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	GNRH Gonadotropin Physiology and Pathology Chapter 8 – Maria Gueorguiev, M.D., Ph.D.,Department of Endocrinology, Barts and the London School of Medicine, Queen Mary University of London, London EC1M 6BQ, United Kingdom Kathleen Prendergast, M.D., Ailleen Heras-Herzig, M.D. Alan Dalkin, M.D. Revised 1 Aug 2008 TO OBTAIN A DOWNLOAD OF THIS CHAPTER IN WORD OR PDF FORMAT, CLICK HERE	Index Contributors Search

PHYSIOLOGY OF THE HYPOTHALAMIC-PITUITARY-GONADAL AXIS

The hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH) is synthesized and released by the hypothalamus into the hypophyseal-portal circulation. GnRH then drives the synthesis and secretion of the two gonadotropin hormones: luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH are produced by the same cells in the anterior pituitary gland, comprising a 5-10% minority of the secretory cell population in that tissue. LH and FSH are released into the systemic circulation, traveling to the target reproductive organs (testes and ovary) where both gonadal hormone and gamete production are controlled. The dynamic nature of the reproductive axis is well recognized and represents the complex interplay of these hormones as well as input from higher central nervous system centers.

While GnRH neurons likely have an intrinsic pulsatile secretory pattern (1), many neurotransmitters may modify the GnRH secretory pattern. The two predominant pathways include the actions of catecholamines and the endogenous opioids (figure 1; 2-4) though dopamine and other neurotransmitters such as NPY and galanin may be involved (5-8). The bulk of data examining the role of catecholamines on the reproductive axis come from studies conducted in rodent models. In the presence of estradiol, intra-cerebral ventricular administration of norepinephrine (pulsatile) stimulates LH secretion (9) whereas treatment with phenoxybenzamine, a potent blocker of catecholamine alpha receptors, inhibits GnRH and gonadotropin release (10,11). Opioids exert an inhibitory effect on GnRH release. Treatment with morphine analogues reduces gonadotropin release in primates and humans while administration of opioid antagonists such as naloxone increases LH (12,13). Indeed, CNS opioid tone may serve to relay, at least in part, feedback inhibition from gonadal steroids on GnRH secretory patterns and be an essential component of dynamic gonadotropin release during the menstrual cycle.

The nature of the GnRH and gonadotropin secretory pattern is a pulsatile burst. Each LH pulse likely reflects a prior GnRH pulse. Direct measurement of GnRH secretory kinetics is not feasible in humans due to the dilution of GnRH (and other hypothalamic factors) from the hypophyseal-portal vessels to the systemic circulation. Hence, the patterns of GnRH release are inferred from data on LH release. However, data from sheep, rodents and other primates confirm this relationship between GnRH and gonadotropins (14-16).

GnRH also has a well recognized "self-priming" effect (17,18). Using various GnRH deficient models, it has been clearly documented that LH secretion increases with sequential GnRH pulses. Regulation of GnRH responsiveness is at least in part via changes in the numbers of GnRH receptors. Data reported from animal models suggest that GnRH receptor numbers decline in the absence of GnRH or following treatment with a GnRH antagonist (19-21). Conversely, pulsatile GnRH restores GnRH receptor expression in a dose, frequency and time-dependent fashion. Loss of receptor expression can occur in the face of either continuous or very high amplitude GnRH, likely reflecting at least one component of the phenomenon of "desensitization" (22). In addition, changes in second messenger systems such as protein kinase A, protein kinase C and calcium/calmodulin appear important in modulating both GnRH signaling and perhaps desensitization (23-26).

Pioneering research from Knobil and colleagues revealed the critical nature of the pulsatile secretory patterns needed for reproductive function (27,28). Using castrated monkeys with hypothalamic lesions to render the animals GnRH deficient, it was reported that only intermittent GnRH maintained LH secretion while continuous GnRH was associated with a rapid decline in LH release. Moreover, the nature of the pulsatile GnRH signal conferred an additional regulatory mechanism. Fast frequency pulsatile GnRH favored LH release while slower frequency pulses resulted in a relatively higher FSH secretion. These findings are supported by data in other animal models of exogenous GnRH treatment suggesting that the genes encoding the gonadotropin subunits are regulated in a similar pattern to LH and FSH release. Thus, the characteristics of the hypothalamic signal are a critical component to reproduction and, when appropriate regulation of GnRH is dysfunctional such as in Polycystic Ovarian Syndrome (to be discussed later in this chapter), alterations in fertility and gonadal functions may ensue.

1. Pubertal Maturation

Immediately after birth, and for the first few months of life, gonadotropin secretion, and presumably hypothalamic release of GnRH, is increased (29). Neither the mechanism behind the hypergonadotropism, nor the subsequent transition to lower levels of gonadotropin secretion, is certain. Perhaps stimulation from maternal steroid hormones such as estradiol induces GnRH release. Thereafter, within the first year of life through the time of puberty, both boys and girls have low levels of gonadotropins and gonadal hormones. GnRH/LH pulses can be detected in prepubertal children, albeit having low amplitude and slow frequency (every 3-4 hours) patterns of release.

In children, pulsatile LH release can be detected using highly sensitive modern hormone assay systems and mathematical techniques to properly identify pulsatile patterns of secretion (30-37). However, pulses are of very small amplitude and slow frequency. The onset of pubertal maturation is heralded by the development of a diurnal pattern of LH secretion (Figure 2, left panels).

2. Menstrual Cycle

By convention, the menstrual cycle is divided into two phases: beginning with the first day of menses and extending through 12-16 days duration is the follicular phase. Development of the dominant follicle with increasing gonadotropin and estradiol secretion, culminating in the mid-cycle gonadotropin surge, broadly characterizes this phase. During the luteal phase, there is reduced gonadotropin secretion in the presence of a corpus luteum and elevated progesterone and inhibin levels that, in turn, also decline with the failure of fertilization resulting in menstruation. It is the complex coordination of the human menstrual cycle that reflects the true dynamic nature of the hypothalamic/pituitary/gonadal axis (Figure 3; 38-40).

The initial portion of the follicular phase is characterized by a relatively high ratio of FSH to LH (41). This is likely the result of a slow GnRH pulse frequency as evidenced by LH release with an approximately 90 minute interpulse interval. This pattern is most prominent during the night-time hours. Data in rodents and primates clearly detail the frequency dependence of gonadotrope function with slow frequency GnRH favors FSH synthesis and secretion while fast frequency GnRH signals selectively increases LH . This period of FSH drive is critical for the recruitment and maturation of ovarian follicles. FSH induces granulosa cell expression of both LH receptors and levels of the enzyme aromatase, needed for the synthesis of estradiol (42-44). By the middle of the follicular phase, LH pulse frequency has increased to a nearly hourly pattern. With this, LH levels rise and ovarian release of estradiol increases significantly toward mid-cycle. The increase in estradiol, and ovarian production of the inhibins (dimeric peptides that selectively inhibit FSH synthesis and secretion) reduce FSH levels, perhaps playing an important role in limiting the final maturation of the non-dominant follicles (45-50).

At the end of the follicular phase, or mid-cycle, the increasing levels of estradiol result in an enhancement of LH responsiveness, thereby inducing the LH surge. Also, progesterone levels begin to increase and may further augment the LH responses to the ongoing GnRH stimulus (51-53). During the surge, LH levels remain increased for 36-48 hours, during which time ovulation occurs, estradiol levels decline and luteinization of the follicle results in increasing production of progesterone. This increase in progesterone plays a critical role in regulating GnRH release by decreasing GnRH pulse frequency (every 2-5 hours). The actions of progesterone are mediated at least in part via endogenous opioid production as opioid antagonist treatment increases GnRH secretion during the luteal phase (54-57).

In addition to gonadal steroids, the corpus luteum releases higher levels of inhibin, resulting in further reductions in FSH release and preventing recruitment of new follicles. In rodents, administration of anti-inhibin antisera results in marked increases in FSH and hyperstimulation of the ovaries with increased numbers of ovulatory follicles.

In the absence of fertilization, the corpus luteum regresses and gonadal steroid and peptide production declines. As the inhibitory actions of progesterone are removed, GnRH pulse frequency increases and LH pulsatility returns to a near hourly pattern. The fall in inhibin and estradiol result in a rise in FSH secretion. Thus, the beginning of follicular recruitment for the subsequent menstrual cycle actually begins during the later portion of the prior luteal phase/menstrual cycle.

In total, the human menstrual cycle represents a complex interplay of peptide and steroid hormones. Dynamic patterns of signaling appear to be critical components in this process. However, much of this process remains to be understood. Indeed, studies in both humans and monkeys have shown that administration of GnRH at a fixed frequency can induce an ovulatory cycle. While these reports have tended to use relatively high doses of GnRH and do not provide insight as to whether this stimulatory pattern would remain efficacious over the long term (i.e. multiple cycles), such data serve to provide a greater impetus to unravel the physiologic control of reproduction. Clearly, altered secretion of gonadotropins is associated with reproductive dysfunction. In that light, a clear understanding of the feedback and feed-forward regulatory mechanisms of the hypothalamic/pituitary/gonadal axis would likely provide useful tools in the management of a variety of common medical conditions.

PATHOLOGY OF THE HYPOTHALAMIC- PITUITARY- GONADAL AXIS

1. Polycystic Ovarian Syndrome

In 1935, Stein and Leventhal described a syndrome comprised of multi-cystic ovaries, irregular menses and hirsutism (58). This disease, ultimately named polycystic ovary syndrome (PCOS) is also associated with infertility, obesity, elevated leutinizing hormone (LH) and insulin resistance; the presence of each of these findings is quite variable among patients. The interaction between abnormal gonadotropin secretion, hyperandrogenemia and insulin resistance is complex, and the relative contributions of each is uncertain. The following will primarily focus on the neuroendocrine aspects of PCOS.

PCOS is common, occurring in 6-8% of women of reproductive age, and is the most common cause of female factor infertility. Certain ethnic groups seem to have a higher prevalence of the disease, as the rate has been found to be higher in women of Greek ethnicity (59) and Caribbean Hispanic women (60). In the US, rates between African-American and Caucasian women are similar at 3.4% and 4.7%, respectively (61).

Among women with PCOS, the risk for co-morbidities such as obesity, diabetes (gestational and type 2), hyperlipidemia, cardiovascular disease, and endometrial cancer are increased. Obesity is found in 40-60% of women with PCOS, depending on ethnicity and geography, and is intimately linked to other co-morbidities associated with PCOS (62,63). The risk of developing diabetes is higher in obese individuals with PCOS when compared to lean women. Hypertension is associated with PCOS in obese, but not lean individuals (64). Significantly higher levels of LDL have been demonstrated in women with PCOS when compared to controls (65). The risk for myocardial infarction has been found to be approximately seven times that of the normal population in one study, however, this figure was not adjusted for BMI(66).

Decreased fertility and early pregnancy loss are common in patients with PCOS. The incidence of infertility is estimated at a mean of 74% (range 35-94%) (67). The rate of spontaneous abortion is approximately 30%, double that of pregnancy loss in normal women (68). However, conception and delivery rates are improving with recent developments in treating women with PCOS (see below).

Pathophysiology

The mechanism(s) by which PCOS develops is controversial, and is likely multi-factorial. It is generally agreed that the primary defect is not ovarian in origin, but the relative roles of hyperinsulinism versus abnormal gonadotropin secretion have not yet been clearly elucidated. Elevated LH levels are found in the majority of women with PCOS and LH stimulation is believed to be the causative factor of excess ovarian androgen production. Treating these women with a GnRH agonist to desensitize LH secretion leads to decreased levels of LH, testosterone, and androstenedione (69).

The primary disorder of gonadotrophs in PCOS has been found to be increased LH pulse frequency and amplitude, however, elevated serum LH levels are not uniformly found among patients. Prior data has demonstrated the presence of elevated LH/FSH ratio, with rates varying from 35 to 90%. (70-74). Several studies have suggested that obese women with PCOS are more likely to have normal LH levels when compared to lean women with the disorder (75-78). This was clearly demonstrated in a study by Taylor, et al, in which LH pulse amplitude was found to be inversely correlated with BMI. Despite this, women with PCOS, regardless of BMI, have been observed to have an LH level greater than the 95th percentile in 75%, and an elevated LH/FSH ratio in 94% (79).

The cause of the abnormal gonadotropin secretion and increased LH levels in women with PCOS is unknown, but possible causes include increased GnRH secretion, or increased responsiveness by the pituitary to GnRH (80). Fast frequency GnRH secretion has been demonstrated in animal models to foster LH production and secretion, while slower frequency GnRH pulses increases FSH production (81, 82). As a surrogate measurement for GnRH, pulse studies have measured LH pulse frequency. Women with PCOS have been consistently found to have faster LH pulse frequency, and this increased pulse frequency is reflected in higher LH levels and LH/FSH ratio (83,84). This suggests that women with PCOS have persistently increased GnRH pulse frequency. Moreover, without the normal slowing of the GnRH pulse generator seen in normal menstrual cycles, FSH production and secretion will be reduced, leading to impaired folliculogenesis and anovulation.

The loss of normal ovarian cyclicity may be self-perpetuating. After the mid-cycle LH surge, production of ovarian progesterone (P) by the corpus luteum will, in normal women, provide feedback to the hypothalamus and foster the initiation of a subsequent menstrual cycle. Indeed, administration of exogenous progesterone to women with PCOS causes a selective increase in FSH and folliculogenesis (85). As an additional mechanism in women with PCOS, an impaired sensitivity of the hypothalamus to P has been demonstrated in plasma P concentrations less than 10 ng/ml, but LH secretion was able to be suppressed with higher P concentrations (13-15 ng/ml)(86). This suppression of LH is also seen in women in PCOS who have recently ovulated. From these data, it is hypothesized that during pubertal maturation, a vicious cycle of: (1) decreased hypothalamic sensitivity to the low levels of P seen during puberty (2) persistent rapid GnRH pulsatility leading to excessive LH secretion (and LH stimulated ovarian androgen production) (3) impaired folliculogenesis and anovulation (4) absence of normal luteal progesterone production and hence no feedback to the hypothalamus, leads to the spectrum of symptoms seen in PCOS (figure 2).

The cause of hypothalamic insensitivity to ovarian steroids is unknown. Hyperinsulinemia has been suggested as the cause, given that it has been well demonstrated that these women are insulin resistant and have elevated insulin levels. However, insulin administration during a hyperinsulinemic, euglycemic clamp has been shown to have no affect on gonadotropin secretion (87). Hyperandrogenemia has also been suggested to be the cause of decreased sensitivity to P. Supporting this theory is data showing that treatment of PCOS women with the anti-androgen flutamide restores GnRH inhibition by low doses of P (88).

The paracrine factors activin, inhibin and follistatin have also been suggested to play a role in the pathogenesis of PCOS, however the contribution of these proteins to the disease process remains to be elucidated.

A genetic component to PCOS has been clearly identified by family studies, with the proportion of first-degree relatives between 15% and 40% (89-91).

Many candidate genes have been studied as genetic determinants in the development of PCOS. However, the controversy regarding the identification of a causal gene originates partly because of the variation of the diagnostic criteria used to select the studied populations, but is also likely because of the variability of the clinical spectrum of this common disease, which like other common complex traits is polygenic.

Among the susceptibility genes studied, a CYP11A gene (encoding a cytochrome P450, family 11, subfamily A polypeptides) promoter variant have been shown to be associated with PCOS (92-94), but this has not been confirmed by other studies (95,96). Yet, different allele associations have been found (97), which makes it an unlikely locus.

Heterozygous carriers of mutations in the 21-hydroxylase (CYP21) gene represent between 20%-30% of adolescent girls or women with hyperandrogenism (98-102), and about 36% among children with premature pubarche (103). Indeed, girls with premature pubarche due to a premature adrenarche have several clinical and biochemical features similar to women with PCOS (Witchel SF Mol Cell Endocrinol 2006)(104). The heterozygosity for CYP21 mutations varies among adolescents and women with premature pubarche and/or PCOS and among different ethnic populations (105, 106); it is possible that this genetic variant may play a role in the pathogenesis of both disorders (104).

Polymorphisms in several genes - the aromatase (CYP19) gene, the IGF-1 receptor (IGF1R) gene, and the insulin receptor substrates 1 and 2 (IRS1 and IRS2) genes - have also been associated with premature pubarche, induced by a premature adrenarche, in girls or young women (107-109).

The PPARγ2 common polymorphism, P12A, has been found to be associated with an increased risk for obesity during adolescence in girls with premature pubarche (110).

The calpain-10 (CAPN10) gene, which encodes the cysteine protease calpain 10, has not generally been found to be associated with PCOS or related phenotypic traits (111-113), although one two-SNP haplotype has been found to be associated with higher insulin levels during OGTT in an African-American population with PCOS or with other clinical features of PCOS (111,114). However, CAPN10 alleles have been found to be associated with PCOS in a Spanish population (115) and with metabolic syndrome among patients with PCOS (116).

Specific haplotypes of calpain-5 (CAPN5) gene, a paralogue (both genes have the same ancestral gene from which they originate by duplication) of CAPN10, and which encodes also a protease, have been found to be associated with PCOS (117). Further replication studies are necessary before the implication of the CAPN5 gene in the pathogenesis of PCOS can be confirmed.

Data referring to the insulin gene VNTR in the 5’ area have been contradictory and inconclusive; some groups having found an association with paternally inherited class III alleles in PCOS patients (118,119) while others did not (113, 120, 121).

Data from studies investigating the role of polymorphisms of the insulin receptor (INSR) gene in the development of PCOS are controversial. One Korean study (122) did not find an association between a SNP and PCOS, but another group found an association (123); the Korean group subsequently found that another SNP in INSR was associated with this disorder (124).

Evidence for linkage between the chromosome 19p13.2 and a marker within this locus (D19S884, which maps to the fibrillin-3 gene, FBN3) with PCOS have been shown by two complementary studies (95,125); a third study showed also association between D19S884 and PCOS (126), but another group failed to replicate it (127). Further, Urbanek et al. found an association between this variant of the FBN3 gene and the metabolic phenotype (markers of insulin resistance and pancreatic β-cell function) in women with PCOS (128).

Variation in the androgen receptor gene (AR), at the length of CAG repeat, has also resulted in controversial data (129-134).

A large number of studies have reported inconsistent data for associations with PCOS and a number of genes, previously shown to be associated with insulin resistance, such as different cytokines - adiponectin, leptin, resistin, TNFα, IL-6, and others (135-139).

Genes involved in the regulation of glucose metabolism, such as the glycogen synthase kinase 3 beta (GSK3β) have provided preliminary associations of haplotypes with PCOS (140).

In thus appears that PCOS is a multifactorial polygenic disorder in which environmental and genetic factors play a determinant role (141,142). Replicating initial results of small studies with large cohorts of patients affected with PCOS will provide new insights in the understanding of the physiopathology of this disorder by identifying new susceptibility and protective genes, and of its treatment and prevention.

In the past, the diagnosis of PCOS has been complicated by the wide variability of presenting symptoms. Some experts have used the criterion of the presence of polycystic ovaries on ultrasound for diagnosis. In fact, polycystic-appearing ovaries on ultrasound are found in 16-25% of normally cycling, non-hyperandrogenemic women (143), and not all women with clinical symptoms suggestive of PCOS have polycystic ovaries on ultrasound. As mentioned above, some series have found that the presence of elevated LH levels in women with this disorder is as low as 35%. Obesity, hirsutism and acne are found with varying frequency, depending on the population studied. Due to this, the NIH consensus conference on PCOS in 1990 determined that the diagnostic criteria require only evidence for chronic anovulation and hyperandrogenemia, and the exclusion of other related disorders such as hyperprolactinemia, thyroid dysfunction, late-onset congenital adrenal hyperplasia or androgen secreting tumors (144).

The treatment for PCOS is dependent upon the concerns of the individual patient. In all women who are obese, weight loss should be strongly encouraged, as this will often ameliorate many symptoms, as well as promote fertility and decrease the risk of diabetes and hypertension. Recently, bariatric surgery as a treatment for morbid obesity has been explored in terms of its effects on women with PCOS (145-147). Indeed, significant weight loss following Roux-en-Y gastric bypass or gastric banding procedure has been associated with improved metabolic and androgen secretory patterns along with an increase in ovulatory function.

For patients who do not desire fertility, maintenance of regular menses either by the administration of medroxyprogesterone or combination oral contraceptives will lower the risk of endometrial hyperplasia and carcinoma. It has also been suggested that oral contraceptives may reduce the risk of ovarian cancer in patients with PCOS (63).

Oral contraceptives will often lower LH and testosterone levels and decrease hirsutism and acne (148). As an alternative or additional approach, blockade of androgen action at the pilosebaceous unit, spironolactone and cyproterone are the two most common anti-androgens used in the treatment of hirsutism (149-151), though cyproterone is not available in the US.

Restoring fertility in women with PCOS has changed dramatically in recent years. Clomiphene citrate has been used as an inducer of ovulation for many years and is believed to act by blocking the effects of estrogen on the hypothalamus. Loss of estrogen inhibition results in increased GnRH release, causing increased levels of FSH and follicular development. One common sequelae of clomiphene therapy is that of functional ovarian cysts. The physician should allow these to regress before initiating a subsequent course of clomiphene. Injectable gonadotropins have also been used to induce ovulation but carry the risk of ovarian hyperstimulation and multiple gestation (152).

Treatment with metformin to reduce insulin resistance has been shown to have great promise in treatment of infertile PCOS patients. Insulin increases LH-induced ovarian androgen production and that treatment with diazoxide (which blocks insulin secretion from the b-cell) results in marked decrease in testosterone levels in PCOS women (153). In women with PCOS, metformin improves insulin sensitivity (154) and has also been found to improve menstrual cyclicity. A number of studies have now been published demonstrating improved rates of ovulation and reduction of androgens in obese women with PCOS (154-159). In one study by Moghetti, et al, a significant reduction in LH and serum androgens was seen during the 6 months of treatment and these effects were maintained for an additional 6 months of open-label treatment (156). Trials investigating pre-treatment of PCOS women with 5 weeks of metformin have demonstrated an eight-fold increase in spontaneous ovulation and a tenfold increase in clomiphene-induced ovulation (159). Furthermore, it has been suggested that hyperstimulation by injected gonadotropins may be decreased if women are pretreated with metformin (155,160).

As mentioned above, early pregnancy loss is not uncommon in women with PCOS. However, a recent study by Jacubowicz et al., demonstrated that the rate of pregnancy loss was decreased in women on metformin therapy through pregnancy (161). Long term safety and efficacy trials are ongoing, but results are promising. A more recent, placebo-controlled, randomized trial has shown that, metformin used on its own compared to clomiphene alone as first line treatment in non-obese women with PCOS, produces similar ovulation rates but a significantly higher conception rate and birth rate compared to clomiphene (although this latter outcome has not been considered as a primary end point) (162). A nonrandomized trial with the same therapeutic modalities in infertile women with PCOS (from the same group) has found similar ovulatory, conception and spontaneous abortion rates for metformin alone versus clomiphene alone (163). Two other controlled randomized trials failed to report any benefit on ovulation (primary end point) and pregnancy (secondary end point) rates for the combination metformin – clomiphene compared to clomiphene alone in untreated women with newly diagnosed PCOS (164). Considering live-birth rates as a primary outcome, metformin was associated with lower rates of ovulation, conception and live births in women with PCOS; the combination metformin - clomiphene resulted in similar effects (regarding ovulation and conception rates) compared to clomiphene alone (165).

Less data are available for the safety and efficacy of other insulin sensitizers such as the thiazolidinediones and D-chiro inositol, but early studies suggest that these treatments decrease the androgen milieu and enhance ovulatory function. Thiazolidinediones (troglitazone previously available but not in use anymore because of hepatic toxicity) have been shown to be effective in reducing free testosterone levels when used in association with metformin (166), while if used alone testosterone levels remain unchanged (167). Studies with other thiazolidinediones – pioglitazone and rosiglitazone – show that similar effects on androgen reduction are also achieved (168,169). The previously used troglitazone as well as the currently available pioglitazone and rosiglitazone have been shown to induce ovulation in women with PCOS compared to placebo (167,170-172). A small trail has shown higher ovulation rates for the combination rosiglitazone-clomiphene versus metformin-clomiphene, but there were no differences in the pregnancy rates in woment with PCOS resistant to clomiphene (173).

Recently, alternative approaches to ovulation induction have been sought for women with PCOS. Specifically, with the development of aromatase inhibitors for the treatment of patients with breast cancer, an alternative method to disrupt estrogen feedback to the GnRH pulse generator has become available. The half life of the aromatase inhibitors is significantly less than that of clomiphene citrate and aromatase inhibitors do not confer partial agonist actions at other estrogen responsive target tissues as is seen with clomiphene. To date, most but not all trials have yielded positive results, suggesting that brief blockade of estrogen synthesis can restore ovulatory function in women with PCOS (174-178).

2. Hypogonadotropic Hypogonadism

Hypogonadism is defined as impaired gonadal function with resultant decreased sex-steroid levels. This condition may result from gonadal failure (primary hypogonadism) or abnormalities of the hypothalamic-pituitary axis (secondary hypogonadism). The distinction between these disorders can usually be made my measurement of follicle stimulating hormone (FSH) and luteinizing hormone (LH). In hypogonadotropic hypogonadism, both the FSH and LH will tend to be inappropriately low given the subnormal sex-steroid levels.

Pulsatile release of gonadotropin-releasing hormone (GnRH) from the hypothalamus is required for the initiation and maintenance of the reproductive axis in the human. Deficient GnRH secretion results in hypogonadotropic hypogonadism. It may occur in isolation [idiopathic hypogonadotropic hypogonadism (IHH)], in association with anosmia [Kallmann's syndrome (KS)] or as a result of a number of structural and functional lesions of the hypothalamus or pituitary. The clinical manifestations of GnRH deficiency depend on the onset and severity of the disorder. The male to female ratio is approximately 4:1 however, among familial cases, this ratio drops to 2:1 (179).

GnRH neurons originate outside the central nervous system in the olfactory placode and migrate along the olfactory nerve, up the nasal septum and through the cribriform plate to the forebrain where they find their place in the arcuate nucleus of the hypothalamus. GnRH has been demonstrated in the human brain as early as 4.5 weeks of gestation. LH and FSH are first measurable in the embryonic brain at 10 weeks of gestation and in the peripheral blood at 12 weeks (180). The levels of LH and FSH continue to rise peaking at mid-gestation and falling thereafter. Although GnRH secretion is clearly present throughout fetal development, the early testicular testosterone production required in utero for normal sexual and genital differentiation in males is maintained by stimulation from maternal human chorionic gonadotropic hormone (hCG). However, GnRH secretion is thought to be necessary late in fetal development and in the early neonatal period for complete descend of the testis and growth of the external genitalia. Thus, patients with hypogonadotropic hypogonadism may present with cryptorchidism and /or microphallus in the neonatal period as a result of inappropriately low and sex-steroid levels during fetal development (179, 181-183).

More commonly, the diagnosis of GnRH deficiency is delayed until adolescence. These patients have failure of normal pubertal development and development of secondary sexual characteristics. In contrast, adrenarche occurs normally in these patients. This disorder is oftentimes difficult to distinguish from constitutional delay of puberty. However, adolescents with GnRH deficiency tend to be of normal height for age and often have a eunuchoidal body habitus due to delayed epiphyseal closure (182). In contrast, patients with constitutional delay have delayed growth, adrenarche and skeletal maturation.

KS is characterized by the presence of anosmia and hypogonadotropic hypogonadism. Other midline craniofacial defects including cleft lip and/or palate and high arched palate represent the next most commonly observed clinical features in this syndrome (181,184,185). In addition, these patients may have sensorineural hearing loss, synkinesia, oculomotor abnormalities, red-green color blindness and cerebellar ataxia (181,183,185,186). Shortened metacarpals, pes cavus deformity of the feet and renal agenesis have also been described (181,185,187).

Both IHH and KS are characterized by: i) complete or partial absence of pulsatile LH secretion (188,189); ii) normal gonadotropin secretion after exogenous physiologic GnRH administration (188-190); iii) normal levels of other hypothalamic-pituitary hormones; iv) normal radiographic imaging of the hypothalamus and pituitary. While the pathogenesis of GnRH deficiency appears to be quite similar, there are several documented patterns of inheritance that may contribute to the variability in presentation and associated abnormalities in different subjects (181,184,191). GnRH deficiency has been associated with autosomal dominant, autosomal recessive and X-linked patterns of inheritance. Furthermore, family members of probands with KS have been found to have IHH without olfactory defects suggesting that the expressed phenotype for any one genetic abnormality may be quite varied (181,184).

The KS gene (KAL1 gene) has been localized to Xp22.3. The protein encoded by the KAL gene has been termed anosmin (192,193). The use of anti-GnRH antibodies in a 19-week human fetus with X-linked KS showed that GnRH neurons migrate from the olfactory placode and cross the perforations in the cribriform plate however, they appear to arrest at the dorsal aspect of the plate without reaching the forebrain or their normal location in the arcuate nucleus of the hypothalamus (194). It has therefore been postulated that anosmin may be crucial for the normal entrance of the GnRH neurons into the olfactory bulb and/or for the establishment of neuronal interactions between the incoming olfactory axons and the central neurons of the bulb.

Intragenic deletions and point-mutations in the KAL1 gene cause the X-linked Kallmann syndrome (192,193,195,196) and are detected in 14%-50% of familial cases and in about 8%-11% of sporadic cases (197,198).

Nonetheless, autosomal genes have also been found to be responsible for GnRH deficiency and it is postulated that these mutations may indeed account for the majority of cases (184). Some of the identities of the genes responsible for GnRH deficiency have been elucidated, while other genes remain yet unknown. No particular mutations of the GnRH gene itself have been described in humans. In contrast, mutations of the GnRH receptor have been identified and the phenotype of the affected subjects varies widely (199-201). Patients have been described with normal pubertal but small testis and abnormal semen analysis while others have been found to have a more complete gonadotropin deficiency with primary amenorrhoea and infertility and delayed puberty with absence of secondary sexual characteristics and cryptorchid testes.

Although a naturally-occurring deletion within the GnRH gene causes an autosomal recessive hypogonadotropic hypogonadism in mice (202), no mutations in this gene have been detected in the human (203-206). Indeed, mutations in the GnRHR gene have been detected in about 40% of patients with familial form of autosomal normosmic recessive hypogonadotropic hypogonadism (HH); in sporadic cases, these mutations have been identified in only 10-17% (205,207). These homozygous or compound heterozygous mutations induce partial loss of function of the receptor in vitro, while the clinical phenotype extends from partial to complete hypogonadism. Partial IHH responds to dose-dependent pulsatile GnRH treatment with induction of gonadotropin secretion and ovulation (208); this treatment is inefficient in female patients with complete IHH (209). Male subjects with partial IHH presenting with apparent constitutional delay of growth and puberty and a slight degree of oligospermia have also been reported (210).

The fibroblast growth factor receptor 1 (FGFR1/KAL2) has been found to interact with anosmin, which is a co-factor for FGFR1. Loss-of-function mutations in FGFR1 gene (chr 8p12) (heterozygous and homozygous) have been associated with hypogonadotropic hypogonadism of autosomal dominant inheritance and anosmia, but the phenotypical spectrum includes cleft palate or lip, dental agenesis, bimanual synkinesis and skeletal anomalies, renal aplasia (211). The reproductive phenotype associated with the same FGFR1 mutation may vary from complete absence of puberty to normal reproductive axis within families, through the fertile eunuch variant of idiopathic hypogonadotropic hypogonadism is spontaneously reversible after testosterone replacement (212). Mutations in the FGFR1 gene have also been associated with a spontaneously reversible form of hypogonadotropic hypogonadism (213). The comparison of the reproductive phenotype between patients with Kallmann syndrome, carrying either KAL1 or FGFR1/KAL2 mutations, revealed that a more severe clinical spectrum with absence of puberty, more frequent cryptorchidism and smaller testicular volume were present in subjects belonging to the first group versus subjects with FGFR1/KAL2 mutation; in this latter group, a few cases has undergone normal puberty, were eugonadal with normal gonadotropins and testosterone levels; gonadotropins and inhibin levels were lower in patients with KAL1 mutations; both groups had nonpulsatile LH secretion (214). Cases with normal olfaction have also been described among the IHH associated with FGFR1 mutations (215-217). The presence of olfactory bulb agenesis or hypoplasia has been found on the MRI imaging in patients with mutations in the FGFR1 gene (211,213,215,218), which suggest that loss-of-function FGFR1 gene mutations may be involved in the abnormal GnRH neuron migrations through alterations within the olfactory bulb morphogenesis (219).

Following hormonal replacement therapy (testosterone alone or combined with gonadotropins or GnRH, or GnRH alone), 10% men with Kallmann syndrome or normosmic idiopathic hypogonadotropic hypogonadism (nIHH) with absent or partial puberty, presented a reversal after discontinuation of their therapy (Raivio T NEJM 2007)(220). Three of these patients harboured mutations: a homozygous GnRHR mutation (sporadic nIHH) and heterozygous FGFR1 gene mutation (familial Kallmann syndrome and sporadic nIHH) (220).

Evidence for linkage on chromosome 19 was obtained from two consanguineous families with siblings affected with IHH and who were found to have a 155 base pair deletion in the GPR54 gene and a loss-of-function mutation (both homozygous), respectively (205,221). Mutations in the GPR54 gene result in impairment of the reproductive function, with failure to undergo puberty, reduced GnRH and LH and FSH secretion, low levels of sex steroids, impaired gametogenesis and lack of menstrual cycles (221-225), in the context of a partial or complete hypogonadotropic hypogonadism. Similar to mutations in the GnRHR gene, mutations in the GPR54 gene cause autosomal recessive normosmic hypogonadotropic hypogonadism and are detected in about 20% of familial cases (221,223,224,226). GPR54-kisspeptin signalling plays a key role in GnRH secretion through direct activation of hypothalamic GnRH neurons that co-express GPR54. GnRH itself is required for the stimulatory effect of kisspeptin on gonadotropin secretion (225,227,228). In support of this are more recent studies, which have showed that central and peripheral administration of kisspeptin induces GnRH release in rodents (229-230). Female patients, homozygote for a loss-of-function mutation in the GPR54 gene, have been able to have a pregnancy with normal progression to term under GnRH therapy, suggestive that GPR54 is not critical for placental function (232,233). Exogenous gonadotropin or long-term pulsatile GnRH infusion results in fertility and normal pregnancy in male and female patients (223,226).

Another genetic cause for hypogonadotropic hypogonadism has been reported: mutations (homozygous, heterozygous or compound heterozygous) in the prokineticin receptor-2 (PROKR2) and its ligand, prokineticin-2 genes (234). The co-existence of a missense mutation in the KAL1 gene with a heterozygous PROKR2 mutation in the same subject suggests a possible dygenic inheritance of the Kallmann syndrome (234). Mice defective for PROKR2 gene display impaired reproductive function and abnormal morphology of the olfactory bulbs, which suggests that this gene is implicated in the pathogeny of the Kallmann syndrome (235), although functional studies with PROKR2 mutants are not yet available (236).

Loss-of-function mutations in the PROK2 gene have been shown to cause both Kallmann syndrome and normosmic hypogonadotropic hypogonadism (237). Recently, truncated PROK2 has been shown to be devoid of biological activity in vitro (237). Knockout mice for PROK2 have also abnormal olfactory bulbs, alterations of the developmental and migrational process of the GnRH neurons, and hypoplasia of the gonads that reflects impairment of the reproductive axis (237,238).

Nasal embryonic LHRH factor (NELF) is expressed during embryonic development, in the olfactory sensory cells and GnRH neurons, and plays a key role in the guidance of these neurons during their migration (239). Knockout mice for NELF show abnormal olfactory axon development and altered migration of GnRH neurons (239). The EBF2 gene is implicated in the migration of GnRH neurons; indeed, mice knockout for EBF2 gene present an abnormal migration of GnRH and have hypogonadotropic hypogonadism (240).

A heterozygous mutation in NELF have been detected in a subject with idiopathic/sporadic case of hypogonadotropic hypogonadism (241). However, molecular analysis of NELF and EBF2 genes failed to identify any mutations in 12 Brazilian patients with Kallmann syndrome or normosmic hypogonadotropic hypogonadism (242).

It is of relevance to note that a gene dosage effect implicating several genes that regulate the same pathway of the reproductive axis, determine the phenotype and thus, may explain its diversity within the pedigrees. Thus, digenic inheritance of IHH genes has been reported for KAL1/anosmin-1-PROKR2, FGFR1-GnRHR, FGFR1-NELF (234,243). These digenic mutations lead to more severe phenotypes of reproductive and olfactory dysfunction, in association with morphological defects in the context of FGFR1 mutations.

An acquired form of GnRH deficiency has recently been described in which subjects have had normal pubertal development but develop erectile dysfunction, decreased libido and impaired fertility during adulthood (244). The biochemical profile is indistinguishable from that of congenital IHH. These patients have an apulsatile pattern of LH secretion, low serum testosterone and respond to a GnRH agonist with a complete normalization of the pattern of gonadotropin secretion. Furthermore, classical factors leading to functional GnRH deficiency such as stress, excessive exercise or weight loss are absent in these subjects. This acquired GnRH deficiency appears to be irreversible.

A series of patients have been described with normal testicular size in the setting of eunuchoid body proportions and secondary sexual characteristics. Men with this syndrome have normal spermatogenesis but have inadequate serum testosterone levels to achieve full secondary virilization (245). They are similar to patients with acquired IHH in that they have normal testicular size but differ in that they have preserved spermatogenesis and can usually be treated with either hCG or testosterone alone (246). This disorder is felt to result from insufficient GnRH secretion in that these patients have a normal response to GnRH agonist but impaired LH secretion in the setting of clomiphene citrate use (247). In addition, as has been described in a subset of patients with IHH or KS (188,189), frequent blood sampling in patients with fertile eunuch syndrome reveals a pattern of nocturnal LH and testosterone secretion similar to that observed during puberty (248).

The prevalence of delayed puberty in relatives of patients with IHH has been estimated at 12% that is in excess of the estimated 1% for the general population (184). These subjects had otherwise normal puberty suggesting therefore that delayed puberty may represent the mildest form of GnRH deficiency.

3. Mutations in gonadotropin/gonadotropin receptors

LH, FSH, hCG and thyroid stimulating hormone (TSH) belong to the family of glycoprotein hormones. These hormones have a heterodimeric structure consisting of a noncovalently associated common a subunit and a unique b subunit. This heterodimerization is crucial for normal hormonal action. The b subunit is responsible for the interaction with hormone-specific receptors and for signal transduction.

Four genetic variants have been identified in the b subunit of LH however only one of these has been found to result in altered activity of the hormone. This mutant LH resulted from a missense mutation in codon 54 causing a Gln to Arg substitution leading to normal serum immunoreactive LH levels but impaired bioactivity (249). The initial case was described in a 17 year-old man with delayed puberty, two-fold elevation of LH, normal FSH levels and decreased serum testosterone. There are several infertile men described in the family of this proband however all the females studied were normal.

A common LH variant has also been identified in which there are two amino acid changing mutations in the b subunit of the LH gene at codon 8 (Trp for Arg) and codon 15 (Ile for Thr) (250,251). The latter is responsible for introducing an altered glycosylation site resulting in abnormal detection by monoclonal antibodies. The prevalence of this variant LH varies greatly among populations with a carrier frequency as high as 53.5% in Australian aborigines (252). Functional differences have been demonstrated between variant and wild type LH. Variant LH has been shown to have a shorter half-life in the circulation (253). On the other hand, there appears to be a 40% higher promoter activity of the gene that may partially compensate for the shortened half-life (254). Homozygotes for the LH variant have been found to have recurrent spontaneous abortions, menstrual irregularities, infertility and polycystic ovarian syndrome in a Japanese population (2557,256). However, the frequency of LH variant was found to be lower in a PCOS population in Finland and the Netherlands (257).

Recently, 3 members of a Brazilian consanguinous family have been found to carry a homozygous mutation at the 5’ splice site in intron 2 of the LHβ gene, which disrupts the splicing of the mRNA and thus its processing (258). The three patients presented with hypogonadism due to LHβ deficiency. The female subject has undergone puberty but had secondary amenorrhea and infertility (low-normal oestradiol and progesterone, normal FSH, undetectable LHβ, which was unresponsive to GnRH, high inhibin, and US of the ovaries has showed multiple follicles at the antral stage (258). Her two affected brothers have not had undergone puberty, and under replacement therapy with testosterone injections the external genitalia have increased in size, one of the siblings had azoospermia and the other subject had absence of mature Leydig cells and spermatogenic arrest revealed by the testicular biopsy; they both had undetectable LH, not stimulatable under GnRH, high FSH and low testosterone levels (258).

A two-nucleotide deletion of the b subunit of FSH has been reported in a subset of boys presenting with delayed puberty, low testosterone and FSH concentrations, and high serum LH (259). Similarly, a female patient homozygous for this mutation presented with primary amenorrhea and decreased FSH levels (260). Another homozygous mutation in the FSH beta-subunit gene has been detected in a Brazilian female with delayed puberty, incomplete pubertal breast development and primary amenorrhea (261).

Men presenting with inactivating mutations of the LH receptor may have variable phenotypes ranging from feminization of the external genitalia to small testis and delayed development of secondary sexual characteristics. In contrast, females with inactivating mutations have normal secondary sexual characteristics but have amenorrhea associated with increased LH concentration (262,263).

The LHR gene is located on chr 2p21 and results in a particular phenotype both alleles of the gene must be inactivated, this either by homozygous mutations or a compound heterozygous loss-of-function mutations, resulting in a complete or a partial inactivation of the LH receptor. Mutations in LHR have been associated with a variable clinical phenotype which extends from complete male pseudohermaphrodism (46XY) to a mild phenotype in female siblings (46XX) compatible with LHR non-responsive function with a hypo-estrogenism (264). The complete male pseudohermaphrodism has external female genitalia, with a Leydig-cell hypoplasia and absence of secondary sex characteristics; biologically low testosterone levels are associated with high LH levels, normal FSH levels, and non-response to LH/hCG stimulation (264).

The milder phenotype in females is characterized by normal female internal gonads and external genitals and normal pubertal maturation, but primary or secondary amenorrhea, hypo-estrogenism, high LH levels, normal FSH levels (265). The ovaries contain follicles at different stages of development, and non-ovulatory follicles may develop into cysts, leading to cystic ovaries (265).

In contrast to the men affected with LHR mutations, those with LH mutations are normally masculinized at birth but do not develop any postnatal sexual differentiation, due to the cessation of placental hCG production after birth – indeed, hCG is not anymore available postnatally to stimulate testosterone production by the Leydig cells if LHβ is inactivated (264). If LHR is mutated, the trophic stimulation of Leydig cells in the fetal testes is not available anymore, and this interferes with the normal untrauterine masculinization that occurs under the stimulus of the fetal testosterone produced by the testes (264).

Inactivating mutations in the FSH receptor (FSHR) gene are reported in a very small number of subjects either as homozygous or compound heterozygous variants. These mutations are associated with hypergonadotropic hypogonadism in a context of a clinical picture that may vary from delayed puberty and amenorrhoea to normal secondary sex characteristics (266,267).

Women harbouring mutations in the FSHR gene have a similar phenotype with those carrying FSH gene mutations – primary or early-onset amenorrhea, lack of follicular maturation and anovulatory infertility. These women do not respond to the administration of exogenous FSH, as opposed to women carrying mutations in the FSH gene (268), but oocyte donation is the best therapeutic option (269). As the ovaries of these women contain a large number of oocytes with incomplete maturation, in vitro maturation could be a potential therapeutic approach when these techniques would be developed.

Five men with inactivating mutations in the FSHR gene have been described – the clinical phenotype is characterized by a variable degree of impaired spermatogenesis, but there is no azoospermia (as it is seen in men with FSHβ mutations), and two of these patients have even fathered two children each (270). It is important to point out that these men were the brothers of women with FSHR mutations, and it is possible that the phenotype could be modulated by a gene dosage effect in these subjects. The milder forms of these FSHR mutations may respond to high-dose FSH stimulation (271), while men with totally inactivating mutations will not respond (272).

Only a small number of FSHR activating mutations have been reported – thus, the case of a hypophysectomized man with spermatogenesis has been published (273) or of young women with ovarian hyperstimulation syndrome (caused by stimulation of FHSR by increased levels of hCG) during naturally conceived pregnancy (274-277).

Steroidogenic factor-1 (SF-1, FTZF1, NR5A1) belongs to the nuclear receptor superfamily and plays a key role in the transcriptional regulation of steroidogenic enzymes within the gonad and adrenal, of hypothalamic and pituitary hormones involved in reproduction (GnRH, FSHR, LHB, GSUA), but is also essential for the formation of the ventromedial hypothalamic nucleus (278-280). There is a co-regulatory interaction between SF-1 and DAX1 as SF-1 transcriptionally modulates DAX1 and DAX-1 inhibits the molecular function of SF-1 (281-285). Mutations in the SF1 gene cause a complete sex-reversal in 46XY individuals with persistent Müllerian structures (uterus, upper vagina) and primary adrenal failure in the early postnatal life (286-289). Patients with testicular dysgenesis (karyotype 46,XY), but without adrenal insufficiency harbouring heterozygous inactivating mutations in the SF-1 gene (leading to SF1 haploinsufficiency) have also been reported (290-293). These individuals have moderate gonadotropin response to GnRH stimulation and lack of testosterone response to exogenous hCG, compatible with the absence of functional gonadal tissue (294). Probably, a gene dosage effect determines the type of inheritance (autosomal dominant or recessive) and the expression of the affected phenotype (294). Only one prepubertal female subject has been reported with mutation in SF1, with apparently preserved ovarian differentiation, but no data are available regarding the functionality of the gonadotroph axis, and with adrenal insufficiency (295).

DAX1 (Dosage-sensitive sex reversal adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1; NROB1) (chr Xp21.3-p21.2) is expressed in the primordium of the adrenal gland, in the fetal and adult adrenal gland, in the hypothalamus and the pituitary in humans, and in the diencephalons and pituitary gonadotropes in mice embryos, which is consistent with a role in gonadotropin production (296,297). DAX1 expression in the mouse gonad during differentiation, follows with a sexually dimorphic expression as DAX1 expression decreases while it persists in the ovary (298) – this suggests a repressor role for testicular development (299). DAX1 encodes for an orphan nuclear receptor and is a repressor of the transcriptional transactivation of SF1. The C-terminal region is the site required for the coregulatory interaction with SF-1. Nonsense or frame-shift mutations in DAX1 gene, resulting in a truncated protein (or deletions/insertions), cause adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism with Sertoli cell hypoplasia and absence of sperm formation in the seminiferous tubules (300, 301). The degree of truncation of DAX-1 protein determines the phenotype (302,303), although modifier genes may have an important effect (304). Cases with either early or late onset of AHC have been reported (305-309). The majority of these mutations affecting the C-terminal region or more rarely, the amino-terminal region of the DAX-1 protein are detected in patients with AHC (289,310). The degree of hypogonadotropic hypogonadism is variable, from the severe picture of gonadal dysgenesis, absence of puberty and infertility (302,306,311-314) to a milder form such as a female patient with a homozygous mutation and normal ovaries and normal adrenal function (315). Spontaneous onset of puberty has also been reported in subjects carrying DAX1 mutations, although pubertal development is incomplete (300,301,316) as well as partial hypogonadism in adulthood (307,308,317). Pulsatile administration of GnRH has not been successful for induction of puberty and fertility (302,307,313,318,319). Administration of gonadotropins has been performed to stimulate testosterone production and induce spermatogenesis, but the results have been difficult to obtain (302,307). The administration of human chorionic gonadotropin (hCG) results in a normal testosterone concentration in most patients, but if necessarily concomitant administration of FSH may give better response (319).

Loss-of-function mutations in the leptin (LEP) gene, leading to congenital leptin deficiency, result in hyperphagia and obesity of early onset in childhood, hypogonadism and altered T-cell mediated immunity (320-324). This phenotype (low LH and FSH levels, loss of LH pulsatility) can be completely reversed under treatment with recombinant human leptin, in particular induction of multiple synchronous nocturnal pulses of LH and FSH with increase in gonadotropins and oestradiol levels (322,325,326); thus, leptin appears as a “permissive factor”, temporally-dependent, which facilitates the induction and progression through puberty, i.e. the activation of the hypothalamo-pituitary-gonadal axis (322). Nonsense or missense mutations in the leptin receptor (LEPR) gene – homozygotes or compound heterozygote – cause a clinical spectrum that is similar, although less severe, to the one due to leptin deficiency (324). In particular, the hypogonadotropic hypogonadism is related to the absence of puberty, but in some adult patients, an extremely delayed onset of puberty – in the third or fourth decade - can occur with irregular menses in presence of normal estradiol, LH and FSH levels (324,327); a similar observation has been reported for a case with leptin deficiency (328). These data suggest that sufficient levels of oestrogen are produced from conversion by aromatase in the excess of adipose tissue, which allow uterine development and irregular menses to occur, but the secondary sexual characteristics remain incompletely developed; the normal LH and FSH levels in this case reflect a functional hypothalamo-pituitary-gonadal axis, although its activation is delayed (324). In a consanguineous family, the truncated leptin receptor was found to be able to bind leptin and to be present, together with leptin, at elevated concentrations in the circulation (329).

Prohormone convertase 1 (PC1) belongs to teh family of serine endoproteases (proprotein convertases) performs the post-translational processing of prohormones and neuropeptides (O’Rahilly 1995; Jackson RS 1997 and 2003)(330,331). Only a small number of individuals carrying compound heterozygote mutations or homozygote missense mutations in the PC1 gene have been reported (330,331). The clinical picture includes severe childhood obesity, adrenocortical insufficiency, postprandial hypoglycemia, and small-intestinal malabsorption (330-332). There is only one adult female patient reported to have hypogonadotropic hypogonadism (330,331).

The Prophet of Pit-1 (PROP1) gene (chr 5q35.3) encodes a transcription factor involved in the ontogeny of the pituitary gland (334). Missense or frameshift mutations in the PROP1 gene are one of the most frequent causes of congenital combined pituitary hormone deficiency (334-337). The associated endocrine deficiencies affect initially the somatotroph, lactotroph and thyreotroph axes, followed by a later onset of gonadotroph, and occasionally corticotroph deficiency of progressive appearance (336). Delayed puberty or absence of puberty and primary amenorrhoea are thus delayed features; GnRH fails to stimulate gonadotropin response and the testosterone response to hCG is absent (336,338). Induction and progress of puberty can be achieved under oral combined treatment of oestrogens and progestins (339). At a further stage, ovulation induction and pregnancy under therapy have been reported (339). Psychomotor retardation and minor kidney malformations could also be part of the clinical spectrum (336).

The paired-like homeodomain TF, encoded by the LHX3/Lim3 gene (chr 9q34), is involved in pituitary gland development. Rare mutations of the LHX3/Lim3 gene have been found to result in combined anterior pituitary deficits involving the somatotroph-mammotroph, thyrotroph, and gonadotroph, which is associated with short cervical spine and limited neck rotation (340-343). Four patients from two families have been found to have mutations of the LHX3/Lim3 gene with combined pituitary deficiency and evidence of impaired gonadotropin response upon functional testing (340). One young boy has presented with micropenis and cryptorchidism at birth, while three elderly patients have failed to develop puberty by the age of 15 (340).

HESX1/Rpx gene (chr 3p21) encodes the paired-like homeodomain TF, and plays a key role in the development of the pituitary gland primordium (for review 344). Two prepubertal patients with homozygote missense mutation of the HESX1 gene have presented with panhypopituitarism and impaired gonadotroph axis, and septo-optical dysplasia (345).

The transcription factor SOX2, a member of the sex-determining region of the Y-chromosome-related (SRY-related) high-mobility group (HMG) box (SOX) family, is expressed at different stages ofembryonic development and cell differentiation, throughout the central nervous system and plays a major role during embryogenesis (346-349). Heterozygous de novo nonsense or loss-of-function mutations or large deletions in the SOX2 gene have been associated with hypogonadotropic hypogonadism (and lack of pubertal development) in mice and humans (350-352). The hypogonadotropic hypogonadism is diagnosed in the context of an anterior pituitary hypoplasia with hypopituitarism, and either isolated or combined hormone deficiency, with or in the absence of defect of the posterior pituitary can be part of the clinical presentation. The affected individuals also present with different malformations such as bilateral or unilateral anophtalmia or severe microphtalmia (septo-optic dysplasia), hypoplasia of the corpus callosum, hippocampal malformation, hypothalamic hamartoma, sensorineural hearing loss, learning difficulties, esophageal atresia (349-354).

4. Hypothalamic Amenorrhea/hypogonadism

Amenorrhea can be subdivided into primary amenorrhea when menarche is delayed beyond age 16 and secondary amenorrhea defined as the absence of menstruation for 3-6 months in a woman with previously regular menses. Oftentimes, hypothalamic amenorrhea (HA) is associated with strenuous exercise, low body weight and body fat, disordered eating or stress. However, functional hypothalamic amenorrhea (FHA) has been described in normal-weight, non-athletic women.

Hypothalamic amenorrhea is believed to arise from a disruption of the normal pulsatile secretion of GnRH from the hypothalamus. GnRH pulsatile release is under the regulation of a pulse generator located in the arcuate nucleus in the medial central hypothalamus. The pulse generator is sensitive to a number of metabolic factors including stress, caloric deficits and particularly weight loss. Disruption of the normal GnRH secretion in HA is oftentimes first manifested by a luteal phase defect with low peak progesterone levels and with luteinization occurring without ovulation. The LH pulse frequency appears to be increased in athletes with a luteal phase defect. Furthermore, strenuous exercise has also been associated with a prolonged follicular phase and an abnormal midcycle LH peak resulting in low estrogen levels and intermittent suppression of menstrual cycles.

Menstrual irregularity is more common in female athletes than in non-athletic women. While the prevalence of menstrual irregularity in the population is estimated at about 5% (355), the prevalence in female athletes is up to 79% (356). Furthermore, up to 25% of female athletes experience exercise-induced amenorrhea.

Amenorrhea is more prevalent in women participating in exercise activities that result in a low body weight such as ballet, gymnastics and long-distance running. Nonetheless, menstrual irregularities are still fairly common in other sports such as swimming. Body composition is a strong predictor of reproductive health. Women with an increased lean-fat ratio appear to be particularly susceptible to menstrual irregularities. Accomplished runners and ballet dancers have a 15% body fat that is in contrast to the relatively high percentage of 20% in swimmers (357). Frisch and McArthur have proposed that menstrual function depends on critical levels of body weight with a 22% body fat being particularly important for normal menses (358). However, it has been observed that athletic amenorrhea can occur despite stable weight and that women who stop exercising but maintain weight can resume normal menstrual cycles (359). It is therefore likely that the metabolic abnormalities resulting from altered body composition contribute to the abnormalities of the GnRH pulse generator. Emerging data suggests a potential link between body composition and the reproductive axis via release of an adipocyte-derived protein hormone called leptin (360-362). Leptin release is pulsatile and correlates with LH and estradiol levels, but female athletes have low leptin levels. Leptin may modulate GnRH release, but this direct action remains controversial as leptin receptors have not been clearly documented on GnRH-secreting neurons. Recent studies have shown that replacement with recombinant leptin treatment, r-metHuLeptin, restored GnRH pulsatility and neuroendocrine and reproductive (ovulation) function (363). None-the-less, in addition to these influences of body mass, it is also recognized that there are other contributing factors such as psychologic disturbances and nutritional deficits.

It is believed that an energy deficit resulting from restrained caloric intake and specifically low number of fat calories leads to a negative energy balance in athletes, anorectics and women with FHA. This energy imbalance leads to a reduced metabolic rate and metabolic adaptations including reduced triiodothyronine (T3)(364,365), leptin (365,366) and insulin-like growth factor (IGF-I) levels (365) and increased mean 24-hour cortisol concentration (367). In addition, the cortisol and corticotropin (ACTH) response to corticotropin releasing hormone (CRH) is blunted in patients with FHA suggesting that the increases in 24-h cortisol concentration results from increases in CRH production despite intact cortisol feedback mechanisms. CRH can inhibit the GnRH-gonadotropin axis leading to appropriate suppression of the reproductive system during states of starvation.

Studies of women with FHA compared to BMI matched controls revealed a lower fat body mass (365,368) and 50% less fat and twice as much fiber consumption when compared to normal controls (368). Furthermore, decreased LH pulse frequency with or without changes in pulse amplitude resulted in lower 24-hour LH concentrations and an increased FSH/LH ratio (365-368). Such changes in LH secretion were completely reversed in exercising women when the caloric intake was increased to meet the energy demands despite continued exercise thus suggesting, that the nutrient restriction and not the increased physical activity per se results in the observed gonadotropin abnormalities (365). In anorexia nervosa, circulating leptin levels are significantly lower compared to constitutionally thin individuals. Treatment of these women with recombinant leptin treatment, r-metHuLeptin, reverses the reproductive and hormonal abnormalities (363).

Psychogenic stress is believed to result in abnormal GnRH secretion and has been postulated as the cause of FHA. Studies using eating disorder and depression inventories have identified higher scores in women with HA however these are both in the subclinical range (368). However, as mentioned above, there also appear to be confounding nutritional derangement in patients with FHA suggesting that the true etiology of this disorder is likely to be multifactorial.

5. Syndromes associated with congenital hypogonadotropic hypogonadism

This complex disorder presents with different manifestations stemming from hypothalamic deficiency. These patients usually have hypogonadotropic hypogonadism in association with other gonadal disorders such as cryptorchidism, hypoplastic external genitalia and delayed or incomplete pubertal developmental. These patients have a various degree of growth hormone (GH) deficiency. Other findings include infantile hypotonia, obesity, glucose intolerance (up to 41%) or diabetes (17%-21%) with insulin resistance (while the normal weight or moderately obese PWS have lower insulin levels and normal glycemia suggesting increased insulin sensitivity), mental retardation, behavioral disorders and short stature. In addition, aberrant control of thermal function and hypersomnolence have also been described. Prader-Willi Syndrome is caused by abnormalities of the imprinted region of the maternal chromosome 15q (chr 15 11q – 13q) (369-370). These abnormalities may result from absent active paternal genes in this region, maternal disomy, or other abnormalities in the imprinting process.

Bardet-Biedl syndrome, also called Laurence-Moon-Bardet-Biedl syndrome, presents as a constellation of hypogonadism (genital hypoplasia/malformation), polydactylie, obesity of very early onset (majority during the first year of life), mental retardation, retinal degeneration, renal cystic disease, but iris coloboma and anosmia have also been described. Males are sterile due to a primary hypogonadism, but female subjects may have hypogonadotroph hypogonadism or elevated LH levels similar to those observed in micropolycystic ovary syndrome (371). This syndrome is inherited in a recessive mode. Genetic studies have identified twelve genes (BBS 1-12) to be responsible for the disease, without much sequence similarities/homology between them or with other proteins. Only a few of the BBS genes encode for known proteins. Three of the BBS genes (BBS6, BBS10, BBS12) which harbour about 30% of all the reported mutations, present strong homology with the type II group of chaperones (372-374). BBS3/ARL6 gene encodes a protein, member of the Ras superfamily of small-GTP binding proteins, while BBS11/TRIM32 encodes for a E3 ubiquitin ligase (375-377). It has been shown that all BBS proteins are located to the cilium/basal body/centrosome complex and that a dysfunction of the primary cilia and the intraflagellar transport are involved in the Bardet-Biedl syndrome pathogenesis (376,378-382).

In this syndrome of autosomal recessive inheritance, classically observed features include hypogonadism, short stature, inconstant preaxial polydactyly, and iris coloboma; hydrocephalus, facial dysostosis and hypospadias may also be present (371). This syndrome is considered as a differential diagnosis of Bardet-Biedl syndrome and overlapping with other rare syndromes exists (371).

CHARGE syndrome includes major abnormalities such as coloboma of the eye, heart defects, atresia choanae, retarded growth and/or development, genital hypoplasia and ear anomalies (including deafness) (383,384). Other abnormalities have also been reported: orofacial cleft, facial palsy or asymmetry, dysmorphic facial features, cranial nerve defects causing severe problems with breathing and feeding, tracheo-esophageal fistula. The sensorineural deafness is due to vestibular abnormalities (semicircular canal dysgenesis) (385). Hypogonadism is common, and manifests with genital hypoplasia and delayed puberty, the first being diagnosed very early in male babies with a micropenis and a hypoplastic scrotum (386). Secondary adrenal insufficiency due to deficient ACTH secretion is an early diagnostic event (387). Functional testing can document a hypogonadotropic hypogonadism, which is associated in the majority of cases with anosmia or hyposmia; the MRI reveals abnormal olfactory bulbs (absent or hypoplastic) (385,388,389). Mutations in the CHD7 gene, which encodes for a protein that is a member of the chromodomain helicase DNA-binding gene family, or in the Semaphorin 3E (SEMA3E) gene have been reported in patients with CHARGE syndrome (385,390,391). As patients with CHARGE syndrome have also features of Kallmann syndrome, the authors speculate for an interaction between FGFR1 signalling and CHD7 signalling, both involved in the development/ differentiation of the olfactory bulbs (388). In addition, growth failure associated with GH deficiency, in some cases, and hypothyroidism has also been reported (388,392,393).

This is a rare autosomal recessive disorder caused by mutations in the ALMS1 gene (chr 2p13), which encodes a constitutive/structural protein of the centrosome, suggesting involvement in ciliary function, and therefore may explain the overlapping phenotype with BBS (394,395). This syndrome affects multiple systems – cone-rod photoreceptor degeneration leading to juvenile blindness, sensorineural hearing loss (cochlear neuronal degeneration) (396-398), early-onset obesity, insulin resistance with hyperinsulinemia or type 2 diabetes mellitus, hypertension, male primary hypogonadism with delayed or absent puberty, testicular atrophy with seminiferous tubules fibrosis and rare Leydig cells, and likely infertility (normal to high LH and FSH and low testosterone), while female have hyperandrogenism with hirsutism, precocious puberty, cystic ovaries, amenorrhoea (with normal FSH and LH but in some cases elevated levels of gonadotropins suggestive of primary hypogonadism); there is also GH deficiency leading to short stature, hypothyroidism, dilated cardiomyopathy, pulmonary fibrosis and restrictive lung disease, progressive hepatic and renal failure)(398-401).

This syndrome associates facial dysmorphic features, microcephaly and mental retardation, ophthalmic abnormalities (myopia and/or pigmentary retinopathy) of childhooood onset, slim tapering extremities with truncal obesity of mid-childhood onset, hypotonia, joint laxity delayed puberty or hypogonadism, short stature, neutropenia (402-404). This rare autosomal recessive disorder can present with variable phenotypic spectrum. The genetic alterations interests/are located within the VPS13B (COH1) gene (chr 8q22-23), the majority of the mutations described are non-sense, resulting in a splice variant with truncated protein (which encodes a potential transmembrane protein which is presumed to be involved in vesicle mediated sorting and intracellular transport (404-406).

This rare X-linked syndrome characteristically associates facial features (a course facial appearance, large fleshy ears), severe cognitive impairment, obesity with early-onset gynecomastia, hypogonadism, hyperextensible tapering fingers. Multiple pituitary hormonal deficiencies (GH, TSH, ACTH and gonadotropin) and optic nerve hypoplasia are also described (407,408). The gene responsible for this syndrome, PHF6 (Plant homeodomain finger gene), encodes a protein localised in the cell nucleus and in the nucleolus, being involved in the ribosome biogenesis, and may play a role in cell growth and proliferation (409).

This syndrome is linked with an autosomal recessive inheritance and the genetic defects is outlined by an increased frequency of sister chromatid exchanges (i.e. the homologous segments from each of the two sister chromatids that constitute a chromosome, are exchanged reciprocally). The phenotype consists of hypogonadism, short stature, photosensitivity and in one reported case presence of bilateral mild lens opacities, immunodeficiency and an increased propensity to develop malignancies (410).

Any tumor or cyst of the hypothalamus or pituitary may lead to abnormal GnRH or pituitary gonadotropin secretion secondary to structural impairment of the normal gland. Malignant tumors of the central nervous system are more likely to affect the hypothalamus (e.g. meningiomas) than the pituitary however metastatic disease form the breast, lung or prostate may present with pituitary dysfunction. Furthermore, prolactin elevation from lactotroph tumors or pituitary stalk deviation from mass effect may also result in hypogonadotropic hypogonadism due to prolactin inhibition of gonadotropin secretion. Similarly, infiltrative diseases such as sarcoidosis and Langerhans cell histiocytosis; hemochromatosis-related iron deposition in the gonadotrophs; and tuberculous meningitis may all result in secondary hypogonadism.

Gonadotropin suppression occurs with estrogens, progestins, androgens and anabolic steroids. Anabolic steroids are androgen like compounds that were once used for the treatment of wasting diseases however, their medical use is now very limited. Nonetheless, abuse of anabolic steroids as performance-enhancing drugs is widespread. These subjects may present with infertility secondary to oligospermia, azoospermia or amenorrhea.

Drugs causing hyperprolactinemia may also result in reproductive dysfunction due to altered gonadotropin secretion. Hyperprolactinemia with the use of dopamine receptor antagonists, opiates and antihypertensives such as verapamil, methyldopa and reserpine has been described.

Critical Illness

Studies of hospitalized patients with a number of different diseases including myocardial infarction, burns and trauma have demonstrated decreased testosterone levels with associated suppression of gonadotropin secretion (411-415). The testosterone reduction appears to correlate with the severity of the illness and likelihood of recovery (416). These hormonal abnormalities are reversible and most likely related to altered GnRH secretion in acute stress (415).

Conversely, individuals with chronic illnesses have low testosterone in association with mildly elevated LH and FSH suggesting a degree of primary testicular failure. However, studies using a GnRH agonist or clomiphene citrate have shown an abnormal LH response suggesting that the hypogonadism often observed in chronic illness may be multifactorial (417).

6. Central precocious puberty

In central precocious puberty (CPP) serum gonadotropins and sex steroid levels correspond to the normal postpubertal range, resulting from the activation of the GnRH pulse generator (for review (418). The most common etiology is idiopathic precocious puberty, but any lesion of the CNS (benign or malignant tumors - hypothalamic hamartomas, rarely craniopharyngiomas, astrocytomas, and other tumours, pituitary adenomas- hydrocephalus or other malformations, traumatism, postinfectious encephalitis, etc.) can result in activation of the hypothalamo-pituitary gonadotropic axis. Genetic defects in GPR54 gene or in other genes causing rare hereditary syndromes with malformations, in some in the context of a hypothalamic hamartoma, can result in central precocious puberty. Some brain lesions require surgical treatment. In the majority of the remaining cases, GnRH analogues are efficient in delaying/stopping progression of puberty until the effective pubertal age is reached.

Severe primary hypothyroidism can cause precocious puberty, despite hyperprolactinemia. In some girls, precocious puberty with elevated levels of gonadotropins has been documented. Alternatively, it has been postulated that the markedly elevated TSH levels seen in chronic primary hypothyroidism may have weak agonistic actions at the gonadotropin receptor. Thyroid hormone replacement thereby reduces the circulating TSH (and hyperprolactinemia) and should arrest the drive for premature sexual maturation.

Several activating mutations of the LHR gene have been identified in boys with gonadotropin-independent precocious puberty, while girls have a normal clinical phenotype (419-423).

A heterozygous gain-of-function mutation in the GPR54 gene have been detected in a girl with precocious puberty, which manifested with thelarche in the neonatal period followed by progressive sexual development, accelerated growth and skeletal maturation (424). At initial evaluation, a GnRH test induced borderline pubertal LH levels (424). Treatment with a depot formulation of GnRH agonist resulted in suppression of the pituitary-gonadotroph axis, which confirms the central activation of this axis in this patient (424). A polymorphism in the promoter of the GPR54 gene has also been suggested to be associated with precocious puberty in Chinese girls but the data need to be further confirmed (425). It is believed that the increased expression of hypothalamic kisspeptin at puberty contributes to the maturation of the reproductive system (426,427).

Pallister-Hall syndrome is inherited in an autosomal dominant manner, and is characterised by the presence of postaxial insertional polydactyly, hypothalamic hamartoma, bifid epiglottis, pituitary dysplasia/dysfunction and precocious puberty, internal organs anomalies (428-430). This syndrome is caused by mutations in the GLI3 gene, a transcription factor involved in Sonic hedgehog (Shh) signalling during development; these mutations are frameshift/nonsense or altering a splicing site, located within the second third of the GLI3 gene (431,432).

Oral-facial-digital syndrome is an autosomal recessive disorder, where in addition to the oro-facial anomalies, developmental anomalies of the posterior fossa such as the cerebellar dysgenesis, and polysyndactyly are common (433). A hypothalamic hamartoma can be an associated finding and, in that setting, may be accompanied by precocious puberty (433).

Precocious puberty of central origin, associated with growth hormone deficiency has been reported in only a few patients with Prader-Willi syndrome (434-440).

Neurofibromatosis type 1 can in very rare cases be associated with hypothalamic hamartoma and central precocious puberty (441).

Tuberous sclerosis, a dominantly inherited condition is characterized by a tendency to cause severe epilepsy, learning difficulties and behaviour disorders, to form benign hamartomas (including the hypothalamus) and cardiac rhabdomyomas, skin lesions, polycystic kidney disease and angiomyolipomas, sub-ependymal and retinal astrocytomas (442,443). The disease can cause central precocious puberty (444).