Review of human genetic and clinical studies directly relevant to GnRH signalling.

Neurokinins are members of the tachykinin family of ancient signalling peptides found both in vertebrates and invertebrates. 64 In humans, the main tachykinins are substance P (SP) and neurokinin A (NKA), both encoded by the same gene, TAC1 , and neurokinin B, encoded by TAC3 . 65 These neurokinins are broadly expressed throughout the CNS. They employ three closely related Gq-coupled receptors to transmit their effects. The genes TACR1 , TACR2 , and TACR3 encode for neurokinin receptors 1, 2, and 3 (NK1R, NK2R, and NK3R), respectively. 66 – 68 All tachykinins have varying degrees of action on each of the three receptors. However, there is a significant degree of selectivity for NKB on NK3R in vivo. 69 , 70 Unlike the identification of KISS1R mutations, a role for neurokinin B in the control of reproduction had been postulated for nearly 20 years, although most findings had been negative or conflicting. However, studies from the Rance laboratory demonstrated dramatically increased NKB expression in infundibular nuclei from postmortem postmenopausal female hypothalami. 71 Moreover, estrogen treatment of ovariectomized monkeys was shown to reduce kisspeptin expression. 72 Subsequently, the Goodman laboratory discovered that both kisspeptin and NKB are coexpressed in the ovine arcuate nucleus, 73 suggesting important interactions between these two peptides in the modulation of reproductive function. Similar to the discovery of KISS1R mutations, autozygosity mapping (utilizing genome-wide SNP genotyping) was performed in 10 multiplex families with IHH. In half of these families, homozygous nonsynonymous mutations were identified within TACR3 (4 families) and TAC3 (1 family). 74 The ligand mutation was predicted to change the terminal methionine of the mature NKB decapeptide to threonine, 74 disrupting the canonical tachykinin motif Phe-X-Gly-Leu-Met-NH2, which is universally conserved among tachykinins. 75 This mutation also compromises the post-translational amidation of the C-terminal which is necessary for full peptide activity. 76 Although mutations in TAC3 are rare in patients with normosmic IHH, mutations in TACR3 are relatively common. 20 , 7 , 77 To date, number of mutations in TAC3 ( n = 9) and TACR3 (>40) have been published or listed in various databases. Micropenis and cryptorchidism in male infants with IHH implies that intact fetal gonadotropin secretion is essential for proper testicular size, descent, and penile growth. 21 Particularly, micropenis has been noted at birth in the majority of infants who have TACR3 mutations, suggesting that intact NKB signalling is required for normal fetal gonadotropin secretion. 20 , 74 , 77 , 78 As with patients who harbour mutations in GnRHR , GNRH1 , KISS1R , and KISS1 , the phenotype patients with mutations in TAC3 or TACR3 appears to be restricted to the reproductive system. Initial reports suggested that TAC3 and TACR3 mutations were associated with fully penetrant nIHH and an autosomal recessive pattern of inheritance. However, a large study of 345 probands with normosmic IHH uncovered many heterozygous cases and a significant rate of clinical recovery. 77 Several of these variants were predicted to be truncating mutations which would lead to haploinsufficiency. It is also possible that these patients might carry an as-yet-unidentified mutation on the opposing allele of TACR3 , or a mutation in another IHH gene. In fact, the presence of more than one IHH-associated-mutant gene in a patient/pedigree (oligogenic aetiology) has been proposed to account for 10%–20% of all IHH cases. 79 – 82 With the increasing use of unbiased genetic studies that are facilitated by whole-exome sequencing, it is now appreciated that oligogenic inheritance is more common than previously appreciated in Mendelian disorders. 83 Further studies will be necessary to determine the true prevalence of oligogenicity in IHH. Clinical reversibility, which is evident by the spontaneous initiation of pubertal development (often after a period of exogenous sex steroid exposure), is observed in 10%–20% of unselected IHH patients. 84 , 85 Patients with mutations in several genes, including ANOS1 , GNRHR , PROKR2 , FGFR1 , CHD7 , TAC3 , and TACR3 , have been reported to recover as evidenced by increases in testicular volume, spontaneous menstruation, normalization of sex steroid levels, and fertility in the absence of fertility medications; 77 , 86 , 87 In one family that was studied in detail, three of four sisters with IHH each of whom carried a novel homozygous null mutation in TAC3 (c.61_61delG p.A21LfsX44) demonstrated reversal as evidenced by pregnancy or spontaneous menstrual periods. 88 Unfortunately, their reversal was not sustained and they reverted back to IHH. In fact, IHH reversibility was observed in 10 of 12 IHH patients (83%) carrying TAC3 / TACR3 mutations, suggesting that the role of the NKB pathway in GnRH secretion may be less critical in adult life than during late gestation and the early neonatal period. 77 With this variably high rate of reversibility, some have hypothesized that CDGP may be a mild form of IHH caused by TAC3 / TACR3 mutations. Although a Finnish cohort of CDGP was devoid of mutations, 89 TAC3 / TACR3 variants were found to be enriched in a different CDGP/delayed puberty cohort, suggesting both CDGP and IHH share a common underlying mechanism. 90 In addition, an SNP immediately upstream of TACR3 (rs3733631) was found to be significantly associated with the age at menarche. 91 Complementary clinical studies have provided valuable insight into the role of NKB in the biology of reproduction. First, Young et al. 78 were able to produce pubertal levels of gonadotropins and gonadal sex steroids with repeated administration of GnRH in patients with null TAC3 mutations, indicating that the site of NKB action is upstream of the GnRH neuron. Second, two sisters with homozygous TAC3 mutations showed clinical evidence of reproductive recovery and conceived spontaneously. Moreover, one of the pregnancies proceeded successfully to term. These observations indicate that NKB, although expressed at high levels in the placenta and other peripheral reproductive organs, is not required for placentation nor pregnancy. 77 Initially, activation of the NKB receptor in vivo gave conflicting results across species. Intravenous administration of 100 μg (approximately 30 nmol/kg) NKB was shown to stimulate LH secretion 3-fold in agonadal juvenile male monkeys. 92 However, NKB appeared to have both excitatory and inhibitory effects on gonadotropin secretion in rodents, depending probably on the hormonal milieu. 66 , 93 – 102 NKB had no significant effect on reproductive hormone (LH, FSH, testosterone, or oestradiol) secretion or LH pulsatility in healthy men, healthy reproductive-age women, and post-menopausal volunteers. 103 , 104 Short-term administration of NKB receptor antagonists, on the other hand, reduced LH secretion and or pulsatility in healthy women. Fezolinetant (ESN364), an NK3R antagonist, produced dose-dependent decreases in LH (with no significant effect on FSH) in healthy female volunteers with regular ovulatory menstrual cycles. 105 Another NK3R antagonist, MLE4901 (Pavinetant), reduced basal LH secretion, without changing pulse frequency, and delayed the LH surge by 7 days in healthy women with regular menses. 106 Elinzanetant is a dual NK1,3R antagonist and therefore has the potential to reduce GnRH pulsatility by blocking the endogenous effects of NKB and SP on the reproductive axis. Elinzanetant has recently been shown to dose-dependently lower serum LH, oestradiol, and luteal-phase progesterone in healthy women. At the highest dose tested, Elinzanetant prolonged the cycle length by a median of 7.0 days. 107 Collectively, these data demonstrate the involvement of NKB-NK3R signalling in the physiological regulation of GnRH/LH secretion in women. Unlike KISS1R or GNRHR , loss-of-function mutations in TACR3 present differential effects on gonadotropin levels, such that plasma LH levels are profoundly diminished while FSH levels are not significantly different from those of healthy individuals, 78 suggesting that this phenotype may arise from a lowered GnRH pulse frequency. 108 In female monkeys, the NK3R antagonist ESN364 prolonged the LH interpulse interval but did not change baseline FSH levels, analogous to the phenotype in patients with deleterious mutations in TAC3 or TACR3 . 109 These observations may reflect the fact that the regulation of FSH secretion is multifactorial while LH release is exclusively dependent upon GnRH. NK3R antagonists have been studied as candidate therapeutic agents for clinical disorders pathophysiologically associated with increased LH secretion, most notably, postmenopausal hot flushes and PCOS. The results so far are promising. About 70% of women globally suffer from postmenopausal hot flushes. Current clinical practice proposes estrogen replacement in severe cases, which is associated with a variety of untoward effects. As mentioned above, the hypertrophied neurons in the hypothalamus of postmenopausal women and ovariectomized monkeys overexpress kisspeptin and NKB; this overexpression is reversed by estrogen replacement. 71 , 110 – 112 KNDy neurons project to the TACR3-expressing median preoptic nucleus within the hypothalamus, an important centre for thermosensory heat-defence. 111 , 113 Based on these observations, the Rance laboratory has shown that KNDy neuron ablation in rats results in reduced tail-skin temperature, indicating that KNDy neurons facilitate cutaneous vasodilatation, a major component of a hot flush. Three independent clinical studies have suggested that NK3R antagonists may be candidates to treat postmenopausal hot flashes. In a randomized, double-blind, placebo-controlled, crossover study, peripheral infusion of NKB intravenously to healthy premenopausal women induced typical hot flushes. 114 Then, a phase 2, randomized, double-blind, placebo-controlled, 4-week crossover trial of an oral neurokinin 3 receptor antagonist, MLE4901 (Pavinetant), formerly also known as AZD4901, demonstrated that this agent significantly reduced the number of hot flushes by 45% compared with the placebo. 115 MLE4901 also showed beneficial effects on sleep quality and mood. MLE4901 was well tolerated with a mild to moderate elevation in liver transaminases. Similarly, in another study, treatment with MLE4901 for 7 days reduced menopausal hot flashes by twothirds. 116 A different compound, Fezolinetant (ESN364), when given orally for 12 weeks, significantly reduced total vasomotor symptoms (VMS) score versus placebo by about one-half and decreased mean frequency of moderate/severe VMSs by five episodes per day versus placebo. Remarkably, the severity and frequency of moderate/severe VMSs were reduced from the first day of treatment. Improvements were achieved in all quality-of-life measures. Mild to moderate transient serum ALT elevations were also observed in this study. As in other studies, NK3R antagonist treatment resulted in lower LH levels while not affecting those of FSH, thus reducing LH/ FSH ratio. Fezolinetant is now in phase 3 development for the treatment of vasomotor symptoms in postmenopausal women. 117 Polycystic ovary syndrome affects approximately 10% of reproductive-aged women globally, making it the most common endocrine disorder. PCOS is the leading cause of anovulatory infertility. 118 , 119 The diagnostic criteria for PCOS include clinical or biochemical hyperandrogenism, which is proposed to be pathophysiogically linked to high-frequency pulses of LH, elevated serum LH, and a high LH/FSH ratio. 120 , 121 In premenopausal women, NK3R antagonism decreases the GnRH pulse frequency leading to reduced basal LH secretion, lower LH/FSH ratio, and the modulation of the temporal dynamics of ovarian sex hormone production over the menstrual cycle. 106 The NK3R antagonist MLE4901 was demonstrated to reduce LH pulse frequency, as well as serum LH and testosterone levels, in women with PCOS. 122 These hormonal findings were echoed in a recent study utilising Fezolinetant; however, there was no improvement in menstrual cycle regularity or PCOSQ scores. The investigators argued that a 12-week duration of treatment in this trial may be inadequate to change these parameters as positive clinical outcomes in PCOS clinical trials are typically detected after 6–9 months of treatment. 117 , 123 Taken together, neurokinin receptor antagonism may negate differential hypersecretion of LH (i.e., excessive secretion of LH in comparison to FSH), and by extension, prevent excessive ovarian androgen production, a cardinal feature of PCOS. In summary, the NKB signalling is an integral part of the GnRH pulse generation. Various NK3R antagonists have been shown to selectively reduce the pituitary LH secretion while being neutral on FSH. This differential effect has been translated into the pharmacological agents to treat the two most common reproductive health problems of the women globally: menopausal hot flushes and PCOS. The results from these clinical studies have been promising.

GnRH is the key molecule in controlling gonadal function in humans, as in other mammalian species. Reproductive disorders stemming from abnormal GnRH neuron action come to clinical attention primarily as either early or delayed puberty. GnRH function is influenced by a multitude of known and still unknown environmental and genetic factors. To date, more than 50 genes have been proposed to modify GnRH neuron function either directly or indirectly. 1 In our understanding, the concept “puberty genes” exclusively refers to those directly pertaining to the regulation of GnRH secretory function. Currently, six genes encoding three ligand-receptor pairs ( GNRH1 / GNRHR , KISS1 / KISS1R , and TAC3 / TACR3 ) have been proven to directly affect GnRH secretion and action. Knowledge regarding these genes has been acquired mainly through performing molecular genetic studies in individuals exhibiting deviation from the expected timing of pubertal development. When these phenotypes are observed more than once in a family (multiplex pedigree), the condition is more likely to be inherited, and by extension, genetic in origin. The contributions from human genetics to our current understanding of GnRH neuron function have been enormous. The identification of kisspeptin and neurokinin B ligandreceptor gene pair mutations in human families with absent puberty have paved the way to the greater understanding of the central regulation of the hypothalamic-pituitary-gonadal (HPG) cascade. This reverse translational pathway of discovery, enhanced by the availability of whole genome sequencing, promises to continue to deliver even more insights into the central control of reproduction. In this article, we aim to review the literature on the genetic and clinical aspects of GnRH and its receptor, as well as the two ligandreceptor sets directly pertinent to the function of GnRH hormone signalling, kisspeptin/kisspeptin receptor and NKB/NK3R.