Leptin, a 16-kDa peptide secreted by adipocytes, was shown to play an important role in reflecting the amount of body energy stores which is known to play a critical role in triggering the neuroendocrine mechanisms involved in the onset of puberty.

Leptin, binding to specific transmembrane receptors, regulates the action of hypothalamic neuropeptides involved in the control of neuroendocrine function and energy intake, and expenditure. It is supposed to signal to the brain the critical amount of fat stores necessary for LHRH secretion, which in turn activates the hypothalamic-pituitary-gonadal axis (60).

Leptin was recently shown to suppress neuropeptide Y (NPY) expression in the arcuate nucleus. Since NPY stimulates appetite, has an inhibitory effect on the gonadotropin axis, and is involved with the inhibition of puberty in conditions of food restriction, it has been hypothesized that leptin might exert its effects by acting on NPY. Under favourable nutritional conditions, the rise in leptin levels would suppress NPY, and in turn release the inhibitory effect of NPY neurons on the GnRH-LH/FSH axis, allowing the initiation of puberty (61). In addition to the central effects, recent in vitro (62) and in vivo (63) data indicate a direct negative effect of leptin on gonadal function through inhibition of the steroidogenic enzymes. Thus, leptin seems to exert a positive central effect on the hypothalamic-pituitary-gonadal axis and a negative peripheral one on the gonads.

Subjects with mutations in the human leptin receptor gene have no pubertal development, while obese patients who are known to have an increased rate of infertility (64) and recurrent spontaneous abortion (65), exhibit high serum leptin levels. Dietary treatment of obese individuals is accompanied by an improvement of endocrine and ovarian function and a concurrent fall in plasma leptin concentrations (66, 67).

During starvation, in conditions of amenorrhea, in subjects with anorexia nervosa, and in strenuously exercising athletes, leptin and E2 levels fall concomitantly. In boys with constitutional delay of puberty, leptin levels are low for the corresponding body mass indexes indicating a potential role of leptin in the initiation and progression of pubertal development (68, 69). In humans and animals, leptin blood concentrations rise with the onset of puberty and normal levels are necessary for the maintenance of menstrual cycles and normal reproductive function. No gender differences were detected in the relationship between leptin serum levels and fat mass in pre-pubertal and early pubertal subjects, while differences were significant in late puberty. At Tanner stages IV and V, in fact, the serum hormone concentrations decrease in males and increase in females. Furthermore, a significant negative correlation between circulating concentrations of testosterone and leptin was described in males only (60). In adolescents of both sexes, the gradual rise in serum leptin levels before puberty together with a decline in circulating levels of soluble leptin receptor suggest that these changes may serve as one of the signals to the central nervous system that metabolic conditions are adequate to support pubertal development and trigger puberty (70). Qualitative and quantitative changes in LH secretion resulting from pulsatile GnRH secretion, occur approximately 2 years before the appearance of secondary sexual characteristics. At puberty, LH pulsatile secretion is characterized by a 28-fold increase in the pulse amplitude, whereas pulse frequency increases only 1.8-fold. During prepubertal years both LH and FSH secretions are preponderant during nighttime. In the peripubertal period the gonadotropin secretions increase during sleep, and stimulation with exogenous GnRH shows an enhanced release of LH from the pituitary gland that may be useful in differentiating a pubertal from a pre-pubertal response. Throughout puberty then, gonadotropin pulses further increase becoming apparent during daytime also. In girls, FSH levels increase during the early stages, and LH levels during the later stages of puberty with a 100-fold increase in hormone concentrations. In boys, FSH levels rise progressively through puberty with an increase in amplitude only, whereas LH levels increase in early puberty reaching shortly a plateau.

Inhibin, activin, and follistatin are also involved in the modulation of the hypophyseal-gonadal axis function, as inhibin and follistatin inhibit and activin stimulates the expression, biosynthesis, and secretion of FSH. These hormones are synthesized mainly in the gonads (2, 71). Inhibin, a heterodimeric glycoprotein, is a member of the TGF-b superfamily produced by the Sertoli cells in the testis and by ovarian granulosa cells. It is composed of an α, and one or two β subunits which form two different products, inhibin A and B, respectively. FSH stimulates the synthesis and secretion of inhibins by the gonads, which in turn are involved in the feedback regulation of FSH secretion. In girls, inhibin A concentrations increase between stage 2 and 3 of puberty, remain constant throughout stages 4 and 5, and correlate positively with bone age, inhibin B and estradiol serum levels (74, 75); in boys, serum levels are undetectable at any pubertal age (72,73). Inhibin B blood concentrations, in girls, increase similarly to inhibin A levels, reaching a plateau at 12 to 18 years, and correlate with estradiol (74,75) and FSH serum levels (76). In boys, hormone concentrations increase from stage 1 and peak at stage 3, decreasing thereafter and correlate with testicular volume (74). In males, this dimer is produced exclusively by the testis and it is considered a valuable index of spermatogenesis (77). Blood levels of activin A were shown both to increase from stage 1 to 3 of puberty (75), and to remain unmodified in females (78). In boys, changes in activin A serum concentrations were not described during pubertal development (76).

Blood concentrations of follistatin decrease slightly from stage 1 to 4 and 5 of puberty in girls (75), whereas no pubertal variations were described to date in males.

In conclusion, at puberty the concentrations of two negative regulators of FSH secretion, inhibin and follistatin, change in opposite directions (75), whereas the blood levels of a positive regulator, activin A, increase, at least in females. All together, these alterations in serum concentrations of FSH-regulatory peptides lead to an increase in FSH secretion.

Other hormones have been shown to undergo significant changes at puberty (79). Growth hormone (GH), insulin, insulin-like growth factor (IGF)-I, and its major binding protein, IGFBP-3, normally rise at puberty. The increase in growth hormone and IGF-I concentrations is probably responsible for most of the metabolic changes observed during puberty, including insulin-resistance, increased β-cell response to glucose, and growth spurt. Data in boys with constitutional delay of puberty treated with either testosterone alone or testosterone in combination with letrozole, a P450-aromatase inhibitor, suggest that GH, and not androgens, directly affects insulin sensitivity regulating the glucose-insulin homeostasis at the time of puberty (80). Circulating levels of adiponectin, an adipocytokine with antidiabetic and antiatherogenic effects, were recently shown to progressively decline in parallel with pubertal development in boys, being inversely related to serum testosterone and dehydroepiandrosterone sulfate levels (81). The role of melatonin in puberty is questioned. In fact,while the marked increase in LH amplitude observed in early puberty at night occurs at precisely the same time of melatonin secretion, precocious puberty associated with pineal tumors and due to ectopic secretion of gonadotropins is independent of melatonin (82).

Premature pubarche refers to the precocious appearance of pubic hair without other signs of puberty or virilization (83, 84). The age limit until recently has been considered 8 years in girls and 9 years in boys. However, the results of a large cross-sectional study carried out in 1997, suggest that the appearance of pubic hair in girls may be considered normal when it occurs after 7 years of age in white subjects, and after 6 years of age in African-Americans (3, 85). Axillary hair, apocrine odor, and acne may or may not be present. Growth velocity may be increased, and slightly advanced bone maturation usually well correlated with the height age, is often present. The transient acceleration of growth and bone maturation has no negative effects on the onset and progression of puberty, and on final height (86, 87).

The precise etiology of premature pubarche is not known. Generally, it has been attributed to an early maturation of the zona reticularis of the adrenal cortex leading to an increase of adrenal androgens to levels normally seen in early puberty and, in turn, to the premature appearance of pubarche (88, 89). Because half of PP patients have normal androgen levels, a hypersensitivity of the hair follicle to steroid hormones has also been proposed. (Fig.5)

The diagnosis of premature pubarche is based on the exclusion of true precocious puberty and the nonclassic forms of congenital adrenal hyperplasia (Fig. 6). The incidence of defective steroidogenesis in children with PA is extremely variable, ranging from 0% in some reports (90) to 40% in others (91), probably due to the varying ethnic background of the populations studied. A high incidence of molecular defects of the CYP21 gene was reported in Greek children with premature pubarche, the majority of whom were heterozygotes for 9 different molecular defects (92). Whether this finding has any general clinical relevance, only long-term prospective studies will be able to establish it. Recently, Type II 3beta hydroxysteroid dehydrogenase gene mutations were also identified in patients with premature pubarche and elevated 17-hydroxypregnenolone ACTH- stimulated plasma levels (93).

Since idiopathic precocious puberty is generally characterized by pubertal progression of the hypothalamic-pituitary-gonadal axis function, it can usually be clinically distinguished from premature pubarche. The plasma concentrations of DHEA, DHEA-S and D4-A as well as the levels of the 17-ketosteroids and their urinary metabolites, are increased for age in children with premature pubarche, and similar to those normally found in children with Tanner stage II of pubertal development (8, 94). ACTH stimulation test rules out nonclassic congenital adrenal hyperplasia but not the carrier state (8). Gonadotropin levels are in the prepubertal normal range both at basal state and after stimulation with gonadotropin-releasing hormone.

Once precocious puberty and nonclassic congenital adrenal hyperplasia are ruled out, no treatment is needed. However, a long-term follow-up of these patients is warranted (Fig. 6). Recent data, in fact, indicate that girls with premature pubarche may not have a benign outcome. Forty percent of postpubertal girls diagnosed with premature pubarche during childhood have an increased frequency of functional ovarian hyperandrogenism (95). Furthermore, hyperinsulinemia is a common feature in adolescent patients with premature pubarche and functional ovarian hyperandrogenism, and is directly related to the degree of androgen excess (96-98). Although the mechanisms interlinking the triad of hyperinsulinemia, premature pubarche, and ovarian hyperandrogenism remain enigmatic, this frequent concurrence may result, at least in part, from a common genetic or early origin, as the result of in utero growth retardation (99). It was suggested that programming of the endocrine axes occurs during critical phases of fetal development and is affected by intrauterine growth retardation (100). Some Authors reported, in a population from a specific part of Spain, a significantly lower birth weight in girls with premature pubarche and ovarian hyperandrogenism (96). It was concluded that girls with premature pubarche born small for gestational age are at a higher risk of getting polycystic ovary syndrome. However, other studies did not confirm these results. Specifically, in a Dutch population of short children born small for gestational age, only 2.2% of the 90 girls examined had premature pubarche (101). This is comparable with the incidence of premature pubarche in the normal population, in which the incidence in white girls is 2.8% (102). Moreover, in a smaller group of Italian patients with premature pubarche all girls had birth weights appropriate for gestational age (103). It was also shown in a cohort of French young women that intrauterine growth retardation predisposes to insulin resistance but not to hyperandrogenism. It is therefore plausible that there are two distinct forms of premature pubarche, one characterized by the association of premature pubarche with low birth weight, hyperinsulinism, and hyperandrogenism, and one by premature pubarche alone in the absence of other clinical and/or biochemical abnormalities. The prevalence of these distinct forms of premature pubarche may vary in the different populations. The pathogenetic mechanisms underlying the two forms of premature pubarche may also be different. In children with premature pubarche and low birth weight, hyperandrogenism, and hyperinsulinism the concurrence of these clinical and biochemical features may result from a common origin as an effect of early exposure of the fetus to poor nutrition leading to permanent changes in insulin metabolism and body fat deposition, according to the thrifty phenotype hypothesis. These are the patients most probably at higher risk of developing PCOS and the metabolic syndrome in adulthood and deserve a careful follow-up. Recent short-term studies suggested that an insulin-sensitizing therapy in these patients may prevent the progression from premature pubarche to PCOS (5). However, since no data regarding safety of long-term use of insulin-sensitizers in children and adolescents, and no long-term studies to document acceptable risk:benefit profile are available, the use of such agents in children with PP is not recommended outside of experimental clinical trials.

In patients with isolated premature pubarche in the absence of biochemical and metabolic abnormalities, a hypersensitivity of the pilosebaceous unit to androgens as a result of increased androgen receptor activity may be responsible for the isolated precocious appearance of pubic hair (6). These are patients who most probably are not at risk of developing endocrine or metabolic abnormalities in adulthood. However, since data on the outcome are not available, a long-term follow-up of these patients is also warranted to ascertain the benign outcome of the disease.

Premature thelarche refers to the precocious appearance of breast development in girls with no other signs of sexual maturation or accelerated growth velocity and bone age advancement. It is most common during the first 2 years of life and its incidence in nursery children from Northern Italy was reported as high as 36.6%. The etiology of premature telarche is still unknown, although different pathogenetic mechanisms have been suggested. Some authors postulated that an increase in breast sensitivity to estrogen might be responsible for the premature development of breast tissue. Others, using an ultrasensitive recombinant cell bioassay, showed that girls with premature thelarche exhibit higher estradiol levels than those of normal pre-pubertal girls (104). Transient estrogen secretion from ovarian follicular cysts (105), increased production of estrogens from adrenal precursors, and transient partial activation of the hypothalamic-pituitary-gonadal axis with increased secretion of FSH, were also claimed as possible causes of premature telarche (106 - 108). An increased prevalence of detectable ovarian microcysts at ultrasound was also reported, but the presence or absence of these cysts did not correlate with basal gonadotropins or estradiol levels (109). Recent studies identified activating mutations of GNAS1 gene in some patients with chronic fluctuating and/or exaggerated thelarche, without other classic signs of McCune-Albright syndrome (110).

In the last years great attention was paid to the effects on pubertal development of the so-called endocrine disruptors. A growing list of chemicals were shown to influence the endocrine system either in vitro or in vivo, but only a few were associated with altered pubertal development. The outbreak of epidemics of premature telarche in some geographical areas was suggested to be linked to exposure to endocrine disruptors. Phthalates were suspected in Puerto Rico, whereas in Michigan polybrominated biphenyls were associated with advanced breast development in children of exposed mothers. Thus, possible exposure to endocrine disruptors should be borne in mind in the diagnostic work-up of premature telarche (111).

Premature telarche is usually a self-limited condition that undergoes spontaneous regression during the first 2-3 years of life. However, in some cases the outcome of premature thelarche is not always entirely benign. It is now clear that when its onset occurs after 2-3 years of age, a certain percentage of patients develop central precocious puberty (112). In an initial Italian series, 14/100 girls diagnosed at a mean age of 5.1 years, progressed to precocious and early puberty (113). In a following retrospective multi-center study on 119 girls, only 60% of the patients who presented with premature breast development before 2 yr of age showed a complete regression during the follow-up period. In 40% of these girls the breast size was unmodified at a follow-up period of 134 months, and 7/38 (18.4%) patients developed central precocious puberty. Furthermore, another subgroup was identified (28.5%) which included patients who showed an accelerated height velocity and/or bone maturation at diagnosis but did not develop precocious puberty (114). In the premature thelarche patients who developed precocious or early puberty, final height was unaffected and normal for mid-parental height, so that the sexual precocity was interpreted as a reflection of early maternal menarcheal age (115). The observation of IGF-I serum concentrations and IGF-I/IGFBP-3 values intermediate between those detected in prepubertal children and in central precocious puberty, suggests that premature thelarche could be considered a very early stage of puberty (116). In addition to clinical and bone age assessment, pelvic ultrasound might be useful to distinguish premature thelarche from precocious puberty. In fact, while no significant differences in uterine and ovarian ultrasound measurements were detected between children with premature thelarche and controls, significant differences in pelvic ultrasound parameters were reported between healthy girls and age-matched girls with CPP. Uterine transverse diameter ("'width"'), uterine length, fundal anteroposterior diameter, uterine volume, ovarian length, ovarian circumference, and mean ovarian volume are all increased in girls with CPP. The calculated cut-off values to predict PP by ultrasound vary among studies. Haber et al (115) found that a uterine volume >1.8 ml, uterine length >3.6 cm, and ovarian volume >1.2 ml were highly predictive for PP. Herter et al (117) reported that the best cut-off points were uterine length 4.0 cm, uterine cross-sectional area 4.5 cm2, uterine volume 3.0 cm3, and ovarian volume 1.0 cm3. Recently de Vries et al. (118) comparing 30 girls with PP and 21 with PT in whom peak luteinizing hormone was < 5 mIU/ml on the GnRH stimulation test, found significant differences in uterine width, fundus diameter, uterine volume, and ovarian circumference. The Authors suggest that increased uterine and ovarian measurements may be an early and sensitive sign of PP (Tab. 1) and that pelvic ultrasound may give the clinician a complementary indication to the GnRH test in distinguishing isolated PT from early-stage PP in girls with early breast budding.

The measurement of baseline estradiol blood concentrations may also be helpful in distinguishing premature thelarche from precocious puberty, although the differential diagnosis is based on the results of the classic gonadotropin releasing-hormone (GnRH) stimulation test (100 mg LH-RH as i.v. bolus). Baseline LH and FSH plasma levels are often higher, and peak LH levels are significantly and constantly elevated in precocious puberty than in premature thelarche patients, whereas peak FSH levels may not be significantly different in the two groups. A stimulated LH/FSH ratio greater than 1, is suggestive of precocious puberty (119). In recent years, the stimulation with an LH-RH analogue such as leuprolide acetate, was proved to be particularly useful in the differential diagnosis of pubertal disorders. Peak LH was shown to be significantly higher and consistently > 8 IU/l in pubertal in contrast to pre-pubertal subjects. Moreover, the gonadal response, which is maximal 24h post-stimulation, was also discriminating between pre-pubertal and pubertal conditions (estradiol in females > 150 pmol/L and testosterone > 3.15 nmol/L in males)(120).

Constitutional delay of growth and puberty (CDGP) is defined as a delay of growth occurring in otherwise healthy adolescents with stature reduced for chronological age, but generally appropriate for bone age and stage of pubertal development, both of which are usually delayed. It is more frequent in boys than in girls with a 10:1 ratio and is the most common cause of delayed puberty (80-90%). The majority of cases (70-80%) are familial. The characteristically retarded linear growth occurs during the early years of life and is followed by regular growth paralleling the normal growth curve throughout the rest of prepubertal years. Pubertal growth spurt is attenuated and occurs after the usual expected time. Bone age is delayed and results in normal predicted adult height (Fig.7). This latter, however, is often in the lower part of the parental height range, with few patients exceeding the target height. When CGDP occurs in the context of familial short stature, it results in short final height (121).

Bone mineral density can be compromised by the low serum steroid concentrations (122, 123). Specifically, the attainment of peak bone mass may be impaired, although recent data do not indicate significant changes in volumetric bone mineral density in young men with previous CDGP compared with appropriate controls (124).

As a consequence of inadequate production of gonadal steroids, acute provocative tests may show a GH response consistent with partial GH deficiency (125). Pre-treatment with gonadal steroids, however, results in the normalization of the GH responses. Serum IGF-I and IGF binding protein-3 levels are normal for bone age, as is the overnight spontaneous GH secretion when the levels in CDGP children are matched with those of appropriate controls (126-128). The sleep-related increase in LH concentrations that characterizes the onset of puberty, is normally present in CDGP children. The LH response to leuprolide acetate, an LH-RH analogue, is intermediate between that of hypogonadal patients and normal pubertal children, and is therefore useful in differentiating CDGP from hypogonadotropic hypogonadism (129).

Treatment of CDGP children is controversial. CDGP is a paraphysiologic condition and as such does not require any specific therapy. Supportive care is most often enough to reassure children that they will achieve a normal adult height and full functional sexual maturation. However, the psychological and social problems faced by CDGP children sometimes force physicians to treat them. In males, testosterone treatment to induce puberty is recommended only if the bone age is greater than 12 years to avoid the risk of inappropriately advance bone age and thus compromise adult height. A 4-6 months course of 50 mg im testosterone enanthate monthly is recommended (121, 129). A rationale approach recently suggested consists in the use of testosterone combined with aromatase inhibitors to reduce estrogen synthesis and action at the growth plate site. Preliminary data are promising as they show an increase in predicted adult height in subjects undergoing this combination treatment (128). In girls, estrogen therapy is recommended only after statural considerations have been carefully taken into account. The administration of estrogen, in fact, even in small amounts, leads to progressive skeletal maturation, and ultimately to epiphyseal fusion. Anabolic steroids might also be used to stimulate growth. Oxandrolone is one anabolic compound that can be utilized, as it was shown to accelerate growth without causing rapid bone maturation or compromising adult height (107). Growth hormone was also reported to transiently improve height velocity. However, given the high costs of GH and the lack of any definite evidence that GH treatment has any beneficial effect on adult height, the use of such growth-promoting agent is not recommended (129 -132).