Interpretation of reproductive hormones before, during and after the pubertal transition—Identifying health and disordered puberty

Abstract Puberty is a process of transition from childhood to adult reproductive capacity, governed by the reactivation of the hypothalamic–pituitary–gonadal axis after a long period of dormancy in mid‐childhood. As such, the reproductive hormones are in a state of flux during the adolescent years, and interpretation of both the onset of healthy, concordant puberty and the differentiation of precocious, delayed or disordered puberty, can be challenging. This review is focused on the description of the endocrine axes in healthy puberty and the markers of disorders of puberty that can aid diagnosis and management for patients with these conditions. It will cover the hypothalamic, pituitary and gonadal hormone systems, the dynamic changes that occur during puberty, conditions leading to precocious, delayed or absent puberty and other syndromes with disordered puberty, and the biochemical diagnosis of these different disorders of puberty.

stimulate their development, gametogenesis and sex steroid and gonadal peptide hormone production.

| Gonadotropin-releasing hormone
GnRH is the master hormone of puberty, via which activation of the HPG axis is controlled. 5 GnRH is a decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly.NH 2 ), which is enzymatically processed from a large 69-amino acid prohormone precursor. GnRH is synthesized in specialized neurons of the hypothalamus, but is not localized to any one discrete nucleus and is also found outside of the hypothalamus in the hippocampus, cingulate cortex and olfactory bulbs.
GnRH release is coordinated through a balance of inhibitory (e.g., GABA) and excitatory (e.g., kisspeptin, glutamate) neuronal and glial inputs ( Figure 1). 6 GnRH is secreted in synchronized pulses into the hypothalamic-pituitary portal system from the nerve endings of about 1000 neurons. 7 The synchrony of these GnRH pulses across multiple GnRH neurons is a complex process, as there is spontaneous electrical activity of the neurons, as well as autocrine regulation through the GnRH receptor. It is likely that the upstream regulation by key peptides, kisspeptin, neurokinin B and dynorphin, produced by neurons in the arcuate nucleus, is ultimately responsible for coordinating GnRH pulse generation. 8 These three peptides act on GnRH neurons to modulate their activity via cell surface receptors and their coordinated action is known as the 'KNDy' model of pulse generation. These pulses occur every 30-120 min and stimulate the processing and secretion of gonadotropins from pituitary gonadotrope cells. 9 2.1.2 | Gonadotropins LH and FSH are produced by the gonadotrope cells of the anterior pituitary in response to GnRH stimulation. 10 GnRH travels to the F I G U R E 1 Schematic of the hypothalamic-pituitary-gonadal axis. GnRH production from hypothalamic GnRH neurons is regulated by kisspeptin, neurokinin, dynorphin (KNDy) and other upstream signalling, including GABAergic and glutamatergic neurons. GnRH travels via the portal circulation to the anterior pituitary where it stimulates LH and FSH production. These gonadotropins act in males on the testes and in females on the ovaries to produce the sex steroids testosterone and estradiol/progesterone (E2/P), respectively. There is negative feedback from inhibin B (IB) on the pituitary, and a mix of negative and positive feedback from sex steroids on the pituitary and on the hypothalamus via kisspeptin neurons. −, negative feedback; +, positive feedback; FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone HOWARD | 703 anterior pituitary via the hypothalamo-hypophyseal portal circulation ( Figure 1) and there binds to its receptor (GnRHR), a typical Gprotein-coupled receptor, on the gonadotrope cell surface. Activation of GnRHR triggers an increase in intracellular calcium concentration and downstream phosphorylation of protein kinase C. Gonadotropes are found throughout the anterior pituitary gland and abut upon the capillary basement membranes to facilitate access to the bloodstream. The same gonadotrope cells produce both LH and FSH, and these cells are small in diameter when the HPG axis is dormant but increase in size in response to GnRH stimulation.
Each GnRH pulse stimulates a pulse of LH, but FSH pulses are less clearly correlated with GnRH release, due to the longer circulatory half-life of FSH. LH is secreted rapidly in response to a bolus of GnRH from pre-existing pools of LH, leading to a rise in circulating LH within minutes, as well as more slowly after further LH processing.
There is also a differential sensitivity between LH and FSH response to GnRH, 11 such that a partial decrease in the activity of the GnRHR can lead to reduced LH and sex steroid hormone production with relatively preserved FSH concentrations. 12 While stimulation by GnRH in an intermittent or pulsatile manner leads to increased gonadotropin secretion, a continuous infusion of GnRH results in a decrease in LH and FSH secretion and a downregulation of the GnRH receptors on the pituitary gonadotrope cells. 13 These alterations in the GnRH receptor have an important role in regulating gonadotrope function.
FSH and LH are glycoproteins that share an identical α-subunit but have distinct 115 amino acid long β-subunits that confer specificity. There are a small number of published cases of mutations in the gonadotropin-specific β-subunits, including an inactivating mutation of beta LH causing pubertal failure with the absence of Leydig cells of the testes, 14 in contrast to inactivating mutations of beta FSH, which lead to lack of follicular maturation and amenorrhoea in females and failure of spermatogenesis in males, although there is some phenotypic variability. 15 Pubertal maturation in women, including breast development and menarche (indicating sufficient oestrogen production for a mitogenic action on the breast tissue and endometrium), can occur in a state of LH deficiency, although LH secretion is obligatory for ovulation. These observations imply that LH is essential for the maturation of Leydig cells and steroidogenesis in men and that its primary role in women is to induce ovulation 16 (Table 1) The majority of circulating testosterone is bound to sexhormone-binding globulin (SHBG) or albumin, with the remaining 'free' testosterone considered to be responsible for biological activity. 2 Testosterone is modulated by prereceptor metabolism through conversion, either by 5α-reductase type 2 (a surface enzyme located on the genital skin and elsewhere) to dihydrotestosterone or by aromatase (CYP19A1) to estradiol. Testosterone and dihydrotestosterone both bind to the androgen receptor but with a greater affinity for dihydrotestosterone. 21 The testosterone/ dihydrotestosterone-receptor complex attaches to the steroidresponsive region of genomic DNA to initiate androgen-dependent transcription and translation.

| Sex steroid feedback on the hypothalamus and pituitary
Once the HPG axis is activated by GnRH stimulation, and sex steroids and gonadal peptide concentrations rise, these then provide negative feedback inhibition to the hypothalamus and pituitary to decrease pituitary LH and FSH secretion. This is seen most profoundly in individuals with gonadal dysgenesis who have high concentrations of LH and FSH during infancy and adolescence. 25,26 In healthy females, positive feedback by estradiol also occurs from mid-puberty onwards, a process that is necessary for ovulation.
This requires an adequate pool of LH for release from the pituitary and priming of the ovarian follicle to reach a sufficient size to produce adequate oestrogen, both of which are kisspeptin-dependent processes. 27 Estradiol also increases pituitary gland sensitivity to GnRH, which, in addition to its action to increase GnRH pulse frequency via positive feedback on the hypothalamic kisspeptin neurons, increases LH secretion. The increase in estradiol also suppresses FSH within the ovary to allow luteinization of the dominant follicle in the presence of LH with subsequent ovulation. Progesterone, the dominant female hormone during the luteal phase, in contrast, slows LH pulse frequency, 28 inhibits proliferation and stimulates differentiation of endometrial cells.

Inhibin and activin
Inhibin B is a heterodimeric glycoprotein member of the transforming growth factor-β family. In males, it is produced exclusively by the Sertoli cells of the testes, and in females by the ovarian granulosa cells and the placenta. Serum inhibin-B concentrations vary during childhood in response to gonadotropin secretion. 29 In boys during the mini-puberty, when Sertoli cells proliferate but do not mature, serum inhibin-B concentrations increase to similar or higher concentrations to those observed in adolescent boys. These levels are sustained until the age of 18-24 months, after which they decline to lower but readily measurable concentrations ( Figure 2). 30 Inhibin B concentrations rise again early in puberty, reaching peak concentrations at Tanner stage G2, but then plateau. 31 In girls, from birth to 6 months, inhibin B levels are approximately 50% lower than at female puberty, when they peak at approximately 50% of the levels seen in male pubertal subjects. 32 Between mini-puberty and puberty inhibin B levels in girls are low. Inhibin B exerts negative feedback on the secretion of FSH from the pituitary gland, which is thought to also contribute to the observed difference in response of LH and FSH to GnRH stimulation. Thus, the absence of inhibin due to gonadal failure causes a greater rise in serum FSH than LH in pubertal and adult subjects. Activin, a subunit of inhibin, has an opposite effect to inhibin B, acting to stimulate the secretion of FSH from the pituitary gland.

Anti-Mullerian hormone
Anti-Müllerian hormone (AMH) belongs to the same family of transforming growth factor-β as inhibin B. In males, it is produced from the testicular Sertoli cells from the time of testicular differentiation to puberty, and in females, it is secreted by the ovarian granulosa cells from birth until menopause. 33,34 In healthy males, AMH is high in the foetus and newborn, peaking at mini-puberty around 2 months of age and then decreases by the age of 1 year. 30 Patients with dysgenetic gonads have low serum AMH while values are elevated in tumours of the Sertoli or granulosa cells. Undetectable AMH and inhibin B are characteristic of congenital anorchia but may also be seen in males with severe hypogonadotropic hypogonadism. A similar pattern in AMH concentrations during the first months of life has also been reported in infant girls, but the concentrations in girls are significantly lower. 30 AMH plateaus during puberty as a sign of androgen action. In girls, concentrations are a marker of ovarian granulosa cell function and are considered a novel marker for follicular reserve. This is because AMH is produced mainly by growing follicles, 5-8-mm diameter, while larger follicles selected for dominance have a marked reduction in their AMH secretion. 35 This has importance, for example, in Turner syndrome, for assessment of potential reproductive capacity. 36 Insulin-like 3

Insulin-like 3 (INSL3) is produced by the Leydig cells and is important
in the male foetus for testicular descent. Concentrations increase in males at puberty and correlate with LH and testosterone. In subjects with Klinefelter syndrome, lower INSL3 concentrations indicate Leydig cell dysfunction from mid-puberty onward. 37 In females, INSL3 is produced by ovarian theca cells of growing antral follicles 38 and is important for androstenedione synthesis, and therefore oestrogen production. It is not detectable in girls until puberty. 39 Knockout studies in mice lead to partial infertility and concentrations are increased in polycystic ovarian syndrome (PCOS) and decreased in women with premature ovarian insufficiency. 40

| Dynamic hormonal changes during pubertal maturation
After periods of activity of the HPG axis in utero, and then during mini-puberty, the axis becomes dormant between the ages of approximately 2 and 8-9 years ( Figure 2). The suppression of the axis is not absolute as LH pulsatility is detectable during this stage using ultrasensitive assays, but pulses are of low amplitude and infrequent.
Likewise, testosterone and estradiol are measurable in the circulation using sensitive assays, thus demonstrating low but definite activity of the prepubertal gonads. Notably, serum gonadotropin concentrations are low in early-mid childhood in the majority of children with gonadal disorders, demonstrating that it is central inhibition rather than negative feedback from sex steroid production that suppresses gonadotropin secretion during this period.
The increasing amplitude of GnRH pulses in early puberty leads to the augmentation of nocturnal LH pulses in children, before any physical development of Tanner genital or breast stage 2 can be observed clinically. 41 This period may thus be seen as the hormonal onset of puberty. The difference between daytime and overnight LH concentrations persists until the late stages of puberty. 42 Mean LH and FSH concentrations both increase gradually through pubertal development in concert with sex steroid concentrations, although LH rises to a greater extent, due to differences in feedback mechanisms and sensitivity for these two hormones, as described above. There is both an increase in basal concentrations of LH and FSH and a greater number and amplitude of LH peaks. 43 The negative feedback from gonadal steroids on the hypothalamic-pituitary production of GnRH and gonadotropins develops by mid-puberty and becomes dominant over the central inhibitory feedback drive.
In males, plasma testosterone concentrations increase dramatically from the onset of puberty to its completion, in parallel with an increase in testes volume, which is not only due to the increasing number of germ cells but also Sertoli cells (Table 2). 44 In early puberty, testosterone may only be detectable in the early morning but this pronounced diurnal rhythm in testosterone in early and

| Precocious puberty
Premature sexual maturation is a frequent cause of concern for parents, and for referral to paediatric clinical services. Precocious puberty is defined as the development of Tanner breast stage 2 in girls before the age of 8 years, and of Tanner genital stage 2 (testes volume > 3 ml) before the age of 9 years in boys. 48  hormonal profiling, as well as monitoring of pubertal development and growth. As a general rule, the earlier the age that puberty commences, the more likely is it to identify an organic cause. 48 Precocious puberty is most commonly gonadotropin-dependent, caused by central activation of hypothalamic GnRH pulsatility. It is around five times more common in girls than boys, 49 and in girls, most commonly due to an unknown or idiopathic aetiology. Much more rarely, peripheral (gonadotropin-independent) precocious puberty occurs due to autonomous activation of gonadal hormone production, for example, due to McCune-Albright syndrome, genetic mutations in the LHCGR causing constitutive activation or ovarian cysts secreting estradiol (  54 The response to GnRH stimulation is considered the gold standard for the diagnosis of central precocious puberty, with a pubertal serum LH concentration after stimulation of ≥5 IU/L. 53 As over recent years the availability of recombinant GnRH has been limited, GnRH analogues have also been considered for the investigation of peak LH and FSH concentrations following stimulation testing. 55,56 FSH is less informative than LH, but a stimulated LH/FSH ratio of more than 0.66 may help to distinguish progressive from nonprogressive cases not requiring intervention. In terms of sex steroid measurements, in boys, testosterone is assessed with a sensitive method, such as radioimmunoassay (RIA) or liquid chromatography with tandem mass spectrometry, which is a good marker of testicular maturation. In girls, estradiol can be uninformative, firstly because unless there is a very sensitive assay such as RIA it may be undetectable even in cases of true precocious puberty, and secondly because there is overlap between the normal range of estradiol for prepubescent and pubescent girls. Thirdly, the increase in estradiol concentration is also highly variable due to the fluctuation and intermittent secretion of this hormone. Oestrogenic exposure can also be assessed by pelvic ultrasound scans to visualize the mitogenic effect of estradiol on the uterus and ovaries.
However, markedly raised estradiol concentrations may be seen in cases of peripheral precocious puberty such as with McCune-Albright syndrome or ovarian disease due to cysts or tumours. 57 In peripheral precocious puberty, the high serum sex steroid concentration is seen in combination with low basal and peak serum LH concentrations after  60 Finally, in older girls, at least 50% of cases of premature sexual maturation will regress or stop progressing without the need for treatment. 61

| Delayed, arrested or absent puberty
Delayed puberty is generally defined in girls by a lack of Tanner stage 2 breast development by the age of 13 years, or by the absence of menarche at the age of 15 years, and in boys by the lack of Tanner genital stage 2 (testicular volume above 3 ml) at the age of 14 years. 62 In an adolescent with delayed puberty the main differential diagnosis is between a central or gonadal cause.

| Hypergonadotropic hypogonadism
Primary gonadal disorders display a biochemical picture of hypergonadotropic hypogonadism at puberty. During mid-childhood, serum gonadotropins may be similar or mildly higher than those in normal controls. In boys, the most common underlying condition is Klinefelter syndrome (47XXY), but in boys with this condition puberty usually starts within a normal timeframe; but tends to arrest in midpuberty with rising FSH and falling testosterone concentrations. 63 In boys, low serum inhibin B reflects primary germ cell failure. Gonadotropin concentrations assessed by basal LH and FSH determination are often increased by 10 years of age in females in primary ovarian insufficiency or in Turner syndrome (45X), but in a prepubertal child hypogonadotropic hypogonadism does not exclude primary gonadal disorders. Women with Turner syndrome without mosaicism commonly do not enter puberty, but overall, 30% of women with a diagnosis of Turner syndrome will undergo some spontaneous pubertal development, and 2%-5% have spontaneous menses and may have the potential to achieve pregnancy without medical intervention. 64 Other causes of hypergonadotropic hypogonadism in adolescence are described in Table 5.

| Hypogonadotropic hypogonadism
A hormonal picture of hypogonadotropic hypogonadism in adolescence is most commonly due to self-limited or constitutional delayed puberty, 65  Delayed puberty with low gonadotropin concentrations may also be due to functional hypogonadism or to congenital or acquired GnRH deficiency, leading to hypogonadotropic hypogonadism.
Functional hypogonadism with delayed puberty, arrested puberty or functional (hypothalamic) amenorrhoea is seen in young people with chronic disease (renal, liver, respiratory, cardiac or inflammatory amongst others), poor nutrition, including anorexia nervosa and excessive exercise. 69 Therapy for the underlying condition can result in normalisation of the HPG axis.
Congenital hypogonadotropic hypogonadism leads to absent, partial or arrested pubertal development in adolescence but may also be apparent in males in infancy with micropenis and cryptorchidism, due to GnRH deficiency during foetal development (Figure 3). Minipuberty provides a window of opportunity for evaluation of the functionality of the HPG axis in males with hypogonadism before puberty. 70

F I G U R E 4
Investigation flow chart for individuals presenting with delayed puberty (DP) in adolescence. BMI, body mass index; FT4, free thyroxine; GH, growth hormone; GHD, growth hormone deficiency; GI, gastrointestinal; PRL, prolactin Despite this, the clinical follow-up to assess spontaneous pubertal development or response to sex steroid therapy is often warranted before a definitive diagnosis can be made. 93 Stimulation tests using more potent GnRH agonists or hCG (in males) may be useful to discriminate these conditions but are not always clinically practical to perform. 94 Basal inhibin B is the most promising biochemical investigation in terms of its sensitivity, with studies reporting a threshold of less than 35 pg/ml in prepubertal boys to discriminate permanent hypogonadotropic hypogonadism from self-limited delayed puberty, 95 but this utility has not been demonstrated in girls. In males, the trio of testes volume (cut-off: 1.1 ml), maximal stimulated LH (cut-off: 4.3 IU/L) and basal inhibin B concentration (cut-off: 61 pg/ml) have been proposed as the most effective discriminator between these two conditions. 65 Most recently, FSH-stimulated inhibin B, at a cut-off of 116.14 pg/ml in males and 116.50 pg/ml in females was shown to have a 100% sensitivity and specificity for prediction of entry into puberty. 96 The diagnostic utility of basal and stimulated inhibin B needs further confirmation in clinical studies.
To assess GnRH production by the hypothalamus, LH measurement in response to stimulation with kisspeptin has been proposed as a useful test to identify individuals with GnRH deficiency and thus permanent hypogonadotropic hypogonadism. Kisspeptin stimulates GnRH pulsatility, and thus promotes LH, and to a lesser extent FSH, secretion. Inactivating mutations of KISS and KISS1R genes have demonstrated the importance of the KISS regulatory system in the regulation of human puberty and fertility, while activating KISS1R mutations have resulted in precocious puberty. 97 A recent clinical study found that maximal LH rise after kisspeptin administration was more accurate for diagnosis of men with GnRH deficiency than GnRH stimulation testing. 98 In a parallel study in adolescents with pubertal delay (3 females and 13 males), peak LH post kisspeptin stimulation was demonstrated to be superior to GnRH stimulation testing for predicting capacity to progress through puberty. 99 All eight study participants with a maximum LH response to kisspeptin of ≤0.4 IU/L reached age 18 years without developing physical signs of puberty, thus confirming the diagnosis of hypogonadotropic hypogonadism.
While further research is required to delineate the parameters of using kisspeptin in clinical paediatric practice, this is a promising area for the biochemical diagnosis of GnRH deficiency in adolescence.

| CONCLUSION
Using the interpretation of reproductive hormones in late childhood and adolescence to distinguish healthy puberty from its disorders is not straightforward. The biochemical parameters must be taken in the context of clinical features, imaging and radiological studies, and monitoring of pubertal progression. Diagnosis may require more extended or resource-intensive investigations, such as measurement of gonadotropin response to stimulation with GnRH or, more recently, kisspeptin, as well as genetic analysis with whole-exome or panel testing. 100 The expertize of the clinical team to put together these pieces of the jigsaw puzzle is key, to allow appropriately directed management in a limited-time window, to minimize negative outcomes and optimize therapeutic care for our patients.