neuronal migration. Two mechanisms have been described: it can act as a membrane-bound signaling molecule or, following proteolytic cleavage, as a soluble factor. Sema7A can interact with two different receptors (plexin C1 and β1-integrin). Binding to plexin C1 decreases integrin-mediated cell attachment and spreading and interacting with β1-integrin induces integrin clustering and the activation of MAPK pathways. The phenotype of the mouse model with GnRH neuron-specific β1-integrin conditional KO showed impaired migration of GnRH neurons, delayed pubertal onset, and impaired fertility in female mice. In addition to its role in the development of the GnRH system, Sema7A appears to mediate the plasticity of GnRH neurons and tanycytes in the adult median eminence.

IGSF10, a member of the immunoglobulin superfamily, is also implicated in GnRH neuronal migration. Tissue expression studies using mouse embryos showed that IGSF10 mRNA expression was localized to embryonic NM during the time that GnRH neurons are migrating through the NM. In a zebrafish IGSF10 knockdown model, loss of IGSF10 led to perturbed migration and failed neurite extension of GnRH3 neurons toward the hypothalamus.

HGF, Axl, and Tyro3 maintain GnRH neuronal survival when the neurons are crossing the cribriform plate region. HGF signals through its receptor, cMet, to promote GnRH neuron migration. Axl and Tyro3 are members of the TAM family of tyrosine kinase receptors and contain a fibronectin domain that binds to heparan sulfate proteoglycans. Mice with targeted Axl/Tyro3 mutations show impaired sex hormone-induced gonadotropin surge, resulting in estrous cycle abnormalities. The protein, growth arrest-specific 6 (Gas6) encoded by Gas6, is a ligand that activates Axl and Tyro3. Gas6 is a heparan sulfate proteoglycan-activated ligand with similarities to the FGFs and HGF. The phenotype of Gas6 knockout mice is characterized by early loss of GnRH neurons during embryonic development. Despite a persistent decrease in GnRH neurons and impaired early stages of sexual maturation, these mice eventually manifested normal fertility.

FEZF1 is a zinc-finger gene encoding a transcriptional repressor that is highly and selectively present during embryogenesis in the olfactory epithelium. Fezf1 -deficient mice have impaired axonal projection of pioneer olfactory receptor neurons that cross the cribriform plate and subsequently innervate the olfactory bulb. These mice have smaller olfactory bulbs and an absence of GnRH neurons in the brain. Thus it appears that the FEZF1 product is required for the olfactory receptor neurons, and hence accompanying GnRH neurons, to enter the brain. ^,

The roles of microRNAs (miRs) in neuronal development and maturation are becoming apparent. Data obtained using mice with targeted deletion of Distal-less-related 5 ( Dlx5 ) gene demonstrated that specific miRs (-9 and -200 class) influence olfactory and GnRH neuron development. Mice with gonadotrope-specific deletion of Dicer exhibited suppressed gonadotropin β-subunits and infertility. Another microRNA, miR-7a2, is expressed in pituitary gonadotropes. The phenotype associated with the genetic deletion of miR-7a2 in mice includes low gonadotropin concentrations and infertility. miR-7a2 is highly expressed in the pituitary but does not appear to influence GnRH neuron migration. ^,

Upon arrival in the hypothalamus, the GnRH neurons extend projections to the median eminence to form a network that can secrete GnRH into the primary plexus of the hypophysial portal circulation. LH and FSH reach detectable levels by the 10th week of gestation in the human pituitary, peak in midgestation, and are higher in female fetuses than male fetuses. Although the hypothalamic control of the fetal pituitary-gonadal axis has not been extensively studied in higher primates, the GnRH pulse generator is clearly driving the gonadotrophs of the fetal pituitary around the 15th week of gestation. Functional activity of this hypothalamic-pituitary system is essential for fetal testicular testosterone synthesis by the Leydig cell and normal male sex development. In contrast to the fetal testis, the ovary at this stage of development is relatively quiescent, and the absence of gonadal feedback signals likely accounts for the higher gonadotropin levels in the female fetus. As gestation progresses, the secretion of estradiol and other steroids by the fetoplacental unit increases dramatically and suppresses gonadotropin secretion from the fetal pituitary by exerting an inhibitory action either directly at the pituitary or indirectly on the hypothalamus to restrain GnRH release.

Postnatal Development of Gonadotropin-Releasing Hormone Pulsatility

Gonadotropin concentrations are typically low in cord blood samples. However, following birth, GnRH pulse generator activity is robustly expressed, presumably due to the loss of placental steroids. The pituitary gonadotrophs of the infant respond with LH and FSH secretion. ^{, ,} Moreover, in the infant boy, the Leydig cells of the testis are stimulated so that circulating testosterone levels are similar to those observed in adult men. Peak testosterone concentrations occur at approximately 2 to 3 months of age and typically decline by 6 months of age. Among preterm male infants, LH and testosterone concentrations are higher than among full-term infants; phallic growth was positively correlated with urinary testosterone levels, and testicular growth was positively correlated with urinary FSH levels. Despite the “adult-like” endocrine milieu of increased gonadotropin secretion and elevated testosterone concentrations during the first few months of life, sexual hair does not develop and gametogenesis is not initiated, presumably due to limited AR signaling in the skin and the immature Sertoli cell. Gonadotropin and testosterone secretion during minipuberty promote anchoring of testes in the scrotum, active Sertoli cell proliferation, and expansion of the pool of germ cells. ^,

In contrast to the brief peak of LH and testosterone secretion within 24 to 48 hours of birth in infant boys, infant girls show a gradual increase in gonadotropin and estradiol concentrations, with estradiol concentrations typically declining around 6 months of age; LH concentrations decline, whereas FSH concentrations tend to remain higher until 3 to 4 years of age. This transient hypothalamic-pituitary-ovarian axis activity is associated with a transient increase in antral follicles. ^,

Utilization of reference ranges for gonadotropins and reproductive hormones during minipuberty can help the management of infants with suspected differences in sex development (DSD) or suspected congenital hypogonadotropic hypogonadism. Although LH and FSH concentrations overlap, LH is higher in boys and FSH is higher in girls. The LH/FSH ratio with a cut-point of 0.32 accurately distinguished boys from girls. AMH concentrations serve as a marker of testicular function and can also separate boys from girls. Girls with Turner syndrome have elevated gonadotropin concentrations. Approximately 25% of men with congenital hypogonadotropic hypogonadism experienced bilateral cryptorchidism. Obtaining LH, FSH, and testosterone concentrations during minipuberty may facilitate early diagnosis of congenital hypogonadotropic hypogonadism. Hence, this brief period of the HPG axis in infancy can be utilized to assess gonadal function in infants with DSD or suspected CHH.

Full hormonal responsivity of the gonad is acquired during childhood. However, the GnRH pulse generator has been brought into check by this stage of development, resulting in the hypogonadotropic state that guarantees continued gonadal quiescence until the prepubertal phase of development is terminated by a resurgence of GnRH pulse generator activity ( Fig. 18.6 ). During childhood and juvenile development, the GnRH neurons, pituitary gonadotrophs, and the cells of the gonads are not limited to the onset of gonadarche. ^{, ,}

Because GnRH is secreted in only picogram quantities into the hypophysial portal circulation, changes in the concentration of this neuropeptide in the peripheral circulation do not reflect hypothalamic activity. Therefore, studies of the dynamics of the pubertal resurgence of GnRH pulse generator activity in humans and other higher primates have generally utilized the high-fidelity relationship that exists between the frequencies of pulsatile GnRH release and episodic LH secretion. The latter may be tracked with relative ease by measuring moment-to-moment changes in LH concentrations in the peripheral circulation. Although the pubertal increase in hypothalamic GnRH drive to the gonadotroph probably involves both frequency and amplitude modulation of the GnRH pulse generator, the relationship between GnRH and LH pulse amplitude is more complex than the relationship between frequency because amplitude modulation of LH release may not always reflect changes in GnRH pulse amplitude. During the initiation of gonadarche in both boys and girls, LH pulse frequency accelerates and LH pulse amplitude increases in association with amplification of a preexisting sleep-related diurnal pattern in release. This change in neuroendocrine activity may occur before the physical changes of gonadarche are manifest. Particularly in boys, LH pulse frequency appears to decline later in pubertal development, probably due to a negative feedback action of rising testosterone concentrations. A longitudinal study of the agonadal monkey suggests that, as in humans, the pubertal acceleration of pulsatile GnRH release is an early neurobiological event in the initiation of gonadarche, and that it is a rapidly completed process. Thus the slow tempo of the overall progression of puberty probably results from mechanisms downstream from the hypothalamus, particularly at the level of the pituitary.

The control system that dictates the up-down-up pattern of GnRH pulse generator activity from birth until puberty may be viewed as a neurobiological “brake” (or central restraint, as it has been previously described in the pediatric literature) that holds GnRH neuronal activity in check during the greater part of prepubertal development. Here it is important to recognize that the notion of a brake is conceptual; that is, the pubertal resurgence in robust GnRH pulsatility could be occasioned either by the removal of an inhibitory input or by the application of a stimulatory signal to the GnRH pulse generator, or a combination of the two. A similar argument may be applied to the earlier transition between infancy and childhood when GnRH pulsatility is markedly diminished. The neurobiological brake on pulsatile GnRH release throughout childhood and juvenile development is imposed in the absence of the ovary or testis. Consequently, the characteristic pattern of gonadotropin secretion observed during postnatal development in humans with robust gonadotropin secretion during infancy and puberty—separated by a prolonged hiatus in LH and FSH secretion—is maintained in the agonadal situation ( Fig. 18.7 ). ^{, ,}

Similarly, LH concentrations are generally elevated in male infants with partial androgen insensitivity, which is associated with higher testosterone levels. Yet, infants with complete androgen insensitivity often fail to demonstrate a postnatal rise in LH and testosterone secretion. The latter finding is counterintuitive; an understanding of the molecular basis of this phenomenon may reveal fundamental insights into the ontogeny of GnRH pulse generation.

In agonadal children, the degree of the prepubertal suppression of gonadotropin release is less than that observed in eugonadal individuals. Interestingly, levels are higher in girls than boys in agonadal children circulating gonadotropin, indicating that the intensity of the neurobiological brake imposed on the GnRH pulse generator during prepubertal development is less in females than in males. As a result, the gonadotropin drive to the prepubertal ovary stimulates a low level of estradiol secretion through which negative feedback action on LH and FSH release, amplifying the relatively weaker neurobiological brake restraining gonadotropin secretion in the prepubertal girl. This sex difference in the strength of the neurobiological brake on prepubertal GnRH release is associated with a shorter duration of the brake in girls, which probably accounts for the relatively earlier age of gonadarche in the female. These and other sex differences in the developmental control of the GnRH pulse generator are presumed to result from greater exposure of the fetal male hypothalamus to testosterone.

Transcriptional regulation of pubertal onset is proposed to be controlled by a system of overlapping gene networks organized in a hierarchical manner. ^, The highest level of control is the transcriptional regulators that direct the expression of subordinate genes leading to pubertal onset. Transcriptional regulators of puberty include the POU-domain gene Oct2 , the homeodomain gene Ttf1/Nkx2.1 , the novel gene Eap1 (Enhanced At Puberty1), and LIN28B . A tumor-related gene (TRG) network has also been identified as transcriptional control of puberty. The TRG network transcriptional regulation of puberty is proposed to function through hubs that are connected to both upper echelon TRG genes ( Oct2, Ttf1, Eap1) and subordinate genes ( KISS1, GPR54, and TSLC1) involved in the regulation of puberty. Kiss1 expression is controlled by both non-TSG transcriptional regulators (TTF1 and Eap1) and TSG transcriptional regulators (CUTL1 and YY1).

Puberty onset is held in check by transcriptional and posttranscriptional repression of genes that are stimulatory to the pubertal process. LIN28A and B block maturation of let-7 MiRNA precursors, whereas excess LIN28B derepresses let-7 miRNA target genes. Other transcriptional repressors include zinc finger (ZNF) genes, POZ-ZF (poxvirus and zinc finger) family of transcriptional regulators, POK (POZ and Krüppel), and PcG (the Polycomb group). The latter three are part of the TSG network.

Nature of the Neurobiological Brake

The discovery in 2003 that loss-of-function mutations in KISS1R in humans were associated with HH and delayed or absent puberty ^, demonstrated the critical role of kisspeptin in regulating GnRH secretion. Subsequent studies using several different experimental models validated this finding. These data led to the proposal that a major component of the neurobiological brake imposed upon pulsatile GnRH release during the greater part of prepubertal development is due to a hiatus in a stimulatory kisspeptin input to the GnRH neuronal network. This proposal was based on findings in the monkey, that hypothalamic expression of KISS1 and release of kisspeptin in the region of the median eminence increase at the time of the pubertal resurgence in GnRH pulsatility. Additionally, intermittent administration of kisspeptin at hourly intervals during juvenile development elicits a precocious and sustained adult-like pulsatile pattern of GnRH and the pubertal increase in GnRH release may be suppressed by the administration of a KISS1R receptor antagonist directly to the median eminence.

The finding in humans that loss-of-function mutations in the NKB signaling pathway are associated with a phenotype similar to that reported earlier for inactivating mutations in KISS1R, together with the observation that these two neuropeptides are coexpressed in the same neurons (KNDy neurons) in the arcuate nucleus, have led to the concept that these KNDy neurons are responsible for the generation of GnRH pulsatility. Thus kisspeptin expressing KNDy neurons in the arcuate nucleus comprises a critical component of the GnRH pulse generator. In the absence of an intact kisspeptin signaling pathway, the output of the GnRH pulse generator will be abrogated and pulsatile GnRH release will be compromised, resulting in a delay or absence of puberty. Overall, available data suggest that the KNDy neurons in the arcuate nucleus themselves do not govern the timing of puberty; rather, these neurons appear to be subservient to upstream regulatory mechanisms that govern the developmental pattern of pulsatile GnRH release and the onset of puberty ( Fig. 18.8 ).

The nature of the upstream pathways that comprise the neurobiological brake on the GnRH pulse generator during childhood and juvenile development remains poorly understood.

Studies of the female rhesus monkey provide evidence that GABA, the major inhibitory neurotransmitter in the brain, is upregulated during juvenile development, and inhibition of GABA tone in the hypothalamus of the prepubertal monkey leads to precocious menarche and ovulation. Interestingly, infusion of the GABA antagonist bicuculline into the median eminence of prepubertal female monkeys stimulates the release of kisspeptin-54 into this region of the hypothalamus in association with that of GnRH, and the bicuculline-induced GnRH release is blocked by simultaneous infusion of a kisspeptin antagonist. However, it is unclear what reduces GABA inhibition prior to puberty, where the relevant GABAergic neurons are physically located, and how GABA signaling interacts with the GnRH pulse generator.

Other transsynaptic signals implicated in the regulation of the pubertal resurgence of GnRH pulse generator activity include glutamate and neuropeptide Y (NPY). Glutamate is the major excitatory neurotransmitter in the brain and, in contrast to GABA, hypothalamic release of this amino acid is increased at the time of puberty in the female monkey. Also, as discussed earlier in the chapter, repetitive activation of glutamate receptors in the juvenile monkey rapidly leads to the onset of precocious gonadarche. NPY neurons are found in the arcuate nucleus and, in the male rhesus monkey, NPY gene expression in the hypothalamus is inversely related to the up-down-up pattern of GnRH pulse generator activity from birth to puberty. NPY receptors are inhibitory G protein receptors, and their activation leads to hyperpolarization and inhibition of neural activity. However, pharmacological approaches failed to demonstrate that inhibition of NPY signaling in the hypothalamus of the juvenile monkey did not promote GnRH release.

While neuroglia have classically been regarded as subserving only a “supporting role” in the central nervous system (CNS), contemporary views hold that these nonneuronal cells play important functional roles within the brain. Moreover, in the context of the hypothalamus, secretion of TGF-α by astroglia has been postulated to provide the GnRH neuronal network with a stimulatory input at the time of puberty. ^,

Attempts to elucidate the neural mechanism dictating the postnatal pattern of GnRH pulse generation have traditionally led investigators to focus on relative isolation on a “favorite” signaling pathway. Using a systems biology approach, global gene discovery has been combined with computational (in silico) biology to identify functional linked networks of hypothalamic genes that are found to be associated with changes in GnRH pulse generator activity. The initial gene discovery approach was conducted without regard to the phenotype of the cells in which the respective genes are expressed and gene networks are operating. Available data indicate that the developmental changes in the transcriptional factors and gene network relevant to GnRH secretion lie upstream of the KISS1 gene and are therefore upstream of GnRH pulse generation. This network of genes serves as a governing hierarchy to orchestrate the resurgence of pubertal GnRH release and, therefore, modulate the timing of puberty. Since such networks of genes are further proposed to function in the absence of signals derived from the periphery, they may conceptually be viewed at a systems level as comprising a pubertal clock (discussed later in the chapter).

Expression of two such transcriptional regulators enhanced at puberty 1 ( EAP1, also known as interferon regulatory factor 2 binding protein-like) and thyroid transcription factor-1 ( TTF-1, also known as NKX2-1 ), increase in the mediobasal hypothalamus of nonhuman primates at puberty. EAP1 is expressed in kisspeptin neurons in the arcuate nucleus of the monkey. Moreover, its expression in the hypothalamus increases at the time of puberty in the female monkey, and the knockdown of EAP1 using a lentivirus approach interrupts menstrual cyclicity in the adult female. Additionally, a single nucleotide polymorphism (SNP) upstream of the EAP1 gene has been associated with irregular menses in the monkey. ^, Conditional deletion of Ttf1 from terminally differentiated hypothalamic neurons was associated with delayed puberty, decreased Kiss1 expression, and subfertility. Genetic analysis failed to identify germline mutations in either EAP1 or TTF-1 in patients with HH. Nevertheless, both EAP1 and TTF-1 are functionally connected to genes identified by genome-wide association studies (GWAS) to influence age at menarche.

Additional evidence supporting the concept of a gene network to determine the level of function of the GnRH pulse generator have identified a cohort of genes that encode for a group of transcriptional suppressor proteins known as the polycomb group. In the pubertal rat, expression of these genes is downregulated by DNA methylation, leading to a reduction in the silencer proteins. Two of the polycomb group genes are expressed in the arcuate nucleus, and overexpression of one of these genes resulted in a decrease in Kiss1 expression associated with delayed vaginal opening and a disruption of GnRH pulsatility in mediobasal hypothalamic (MBH) explants. A global inhibition of DNA methylation in prepubertal rats resulted in the delay of the vaginal opening, indicating that epigenetic regulation of gene expression may be important in timing puberty. Similarly, data obtained in monkeys indicate that some zinc-finger transcriptional repressors restrain puberty by epigenetically repressing a gene network that operates in the arcuate nucleus and controls puberty by governing pulsatile GnRH release.

Additional support for a zinc-finger motif containing a suppressor gene holding puberty in check has come from human mutation studies. Using whole exome sequencing (WES), inactivating mutations in the gene encoding for makorin RING finger protein 3, MKRN3 , are the most common genetic defects associated with CPP to date. The MKRN3 protein is the first protein to be identified with an inhibitory role in GnRH secretion identified in humans to have reported mutations. It is a maternally imprinted gene on chromosome 15q11.2 in the Prader-Willi critical region. Whereas the maternally inherited allele is methylated in the central nervous system, loss of function mutations are paternally inherited. The makorin family of proteins contains a particular zinc-finger motif that has been associated with ubiquitination, a process that is involved in protein trafficking, which in some cases leads to protein degradation. The relevance of this gene to puberty is further supported by demonstrating declining levels of circulating MKRN3 product prior to pubertal onset in Danish girls and boys. ^, Expression of this gene decreases immediately before the onset of puberty in the mouse hypothalamic arcuate nucleus. Thus, in mice, makorin-3 appears to contribute to the neurobiological brake on GnRH secretion via neurons producing kisspeptin and neurokinin B. MKRN3 is expressed in KISS1 neurons in the hypothalamic regions critical for puberty initiation and reproduction in rodents and nonhuman primates and associates with KISS1 and TAC3 gene promoters to repress transcriptional activity. In female mice, the prepubertal decline in MKRN3 was shown to be independent of gonadal activation. Together, these studies support the concept that transcriptional repression is a core component of the neuroendocrine circuitry that regulates the timing of puberty.

Recently, genome-wide association studies indicate that several specific loci on the human genome are associated with variations in the age at menarche. ^, An SNP that was consistently and strongly associated with an earlier age at menarche was found on chromosome 6 near LIN28B, which encodes for a micro-RNA binding protein. The differences in age at menarche in subjects with and without variants in the region of LIN28B are small (1–2 months) compared to the recognized range for the age at menarche in the population at large that may extend from 10 to 16 years of age. A genetic study on girls with early puberty did not find any potentially responsible LIN28B mutations. It is currently unclear the exact causal role of LIN28-Let7, if any, in timing of human puberty. A follow-up meta-analysis study using genome-wide and custom-genotyping arrays in more than 180,000 European women found strong evidence for 123 SNPs at 106 gene loci to be associated with earlier age at menarche. Surprisingly, only a few known puberty genes overlap with the genes associated with age at menarche. These include MKRN3 , LEPR , IGSF1 , and TACR3 , further implicating biological significance of these genes in pubertal development.

Regardless of the components of the neurobiological brake, which dictate the up-down-up pattern of pulsatile GnRH release from birth until puberty, it is to be anticipated that application and withdrawal of the brake will be associated with a corresponding structural remodeling in those hypothalamic neuronal and glial circuits involved. In this regard, the hypothalamus of the postnatal brain retains its capacity for plasticity, as reflected by the expression of polysialic acid-neural cell adhesion molecule (NCAM).

A significant number of boys and girls with delayed puberty do not have a readily recognizable diagnosis, such as an anatomic lesion of the hypothalamus or pituitary, primary gonadal insufficiency, or functional hypogonadotropic hypogonadism due to illness, stress, or negative energy balance. A study of 16 children with delayed puberty showed that unstimulated LH measurements, the presence of overnight LH pulses, and GnRH-stimulated LH secretion did not distinguish between children who would and would not progress through puberty; however, LH response to kisspeptin did distinguish between the two groups. All participants who had a rise in LH of 0.8 mIU/mL or greater in response to kisspeptin progressed through puberty and those with an LH response less than or equal to 0.4 mIU/mL reached age 18 years without developing signs of puberty. The sensitivity and specificity of the kisspeptin stimulation test were both 100%.

Nutrition and Diet

Adequate energy stores are essential for the accelerated linear growth and achievement of reproductive competence at puberty. Neuropeptides and hormones provide information regarding energy status. The nutritional state modulates the onset of gonadarche in girls, as reflected by the findings that menarche is delayed in malnourished girls, and menarche tends to occur at a particular or “critical” body weight rather than at a set age. Obesity is associated with early breast development and menarche. Rapid weight gain during the first year of life is associated with obesity and early menarche in a prospective UK cohort study. In addition, the timing of adrenarche may also be influenced by nutritional status. In the female rhesus monkey, high-calorie diets were associated with a precocious increase in nipple volume, sex-skin swelling, and menarche in association with increased concentrations of leptin and IGF-1 concentrations, and an increased BMI. ^, While the impact of undernutrition on gonadarche in boys has received less attention, there is no reason to suspect that the male axis is unaffected in this regard.

Leptin and osteocalcin appear to communicate energy status to several tissues including adipose tissue, bone, CNS, and gonads. In its undercarboxylated (active) form, osteocalcin promotes insulin secretion, improves insulin sensitivity, induces β-cell proliferation, increases adiponectin secretion, and decreases lipolysis. In osteoblasts, insulin signaling increases bone formation, bone resorption, and secretion of active osteocalcin production.

Differences in the timing and tempo of gonadarche unrelated to racial or ethnic background have been reported even among well-nourished girls. Researchers have suggested that diet may account for such variations. Attention has been drawn to subtle relationships between diets high in animal protein and early menarche, and between diets high in vegetable grains and delayed onset or tempo of gonadarche. Moderate to vigorous exercise in the absence of weight restriction appears to have a negligible impact on the timing and tempo of puberty in either sex. However, breast development, menarche, and skeletal maturation can be delayed in girls involved in strenuous physical training. The association between extreme energy expenditure and delayed gonadarche in the female is particularly marked in ballerinas, long-distance runners, and figure skaters, who must maintain their body weight within strict limits. Such girls may experience amenorrhea secondary to HH. The factors responsible for compromising pulsatile GnRH release in pubertal children who exercise vigorously are presumed to be similar to those resulting from undernutrition. The life of the young female dancer or athlete is stressful; therefore stress may also represent a contributing factor underlying exercise-induced delayed gonadarche. Because certain physical and psychological characteristics are necessary for outstanding athletic performances, there may be a significant contribution of self-selection involved in the decision of girls to participate at such an exceptional level. This can be complicated by eating disorders, which are more common in elite artistic athletes such as gymnasts. ^, In a study of young female gymnasts and their parents, menarche in the mothers was found to be delayed relative to that of mothers of sedentary girls. Therefore, the possibility of a genetic contribution to this phenomenon cannot be totally excluded. The effect of physical training on the timing of pubarche in girls has been less studied, and conclusive data are lacking.

Although the relationship between strenuous physical training and male puberty has received less attention, this developmental process is apparently less susceptible in boys than it is in girls. This may be related, in part, to sex differences in the age at which training is initiated, and to the intensity of the exercise. Nevertheless, in sports like wrestling that require weight control achieved with a combination of strenuous exercise and dietary restriction, impaired testosterone secretion may occur.

Environmental Disruptors

Increased attention has been focused on EDCs as modulators of pubertal timing. Most EDCs have estrogenic or antiandrogenic activities. Many occur as mixtures rather than single agents and may have multiple actions. Sexually dimorphic actions can occur. Dioxins, PCB, PBB, BPA, plasticizers (phthalates), pesticides, fungicides (vinclozolin), alcohol, tobacco, and pharmaceutical agents (diethylstilbestrol) are considered to be EDCs. ^, Phytoestrogens, which can function as selective estrogen receptor modulators (SERMS), are found in soybeans, flaxseed, peanuts, and some vegetables. Most are diphenolic compounds with structural features common to estrogenic steroid agonists and antagonists. EDCs can affect peripheral reproductive systems, such as inducing breast development. These chemicals may also exert neuroendocrine effects to influence hypothalamic and pituitary function. EDCs can act through hormone receptors, can affect enzymatic processes, and may disrupt the complex interactions of endogenous hormones at multiple levels. Available data suggest that EDCs are associated with both early puberty and delayed completion of puberty. The specific consequences of EDCs may depend on age and duration of exposure. Further investigations into EDCs, defining mechanisms of action and consequences, are necessary.

Prenatal Influences

The roles of prenatal influences and environment on postnatal development including puberty are increasingly recognized. ^, Children with intrauterine growth retardation (IUGR) or born small for gestational age (SGA) have increased risks to develop insulin resistance, hypertension, diabetes mellitus, metabolic syndrome, and coronary artery disease in adulthood. Increased weight gain during the first few years of life exaggerates these risks. Increased adrenal androgen levels and in some cases precocious or exaggerated adrenarche has been reported in both boys and girls born SGA. ^, Some, but not all, girls with premature adrenarche have an increased risk to develop polycystic ovary syndrome (PCOS) during adolescence. Reports are inconsistent regarding the timing of menarche in girls born SGA compared to girls born at an appropriate size for gestational age. ^, The timing of puberty in boys born SGA appears to be normal , although subfertility has been reported after they reached adulthood. Perinatal HIV infection is associated with later onset of puberty. ^, Potential mechanisms responsible for the consequences of abnormal fetal growth are multifactorial and not mutually exclusive. Suggested causes include genetic variations, differential methylation, alterations in microRNA, altered histone acetylation, and other long-range regulatory effectors.

The neuroendocrine consequences of prenatal androgen exposure have been best characterized in sheep. Findings include decreased sensitivity to estradiol and progesterone feedback, decreased content of dynorphin and NKB in KNDy neurons, and altered morphology of KNDy neurons. Whether these data are applicable to humans and whether prenatal androgen exposure alters GnRH pulse generator function is unclear.

Adoption or Migration From Developing to Developed Countries

Several European countries have reported precocious gonadarche in a relatively dramatic proportion of children—particularly girls—adopted from developing world regions like Asia and South America. ^, This increased endocrine activity in the hypothalamic-pituitary-ovarian axis prior to the onset of physical manifestations of puberty indicates a central origin to the condition. The cause of the precocity appears to be complex, but improved nutritional and social conditions may contribute to this phenomenon. Precocious gonadarche is also seen in immigrant girls arriving with their parents and lacking evidence of earlier compromised growth and nutrition. Moreover, ethnic background does not appear to influence this trend. A study of immigrant girls in Belgium with premature gonadarche reported an association with previous exposure to organochlorine pesticides. A study in Spain found an increased relative risk for precocious gonadarche in adopted girls (both national and international), but not in immigrant girls. However, this outcome is inconsistent because adopted Chinese girls experienced menarche at ages comparable to nonadopted Chinese girls.

Gonadotropin-Releasing Hormone-Dependent Precocious Pubertal Development

Progressive Precocious Gonadarche or Central Precocious Puberty

Although this disorder is labeled GnRH-dependent precocious puberty or puberty (central precocious puberty [CPP]), it represents precocious gonadarche due to either premature resurgence or incomplete suppression of the hypothalamic GnRH pulse generator. It occurs more often in girls than in boys. This sex difference is probably related to the lesser prepubertal suppression of the GnRH pulse generator in girls than in boys. The sequence of pubertal development is typical of a normal puberty including adrenarche in some cases, but it begins at an earlier-than-normal age. Observational data collected from 2008 to 2010 through a stringent Spanish hospital-based registry reported an annual incidence ranging from 0.02 to 1.07 cases per million.

Approximately 90% of cases of CPP in girls are considered to be idiopathic, whereas 50% to 70% of cases in boys are associated with identifiable etiology. The discovery of genes involved in GnRH and gonadotropin secretion has allowed for identification of specific gene mutations in patients with CPP. A heterozygous missense mutation, p.Arg386Pro, in the KISS1R gene was associated with precocious puberty in a girl. This mutation located in the C-terminal tail of the receptor was associated with delayed degradation of the receptor yielding a prolonged duration of action. In addition to this activating mutation in the receptor, a mutation in the KISS1 gene was identified in a young boy who presented at 17 months of age with CPP. This variant, p.Pro74Ser, appears to prolong the half-life due to resistance to degradation. To date, KISS1 and KISS1R mutations are extremely rare in children with CPP.

As described above, paternally inherited MKRN3 mutations are associated with CPP in boys and girls. This gene is mapped to chromosome 15q11.2 in the Prader-Willi Syndrome critical region. The maternal allele is methylated and silenced resulting in expression of the paternal allele. The gene encodes a protein involved in ubiquitination and cell signaling. Several unique mutations have been reported among different ethnic groups. Mutations in this gene appear to be the most common familial etiology of CPP. The median age of pubertal onset is 6.0 years (3.0–7.5) in girls and 8.25 years (5.9–9.0) in boys. No abnormalities of the CNS have been reported. Curiously, despite the physical proximity of MKRN3 to the Prader-Willi syndrome locus, no major signs of Prader-Willi syndrome have been described. A GWAS study looking at the parent of origin effects on puberty identified a signal in the region of MKRN3 gene; the paternal allele of a specific SNP (rs12148769, G>A) affects age at menarche in healthy girls suggesting that variants in this region affect pubertal timing within the normal range. ^, Circulating makorin-3 concentrations were found to decline prior to puberty among healthy girls and boys ( Figs. 18.10 and 18.11 ). ^{, ,}

CPP has been described in the Williams-Beuren syndrome, histiocytosis X, and maternal uniparental disomy for chromosome 14. Evaluation of a family with CPP led to identification of a deletion/duplication mutation in the delta-like 1 homologue ( DLK1 ) gene located at chromosome 14q32. The four affected individuals presented with thelarche between the ages of 6.4 to 8.0 years of age. An additional investigation revealed undetectable serum DLK1 concentrations (<0.4 ng/mL) compared to control group (1.9–20 ng/mL). DLK1 is a paternally expressed gene located within the genetic locus associated with Temple and Kagami-Ogata syndromes. Temple syndrome is characterized by IUGR, hypotonia in infancy, CPP, and short stature. Genetic findings in Temple syndrome include maternal uniparental disomy, paternal deletion, or loss of differential methylation at the DLK1/MEG3 region on chromosome 14. Patients with Kagami-Ogata syndrome is associated with prenatal overgrowth, developmental delay, abdominal wall defects, but disorders of pubertal timing do not appear to be a prominent feature. Based on available animal and human data, the opposing directions of regulation of MKRN3 and DLK1 expression prior to puberty may underlie interactions between these factors that modulate the timing of puberty.

Both DLK1 and MKRN3 are maternally imprinted and paternally expressed genes. The role of DLK1 in pubertal timing has been implicated by a GWAS that showed SNPs near paternally inherited DLK1 were associated with a significantly earlier age of menarche. Temple Syndrome occurs with loss of DLK1 expression in conjunction with two other genes paternally inherited genes, RTL1 and DIO2. Polymorphisms in other candidate genes such as LIN28B and the paternally imprinted KCNK9 have also been associated with age of menarche and are candidates of monogenic causes of central precocious puberty. To date, there have been no described patients with central precocious puberty with this mutation. The relatively high frequency of imprinting mutations associated with monogenic causes of central precocious puberty suggests that imprinting may play an important role in the regulation of puberty and that pubertal onset may be associated with specific changes in the regulatory control of epigenetic modification.

Hypothalamic hamartomas, congenital malformations composed of a heterotropic gray matter, neurons, and glial cells usually located on the floor of the third ventricle or attached to the tuber cinereum, are a common etiology of CPP. Two potential mechanisms have been hypothesized for the association between CPP and hypothalamic hamartomas; one is increased GnRH secretion from tissue emancipated from suppression by the prepubertal brake and the other is that factors such as TGF-α provide an ectopic drive to GnRH neurons with a normal distribution in the hypothalamus. The hamartomas can be classified as parahypothalamic , 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) and α-fetoprotein. Extreme precocity suggests a hamartoma. Although gelastic or laughing seizures can be associated with precocious puberty due to hypothalamic hamartomas, the majority of patients with hypothalamic hamartomas do not exhibit neurological symptoms. ^, Among patients in whom hamartomas were removed to treat intractable seizures, hamartomas associated with CPP were more likely to contact the infundibulum or tuber cinereum and were larger than hamartomas not associated with CPP. All hamartomas expressed GnRH, TGF-α, and GnRHR. No differences were detected in the expression of KISS1 and GPR54 between hamartomas associated with CPP and those not associated with CPP. Gene expression profiling of hypothalamic hamartomas associated with precocity may provide clues regarding genes, proteins, and regulatory pathways associated with the timing of puberty.

Hypothalamic hamartomas are generally sporadic but may occur as a feature of several dysmorphic syndrome including Pallister-Hall syndrome and oral-facial-digital syndrome (OFD) types I and VI. Pallister-Hall syndrome is an autosomal-dominant disorder associated with mutations in the GLI3 gene located at chromosome 7p13. Additional features include pituitary anomalies, hypopituitarism, imperforate anus, and polydactyly. In the presence of sonic hedgehog (SHH), the full-length GLI3 translocates to the nucleus, where it functions as a transcriptional activator. When SHH is absent, GLI3 is phosphorylated and cleaved; the cleaved protein translocates to the nucleus to repress specific target genes. The truncated GLI3 protein showed stronger repressor activity than the wild type of protein, suggesting that these mutations are associated with constitutive repression of SHH signaling. Mutations in additional genes in the SHH pathway, especially PRKACA, have been detected in HH associated with epilepsy.

In addition to hypothalamic hamartomas, optic gliomas, suprasellar cysts, arachnoid cysts, previous head trauma, static cerebral encephalopathy, CNS infections, CNS radiation, hydrocephalus, meningomyelocele, and neurodevelopmental disabilities may also be associated with CPP. For hypothalamic-pituitary disorders, the endocrine symptoms of precocious puberty may precede neurological symptoms. Three predictors associated with CNS lesions in girls are: (1) age younger than 6 years; (2) absence of pubic hair; and (3) estradiol concentrations greater than 110 pmol/L. The type of CNS lesion influences the presentation of GnRH-dependent precocious puberty presumably due to differences in the mechanisms inducing puberty and to the hypothalamic-pituitary deficiencies associated with the initial lesion or its treatment.

Optic gliomas are associated with neurofibromatosis type 1 (NF1), an autosomal-dominant disorder diagnosed based on clinical features that include size and number of café-au-lait spots, macrocephaly, and family history of the disorder. The mechanism of action has been presumed to be a mass effect. Pineal tumors can be associated with precocious puberty due to tumor-induced hydrocephalus or germ cell tumors. CNS radiation, dose range 18 to 50 Gray, used to treat intracranial tumors or used prophylactically for malignancies, can induce precocious gonadarche, perhaps as a result of an astroglial response with increased TGFα production. In the situation of CPP, simultaneous GH deficiency (GHD) may be masked by the accelerated growth velocity associated with increased gonadal sex steroid secretion.

Other situations associated with progressive CPP include children with virilizing disorders (such as CAH) and familial male-limited precocious puberty (testotoxicosis) in whom skeletal maturation is usually markedly advanced. In these situations, the precocious gonadarche is considered to be secondary to the virilizing disorder, but the mechanism through which the GnRH pulse generator is prematurely activated is unclear.

Gonadotropin-Releasing Hormone-Independent Precocious Pubertal Development

Precocious pubertal development may occur independently of pulsatile GnRH secretion. In these situations of peripheral precocious puberty, inappropriate gonadal or adrenal steroid secretion or exposure to exogenous steroids induces the physical signs of puberty. In most instances, pubertal development is incomplete and fertility is not attained.

Feminizing Disorders

Virilizing Disorders

Premature pubarche is defined as the development of pubic hair, axillary hair, and apocrine body odor prior to 8 years of age in girls and age 9.5 years of age in boys. The differential diagnosis includes premature adrenarche, CAH, and androgen-secreting tumors.

Disorders of Steroidogenesis

Other Disorders Affecting Steroidogenesis