{"paper_id":"685aa552-2c6c-4673-8e26-7d84d9e7ae10","body_text":"Successful reproduction requires sufficient energy and thus there is\ndynamic communication between homeostatic signals and hypothalamic GnRH\nfunction. The hypothalamic-pituitary-gonadal (HPG) axis is activated at puberty\nafter a period of dormancy during childhood, marking the acquisition of\nreproductive capacity. At the apex of the HPG axis, kisspeptin neurons regulate\npulsatile secretion of gonadotropin-releasing hormone (GnRH) from GnRH neurons\ninto the hypophyseal portal circulation to stimulate pituitary gonadotrophs to\nrelease gonadotropins: i.e., luteinizing hormone (LH) and follicle stimulating\nhormone (FSH). In males, LH stimulates Leydig cells to produce testosterone,\nwhereas FSH acts on Sertoli cells to support spermatogenesis. In females, the\ncomplex interplay between gonadotropins and sex-steroids are characteristic\nfeatures of the ovarian cycle, encompassing folliculogenesis, ovulation, and\nluteal progesterone production.\nPioneering studies from Ernst Knobil were instrumental in establishing\nthat pulsatile secretion of the decapeptide GnRH is requisite for the\nhypophysiotropic stimulation of pituitary gonadotrophs ( 1 ). In ovariectomized rhesus monkeys who were rendered GnRH\ndeficient by lesioning the hypothalamic arcuate nucleus, chronic constant\ninfusion of GnRH led to decline of serum gonadotropins, despite a brief initial\nflare of gonadotropins. By contrast, intermittent pulsatile administration of\nGnRH could stimulate gonadotropin secretion long-term. This concept was\nsubsequently translated to women with GnRH deficient states such as functional\nhypothalamic amenorrhea (FHA) and hyperprolactinemia, whereby chronic\nintermittent administration of GnRH was able to normalize serum gonadotropin\nlevels, restore menstrual cyclicity, and ovulation ( 2 , 3 ). The precise\nsynchrony between GnRH and LH pulses, and the lack of LH pulses not preceded by\nGnRH pulses, confirmed the pulsatile nature of GnRH and in turn LH secretion\n( 4 , 5 ). For many years, GnRH was believed to reside at the apex of the\nHPG axis, but GnRH neurons lack receptors for peripheral signals to modulate the\nHPG axis including estradiol (E2) or leptin receptors. Thus, the presence of the\n‘GnRH pulse generator’, an upstream neuronal population that regulates GnRH\nneuronal function and could integrate peripheral metabolic and sex-steroid\nsignals was posited.\nIn 2003, the seminal findings from two reports indicated that\ninactivating variants of the kisspeptin receptor gene ( KISS1R )\nled to congenital hypogonadotropic hypogonadism (CHH) and a failure of the HPG\naxis ( 6 , 7 ). It was later discovered that these kisspeptin-expressing neurons\nin the infundibular nucleus (arcuate nucleus in animals), co-express neurokinin\nB (NKB), and regulate GnRH neuronal function, thus likely representing the ‘GnRH\npulse generator’. Kisspeptin and NKB are now established to be hypothalamic\nneuropeptides that play a key role in regulating GnRH neuronal secretion.\nIn this review, we discuss the physiological roles of kisspeptin and NKB\nacross the reproductive lifespan including during puberty, menstrual cyclicity,\nreproductive behavior, pregnancy, menopause, and bone homeostasis. We describe\nhow perturbations of these key physiological processes can result in disease\nstates, and we consider how kisspeptin and NKB could be exploited\ntherapeutically, for example during  in vitro  fertilization\n(IVF), and the treatment for menopausal vasomotor symptoms.\n\nThe gene encoding kisspeptin,  KISS1 , and its transcript\nwas first identified in 1996 through subtractive hybridization of micro-cell\nmediated transfer in melanoma and breast cancer cell lines as a\nmetastasis-suppressor gene ( 8 – 10 ). The original complementary DNA (cDNA)\ndesignation,  Ki SS 1 , combined the\nnomenclature for putative  S uppressor\n S equences and paid homage to Hershey, Pennsylvania,\nwhere the gene was first discovered ( 10 )\nand where the famous chocolate ‘ kisses ’ are produced. Using\nfluorescence in situ hybridization (FISH), the  KISS1  gene was\nmapped to the long arm of chromosome 1 (1q32-q41), consisting of four exons with\ntwo 5’ untranslated exons and two partially translated exons ( 11 ).\nThe cognate receptor for kisspeptin was discovered in an entirely\nunrelated context. Discovered in 1999, the cDNA clone encoding a G-protein\ncoupled receptor, GPR54 in rats was noted to bear resemblance (40-50%) to the\ngalanin receptor gene family ( 12 ). This\ngene mapped to chromosome 19q13.3 and had an open reading frame of 1191 base\npair encoding a receptor consisting of 396 amino acids ( 12 ). In 2001, three separate groups working on orphan\nG-protein coupled receptors confirmed the human ortholog of the rat GPR54.\nInitially named hOT7T175 ( 13 ), AXOR12\n( 14 ) or GPR54 ( 15 ) the mRNA consisted of five coding exons, interrupted by\n4 introns, and encodes a 398 amino acid protein in humans (cf. 396 amino acid in\nrats). In congruence with other G-protein coupled receptors, GPR54, now known as\nthe kisspeptin receptor, has 7 hydrophobic putative transmembrane domains.\nKisspeptin peptide was confirmed to be the cognate ligand of the GPR54 receptor\n( 14 ) using peptides from human\nplacental isolates ( 13 , 15 ).\nKISS1  encodes the 145 amino acid prepolypeptide now\nknown as kisspeptin (previously termed metastin), which undergoes proteolytic\ncleavage into N-terminally truncated segments including kisspeptin-54,\nkisspeptin-14, kisspeptin-13 and kisspeptin-10; the suffix denoting their\nrespective amino acid length ( 15 , 16 ). All native kisspeptin isoforms share a\ncommon C-terminal decapeptide equivalent to kisspeptin-10 including an\nArg-Phe-NH 2  motif characteristic of the RF-amide peptide family\n( 13 – 15 ) ( Figure 1 ). In binding- and\nfunctional-assays, kisspeptin-10 resulted in the highest potency in kisspeptin\nreceptor activation, but the un-amidated C-terminal form had only very weak\nactivity, indicating that the amidated RF-amide moiety of the C-terminal portion\nis required for binding and subsequent activation of the kisspeptin receptor\n( 13 – 15 ). The kisspeptin receptor demonstrated selective activation by\nkisspeptin peptides, as other RF-amide peptides including neuropeptide FF and\nAF, RF-amide-like peptide-1 and 3 and prolactin-releasing peptide failed to\nactivate the kisspeptin receptor ( 14 , 16 ).\nThe pivotal role of kisspeptin in reproductive physiology emerged in\n2003 following two landmark papers reporting that inactivating variants in the\nGPR54 gene, now commonly known as the kisspeptin receptor (encoded by\n KISS1R  gene in humans and  Kiss1r  in\nnon-humans ( 17 )), caused congenital\nhypogonadotropic hypogonadism (CHH) in human ( 6 , 7 ). Moreover, phenocopies\nwere also achieved in mouse  Kiss1r  knockout models ( 18 ). In 2012, an inactivating variant in\nthe gene encoding kisspeptin ( KISS1  in humans,\n Kiss1  in non-humans ( 17 )) was shown to also result in failure of pubertal initiation\n( 19 ). Similarly, a\n Kiss1  knockout mouse model also demonstrated lack of\npuberty, gonadal failure and infertility, albeit the phenotype was less severe\nthan  Kiss1r  knockout models ( 20 , 21 ). By contrast,\nactivating variants of both  KISS1  ( 22 ) and  KISS1R  ( 23 ) resulted in central precocious puberty (CPP). These\nrare cases and concordant animal models consolidated kisspeptin’s essential role\nas a regulator of the HPG axis.\nAs expected, kisspeptin-10 could not elicit an LH response in\n Kiss1r  knockout models, however in these models, GnRH\nneuronal migration, GnRH synthesis and pituitary responsiveness to GnRH were\npreserved ( 7 , 21 ). Exogenous kisspeptin administration has since been\ndemonstrated to stimulate gonadotropin secretion across multiple mammalian\nspecies through various administration modalities including both central\n(intracerebroventricular) and peripheral routes (intravenous, intraperitoneal\nand subcutaneous) ( 24 – 31 ). Moreover, kisspeptin had a direct\neffect on GnRH neurons in hypothalamic explants  ex vivo  ( 32 ). Mechanistically, kisspeptin’s ability\nto stimulate gonadotropin release is dependent on GnRH secretion, indeed\nadministration of GnRH antagonist blocked its effect on gonadotropin secretion\nin animal models ( 24 – 26 ). Anatomically, kisspeptin\nimmunoreactive fibers are in close apposition with GnRH neurons ( 30 , 33 , 34 ) and ~90% of GnRH\nneurons express  Kiss1r  mRNA in both juvenile and adult mice\n( 24 , 35 ). Functionally, kisspeptin induced c-Fos expression in 86% of\nGnRH neurons ( 25 ), moreover\nelectrophysiological studies demonstrated that kisspeptin directly depolarized\nGnRH neurons in murine brain slices ( 36 ).\nAltogether, these data suggest that kisspeptin acts upstream of GnRH and\nregulates GnRH neuronal function.\nKisspeptin-expressing neurons are largely distributed in two discrete\nhypothalamic nuclei: the arcuate (analogous to the infundibular nuclei in\nprimates) and the anteroventral periventricular nucleus (AVPV) in the rostral\nhypothalamus, the latter extending into the preoptic periventricular nucleus\n(PeN) collectively termed the rostral periventricular area of the third\nventricle (RP3V) ( 37 ). The RP3V neuronal\npopulation has female-dominant kisspeptin expression with a 10-fold sex\ndifference, whilst the arcuate nucleus does not exhibit any discernible sexual\ndimorphism in rodents ( 37 ) ( Figure 2 ).\nArcuate  k isspeptin neurons co-express\n N KB and  dy norphin collectively\nknown as KNDy neurons; NKB stimulates whereas dynorphin inhibits kisspeptin\nrelease. These KNDy neurons (right panel) exhibit episodic activity that induce\npulsatile GnRH, and in turn LH secretion, and are recognized to be the ‘GnRH\npulse generator’ (at least in rodents). These arcuate kisspeptin expressing\nneurons are susceptible to E2 mediated negative feedback. By contrast, RP3V\nkisspeptin neurons (left panel) induce an LH surge through E2 mediated positive\nfeedback to induce ovulation in females.\nE2 receptors are associated with or embedded in the plasma membrane, E2\nreceptors are also located in the cytoplasm or the nucleus of target cells.\nHowever, within the limit of the schematic nature, this graphical depiction may\nnot have fully captured this complex expression pattern.\nAVPV, anteroventral periventricular nucleus; FSH, follicle stimulating\nhormone; GnRH, gonadotropin-releasing hormone; KNDy, kisspeptin, neurokinin B\nand dynorphin; LH, luteinizing hormone; PeN, periventricular nucleus; RP3V,\nrostral periventricular area of the third ventricle. Figure created with\n BioRender.com .\nArcuate  k isspeptin neurons co-express\nneurotransmitters including  N KB and\n dy norphin, and are thus termed ‘KNDy neurons’ ( 38 ). This expression pattern is highly\nconserved across various mammalian species including ewe ( 38 ), mouse ( 39 ), rat\n( 40 ) and goat ( 41 ). In addition to appositions to GnRH neurons,\ninterconnected networks of reciprocal KNDy-KNDy connections are capable of\nmodulating and eliciting synchronized neuronal firing. KNDy neurons do not\nexpress  Kiss1r  but do express the NKB receptor (NK3R) and\n κ -opioid receptor. Whilst\ndynorphin/ κ -opioid receptor agonist abrogated the slow\nexcitatory postsynaptic potential, blockade of κ-opioid was able to revert this\n( 42 ). By contrast, NKB and neurokinin\n3 receptor agonist evoked stimulation of arcuate kisspeptin neurons ( 42 ). The regulation of KNDy neurons\ntherefore occurs in an autocrine/paracrine manner with stimulatory NKB and\ninhibitory dynorphin signaling, whilst kisspeptin acts predominantly on\ndownstream GnRH neurons ( 42 ). Indeed,\nadministration of exogenous kisspeptin in patients with an inactivating variant\nof NKB signaling could restore LH pulse frequency, thus signifying the\nfunctional hierarchy in that NKB signaling functions upstream of kisspeptin\n( 43 ). Interestingly, continuous\ninfusions of kisspeptin can result in generation of pulsatile LH secretion in\nvarious species including in women with FHA ( 43 – 46 ). This could be due to\nkisspeptin amplifying and revealing previously undetectable low volume pulses\nthat had still been generated by the GnRH pulse generator. Additionally, data\nsuggests that GnRH neurons possess inherent pulsatility that could contribute to\nthis curious clinical observation. Moreover, conflicting theories on the GnRH\npulse generator are described further below.\nThe first systematic study in humans utilized hybridization\nhistochemistry and computer-assisted microscopy to quantify and localize\nkisspeptin-expressing neurons in postmortem hypothalamic tissues ( 47 ).  KISS1  gene\ntranscripts were identified predominantly within the hypothalamic infundibular\nnucleus (analogous to arcuate nucleus). KNDy neurons were demonstrated in the\ninfundibular nucleus of post-mortem tissues from women ( 48 ) but the lack of colocalization in young men ( 49 ) suggests that age, sex and species\ndifferences may contribute to this variation. Moreover, the medial hypothalamic\nsections did not demonstrate a population of  KISS1  neurons in\nthe RP3V as described in rodent models, although labeled neurons were noted to\nscatter sparsely within the medial preoptic area ( 47 ). Further immunohistochemical studies from postmortem\nhuman hypothalamic tissue corroborated these earlier findings, demonstrating\nthat highest numbers of kisspeptin-54 immunoreactive cell bodies resided in the\ninfundibular nucleus ( 50 ). Kisspeptin\ncell bodies were also observed in the rostral periventricular zone in female\nhypothalami and were hypothesized to anatomically represent the kisspeptin\nneurons of the RP3V observed in rodents, although the precise function of this\nneuronal population in human remains unclear ( 50 ). Recently, highly specific pre-prokisspeptin antibody-based\nimmunohistochemical and immunofluorescent techniques enabled visualization of\nimmunoreactive cell bodies in rostral hypothalamic sections in humans. These\ncell bodies were devoid of NKB, substance P, and cocaine and\namphetamine-regulated transcript (CART) ( 51 ). Unlike kisspeptin neurons in the rodent RP3V, these kisspeptin\nneurons did not express neurotransmitters such as enkephalins, galanin and\ntyrosine hydroxylase ( 51 ). The\nidentification of kisspeptin neurons in the human rostral hypothalamus and\npositive estrogenic regulation of this neuronal population challenge the\nparadigm that positive estrogen feedback is restricted to the mediobasal\nhypothalamus in primates ( 51 ).\nThe concept of the ‘GnRH pulse generator’ was coined in the 1980s,\npositing that pulsatile LH secretion is controlled by a hypothalamic pulse\ngenerator that regulates GnRH neurons. Selective lesioning of the arcuate\nnucleus resulted in cessation of gonadotropin secretion whilst function of the\nbasal thyroid, adrenocortical, and growth hormone axes were preserved ( 52 ). By comparison, complete\ndeafferentation of the mediobasal hypothalamus (MBH) with sparing of the arcuate\nnucleus did not impact the pulsatile rhythm of gonadotropins in rats ( 53 ) or monkeys ( 54 ), and the positive feedback action of E2 on gonadotropin\nrelease was also preserved. Early electrophysiological studies recorded from the\nvicinity of the arcuate nucleus provided evidence that multiunit electrical\nactivity (MUA) volleys were invariably associated with initiation of LH\nsecretion ( 55 ). These anatomical and\nelectrophysiological studies therefore suggested that the arcuate region was the\nprimary structure mediating the hypothalamic control of gonadotropin secretion\nin the rhesus monkey and suggested that the pulse generator exists in this area,\nthus initially named the ‘arcuate oscillator’.\nWhether the pulse generator was inherent within GnRH neurons or whether\nthis was mediated extrinsically via afferent neurons located in the arcuate\nnucleus was debated at the time. Evidence for the concept that the pulsatility\nwas intrinsic to GnRH neurons stem largely from  in vitro \nimmortalized ( 56 ) and subsequently\nembryonic GnRH cell lines (rhesus monkeys ( 57 ); rats ( 58 ); sheep ( 59 ); mouse ( 60 )). These cell lines demonstrated GnRH pulsatility profile and\ninterpulse frequency reminiscent of those observed in castrated rodents\nsuggesting that synchronization could be mediated cell-to-cell or through a\ndiffusible mediator in a paracrine manner. However, the diffuse localization of\nGnRH perikarya  in vivo  questions the applicability and\ntranslatability of these findings. Furthermore, the ability of isolated median\neminence explants devoid of GnRH cell bodies to also elicit GnRH pulsatility,\nsuggests that synchronization between GnRH cell bodies may not be necessary to\nsynchronize pulsatile GnRH secretion ( 61 ).\nVariation in the frequency of pulsatile GnRH secretion leads to\ndifferential LH and FSH secretion ( 62 , 63 ). In the early\nfollicular phase, low amplitude pulses occur approximately every 1-1.5hrs, with\ntransitions to high amplitude pulses every 3-4hrs during most of the luteal\nphase ( 61 ). This variation is purported\nto be secondary to homeostatic cues including E2. E2 predominantly exerts its\naction via estrogen receptor-alpha (ERα), however, as GnRH neurons lack ERα as\nwell as androgen and progesterone receptors, sex-steroid feedback is therefore\nlikely mediated indirectly via an intermediary neuronal population ( 64 ). Together this evidence suggests that\nthe GnRH pulse generator is likely to be extrinsic to the GnRH neurons.\nIn 2017, optogenetic approaches captured near-perfect correlation\nbetween pulsatile LH secretion (proxy of GnRH secretion) and brief repetitive\nepisodes of elevated calcium within the arcuate neuronal population ( 65 ). Selective activation and inhibition of\nthese arcuate KNDy neurons could stimulate and suppress pulsatile LH secretion\nrespectively, thus providing functional evidence that arcuate KNDy neurons are\nindeed the GnRH pulse generator, at least in rodents ( 65 ). Recently, fiber photometry and  in\nvivo  calcium recordings of KNDy cells demonstrated that\nsynchronized activity of KNDy cells preceded LH pulses ( 66 ). Studies of KNDy cellular activity at a single-cell\nlevel provided the required granularity to reveal that synchronized episodes in\nKNDy cells occur in a predictable temporal order with ‘leader cells’ capable of\ninitiating episodic LH pulses ( 67 ). The\ntight temporal relationship between LH and corresponding synchronized KNDy cell\nactivity in rodent studies supports the hypothesis that KNDy neurons are\nimportant components of the GnRH pulse generator.\nGnRH neurons have a bipolar morphology consisting of the soma and\nproximal dendrites with few dendritic processes possessing blended dendritic-\nand axonal-like properties termed ‘dendrons’ ( 68 ). Morphologically, these dendrons are interconnected, receive\nshared inputs from afferent neurons, with KNDy neurons forming appositions at\nthe distal dendrons to enable synchronized GnRH secretion into the portal\nvasculature ( 69 ). Surprisingly, using\nexpansion microscopy it was shown that unlike the classical synapses observed\nbetween KNDy neurons, GnRH somata and proximal dendrites; KNDy neurons make\nnon-synaptic appositions with GnRH neuronal dendrons and activate them via\nshort-distance volume transmission ( 69 ).\nThrough selective inhibition of proximal and distal GnRH neuronal dendritic\ncompartments, recent chemogenetic studies demonstrated that the distal dendritic\nzones are important during both pulsatile secretion and generation of GnRH/LH\nsurge, whilst the soma-proximal dendritic compartment appears to be critical for\nthe generation of the GnRH/LH surge ( 70 ).\nIn addition to the hypothalamic kisspeptin neuronal populations,\n Kiss1/KISS1  mRNA was also detected in several areas of the\ncentral nervous system (CNS) (including the pituitary gland, basal ganglia,\namygdala, substantia nigra and hippocampus), the placenta, pancreas, and bone.\n KISS1R  mRNA is expressed in similar regions as\n KISS1  mRNA, abundantly expressed in the placenta, pituitary\ngland, spinal cord, and pancreas with lower expression in extrahypothalamic\nbrain regions and various tissues such as the stomach and small intestine ( 14 , 15 ).\nWhilst the hypothalamic action of kisspeptin is well recognized, the\nexpression of kisspeptin receptor and its cognate ligand in the pituitary\nsuggests a potential direct action of kisspeptin at the level of the pituitary\ngland. Specifically, the expression of  Kiss1  and\n Kiss1r  in the pituitary is differentially regulated by the\nsex-steroid milieu;  Kiss1  expression decreased following\novariectomy but pre-treatment with E2 was able to prevent this, whilst\n Kiss1r  expression increased following ovariectomy, but this\neffect was also negated with E2 treatment ( 71 ). In rhesus monkeys, kisspeptin-positive cells were observed in\nthe intermediate and anterior lobe of the pituitary gland ( 72 ) and push-pull perfusate samples from the median\neminence of pubertal monkeys indicate significant kisspeptin increments in\nassociation with GnRH ( 73 ). Similarly,\nthe presence of kisspeptin in the ovine hypophysial portal blood also supported\na potential role of kisspeptin on the anterior pituitary gland ( 74 ).\nDespite the anatomical and functional evidence for possible direct\naction of kisspeptin at the level of the pituitary, the importance of the\nanterior pituitary in mediating kisspeptin action remains contestable. For\nexample, even though low levels of kisspeptin are detected in the hypophysial\nportal circulation, no temporal rise in kisspeptin levels were seen with the\nE2-induced GnRH/LH surge in ewes ( 74 ).\nSurgical disconnection of the hypothalamic-pituitary unit abolished the LH rise\nfollowing kisspeptin stimulation ( 74 )\nsimilar to that observed following pre-treatment with GnRH antagonists ( 24 , 35 ). Most recently selective  Kiss1r  knockout in the\npituitary gonadotrophs using a PKiRKO mouse model which achieved an 88% and 64%\nreduction in  Kiss1r  mRNA in the pituitary of male and female\nmice, respectively, have been used to delineate the contribution of the\npituitary kisspeptin signaling ( 75 ).\nPhenotypically there were no differences in pubertal timing, gonadal weight and\nbasal gonadotropin levels observed in the PKiRKO models, which although do not\nnegate the potential direct pituitary actions of kisspeptin, suggests that\nkisspeptin signaling at the level of the pituitary does not play a major role in\nthe control of the HPG axis( 75 ).\nFinally, reproductive tissues including the ovaries express\nkisspeptin/KISS1R and NKB/NK3R locally, and their putative roles will be further\ndescribed in the subsequent section. Altogether, these findings strongly\nsuggested the potential involvement of the kisspeptin system in the control of\ndiverse physiological systems ( 76 ), which\nare explored further in the relevant subsequent sections below.\n\nPuberty is characterized by sexual maturation and acquisition of\nreproductive capacity following activation of the HPG axis from its quiescent\nprepubertal state ( 77 ). In most mammals,\nduring late prenatal or early postnatal life, a period of transient activation of\nthe HPG axis known as ‘ mini puberty ’ believed to be important for\npriming reproductive organs is followed by period of relative quiescence until the\nonset of puberty ( 77 , 78 ). The resurgence of pulsatile GnRH release is recognized as\nthe key neuroendocrine initiator central to the onset of puberty ( 79 ).\nThe re-awakening of the gonadotropic axis and attainment of reproductive\ncapacity during puberty involves the complex interplay of enhanced excitatory,\nlowered inhibitory signals and permissive signals that integrate genetic,\nenvironmental, and metabolic factors on GnRH neurons ( Figure 3 ). In recent years, through studies in patients with disordered\npuberty i.e., precocious or delayed/absent puberty, key neuroendocrine players that\ncontribute to regulating hypothalamic function have enhanced our understanding of\nthe physiological regulation of puberty and the HPG axis.\nFSH, follicle stimulating hormone; GnRH, gonadotropin-releasing hormone; LH,\nluteinizing hormone; MKRN3, makorin RING finger protein 3; NKB, neurokinin B. Figure\ncreated with  BioRender.com .\nData from electrophysiological studies mark kisspeptin as one of the\nmost potent excitatory stimuli to GnRH neurons yet discovered ( 80 ). The proposed involvement of kisspeptin\nin the timing of puberty onset was based on loss-of-function variants in\nkisspeptin signaling leading to delayed or absent puberty, whilst\ngain-of-function variants led to precocious puberty ( 6 , 7 , 19 , 22 , 23 ).\nExpression of hypothalamic  Kiss1  mRNA/kisspeptin\ncontent in rodents and rhesus monkeys increases throughout the pubertal\ntransition ( 28 , 81 ). Intriguingly, whilst the number of\n Kiss1  expressing cells increases by 7-fold in the RP3V,\n Kiss1r  expression on GnRH neurons appeared unaltered across\npuberty ( 35 ) suggesting that increased\nRP3V kisspeptin neuronal projection to GnRH neurons may contribute to\nreactivation of GnRH neurons peripubertally. Indeed, appositions between\nkisspeptin fibers and GnRH neuron somata was first apparent at postnatal day 25\nand increased throughout pubertal development in rodents ( 37 ). Thus, kisspeptin appears to be an important instigator\nof pubertal onset and reactivation of the HPG axis.\nCentral kisspeptin administration to prepubertal male and female rats\ncould induce GnRH release, increasing low prepubertal LH levels to adult levels\n( 81 ). However, when compared to adult\nmice with robust LH responses to kisspeptin, juvenile mice required higher doses\nof kisspeptin stimulation to elicit an LH response ( 35 ). Likewise, electrophysiological studies have\ndemonstrated that GnRH neurons acquire sensitivity to the excitatory actions of\nkisspeptin during the pubertal transition, with least GnRH neuron depolarization\nseen in juvenile mice and maximum depolarization seen in adult mice ( 35 ). Functionally, chronic central\nkisspeptin administration to prepubertal female rats resulted in a precocious\npubertal phenotype, evidenced by premature vaginal opening, increased uterine\nweight, LH and E2 levels ( 82 ).\nConversely, central infusion of the kisspeptin antagonist, p234, to peripubertal\nfemale rats led to a marked delay in pubertal timing with significantly delayed\nvaginal opening, reduced ovarian and uterine weight ( 83 ). Similarly, kisspeptin-10 administration resulted in\nsignificant LH and testosterone increase in boys at Tanner stage V and adult men\nbut not in boys at Tanner stages I-IV ( 84 ). Female adult rats demonstrated failure to sustain LH secretion\nafter 48hrs of chronic kisspeptin-10 infusion in keeping with desensitization\n( 85 ). In contrast, at the time of\npuberty, LH concentrations were persistently elevated even at 7 days after\nconstant kisspeptin-10 administration, suggesting persistent stimulation ( 85 ). All in all, increased numbers of\n Kiss1  neurons and their projections to GnRH neurons,\nenhanced excitatory action of kisspeptin and improved kisspeptin receptor\nsignaling efficiency across the pubertal transition provide further evidence for\nkisspeptin’s role in the instigation and maintenance of puberty ( Figure 3 ).\nIn 2011, a series of toxin-based genetic studies achieved over 90%\ntargeted ablation of neurons expressing kisspeptin and kisspeptin receptors\n( 86 ). In the absence of kisspeptin or\nkisspeptin receptor expressing neurons, mice exhibited reduced ovarian size,\nhowever these mice did not exhibit impaired pubertal maturation or fertility\nsuggesting that kisspeptin neurons are needed for full gonadal maturation but\nmay be dispensable with regards to the timing of puberty onset ( 86 ). By contrast, ablation of kisspeptin\nexpressing neurons in adult female mice led to acyclicity and infertility,\nhowever this phenotype was not replicated by conditional ablation of kisspeptin\nreceptors ( 86 ). The authors concluded\nthat the phenotypic discrepancies between congenital and conditional ablation of\nkisspeptin-expressing neurons may be due to developmental compensation; thus,\nablation of kisspeptin-expressing cell in adulthood appears to preclude\nactivation or formation of these alternative reproductive circuits ( 86 ). Furthermore, following ablation, the\nremaining 7% of the GnRH neuronal population (i.e. the non-kisspeptin receptor\nexpressing GnRH population) was sufficient to mediate reproductive function\nsuggesting considerable redundancies in the HPG axis ( 86 ) consistent with prior data ( 87 ). Further exploration of the plasticity and redundancy\nof the kisspeptin system in the reproductive axis demonstrated that a 95%\nreduction in  Kiss1  transcript levels in mice still allowed a\nnormal reproductive phenotype in male mice, whereas females are subfertile,\nsuggesting that females require higher levels of kisspeptin expression for\nreproductive capacity ( 88 ). Recently,\nusing female global  Kiss1  knockout rats with no gonadotropin\nactivity, reintroduction of kisspeptin to only 20% of arcuate KNDy neurons was\nable to rescue folliculogenesis and pulsatile LH release ( 66 ). Therefore, akin to GnRH, the kisspeptin system\ndisplays considerable redundancy with only a limited number of functional KNDy\ncells required to generate sufficient LH pulses to maintain downstream\nactivation of the HPG axis, with this being greater in females than males.\nNKB and dynorphin are neuropeptides co-expressed in arcuate KNDy\nneurons. These neuropeptides act in a concerted auto/paracrine manner to\nregulate the pulsatile GnRH release, and represent the ‘GnRH pulse generator’.\nNKB, encoded by  Tac2  in rodents and  TAC3  in\nhumans, binds preferentially to the neurokinin 3 receptor (NK3R) encoded by\n Tac3r  in rodents and  TACR3  in humans. In\n2009, seminal papers reported that inactivating variants of\n TAC3  and  TACR3  led to hypogonadotropic\nhypogonadism in humans ( 89 , 90 ) and in female mice ( 91 ), identifying the critical role of NKB\nfor normal pubertal timing.\nHypothalamic expression of  Tac2  and\n Tac3r  mRNA increases progressively through postnatal ages\nwith maximal expression seen at time of puberty onset for  Tac2 .\nIn contrast to  Kiss1 , which demonstrates increased expression\nwithin the RP3V postnatally until puberty with no discernible change within the\narcuate nucleus,  Tac3r  expression was significantly increased\nwithin the arcuate ( 92 ). Central and\nperipheral administration of NKB, and the NK3R agonist, senktide, have been\nshown to stimulate GnRH neurons and downstream LH release in several mammalian\nspecies, likely through inducing kisspeptin release ( 93 – 95 ). Senktide\ncould also stimulate LH release in pre-pubertal rodents and the amplitude of LH\nresponse increased with advancing post-pubertal age ( 92 ). Conversely, chronic infusion of an NK3R antagonist\nduring puberty led to mildly decreased LH levels and delayed vaginal opening in\nfemale mice ( 92 ).\nHowever, unlike disruption in kisspeptin signaling, the reproductive\nphenotype of deficient NKB signaling is notably milder. For instance, humans\nharboring inactivating variants in  TAC3/TACR3 , could\nsubsequently proceed to achieve HPG axis activation in adulthood, a phenomenon\ntermed ‘reversal’ ( 96 ). In rodent models,\n Tac2 -/-  knockout male mice exhibit no delay in\nsexual maturation or fertility however, in contrast,\n Tac2 -/-  females had profound delays in sexual\nmaturation and initial abnormalities in estrous cycles which then recovered\nduring adulthood and these females were ultimately fertile ( 91 ).  Tac3r  null mice\ndemonstrated normal markers of sexual maturation, however males had lower\ntesticular weight and females had lower uterine and abnormal estrous cycles with\nprolonged time spent in diestrus, nonetheless, these mice were fertile ( 97 ). Overall, this suggests that NKB\nsignaling is important in early sexual development and timing of puberty.\nThe tempo of puberty is orchestrated by the dynamic interactions between\ngenetic and environmental factors. As both puberty and reproduction are energy\ndemanding physiological processes, endogenous signals reflective of the body’s\nenergy availability are essential for successful reproduction and subsequent\nlactation. Metabolic perturbations, both under- and over-nutrition, can affect\npubertal onset. Leptin, an anorexigenic adipokine secreted by white adipose\ntissue reflective of total body energy stores ( 98 , 99 ), is a permissive\nsignal requisite for healthy GnRH neuronal function. Leptin has an unequivocal\nrole in puberty wherein leptin deficient mice and humans have absent puberty and\nare infertile ( 100 ).\nChanges in leptin levels reflective of energy availability are\npostulated to impact on kisspeptin and NKB expression/function. Through\nmanipulating postnatal feeds, pubertal timing has been shown to be sensitive to\nearly nutritional availability. Overfeeding resulted in higher levels of leptin,\n Kiss1  mRNA expression and earlier vaginal opening whilst\nsubnutrition led to lower levels of leptin and  Kiss1  mRNA\nparalleled by delayed vaginal opening ( 101 ). In prepubertal rats, chronic administration of kisspeptin was\nsufficient to stimulate gonadotropin and estrogen secretion and restore vaginal\nopening despite reductions in  Kiss1  mRNA expression induced by\ncaloric restriction ( 102 ). Continuous\nkisspeptin-10 infusion to underfed pubertal female rodents with marked\nhypo-leptinemia significantly increased serum LH, uterine weight and restored\nvaginal opening in >62% of the animals over a 7-day treatment period despite\nno change in body weight ( 85 ). In\ncontrast, chronic leptin infusion, which led to further body weight reduction,\nfailed to persistently elevate gonadotropins and only rescued vaginal opening in\n25% of animals ( 85 ) consolidating\nkisspeptin’s key role in maturation of the HPG axis.\nLikewise, rodent models subjected to caloric restriction, leading to\n>25% reduction in body weight demonstrated decreased arcuate\n Tac3r  mRNA expression. LH response to senktide in caloric\nrestriction model was enhanced in comparison to age-matched controls fed\n ad libitum , with some caloric restricted animals\ndemonstrating complete vaginal opening ( 91 ). These findings reaffirm the intricate links between metabolic\nstatus and reproductive health mediated through kisspeptin and NKB\nexpression/function, culminating in the downstream effects on GnRH output and\nthe resultant reproductive phenotypes.\nThe effect of leptin on kisspeptin neurons is likely to be mediated\nindirectly through an intermediary neuronal population as selective deletion of\nleptin receptor from kisspeptin neurons ( 103 ) in mice had no effect on fertility. Arcuate agouti-related\npeptide (AgRP) neurons, which co-express neuropeptide Y (NPY) and GABA; and\npro-opiomelanocortin (POMC) neurons which co-express cocaine- and\namphetamine-regulated transcript (CART), are likely candidates given their\nabundant expression of leptin receptors as well as their well-recognized role in\nenergy homeostasis. In the hypoleptinemic state, orexigenic AgRP/NPY neurons are\nhyperactivated ( 104 ), whilst\nanorexigenic POMC neurons are suppressed ( 105 ).\nThe neuropeptides AgRP secreted by AgRP neurons, and alpha-melanocyte\nstimulating hormone (α-MSH), the main secretory product of POMC neurons, are\nrespective antagonist and agonist of the melanocortin 3 receptor (MC3R) and\nmelanocortin 4 receptor (MC4R) ( 106 ).\nThese neuropeptides have been shown  in vitro  to act directly on\nGnRH and kisspeptin neurons ( 106 ).\nAgRP neurons form physical connections with arcuate and RP3V kisspeptin\nneurons ( 107 ). In female mice lacking\nleptin receptors, AgRP neuronal ablation was able to restore reproductive\nfunction ( 108 ). Furthermore, selective\nAgRP neuron-specific rescue of leptin receptors in leptin receptor null mice\ncould partially or fully restore reproductive function despite persisting\nmetabolic effects ( 109 ) thus\ndemonstrating AgRP neuron’s role in integrating the metabolic effects of leptin\nand reproductive phenotype.\nNeuroanatomically, POMC neurons send projections to GnRH cell bodies and\nterminals ( 106 ) and immunoreactive α-MSH\nfibers were identified in close apposition to  Kiss1  cell bodies\nof pubertal rats ( 110 ). Chronic MC3R and\nMC4R blockade in peripubertal female rat induced significant suppression of\narcuate  Kiss1  resulting in delayed puberty. Moreover, in\n Kiss1r  knockout rodent model, activation of MC3R and MC4R\nfailed to stimulate an LH response ( 111 ). These preclinical studies suggest that the α-MSH/melanocortin\nsystem acts upstream and is dependent of kisspeptin to exert its effect on the\nreproductive phenotype. In corroboration with preclinical data, a homozygous\nloss of function variant in MC3R in humans was associated with reduced linear\ngrowth, reduced lean mass, raised body mass index (BMI), and delayed puberty\n( 112 , 113 ).\nEpigenetic regulation governs gene expression through DNA methylation\nand hydroxymethylation, post-translational modifications of histones, and\nnon-coding RNAs such as microRNAs (miRNA). DNA methyltransferases (DNMTs) and\nten-eleven translocation (TET) enzymes mediate DNA methylation and\ndemethylation, respectively ( 114 ). The\nbalance between methylated and demethylated DNA influences chromatin structure,\nwhich in turn determines transcriptional activity through conformational changes\nof chromatin ( 115 ). Histones are key\nstructures in nucleosomes, post-translational modifications of histones through\nprocesses including acetylation, methylation, phosphorylation, ubiquitination\nand sumoylation, can thereby influence transcriptional activity ( 114 ). For example, the role of epigenetic\nsilencers and activators in fine-tuning pubertal timing has been noted through\nactivation or repression of  Kiss1  expression. Prepubertally\n Kiss1  expression is low, in part due to expression of\nPolycomb Group (PcG) epigenetic silencers, whereas during pubertal progression,\n Kiss1  expression is enhanced following recruitment of\nepigenetic activators, such as the Trithorax group to the promoter region of\n Kiss1  ( 116 ). This\nsubsequently affects chromatin structure (i.e., epigenetic silencer induces\ncompaction whereas activator induces open conformation), which affects access of\nthe transcriptional machinery and thereby final  Kiss1  gene\nexpression ( 116 ).\nSpecifically, within the PcG system, hypothalamic mRNA expressions of\n Cbx7  and  Eed , two PcG genes required for\nPcG action, decrease at the initiation of puberty. Arcuate Kiss1 neurons\nco-express both  Cbx7  and  Eed7  gene; indeed,\nthe increase in  Kiss1  expression observed during the pubertal\ntransition was accompanied by eviction of EED from  Kiss1 \npromoter, highlighting a putative mechanism of PcG mediated repression in the\nregulation of pubertal timing ( 117 ).\nConversely, mixed-lineage leukemia 1 (MLL1) and 3 (MLL3), two members of the\nTrithorax group, have been shown to counteract the repressive actions of PcG by\nfacilitating the configuration chromatin changes from repressive to permissive\nby acting at the  Kiss1  and  Tac3  promoter\nregions ( 118 ).\nEpigenetic modifications also provide a potential link between\nnutritional status and pubertal development. Sirtuin 1 (SIRT1), an\nenergy-sensing deacetylase, is abundantly expressed in arcuate/MBH Kiss1 neurons\n( 119 ). Hypothalamic SIRT1 content\ndecreased during the pubertal transition, which coincided with increased\n Kiss1  and  Tac3  expression. Overnutrition\nleads to earlier pubertal development in rodent models and is associated with a\nreduction in  SIRT1  and elevation of both  Kiss1 \nand  Tac3  expression; whilst the converse phenotypic and\nexpression profiles are observed in undernutrition; moreover, transgenic\noverexpression of  SIRT1  led to delayed pubertal maturation\n( 119 ). Mechanistically SIRT1 have\nbeen shown to repress Kiss1 expression through interaction with the PcG complex\nat the  Kiss1  promoter region and acts synergistically with EED\nto induce a repressive chromatin configuration and thereby reduces\n Kiss1  transcription ( 119 ).\nSeveral hypothalamic zinc finger genes (ZNF) are downregulated in the\nMBH during the juvenile-pubertal transition in monkeys. Notably\n GATAD1  and  ZNF573  overexpression delayed\npubertal onset and GATAD1 have been shown to repress  KISS1  and\n TAC3  promoter activity ( 120 ). Another zinc finger protein, initially termed zinc finger\nprotein 127 ( ZNF127 ), now renamed makorin RING finger protein 3\n(MKRN3) is recognized as a puberty-suppressing factor acting upstream of GnRH\nsecretion encoded by the gene located in the Prader-Willi syndrome critical\nregion ( 121 ). MKRN3 belongs to a family\nof E3 ubiquitin ligases involved in the ubiquitination process important in\nregulation of protein degradation ( 121 ).\nWhole-exome sequencing in patients from 15 families with central precocious\npuberty demonstrated loss-of-function variants in  MKRN3  ( 121 ). Moreover, in rodent models,\n Mkrn3  mRNA was expressed in the arcuate nucleus with\nheightened juvenile expression and with striking reduction immediately prior to\npuberty ( 121 ). This pubertally regulated\ntransition was also replicated in female rats and rhesus monkeys irrespective of\nthe sex-steroid milieu ( 122 ).\n MKRN3  is most strongly expressed in the ventromedial and\narcuate nuclei and colocalizes with arcuate kisspeptin-expressing neurons ( 122 ). Mechanistically, MKRN3 selectively\ninhibits  KISS1  and  TAC3  promoter activity,\nthereby inhibiting kisspeptin and NKB expression without affecting the promoter\nregion of  PDYN  (which encodes prodynorphin) ( 122 ). Lastly, MKRN3 represses\n KISS1  and  TAC3  gene promoter activities\nthrough its action as an E3 ubiquitin ligase with reduced activity in pathogenic\nvariants affecting the RING finger domain of the protein ( 122 ) and may also target neuropeptides of Kiss1 neurons to\nubiquitination and degradation pathways ( 123 ). Kisspeptin therefore mediates the final common pathway\ndownstream of MKRN3 to determine pubertal timing.\nFinally, non-coding mRNAs such as miRNAs, largely through translation\nsuppression or RNA degradation, have been shown to be important in the\nregulation of puberty. The RNAase III enzyme, Dicer, is important in the final\nstep of mature miRNA biosynthesis. Selective inactivation of Dicer in GnRH\nneurons led to central hypogonadism and failure of pubertal completion in mice\nthrough involvement of miR-200/429 family and miR-155 ( 124 ). Likewise female mice engineered to lack miRNA\nsynthesis in  Kiss1  neurons failed to complete puberty and\nattain fertility ( 125 ).\nDelayed puberty is defined as the absence of testicular enlargement\n(testicular volume < 4ml, Tanner stage 2) in boys and breast development in\ngirls (Tanner stage 2) at an age that is 2 standard deviations later than the\npopulation mean, traditionally defined as the age of 14 years in boys and 13\nyears in girls ( 126 ). Biochemically,\nmost boys (95%) and girls (75-85%) with delayed puberty will have low levels of\nsex-steroids and inappropriately normal or low levels of gonadotropins\nconsistent with hypogonadotropic hypogonadism ( 127 ). Constitutional delay of growth and puberty (CDGP) is the most\ncommon cause of delayed puberty, which affects 60-80% of boys and 30-55% of\ngirls with biochemical hypogonadotropic hypogonadism ( 127 ). CDGP represents a variant of the normal spectrum of\npubertal timing, and affected adolescents will proceed through puberty\nspontaneously without treatment albeit delayed to their peers ( 126 – 129 ). An important but less common cause of delayed puberty with\nsimilar presentation and biochemical profile to CDGP is CHH. CHH is caused by\ngenetic variants causing impaired hypothalamic GnRH neuronal migration or\nfunction, affecting 10% of younger adolescent boys and 10-20% of girls ( 127 ). Whilst CDGP can usually be managed\nconservatively, patients with CHH benefit from treatment with pubertal induction\nto safeguard future reproductive, sexual, bone, metabolic and psychological\nhealth ( 130 ). Timely and accurate\ndistinction between CDGP and CHH is challenging; due to the overlapping clinical\nfeatures, biochemical profiles and the absence of a ‘gold standard’ diagnostic\ntest ( 131 ).\nPrecocious puberty is defined as sex-hormone production or exposure\noccurring earlier than that which is expected for gender, ethnicity and race;\ntypically with female preponderance ( 132 ). In girls, this is defined as the onset of breast development\nbefore the age of 8 years and in boys as increased testicular volume (>4ml)\nbefore the age of 9 years, accompanied by acceleration of linear growth and bone\nage. Precocious puberty can be classified as GnRH-dependent or GnRH-independent\nprocesses. GnRH-dependent or central precocious puberty (CPP) results from the\npremature activation of the HPG axis by CNS abnormalities, whilst\nGnRH-independent or peripheral precocious puberty results from the unregulated\ngonadal production of sex-steroids ( 132 ).\nThe ability of kisspeptin to directly stimulate hypothalamic GnRH\nrelease offers an opportunity to use it as a test to evaluate hypothalamic\nfunction ( 133 – 136 ). Kisspeptin administration has been utilized as\nan  in vivo  interrogator of the GnRH neuronal function in\nCHH ( 133 – 136 ). As hypothalamic GnRH neuronal\nmigration/secretion/function is impaired in CHH, patients with CHH have\nminimal gonadotropin response to kisspeptin ( 133 – 136 ).\nIntravenous boluses of kisspeptin-10 or kisspeptin-54 result in lower LH\nlevels than in healthy controls in both adult and pediatric cohorts ( 133 – 136 ).\nIn adults, kisspeptin-54 led to maximal LH rise of 12.5 IU/L in\neugonadal men compared to 0.4 IU/L in men with CHH ( 135 ). When compared to a GnRH test, kisspeptin-54 more\naccurately differentiated men with CHH from eugonadal men, with no overlap\nbetween the two cohorts (area under receiver operating characteristic curve\n(AUROC) kisspeptin-54: 1.0, 95% confidence interval (CI) 1.0–1.0; GnRH:\n0.88, 95% CI 0.76–0.99) ( 135 ).\nWithin the CHH cohort, LH rises after kisspeptin-54 were also significantly\nlower in those with anosmia as compared to normosmic patients. Likewise,\npatients with CHH who had identified pathogenic/likely pathogenic variants\nin CHH genes had even lower LH rises after kisspeptin compared to other men\nwith CHH ( 135 ). Furthermore, in a\nsmall cohort of patients with sustained CHH reversal, response to\nkisspeptin-10 was regained suggesting that a kisspeptin test could serve as\na useful test for current hypothalamic function ( 133 ).\nIn the pediatric cohort, the participants’ responses to\nkisspeptin-10 could accurately predict those who later progressed through\npuberty denoted “kisspeptin-responders (LH ≥ 0.8 mIU/mL)” compared to\n“kisspeptin non-responders (LH ≤ 0.4 mIU/mL)” who did not progress through\npuberty. Sensitivity and specificity for the kisspeptin-stimulation test\nwere both 100% (95% CI 74%-100%) which predicted outcomes more accurately\nthan previously described basal/stimulated hormonal markers and genetic\ntesting ( 136 ). Data from these\nstudies demonstrate the promise of a kisspeptin test of hypothalamic\nfunction in the context of delayed puberty and warrant larger studies ( Figure 4 ).\nThe ability of kisspeptin to directly stimulate hypothalamic GnRH\nrelease offers novel insight into the physiology of the hypothalamic GnRH\nneuronal network. Exogenous kisspeptin was utilized in studies as an\n in vivo  interrogator of the GnRH neuronal function in\nCHH and CDGP. Serum kisspeptin levels can distinguish between different\ncauses of CPP.\nCDGP, constitutional delay of growth and puberty; CHH, congenital\nhypogonadotropic hypogonadism; CPP, central precocious puberty; LH,\nluteinizing hormone. Figure created with  BioRender.com .\nSerum kisspeptin levels were first measured as a potential marker of\nprecocious puberty in 2009 ( 137 ). In\ngirls with CPP, serum kisspeptin levels were found to be significantly\nhigher than in age-matched prepubertal controls (14.62 ± 10.2 pmol/l\n vs  8.35 ± 2.98 pmol/l) ( 137 ), however there was some overlap between the two\ngroups. A recent systematic review and meta-analysis included 316 CPP\npatients and 251 controls from 11 studies ( 138 ). Consistent with the first study ( 137 ), kisspeptin levels were found to be higher in the\nCPP compared to controls; the bias-corrected standardized mean difference\n(SMD) was 1.53 (95% CI 0.56-2.51) ( 138 ). Subgroup analyses showed a positive correlation between\nserum kisspeptin and age in the CPP cohort, and an association between serum\nkisspeptin levels and precocious thelarche ( 138 ). However, as noted previously there are overlaps between\nthe two cohorts. Kisspeptin levels could therefore complement current\ndiagnostic tools in precocious puberty ( Figure\n4 ).\n\nFollowing pubertal transition and attainment of reproductive capacity,\nmaintenance of the reactivated HPG axis function is indispensable for fertility and\nsuccessful reproduction. Key physiological processes in the menstrual cycle such as\nfolliculogenesis and ovulation are tightly regulated by intricate negative and\npositive feedback mechanisms in response to sex-steroids (and other signals) with\npatterns of GnRH and subsequent LH release varying during different phases of the\nmenstrual cycle. During follicular development, pulsatile GnRH secretion is\nmodulated by negative feedback from circulating E2 with LH pulses occurring\napproximately every hour, whilst during the preovulatory stage, high E2\nconcentrations exert positive feedback to result in the mid-cycle LH surge and\novulation following which LH pulse frequency progressively lengthens to every 2 to\n4hrs during the luteal phase ( 61 ).\nThe activity of kisspeptin neurons varies throughout the menstrual cycle\nmodulated by E2 levels. Whilst both the arcuate KNDy neurons and RP3V kisspeptin\nneurons express receptors for sex-steroids including estrogen receptor alpha (ERα)\n( 139 ), progesterone ( 140 ) and androgen receptors ( 141 ) these two kisspeptin-expressing neuronal\npopulations are associated with disparate functions. During the majority of the\nfollicular and luteal phases, E2 exerts negative feedback and inhibits arcuate KNDy\nneurons, thus plays a role in the maintenance of pulsatile GnRH secretion. In the\nlate follicular phase, high E2 stimulates RP3V kisspeptin neurons through positive\nfeedback resulting in the GnRH/LH surge responsible for ovulation. This was\nsupported by the differential regulation effects of E2 on RP3V\n Kiss1  gene expression (stimulatory) and arcuate\n Kiss1  expression (inhibitory) ( 142 – 144 ). Correspondingly, as\nthe GnRH/LH surge, which is essential for ovulation, occurs exclusively in females,\nRP3V kisspeptin neurons demonstrate marked sexual dimorphism with increased\n Kiss1  in females compared to males. During the luteal phase,\nprogesterone from corpora lutea acts to slow pulsatile GnRH and LH secretion.\nFollowing exogenous progesterone administration, the ovine  Kiss1 \nmRNA expression is reduced ( 145 ) whilst\n Pdyn  mRNA expression is increased ( 146 ), thus supporting the potential role of KNDy neurons in\nmediating this homeostatic-negative feedback to regulate GnRH and LH pulse\ngeneration. This putative role was recently investigated using mice with conditional\nprogesterone receptor deletion from KNDy neurons which demonstrated that whilst\nfemales have significantly fewer pups, there were no observable effects on estrous\ncyclicity, LH pulse parameters, or the ability of exogenous progesterone to mediate\nLH suppression ( 147 ). Thus, the loss of\nprogesterone receptor from arcuate KNDy neurons (89% knockout) is insufficient to\ndisrupt negative feedback regulation of GnRH pulses in female mice, suggesting that\nthe small number of remaining KNDy neurons may be sufficient or indeed that other\ncells may be implicated to regulate GnRH pulse generation ( 147 ).\nIn premenopausal women, the gonadotropin response to exogenous kisspeptin is\ndependent on the endogenous sex-steroid milieu. During most phases of the cycle, the\nLH response to kisspeptin is modest and less than that in eugonadal men\n(27,44,148–150). In healthy adult men, a 90 minute infusion of kisspeptin-54\n(4pmol/kg·min) led to mean stimulated LH of 10.8 ± 1.5 IU/L  vs  4.2\n± 0.5 IU/L following saline control ( 27 ).\nSimilarly, an infusion of kisspeptin-10 (4μg/kg·hr) led to a robust rise in LH from\na mean of 5.4 ± 0.7 to 20.8 ± 4.9 IU/L ( 44 ).\nIn females, a subcutaneous bolus of kisspeptin-54 increased plasma LH compared with\nsaline in all phases of the cycle, however the greatest LH rise was seen in the\npreovulatory and least in the follicular phase (mean increase in LH over baseline in\nfollicular phase: 0.12 ± 0.17 IU/L; preovulatory phase: 20.64 ± 2.91 IU/L and luteal\nphase: 2.17 ± 0.79 IU/L) ( 29 ). This\ndifferential response is also evident following kisspeptin-10, where during the\nfollicular phase, no rise in serum gonadotropins was observed, whereas in contrast\nduring the preovulatory phase, serum LH and FSH were elevated after an intravenous\nbolus of kisspeptin-10 (10 nmol/kg) with a mean LH area-under-the-curve (AUC)\nincrease of 30.3 ± 7.7 h·IU/L) ( 149 ). The\nincremental LH response to kisspeptin solely in the late follicular/preovulatory\nphase of the menstrual cycle evidences the role for kisspeptin in the preovulatory\npositive estrogenic drive to GnRH/LH secretion ( 29 , 31 , 45 , 149 , 151 , 152 ).\nIn ovariectomized mice, during an LH surge induced with exogenous\ngonadal steroids, ~30% of RP3V kisspeptin neurons expressed c-Fos compared to\nnone in non-surging controls ( 153 ).\nNotably, there was a strong correlation between the percentage of c-Fos-positive\nkisspeptin neurons and the percentage of c-Fos-positive GnRH neurons ( 153 ). The LH surge was absent in\n Kiss1  ( 154 ) and\n Kiss1r  knockout rodent models with low functional\nc-Fos-GnRH activity ( 154 ). Moreover,\ncontinuous intracerebroventricular injection of a kisspeptin receptor antagonist\nprevented the preovulatory LH surge in adult cycling female rats in the\nproestrus phase and in the sheep ( 83 , 155 ). Thus, kisspeptin\nsignaling is essential for GnRH neuronal activation that initiates\novulation.\nTo investigate the role of kisspeptin in the physiological positive\nestrogen feedback that induces the ovulatory LH surge in women ( 156 ), exogenous estrogen was administered\nto achieve sufficient plasma E2 levels to induce ovulation. Treatment with\nexogenous estrogen for 32hrs increased serum E2 and serum LH at 48hrs, which\ncontinued to be elevated at 72hrs. Kisspeptin-10 infusion was able to stimulate\nLH secretion with the degree of LH rise proportional to serum E2 concentrations\nat the start of the infusion ( 156 ).\nCongruous with preclinical data, kisspeptin appears to be a key component of the\npreovulatory LH surge through direct GnRH stimulation.\nThe potential role of locally expressed kisspeptin, NKB and their\ncognate receptors in the ovaries is increasingly recognized. Within the ovaries\nNKB/NK3R and kisspeptin/KISS1R is expressed in the uterus, ovary, oviduct ( 157 ) and within ovarian granulosa cells\n( 158 ). Studies using\n Kiss1r  haplo-insufficient mice model led to premature\novarian insufficiency, progressive loss of developing follicles despite\npreserved gonadotropin levels thus substantiating the importance of direct\nkisspeptin signaling in the ovaries ( 159 ).  In vitro  applications of kisspeptin and NKB in\nfollicular cells induced expression of steroidogenic enzymes ( 160 ), local growth factors to regulate\novarian cells’ viability, proliferation, apoptosis, and hormone release ( 161 ). The NKB/NK3R and kisspeptin/KISS1R\nsystem may therefore be important in the autocrine/paracrine regulation of\nfollicular development, oocyte maturation, ovulation and ovarian steroidogenesis\n( 162 ). In women with PCOS,\nexpression studies demonstrated upregulation of  KISS1  and\n KISS1R  ( 163 ) and\ndownregulation of  NK3R  mRNA ( 160 ) in granulosa cells compared to eumenorrheic controls. Further\nunderstanding of the physiology of NKB/NK3R and kisspeptin/KISS1R systems in the\novary may therefore advance our knowledge of the pathogenesis of ovulatory and\nreproductive disorders.\nOvulatory disorders are common causes of oligo/amenorrhea and\nsubfertility. The International Federation of Gynecology and Obstetrics (FIGO)\nguidelines primarily classifies ovulatory disorders into four groups: Type I:\nHypothalamic; Type II: Pituitary; Type III: Ovarian and Type IV: polycystic\novary syndrome (PCOS) ( 164 ). With the\nexception of Type III: Ovarian, the other causes of ovulatory disorders involve\nthe neuroendocrine control of GnRH function.\nFunctional hypothalamic amenorrhea (FHA) is one of the most common\ncauses of amenorrhea and ovulatory dysfunction being present in 53% and 72% of\nprimary and secondary amenorrhea, respectively ( 165 ). FHA is characterized by low body-weight, excessive exercise,\nand stress, on a background of genetic susceptibility. The resultant reduced\nenergy availability associated with hypoleptinemia, results in reduced GnRH\nneuronal function and a top-down disruption of the HPG axis with detrimental\nimpact on fertility, bone, and cardiovascular health ( 166 ).\nCaloric restriction models are frequently employed to evaluate the\nimpact of low body weight and hypoleptinemia on reproductive phenotypes. Under\nchronic undernutrition, gonadally intact female mice experienced rapid weight\nloss, cessation of estrus cyclicity, a significant decrease in uterine/ovarian\nweight and number of corpora lutea.  Kiss1  mRNA expression in\nthe arcuate and RP3V nucleus were reduced resulting in marked suppression of\npulsatile LH secretion and E2-induced LH surge ( 167 ). As discussed above, the effect of low circulating leptin\nlevels on the reproductive axis in FHA is likely mediated through intermediary\nneurons including AgRP/NPY and POMC/CART neurons that abundantly express LepR as\nwell as insulin receptor and growth hormone secretagogue receptor ( 168 – 171 ), which are cognate receptors for leptin, insulin, and ghrelin,\nrespectively. These metabolic hormones are major endocrine signals of energy\nreserves. In addition to their key roles as gatekeepers of pubertal development\nthese neurons also integrate and finetune the metabolic hormones’ permissive\nsignals for healthy cyclicity and ovulation. For example, chemogenetic\nactivation of AgRP neurons disrupts rodent estrus cyclicity, increases duration\nof diestrus phase and time to conception ( 107 ) as well as decreasing LH secretion post-gonadectomy ( 172 ), mimicking the FHA phenotype.\nPCOS affects 2-13% of reproductive age women and is traditionally\ndiagnosed based on the presence of 2 of the following 3 criteria: (i) menstrual\nirregularity, (ii) hyperandrogenism (clinical or biochemical) and (iii)\npolycystic ovarian morphology ( 173 ).\nPCOS is characterized by elevated LH pulse frequency, androgen excess, which\ncauses impaired suppression of GnRH secretion in response to sex-steroid induced\nnegative feedback ( 174 ). The raised LH\nto FSH ratio (due to increased GnRH pulsatility) gives rise to the reproductive\nphenotypes through stimulation of androgen secretion from thecal cells and\npreovulatory follicle arrest. In PCOS, impaired negative feedback to E2 and\nprogesterone indicates neuroendocrine disruption which impair the ability of\nsteroid hormones to restrain GnRH/LH pulse frequency ( 175 ). Indeed, antagonizing the androgen receptor could\nrestore sensitivity to sex-steroid mediated feedback ( 176 ). The lack of androgen receptors on GnRH neurons\nimplies involvement of afferent intermediary neurons such as\nkisspeptin-expressing neurons. A prenatal androgen-treated mouse model of PCOS\ndemonstrated elevated androgen receptor gene expression in KNDy cells whilst\nsignificant reductions in progesterone receptor and dynorphin gene expression\nwere observed suggesting impaired negative feedback to KNDy cells ( 141 ). Furthermore, synaptic inputs from\nhypothalamic regions sensitive to sex-steroids to KNDy neurons were reduced\n( 141 ).\nAs the pathogenesis underlying both FHA and PCOS involve neuroendocrine\ndysregulation of GnRH pulsatility and ovulation; the kisspeptin/NKB system, with\nits key physiological role as the GnRH pulse generator, has therefore emerged as\na prime neuronal population to integrate internal homeostatic factors to\nfine-tune the final neuronal output ( Figure\n5 ). Considerable research in the clinical application of kisspeptin\nand NKB has therefore been undertaken as detailed below.\nCHH, congenital hypogonadotropic hypogonadism; FHA, functional\nhypothalamic amenorrhea; GnRH, gonadotropin-releasing hormone; LH, luteinizing\nhormone; NKB, neurokinin B; NK3R, neurokinin 3 receptor; PCOS, polycystic ovary\nsyndrome. Figure created with  BioRender.com .\nAssessment of circulating kisspeptin levels may have diagnostic\nutility and be used to differentiate ovulatory disorders. Circulating\nkisspeptin levels are lower in women with FHA, particularly in those with a\nreduced LH, compared to healthy women on days 11-13 of the menstrual cycle\n( 177 , 178 ). In keeping with this, kisspeptin levels also\nnegatively correlated with physical activity ( 179 ). Conversely, a meta-analysis of 12 studies\nreported that circulating kisspeptin levels were higher in women with PCOS\nthan in healthy controls with a pooled AUC of 0.835 and pooled odds ratio\n(OR) of 13.71 when differentiating women with PCOS from BMI-matched controls\n( 180 ). A further case-control\nstudy also demonstrated that PCOS was associated with increased kisspeptin\nlevels ( 181 ). However, at present\nthe challenges of accurately detecting low serum kisspeptin levels using\ncurrent assays limit its potential clinical use.\nThe ability of kisspeptin to directly stimulate GnRH secretion\nenables its potential use as a diagnostic test to interrogate hypothalamic\nGnRH neuronal functioning. Men with CHH demonstrated a reduced LH and FSH\nresponse to an intravenous bolus of kisspeptin-54 compared to eugonadal men\n( 135 ). A subcutaneous bolus of\nkisspeptin-54 stimulates a greater rise in LH in women with FHA compared to\neumenorrheic controls ( 182 ). The\nkisspeptin receptor agonist, MVT-602, demonstrated a similar degree of LH\nincrease in both healthy women and those with PCOS, but an augmented and\nexpedited rise in women with FHA ( 152 ). These differential responses offer the possibility of\nkisspeptin being utilized as a diagnostic test to differentiate between\novulatory disorders pending further studies.\nKisspeptin could have therapeutic utility for functional\nhypogonadal disorders with hypothalamic dysfunction such as FHA given\nits ability to directly stimulate the hypothalamus. FHA is characterized\nby a loss of the physiological pulsatile release pattern of GnRH and LH\nsubsequently leading to reduced folliculogenesis, low E2 production and\nanovulation ( 183 ). The\nrecommended first-line treatment for FHA following lifestyle\nmodification is pulsatile GnRH pump therapy to replace the lack of\npulsatile GnRH release in FHA, but limited availability of these pumps\nprecludes its clinical utility ( 31 , 166 , 184 ). Estrogen supplementation\nprovides symptomatic relief and benefits to bone mineral density (BMD)\nbut does not address fertility issues ( 31 , 184 ).\nFurthermore, the use of clomiphene citrate, a selective estrogen\nreceptor modulator, which reduces the E2 mediated negative feedback to\nincrease endogenous gonadotropin secretion, often has limited effect due\nto the inherently hypoestrogenic state associated with FHA ( 31 , 184 ). Recombinant leptin treatment is also not desirable for\nuse in women with FHA as it can cause weight loss ( 99 , 166 ).\nKisspeptin-54 administration can increase LH pulsatility in women with\nFHA even when administered in a non-pulsatile manner with a greater LH\nrise than in healthy women ( 31 , 182 ). Indeed, the\nresponse to kisspeptin is increased in FHA, which could be by a\ncompensatory increase in the kisspeptin receptor, as observed in\ncaloric-restricted rodent models ( 102 ). However, chronic administration protocols are required\nto facilitate its therapeutic use in FHA.\nChronic administration of twice daily kisspeptin-54 in FHA\nresulted in tachyphylaxis, with the LH response to kisspeptin-54 being\nsignificantly diminished by day 14 of administration ( 182 ). This phenomenon is\nhypothesized to be secondary to kisspeptin receptor desensitization,\nwith response to GnRH maintained after kisspeptin treatment,\ndemonstrating intact pituitary response ( 182 ). Tachyphylaxis with chronic kisspeptin\nadministration has been demonstrated across several species and is more\nlikely to occur with frequent and high dose administration ( 185 ). Tachyphylaxis also appears\nto be influenced by sensitivity to kisspeptin, with the same dosing\nschedule used in women with FHA, not inducing tachyphylaxis during the\nfollicular phase of healthy women when they are less sensitive to\nkisspeptin ( 29 , 149 ). Extending the dosing\ninterval of kisspeptin-54 to twice weekly enabled persistent LH\nstimulation over 8 weeks of treatment, although menstrual cyclicity was\nnot restored ( 186 ). Thus, an\nintermediate dosing regimen between twice daily and twice weekly could\nbe required for this indication.\nContinuous infusion of kisspeptin could provide an alternative\nmethod for administration for chronic stimulation in women with FHA.\nAdministration of kisspeptin-54 to women with FHA via continuous\nintravenous infusion over 8 hours led to a dose-dependent increase in\nmean LH and FSH levels, with an intermediate dose having the greatest\neffect on LH pulsatility ( 187 ).\nThus, continuous administration of kisspeptin at lower doses could\nmaintain LH pulsatility but avoid causing desensitization. However,\nstudies utilising chronic continuous kisspeptin administration to\nidentify the optimal dose to achieve stimulation persistently in this\npopulation are still required.\nKisspeptin receptor agonists also provide a potential\ntherapeutic option in FHA. MVT-602 (previously known as TAK-448) was\ndeveloped through modification of kisspeptin-10 to form a nonapeptide\nthat has increased stability, water solubility, and potency ( 152 ). MVT-602 has a similar\npharmacokinetic profile to kisspeptin-54, with a half-life after\nsubcutaneous injection of 1.5hrs, however in healthy women in the\nfollicular phase it caused a more sustained rise in LH ( 152 ). When administered to women\nwith FHA, MVT-602 resulted in an advanced LH response compared with\nhealthy women with a greater rise in FSH than in the follicular phase of\nhealthy women ( 152 ). MVT-602\nadministration has exhibited tachyphylaxis when given as a high-dose\nsubcutaneous infusion in men ( 188 ). However, the response to a single subcutaneous bolus\nof MVT-602 in women with FHA was sustained for 48hrs and could\nfacilitate infrequent low-dose bolus chronic administration protocols\nfor FHA treatment to mitigate against tachyphylaxis ( 152 ). Further studies are needed\nto evaluate whether low dose intermittent administration of MVT-602 can\nachieve persistent stimulation and therefore be appropriate for a\nchronic administration protocol, especially given its increased potency\n( 152 ).\nKisspeptin could also have a therapeutic role in other causes of\nfunctional hypogonadism due to hypothalamic dysfunction such as\nhyperprolactinemia. Hyperprolactinemia results in hypogonadotropic\nhypogonadism through suppression of kisspeptin afferents to GnRH neurons\nresulting in reduced GnRH/LH pulse frequency and amplitude ( 189 – 191 ). An intravenous infusion of kisspeptin-10 for\n12hrs significantly increased LH, FSH and E2 levels, and increased LH\npulsatility in women with chronic hyperprolactinemia-induced\nhypogonadotropic amenorrhea with cabergoline-resistant\nmicroprolactinomas ( 190 ).\nLikewise, repeated intravenous bolus administration of kisspeptin-10\nalso increased LH levels in women with hyperprolactinemia ( 189 ). This may offer a treatment\noption to restore ovarian function in cases of hyperprolactinemia where\ndopamine agonists are ineffective or not tolerated and pituitary surgery\nis not an option. However, kisspeptin administration would not be\nexpected to affect prolactinoma size, and safety of restoring ovulation\nshould be carefully considered by the multidisciplinary team.\nCurrent treatment strategies for PCOS target specific symptoms\nrather than the underlying pathophysiological process. PCOS is\ncharacterized by an abnormally increased GnRH pulse frequency, which is\nreflected by LH predominant secretion with arrested follicle development\nresulting from the relative FSH deficiency ( 192 ). This increased LH promotes ovarian\nhyperandrogenism, which in turn reduces sex-steroid negative feedback to\nhypothalamic kisspeptin neurons, to further drive increased LH\nsecretion, establishing a vicious cycle. When kisspeptin-10 was\nadministered to women with PCOS, the response was LH predominant,\ndemonstrating a positive association with pre-treatment E2 concentration\nbut with little FSH response ( 193 ). However, following pre-treatment for 1 week with an\noral neurokinin-3 receptor antagonist an FSH response was observed\nfollowing kisspeptin-10 administration with a maintained LH response.\nNeurokinin-3 receptor antagonist administration reduced FSH and LH\nsecretion and LH pulse frequency, which could facilitate the\ndifferential response to kisspeptin-10 observed and help restore\nfolliculogenesis ( 193 ). The\nkisspeptin receptor agonist MVT-602 may also provide a promising\nalternative option. When administered to women with PCOS, MVT-602 causes\nboth an LH and FSH rise to a similar degree as healthy women in the\nfollicular phase, and could be used to trigger ovulation during\novulation induction cycles pending further studies ( 152 ).\nThe efficacy of chronic administration of kisspeptin to\noligo/anovulatory women with PCOS has been investigated. Twice daily\nsubcutaneous kisspeptin-54 for 3 weeks resulted in an overall small rise\nin LH, but no rise in FSH ( 194 ).\nFurthermore, only 2 women with oligo/anovulatory PCOS subsequently\novulated. A similar finding was demonstrated using rodent models of PCOS\nwhere in anovulatory rats with neonatal androgen exposure, a bolus of\nkisspeptin-54 resulted in marked LH and FSH responses and rescued\novulation. However, in post-weaning androgenized rats with persistently\nraised androgen levels, the LH response to kisspeptin-54 was blunted and\nthere was no resulting ovulation ( 194 ). This study evidences the variability of endocrine\nprofile in women with PCOS and how it will likely influence the\nsubsequent response to kisspeptin. Therefore, an individualized approach\nis required when approaching the management of patients with PCOS.\nNKB antagonism has also been an area of interest in PCOS treatment.\nWomen with PCOS with inactivating variants of genes encoding for NKB or its\nreceptor, have low baseline LH and LH pulsatility ( 89 , 90 ). However,\nmouse models with absent NKB signaling can still generate LH pulses ( 195 ). Thus, antagonism of NKB action could\ntarget the pathophysiological process underlying the increased LH secretion and\nhyperandrogenism observed in PCOS through normalization of GnRH pulsatility.\nWomen with PCOS who received the oral neurokinin-3 receptor antagonist MLE4901\nat 80mg/day had a 52% baseline-adjusted reduction in the AUC of LH, a 79%\nreduction in basal LH secretion, and an LH pulse decrease of 3.6 pulses/8hrs\nafter 7 days of treatment compared to placebo ( 196 ). Furthermore, total testosterone and free testosterone levels\nwere reduced by 29% and 19% respectively ( 196 ). A subsequent study administered MLE4901 at 40mg twice daily to\nwomen with PCOS and showed a reduction in LH secretion (from 6.5 to 4.0 IU/L),\nas well as in LH pulse frequency and FSH levels compared to placebo ( 193 ). Another neurokinin-3 receptor\nantagonist, Fezolinetant, has also been trialed in women with PCOS at dose of\n60mg and 180mg for 12 weeks, causing reduced LH, FSH, total testosterone levels\nand LH:FSH ratio in a dose-dependent manner ( 197 ). No changes were observed in E2 and progesterone levels,\nendometrial thickness, follicle development, or menstrual cycle irregularity in\nthis study.\nSubfertility affects 1 in 6 couples and is defined as the inability to\nconceive following 12 months of regular unprotected sexual intercourse ( 198 ).  In vitro \nfertilization (IVF) is one of the main treatment options for infertility, with a\n4-5% annual increase per year in the number of IVF cycles undertaken in the UK\n( 199 ). During IVF,\nsupraphysiological doses of FSH are used to induce multi-follicular growth in\nthe ovaries. Premature ovulation is prevented by administration of competitive\nGnRH antagonist or by chronic administration of GnRH agonist (short\n vs  long protocol respectively). Once the follicles reach a\nsize threshold of 17-18mm, LH receptor agonism is provided to induce oocyte\nmaturation (resumption of the first meiotic division and luteinization of\ngranulosa cells) and ovulation. Human chorionic gonadotropin (hCG) or GnRH\nagonists are usually used in current clinical practice to provide this LH\nreceptor agonism ( 200 ).\nThe physiological LH surge during the natural menstrual cycle has a mean\nduration of 48hrs with three phases; firstly a short ascending phase lasting\n14hrs, secondly a peak plateau phase reaching an average amplitude of 56.5 IU/L\nwith a standard deviation of 23.4 (range 25-114 IU/L) ( 201 ) lasting 14hrs, and lastly a long descending phase of\n20hrs ( 202 ). Kisspeptin has been shown\nto induce an amplitude of LH rise more in keeping with that of the physiological\nmid-cycle LH surge compared to either hCG or GnRH agonists ( 203 – 205 ). The peak LH level following a kisspeptin-54 trigger was 41.4\nIU/L at 4hrs post-administration ( 206 , 207 ). The kisspeptin\nreceptor agonist, MVT-602, has recently been characterized in healthy women and\nin women with reproductive disorders ( 152 ). In the healthy follicular phase, the amplitude of LH rise was\nsimilar to that after kisspeptin-54, however the duration was markedly prolonged\ncompared to kisspeptin-54 (time of peak LH: MVT-602 21-22hrs  vs \nkisspeptin-54 4.7hrs) leading to a more than four-fold increase in the area\nunder the curve of the LH exposure ( 152 ). In a minimal stimulation cycle, the mean increase in LH from\nbaseline was 82.4 IU/L at ~25hrs following administration of 3 μg MVT-602 and\nremained elevated to >15 IU/L for 33hrs ( 208 ). Thus, MVT-602 appears to induce an LH profile that is most\nsimilar to that of the endogenous LH surge. In contrast, GnRH agonist trigger\nresults in a supraphysiological peak LH level of 140.4 IU/L at 4hrs\npost-administration, and hCG levels peak ~24hrs post administration at 121.0\nIU/L ( Figure 6 ) ( 207 ). Therefore, kisspeptin appears to be a promising\nalternative agent providing a more physiological LH profile for induction of\noocyte maturation in IVF protocols.\nKisspeptin/MVT-602 acts at the level of the hypothalamus to stimulate\nkisspeptin receptors on GnRH neurons leading to GnRH release. Kisspeptin induces\na peak LH rise of ~45 IU/L at ~5hrs, returning to pre-trigger levels at\n12-14hrs. MVT-602 induces a peak rise in LH of similar amplitude to that of\nkisspeptin-54, however the duration of the LH rise was markedly prolonged, with\npeak LH occurring at ~21-22hrs. GnRHa acts at the level of the anterior\npituitary gonadotrophs to stimulate endogenous LH and FSH secretion. GnRHa\ninduces a peak LH level of 140.4 IU/L at 4-6hrs after administration.\nhCG and rLH act at the level of the ovary, directly on LH receptors. A\nsubcutaneous bolus of hCG results in a peak hCG level of 121.0 IU/L at 24hrs\nafter administration.\nGnRHa, gonadotropin-releasing hormone agonist; hCG, human chorionic\ngonadotropin; LH, luteinizing hormone; rLH, recombinant luteinizing hormone.\nFigure created with  BioRender.com .\nIndeed, kisspeptin-54 was used as a trigger for oocyte maturation in a\nproof-of-concept study in 2014 ( 203 ). A\nsingle subcutaneous bolus injection of kisspeptin-54 (dose-range between 1.6 and\n12.8 nmol/kg) in 53 women with infertility undergoing a GnRH antagonist\nco-treated IVF cycle ( 203 ). This\nresulted in the successful retrieval of at least one mature oocyte in 51 of 53\nwomen (96.2%), one embryo for implantation in 49 of 53 women (92.5%), and the\nbirth of 12 healthy babies ( 203 ).\nA major complication of established protocols using hCG as the oocyte\nmaturation trigger is the risk of ovarian hyperstimulation syndrome (OHSS),\nwhich can be life-threatening with severe forms occurring in 2-6% of IVF cycles\n( 209 – 211 ). This can be attributed to the prolonged duration of\naction of exogenously administered hCG lasting ~10 days ( 212 ). OHSS occurs due to excessive ovarian stimulation\ncausing release of vascular endothelial growth factor (VEGF) and increased\nvascular permeability, resulting in fluid shifts from the intravascular to the\nthird space compartments ( 209 ). This can\nlead to ascites, pleural effusions, and renal failure ( 209 ). Current strategies employed in IVF practice to\nreduce OHSS risk include the use of GnRH antagonist protocol to prevent\npremature ovulation during ovarian stimulation and the use of a GnRH agonist to\ntrigger final oocyte maturation, however these techniques can have unwanted\neffects on efficacy, such as lengthening the time to pregnancy, or increasing\nthe risk of late pregnancy complications such as pre-eclampsia ( 209 ).\nKisspeptin-54 was investigated as an oocyte maturation trigger in women\nat high risk of OHSS defined as total antral follicle count of >23 or serum\nAMH level ≥40 pmol/L ( 204 ). In 60 women\nat high risk of OHSS, kisspeptin safely induced oocyte maturation with no cases\nof moderate, severe, or critical OHSS ( 204 ). A single subcutaneous bolus of kisspeptin-54 (dose range\n3.2-12.8 nmol/kg) resulted in oocyte maturation in 95% of women and this rate\nincreased in a dose-dependent manner: 53% at 3.2 nmol/kg, 86% at 6.4 nmol/kg and\n9.6 nmol/kg, and 121% at 12.8 nmol/kg ( 204 ). Embryo formation occurred in 90% of women with resulting\nbiochemical pregnancy, clinical pregnancy, and live birth rates per transfer of\n63%, 53%, and 45% respectively ( 204 ). In\na single-center retrospective study, the risk of OHSS was markedly increased\nwith hCG (OR 33.6; CI, 12.6-89.5) or a GnRH agonist (OR 3.6; CI 1.8-7.1) than\nkisspeptin-54 ( 213 ).\nEven though the amplitude of LH exposure induced by kisspeptin-54 (~45\nIU/L) is similar to that of the physiological midcycle LH surge (56.5 IU/L); the\nduration of LH exposure induced by kisspeptin-54 is shorter than the triphasic\nphysiological LH surge. To determine if the duration of LH-exposure impacts on\nIVF outcomes, 62 women at high risk of OHSS were randomized to receive either\none or two doses of kisspeptin-54 to trigger oocyte maturation ( 205 ). This second dose of kisspeptin-54\ninduced further LH secretion at 4hrs after the second injection, providing a\n‘rescue’ response in those who had a lower LH rise following the first\nkisspeptin-54 dose ( 205 ). This prolonged\nLH-exposure improved the number of women achieving at least 60% oocyte yield\n(71%  vs  45%), implantation rates (37%  vs  23%),\nand live birth rates (39%  vs  19%) compared to those receiving a\nsingle dose ( 205 ). Importantly, this\nprolonged LH exposure did not cause an increase in clinically significant OHSS\nin these high-risk women ( 205 ). Besides\nits shorter duration of action, kisspeptin also appears to have direct action on\novarian kisspeptin receptors to suppress VEGF release which may contribute to\nthe reduction in the incidence of OHSS ( 204 ).\nLive birth rate per embryo transfer following all kisspeptin doses\ntested appears to be at least comparable to currently used triggers: 32%\n(51/160) ( 200 ) and up to 45% (23/51)\n( 204 ) in high responders with\ncontemporaneous live birth rate per transfer in women <35 years treated with\nfresh embryo transfer being 32.8% ( 214 ).\nThus, a prospective comparison of the safety and efficacy of kisspeptin against\ncurrent agents is warranted.\nEndometriosis and uterine fibroids are common disorders affecting women\nof reproductive age and are leading causes of pelvic pain, abnormal uterine\nbleeding, and subfertility ( 215 , 216 ). Early age of menarche and short\nmenstrual length are both associated with development of uterine fibroids and\nendometriosis ( 215 , 216 ) consistent with prolonged endometrial and myometrial\nexposure to estrogen being an important factor in the pathogenesis of these\ndisorders ( 215 , 216 ). Lowering E2 levels to between 110 to 184 pmol/L has\nbeen recommended as being effective in reducing the symptoms of uterine fibroids\nand endometriosis ( 217 , 218 ). GnRH modulators are able to suppress\nestrogen levels by shutting down the HPG axis top-down and are currently\nvalidated treatment for both conditions on this basis ( 219 , 220 ). However,\nthese agents typically induce lower E2 and require add back sex-steroid\nreplacement to avoid the associated adverse effects ( 221 , 222 ). NKB\nreceptor antagonists could potentially be dosed to reduce E2 levels into the\nrecommended therapeutic range sufficient to moderate endometriosis and uterine\nfibroid development without the adverse effects of undetectable E2 levels.\nSeveral NKB receptor antagonists have demonstrated the ability to reduce\nLH secretion whilst preserving FSH secretion. The NK3R antagonist MLE4901 (also\nknown as AZD4901, formerly AZD2624) reduced basal LH secretion in healthy women\nwithout affecting the LH pulse frequency during the early-mid follicular phase\nand delayed the LH surge by 7 days, whilst FSH secretion was unaffected ( 223 ). MLE4901 has also been shown to\nreduce E2 secretion, endometrial thickness and folliculogenesis during the\nfollicular phase ( 224 ). Another NK3R\nantagonist, Fezolinetant (ESN364), also led to a dose-dependent reduction in\nserum LH and delayed the LH surge in healthy women, with no significant effect\non FSH ( 225 ). Fezolinetant subsequently\ncaused a dose-dependent delay to the rise in serum E2 levels during the\nfollicular phase, but the serum E2 trough level remained above the 110 pmol/L, a\nthreshold considered significant for inducing menopause-like symptoms ( 225 ). The dual neurokinin 1, 3 receptor\nantagonist Elinzanetant induced a trend towards a dose-dependent reduction in\nserum LH when administered once daily to healthy women for 21 days with no\nsafety concerns or issues with tolerance ( 226 ). Elinzanetant also caused a dose-dependent reduction in serum\nE2 and progesterone levels, with 120mg/day causing a median reduction in serum\nE2 to 141 pmol/L, which is within the recommended range to effectively treat\nuterine disorders ( 226 ). Furthermore,\nthe 120mg Elinzanetant dose increased the menstrual cycle length by a median of\n7 days. This suggests that NKB antagonism may provide a novel therapeutic\napproach, and further study is required to evaluate its use in uterine\ndisorders.\n\nEfficient reproductive strategies are crucial cornerstones for the\ncontinuity of survival of any species. The timing of reproductive activity in many\nspecies results from the integration of internal physiological processes and\nbehavioral cues, aligning precisely with the optimal window for fertilization. The\nevolution of sexual desire, deriving pleasure, and arousal from these experiences\nserves as a compelling force for many species, including humans, urging individuals\nto engage in sexual encounters and, consequently, frequently facilitating\nreproduction. Indeed, this orchestration of reproductive function and sexual\nbehavior appears to be mediated by common hormonal factors, among which estrogen,\ntestosterone and kisspeptin prominently feature with the role of NKB in these\nprocesses yet to be fully explored.\nIn addition to its reproductive roles mediated largely by\nhypothalamic kisspeptin neurons, kisspeptin and its cognate receptor are\nalso expressed in extra-hypothalamic regions, particularly within the\nmammalian limbic and paralimbic system, which consists of areas that are\nimplicated in mood, behavior, sexual desire, and function ( 227 ).\nIn rodents,  Kiss1r  mRNA was detected in the\namygdala, thalamus (caudate nucleus, globus pallidus, putamen), hippocampus,\npara-hippocampal gyrus, medial and superior frontal gyrus, and striatum of\nrats ( 12 , 13 , 15 ).\n Kiss1r  mRNA is similarly expressed in the limbic and\nparalimbic areas of mice, with highest expression in the dentate gyrus of\nthe hippocampus ( 228 ). In both rats\nand mice, there is a well-established kisspeptin neuronal population in the\nposterodorsal medial amygdala (mePD), a neural locus in the limbic brain\ninvolved in the regulation of sexual behaviors including investigation and\nattraction towards opposite sex-conspecifics, as well as emotion, such as\nfear and anxiety. This is indeed a critical area in the modulation of sexual\nbehavior through both sex-steroids and kisspeptin ( 229 ).\nSimilarly in humans, extrahypothalamic expression of\n KISS1R  mRNA and protein has been detected by reverse\ntranscription polymerase chain reaction (rtPCR) in the amygdala, caudate\nnucleus, cingulate gyrus, globus pallidus, hippocampus, medial frontal\ngyrus, nucleus accumbens, para-hippocampal gyrus, putamen, striatum,\nsubstantia nigra, superior frontal gyrus and thalamus ( 14 , 15 ).\nKisspeptin expression is also present throughout the aforementioned areas in\nboth rodents and humans, albeit at lower levels ( 14 , 230 ).\nThe initiating step to rodent reproduction is detection,\nrecognition, and selection of potential reproductive partner. Sexually\nactive male rodents pursue female conspecifics, while females in estrous\nphase attempt to engage with males ( 231 ). This behavior, recognized as ‘sexual mate preference’,\ndepends partly on the recognition and utilization of various olfactory and\nacoustic stimuli to maximize their chances of reproduction and species\npropagation.\nOlfactory mediated sexual behavior relies on the detection of\npheromones from conspecific of the opposite sex ( 232 ). In rodents, pheromones are detected and\nprocessed by a highly specialized neural circuit, within the accessory\nolfactory system, initiating in the vomeronasal organ (VNO) in the nasal\nseptum ( 233 ). Wild-type male mice\nexhibit a significant olfactory preference for female stimuli by spending\nover 70% of their investigatory time with females. However,\n Kiss1r  knockout males displayed no preference for\neither sex, and allocated equal investigatory time to both males and females\nirrespective of sex-steroid replacement/milieu ( 233 ). Notably, these findings persist, even though\n Kiss1r  knockout male mice maintain normosmia, as\nassessed through a ‘hidden cookie test’, hence demonstrating that the defect\nwas not in distal olfaction ( 233 ).\nImportantly, the olfactory partner preference of  Kiss1r \nknockout males remained unaltered by replenishing physiological testosterone\nlevels with treatment, thereby attributing the observations to the absence\nof intact kisspeptin receptor, rather than any sex-steroid confounders\n( 233 ).\nKisspeptin’s regulatory role in olfaction-induced sexual partner\npreference in male mice, appears to be site-specific, with the posterodorsal\nmedial amygdala (MePD) particularly implicated ( 234 ). In male mandarin voles, opposite sex pheromonal\ncues conveyed via the accessory olfactory bulb (AOB) induce c-Fos activation\nin the MePD kisspeptin neurons, thus suggesting a reciprocal synaptic\ninnervation between the two ( 235 ).\nSimilarly, in adult male rats, kisspeptin neuronal fibers in the mitral cell\nlayer of the AOB connect bi-directionally with kisspeptin neuronal fibers in\nthe medial amygdala (MeA), which in turn, make connections with the preoptic\narea of the hypothalamus ( 236 ).Therefore, kisspeptin has a dual role of integrating olfactory\npheromonal cues in sexual behavior centers and the HPG axis.\nChemogenetic stimulation of MePD  Kiss1  neurons in\nmale mice presented with a choice between an estrous female and a male, led\nthem to spend twice as long investigating estrus females compared to\ncontrols, thereby confirming that kisspeptin activation is implicated in the\nenhancement of sexual partner preference ( 234 ). Furthermore, when wild-type male mice are exposed to\nfemale olfactory stimuli, there is a two-fold rise in the number of c-Fos\npositive MePD kisspeptin neurons, accompanied by a concomitant rise in LH\nrelease ( 237 ).\nInterestingly, the total time spent investigating both conspecifics\nwas also significantly greater suggesting a potential increase in\nsociability ( 234 ). In a\ncomprehensive investigation into sexual motivation, three groups of adult\nmale rats were subjected to appetitive behavioral testing, which focused on\nmeasures, such as the frequency of attempts to approach the female and the\nlatency period preceding these attempts, in the presence of an estrous\nfemale. Each group received either intranasal GnRH analogue (buserelin),\nintranasal kisspeptin-10 or intraperitoneal kisspeptin-10 ( 238 ). GnRH analogue administration\nresulted in a three-fold increase in testosterone levels but had no effects\non the number of attempts to approach the estrous female, nor influenced the\nlatency period between the attempts. Conversely, intraperitoneal\nkisspeptin-10 led to modest rise in testosterone levels, while also\nsignificantly increasing the number of attempts and decreasing the latency\ntime. Intranasal kisspeptin-10 did not affect testosterone levels, but akin\nto intraperitoneal kisspeptin-10, led to a significant increase in the\nnumber of attempts and a decrease in latency time. This study highlights the\ntestosterone-independent effects of kisspeptin in enhancing sexual\nmotivation in male rodents ( 238 ).\nWhen female mice are exposed to male odors, such as male urine or\nsoiled bedding, the RP3V kisspeptin neurons are specifically activated\n( 239 ) via the VNO ( 240 ). Conversely RP3V\n Kiss1  activation does not occur when wild type female\nmice are exposed to same-sex (female) pheromones ( 239 ). When ovariectomized female rats are presented\nwith male (but not female) odors, they exhibit increased kisspeptin activity\nin the RP3V, as well as an augmented LH surge ( 241 ).  Kiss1r  knockout female mice,\ndespite intact olfaction, displayed no olfactory preference for either sex,\nirrespective of their sex-steroid milieu ( 233 ). When  Kiss1  expressing RP3V cells are\nablated in ovariectomized female mice on adequate estrogen and progesterone\nreplacement (OVX+E+P), they no longer display male-directed preference\n( 240 ). Notably, male-directed\npreference is restored on administration of subcutaneous kisspeptin-10\n( 240 ). Taken together, these\ndata suggest that RP3V kisspeptin neurons are an essential component of the\nneural circuits downstream of the VNO mediating olfactory-driven mate\npreference in female mice. Indeed, in female mice, only 36% of kisspeptin\nneurons in the RP3V send projections to GnRH neurons, suggesting that a\nsignificant proportion of the kisspeptin neurons are implicated in other\nfunctions ( 240 ). Transgenic female\nmice incapable of GnRH secretion failed to show male-directed preferences. A\nsingle subcutaneous injection of GnRH restored their physiological behavior,\nwhereas a subcutaneous injection of kisspeptin-10 failed to elicit a\nmale-directed preference in these mouse models ( 240 ). Collectively, these data suggest that olfactory\nmate preference in female mice is mediated through RP3V kisspeptin via GnRH\nneurons.\nExposure of ovariectomized goats to a sexually mature male goat\nincreases arcuate kisspeptin neuronal activity which leads to simultaneous\nLH pulse generation ( 242 ). Anestrous\newes introduced to a male potential sexual partner, display increased c-Fos\nactivity within the arcuate kisspeptin neurons with concomitant rise in LH\namplitude and pulse frequency. Interestingly, this effect is abolished when\nthe female ewes are pre-administered a kisspeptin antagonist ( 243 ).\nBeyond olfaction, kisspeptin also integrates auditory pathways\nregulating reproductive behavior in female mice. For instance, males emit\nsong-like ultrasonic vocalizations (USVs) to exhibit sexual intentions and\nattract a receptive female. When female mice were exposed to an audio file\nof repeated male USVs for 20 minutes, there was an increase in arcuate\nkisspeptin neuronal activity compared to control noises. Interestingly,\nexposure to male USVs did not lead to an increase in the RP3V neuronal\nactivity ( 244 ). Putting these\nfindings together, it appears that kisspeptin neurons signaling plays a\ncrucial role in conveying olfactory cues in both female and male\nnon-primates, to stimulate opposite sex-directed partner preference and\nincrease LH responses, ultimately maximizing reproductive success. The brain\nregions involved in non-primate olfactory partner preference include the\nMePd, the RP3V and the ARC. Additionally, in female mice, acoustic male cues\nappear to be related to the arcuate (rather than RP3V) kisspeptin\nneurons.\nFollowing sexual mate selection, rodents engage in distinct\ncopulatory actions. In males, these encompass mounting, thrusting,\nintromission, and ejaculation ( 245 ).\nFemale rodents typically control the initiation and timing of copulatory\ncontacts to ensure synchronization with ovulation and thus optimize\nprobability of successful fertilization ( 246 ). Once partner preference is established, females display\nreceptive behaviors, such as lordosis, essential for intromission ( 247 ).\nThe MeA, a key area in mediating male copulatory behaviors (e.g.,\nerections and intromission), expresses androgen receptors, yet direct\nadministration of androgen in the MeA did not induce spontaneous erections,\nthereby implicating involvement of other pathways ( 248 ). Notably, bilateral radiofrequency ablation of\nthe MePD in reproductive-age male rats abolished non-contact erection\n(ex-copula) observed when males are placed in proximity to inaccessible\nestrous females. However, normal male copulatory behavior remained intact\nwhen placed with receptive females, albeit with significantly longer\nintervals between intromissions ( 249 ). Consistently, intracerebral microinjections of kisspeptin\ndirectly into the MeA stimulate ex-copula erections in a dose-dependent\nmanner, an effect blocked by pre-treatment with a kisspeptin receptor\nantagonist ( 250 ). These findings\nsuggest that kisspeptin signaling within the MeA mediates ex-copula, while\nother factors likely contribute to behaviors observed upon opposite-sex\nsexual contact and copulation. Furthermore, kisspeptin’s effects on\nex-copula erections appear to be specific to the MeA, as demonstrated by the\nabsence of any discernible effects when kisspeptin is administered\nintracerebroventricularly, despite comparable LH responses ( 250 ).\nIn female rodents, the RP3V, which is also central to sexual\nmotivation and partner selection, influences pre-copulatory and\nintra-copulatory behaviors ( 240 ).\nAblation of 70% of kisspeptin neurons in the RP3V impaired lordosis, which\nis rescued by a single peripheral kisspeptin-10 injection ( 240 ). Consistently, optogenetic\nstimulation of RP3V kisspeptin neurons during male mounting enhanced\nlordosis. Utilizing female mice with genetic manipulation resulting in the\nabsence of GnRH secretion during adulthood demonstrates that while\nmale-directed preference is eliminated in the absence of GnRH signaling,\nlordosis behavior remains unaffected ( 240 ). This suggests that lordosis operates independently of GnRH\nsignaling.\nTracing studies identified a subset of nitric oxide synthase (NOS)\nexpressing neurons in the ventromedial hypothalamus (VMHvl) that form\ncommunications with RP3V kisspeptin neurons. Kisspeptin-10 injection\ndirectly into the VMHvl induced a significant increase in lordosis, whilst\nadministration into the PVN had no significant effect on lordosis expression\n( 251 ), thereby confirming site\nspecificity for kisspeptin’s actions. Consistent with the concept that\nnitric oxide (NO) is a key neurotransmitter downstream of kisspeptin\nneurons, nNOS knockout female mice (nNOS −/− ; OVX+E+P) experience\nattenuated lordosis, not restored with subcutaneous kisspeptin-10 injection.\nBy contrast,  Kiss1 −/−  (OVX+E+P) females injected\nwith the NO-donor, SNAP, showed wild type-levels of lordosis. Furthermore,\nbilateral administration of the nNOS inhibitor, L-NAME, in the VMHvl of\nfemale mice led to a strong deficit in lordosis behavior. Consistently, SNAP\nadministration into the VMHvl induced a significant increase in lordosis\nbehavior ( 251 ). Taken together,\nthese findings highlight NO as a key neurotransmitter downstream of\nkisspeptin neurons mediating both mate preference and lordosis behavior\n( 240 ).\nAll in all, in female rodents, sexual partner preference seems to\ndepend on downstream GnRH-signaling ( 240 ). However, lordosis behavior, while independent of\nGnRH-signaling, relies on the synergistic action of NO within the\nventromedial hypothalamus ( 251 ).\nWhile there are no direct reports of sexual behaviors in humans with\nkisspeptin variants, animal studies have provided valuable insights discussed\npreviously in the manuscript. For instance,  Kiss1  knockout male\nrats exhibited reduced sexual behavior, irrespective of sex-steroid milieu\n( 233 ), which was restored upon\nkisspeptin replacement ( 238 ). Similarly,\n Kiss1  knockout female mice failed to show male-directed\npreference in mate choice tests, a behavior also rescued by kisspeptin\nadministration ( 240 ). These animal\nstudies suggest a significant role for kisspeptin in regulating sexual behavior,\nand further research in humans would be highly informative\nMuch like in non-human species, in humans, kisspeptin plays an\nimportant role in modulating signaling pathways involved in attraction, desire\nand arousal through integration of external stimuli, such as olfactory and\nfacial recognition cues, thereby regulating sexual attraction and reproductive\nbehavior towards potential mates ( 252 ).\nThe primary olfactory network in human projects to key limbic areas involved in\nsexual and emotional processing ( 253 )\nand functional neuroimaging studies indicate that exposure to feminine scents\nincreases brain activity in limbic regions associated with sexual desire and\narousal in heterosexual men ( 254 ).\nA randomized, placebo-controlled study employing functional\nneuroimaging, hormonal assessments, and psychometric evaluations revealed that\nintravenous kisspeptin-54 (1 nmol/kg/hr over 75-minutes) in healthy heterosexual\nmen exposed to a validated pleasant feminine scent delivered nasally (Chanel\nNo5) resulted in heightened brain activity within olfactory and limbic circuits,\nincluding the amygdala and thalamus, compared to vehicle administration ( 255 ). These activated regions are\nrecognized for their involvement in olfactory processing, the hedonic valuation\nof olfactory stimuli, and sexual arousal in humans. Notably, kisspeptin\nexhibited no impact on brain activity in control motor areas, thereby\nhighlighting the specificity of its effects within olfactory and limbic circuits\nassociated with sexual behavior in men exposed to feminine olfactory feminine\ncues ( 255 ).\nSimilarly, during a facial attractiveness task, kisspeptin-54\nselectively increased activity in frontal brain regions implicated in human\nperception of beauty, including the medial pre-frontal cortex (mPFC) and\nsuperior frontal gyrus in response to attractive female faces ( 255 ). Importantly, kisspeptin-54 activated\naesthetic brain regions, such as the anterior cingulate cortex (ACC) and insula,\nmore robustly in men with lower baseline sexual quality of life ( 255 ). These regions are implicated in\nsexual arousal, facial attraction, and motivation towards reward, thereby\nsuggesting that the enhanced effects of kisspeptin in these individuals might\nserve to accentuate attraction and motivation to engage in sexual contact,\nparticularly in individuals experiencing lower sexual quality of life ( 255 ). Crucially, all these effects\noccurred in the absence of any downstream sex-steroid changes. Therefore, this\ndata emphasizes the targeted region-specific effects of kisspeptin, contingent\non the nature of the attraction cue, whether it be olfactory or visual ( 255 ).\nKisspeptin also seems to be implicated in enhancing sexual desire and\narousal in humans, in response to external erotic stimuli ( 256 ). In a functional neuroimaging study, intravenous\nkisspeptin-54 was administered to healthy heterosexual men while viewing erotic\nimages. This led to enhanced brain activity in the anterior and posterior\ncingulate as well as the left amygdala during kisspeptin administration compared\nto placebo ( 256 ). These regions are\nknown to express kisspeptin and kisspeptin receptors ( 14 , 15 ), and are\nareas that are known to be activated by sexual stimuli in humans ( 257 , 258 ). Furthermore, the left amygdala has been well described as the\ncenter involved in sexual and emotional processing in men. The activations\nobserved correlated well with responses to psychometric questionnaires,\nproviding further functional relevance of these findings. An inverse correlation\nbetween the degree of sexual-processing network enhancement (including the\ncingulate, putamen, and globus pallidus), and the level of aversion to sex that\nthe men displayed was evident, suggesting a role for kisspeptin in sexual\ndisinhibition. Moreover, in response to erotic stimuli, kisspeptin enhanced\nactivity in limbic structures (including the hippocampus, amygdala, and\ncingulate) more in men with lower baseline reward behavioral scores. As human\nsexual behavior is closely associated with pleasure and reward, this implies\nthat kisspeptin’s effects might be accentuated in individuals experiencing\npsychosexual disorders where reward behavior may be suppressed.\nIn mammalian reproduction, emotional attachment towards a partner often\nserves as a prerequisite for engaging in sexual encounters, a phenomenon\ncommonly referred to as ‘bonding’. Bonding can manifest in various forms,\nincluding romantic love, maternal love, or unconditional love. The role of\nkisspeptin in romantic love bonding has been studied in humans using functional\nmagnetic resonance imaging (fMRI) ( 256 ).\nIn heterosexual healthy men intravenous kisspeptin-54 modulated the response to\ncouple-bonding images in regions similar to those activated in response to\nsexual images, such as the anterior and posterior cingulate and the amygdala\n( 256 ). Kisspeptin-54 also enhanced\nactivity in the thalamus and globus pallidus known to express kisspeptin and\nkisspeptin receptors in humans and were regions not previously activated in\nresponse to sexual images but are implicated in “romantic love” ( 259 ). Importantly, the enhancement of\namygdala activity, also associated with bonding behaviors ( 259 ), in response to the bonding images, correlated with\nreported improvements in positive mood ( 256 ). All in all, these data demonstrate that kisspeptin plays an\nimportant role in processing both sexual and bonding stimuli in humans, both of\nwhich are essential drivers of sexual behavior. Furthermore, a study using the\nsame administration protocol in healthy heterosexual men showed that kisspeptin\ndid not modulate brain regions in response to visual food stimuli or appetite\nparameters, as assessed by fMRI and psychometric tests, respectively ( 260 ).This is significant as it proves that\nkisspeptin’s actions on the limbic system are confined to sexual and emotional\nstimuli. Collectively, these data suggest that kisspeptin not only enhances\nactivation in established structures of sexual arousal and bonding, but that\nthis activation also correlates with behavioral measures of reward, drive, and\nsexual aversion.\nTo derive further mechanistic insights on how kisspeptin integrates\nexternal cues to modulate sexual behavior in humans, it is important to\nunderstand kisspeptin’s effects on resting functional connectivity in humans and\nhow this correlates with subsequent processes occurring in response to sexual\nand emotional stimuli. Therefore, a further fMRI study employing the same\nexperimental protocol, demonstrated that peripherally administered kisspeptin-54\nmodulates the default mode network (DMN), known to be associated with social and\nemotional internal processing, and frequently disrupted in psychosexual\ndysfunction. Importantly, DMN activations correlated with subsequent enhanced\nactivity in limbic brain regions, including the globus pallidus and cingulate\ngyrus, in response to visual sexual images. Additionally, in response to\nkisspeptin, enhancement of the DMNs was stronger in individuals with lower\nreward drive and resulted in reduced sexual aversion ( 261 ).\nExpanding upon these discoveries, considering that kisspeptin not only\nenhances activity in sexual and emotional brain centers but also appears to be\nmore effective in individuals with lower quality of life or sex-drive, the\ninvestigation into kisspeptin's impact on patients with psychosexual dysfunction\nhas ensued (discussed in later sections). Recognizing desire for sexual\nstimulation as a fundamental element of the human sexual response, these\nfindings from rodents to humans pave the way for potential clinical utilization\nof kisspeptin in addressing such issues.\nHypoactive sexual desire disorder (HSDD) is a common cause of reduced\nsexual desire in both men and women, impacting approximately 8% of men ( 262 ) and 10% of women ( 263 ). In both sexes, HSDD manifests as low\nsexual desire with associated distress and significant impact on quality of\nlife. Presently, there are no licensed pharmacotherapies available for men or\npost-menopausal women, while treatments for pre-menopausal women such as\nbremelanotide and flibanserin are limited by their effectiveness or side effects\n( 264 ). While meta-analysis of\nrandomized controlled trials has shown small but positive effects of\ntestosterone in postmenopausal women with HSDD, testosterone’s impact on\nindividuals’ wellbeing, musculoskeletal, and cognitive health, as well as its\nlong-term safety in women, warrant further research ( 265 ). Thus, there exists a pressing clinical need for\nnovel, safe, and effective therapies to address the considerable burden posed by\nHSDD.\nHSDD is characterized by the top-down theory, which posits that\nincreased activity in higher cortical, cognitive brain regions typically\ninvolved in introspection, self-criticism and guilt inhibits lower limbic and\nemotional regions associated with sexual desire and arousal ( 266 ). In a study involving heterosexual\nmen with HSDD, intravenous kisspeptin-54 administration modulated brain regions\nknown to express the kisspeptin receptor in humans when exposed to erotic\nvideos. Specifically, kisspeptin enhanced activity in sexual arousal centers,\nincluding the left ACC, as well as the left middle frontal gyrus (MFG), a\nrecognized executive attention center. Furthermore, kisspeptin administration\nled to a significant deactivation of brain regions involved in self-monitoring\nand introspection, such as the bilateral parahippocampus, frontal pole and\nprecuneus ( 267 ). Remarkably, kisspeptin\nadministration led to a 56% increase in penile tumescence (akin to sildenafil),\ncompared to placebo, while watching erotic videos. Additionally, male\nparticipants reported increased ‘happiness about sex’ in response to kisspeptin.\nNotably, all the aforementioned effects of kisspeptin occurred despite stable\ntestosterone levels, which typically rise much later in response to kisspeptin\n( 267 ). Hence, kisspeptin's capacity\nto heighten sexual arousal and penile tumescence in heterosexual men with HSDD\nmay stem from its capability to deactivate brain regions responsible for\nself-monitoring and self-control. This deactivation, in turn, alleviates\ninhibitory constraints on sexual arousal centers in the brain, thereby\nintensifying arousal levels.\nSimilarly, kisspeptin demonstrated efficacy in pre-menopausal women\nwith HSDD ( 264 ). When viewing erotic\ncontent, intravenous kisspeptin-54 infusion in women with HSDD resulted in the\ndeactivation of higher cortical brain centers, such as the left inferior and\nmiddle frontal gyri. These areas are implicated in the ‘internal monologue’,\nfeelings of guilt and inhibitory control and are hyperactivated in women with\nHSDD. Deactivation of the left inferior frontal and middle frontal gyri by\nkisspeptin can therefore allow lower-level responses to be expressed. Indeed,\nwomen with HSDD also experienced hyperactivation of brain centers involved in\nsexual arousal following kisspeptin administration, including the supramarginal\nand postcentral gyri. Kisspeptin-induced enhancement of the posterior cingulate\nactivity correlated with reduced sexual aversion to male faces. Women\nexperiencing higher distress levels regarding sexual function at baseline showed\nmore enhanced brain activity in sexual centers, including the hippocampus, when\nexposed to erotic stimuli. Kisspeptin administration also deactivated the\ntemporoparietal junction associated with negative perception of others and\nreduced self-consciousness in response to viewing male faces ( 264 ).\nIn both men and pre-menopausal women with HSDD, kisspeptin appeared to\nmodulate brain regions known to express the kisspeptin receptor, suggesting\npotential direct receptor-mediated actions of kisspeptin in these brain regions\nin humans. Interestingly, kisspeptin also demonstrated effects in brain regions\nlacking identified kisspeptin receptors in humans, indicating additional\nindirect mechanisms. Taken together, this suggests that kisspeptin can act\ndirectly or indirectly on various brain regions to essentially decrease activity\nin higher brain centers to relieve the inhibition on downstream sexual brain\nareas, ultimately enhancing sexual behavior. Importantly, all the aforementioned\nbehavioral kisspeptin trials employing fMRI techniques and assessing sexual\nbehavior in men and pre-menopausal women have utilized kisspeptin-54, as opposed\nto kisspeptin-10. Kisspeptin-54’s ability to cross the blood-brain barrier\n( 268 ), and thus having access to act\ndirectly on deep brain structures expressing  KISS1R  could be\nhighly relevant for this action ( 256 ).\nCurrent evidence suggests that kisspeptin might act directly via\nkisspeptin receptors or interact with various neurotransmitters and signaling\npathways to modulate reproductive behavior, mood, and cognition across different\nspecies. These include dopamine ( 236 ),\nserotonin ( 269 ), glutamate ( 270 ), nitric oxide ( 240 ) and gamma-aminobutyric acid (GABA) ( 271 ). Notably, pre-clinical studies\nconfirm GABAb1 subunit co-expression in the majority of Kiss1 neurons within the\nMeA and GABAb1 knockout mice have intact  Kiss1  expression in\nthe RP3V and arcuate nucleus, but increased expression in the MeA, the bed\nnucleus of the stria terminalis (BNST) and lateral septum in both adult male and\nfemale mice, despite normal sex-steroid levels ( 237 ). The MeA and BNST, known for their involvement in anxiety-like\nbehaviors and social interactions, are influenced by GABAergic neurons. Given\nGABA's role in mood and behavioral disorders, it is possible that GABA mediates\nsome of kisspeptin's effects, possibly through inhibiting  Kiss1 \nexpression in the BNST and MeA ( 272 ).\nCorroborating data in rodent models, a study in humans using magnetic\nresonance spectroscopy (MRS), demonstrated up to 16% decrease in total\nendogenous GABA levels in the ACC (which enhances in response to sexual and\ncouple bonding stimuli ( 256 )) following\nan intravenous infusion of kisspeptin-54 (1 nmol/kg/hr) in healthy men in the\nabsence of any changes in testosterone levels ( 271 ). The magnitude in GABA reduction is similar to that reported in\npsychological studies with functional significance (in\nattention-deficit/hyperactivity disorder (ADHD) ( 272 ) and in response to pain ( 273 )). Collectively, the aforementioned data suggests the\nkisspeptin’s effects on stimulation of the ACC and other limbic brain structures\nin response to sexual and couple-bonding stimuli in humans are, at least in\npart, mediated through central GABA inhibition. Whether the interaction with\nGABA is indirect via modulations of other pathways (serotonin, dopamine,\nvasopressin, glutamate, nitric oxide) or via direct binding to kisspeptin\nreceptors in the ACC is still to be elucidated.\n\nThe process of embryo implantation and placentation are tightly\ncoordinated both temporally and spatially; indeed, dysregulation of trophoblast\ninvasion underlies the pathogenesis of multiple pregnancy-related disorders.\nSeveral factors secreted by the feto-placental unit contribute to the autocrine\nand paracrine regulation of trophoblast invasion. Kisspeptin, originally\nidentified as a metastasis suppressor in various types of cancer called\n‘metastin’, has emerged as a key player in implantation and placentation.\nDuring pregnancy, the placenta is the predominant source of circulating\nkisspeptin. Once the blastocyst penetrates the endometrium, the trophoblastic\nstem cells form into an inner layer of undifferentiated cytotrophoblasts, and an\nouter layer of terminally differentiated syncytiotrophoblasts by fusion of\nnon-migratory villous cytotrophoblasts ( 274 ). The extravillous cytotrophoblasts eventually differentiate\ninto the placental bed, responsible for the migration of the embryo into the\ndecidua through degradation of extracellular matrix proteins, and the\nendovascular trophoblasts, responsible for the uterine spiral artery remodeling\n( 274 ).  KISS1  is\nexpressed in syncytiotrophoblasts, but  KISS1R  is expressed by\nboth cytotrophoblasts and syncytiotrophoblasts ( 274 ).  KISS1R  is expressed at higher levels in first\ntrimester trophoblasts than in trophoblasts from the placenta at term, inferring\nthe importance of kisspeptin signaling in implantation and placentation ( 275 ).\nInteractions between kisspeptin and cell adhesion molecules promote\nembryo attachment to the endometrium and upregulate leukemia-inhibitory factor\n(LIF) to promote stromal decidualization ( 274 ). Kisspeptin inhibits placental trophoblast cell migration to\nregulate implantation and placentation; preventing excessive trophoblastic\ninvasion of the endometrium ( 275 – 277 ). Mechanistically kisspeptin directly\nactivates the ERK1/2 signaling pathway to increase cell adhesion and inhibit\nfirst trimester trophoblast cell migration as well as through downregulation of\nmatrix metalloproteinase (MMP) system which inhibits extracellular matrix\nbreakdown ( 276 , 277 ). Kisspeptin-10 stimulated intracellular\nCa 2+  release in primary trophoblasts of the early placenta and\nsubsequently inhibited trophoblast migration, suggesting that this form is the\nphysiological activator of kisspeptin receptor in the human placenta ( 275 ). Several studies suggest that\nkisspeptin may restrain trophoblastic growth by acting as a placental\npro-apoptotic agent in a dose-dependent manner ( 278 – 280 ). Kisspeptin is also\nimplicated in uterine spiral artery remodeling and regulating angiogenesis\nrequired for placentation, with VEGF-A expression from human placental\ntrophoblasts being downregulated by kisspeptin ( 274 , 276 , 281 – 283 ).\nFurthermore, kisspeptin appears to be important in regulating maternal\nimmune tolerance and thus preventing rejection of the developing fetus.\n In vitro  studies have demonstrated that there is increased\ndifferentiation of naïve human CD4+ lymphocytes into adaptive T-regulatory cells\nand when incubated with kisspeptin-54 at levels similar to those during\npregnancy, with inhibition of T-helper 17 lymphocytes induction ( 284 , 285 ). Natural killer cells also specialize into a regulatory subtype\nwith reduced cytotoxic activity against fetal cells when incubated with\nkisspeptin-54 ( 286 ). Kisspeptin-54\nconcentrations in pregnancy also reduce the functional activity of neutrophils\nand increase the functional activity of monocytes; potentially to also\nfacilitate the development of immune tolerance to the fetus whilst balancing the\nneed for maternal immunity ( 287 ) ( Figure 7 ).\nKisspeptin has emerged as a promising biomarker to predict several\nadverse pregnancy complications. Circulating kisspeptin levels increase linearly\nin healthy pregnancy but are reduced in miscarriage during early pregnancy.\nKisspeptin levels are reduced in ectopic pregnancy, fetal growth restriction,\ngestational diabetes and early onset pre-eclampsia. Kisspeptin levels are raised\nin pre-eclampsia during the later stages of pregnancy and gestational\ntrophoblastic neoplasia. Figure created with  BioRender.com .\nPlasma kisspeptin levels increase linearly and dramatically during a\nhealthy pregnancy with gestational age, being over 200-fold greater in the third\ntrimester of pregnancy than in non-pregnant women and returning to non-pregnant\nlevels soon after birth ( 288 – 290 ). Circulating kisspeptin levels in\nhealthy pregnancy are influenced by several variables; advanced maternal and\ngestational age increase circulating kisspeptin levels, whilst Afro-Caribbean\nethnicity, smoking, and high BMI were all associated with lower circulating\nkisspeptin levels during pregnancy ( 291 ).\nGestational or pregnancy-induced hypertension is defined as\nhypertension (systolic blood pressure ≥140 mmHg and/or diastolic blood\npressure ≥90 mmHg) that occurs after 20-week gestation ( 292 ). Pre-eclampsia occurs when\ngestational hypertension is accompanied by proteinuria, maternal\nend-organ or utero-placental dysfunction ( 292 ). Early onset pre-eclampsia is attributed to\npoor placentation and impaired uterine spiral arteries remodeling\nleading to disruption in placental perfusion, whilst late onset\npre-eclampsia occurs when the placenta outgrows the uterine circulatory\ncapacity leading to placental malperfusion ( 293 ). Both etiologies result in placental hypoxic\nstress, however fetal growth restriction (FGR) is more associated with\nearly onset pre-eclampsia given the longer duration of dysfunctional\nuteroplacental perfusion ( 293 ).\nKISS1  expression was increased in the placenta\nof pre-eclamptic pregnancies according to several reports ( 294 – 297 ). Placental  KISS1  expression\ninhibits trophoblast invasion and angiogenesis with resulting defective\nremodeling of the uterine spiral arteries, inferring a role in the\npathophysiology of pre-eclampsia ( 298 ). Conversely, there have also been reports of reduced\nkisspeptin expression in pre-eclamptic placentae ( 299 ). However,  KISS1R  expression\nhas also been found to be increased in pre-eclampsia compared to normal\nhealthy pregnancy and this may facilitate increased functional activity\nof kisspeptin in pre-eclampsia ( 299 ).\nVarying levels of circulating kisspeptin have been reported in\nhypertensive disorders of pregnancy according to the subtype, severity,\nand timing of onset. Most studies report reduced kisspeptin levels in\ngestational hypertension compared to normotensive pregnancy with levels\ndeclining further with severe disease and in pre-eclampsia ( 294 , 300 – 306 ),\nwhich may reflect reduced placental mass ( 303 , 304 ).\nCorrespondingly, both circulating kisspeptin levels and placental mass\nare lower in early onset pre-eclampsia compared to late onset\npre-eclampsia ( 303 , 307 , 308 ).\nFGR describes both intra-uterine growth restriction (fetal\nweight <10 th  centile for gestational age with abnormal\numbilical artery doppler flow) and small for gestational age (delivery\nweight <10 th  centile for gestational age) ( 309 , 310 ). FGR is postulated to be the result of\nabnormal trophoblast invasion and spiral artery remodeling leading to\ndeprivation of adequate oxygen supply to the placenta ( 311 , 312 ). Ischemic injury from this inadequate oxygen\nsupply leads to generation of reactive oxygen species, subsequent\napoptosis, and restriction of placental and fetal growth ( 311 , 312 ). As kisspeptin has been implicated in\nregulating trophoblast invasion and spiral artery remodeling, kisspeptin\nlevels have been demonstrated to be lower in FGR across all three\ntrimesters compared to healthy pregnancy ( 300 , 308 , 313 , 314 ) and may contribute to the\npathophysiology of FGR. The low circulating kisspeptin levels in FGR may\nbe representative of the low placental mass.\nHowever, recently kisspeptin levels have been demonstrated to\nbear no association to the severity of pre-eclampsia and are increased\nin hypertensive disorders of pregnancy during the third trimester ( 308 ). Likewise, another study\nfound that plasma kisspeptin was higher in late-onset pre-eclampsia\n(>34 weeks gestational age) than in gestation-matched controls, but\nthis was not observed for early-onset pre-eclampsia (≤34 weeks\ngestational age) ( 315 ).\nDiscrepancies between the findings of existing studies may be attributed\nto confounding variables such as BMI and gestational age as well as the\ncomplexities of different disorder subsets as well as analytical issues\n( 308 ). These would need to\nbe considered in future larger observational studies to address these\ninconsistencies, assessing each subset of hypertensive disorders of\npregnancy and pre-eclampsia separately throughout pregnancy and using\nrobust assays.\nSimilarly, the placenta has also been shown to express high\nlevels of  TAC3  ( 316 ) particularly in the outer syncytiotrophoblast layer\n( 317 ), facilitating\nsecretion of NKB into the maternal circulation ( 318 ). NKB is also present in cord blood ( 318 ), consistent with a potential\nrole in the physiology of feto-placental circulation. High dose NKB\ninfusion in female rats increased uterine weight by 37%; moreover, NKB\nlevels correlated with nitric oxide metabolites, with higher circulating\nlevels observed in women with pre-eclampsia and IUGR ( 319 ). Therefore, suggesting that\nNKB, together with locally secreted metabolites such as nitric oxide,\ncould be important in the hemodynamic adaptation during pregnancy to\nensure adequate fetal blood supply. As pre-eclampsia is a disorder of\ntrophoblastic invasion, NKB is postulated to be a potential biomarker of\npre-eclampsia, with plasma NKB concentrations becoming detectable by the\nninth week of gestation ( 320 ).\nNKB plasma concentrations are low and increase throughout normotensive\npregnancies, with highest concentrations at term before reducing\npostpartum, consistent with NKB being reflective of placental mass\n( 319 , 321 ). In contrast, pre-eclamptic women demonstrate\na significantly higher level of plasma NKB and NKB placental expression\nduring the third trimester ( 319 , 322 – 325 ). NKB, as a marker, appears\nunique to pre-eclampsia and has no demonstrated association with other\nhypertensive disorders ( 326 ).\nHowever, whether plasma NKB levels have predictive value in early\npregnancy for subsequent pre-eclampsia development needs to be clarified\nwith further longitudinal studies.\nGestational trophoblastic disease (GTD) is characterized by\nabnormal proliferation of placental tissue, including premalignant\ncomplete or partial hydatid moles and malignant gestational\ntrophoblastic neoplasia (GTN). GTN is the result of malignant lesions\n( 327 ) that arise from\nplacental villous and extravillous trophoblasts, comprising\nchoriocarcinoma, invasive mole, placental site trophoblastic tumor and\nepithelioid trophoblastic tumor ( 328 ). Serum β-hCG levels are utilized for the diagnosis,\nstaging and prognostication of GTN ( 328 , 329 ).\n KISS1  and  KISS1R  expression are\nreduced in choriocarcinoma cells compared to healthy placentae and\nbenign hydatidiform molar pregnancies ( 330 ). Reduced KISS1/KISS1R signaling locally may\nmechanistically result in dysregulation of the placentation process thus\nfacilitating trophoblastic invasion in GTN, and so could have diagnostic\nutility in distinguishing metastatic from non-metastatic forms of GTD\n( 289 ). Conversely,\ncirculating kisspeptin levels are elevated in GTN compared to healthy\npregnancy likely as a reflection of the increased malignant\ntrophoblastic mass in GTN ( 289 ),\nwith levels significantly declining post-chemotherapy.\nMiscarriage is the spontaneous loss of an intrauterine\npregnancy before 24 weeks of completed gestation ( 331 ). Establishing the diagnosis of miscarriage\ncan be challenging, and pregnancy can be failing for some time before\npregnancy loss is conclusively confirmed. This prolonged phase of\nuncertainty and the need for serial investigations poses significant\npsychological burden ( 331 , 332 ).\nPlasma kisspeptin levels, corrected for gestational age, are\nreduced by 60-79% in pregnancies that ended with miscarriage compared to\nhealthy pregnancies ( 291 , 333 – 337 ). Likewise, trophoblastic kisspeptin\nexpression was lower in pregnancies that ended with miscarriage compared\nto those seen following elective termination of pregnancy ( 338 ). Plasma kisspeptin levels can\nreflect the type of miscarriage, with lower levels reported in complete\nmiscarriage compared to incomplete or missed miscarriage ( 291 ).\nThe diagnostic performance of kisspeptin in identifying cases\nof miscarriage remains high into the late first trimester (>8 weeks\ngestation), unlike the alternative biomarker β-human chorionic\ngonadotropin (β-hCG) ( 291 ).\nPrevious studies in women undergoing assisted reproduction showed that\nβ-hCG levels were a better predictor of pregnancy outcome than\nkisspeptin levels in early first trimester ( 339 , 340 ).\nThese findings may be attributed to the very early gestation at which\nkisspeptin levels were being measured (12-21 days after embryo\ntransfer); indeed, the performance of kisspeptin for predicting\nmiscarriage improves with gestation ( 291 , 339 , 340 ). A combination approach,\nutilizing both plasma kisspeptin and β-hCG levels can provide a higher\ndiagnostic accuracy of miscarriage at all gestations (AUCROC 0.92, 95%\nCI 0.89-0.95) ( 291 , 334 , 336 ).\nEctopic pregnancy refers to an embryo implanting and developing\noutside of the uterine cavity most commonly in the fallopian tubes and\ntherefore poses the risk of potentially life-threatening complication of\ntubal rupture ( 341 ). Ectopic\npregnancy is currently diagnosed using ultrasonographic evaluation and\nserialized serum β-hCG level measurements ( 342 , 343 ).\nHowever, about 1 in 5 extra-uterine pregnancies have a β-hCG rise in\nkeeping with an intra-uterine pregnancy. Furthermore, a viable\nintra-uterine pregnancy may not be visualized on ultrasound if β-hCG\nlevels are less than 1000 IU/L ( 342 ). Kisspeptin has been investigated as a potential\nbiomarker of ectopic pregnancy. Kisspeptin levels are lower in ectopic\npregnancy than healthy pregnancy but are higher than the levels seen in\nmiscarriage ( 344 ). Lower\ncirculating kisspeptin levels at early gestational ages can challenge\ndetection and therefore, larger studies are needed to determine the\nutility of kisspeptin for diagnosing ectopic pregnancy at early\ngestational ages.\nGestational diabetes mellitus (GDM) affects 20% of pregnancies\nworldwide ( 345 ). During\npregnancy there is a physiological increase in maternal insulin\nresistance to provide glucose to the developing fetus; GDM occurs when\nmaternal pancreatic β-cells fail to adapt to this demand ( 346 , 347 ).\nIn vitro  and  in vivo  studies\nhave revealed various physiological effects of kisspeptin in\nglucose-dependent pancreatic β-cell regulation. Kisspeptin-10,\nkisspeptin-13 and kisspeptin-54 all potentiate glucose-stimulated\ninsulin secretion (GSIS) in mouse and human islet cells  in\nvitro  ( 348 – 351 ). At lower glucose\nconcentrations (2.8-11.1 mmol/L) kisspeptin-13 and kisspeptin-54\nparadoxically demonstrate a dose-dependent inhibition of insulin\nsecretion in mouse islets, an effect not seen at higher glucose\nconcentrations ( 352 ).\nKisspeptin-54 has also been shown to increase GSIS  in\nvivo  when administered to healthy men following an\nintravenous glucose tolerance test ( 353 ). In addition, chronic administration of kisspeptin-10\nto non-pregnant mice potentiated GSIS and improved glucose tolerance\n( 354 ). These data suggest\nthat kisspeptin can enhance GSIS and glucose tolerance in the context of\nhyperglycemia.\nIndeed  Kiss1r -null female mice demonstrated\nimpaired glucose tolerance, hyperlipidemia, and weight gain ( 355 ). β-cell specific\n Kiss1r  knockout and pharmacological inhibition of\n Kiss1r  causes reduced β-cell proliferation compared\nto what is expected in murine pregnancy, thus reducing GSIS and\nimpairing glucose tolerance ( 354 ). Nonetheless β-cell mass is not reduced to pre-pregnancy\nlevels, suggesting that kisspeptin mediated pathways are not the sole\nsignal to drive β-cell proliferation during pregnancy ( 354 ).\nIn GDM, human placental  KISS1  and\n KISS1R  expression is elevated in the third\ntrimester ( 356 , 357 ), however, circulating\nkisspeptin levels are either reduced ( 301 , 354 ) or not\nsignificantly altered ( 308 , 358 ). Significant positive\ncorrelations between kisspeptin and AUC plasma insulin levels following\nan oral glucose tolerance test, glucose-stimulated insulin levels, and\nβ-cell secretory function have been demonstrated in the third trimester.\nHowever, no significant correlations were observed between plasma\nkisspeptin and fasting plasma insulin or markers of insulin resistance\n( 354 ). This suggests that\nthe described positive correlation between kisspeptin and\nglucose-stimulated insulin levels are not attributable to higher\ncirculating glucose levels or altered insulin resistance ( 354 ). In this study, women with\nGDM also had significantly lower plasma kisspeptin levels than women\nwithout GDM ( 354 ) thus\nimplicating a role for kisspeptin in the adaptive response to glucose\nhomeostasis during pregnancy.\nPreterm birth is defined as births before 37 completed weeks of\ngestation ( 359 ). Kisspeptin may\nplay a role in preterm birth and has been proposed to initiate labor\nthrough increased oxytocin neuronal firing rates in pregnant rats ( 360 ). Circulating kisspeptin\nlevels, adjusted for gestation, are higher in pregnancies affected by\npreterm birth than in controls during the late-first trimester ( 308 ). The adjusted odds of preterm\nbirth are increased by 20% (95% CI, 1-42%) for every 1 nmol/L increase\nin plasma kisspeptin ( 308 ).\nPlacental  KISS1  mRNA expression is increased in preterm\nplacentae than in term placentae, placentae from with term vaginal\ndelivery having a higher  KISS1  mRNA expression than\nterm cesarean delivery ( 361 ).\nThis implicates increased placental kisspeptin expression in the\ninduction of labor. However, no differences have been found in the third\ntrimester in circulating kisspeptin levels between pregnancies reaching\nterm and preterm birth ( 308 , 361 ). Further\nstudies are needed to evaluate if there are any differences in\nkisspeptin levels preceding onset of labor.\n\nMenopause marks the permanent cessation of menstruation and\nreproductive capacity in women and is characterized by depletion of ovarian\nfollicles, reductions in sex-steroids and inhibin B levels. The loss of\ninhibitory actions of sex-steroids and inhibin result in marked increase in GnRH\nand downstream gonadotropin activity. As described in previous sections, KNDy\nneurons are key intermediary neurons which mediate sex-steroid feedback on GnRH\nneurons; thus, the hypoestrogenic state in menopause leads to increased KNDy\nneuronal activity.\nEvidence from animal studies, largely using ovariectomized models, has\nfurthered our understanding of the neuroendocrine changes in menopause.\nOvariectomized cynomolgus monkeys demonstrated marked neuronal hypertrophy and\nincreased NKB and kisspeptin gene expression in the arcuate nucleus ( 47 ). Similarly, increased KNDy neuronal\ncell size was evident in ovariectomized mice ( 362 ). Estrogen replacement in these models was able to revert the\nheightened NKB and kisspeptin gene expression and KNDy neuronal size to their\nbaseline levels ( 47 , 362 ). This indicated that the hypertrophic morphological\nchanges and alterations in KNDy gene expression specifically resulted from\nestrogen withdrawal as opposed to being part of the ageing process ( 47 ).\nMorphological changes observed within human hypothalamus post-menopause\nwere first described in 1966 ( 363 ).\nPioneering studies using postmortem hypothalamic tissues from postmenopausal\nwomen demonstrated that  ERα  ( 364 ),  NKB, substance P  ( 365 ), and  KISS1  mRNA ( 47 ) expressing neurons within the\ninfundibular nucleus were hypertrophic.\nImportantly  KISS1  and  TAC3  mRNA\nexpression was increased, conversely  prodynorphin  mRNA\nexpression was reduced ( 48 ). This\nexpression profile suggests increased stimulatory action of kisspeptin and NKB\nand reduced basal inhibitory action from dynorphin thus resulting in increased\nKNDy neuronal activity and downstream GnRH and gonadotropin secretion following\nmenopause ( 48 ).\nVasomotor symptoms (VMS), a collective term to describe hot flashes and\nsweats, are commonly experienced by over 75% of women during the menopausal\ntransition ( 366 ). The average duration\nof symptoms is 7 years ( 367 ), but\nsymptoms last longer in a third of women, with 10% of women experiencing\nsymptoms for up to 12 years ( 368 ).\nIn rodent models, the tail functions as the primary heat exchange\norgan, thus measurement of skin-tail temperature ( 369 ) was used as a model to portray cutaneous\nvasodilation, the primary mechanism of hot flashes in humans ( 370 ). In ovariectomized rodents with low\nE2 levels, the skin-tail temperature is increased to facilitate heat dissipation\n( 371 ) and the temperature threshold\nat which heat-defence mechanisms are activated is reduced ( 372 ); both mechanisms to facilitate heat exchange.\nAblation of KNDy neurons led to consistently decreased tail skin\nvasodilatation supporting the role of KNDy neurons in modulation of body\ntemperature in rodents ( 369 ).\nAnatomically, in preclinical models, arcuate KNDy neurons project to key\npreoptic thermoregulatory areas including the median preoptic nucleus (MnPO) and\nmedial preoptic area (MPA) ( 370 ). The\nMnPO and MPA integrate thermosensory information from warm-sensitive cutaneous\nsensors and ultimately influence thermoeffectors such as autonomic cutaneous\nvasodilation and cold-seeking behavior ( 370 ). Furthermore, NK3R expression has been demonstrated in the\nMnPO. Taken together this intricate anatomical and functional connection between\nthe arcuate nucleus and preoptic area suggests that a possible mechanism\nunderlying the pathogenesis of hot flashes in menopause occur at the level of\nthe hypothalamus through increased NKB signaling ( 370 ) ( Figure 8 ).\nWithin the arcuate nucleus (analogous to the infundibular nucleus in\nhuman), KNDy neurons have hypertrophic morphology compared to in the\npremenopausal state. Due to the loss of negative feedback, there are\ncompensatory increments in the secretion of kisspeptin and NKB and reduced basal\ninhibitory action from dynorphin. In preclinical models, KNDy neurons project to\nthe hypothalamic median preoptic nucleus (MnPO), which is important for\nintegration of thermosensory information from warm-sensitive cutaneous sensors,\nmediation of efferent neural pathways controlling heat-defense effectors and\nultimately control of thermoeffectors such as autonomic cutaneous vasodilation\nand cold-seeking behavior. NK3R are expressed by neurons within the MnPO and\narcuate nucleus. Thus, antagonism of these NK3Rs offers a novel therapeutic\noption for the management of VMS.\nE2, estradiol; GnRH, gonadotropin-releasing hormone; FSH, follicle\nstimulating hormone; KNDy; kisspeptin, neurokinin, dynorphin; LH, luteinizing\nhormone; MnPO, median preoptic nucleus; NK3R, neurokinin 3 receptor; PRG,\nprogesterone; VMS, vasomotor symptoms. Figure created with  BioRender.com .\nFocal microinfusion of a selective NK3R agonist, senktide, into the\nhypothalamic MnPO resulted in a rapid, dose-dependent reduction in core\ntemperature ( 373 ). Additionally,\nadministration of NK3R agonist increased skin-tail temperature and reduced core\ntemperature in mice, in keeping with heat dissipation effector activation ( 374 ) these effects are particularly\npronounced in hypoestrogenic states e.g. post-ovariectomy ( 375 ). Furthermore, administration of a neurokinin receptor\nantagonist cocktail (with affinity for NK1R, NK2R and NK3R) into the MnPO\nprevented the increase in skin-tail temperature heat dissipation response in\nmice ( 375 ). These findings highlight\nthat the NKB/NK3R/NK1R signaling pathway plays a key role in the pathogenesis of\nhot flashes, and therefore antagonizing the action of NKB in these signaling\npathways is delivering a novel therapeutic avenue for VMS ( 376 ).\nSome studies have shown that there is a close temporal relationship\nbetween the onset of VMS and LH pulsatile secretions ( 377 ), and significant positive correlations between skin\ntemperature measurements and circulating LH levels ( 378 ), however data to support this notion is sparse with\nsmall number of subjects included. Women with previous hypophysectomy and FHA\nwith low circulating concentrations of serum gonadotropins challenged this dogma\nand provided clinical evidence for the uncoupling of LH pulses from the onset of\nVMS ( 379 ). Women with isolated\ngonadotropin deficiency (representative of defective GnRH secretion or\nfunction), experienced similar rates of hot flashes compared to postmenopausal\nwomen thus suggesting that alteration of GnRH synthesis and/or release is not\ninvolved in the generation of hot flashes ( 380 ). Indeed, blinded deconvolution and Bayesian spectrum analysis\ndemonstrated no clear association between LH pulse and hot flashes interval and\nchallenged the long-held dogma regarding the synchronicity of hot flashes and LH\npulses ( 381 ). Finally, women affected by\nFHA (representative of dysfunctional neurotransmitter input to GnRH neurons),\ndespite marked hypoestrogenism, reported no symptoms resembling hot flashes\n( 380 ). These findings substantiate\nthe hypothesis that menopausal VMS occurs secondary to estrogen withdrawal and\nis mediated through functional changes of estrogen-sensitive afferent\nintermediary neurons such as KNDy neurons that provide input and regulate GnRH\nneuronal secretion.\nTo evaluate the contribution of NKB/NK3R pathway in reproduction,\nperipheral intravenous NKB infusion was administered to healthy men and\npremenopausal women during the follicular phase ( 382 ). In healthy men and women, NKB infusions did not\nsignificantly alter downstream serum gonadotropin or sex-steroid levels compared\nto vehicle ( 383 ). In addition,\nco-administration of kisspeptin-54 and NKB resulted in significantly lower\nincreases in gonadotropins compared with kisspeptin alone or when kisspeptin-54\nand naltrexone were co-administered ( 384 ). Intriguingly at higher doses, three subjects experienced a hot\nsensation and appeared flushed with mild increased in heart rate which stopped\npromptly following cessation of NKB infusion. Indeed, this was the first\nobservation providing direct evidence of NKB stimulation and induction of hot\nflashes in humans and ignited interests in antagonizing this pathway as a\npotential treatment for menopausal hot flashes.\nFollowing the above study, the ability of NKB to elicit hot flash-like\nepisodes was tested in premenopausal women. Intravenous NKB induced hot flashes,\nboth subjectively and objectively (mean heart rate, skin temperature and skin\nconductance increased by similar magnitudes to changes observed during natural\nmenopausal flashes) ( 382 ). Meta-analysis\nof three genome-wide association studies (GWAS) of postmenopausal women of\nEuropean American, African American, and Hispanic American descent identified 14\nsingle-nucleotide polymorphisms (SNPs) associated with VMS ( 385 ). Intriguingly, these SNPs were all\nlocated on chromosome 4 in the  TACR3  locus, which suggested\nthat genetic variation in  TACR3  may contribute to an increased\nrisk of VMS ( 385 ). Collectively, these\ndata provided functional and genetic evidence to support the pivotal role of NKB\nsignaling as a mediator of menopausal flushing and sparked interests in\nantagonism of the NKB through the use of NK3R antagonists to alleviate VMS\n( 376 ).\nSubstance P is the endogenous ligand that activates NK1R ( 365 ). Central infusion of substance P in\nmice led to sleep disturbance, an effect blocked by NK1R antagonist\npre-treatment ( 386 ). Similarly,\nintravenous infusion of substance P in healthy men led to worsened mood,\nincreased rapid eye movement (REM) latency, and disturbed sleeping patterns\n( 387 ). Furthermore, substance P\ninfusion caused hot flashes ( 388 ) and\nNK1R has been demonstrated to facilitate heat dissipation through active\nvasodilatation ( 389 ). Therefore, NK1R\nantagonism may offer beneficial effects not only on the control of VMS but could\nalso improve sleep quality which is frequently disrupted following\nmenopause.\nTo date, four novel neurokinin receptor antagonists that target the\nNK3R and NK1R have been evaluated in human clinical trials; Pavinetant\n(MLE4901), Fezolinetant (ESN364), Elinzanetant (NT-814) and SJX-653.\nThe first proof-of-concept trial of NK3R antagonism in postmenopausal\nwomen affected by hot flashes was published in 2017 ( 390 ). MLE4901 (Pavinetant) was administered to 28 healthy\npost-menopausal women aged 40-62 years who experienced ≥7 hot flushes per day in\na phase 2, randomized, double-blinded, placebo-controlled, crossover trial\n( 390 ). MLE4901 resulted in a\nsignificant reduction in the number and frequency of hot flashes with 45%\nreduction in subjective report of hot flashes, 41% and 58% reduction in the\nseverity and interference of weekly hot flashes respectively compared to placebo\n( 390 ). The positive symptomatic\nrelief was appreciable from the second day of treatment ( 391 , 392 ) thus\nhighlighting the potential to induce rapid relief for VMS.\nThe effects of MLE4901 on LH pulse profile appeared to be heterogenous\nwith one report describing no significant changes in the number of LH pulses but\nMLE4901 interestingly increased LH pulse amplitude, and improved orderliness of\nLH pulses, compared to placebo ( 390 ).\nConversely in another report, MLE4901 treatment for 7 days resulted in reduction\nin basal LH secretion with an overall suppressive effect of LH secretion ( 391 ). No change in E2 ( 390 ) or FSH ( 391 ) levels were observed. Changes in hypothalamic GnRH\npulse release were postulated to account for reductions in LH pulses following\nadministration of NK3R antagonists and that this could independently suppress\nVMS. Due to a rise in transaminase levels up to 6x upper limit of normal\nassociated with MLE4901 treatment its development has subsequently been\ndiscontinued although other NK3R antagonists have emerged.\nFezolinetant (ESN364) is a selective and reversible antagonist of NK3R.\nTwice daily Fezolinetant significantly reduced total VMS scores, frequency of\nmoderate/severe VMS and led to a 93% reduction in VMS frequency from baseline to\nweek 12  vs  46% with placebo ( 393 ). At 3hrs post Fezolinetant dose (during peak drug levels),\nplasma LH levels decreased by 49.8%  vs  16.4% with placebo\nrelative to baseline consistent with the proposed mechanism of action of KNDy\nneuron inhibition ( 393 ). No effect on\nE2, FSH and sex hormone binding globulin (SHBG) levels were observed ( 393 ).\nSKYLIGHT 1, 2 and 4 are phase 3 multicenter trials of Fezolinetant\ntreatment that resulted in significant reduction in VMS frequency and severity\ncompared to placebo ( 394 ) with\nbeneficial effects maintained for 52-weeks ( 395 ). Interestingly, the MOONLIGHT trial, conducted in Asian women\nacross 48 Asian countries demonstrated no significant difference in\nmoderate-severe VMS frequency and severity with Fezolinetant use compared to\nplacebo ( 396 ). This may be due to\ngenetic differences and environmental influences.\nThere were no significant changes from baseline endometrial thickness\nbetween Fezolinetant and placebo-treated participants, and no effect on bone\nhealth after a year of treatment ( 397 ).\nNotably, hepatic safety profile was assessed in participants with relevant\nhepatic risk factors such as obesity and non-alcoholic fatty liver disease, and\nthere were no evidence of liver function impairment or liver-associated\nsymptoms, including no Hy's law cases to indicate drug-induced liver injury\nfollowing Fezolinetant use ( 397 ).\nElinzanetant is a dual NK1R and NK3R antagonist. Given its dual\nNK1R/NK3R antagonism it has the potential to decrease GnRH pulse frequency by\nblocking the effects of endogenous substance P and NKB on the reproductive axis,\nas the cognate receptors for substance P and NKB are NK1R and NK3R respectively\n( 226 ). Although NK1R has been\nproposed to contribute to the pathogenesis of VMS, the predominant action on VMS\nassociated with Elinzanetant is likely to be mediated via NK3R antagonism as\nNK1R antagonism alone is unlikely to fully alleviate VMS but may provide\nadditional benefits on sleep and anxiolytic effects in postmenopausal women\n( 398 ). Higher dose regimens of\nElinzanetant were associated with significant reductions in VMS frequency\n(150mg: 84% reduction, 300mg: 66% reduction, placebo: 37% reduction) and in\nnight-time awakening due to night sweats (150mg: 81% reduction, 300mg: 63%\nreduction, placebo: 32% reduction) at the end of the 14 day period ( 399 ) with significant improvements in\nsleep and quality of life with return to baseline 4 weeks after discontinuation\nof treatment ( 400 ). The OASIS 1 and 2\ntrials evaluated the efficacy and safety of Elinzanetant 120mg for the treatment\nof VMS. Elinzanetant use resulted in significant reductions in VMS frequency\n(55-67% reduction) and severity, evident by 4 weeks of treatment. Furthermore,\nElinzanetant improved menopause-related quality of life and reduced sleep\ndisturbances by the end of 12 weeks. ( 401 ).\nSJX-653 resulted in a reversible suppression (up to 70%) of\ngonadotropins and testosterone after a single oral dose (dose range 0.5-90mg) in\nhealthy men aged 18-45 years ( 402 ). As\nother NK3R antagonists after achieving similar reductions in LH and testosterone\nin men have subsequently been shown to demonstrate reduction in menopausal hot\nflushes, this led to the extrapolation that SJX-653 may be an effective\ntreatment of menopausal hot flushes ( 402 ). Subsequently, this prompted a phase 2 trial to assess the efficacy\nof SJX-653 in postmenopausal women with moderate to severe VMS ( 403 ). However, as the primary outcome of\nsafe and efficacious treatment of VMS was not met, the trial was terminated\nearly and its further development since discontinued ( 403 ).\nData from animal models and post-mortem hypothalamic studies from\npostmenopausal subjects paved our understanding of the role of the NKB pathway\nin the pathogenesis of VMS. Antagonism of the NKB pathway resulted in rapid and\nsustained relief of VMS and improvements in sleep quality, and overall markers\nof quality of life. Antagonism of the NKB pathway demonstrates clear\neffectiveness for the management of menopausal hot flushes and is likely to\nprovide an effective, non-hormonal treatment strategy for the management of VMS.\nRecently in 2023, the US Food and Drug Administration (FDA) has approved\nFezolinetant for use to reduce the frequency and severity of post-menopausal hot\nflashes marking the first translational application of therapeutic agent\ntargeting the NKB pathway ( 376 ).\n\nSkeletal homeostasis in mammals refers to the dynamic equilibrium\nwhereby bone formation and bone resorption are meticulously balanced to maintain\nstable and optimal bone mass. This equilibrium is essential for promoting growth\nand ensuring the skeleton’s resilience to mechanical stressors and thereby\npreventing fractures. Skeletal homeostasis is achieved by the process of bone\nremodeling which comprises two essential components; osteoclastic bone\nresorption and osteoblastic bone formation.\nOsteoclasts are specialized cells that differentiate from hematopoietic\nstem cells under the control of receptor activator of nuclear factor kappa beta\nligand (RANKL) signaling and are responsible for resorbing old or damaged bone\ntissue. Enzymatic activity during bone resorption releases fragments of type 1\ncollagen, including C-terminal telopeptide of type 1 collagen (CTX) and\nN-terminal telopeptide of type 1 collagen (NTX), into the bloodstream, serving\nas measurable biochemical markers of bone resorption. On the other hand,\nosteoblasts differentiate from mesenchymal stem cells, and are the bone forming\ncells involved in new bone matrix synthesis and mineralization to achieve bone\nstrengthening and facilitate microdamage repair. Osteoblasts release proteins\nincluding osteocalcin and cleavage fragments from type 1 procollagen synthesis,\nsuch as procollagen type 1 N-terminal propeptide (P1NP), which can be detected\nin the circulation as biochemical markers of bone formation. Similarly, bone\nspecific iso-enzymes such as alkaline phosphatase (B-ALP) are released by\nosteoblasts to aid bone mineralization and are also measured as markers of bone\nformation. Once mineralization is complete, a proportion of mature osteoblasts\ndifferentiate into osteocytes, which reside within the mineralized bone.\nOsteocytes secrete important hormones and chemicals that regulate bone\nremodeling, including fibroblast growth factor-23 (FGF23; phosphate regulating\nhormone), RANKL and sclerostin (an antagonist of the osteoblast activator, Wnt).\nOsteocytes may also act as sensory cells, translating mechanical cues into\nsignals that stimulate bone formation ( 404 ).\nBone remodeling is orchestrated by the coordinated action of hormones,\nsuch as parathyroid hormone (PTH), vitamin D, growth hormone, insulin-like\ngrowth factor-1, sex-steroids and adequate nutrients ( 405 ). Crucially, there exists a well-established\nconnection between the HPG axis and bone health. Bone expresses receptors for\nthe action of several reproductive hormones ( 405 ), with the predominant action from testosterone and estrogen\n( 406 ). One of the main secondary\ncauses of skeletal disease such as osteoporosis are reproductive disorders\ncausing sex-steroid deficiency in women and men. Physiologically, androgens\ninfluence bone health by directly binding to androgen receptors on bone or\nindirectly through aromatization to estrogen ( 406 ). Estrogen suppresses osteoclast formation and resorption\nactivity, partly through antagonistic effects on the RANKL pathway ( 407 ). Furthermore, estrogen induces\nsecretion of semaphoring-3A from osteocytes, a protein that also reduces bone\nresorption and increases bone formation ( 408 ) and exerts anti-apoptotic effects on osteoblasts ( 409 ). Therefore, in the absence of\nadequate levels of sex-steroids, there is significant bone compromise. Indeed,\nfollowing menopause, a decline in circulating estrogen enhances bone resorption,\nwhich is followed by a lesser increase in bone formation due to coupling,\nresulting in a net loss of bone ( 410 )\nsuch that during early menopause there is up to a 100% increase in bone turnover\n( 411 ). The significance of estrogen\ndeficiency is also highlighted in eumenorrheic (eugonadal) women with anorexia\nnervosa exhibiting higher BMD than amenorrheic (hypogonadal) women with lower\nestrogen levels (T-score -1.2 in eumenorrheic  vs  -2.3 in\namenorrhoeic women) ( 412 ). Whilst\noptimal sex-steroids are indispensable for bone health, disruptions to the\nnormal process of bone remodeling can occur even in the presence of normal\ncirculating sex-steroid levels. This is evidenced by peri-menopausal women\nexperiencing declines in BMD ( 413 ) and\nwomen with prolactin-secreting adenomas exhibiting a higher prevalence of\nfractures, despite a normal sex-steroid milieu ( 414 ). Additionally, women with anorexia nervosa suffer bone\nconsequences due to disturbances in leptin, IGF-1, and cortisol, even in the\nabsence of amenorrhea (and thereby intact sex-steroid levels) ( 415 ).\nEmerging evidence suggests that kisspeptin is also a significant player\nin bone homeostasis. Individuals with loss of function  KISS1R \nvariants demonstrated delayed bone maturation in the absence of kisspeptin\nsignaling ( 6 ). This finding first marked\nthe potential involvement of kisspeptin in bone homeostasis ( 6 ). Similarly, CPP from\n KISS1R  gain of function variant was associated with\naccelerated growth and skeletal maturation ( 23 ). Admittedly due to the observed significant alterations in sex\nhormones associated with these conditions (hypogonadotropic hypogonadism or\nprecocious puberty), it is not possible to assert a direct relationship between\nkisspeptin itself and the observed effects on bone; nonetheless these\nobservations contributed to the interest in the potential role of kisspeptin in\nbone homeostasis.\nThe link between kisspeptin and bone homeostasis is increasingly\nevident, following studies demonstrating significant\n Kiss1/KISS1  and  Kiss1r/KISS1R  expression\non key cells involved in bone homeostasis, suggesting possible direct effects of\nkisspeptin on bone.\nKISS1R  has been identified as one of the\nhypoxia-inducible genes in the osteoclast microarray ( 416 ). In fact, in humans,  KISS1R  mRNA\nis expressed throughout the process of osteoclastogenesis  in\nvitro , at multiple differentiation stages, ranging from the\nCD14-monocyte stage to mature osteoclast stage ( 417 ).\nThe osteoanabolic effects of kisspeptin have been demonstrated\npredominantly in  in vitro  studies in cultured cell lines or\nosteoprogenitor cells. Using cDNA microarray technology,\n KISS1  gene was first identified in osteosarcoma U-2\ncell lines in 2003 ( 418 ). Further\nexpression studies in osteosarcoma cell lines, commonly used as osteoblastic\nmodel, confirmed moderate  KISS1  mRNA and protein expression\nin U-2 cell lines, weak expression in Saos-2 cell lines, and absent\nexpression in MG-63 cell lines ( 419 ).  KISS1  mRNA expression was most notable in\nimmortalized human fetal osteoblastic cells transformed by expression of\nSV40 large T antigen (hFOB1.19) ( 419 ). Interestingly, weaker  KISS1  expression was\nassociated with greater invasive capability in an osteosarcoma cell line\n in vitro , whilst in osteosarcoma elevated  KISS1\nin vivo  was associated with relapse or early metastasis ( 419 ). Low level heterogeneous\nexpression of  KISS1  was also detected in primary\nmesenchymal stem cells (MSC) and MSC-derived osteoprogenitor cells\n(undifferentiated osteoblastic cells) from healthy donors ( 420 ). Furthermore,\n Kiss1  and  Kiss1r  are expressed in\ncanine osteosarcoma cell lines (COS, POS) ( 421 ). Application of kisspeptin to these cell lines increases\nCOS proliferation and expression of bone remodeling factors, such as RANKL\nand specific serotonin (5HT) receptor (HTR2a) ( 421 ).\nExpanding upon these expression profiles,  in vitro \ndata in rodents has recently confirmed kisspeptin’s involvement in\nosteoblast differentiation. Kisspeptin-10 induced a dose-dependent\nupregulation of early osteogenic factors mRNA and protein expression such as\ndistal-less homeobox 5 (Dlx5), runt-related transcription factor 2 (Runx2)\nand ALP, known to be important in osteoblast differentiation in murine\nfibroblastic mesenchymal stem cell–like osteoblast cell lines (C3H10T1/2)\n( 422 ). As kisspeptin-10 only\nexerts its effects in  Kiss1r  expressing cells, this\nprovided evidence that these effects occur via  Kiss1r \nexpressed on C3H10T/2 cells ( 422 ).\nIn rodents, bone morphogenetic proteins (BMPs) that belong to the\ntransforming growth factor-β superfamily, appear to mediate the\nosteoanabolic effects of kisspeptin-10 through the activation of\ntranscription factors including NFATc4 (in osteoblasts and the embryonic\nkidney) ( 422 ) and Sp1 (in the\nembryonic kidney) ( 423 ). The BMPs\nimplicated in this pathway to date are BMP-2 ( 422 – 424 ) and\nBMP-7 ( 423 ). Therefore, kisspeptin,\nat least  in vitro , may act as an autocrine growth factor\nwith pro-proliferative effects on bone ( 421 ).\nFurther pivotal data on the direct effects of kisspeptin on human\nbone metabolism have emerged more recently. Immortalized human mesenchymal\nstem cells (hMSC-TERT4) were incubated with kisspeptin-54 for 7 days, to\nevaluate the effects on osteoblastogenesis ( 417 ). This led to an increase in ALP activity, a surrogate for\nosteoblast activity, by 41%, therefore suggesting enhancement of\nosteoblastogenesis in this human cell line. Kisspeptin addition to mature\nosteoblasts did not alter ALP activity, thereby suggesting selective effects\non osteoblast precursors ( 417 ).\nDuring bone resorption assays, infusion of kisspeptin with\nosteoclast monocultures revealed potent antiresorptive effects. Microscopic\nevaluation of the percentage of eroded surface per bone surface demonstrated\na dose-dependent effect ranging from 29.6-48.1% reflective of inhibition of\nosteoclast activity. Kisspeptin exhibited similar effects in\nosteoblast/osteoclast co-cultures, which represents a more realistic\n in vivo  bone remodeling environment, with suppressive\neffects ranging from 26.2% to 53.4% ( 417 ). To translate kisspeptin’s positive effects on bone\nhomeostasis observed  in vitro , a tentative indirect\ncomparison with established osteoporosis treatments revealed that\nkisspeptin’s osteoanabolic effects compare favorably with those of\nteriparatide ( 425 ), and its\nosteoclastic effects with zoledronic acid ( 426 ). To date, no studies have explored whether kisspeptin\nsignaling occurs in osteocytes.\nThe identification of kisspeptin and its receptor expression in\nbone, coupled with compelling findings that identify kisspeptin’s role in\npromoting osteoblastogenesis and inhibiting osteoclastic activity  in\nvitro , spurred additional research. Further exploration has\nprovided better insights into kisspeptin’s role in the bone  in\nvivo , suggesting promising applications for translational\nresearch in addressing disorders of skeletal homeostasis, including\nosteoporosis of various etiologies.\nIn both male and female (intact or gonadectomized) rodents, chronic\nadministration of 17β-estradiol has been shown to increase trabecular bone\nmass through ERα. Intriguingly, the action via hypothalamic ERα seems to\nhave the opposite effect on bone ( 427 ). Ablation of ERα in the medial basal hypothalamus (MBH) in\nEsr1Nkx2-1Cre models, results in a robust bone phenotype characterized by\nincreased BMD in trabecular and cortical bones. Stereotaxic-guided ablation\nof ERα specifically in the arcuate nucleus (ERαKOARC), mirrored the\nsignificant increases in BMD observed exhibiting an impressive up to 700%\nincrease in trabecular bone mass and an 80% increase in bone volume/total\nvolume (BV/TV) in female mice. Importantly, these effects manifest\nindependently of alterations in food intake and other circulating hormones\nknown to have osteoanabolic effects, such as leptin, thyroxine, LH, FSH,\ntestosterone and estrogen. Crucially, even after ovariectomy, female mice\nsubjected to ERαKOARC still demonstrate a 50% increase in BMD, suggesting\nthe presence of an intact brain-bone circuit irrespective of the sex-steroid\nmilieu ( 427 ). This finding holds\nsignificance, suggesting a potential therapeutic avenue for addressing bone\nmass loss in women due to menopause or other estrogen deficient states.\nSimilarly, striking increases in bone mass were observed in female mice when\nERα was stereotactically ablated in arcuate Kiss1-expressing cells\n(Esr1Kiss1-Cre) using a Kiss1-Cre-GFP knock-in allele. These sexually\ndimorphic effects appeared to be independent of high E2 levels. A BV/TV\nincrease of approximately 88% at the distal femur, along with similar\nchanges in the L5 vertebra and the cortical bone mass, were also observed.\nImportantly, ERα ablation in POMC expressing neurons (Esr1POMC-Cre), which\nshare a common lineage with most  Kiss1  neurons, did not\nlead to skeletal improvements ( 427 ).\nCollectively, these findings suggest that a female-specific brain-to-bone\npathway is mediated by a subset of  Kiss1  neurons.\nFurthermore, transcriptional profiling demonstrated that the observed\neffects were mediated via BMP signaling and enhanced osteoblast\ndifferentiation ( 427 ). This aligns\nwith previous  in vitro  findings on the mechanism of action\nof kisspeptin in the bone.\nA recent study utilized parabiosis and bone transplant methods to\nreveal that a circulating factor, identified as Cellular Communication\nNetwork Factor 3 (CCN3), accounts for the high bone mass observed\nspecifically in females, after the deletion of ERα from arcuate Kiss1\nneurons in the Esr1NKx2-1 Cre model ( 428 ). CCN3’s osteoanabolic effects were demonstrated  in\nvitro  in mouse and human skeletal stem cells (SSCs) when\nadministered at low concentrations, as well as  in vivo  in\ngain-of-function and loss-of-function studies. Importantly, CCN3 is\nco-expressed with kisspeptin in arcuate KNDy neurons, but its expression in\narcuate Kiss1 neurons was only significantly increased in both\nestrogen-depleted lactating females and lactating dams. In lactating dams,\nremoval of pups (forced weaning) led to reduction in CCN3 expression, thus\nsuggesting that CCN3’s bone-promoting property lessens with cessation of\nlactation ( 428 ). Further studies\nthat will help elucidate its interaction with kisspeptin, and how this might\nbe utilized in post-menopausal bone loss and osteoporosis, are warranted.\nWhile the exact underlying mechanisms, beyond CCN3, that promote the high\nmass bone phenotype in Esr1Nkx2-1Cre, Esr1Kiss1-Cre, and ERαKOARC female\nmice remain undetermined, the findings of the aforementioned studies further\nimplicate the involvement of Kiss1-Kiss1r signaling in skeletal homeostasis,\nhere demonstrated in lactating mice ( 427 ). This complex neuro-skeletal circuit may have evolved as an\nenergy conserving mechanism during periods of negative energy balance to\nregulate metabolic demands of the bone and to preserve reproductive\ncapacity. While these findings imply a potential link, they do not confirm a\ndirect bone effect attributed to peripheral kisspeptin signaling. In\naddition to offering deeper insights into the physiology of skeletal\nhomeostasis and the pathophysiology of skeletal disorders, these findings\nsuggest the possibility of pharmacologically targeting kisspeptin pathways\nin humans to improve bone health.\nMoreover, a recent study in mice has shown results implicating\nkisspeptin-10 signaling as a negative osteoclast modulator, thereby\nconferring bone protective effects. The resorptive effects of osteoclasts\nrely on the phosphorylation and activation of Src kinase.  In\nvitro  kisspeptin-10 dose-dependently upregulated the expression\nof a phosphatase (Dusp18), which phosphorylates and inactivates Src.\n In vivo , both whole-body\n( Kiss1 − / − ,\n Gpr54 − / − ,\n Dusp18 − / − ) and osteoclast\nconditional knockout ( Kiss1  cKO,  Gpr54 \ncKO) mice exhibited bone loss and osteoclast hyperactivation ( 429 ). These effects were paralleled by\nincreased osteoblast differentiation in the\n Kiss1 − / −  and\n Gpr54 − / −  models. The\nobserved phenomenon may be attributed to a mechanism known as 'bone\ncoupling', wherein the activation of osteoclasts is reliant on cytokines\nderived from osteoblasts, such as M-CSF and RANKL. Moreover, in\novariectomized mice, both intravenous injections of kisspeptin-10 and\nbone-targeting kisspeptin-10 ((DSS)*6-Kp-10) administered twice weekly for\ntwo months exhibited bone-protective effects ( 429 ). The bone-targeting (DSS)*6-Kp-10 was designed\nusing six repetitive sequences of the amino acids aspartate, serine, and\nserine, ensuring an effective bone surface-targeting delivery system ( 430 ). Indeed, the bone-protective\neffects of (DSS)*6-Kp-10 were far superior compared to those receiving\nequivalent doses of kisspeptin-10, as determined by Von Kossa staining and\nparameters of trabecular bone analysis. This difference was observed despite\nsimilar gonadotropin levels. Treatment with a higher dose of bone-targeted\n(DSS)*6-Kp-10 led to increased bone mass in both ovariectomized and\ngonad-intact mice, as shown using micro-CT analysis, and TRAP staining\nrevealed suppressed osteoclast activation in both groups, supported by\nmeasurements of osteoclast parameters. Collectively, these data suggest that\nthe kisspeptin-10/Gpr54 signaling axis plays a bone-protective role and\nimproves bone health both in vitro and in vivo by mechanisms which include\ninhibiting osteoclastic bone resorption ( Figure 9 ). Kisspeptin-10 could, therefore, be utilized as a\npotent therapeutic target for the treatment of osteoclast-associated bone\nloss, such as age-related osteoporosis ( 429 ).\nBALP, bone alkaline phosphatase; CTX, C-terminal telopeptide of\ntype I collagen; E2, estradiol; FSH, follicle stimulating hormone; INH,\ninhibin; KO, knock-out; MSC, mesenchymal stem cells; NTX, N-terminal\ntelopeptide of type I collagen; OVX, ovariectomized; P1NP, procollagen type\n1 N-terminal propeptide; P1CP; carboxy-terminal propeptide of type 1\nprocollagen; PRL, prolactin; RANK; receptor activator of nuclear factor\nkappa-B; RANKL, receptor activator of nuclear factor kappa-B ligand; T,\ntestosterone; TRAP, tartrate-resistant acid phosphatase. Figure created with\n BioRender.com .\nThe only study to demonstrate that kisspeptin’s  in\nvitro  effects on bone metabolism could be translated into humans\nwas performed recently ( 417 ). When\nintravenous kisspeptin-54 was administered to 26 healthy men over 90 minutes, it\nelicited a 20.3% rise in total osteocalcin and a 24% rise in carboxylated\nosteocalcin levels, compared to placebo. Carboxylated osteocalcin is the\npredominant form of osteocalcin involved in bone remodeling. Therefore, this\nacute rise following only 90 minutes of kisspeptin and in the absence of any\nsex-hormone changes, suggests potent positive bone effects of kisspeptin\n in vivo . In contrast to the  in vitro \nfindings where kisspeptin incubation with mature osteoblasts did not increase\nALP, the observed osteocalcin rise suggests effects on more mature osteoblasts\n in vivo  ( 417 ). The\nshort infusion of kisspeptin-54 elicited no discernible impact on bone\nresorption (CTX) potentially due to the short (90 minute) exposure to\nkisspeptin. To potentially observe significant alterations in bone turnover\nmarkers, it is plausible that sustained administration of kisspeptin-54 or its\nreceptor analogues, TAK448/MVT602, may translate the suppression of osteoclast\nactivity seen  in vitro  and in non-human  in\nvivo  studies. Further investigations with extended exposure periods\nand continuous administration may reveal the full spectrum of kisspeptin’s\nimpact on bone turnover. All in all, these findings indicate that acute\nadministration of kisspeptin may have direct beneficial effects on skeletal\nhomeostasis in humans (likely via KISS1R on mature osteoblasts), independent of\neffects on downstream sex-steroids.\nKisspeptin emerges as a promising therapeutic option for preventing and\ntreating skeletal complications associated with conditions characterized by\nsex-steroid deficiency, such as hypogonadotropic hypogonadism, POI, and\nmenopause. In women experiencing FHA, BMD is significantly reduced at various\nskeletal sites, in both trabecular and cortical bone ( 415 ). This decline in BMD correlates with an increased\nrisk of stress fractures, extending beyond the effects of estrogen deficiency\n( 415 ). Notably, reduced kisspeptin\nsignaling is a recognized factor in FHA as described previously. Kisspeptin’s\nfavorable safety profile, along with its potential to restore menstrual\ncyclicity and potentially ovulation in women with FHA, positions it as a\ncompetitive agent for both bone, menstrual health, and fertility in FHA. This\nadvantage is particularly noteworthy as it may aid bone health without the\nassociated side-effects and risks associated with HRT ( 415 ). Alternatives to HRT for bone health in these women\nare limited. Leptin levels are reduced in FHA and although subcutaneous\nmetreleptin over two years was associated with 4-6% BMD gain in the lumbar spine\nin exercising women, it was also associated with 3% weight loss which has\ntherefore dampened its viability for FHA treatment ( 431 ). By contrast, current evidence in humans does not\nsuggest an association between kisspeptin administration and weight loss.\nFurther studies investigating the impact of kisspeptin signaling on the bone in\ncases of osteoporosis, particularly in women with FHA or menopause are eagerly\nanticipated. Further exploration is warranted to assess its viability as a\nprospective therapeutic target for clinical conditions related to bone\nmetabolism such as osteoporosis, where current treatments have\ncontraindications, frequently cause side-effects and are limited in\nduration.\n\nIn addition to the future research directions highlighted in the\ncorresponding sections, we have included below some additional avenues for\nkisspeptin and NKB research and considerations of clinical applications of compounds\ntargeting the kisspeptin/NKB pathways.\nKisspeptin presents significant diagnostic and therapeutic potential\nthat could revolutionize the fields of reproductive endocrinology, metabolic and\nbone health. To date, current data have predominantly investigated subcutaneous\nor intravenous routes of administration, which may hinder its development as a\ndiagnostic or therapeutic option. Exploring alternative administration methods,\nsuch as intranasal administration, could present an alternative non-invasive\ndelivery route. Recent data in rodents have shown that up to 25% of the total\nGnRH population resides within the olfactory bulb ( 432 ). These GnRH neurons express olfactory and vomeronasal\nreceptors activated by opposite-sex odors and maintain functional connections\nwith olfactory and vomeronasal structures. Thus, GnRH neurons are well-equipped\nto detect and respond to social cues, such as pheromones, potentially impacting\nmating behaviors. Indeed, chemogenetic activation of GnRH neurons in the\nolfactory bulb of male mice increases the firing rate of GnRH neurons in the\npreoptic area, stimulating downstream LH and testosterone production ( 432 ). These findings suggest that the\nolfactory system could enable novel therapeutic approaches for modulating\nreproductive and behavioral functions, potentially through intranasal kisspeptin\nadministration. Studies involving rodent and human intranasal kisspeptin-54\nadministration are currently underway, with promising preliminary results ( 433 ). In future, non-peptide agonists of\nthe kisspeptin receptor could enable additional non-invasive routes of\nadministration such as via the oral route.\nAs tachykinins and their receptors are expressed in multiple\nextrahypothalamic tissues, antagonism of NKB action in the management of VMS\ncould have additional effects beyond its thermoregulatory role. For instance,\ntachykinins are expressed within the enteric nervous system and within the\nmucosa of the gastrointestinal tract, albeit with marked interspecies variation\n( 434 ). Data from animal studies\ndemonstrated a role of tachykinins in gastrointestinal motility/peristalsis\nregulation, secreto-motor response and in the modulation of gastrointestinal\ninflammatory, immune, and sensory response. Thus, this has sparked interest in\nits therapeutic potential in gastrointestinal disorders. Conversely, the NK3R\nantagonist, Talnetant, did not demonstrate efficacy over placebo ( 435 ) in the control of irritable bowel\nsyndrome (IBS) related discomfort, which did not corroborate fully with\npre-clinical data. Moreover, Aprepitant and later Fosaprepitant (both NK1R\nantagonists), have been approved by the FDA in the treatment of\nchemotherapy-induced and postoperative emesis through its central actions on NK1\nreceptors ( 434 , 436 ). Overall, it will be informative to monitor for\nextrahypothalamic effects of NK3R antagonists, as well as the combination of\nNK3R and NK1R antagonists, currently being investigated in the management of\nmenopausal VMS in the Phase 3 trials.\nWhilst vasomotor symptoms are the hallmark symptoms associated with the\nperimenopause and early postmenopausal years; women may experience a number of\nother symptoms that can negatively impact on quality of life. These include an\naltered metabolic rate that can be associated with weight gain ( 437 ), cognitive impairment ( 438 ), and mood disturbances ( 439 ). The changes in hypothalamic\nneuropeptide expression observed in post-mortem tissue from post-menopausal\nwomen and ovariectomized animal models raise the question if kisspeptin or NKB\naction contribute to these observed changes.\nRodent studies using global  Kiss1r  knockout models\nhave consistently demonstrated that adult female  Kiss1r \nknockout mice (at 18 weeks) exhibit an increase in body weight of up to 30%\ncompared to wild-type controls ( 440 – 442 ), comprising mainly\nof increased adiposity ( 442 ). Notably,\nkisspeptin's effects on body weight appear to be sexually dimorphic, as adult\nmale  Kiss1r  knockout mice show no significant changes in body\nweight ( 440 , 442 ). Some of the weight changes have been attributed to\nthe hypogonadal state or inadequate sex-steroid replacement in\n Kiss1r  knockout mice. However following ovariectomy,\n Kiss1r  knockout mice had higher body weight, leptin levels,\nand adiposity compared to ovariectomized control females with similar\nsex-steroid milieu ( 440 , 442 ). Therefore, the effect of kisspeptin\non energy homeostasis is likely mediated through direct effects on energy\nexpenditure and indirectly through changes in reproductive hormone levels ( 440 , 442 ). In rats, a 25% gain in body weight was observed by three weeks\npost-ovariectomy, however this increase in body weight was abolished following\nselective ablation of the arcuate KNDy neurons ( 443 ). Furthermore, E2 replacement in control rats induced weight\nloss, an effect lost in KNDy ablated rats, thus suggesting the essential role of\nKNDy neurons in mediating the effects of E2 on body weight, at least in rats\n( 443 ). However, at present\ncorroborating data in humans is lacking, and the long-term effects of NK3R\nantagonism (which currently is entering clinical use for treatment of hot\nflashes) on body weight will be of interest.\nKisspeptin and its receptor are expressed in critical brain regions\nassociated with memory and learning, including the amygdala and hippocampus\n( 444 ). This anatomical localization\nsupports the proposed cognitive roles of kisspeptin signaling. Experimental\nstudies in mice suggest that stimulating kisspeptin signaling within the\nhippocampus can enhance learning and memory and exert neuroprotective effects,\nparticularly concerning amyloid-β accumulation in the hippocampus, as observed\nin preclinical models of Alzheimer's disease ( 445 ). Kisspeptin-13 has been shown to improve memory consolidation\nin passive avoidance learning in male rodents ( 446 ) and enhance learning in zebrafish ( 447 ). Additionally, the administration of kisspeptin-13\nvia intracerebroventricular and intra-hippocampal routes in mice enhances memory\nretention and facilitates the formation of object and location recognition\nmemory ( 448 ). These effects are\nabolished by the kisspeptin receptor antagonist, kisspeptin-234, indicating that\nkisspeptin signaling within the hippocampus is mediating these cognitive\nbenefits ( 448 ).\nFurthermore, preclinical studies on kisspeptin have reported a range of\neffects on anxiety, including anxiogenic ( 449 – 451 ), anxiolytic ( 452 , 453 ), and neutral outcomes ( 454 ). Central administration of kisspeptin to male rats also had no\neffect on other stress-related behaviors, including locomotion, sleep, and\ngrooming ( 32 ). Acute administration of\nintravenous kisspeptin in clinical studies involving healthy heterosexual men\n( 261 ), as well as studies involving\nmen ( 267 ) and premenopausal women ( 264 ) with HSDD did not result in\nsignificant changes in psychometric measures of anxiety or circulating cortisol\nlevels. In healthy men, kisspeptin administration led to reductions in negative\nmood as assessed by psychometric testing, mirroring findings seen in rodents\n( 455 ), and enhanced activity in\nbrain structures related to the reward system, such as the hippocampus,\namygdala, and cingulate, in response to sexual images ( 261 ). Collectively, the evidence in humans suggests that\nkisspeptin might have antidepressant effects and a neutral impact on anxiety.\nTherefore, beyond its potential use in addressing symptoms in postmenopausal\nwomen, kisspeptin could potentially be used to treat patients experiencing mood\ndisorders and sexual dysfunction, which often co-exist. Preclinical data on the\nNKB pathway on anxiety remains incongruous, with some studies suggesting\npro-anxiety effects ( 456 ) and others\nindicating anxiolytic-like effects ( 457 , 458 ) in rodent models.\nRegarding depression, the NK3R agonist, aminosenktide, has demonstrated\nantidepressant activity in mice ( 459 ).\nIn conclusion, the potential roles of kisspeptin in modulating\ncognition, mood, and body weight represent a promising area for future research.\nA deeper understanding of these mechanisms may pave the way for targeted\ntherapies aimed at alleviating some of the most challenging symptoms of\nmenopause, as well as other psychosexual, mood, or cognitive conditions.\n\nSince the key physiological roles of kisspeptin and NKB in reproduction\ncame to light in 2003, developments in pre-clinical and translational research have\nsignificantly advanced our understanding of the contributions of these two\nneuropeptides to the functioning of the HPG axis. The unique position of kisspeptin\nand NKB within the hypothalamic neuroendocrine circuitry has garnered significant\ninterest for their potential applications in diagnosing and treating pubertal and\nreproductive disorders. Kisspeptin holds promise in distinguishing the causes of\ndelayed puberty, while measurement of serum kisspeptin levels can aid in diagnosing\nprecocious puberty and serve as marker for risk-stratification of pregnancy\ncomplications. Kisspeptin-based therapeutic avenues encompass various possibilities,\nincluding the restoration of pulsatile GnRH secretion in hypogonadal disorders like\nCHH, FHA, and hyperprolactinemia. Additionally, kisspeptin may serve as a safer\noocyte maturation trigger in IVF treatment and offer a treatment option for\ndistressing low sexual desire in HSDD.\nSimilarly, antagonism of the NKB pathway has emerged as a potential\ntherapeutic target for uterine disorders including uterine fibroids and\nendometriosis, and for PCOS given their ability to reversibly suppress, but not\nabolish, the HPG axis. Most significantly, within the last year, we have seen the\nbench to bedside translation of NK3R antagonist, Fezolinetant, for treatment of hot\nflashes in menopause.\nDespite the impressive advancements over the past two decades ( Figure 10 ) and the successful translation of the\nfirst-in-class NK3R antagonist, the reproductive biology of kisspeptin and NKB\nremains an intriguing research area with exciting potential therapeutic discoveries.\nThe rapid technological advancements in optogenetics, fiber photometry and\n in vivo  recordings of KNDy neuronal activity in freely moving\nanimals have provided detailed insights into KNDy neurons at a cellular level. The\nidentification of kisspeptin neurons in the human rostral hypothalamus and the\npositive estrogenic regulation of this neuronal population challenge long-held views\nregarding estrogen feedback in humans. Advancements in genetics and epigenetics have\nrevealed novel silencers and activators that fine-tune pubertal timing through\nactivation or repression of kisspeptin gene expression. Additionally, the\nintegration of metabolic homeostatic cues and the involvement of afferent,\nintermediary neurons have deepened our understanding of the intricate connections\nbetween metabolic states and the HPG axis, which hold broader physiological\nimplications beyond reproductive physiology.\nIn the field of reproductive endocrinology, significant unmet treatment\nchallenges persist including therapeutic options for hypogonadotropic hypogonadism\n(FHA). There is also a pressing need for oocyte maturation trigger with favorable\nOHSS safety profile and new osteoporosis treatments. Addressing and understanding\nthe distressing low sexual desire as seen in HSDD remains a challenge, while ongoing\ndevelopment and evaluation of different NK3R antagonists in menopausal hot flashes\nwill provide data on their long-term safety and efficacy profile in different\npatient groups. These challenges are driving ongoing translational research efforts,\nparticularly focusing on the kisspeptin and NKB pathways, paving the way for more\nadvancements in the years to come.","source_license":"CC-BY-4.0","license_restricted":false}