Current pharmacotherapy and future directions for neuroendocrine causes of female infertility

ABSTRACT Introduction Infertility is recognized as a major global health issue, often associated with significant psychological distress for affected couples. Causes of female infertility include endocrine conditions leading to oligo/anovulation, in addition to structural causes such as tubal, uterine, or peritoneal disorders. Pharmacological treatments, targeting pathways in the hypothalamic-pituitary-ovarian axis, can improve rates of ovulation, conception, pregnancy, and birth. Some existing therapeutic options are hindered by limited efficacy or by a non-physiological mechanism, which can risk excessive stimulation and treatment-related adverse effects. Therefore, there is a continued need for novel therapies to improve care for patients suffering with infertility. Areas covered In this review, the authors focus on endocrine causes of oligo/anovulation in women and on advances in assisted reproductive technology. Current pharmacological treatments and putative future therapeutic avenues in development to aid fertility in women are outlined. Expert opinion A deeper understanding of the reproductive neuroendocrine network governing hypothalamic gonadotropin-releasing hormone release can offer novel therapeutic targets for the treatment of female subfertility, leading to improved clinical outcomes, less invasive routes of administration, and decreased treatment-related side-effects. The ultimate aim of development in female subfertility is to offer therapeutic interventions that are effective, reproducible, associated with minimal risks, and have an acceptable route of administration.


Introduction
Infertility is defined as the inability to conceive after 12 months or more of regular unprotected sexual intercourse [1]. Infertility is recognized as a global health problem and is estimated to affect 12-30% of couples attempting to conceive [2] and may be attributed predominantly to the male (30%), the female (55%), or to a combination of both partners (40%) [3]. In a significant minority, despite investigations, the cause remains "unexplained" with no identified male or female cause (25%) [3].
Female factors leading to infertility are classed as ovulatory disorders (25%), tubal disorders (20%), and uterine or peritoneal disorders (10%) [3]. The National Institute for Health and Care Excellence (NICE) recommended that a couple who has not conceived after 12 months of unprotected sexual intercourse should be offered clinical assessment and investigation. Expedited specialist referral can be offered in cases where the woman is older than 36 years, where there are known clinical causes or predisposing factors for infertility, or where planned treatment may result in infertility e.g. cancer treatments [3].
Evaluation of infertility requires careful history and physical examination carried out in a suitably sensitive setting [3]. General advice for both partners includes limiting alcohol consumption, smoking cessation, maintenance of a healthy body weight, avoidance of tight-fitting underwear in men, and folic acid supplementation in women [3]. Investigations aim to establish the presence of ovulation, normal uterine anatomy, and patent fallopian tubes in women and normal semen parameters in men [4].
This review aims to focus on endocrine causes of oligo/ anovulation in women, describe current pharmacological treatment, and highlight potential future therapeutic avenues in development.

Ovulatory disorders
Ovulatory disorders account for one quarter of female infertility cases. The World Health Organization (WHO) classifies ovulatory disorders into three groups based on the measurement of serum gonadotropins (luteinizing hormone (LH) and follicle-stimulating hormone (FSH)) and sex-steroid levels (estradiol (E2) and progesterone) [5] [ Figure 1].

Group I: hypogonadotropic hypogonadism (HH)hypothalamic or pituitary failure
Hypogonadotropic hypogonadism (HH) accounts for ~10% of ovulatory disorders and is characterized by levels of gonadotropins (LH and FSH) that are insufficient to stimulate follicular development, thus leading to low E2 levels [6]. Three conditions included in WHO Group I ovulation disorders include functional hypothalamic amenorrhea (HA), congenital HH, and hypopituitarism [7]. HA is a functional condition, with normal anatomical assessment of the hypothalamus on imaging, in which there is insufficient energy available for healthy gonadotropin-releasing hormone (GnRH) pulsatile secretion. It is characterized by the triumvirate of low body weight, excessive exercise, and psychological stress, on a background of genetic susceptibility [8]. Congenital HH occurs due to variants in genes controlling GnRH neuronal migration or function. Hypopituitarism may occur in the setting of a pituitary adenoma, or hyperprolactinemia, which inhibits GnRH pulsatility [9]. If HH due to one of these causes occurs prior to completion of puberty, this may lead to pubertal delay and girls may present with primary amenorrhea. Cessation of periods during adulthood, after having initially been established, is termed "secondary amenorrhea."

Lifestyle interventions
Lifestyle interventions, including relaxation of dietary restrictions and/or reduction of exercise levels, can improve ovulation and conception rates in women with HA. The exact threshold of body weight at which HA occurs varies between individuals and is genetically predisposed but becomes progressively less prevalent at body mass index (BMI) of >24 kg/ m 2 [8]. Furthermore, a woman with HA may need to achieve a higher body weight to regain periods than that at which menses were lost [10]. Current guidance from the Endocrine Society recommends that ovulation induction (OI) should only be attempted when BMI is greater than 18.5 kg/m 2 in order to reduce risk of pregnancy complications including miscarriage, small for gestational age, prematurity, and delivery by Cesarean section [11].

Pulsatile GnRH ('GnRH pump')
GnRH is a decapeptide that stimulates GnRH receptors on the pituitary gland when given in a pulsatile manner but induces desensitization if administered persistently [12]. Thus, long-acting GnRH receptor agonists can be used to induce castrate levels of gonadotropins and sex steroids, of use in treatment of prostate cancer for example. However, to restore ovulation in GnRHdeficient states such as HA or congenital HH, GnRH needs to be given in a pulsatile manner as first described in 1978 [12]. Although the Endocrine Society guidelines recommend pulsatile GnRH therapy as the first-line treatment to induce ovulation in HH and HA [11], in practice, the specialist pumps required to provide pulsatile administration are not widely available [7,11].
Pulsatile GnRH typically results in ovulation in more than 90% of cycles, with pregnancy rates per cycle ranging from 18% to 32% [13]. As the pituitary-ovarian feedback mechanisms remain intact, pulsatile GnRH is more likely to avoid excessive stimulation and induce mono-follicular development, thereby reducing the risk of ovarian hyperstimulation syndrome (OHSS), or multiple pregnancies, in comparison to exogenous gonadotropin treatment [14].

Gonadotropin therapy
Injectable gonadotropin preparations are an alternative method to induce folliculogenesis, including in pituitary disorders, where pulsatile GnRH may be ineffective due to gonadotropin deficiency. Typically, in HA, LH levels are disproportionately reduced compared to FSH levels reflecting decreased GnRH pulsatility. According to the two-cell-two gonadotropin theory [15], both LH and FSH levels are needed to induce follicular growth with physiological sex-steroid production. Thus, in HA, gonadotropin preparations containing LH are preferred to sole FSH preparations [11,[16][17][18]. For example, human menopausal gonadotropin (hMG), which contains a mixture of FSH and LH in a 1:1 ratio provides higher E2 production, improved endometrial thickness, and ovulation rates compared to preparations that contain FSH alone [19,20]. Alternatively, recombinant LH (rLH) can be added if using a preparation with sole FSH action. The FSH to LH ratio can be prescribed in a 2:1 ratio to mimic physiological levels [19,21].

Clomiphene citrate (CC)
CC is a selective estrogen receptor modulator and antagonizes estrogen-induced negative feedback on the hypothalamus and pituitary gland leading to increased FSH secretion and thus restoration of folliculogenesis [22]. The estrogen-deficient state, already present in HA, means that there is less estrogen action to antagonize by CC and often leads to a poorer success rate in these women [23]. However, milder forms of HA with low normal LH and FSH levels along with detectable endogenous estrogen production can respond to prolonged CC protocols [7,11,24]. At present, there are no randomized trials that have evaluated CC use in infertility treatment in HH, but CC is less likely to be a suitable therapy for some such patients [11].
Dopamine agonists (DA) for hyperprolactinemia DA are first-line medical therapy in women with prolactinoma and those with ovulatory disorders secondary to hyperprolactinemia [3]. The Endocrine Society recommends cabergoline as the DA of choice as it is efficacious in normalizing prolactin levels and results in pituitary tumor shrinkage [25]. Systematic review and meta-analyses of hyperprolactinemic patients demonstrate that DAs are effective in resolving amenorrhea (median 78%; range 40-100%) and achieving pregnancy (53%; 10-100%) [25].

Article highlights
• Infertility is recognized as a global health problem and is estimated to affect 12-30% of couples attempting to conceive. • Some existing therapeutic options are hindered by limited efficacy, or by a non-physiological mechanism, which can risk excessive stimulation and treatment-related adverse effects. • Improved understanding of the reproductive neuroendocrine network governing hypothalamic GnRH release has offered novel therapeutic targets for the treatment of female subfertility. • Kisspeptin has been shown to increase gonadotropin levels and sex steroids in both men and women. Kisspeptin use has been investigated in the context of hypothalamic amenorrhoea, hyperprolactinemia, polycystic ovary syndrome, and as oocyte maturation trigger. As kisspeptin acts at a level higher than GnRH, even constant infusion of kisspeptin can induce pulsatile secretion of GnRH. Thus, the use of kisspeptin could relieve the technological challenge faced by the need to administer GnRH in a pulsatile manner. • The ultimate aim of development in female subfertility is to offer therapeutic interventions that are effective, reproducible, associated with minimal risks, and have an acceptable route of administration.
The most frequent adverse effects associated with DAs include nausea, vomiting, dizziness, orthostatic hypotension, and headaches [25]. In recent years, neuropsychiatric disturbances with the use of DAs especially with regards to impulse control disorders, which may include gambling, hypersexuality, compulsive shopping, and bingeeating, are increasingly recognized as being more common than previously acknowledged, affecting one in six patients with prolactinoma treated with DA [26]. These effects are postulated to result from DAs binding to dopamine receptors (D3) in the mesocorticolimbic pathway, which regulates behavior, pleasure, and addiction [27]. These neuropsychiatric side-effects appear not to be doserelated and if intolerant to DA, some patients can be offered surgical management for their prolactinoma.

Future therapeutic options
Kisspeptin A loss of function variant in the gene encoding for the kisspeptin receptor (KISS1R; previously known as GPR54) caused subfertility due to congenital HH was first described in two seminal papers in 2003 [28,29]. Its role in regulation of the hypothalamic-pituitarygonadal (HPG) axis was further established when a gain of function variant in KISS1R was shown to lead to central precocious puberty [30].
The KISS1 gene encodes a 145 amino acid precursor peptide that is proteolytically cleaved into shorter peptides, whereby the numbers of amino acids are denoted by their suffix e.g. kisspeptin-54 (KP54), kisspeptin-14 (KP14), and kisspeptin-13 (KP13) [31]. All these isoforms share a common C-terminal decapeptide sequence (KP10), which is necessary to activate the kisspeptin receptor [32][33][34]. Since then, exogenous administration of kisspeptin has been shown to increase gonadotropin levels and sex steroids in both men and women [35,36]. The hormonal milieu modulates the effect of kisspeptin on gonadotropin release, with the greatest LH rise being observed in women in the preovulatory phase of the menstrual cycle [36,37].
Animal models of HA suggest that KISS1 expression is decreased in the arcuate nucleus of the hypothalamus, which could represent part of the pathophysiology of HA. This in turn leads to decreased GnRH pulsatility and low LH and E2 levels. Thus, administration of kisspeptin could be used to treat women with HA in order to restore physiological GnRH secretion and reproductive health. On the first day of treatment with twice daily KP54 (6.4 nmol/kg) subcutaneous injections in women with HA, there was a potent increase in serum gonadotropin levels and E2, but this stimulation was markedly diminished by 2 weeks of treatment [38]. The reduction in endocrine stimulation with repeated administration suggests tachyphylaxis at the kisspeptin receptor (women remained responsive to GnRH) [38]. The tachyphylaxis appears to relate to both the dose and frequency of administration, such that extending the dosing interval to twice-weekly administration enabled persistent stimulation of the reproductive axis for 8 weeks [39]. Furthermore, as kisspeptin acts at a level higher than GnRH, even constant infusion of kisspeptin can induce pulsatile secretion of GnRH [40]. As continuous administration of kisspeptin can induce pulsatile GnRH secretion, many pumps currently used to provide basal insulin in patients with diabetes can be utilized for this purpose [41]. Thus, the use of kisspeptin could relieve the technological challenge faced by the need to administer GnRH in a pulsatile manner.
MVT-602 (previously known as TAK-448) is a nanopeptide kisspeptin receptor agonist developed through modification of KP10 to have increased stability, potency, and water solubility [42]. Recently, the pharmacokinetic, pharmacodynamic and endocrine profiles of MVT-602 were evaluated in women for the first time [43]. MVT-602 induced a prolonged duration of gonadotropin release in comparison to native KP54 but to a similar peak amplitude of LH secretion. Thus, overall, this resulted in a four-fold increase in the area under the curve of the LH rise [43]. This longer duration of gonadotropin release could mean that less frequent injections of MVT-602 could be sufficient and thus could potentially limit tachyphylaxis at the kisspeptin receptor during chronic stimulation protocols; however, further studies are required to demonstrate this. This, together with the observation that the gonadotropin response to MVT-602 was expedited and augmented in women with HA in comparison to healthy controls or those with PCOS, advocates its potential to induce folliculogenesis in women with HA. Further data is required before kisspeptin can be recommended for clinical use [11]; however, it could in future be used to offer an alternative method for restoring reproductive health in women with HA.
Daily kisspeptin administration has been shown to restore hypothalamic GnRH release, gonadotropin secretion, and ovulation, in a hyperprolactinemic mouse model [44]. This, together with diminished kisspeptin expression found in these models, suggests that kisspeptin neurons could form part of the pathogenesis of hyperprolactinemia-induced hypogonadotropic anovulation [44]. The same group studied the effect of KP10 in two women with chronic hyperprolactinemiainduced hypogonadotropic amenorrhea secondary to microprolactinomas that were resistant to cabergoline. This exploratory study demonstrated that KP-10 administration resulted in a significant increase in LH pulsatility, gonadotropin, E2, and inhibin B levels [45]. Kisspeptin infusion in the two patients studied was well tolerated, with no evidence of pituitary adenoma necrosis or hemorrhage, prolactin levels, and hypophyseal appearances on imaging both remained static following kisspeptin treatment [45]. Kisspeptin may therefore be therapeutic option for hyperprolactinemic women, such as those with a prolactinoma, who would like to conceive but are resistant or intolerant to dopamine agonists.

Recombinant leptin
Leptin is an adipokine hormone secreted in proportion to fat mass. Low levels of leptin are present in women with HA, due to low fat mass, and leptin deficiency impairs GnRH secretion. This finding led to interest in the use of exogenous recombinant leptin replacement to restore the reproductive axis in women with HA. Recombinant methionyl human leptin (r-metHuLeptin) administered to eight women with HA over a 3-month period resulted in an increase in LH pulse frequency, mean LH levels, E2 levels, inhibin B level, ovarian volume, follicular diameter, and number of dominant follicles [46]. Furthermore, three women (43%) had an ovulatory menstrual cycle and two (29%) developed preovulatory follicles with subsequent withdrawal bleeds [46]. Improvements in other hormone levels including fT 4 , fT 3 , IGF-1, and bone formation markers were also observed.
The same group conducted a further randomized, doubleblinded, placebo-controlled trial comparing daily subcutaneous injection of recombinant methionyl human leptin (metreleptin) treatment in women with HA over a 36-week study period [47]. Restoration of menses occurred in seven of ten women treated with metreleptin compared to two of nine subjects in the placebo group [47]. Ovulation occurred in over half of the women with restored menses in the metreleptin group and one subject became pregnant [47]. E2 and progesterone levels were significantly higher in the metreleptin-treated arm, but there were, however, no significant difference in testosterone, LH, FSH, and inhibin B levels between two groups [47].
The main limitations associated with recombinant leptin therapy were appetite suppression and weight loss, which is a particular concern when used in women with HA [48]. This drawback was reflected in the trial where dose adjustment of metreleptin was necessary to maintain stable body weights and one participant had to withdraw from the study because of persistent weight loss despite dose adjustments [47]. Furthermore, the ovulation rate in leptin treatment was still lower compared to GnRH and gonadotropin therapy [48], and therefore, metreleptin therapy has not gained traction for the treatment of subfertility in women with HA [11].

Naltrexone
Chronic stress in HA results in activation of the hypothalamicpituitary-adrenal (HPA) axis. Increased corticotropin-releasing hormone secretion from activation of the HPA axis leads to increased endogenous opioid and endorphins secretion, which in turn impairs pulsatile GnRH secretion [49]. Exogenous opiates, when administered, also result in impaired gonadotropin secretion [50]. Opiate antagonists such as naltrexone and naloxone have been shown to increase frequency and amplitude of pulsatile gonadotropin release in women with HA [51]. The transient rise in GnRH pulsatility and only occasional ovulation, however, limits the clinical applicability of long-acting opioid blockade in restoration of ovulation [52] .

Group II: Normogonadotropic anovulationhypothalamic pituitary dysfunction
Group II disorders involve hypothalamic-pituitary-ovarian axis dysfunction and are the most common type of oligo/anovulatory infertility [53], with polycystic ovary syndrome (PCOS) being the most common cause [54]. Women with PCOS have a relative FSH deficiency, such that increasing FSH levels may aid in the restoration of folliculogenesis and ovulation. This can be achieved by supplementation with preparations containing FSH, or alternatively by reducing negative feedback on the hypothalamus and pituitary from E2, by using estradiol receptor modulators or aromatase inhibitors, to thus increase gonadotropin levels [53]. Insulin sensitizers such as metformin or inositol may also alleviate insulin resistance and improve ovulation rates.

Lifestyle interventions
Modest weight loss (5-10%) for women with PCOS who have a BMI of greater than 30 kg/m 2 has been shown to restore ovulation, improve response to ovulation induction (OI) treatment, and improve pregnancy rates [3,53]. A Cochrane review concluded that lifestyle intervention improves the free androgen index, body weight, and BMI in women with PCOS. However, at present there are still no studies to demonstrate the effect of lifestyle changes on live birth, miscarriage, or menstrual regularity [55].

Aromatase inhibitors
Aromatase inhibitors block the conversion of testosterone to E2, thus reducing E2-induced negative feedback on the hypothalamus and pituitary and thereby increasing FSH production [56]. A 5-day course of once daily letrozole at 2.5-7.5 mg can be used for OI in PCOS [56]. Historical concerns have suggested an increased risk of congenital abnormalities with letrozole, however recent studies have allayed these fears [57]. Indeed, higher pregnancy rates, lower rates of multiple pregnancies, and OHSS have been reported with letrozole than CC [57]. Thus, letrozole is increasingly recommended as first-line agent for OI in PCOS, including in the 2018 international evidence-based guidelines on PCOS management [58]. However, at present the use of letrozole for OI is off-license and is yet to be approved by the US Food and Drug Administration (FDA). A Cochrane review of 42 randomized clinical trial on the use of letrozole as OI agent in PCOS demonstrated that the use of letrozole resulted in higher birth rates (odds ratio (OR) 1.68, 95% confidence interval (CI) 1.42 to 1.99; n = 2954), pregnancy rates (OR 1.56, 95% CI 1.37 to 1.78; n = 4629), but with no difference in OHSS rates, miscarriage rates, and multiple pregnancy rates compared to CC [59].

Anti-estrogens
Experience with CC use for OI in PCOS dates back nearly 60 years [60]. A 5-day course of CC of 50-150 mg daily, incrementing with each cycle until ovulation is achieved, remains the first-line treatment of anovulatory PCOS in multiple guidelines and consensus statements [3,54,61], although as mentioned above, many are now transitioning to recommending letrozole as first-line [58]. CC has selective estrogen receptor modulating activity and has mixed agonistic and antagonistic activity. CC is a racemic compound of two stereoisomers: zuclomiphene and euclomiphene, the first has a relatively short half-life (5 days), while the latter has extended clearance of at least 6 weeks [22]. CC binding to E2 alpha receptors in the hypothalamus and pituitary gland results in reduced estrogen negative feedback and leads to increased FSH secretion, thus promoting follicular growth [62].
Ovulation rates and pregnancy rates with CC use are estimated around 73% and 36%, respectively, per cycle [63]. An unwanted impact on implantation by CC may be attributable to its anti-estrogenic action on the endometrium, cervical mucus, and LH hypersecretion [62]. Multiple follicle development is a risk with CC if FSH stimulation is excessive and higher order pregnancies are estimated at around 8-13% [63], which carries additional risks to both mother and baby. NICE guideline recommends that ultrasound monitoring during at least the first cycle of CC treatment is performed to ensure that risk of multiple pregnancies is minimized [3].
After the dose of CC that can induce ovulation is identified, treatment is continued for six cycles [3], and the dose is not altered unless ovulatory response is lost [62]. Failure to ovulate after CC termed "clomiphene resistance" occurs in 15-40% [64]. Factors affecting CC success in inducing ovulation and resultant live birth include BMI, hyperandrogenemia, and cycle history (oligomenorrhea have better success than amenorrhea) [65]. Pre-treatment (with agents such as ketoconazole and oral contraceptive pill) and medical adjuncts (such as bromocriptine, hormonal supplementation, hCG, and dexamethasone) have been trialed to address clomiphene resistance. However, as these are usually single randomized controlled trial, there are insufficient data to support their use, although addition of dexamethasone as medical adjunct to CC in CC-resistant women has demonstrated benefit to both ovulation and pregnancy rates [66,67]. Tamoxifen has also been used for OI; it has a similar mode of action to CC but is used much less frequently than CC [64]. Second-line treatments as recommended by NICE after CC include combined treatment with CC and metformin, gonadotropin treatment, or laparoscopic ovarian drilling [3].

Insulin sensitizers
Metformin, an oral biguanide, is widely used to improve insulin resistance and the resultant hyperinsulinaemia [56]. Systematic review and meta-analysis of randomized controlled trials demonstrate that metformin monotherapy is effective at inducing ovulation compared to placebo with OR of 3.88 (95% CI 2.25 to 6.69) [68]. Combined CC and metformin treatment leads to improved ovulation rates compared to CC monotherapy alone, with an OR of 4.41 (95% CI 2.37 to 8.22) [68]. Analysis of live birth rates shows that metformin may improve live birth rates compared to placebo (OR 1.59, 95% CI 1.00 to 2.51), but it is unclear if this effect is still seen when metformin is used in combination with CC [69]. NICE recommends the use of metformin, both as monotherapy, or in combination with CC, as first-line therapy [3].

Neurokinin 3 (NK3) receptor antagonists
The hypothalamic neurons expressing Kisspeptin, Neurokinin B, and Dynorphin A (KNDy) act in an auto-/para-crine manner to modulate pulsatile GnRH secretion [70]. KNDy neurons are believed to be the GnRH pulse generator governing the release of GnRH and in turn gonadotropins. Hypothalamic GnRH pulsatility is increased in PCOS, from every 90 minutes to every 60 minutes [70], independent from alteration in sexsteroid levels. Increased GnRH pulsatility favors LH secretion from pituitary gonadotrophs and ultimately results in elevated androgen levels [8]. NK3 receptor antagonists are postulated to reduce GnRH pulsatility, and in turn LH levels and subsequently androgen levels [70]. A recent proof-of-concept study demonstrated that treatment with NK3 receptor antagonist, fezolinetant, for 12 weeks in women with PCOS resulted in significant reductions of serum testosterone, LH, FSH, and LH: FSH ratio compared to placebo [71]. The relatively short study duration likely contributed to the inability to assess changes in clinical outcome measures such as regulation of menses or ovulation [71]. Nonetheless, the biochemical and hormonal changes, including improved LH:FSH ratio, makes NK3 receptor antagonists a potential therapeutic option to address the increased LH pulsatility central to PCOS pathogenesis and thus provides an additional potential avenue for PCOS treatment.

Group III: hypergonadotropic hypogonadism
Premature ovarian insufficiency (POI) is the primary ovarian dysfunction associated with oligo/amenorrhea and hypergonadotropic hypogonadism occurring in women under the age of 40 years [72]. Ovarian function is intermittent and unpredictable, such that ovulation and spontaneous pregnancies remain possible but overall conception rates with treatment are low [73]. Oocyte donation is an established option for fertility in POI in both NICE and the European Society of Human Reproduction and Embryology (ESHRE) guidelines, as at present there are no interventions that have reliably increased ovarian activity and natural conception rates [3,74]. Treatments including hormonal replacement therapy (for estrogen deficiency and bone protection), in vitro fertilization (IVF), in vitro maturation, and stem cell therapy have been explored; however, overall treatment of subfertility in women with POI remains challenging [75]. Table 1 and Figure 2 summarize current treatment and future therapeutic agents in ovulation disorders.

Endometriosis
Endometriosis is a chronic, estrogen-dependent, inflammatory disorder that causes chronic non-menstrual pelvic pain, dysmenorrhea, dyspareunia, and subfertility [76]. Current available medical therapy including analgesia (nonsteroidal antiinflammatory drugs), progestin-containing oral contraceptives, injectable depot formulations of GnRH agonists, and the emerging group or oral GnRH antagonists (elagolix [76], linzagolix [77], and relugolix [78]) aims to alleviate endometriosis-related pain rather than to improve fertility [79]. The management of endometriosis-related subfertility as recommended by NICE includes involvement of a multidisciplinary team and for surgical treatment if needed [80].

ASSISTED REPRODUCTIVE TECHNOLOGY (ART)
IVF is an effective tool to achieve conception with ~69,000 IVF cycles conducted in the UK in 2019 [81]. NICE recommends against the use of OI agents in women with unexplained infertility and states that IVF should be considered and offered to couples with unexplained infertility who are still not able to conceive after 2 years [3].
During IVF treatment, a pharmacological dose of FSH is administered to induce folliculogenesis [82]. Avoidance of premature ovulation is achieved through the use of a GnRH antagonist or continuous administration of a GnRH agonist (GnRHa) to downregulate the GnRH receptor [82]. Oocyte maturation and subsequent ovulation are controlled by the precise timing of LH-like exposure by the "trigger" to simulate the physiological mid-cycle LH surge. The triggers of oocyte maturation currently used in IVF protocols include human chorionic gonadotropin (hCG) or GnRH agonist (GnRHa).
The protocol used to prevent premature ovulation dictates the choice of trigger that can be used to induce oocyte maturation. GnRH antagonist co-treated protocols utilize competitive antagonism of the GnRH receptor, such that its inhibitory effect can be overcome by a GnRH agonist which can therefore be used as a trigger of oocyte maturation [82]. By contrast, in GnRH agonist co-treated protocols, only hCG can be used to induce oocyte maturation, as the GnRH receptor remains desensitized [82]. LHlike exposure not only provides a critical step in IVF protocols, but the choice of trigger also determines the risk of OHSS [83]. OHSS is a serious and potentially fatal iatrogenic complication, with a broad spectrum of clinical manifestations ranging from ovarian enlargement, to third space extravasation, intravascular volume depletion, hemoconcentration, and ultimately multi-organ failure [84]. The pharmacological use of hCG (used in more than three quarters of IVF cycles) [85], in particular is the major cause of OHSS  [83], due to its long half-life, high LH receptor activity, and long duration of intracellular effect [82]. Alternative trigger agents capable of inducing oocyte maturation in IVF protocols by mimicking the physiological LH surge are therefore needed to improve safety of treatment, particularly in populations at high risk of OHSS, such as those with PCOS [86].

GnRHa as oocyte maturation trigger
GnRHa, through induction of LH and FSH secretion, have also been used as a single agent or administered in addition to hCG in 'double' or 'dual' trigger protocols. The LH surge attained through GnRHa alone results in a short duration of LH secretion, which although safer with regards to the risk of OHSS, also leads to corpus luteum deficiency and a defective luteal phase [87]. Intensive E2 and progesterone [88], adjuvant low dose hCG [89,90] and recombinant LH [87], have been used for luteal phase support to try to maintain pregnancy rates [87].
Kisspeptin as oocyte maturation trigger Administration of kisspeptin was shown to induce an LH surge in gonadotropin-treated rats sufficient to induce ovulation to a similar degree as hCG [91]. A proof-of-concept study in women demonstrated that a single subcutaneous injection of kisspeptin-54 (KP54) is able to trigger oocyte maturation in infertile women and successfully result in live birth [92]. KP54 has also been successfully used to induce ovulation in women at high risk of OHSS, with favorable oocyte maturation, implantation rates, clinical pregnancy rate, and live birth, with no clinically significant OHSS [86,93]. The odds of severe OHSS were reduced by 33.6 times when kisspeptin was used to induce oocyte maturation compared with that of hCG [94]. Furthermore, two injections of KP54 administered 10 hours apart further improved oocyte maturation without inducing OHSS. Therefore, longer acting kisspeptin receptor agonists could be suitable as triggers of oocyte maturation.

Future patient-friendly IVF protocols
At present, IVF treatment involves multiple injections usually on an at least once daily basis for several weeks, especially if injectable progesterone preparations are used for luteal phase support. In future, with the advent of oral FSH analogues [95], oral GnRH antagonists [77], and the demonstration of efficacy of oral dydrogesterone for luteal phase support [96], the number of injections needed to complete an IVF protocol can be significantly reduced.

EXPERT OPINION
Development of pharmacological treatments and assisted reproductive techniques have contributed to improvements in ovulation, conception, and live birth rates in women with subfertility. Existing therapeutic options can be hindered by limited efficacy, side-effects, e.g. impulse control disorder with DA use [26], and OHSS with hCG to trigger oocyte maturation [84]. Limited commercial availability means that more physiological treatments such as pulsatile GnRH therapy, although recommended as the first-line treatment to induce ovulation in HH and HA [11], in practice is not widely available. Current hormonal treatments, including GnRH pump therapy, gonadotropins, and those used as oocyte maturation triggers in IVF treatment, use subcutaneous injection as the predominant route of administration, which can be uncomfortable for patients and affect adherence to treatment. The ultimate aim of development in female subfertility is therefore to offer therapeutic interventions that are effective, reproducible, associated with minimal risks, and have an acceptable route of administration. Over the last 20 years, improved understanding of the reproductive neuroendocrine networks [97] have offered novel therapeutic agents for the treatment of subfertility, utilizing the kisspeptin and neurokinin B pathways. Therapeutic interventions in development aim to improve clinical outcomes and address the aforementioned issues particularly around safety profile with regards to OHSS [94]. Their clinical development and the use of alternate agents acting on their receptors, such as kisspeptin receptor agonists, can aid in the translation of these peptides for patient benefit.
The heterogeneity of the conditions underlying female subfertility makes therapeutic intervention in this area challenging. Nonetheless improved understanding of the reproductive axis has led to a plethora of new research resulting in the development of novel potential therapeutic avenues, with the aim to improve clinical outcomes and alleviate the psychological distress associated with subfertility in the coming years.

Funding
This work is supported by grants from the National Institute for Health Research (NIHR) and supported by the NIHR/Wellcome Trust Imperial Clinical Research Facility and the NIHR Imperial Biomedical Research Centre. The Section of Endocrinology and Investigative Medicine of Imperial College London was funded by grants from the Medical Research Council (MRC), the Biotechnology and Biological Sciences Research Council (BBSRC), the NIHR and was supported by the NIHR Biomedical Research Centre Funding Scheme. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.