In the general population, there is a near-normal distribution of the timing of puberty, with the mean age of onset of G2 at 11.5 years in boys (Fig. 4.2). In healthy boys, the normal age limits for G2 development are between 9.5 and 14 years [4]. While a large variability in the timing of pubertal onset exists, clear age cutoffs for normal pubertal development have been established. However, the age limits for identifying children who need evaluation for precocious or delayed puberty may vary among different ethnic groups. Within this distribution, there has been in recent years an increasing degree of skew at both ends of the spectrum, as an increasing prevalence of an earlier age of pubertal onset (G2) has been documented in some populations as well as an increase in the number of children completing puberty at a later age [5].

Peak height velocity (PHV) and peak pubertal growth hormone production coincide approximately with the midpoint of pubertal development (Fig. 4.3). In boys, the PHV coincides with G3-4 at an average age of 13.5 years and achieves an incremental rate of 9.5 cm/yr [6]. Up to 25% of total adult height is achieved from growth during puberty, but the amplitude and peak velocity of the pubertal growth spurt are not fixed and vary with age at onset of puberty [7]. Early puberty is associated with a large pubertal growth spurt, while late maturers, who have a longer prepubertal period of growth, in turn experience a less pronounced pubertal growth spurt [8]. Therefore, although extremes of pubertal timing may lead to a small degree of final height reduction due to the reduced overall period of growth in precocious puberty, or poor PHV in delayed puberty [9], in general within wide limits, the timing of puberty does not influence adult height.

Variability in the timing of puberty in healthy adolescents is governed by complex regulatory mechanisms involving genetic, environmental, and other factors [10]. Nutritional status, adoption, geographical migration, and emotional well-being have all been shown to affect pubertal timing [11–13]. While the timing of pubertal onset in girls in most countries in the developed world exhibited a rapid decrease in the first half of the twentieth century [14, 15], in boys, these trends are less clear. A small but significant change in the normal spectrum of timing of G2 development was documented in a European cohort [16] and in the USA [17] but remains controversial [18]. Much has been postulated about this observed secular trend toward an earlier age of pubertal onset in the developed world [19]. It also follows that it is difficult to understand the pathophysiology of delayed puberty before we can interpret this population level advancement in pubertal age.

Nutritional changes clearly have an important role, as shown by the positive correlation between age at puberty onset and childhood body size [20], most markedly in girls [11]. He and Karlberg demonstrated in a large dataset (n = 3650) that one BMI unit increase between the ages of 2 and 8 years is associated with a 0.11 year advancement in the timing of puberty as measured by peak height velocity in both genders [21]. In boys, however, the data are less consistent, with some studies documenting an earlier onset of puberty with greater adiposity, and some a later onset. In particular, more European studies have noted the former trend, while US studies have more often shown the latter [22]. A recent study from the USA reported a far more complex relationship between fat mass and pubertal timing, with overweight status being associated with earlier pubertal onset, while obesity was associated with later onset. These effects also vary between ethnic groups [23]. Thus, one hypothesis in boys is that greater BMI leads to earlier pubertal timing up to the threshold at which obesity occurs. Obesity may delay pubertal timing in boys due to the suppression of the HPG axis with adiposity leading to excess aromatase activity and thus increased estrogen production.

Additional data point to an earlier trend in the age of puberty onset that is independent of BMI [24]. As detailed above, some studies suggest that over the last decade, the age of completion of puberty in males in some populations has become skewed toward later ages [5]. The effect of possible endocrine-disrupting chemicals (EDCs) on the timing of puberty is also an ongoing concern [5, 25]. Polybrominated biphenyls, bisphenol A, atrazine (herbicides), and phthalates, among others, have been suggested as possible EDCs responsible for contributing to this observed trend [26]. For example, children migrating for international adoption, and previously exposed to the estrogenic insecticide DTT in their country of origin, displayed early or precocious pubertal timing [27]. However, a mechanism of action for EDCs through the early initiation of the pulsatility of GnRH has not been conclusively demonstrated. Studies are complicated by the likely differing and possibly divergent influence of variable doses and mixtures of EDCs, and differing effects depending on age and length of exposure [5, 27, 28].

Epigenetic regulators are potential mediators of the effects of the environment on the hypothalamic control of puberty. However, while experimental data from rats provide evidence for changes in histone acetylation and gene methylation leading to altered gene expression during sexual development, the link between environmental factors and the epigenetic control of puberty has not been established. Although the window of opportunity for the effects of EDC exposure was historically considered to occur in the late prepubertal period, evidence of a fetal and neonatal origin for changes in pubertal timing counters this dogma. A multicenter US study found that prenatal exposure of boys to EDCs such as phthalates, based on the concentrations of four phthalate metabolites in a sample of urine, was associated with a short anogenital index, a marker for reduced masculinization of genital structures [29] (see Chap. 14). Epigenetic changes during fetal life are a potential mechanism for the effects of EDCs in utero [5]. Recent evidence suggests that effects of EDCs may persist in pregnant rats in not only their unborn fetus but into the next generation [30].

Despite the demonstrated importance of environmental factors, the genetic influence on pubertal timing is clearly fundamental. While the timing of pubertal onset varies within and between different populations, it is a highly heritable trait. The timing of sexual maturation is highly correlated within families and in twin studies, suggesting strong genetic determinants [31]. Previous epidemiological studies and genetic approaches estimate that 60–80% of the variation in pubertal onset is under genetic regulation [14, 32, 33]. Despite this strong heritability, however, little is known about the genetic control of human puberty either in the normal population or in cases of disturbed pubertal timing [4]. A lack of clear understanding of the genetic factors that control and trigger the onset of puberty is an important barrier to deciphering the mystery of EDC and other environment/external cues to understand the secular trend toward earlier puberty [34].

LH controls sex steroid production by upregulating expression of the steroidogenic enzymes and by controlling metabolic activity of the steroid-producing Leydig cells (see Chap. 2). LH may also stimulate Leydig cell proliferation and differentiation [44]. During puberty, plasma testosterone levels increase dramatically. Table 4.2 summarizes the levels of testosterone at various developmental stages with respective testis sizes. The pubertal increase in testis size results primarily from an increased number of proliferating and differentiating germ cells and to an increase in the number of Sertoli cells [45]. In early and mid-puberty, there is a pronounced diurnal rhythm with a morning peak in testosterone levels, but this is less pronounced in later puberty, and declines gradually with age [46], probably due to decreased day–night ratios of gonadotropins [47].

Inhibin-B is a heterodimeric glycoprotein produced by Sertoli cells beginning in the fetus. Several studies show that serum inhibin-B levels in children change in concert with the secretion of gonadotropins [48–50]. During the ‘mini-puberty,’ serum inhibin-B levels increase to similar [51, 52] or even higher [49] levels than in adolescent boys and adult men. This early inhibin-B secretion is sustained until the age of 18–24 mo; thereafter, serum concentrations decline to lower but readily measurable levels [49]. Early in puberty, between Tanner stages G1 and G2, serum inhibin-B concentrations again increase to reach peak levels at the Tanner stage G2, but then the levels plateau [48, 50].

GnRH pulsatility is coordinated by a balancing act between a number of inhibitory and excitatory neuronal and glial inputs (Fig. 4.5) [53]. Inhibitory inputs are primarily from GABAergic and opiatergic neurons, while glutamate and kisspeptin are the central excitatory neuronal signals. Glial cells additionally facilitate GnRH secretion via growth factor-derived signaling [54]. The onset of puberty is triggered by a decline in these inhibitory signals and an amplification of excitatory inputs, leading to increased frequency and amplitude of GnRH pulses [55]. Before the onset of puberty, GABA release in the preoptic area decreases in female rats [56], and in the rhesus monkey, GABA release into the median eminence decreases concomitant with the pubertal increase of GnRH secretion [57]. In female rats, glutamine synthase is downregulated, and glutamate dehydrogenase becomes more abundant in the hypothalamus at puberty, both leading to increased availability of glutamate [58]. Glutamate agonists are potent stimulators of GnRH secretion, and administration in prepubertal primates can stimulate LH and testosterone secretion [59].

Another vital piece in the puzzle of the central control of GnRH release came with the discovery of Kisspeptin. This excitatory neuropeptide was identified as a permissive factor in puberty onset by the discovery of patients with GnRH deficiency who had loss-of-function mutations in the KISS1 receptor, KISS1R (previously known as GPR54) [60, 61]. Mice with knockout of kiss1r were simultaneously discovered to be infertile despite anatomically normal GnRH neurons and normal hypothalamic GnRH content [61]. Their phenotype can be rescued by exogenous delivery of GnRH. Kiss1 knockout mice also have a phenotype consistent with normosmic GnRH deficiency. However, to date, only very rarely have mutations in KISS1 been found in patients with delayed or absent puberty [62], although two potential activating mutations were identified in patients with central precocious puberty (CPP) [63].

Kisspeptin signals directly to GnRH neurones to control pulsatile GnRH release. Kisspeptin is upregulated in both primates and mice in the peri-pubertal period, and its administration in prepubertal rodents advances the onset of puberty [64]. Kisspeptin also appears to be downregulated in functional hypogonadism, suggesting its role as a mediator of the action of environmental factors such as nutritional status and emotional well-being on puberty and reproductive capacity. Kisspeptin signaling is an important element of both positive and negative feedback loops in the HPG axis. While kisspeptin has been identified as a pivotal upstream regulator of GnRH neurons, whether kisspeptin is the key factor that triggers the onset of puberty remains unclear.

An additional excitatory neuropeptide, neurokinin B, has been implicated in the upstream control of GnRH secretion. Identification of this pathway was via the discovery of loss-of-function mutations in TAC3, encoding neurokinin B, and its receptor TACR3, in patients with normosmic GnRH deficiency and pubertal failure [65, 66]. Both KISS1 and TAC3 are expressed by neurons in the arcuate nucleus of the hypothalamus that project to GnRH neurons, and their expression is downregulated by estrogen [67]. However, studies of the effects of neurokinin B administration have provided conflicting results. While central administration of neurokinin B agonists failed to stimulate GnRH release in rodents, and Tacr3 knockout mice have grossly normal fertility [68, 69], primate studies showed that neurokinin B can act to stimulate GnRH release via kisspeptin signaling [70]. Additionally, neurokinin B is expressed more widely in the central nervous system than in kisspeptin, suggesting some differences in the roles of these two neuropeptides in the control of pubertal onset.

Dynorphin, an opioid peptide that is coexpressed with kisspeptin and neurokinin B in so-called KNDy neurons, inhibits the release of GnRH, and together, these peptides are currently believed to play a fundamental role in the GnRH pulse generator. Other neurons regulate GnRH including GABAergic signaling pathways which function in the stress-induced suppression of LH, and RFamide–related peptide gene (RFRP), the mammalian ortholog of the avian peptide gonadotrophin–inhibiting hormone (GnIH) [71].

Glial inputs appear to be predominantly facilitory during puberty, acting via growth factors and small diffusible molecules, including TGFβ1, IGF-1, and neuregulins that directly or indirectly stimulate GnRH secretion [72]. Glial cells in the median eminence regulate GnRH secretion through the production of growth factors that activate receptors with tyrosine kinase activity. FGF signaling is required for GnRH neurons to reach their final destination in the hypothalamus [73], as well as for GnRH neuronal differentiation and survival [74]. Additionally, GnRH neuron secretory activity is facilitated by IGF-1 and by members of the epidermal growth factor family such as neuregulin 1β [54, 75]. Moreover, plastic rearrangements of glia–GnRH neuron adhesiveness, mediated by soluble molecules such as neuronal cell adhesion molecule (NCAM) and synaptic cell adhesion molecule (SynCAM), coordinate the controlled delivery of GnRH to the portal vasculature [72], a process that is subject to sex steroid regulation [76].

The interplay between puberty and metabolism has been the focus of intense study. The role of fat mass in pubertal timing is thought to be mediated, at least in part, through the permissive actions of the metabolic hormone leptin, a key regulator of body mass, secreted from white adipose tissue (WAT) [77]. Absence of leptin signaling results in obesity and infertility, whereas leptin treatment decreases food intake and restores reproductive functions. During fasting, leptin levels decrease and gonadotropin secretion is suppressed. These findings imply that nutritional status, especially fat tissue and leptin, could contribute to pubertal development. Clinical observations have further supported a role for leptin signaling in the onset of puberty, e.g., humans and mice lacking leptin (Lep ob/ob) or the leptin receptor (LepR db/db) fail to complete puberty and are infertile [78], and in a 12-yr-old girl with congenital leptin deficiency, treatment with recombinant leptin was followed by a pubertal pattern of LH release [79].

GnRH neurons do not express the LepR. Instead, leptin appears to indirectly regulate GnRH via actions on cells that are afferent to GnRH neurons such as LEPR-expressing GABA neurons from the arcuate nucleus [80], or via cells that interact morphologically with GnRH neurons, partly through nitric oxide signaling, which is required for leptin action [81], and via kisspeptin/neuropeptide Y neurons [82, 83]. Cross-sectional studies showed that serum leptin levels rise in girls in early puberty [84] but not in boys. In careful longitudinal studies in male rhesus monkeys, leptin levels decreased during the juvenile period and were unchanged during puberty [85, 86], and leptin gene expression in the hypothalamus is unchanged during the development in the rat [87, 88]. Therefore, leptin is probably not the requisite metabolic trigger for the onset of puberty, but rather is a permissive factor signaling healthy energy balance.

Neuropeptide Y (NPY) is involved in many CNS functions, including appetite control and reproduction. Evidence from primate studies suggests that NPY may also have a contributory role in the break restraining the onset of puberty in primates [68, 89]. Ghrelin and other gut-derived peptides may also form part of the mechanism by which energy homeostasis regulates reproductive development [90]. Both low birthweight and prematurity are associated with earlier onset of puberty [91, 92], particularly in those children with rapid increase in length or weight in the first two years of life [93]. It remains unclear, however, whether childhood obesity, insulin resistance, excess androgens, or other factors may explain this association [94, 95].

While GnRH pulsatility is clearly the central driver of pubertal onset, and aberrant GnRH neuronal development and function lead to hypogonadotropic hypogonadism (HH), our collective understanding of the upstream neurocircuitry regulating GnRH neurons remains incomplete. Data pointing to hypothalamic regulation via a hierarchical network of genes (Fig. 4.6) have mainly come from a systems biology approach and from animal models, with little data from human subjects [96]. Candidate transcriptional regulators that have been identified via these approaches include Oct–2, TTF–1, and EAP1 [97–99].

The cutoff age for boys who need an evaluation for delayed puberty may vary in different ethnic groups, but in most populations, early signs of secondary sexual development should be present by age 14 years. The evaluation of testicular volume is vital, as an accurate diagnosis of delayed puberty cannot be made without assessment of Tanner genital stage. A thorough medical history should note the symptoms and signs of anorexia nervosa, the intensity of athletic training, and the timing of puberty of both parents, as there is often a family history of DP in cases of self-limited DP (Fig. 4.6). A history of chronic illness, such as celiac disease or inflammatory bowel disease, suggests a temporary or secondary delay of puberty. Stature and height velocity should be evaluated using appropriate growth charts. Bone age (X-ray film of left hand and wrist read according to standards such as Greulich and Pyle) delay provides useful information in the growth analysis but contributes little to the differential diagnosis. It may be very difficult to distinguish clinically between the diagnosis of self-limited DP and congenital HH in the teenage years [141]. While gonadotropin levels are generally increased in primary testicular failure or in Klinefelter syndrome, single basal serum LH and FSH determinations are not useful in the differential diagnosis of self-limited delay vs hypogonadotropic hypogonadism.

In some cases, the diagnosis of permanent HH can be suspected before the age of pubertal onset, and importantly, if this suspicion arises in the first six months of life, it can be confirmed on the basis of low testosterone and gonadotropin levels, indicating an absence of the normal ‘mini-puberty.’ The presence or absence of ‘red flag’ features remains the strongest discriminator between self-limited DP and IHH. These red flags include cryptorchidism or micropenis, or the presence of the other components of the Kallmann syndrome which include anosmia or hyposmia due to hypoplasia of the olfactory bulbs, as well as cleft lip and palate, unilateral renal agenesis, short metacarpals, sensorineural hearing loss, synkinesia, and color blindness.

Investigation of the differential diagnosis of the two conditions may involve a number of physiological and dynamic tests including assessment of LH pulsatility by frequent blood sampling [42], the prolactin response to provocation [142, 143], the gonadotropin response to GnRH [144, 145] and analogs [146], the testosterone response to hCG [146–148], and first morning-voided urinary FSH and LH levels [149]. Boys with self-limited delay who are destined to undergo spontaneous pubertal development within 6 to 12 months may have a pubertal pattern of response to GnRH (post-GnRH maximum LH levels higher than maximum FSH levels). However, a low prepubertal LH response to GnRH is usually found in boys with self-limited DP who will develop later than that, as well as in boys with permanent HH. Most recently, a single measurement of inhibin-B <35 pg/mL has been shown to help discriminate prepubertal boys with IHH from DP with high sensitivity [150]; nevertheless, patient follow-up is necessary for a definitive diagnosis in the majority of cases. With the discovery in the last two decades of genes causing HH, genetic testing will no doubt modify the management of many of our male delayed puberty patients [151].