Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter produced by specialized neurons in the hypo thalamus and has an important role in regulating the activity of the GnRH pulse generator. Elegant hypothalamic perfusion studies have revealed that release of GABA into the median eminence decreases as pulsatile GnRH secretion increases at the onset of puberty.³¹ Conversely, central perfusion with a GABA_A receptor antagonist (bicuculline) or the antisense oligodeoxynucleotide for the mRNA coding the GABA_A synthesizing enzyme (glutamate acid decarboxylase) stimulates GnRH release.^31,32 Chronic administration of bicuculline into the third ventricle induces precocious puberty and menarche in prepubertal female monkeys.³³ Evidence suggests that changes in the
subunit composition of GABA_A receptors may contribute to the disinhibition of pulsatile GnRH secretion at the onset of puberty.³⁴ These observations suggest that central GABA signaling is one of the factors that restrains GnRH neuronal activity during childhood.

Neuropeptide NPY (NPY) is a hypothalamic peptide involved in the control of food intake behavior and reproductive function in adults. In castrate adult female monkeys, intracerebroventricular administration of NPY inhibits pulsatile GnRH release.³⁵ In males, the postnatal pattern of GnRH pulse generator activity is inversely related to NPY gene and protein expression in the medial basal hypothalamus and central administration of an NPY receptor antagonist stimulates GnRH release in juveniles.³⁶ These observations suggest that NPY, like GABA, is an important component of the “neurobiologic brake” that restrains the GnRH pulse generator in prepubertal primates. However, others have observed that NPY levels increase in the median eminence at the onset of puberty,³⁷ that infusion of NPY into the median eminence increases GnRH release,³⁸ and that infusion of NPY antiserum into the median eminence did not stimulate GnRH secretion in prepuberal monkeys.³⁷ The varying effects of NPY appear to relate to the site of infusion within the brain and not on the steroid milieu.³⁵ Additional work will be required to clarify the role of NPY in regulation of the hypothalamic pulse generator and the onset of puberty.

Glutamate is an excitatory neurotransmitter in the hypothalamus and stimulates GnRH release via N-methyl-D-aspartate (NMDA) receptors both in vivo and in vitro.³⁹ An intravenous bolus of NMDA stimulates hypothalamic GnRH release,⁴⁰ and treatment with a specific glutamate receptor antagonist blocks the effect in nonhuman primates. Moreover, prolonged (16-30 weeks) intermittent NMDA stimulation (1 minute every 3 hours) activates the hypothalamic-pituitary-gonadal axis and stimulates precocious puberty and the initiation of spermatogenesis in juvenile males.⁴¹ These observations suggest that glutamate signaling may play a role in the resurgence of pulsatile GnRH secretion at the onset of puberty.

In just the past few years, kisspeptins have emerged as a critical component of the system that controls the level of GnRH neuronal activity between infancy and puberty. Kisspeptins are neuropeptides (encoded by the KISS1 gene) that signal via the G-protein coupled receptor, GPR54 (encoded by the KISS1R gene).⁴² Interestingly, the first evidence of their importance in the regulation of the hypothalamic-pituitary-gonadal axis came from observations in humans. Several members of a large consanguineous family with hypogonadotropic hypogonadism and delayed puberty were found to harbor homozygous inactivating mutations for GPR54.^43,44 One affected compound heterozygote exhibited an exaggerated pituitary response to exogenous pulsatile GnRH administration, suggesting a hypothalamic locus for the disorder.⁴⁴ More importantly, the observation also suggested that kisspeptin signaling via GPR54 might play a major role in the resurgence of pulsatile GnRH secretion at puberty in primates. The results of subsequent studies in nonhuman primates and humans strongly support that interpretation.

Neurons expressing KISS1 are located exclusively in the arcuate nucleus,^45,46 where GnRH neurons also express GPR54.⁴² In castrate male and intact female monkeys, the pubertal resurgence of pulsatile GnRH secretion is associated with a nearly 5-fold increase in KISS1 expression and, in females, also with an increase in KISS1R expression.⁴⁷ Hypothalamic kisspeptin secretion is distinctly pulsatile and highly correlated with that of GnRH.⁴⁸ An intermittent kisspeptin infusion can sustain pulsatile LH secretion in castrate juvenile animals after discontinuation of a priming pulsatile infusion of exogenous GnRH, but not in the presence of a GnRH receptor antagonist, indicating that the effect of kisspeptin is mediated via pulsatile GnRH secretion.⁴⁷ The observation that patients with inactivating mutations of GPR54 exhibit pulsatile LH secretion with low amplitude and normal
frequency suggested that kisspeptin might only amplify and not stimulate GnRH pulse generator activity directly.^44,49 However, a continuous kisspeptin infusion, which downregulates GPR54, suppresses both LH pulse amplitude and frequency, implying that kisspeptin has similar effects on pulsatile GnRH secretion.^50,51 Finally, an activating KISS1R mutation resulting in prolonged activation of the KISS1R signal transduction pathway has been described in a young girl with GnRH-dependent (central) precocious puberty.⁵² Taken together, these observations indicate that hypothalamic kisspeptin-GPR54 signaling is a key component of the neurobiologic mechanism that triggers the onset of puberty. They further suggest that kisspeptin neurons may provide the fuel for the hypothalamic GnRH pulse generator. The report of undetectable serum gonadotropins in an infant boy bearing a loss-of-function mutation in the KISS1R gene suggests that kisspeptin input to the GnRH neuronal network also is necessary for the increased pulsatile GnRH secretion normally observed during early infancy.⁵³

There is increasing evidence from studies in nonhuman primates that kisspeptin neurons also are involved in mediating the feedback actions of both testicular and ovarian hormones. In males, testosterone negative feedback, which regulates LH secretion by slowing the pace of pulsatile GnRH secretion,⁵⁴ is associated with a decrease in hypothalamic KISS1 mRNA levels.⁵⁵ The observation suggests that kisspeptin neurons play an important role in the negative feedback loop that regulates LH secretion in the male, which also involves opioid and GABA neuronal input.⁵⁶ Gonadal steroids also suppress hypothalamic KISS1 expression in females. In postmenopausal women and ovariectomized monkeys, the density of neurons expressing KISS1 mRNA is significantly higher than in premenopausal women and in intact females monkeys, and treatment with estrogen and progesterone markedly decreases KISS1 expression,⁴⁶ suggesting that kisspeptin neurons also participate in mediating the hypothalamic negative feedback actions of ovarian steroid hormones. The collective body of evidence thus indicates that kisspeptin neurons are a critical component of the neurobiologic mechanism that regulates the activity of the hypothalamic pulse generator.

In some way, the system that controls the ontogeny of pulsatile GnRH secretion integrates kisspeptin signaling with that of other neuotransmitters (glutamate, GABA) and neuropeptides (NPY). Whether kisspeptin neurons in the arcuate nucleus function as a “pubertal clock,” as a growth-tracking “somatometer,” or simply relay information from such centers to the GnRH neuronal network is unknown.⁵⁷ Regardless, kisspeptin neurons have emerged as one of the primary transducers of the internal and external environmental cues that regulate the neuroendocrine reproductive axis.

Leptin is produced by adipocytes and serum concentrations are strongly associated with body fat and changes in body fat content. Not surprisingly, leptin has been implicated as one way in which metabolic signals might be communicated to the higher centers controlling the activity of the hypothalamic pulse generator at the onset of puberty.
Leptin-deficient mice and rats fail to enter puberty, and treatment with leptin induces the onset of puberty.⁶⁵ In humans, serum leptin concentrations in boys and girls diverge at puberty. In males, leptin levels first increase then decrease again to prepubertal concentrations, whereas in females leptin concentrations rise throughout puberty.^66,67 One study found that serum leptin levels were directly related to the amount of subcutaneous fat and inversely related to androgen levels.⁶⁸ Another in girls observed that an increase in the mean serum leptin concentration to 12.2 ng/mL, corresponding to 29.7% body fat and a body mass index (BMI) of 22.3, was associated with a decrease in age at menarche, and that a 1 ng/mL increase in serum leptin lowered the age at menarche by 1 month.⁶⁹

Evidence from studies in children with congenital leptin deficiency has provided insights into the potential importance of leptin as a somatic stimulus for the onset of puberty. In affected pubertal age children, treatment with recombinant leptin has been associated with endocrine changes consistent with the onset of puberty, whereas all adults with congenital leptin or leptin receptor deficiency described have had severe hypogonadotropic hypogonadism.⁷⁰ However, similar treatment in younger children has not induced premature puberty.⁷¹ These clinical observations suggest that leptin plays an important, but only permissive, role in the onset of puberty. Nonetheless, they are consistent with the idea that a circulating somatic hormone might have the ability to influence or modulate the activity of the hypothalamic GnRH pulse generator.¹⁷

Numerous other metabolic signals have been suggested as playing a role in the nutritional regulation of reproduction, such as insulin, ghrelin (the endogenous ligand of the growth hormone secretagogue with a putative role in energy balance)⁷² galanin-like peptide (a potential neuronal target of leptin),⁷³ and free fatty acids.⁷⁴ However, the manner in which these signals might interact with inhibitory and excitatory hypothalamic neurotransmitters and peptides and any role they may have in the onset of puberty remain to be established.

Premature adrenarche is the most common cause of premature pubarche, describing other-wise unexplained early growth of genital hair associated with increased levels of adrenal androgens.²¹⁵ Generally, the best indicator of adrenarche is a serum DHEA-S concentration greater than 40 μg/dL, which is higher than that normally seen in children 1-5 years of age (5-35 μg/dL). In children with premature adrenarche, the growth rate and bone age often are above average but still within normal ranges. Exaggerated adrenarche is the term used to describe the clinical extreme of premature adrenarche, wherein the serum DHEA-S
level exceeds that typical of adrenarche or for age and usually, but not always, is associated with a somewhat early onset of true puberty.²¹⁶

The cause of premature adrenarche is unknown. The condition traditionally has been considered an early variant of normal development and, as such, generally has no serious consequences. However, up to 20% of girls with premature adrenarche may subsequently develop gonadotropin-dependent precocious puberty and close follow-up therefore is recommended.^77,217 Other evidence indicates that girls with premature adrenarche also are at increased risk for developing polycystic ovary syndrome, suggesting that premature adrenarche may be an early manifestation of the disorder.^{218,219,220,221,222} and ²²³ In many, premature pubarche is preceded by low birth weight and is followed by hyperandrogenism, hirsutism, and oligomenorrhea in adolescence, often accompanied by hyperinsulinemia and dyslipidemia. These observations suggest that insulin resistance may be the underlying metabolic disorder, causing decreased growth during fetal life, premature pubarche, and hyperandrogenism that worsens during late puberty or the early postmenarcheal years.²²⁴ In those affected, metformin treatment can decrease insulin resistance and hyperandrogenism, improve the lipid profile, often restore cyclic menses, and may help to prevent later development of diabetes and cardiovascular disease.²²⁵

Premature pubarche usually results from a premature adrenarche but also has other causes. Idiopathic premature pubarche, unassociated with any demonstrable increase in adrenal androgen production, probably reflects an increased sensitivity of hair follicles to normal androgen concentrations. Premature pubarche sometimes can be the only clinical manifestation of a mild form of congenital adrenal hyperplasia (CAH).^226,227 Other rare causes of ACTH-dependent childhood virilization include Cushing syndrome, glucocoticoid resistance, cortisone reductase deficiency, and androgen-producing neoplasms of the adrenal gland or ovary.

The evaluation of premature pubarche should focus first on determining whether the growth of pubic hair is an isolated phenomenon or may be associated with other signs and symptoms suggesting another of the diagnoses mentioned above. The single most important and useful test is an x-ray of the left hand and wrist for bone age. If sexual hair is small in amount and slow-growing and bone age is normal, precocious puberty is unlikely and expectant management is appropriate, with re-evaluation at 6 months and periodically thereafter. A limited endocrine evaluation should include measurements of serum testosterone and DHEA-S, for comparison to age-adjusted normal values. The presumptive diagnosis of premature adrenarche can be made when both are appropriate for pubarche, bone age is normal, and predicted adult height is within the range expected for the family.²²⁸ More extensive endocrine evaluation can be reserved for those children having other signs suggesting true precocious puberty or a virilizing disorder.

An ACTH stimulation test to exclude the diagnosis of CAH is indicated when bone age is advanced abnormally, the predicted adult height is abnormally low, or when the serum testosterone and DHEA-S concentrations are elevated above the ranges typical of premature adrenarche. The test is performed by obtaining blood samples before and 60 minutes after administering cosyntropin (synthetic ACTH 1-24; 1 μg/m² or 0.25 mg). A stimulated serum 17-OHP concentration greater than 1,000 ng/dL generally indicates 21-hydroxylase deficiency.²²⁹ In children with premature pubarche, diagnosis of the rare 3β-HSD deficiency requires a stimulated 17a-hydroxpregnenolone level greater than 9,790 ng/dL.²²⁷

Premature adrenarche is a benign condition and requires no specific treatment. Parents can be reassured that the condition is a normal variant relating to increased sensitivity of hair follicles to low levels of androgen, or an early occurring incomplete form of puberty. However, children with a diagnosis of premature adrenarche merit periodic re-evaluation for evidence of progressive virilization.

Premature thelarche generally is defined as isolated breast development in girls before the

age of 8 years. In girls, premature thelarche usually is a benign condition considered a variant of normal puberty. Early breast development is particularly common during the first year of life when the hypothalamic-pituitary-gonadal axis is still active.^230,231 Studies using ultrasensitive bioassays for estrogen have detected higher estrogen levels in many, but not all, girls with premature thelarche than in normal controls.²³² The breast also may be more sensitive to estradiol than normal in some girls.²³³ Although most affected children subsequently experience normal puberty and growth,^234,235 and ²³⁶ a significant proportion experiences an earlier than average menarche.²³⁷

Physical examination typically reveals a light pink areola with an infantile appearance, with Tanner stage 2 or 3 breast development; frequently, the change may be unilateral or asymmetrical. There is no sign of androgen exposure.

Exaggerated thelarche describes those with premature thelarche who also exhibit increased growth velocity and/or advanced bone age and may represent an intermediate between premature thelarache and precocious puberty.^232,238 Even girls with exaggerated thelarche exhibit a prepubertal pattern of response to acute stimulation with GnRH or a GnRH agonist; FSH levels rise more than LH, which remains below 5 IU/L.²³⁹ Some cases have been associated with the presence of functional ovarian cysts.¹⁹⁷ Genetic studies in girls with exaggerated thelarche have revealed that some harbor a mutation in the GNAS1 gene, suggesting that the disorder can be an early or the only sign of McCune-Albright syndrome.^232,240

Premature thelarche also has been related to exposure to exogenous estrogen, including environmental chemicals that degrade slowly in the environment and can accumulate in the food chain, but no clear relationship with premature thelarche has been established.

The evaluation of premature thelarche, like that of premature adrenarche, should focus on determining whether breast development is an isolated phenomenon or associated with other signs of precocious puberty; here again, the most important initial test is an evaluation of bone age. In children with Tanner stage 2 breast development and normal bone age, precocious puberty is unlikely and expectant management is appropriate, with reevaluation at 6 months and periodically thereafter.

Approximately 15-20% of girls with premature thelarche subsequently develop gonadotropin-dependentprecocious puberty, at a mean age of 7.1 ± 0.7years and mean bone age of 9.0 ± 1.1 years.^241,242 A longitudinal study involving more than 150 girls with premature thelarche observed that 69% had complete regression of breast development (13% of these later developing true precocious puberty), 21% had recurrent episodes of breast development (32% later developing true precocious puberty), and 10% had persistent breast development (57% later developing true precocious puberty).²⁴²

Long-acting GnRH agonists have proven both safe and effective for the treatment of idiopathic gonadotropin-dependent precocious puberty.^{252,253,254,255,256,257,258} and ²⁵⁹ GnRH agonist treatment causes a brief initial “flare” of gonadotropin release, followed by pituitary desensitization (exhaustion of available stores of releasable gonadotropins), and down-regulation (decrease in GnRH receptors). By suppressing the pituitary-gonadal axis, GnRH agonist therapy can prevent progressive pubertal development, and increase final adult height, compared to pretreatment predictions. Young children and those who exhibit rapidly progressive development can be expected to have early epiphyseal fusion, are at greatest risk for compromised adult height, and can benefit most from treatment.²⁴⁹

In girls under 6 years of age with idiopathic gonadotropin-dependent precocious puberty, treatment with a GnRH agonist can be expected to add 9-10 cm to adult height. In older children already past their peak with slowing growth velocity, treatment can be expected to slow it further, to delay epiphyseal fusion, and to yield slow but steady increases in predicted adult height. In girls between 6 and 8 years of age, GnRH agonist treatment typically results in a gain of 4-7 cm in height, less if bone age is significantly advanced.²⁴⁹ Girls already close to the age of normal puberty, those with slowly progressive maturation, and girls with a predicted height above 150 cm have less to gain and may not benefit significantly from treatment.^260,261 and ²⁶²

The choice among the available GnRH agonist formulations depends mostly on physician preference and availability. Depot preparations generally are preferred because of improved compliance. Direct comparisons in randomized trials have not been made,
but any of the following treatment regimens generally can be expected to suppress the pituitary-gonadal axis:^263,264 and ²⁶⁵

Nonetheless, the dose of GnRH agonist treatment required can vary significantly.²⁶⁶ Inadequate treatment can permit progressive sexual development and bone maturation. Conversely, over-treatment can suppress endogenous GH and decrease growth velocity and bone mineral accumulation to levels below those normally expected during the prepubertal years.²⁶⁷ The adequacy of GnRH agonist treatment can be monitored simply by measuring the serum LH concentration 30-60 minutes after each repeated injection of the agonist; the LH level should be less than 3.0 IU/L, consistent with prepubertal norms after acute GnRH agonist stimulation.²⁶⁸

GnRH agonist treatment should be monitored at 3-6 month intervals with serial physical examinations to detect any progressive pubertal development; bone age also should be evaluated periodically.²⁴⁹ Breast development should cease and growth velocity and the pace of advancing bone age should decrease. Pubic hair development may continue due to normal adrenarche.²⁶⁹ Although bone density may decline during longer durations of treatment, bone mass is regained after treatment ends and peak bone mass is normal; consequently, there is no reason or need to monitor bone density.²⁴⁹

GnRH agonist therapy also is recommended for treatment of GnRH-secreting hypothalamic hamartomas^187,272; the tumor can be monitored by serial imaging and risky surgery can be avoided. Treatment for other hypothalamic, pituitary, cerebral, or pineal tumors must be individualized. Many that are small and do not extend around or into vital structures can be excised successfully.