madman
Super Moderator
Male infertility and gonadotropin treatment: What can we learn from real-world data? (2022)
Sandro C. Esteves, Arnold P.P. Achermann, Manuela Simoni, Daniele Santi, Livio Casarini
Abstract
Gonadotropin therapy to treat specific male infertility disorders associated with hypogonadotropic hypogonadism is evidence-based and effective in restoring spermatogenesis and fertility. In contrast, its use to improve fertility in men with idiopathic oligozoospermia or nonobstructive azoospermia remains controversial, despite being widely practiced. The existence of two major inter-related pathways for spermatogenesis, including FSH and intratesticular testosterone, provides a rationale for empiric hormone stimulation therapy in both eugonadal and hypogonadal males with idiopathic oligozoospermia or nonobstructive azoospermia. Real-world data (RWD) on gonadotropin stimulating for these patient subsets, mainly using human chorionic gonadotropin and follicle-stimulating hormone, accumulated gradually, showing a positive therapeutic effect in some patients, translated by increased sperm production, sperm quality, and sperm retrieval rates. Although more evidence is needed, current insights from RWD research indicate that selected male infertility patients might be managed more effectively using gonadotropin therapy, with potential gains for all parties involved.
Introduction
Male infertility is a disease of the male reproductive system with various non-mutually exclusive causes and contributory factors, encompassing congenital and genetic factors, anatomical dysfunctions, hormonal disorders, ejaculatory dysfunctions, genital tract infections, immunological abnormalities, chronic diseases, cancer and related treatments, gonadotoxin exposure, inadequate lifestyle, and sexual disorders incompatible with intercourse [1-9].
Global estimates indicate that male factors, alone or combined with female factors, contribute to at least 50% of reported infertility cases and that over 50 million men have infertility worldwide [10]. In many cases, the factor(s) impairing male fertility can be identified through an andrological evaluation and treated or alleviated, positively impacting the couple's pregnancy prospects [11-15]. However, only about half of the men facing infertility seek medical assistance in developed countries [16]. A well-conducted andrological evaluation is critical to achieving these goals and includes a detailed medical and reproductive history of the patient, a physical examination, and at least two routine semen analyses carried out in a specialized andrology laboratory [1,7,17]. The second-line investigations (such as hormonal assessment, sperm functional tests, genetic analysis, and imaging studies) might be necessary and are based on the clinical and semen analysis findings [2,9,15,18-22].
Hormonal therapy has been integral to male infertility treatment options [23]. The rationale of this approach relates to the critical regulatory role of the hypothalamic-pituitary-gonadal (HPG) axis on spermatogenesis and the common knowledge that hormonal abnormalities are potentially treatable causes of male infertility [7,11,23-25]. Indeed, the use of hormonal therapy to treat specific endocrine disorders (such as hypogonadotropic hypogonadism and hyperprolactinemia) is well-established and evidence-based [7,24]. The administration of exogenous human chorionic gonadotropin (hCG), alone or combined with exogenous follicle-stimulating hormone (FSH), restores spermatogenesis to varying degrees in up to 90% of patients with hypogonadotropic hypogonadism, with reported pregnancy rates of up to 65% (natural or assisted) [7,26,27]. In these patients, sperm output is usually higher when hCG is combined with FSH (vs. hCG alone), particularly in men with congenital forms of hypogonadotropic hypogonadism [7,26-28]. Despite that, data from randomized controlled trials (RCTs) comparing drugs and regimens in men with hypogonadotropic hypogonadism are lacking.
By contrast, the use of hormonal therapy to improve fertility in men with idiopathic oligozoospermia or nonobstructive azoospermia (NOA) remains controversial [23], despite being widely practiced. Surveys conducted among urologists indicate that 65-87% of practitioners routinely prescribe drugs targeting the HPG axis, including selective estrogen receptor modulators (SERMs), aromatase inhibitors, hCG, FSH, and human menopausal gonadotropin (hMG) [29,30]. Moreover, a multicenter study conducted in Italy where the National Medicines Agency approves FSH therapy for infertile men with idiopathic infertility and FSH levels <8 IU/L, independently of semen parameters showed that hormonal therapy is prescribed to 55% of eligible patients [31]. The existence of two major pathways for spermatogenesis, including the supraphysiological FSH stimulation and intratesticular testosterone (ITT) production boosting, has provided a rationale for empiric hormone stimulation therapy in both eugonadal and hypogonadal males with idiopathic oligozoospermia or NOA [28,32].
Notwithstanding these observations, large-scale RCTs on the matter concerned are lacking, and knowledge about side effects and reproductive outcomes after the treatment is scanty, precluding conclusive clinical recommendations. Accordingly, major urological/andrological guidelines do not currently advocate the routine use of empiric hormonal therapy for spermatogenesis stimulation in men with idiopathic oligozoospermia or NOA [23,33]. Despite that, real-world data (RWD) on the use of hormonal stimulation for male infertility treatment, mainly through exogenous gonadotropins, have accumulated gradually.
Data from observational studies may represent a valid study type to acquire knowledge about the potential role of hormonal therapy in specific male infertility conditions [34]. RWD may also present an opportunity for obtaining data on patients with characteristics outside those typically required for trial eligibility [35-37]. RWD allows for assessing how therapy affects a heterogeneous population, generating hypotheses for testing in RCTs. Also, it adds information for evaluating trial feasibility by examining the impact of planned inclusion/exclusion criteria in the relevant population, both within a geographical area and at a particular trial site [35]. Informing prior probability distributions and identifying prognostic indicators or patient baseline characteristics for enrichment or stratification are other advantages of RWD observational studies [38,39].
In this review, we summarize the evidence concerning the use of hormonal therapy with exogenous gonadotropins for male infertility treatment, focusing on men with idiopathic oligozoospermia and those with NOA. We also review the essential knowledge about spermatogenesis hormonal control, summarize the recent animal and human data on the molecular mechanisms of gonadotropin action, and discuss the biological plausibility of gonadotropin therapy and its potential clinical utility, focusing on semen and pregnancy data. Lastly, we appraise the knowledge gaps and suggest a path for future research.
Spermatogenesis hormonal regulation
Spermatogenesis consists of the diploid spermatogonial proliferation and differentiation into haploid spermatozoa [25,40]. The process occurs in close contact with the Sertoli cells, the male counterpart of granulosa cells, located within testicular seminiferous tubules, and is regulated primarily by FSH and luteinizing hormone (LH)-driven testosterone [41]. The pulsatile secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus stimulates the release of pituitary FSH and LH. Inhibin B and estradiol are the primary inhibitors of FSH secretion, but many other pituitary-regulatory proteins, such as activin and follistatin, have been implicated in FSH secretion and action [25,28,40].
Follicle-stimulating hormone
The FSH is a heterodimer displaying an a subunit, common to all gonadotropins and the cognate molecule thyroid-stimulating hormone (TSH), and a b subunit specific for its receptor (FSHR) [42]. The a subunit is coded by the CGA gene located in chromosome 6 [43], while the FSHB gene codes the b subunit and is in chromosome 11 [44,45]. The gene transcriptional activity is primarily modulated by the presence of a single-nucleotide variation (SNV; formerly single-nucleotide polymorphism [SNP]) falling within the promoter FSHB gene [46], 211 base pairs upstream from the transcription start site. It is characterized by the G to T nucleotide change (211 G/T; rs10835638) and is associated with reduced serum FSH levels, decreased sperm count, and reduced testicular volume; patients carrying the T allele do not adequately upregulate FSH circulating levels to achieve full spermatogenesis [47]. Other SNVs, also located in chromosome 11 and within the FSHB gene, might also affect the FSH serum level and its action on spermatogenesis in men with idiopathic or unexplained infertility [48].
The number of spermatogonia modulates pituitary FSH secretion [49]. Normal FSH levels are typically associated with adequate spermatogonial quantity. By contrast, when spermatogonia are absent or their number is markedly reduced, the endogenous FSH levels increase [50]. Furthermore, low FSH levels are typically observed in men with primary pituitary dysfunction (e.g., primary hypogonadotropic hypogonadism) or in those using testosterone replacement therapy [7,49]. Along these lines, glycosylation partially regulates FSH activity, which provides several hormone variants naturally released by the pituitary [51]. In particular, hypoglycosylated FSH molecules have a higher bioactivity than the fully glycosylated variants [52], a feature linked to different glycoproteinereceptor interactions on the cell surface [53].
The FSH provides indirect structural and metabolic support for spermatogenesis via its receptors in the Sertoli cells [28,40,41]. It regulates structural genes involved in the organization of cell-cell junctions and genes required for the metabolism and transport of regulatory and nutritive substances from Sertoli to germ cells [40]. In the testis, endothelial FSHRs mediate FSH transport across the gonadal endothelial barrier [54]. The FSH also has a regulatory role in the Sertoli cell number that is critical to maintaining spermatogenesis. Specifically, FSH regulates the mitotic proliferation of Sertoli cells, supports their growth and maturation, and prompts the release of androgen-binding protein [41].
The amplitude of the Sertoli cell response to FSH is modulated by a common FSHR SNP, characterized by the A to G nucleotide change at position 2039 of the transcription start site, resulting in the asparagine to serine change at position 680 of the protein chain [54]. In particular, it is known that homozygous women for the serine receptor phenotype undergoing medically assisted reproduction are less sensitive to ovarian stimulation with exogenous FSH [55], and some evidence is also rising in men [56]. Another FSHR SNP likely modulating the response of Sertoli cells to FSH is the G to A nucleotide change at position 29 of the FSHR gene [57]. An in vitro study suggested that this SNP influences receptor mRNA transcriptional levels [58], although some clinical observations found no association with serum FSH levels and sperm parameters [59]. Reasonably, the pharmacogenomic response to FSH should account for the combined effect of several FSHB and FSHR SNPs [60].
*In summary, the primary role of FSH is to increase sperm quantity in synergy with ITT. Although an adequate FSH level is not mandatory for the completion of spermatogenesis in humans, its deficiency markedly reduces sperm production.
Luteinizing hormone
LH is a glycoprotein released by pituitary gonadotrope cells in a pulsatile fashion upon assembly of the a and LHb subunits; the molecule carries two glycosylations in the a and one in the b subunits [42,61]. The latter originated from mRNA transcripts coded by the LHB gene located in chromosome 19q13.32. The LHB gene is embedded in a genetic cluster containing six highly similar genes and pseudogenes (CGBs) coding the b subunit of hCG in pregnant women [62]. LHb and hCGb coding genes share about 95% of their identities and differ mainly for an additional sequence of CGBs, resulting in a 28-amino acid extension of the molecule and five additional glycosylation sites [63].
LH's primary function is to stimulate testosterone production by the Leydig cells. It acts through its transmembrane receptors (LHCGR) located in the Leydig cells [25]. It is common knowledge that, upon hormone binding, the receptor undergoes a conformational change linked to Gas protein dissociation from the bg dimer and triggers the activation of multiple signaling pathways. However, several decades ago, pioneering experiments performed with hCG used as an LHCGR ligand demonstrated that treatment with hCG leads to the intracellular increase in the cyclic adenosine monophosphate (cAMP) [64]. The second messenger, cAMP, is one of the critical players triggering steroidogenesis, which occurs as an event involving upstream activation of protein kinase A (PKA), phosphorylation of the transcription factor, cAMP-responsive element-binding protein (CREB), and upregulation of target genes coding steroidogenic enzymes [65].
Furthermore, recent studies have elucidated the role of other LHCGR intracellular interactors, such as Gq and Gai proteins and b arrestins [66]. These molecules mediate relatively marked LH-induced phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2), leading to survival and mitogenic signals [67,68]. Moreover, they support steroidogenesis [69] and are involved in receptor internalization and trafficking [70], inducing the intracellular routing of the receptor and transmitting sustained cAMP signaling [71]. Therefore, LH controls its receptor, inducing the sequestration from the cell membrane and changing its mode of action. Downstream LH-mediated events account for the production of growth factors (e.g., EGF-like) by Leydig cells and play a critical role in spermatogonial proliferation [72].
Testosterone
In males, testosterone is the primary circulating androgen. Over 95% of testosterone is secreted by the Leydig cells in the testes, which produce approximately 6-7 mg of testosterone daily [73]. The remaining circulating testosterone comes from the adrenal gland. The major substrate for testosterone synthesis is cholesterol [25]. Testosterone can be converted to estradiol by aromatase or dihydrotestosterone by 5a-reductase [74]. LH stimulates the transcription of genes that encode the enzymes involved in the steroidogenic pathways.
LH-driven testosterone is critical for spermatogenesis. ITT acts via its intracellular androgen receptors, present in the Sertoli cells, which secrete testosterone-dependent paracrine stimuli for the development of germ cells [73,75,76]. The primary function of ITT relates to the post-meiotic progression of round spermatids to mature sperm (spermiogenesis) [32,40,73,75]. ITT is also needed to transition from type A to type B spermatogonia and androgen receptor up-regulation, ultimately enhancing Sertoli cell function [73,75].
Biological plausibility of gonadotropin therapy for male infertility treatment in nonobstructive azoospermia and idiopathic oligozoospermia
Imbalances of reproductive hormones are common in men with infertility. Primary testicular disorders caused by congenital (cryptorchidism and spermatogenic failure), genetic (Klinefelter syndrome and Y chromosome microdeletions), anatomical (varicocele, testicular trauma, or torsion or infection), and neoplasms (e.g., testicular cancer and related gonadotoxic treatments) are associated with oligozoospermia or azoospermia; these factors can impair testosterone synthesis [2-4,23,77]. Indeed, biochemical hypogonadism, defined by low serum testosterone levels (e.g., <300-350 ng/dL or <10-12 nmol/L) [78], affects up to 50% of males with NOA [79,80]. As a result, the pituitary gland typically responds by increasing FSH and LH secretion to stimulate sperm and androgen production. A hormonal assessment, including FSH and testosterone levels as minimum standards, is recommended for patients presenting with oligozoospermia or azoospermia, erectile dysfunction, hypospermia (ejaculate volume <1 ml), endocrinopathies (current or previous), or testicular hypotrophy [1,4,11,23,77].
*Lessons from mouse knockout models
Gonadotropins’ molecular properties, action, and preparations
Effect of FSH on Sertoli cells
In Sertoli cells, FSH binding to its receptor triggers the activation of Gas protein and, in turn, of the adenylyl cyclase enzyme. These events lead to the intracellular increase in the second messenger cAMP [91], achieving a new equilibrium between its production and the degradation rate operated by phosphodiesterases (PDEs) [92]. cAMP binds PKA, resulting in the release of PKA catalytic subunits and indirectly mediating the phosphorylation of the ERK1/2 mitogen-activated protein kinase (MAPK) and the transcription factor CREB [91] (Fig. 1). The activation of these molecules is associated with mitogenic signals at the postnatal age [93]. Thus, FSH is a central regulator of intracellular cAMP concentration and a critical player in promoting Sertoli cell proliferation, synergically complemented by the action of androgens, activin A, and insulin-like growth factor (IGF)-I [94].
Interestingly, intracellular levels of cAMP are developmentally stage-dependent, because FSH efficacy in inducing second messenger production increases from birth to puberty [93]. The age-related rise of the Sertoli cell dependence from cAMP could be a requirement to maintain the effectiveness of FSH in activating proliferative signals. These events are accompanied by the decline in sensitivity to FSH, over time, inducing the production of other molecules associated with survival signals at the prenatal stage, such as phosphatidyl-inositide-3 kinase (PI3K), AKT, mammalian target of rapamycin (mTOR), and ribosomal protein S6 kinase beta-1 (p70S6K) [95].
Besides the well-known cAMP/PKA pathway, the last two decades provided new insights into the FSH mode of action in Sertoli cells, revealing a complex network of intracellular signaling cascades activated by the hormone. The FSHR coupling to Gai protein mediates the phosphorylation of ERK1/2 (pERK1/2) independently of cAMP, further supporting the FSH-dependent mitogenic signal [93,96]. Moreover, G protein-dependent pERK1/2 activation would occur very rapidly, within 1-5 min upon hormone-receptor binding, while prolonged ERK1/2 phosphorylation is sustained by other FSHR intracellular interactors, such as b-arrestins [97]. These molecules are scaffold proteins that sequentially dictate the kinetics of the ERK1/2 phosphorylation profile, cooperate with Gas protein in triggering p70S6K activation, and are involved in the trafficking of the receptor [98]. Therefore, b-arrestins exert several actions related to FSH functioning, thus playing an essential role in supporting male fertility. These issues are demonstrated by clinical observations revealing that b-arrestin-, but not cAMP-dependent signaling, is maintained in oligozoospermic men bearing the inactivating Ala189Val FSHR mutation [99]. Notably, after binding its ligand, FSHR undergoes internalization into intracellular compartments via pathways not involving b-arrestins and with regulatory functions of cAMP signaling [100].
Additionally, recent investigations found increasing relevance of micro-RNAs (miRNAs) in regulating FSH action. Studies in rats with suppressed FSH and testosterone activity described the miRNA network at spermiation as susceptible to hormonal control [101]. Two of the transcripts identified in this model were attributable to the phosphatase and tensin homolog (PTEN) enzyme mRNA, suggesting that they would be a target of FSH and/or androgen action. Indeed, Sertoli cell treatment with FSH in vitro enhanced PTEN protein levels massively within minutes, restoring terminal cell differentiation and proliferation [102]. Moreover, FSH treatment induced the decline of miR-92a-3p levels, which could be linked to increased FSHR expression [103]. These data suggest that the miRNA network might have a crucial role in the modulation of the FSH signaling in Sertoli cells, although knowledge about the miRNA-mediated regulation of Sertoli cell functioning remains limited.
Currently, both urinary and recombinant FSH preparations are available for clinical use. Urinary FSH preparations are marketed in lyophilized vials (typically containing 75 IU), whereas recombinant FSH preparations are available in both lyophilized vials (typically containing 75 IU) and pen devices (with doses varying from 300 IU to 900 IU, for fractionated use) [42]. Both preparations are administered subcutaneously; the lyophilized products must be reconstituted using sterile water before injection, whereas the pen devices are manufactured ready-to-use. After each injection, the peak serum FSH levels are reached within 10-12 h; the FSH levels then decline until the next injection. Recently, a long-acting recombinant FSH preparation was developed for subcutaneous use by combining recombinant human FSH with the hCG C-terminal peptide (CTP) [42]; the preparation has a plasma half-life of 65 h. Menotropins (e.g., hMG) extracted from the urine of postmenopausal women also provide lyophilized FSH for daily subcutaneous or intramuscular administrations; these preparations contain FSH and LH activity (mainly derived from the addition of hCG) in a 1:1 ratio, i.e., 75 of IU FSH and 75 IU of LH activity [42].
Recombinant FSH has a better safety and quality profile than its urinary counterparts [42,104]. In general, recombinant FSH preparations are purer than urine-derived FSH, and the incorporation of vial filling by mass virtually eliminated batch-to-batch variations and enabled accurate dosing. While FSH preparations have been extensively used for ovarian stimulation in women undergoing fertility treatments [38,39,42,43], data concerning their use in the context of male infertility treatment are less robust. The label indication for FSH use in males is typically restricted to hypogonadotropic hypogonadism (HH), which must be combined with hCG. In HH males, the doses vary from 75 IU to 225 IU, administered twice or thrice a week [7,26,27]. Similar doses have been prescribed for stimulating spermatogenesis in idiopathic oligozoospermic and hypergonadotropic hypogonadal or eugonadal NOA patients [3,27-29,32,56,105], as an off-label prescription in most countries.
Effect of LH (and hCG) on Leydig cells
Given the steroidogenic fingerprint of Leydig cells, which express LHCGRs to which both LH and hCG can bind, it is understandable that hCG found clinical utility in replacing LH functions [66]. Both glycoproteins share a common a and a specific b subunit, assembled to form a noncovalently linked heterodimer acting on specific leucine-rich repeats (LRRs) and rhodopsin-like G protein-coupled receptors (GPCRs). However, hCG is easily purified in high concentrations from the urine of pregnant women. In contrast, human LH of pituitary origin is difficult to obtain and lacks full biological activity because it embeds a proteolytic site leading to internally cleaved hormones [66]. Recombinant technology allowed the production of human recombinant LH, but it has only recently become available for clinical use in ovarian stimulation, and its use in male infertility has been anecdotal [42,66]. Additionally, hCG has a longer half-life than LH, making it more patient-friendly than LH, as less frequent injections would be required [42]. The differences in the half-lives of the two molecules relate mainly to oligosaccharide structures, especially O-linked [42,66]. Notably, hCG has a long carboxy-terminal segment with 24 amino acids containing four O-linked oligosaccharide sites, whereas LH has just one [42]. After intravenous administration, hCG has a terminal half-life of 23-31 h compared to 10-12 h of recombinant LH [106,107].
It is well known that hCG binds LHCGRs and has a higher steroidogenic potential than LH [66]. In vitro data from several primary and engineered cell models demonstrated that hCG acts preferentially as an inducer of cAMP [66,67,108] and intracellular Ca2þ increase [109] rather than ERK1/ 2 or AKT phosphorylation, markedly upregulating the transcription of genes coding steroidogenic enzymes. For example, in a mouse Leydig tumor cell line, the activation of gonadal steroid synthesis is more pronounced using hCG than LH [110]. Another in vitro study confirmed the higher cAMP production after the treatment of mouse Leydig cells with hCG vs. LH, although no differences were found in testosterone production [68]. Such experiments are characterized by cell lines expressing nonhuman receptors and need a prudent interpretation. Nevertheless, these data support the use of hCG for testosterone biosynthesis and spermatogenesis.
Given the relatively low availability of human Leydig cells for in vitro studies, assessing the differential action of LH and hCG has been provided by in vivo clinical studies. A trial conducted on 19 healthy men undergoing pituitary suppression with GnRH antagonist indicated that Leydig cell stimulation might be exerted by relatively low exogenous LH doses (787.5 IU/week), which induced similar testosterone levels to those obtained by administration of 5000 IU/week of hCG [111]. This finding may lead to the speculation that LH might be used at a lower dosage than hCG to induce androgen production in vivo, even if it is non-pulsatile, indicating a differential action of the two molecules in human males.
The results of the study mentioned above may be explained by the fact that LH exerts a preferential kinase-dependent activation of mitogenic signals [67,108], which could improve the metabolic state of the cell, positively impacting steroidogenesis (Fig. 1). This effect would be detectable in cells expressing the human receptor, which may discriminate between LH and hCG binding [112]. By contrast, in vitro studies using mouse receptor-expressing cells are unable to provide the complete pattern of LH- and hCG-specific intracellular pathways [68].
Three additional studies are noteworthy as they pioneered the comparisons between hCG and LH in the human male setting. Two decades ago, a case of an 18-year-old boy with a deletion of the exon 10- coded portion of the LHCGR gene was described [113]. The patient had Leydig cell hypoplasia, delayed pubertal development, small testes, high serum LH, and low testosterone levels. Interestingly, he was unresponsive to endogenous LH, but exogenous hCG administration restored androgen biosynthesis and spermatogenesis (Fig. 2). In vitro analysis confirmed the lack of LH-induced cAMP increase, while hCG activated the signal transduction pathway upon mutant receptor binding [114]. Although the clinical scenario described above is very rare, it highlights the capability of the LHCGR to discriminate between the two natural ligands (Fig. 2), possibly reflecting a different regulation of gametogenesis and placentation in humans. Most importantly, this case report suggested that hCG may have different actions than LH in male gonads.
Subsequently, in 2008, a randomized, single-blind study including 20 healthy eugonadal men aged 18-30 evaluated hCG's and LH's effect on Leydig cell function [115]. A subgroup received either vehicle alone or urinary hCG intramuscularly in doses varying from 50 to 5000 IU, while another group received recombinant hCG intramuscularly in a dose of 250 mcg (6500 IU) or recombinant LH intravenously in doses varying from 75 to 225 IU. A serial assessment of serum testosterone and estradiol was carried out to determine the response to stimulation. There was a dose-dependent increase in testosterone and estradiol levels with urinary hCG administration, with a peak observed at 48 h and 72 h after the injection of 500 IU and 5000 IU, respectively. The peaks in testosterone and estradiol obtained with urinary hCG (5000 IU) and recombinant hCG (6500 IU) did not differ. Serum LH levels increased dose-dependently after injection of recombinant LH; the peak value was obtained 30 min after injections, progressively dropping to reach basal values 6 h later. Similarly, a dose-dependent increase in testosterone (but not estradiol) was observed after recombinant LH administration, with a peak observed 6 h after administering 225 IU. The lack of a stimulatory effect on estradiol levels by recombinant LH is attributed to its lesser steroidogenic potency than hCG, probably explained by its short plasma half-life and the reversibility of its binding to the LHCGR. In this study, the highest serum testosterone levels attained within 5 h after the injection of either 150 or 225 IU recombinant LH correlated with the 48-h peak increase in testosterone in response to the injection of 50 IU recombinant hCG [115]. The results of this study confirm the ability of both recombinant hCG and recombinant LH to promote adequate androgen production in normal men.
Along these lines, a study comparing the effects of a daily, low (75 IU) dose of recombinant LH versus 75 IU hCG in a hypogonadotropic hypogonadal hypophysectomized man found no differences between the two hormones in terms of restoring the eugonadal status after 12-14 days of treatment [116]. These findings may be explained by the known mitotic effects of testosterone demonstrated in seminal vesicles of animal models [117], and other growth factors, such as estrogens, produced in vivo, which could have covered the gonadotropin-specific signal. In any case, more data are needed to elucidate the gonadotropin-specific action in human Leydig cells.
Historically, only hCG has been used in the context of male infertility, mainly for males with hypogonadism [7,26,118]. Urinary hCG preparations are currently marketed in lyophilized vials with 5000 or 10,000 IU for intramuscular use. In contrast, recombinant hCG is available in prefilled syringes or pen devices containing 250 mcg of pure hCGdequivalent to approximately 6750 IU of urinary hCG [42]. Like recombinant FSH preparations, recombinant hCG is purer than urine-derived hCG and has a better quality and safety profile than its urinary counterparts [42,104]. Nevertheless, data concerning recombinant hCG in the context of male infertility treatment are minimal, and its use remains off-label. By contrast, urinary hCG has a label indication to treat males with hypogonadism, including those with oligozoospermia. Given the shortage of urinary hCG availability in many countries, recombinant hCG must receive approval from regulatory agencies for use in male hypogonadism.
Clinical application
The rationale for using hCG therapy (as a surrogate for LH) off-label for treating male infertility conditions associated with hypogonadism relies on the fact that low ITT concentrations cause spermatogenesis disruption. In rodents, reductions of over 75% of the ITT concentration are incompatible with sperm maturation [119-121]. Furthermore, gonadotropin action is determined by its secretory pulses' frequency, amplitude, and duration [32,122-124]. In NOA males with elevated circulating levels of endogenous FSH and LH, the relative amplitudes of FSH and LH are low, leading to a paradoxically weak stimulation of Leydig and Sertoli cells [28,32,79,122-124]. Human studies have shown that hormonal therapy with hCG increases ITT and circulating testosterone in men with nonobstructive azoospermia [122,123]. hCG treatment also promotes spermatogonial DNA synthesis, as assessed by the expression of proliferating cell nuclear antigen (PCNA) [122]. Expression of the PCNA genes is associated with cell proliferation and, thus, with DNA synthesis during genome replication in the S phase of the cell cycle [122].
Nevertheless, it remains unclear what the optimal testosterone levels are for stimulating spermatogenesis and improving sperm retrieval rates (SRR) in NOA males. The assessment of ITT levels is challenging because measurements require testicular aspiration, which is an invasive procedure [76]. Measurement of circulating testosterone levels has been used as a proxy of ITT assessment because of the high correlation (r = 0.82) in the concentrations of both hormones in normal men [125]. It should be noted that ITT concentrations are markedly higher (~100-200x) than circulating testosterone concentrations [76,125], making direct extrapolations between intratesticular and circulating testosterone levels potentially inaccurate. For example, the correlations between ITT and testosterone levels are poor in men receiving testosterone replacement therapy because of its negative feedback on the hypothalamic-pituitary-gonadal axis [126]. Serum 17-hydroxyprogesteronedan intermediate steroid produced by the testicles and adrenal glands have been proposed as an alternative biomarker for evaluating ITT levels [127].
Furthermore, human and animal data suggest pathological desensitization of the FSHR caused by high circulating FSH levels [128-131]. It has been postulated that hormone therapy using GnRH or hCG could benefit these patients by suppressing the endogenous gonadotrophin levels and thereby overcoming Sertoli cell receptor desensitization caused by chronically raised circulating FSH levels [132,133].
Lastly, as previously discussed, FSH promotes cell proliferation when acting in synergy with ITT [40]. Transgenic murine studies have suggested that spermatogenesis can be stimulated by high FSH concentrations using exogenous FSH administration, even in the presence of low ITT [40,134]. Moreover, it has been suggested that a subset of men with idiopathic oligozoospermia and low-to-normal circulating FSH levels are FSH deficient as a consequence of reduced FSH activity, which depends on the amount of circulating FSH, its glycosylation, as well as the genetically determined expression levels and function of the FSHR [56,60,134,135]. Therefore, hormonal therapy with FSH might help improve sperm parameters in some patients, as shown in carriers of the FSHR p. N680S homozygous N [56]. Although limited, these data have provided a rationale for the off-label administration of exogenous FSH to males with idiopathic oligozoospermia.
Real-world data
Idiopathic oligozoospermia
Collectively, modest evidence indicates that exogenous FSH therapy might increase sperm quantity in men with idiopathic oligozoospermia, with an apparent positive effect on pregnancy rates. The current evidence is based on studies including few participants and using different treatment protocols and follow-up periods. Further high-quality research is needed to clarify the potential clinical role of FSH therapy in overcoming oligozoospermia and improving pregnancy rates. The optimal treatment regimen and duration and the role of pharmacogenomics in identifying the best candidates for treatment have also to be determined.
Nonobstructive azoospermia
The treatment protocol used at the ANDROFERT Clinic relies primarily on recombinant hCG used off-label to boost ITT production (Fig. 4). Our experience shows that most patients' circulating testosterone levels increase after hCG treatment. The increased testosterone levels reset some patients' elevated baseline FSH levels to normal levels. This is beneficial as the FSH reset might increase FSH receptors' expression and improve Sertoli cell function [123,123,132]. Notably, some patients exhibit a remarkable decline in circulating FSH levels during hCG treatment; we add recombinant FSH to the hCG regimen when the FSH levels drop below 1.5 IU/L. Patients are followed up with a monthly hormonal assessment, and an aromatase inhibitor is added during the treatment when the testosterone-to-estradiol ratio turns less than 10 [3,77,188,193].
Collectively, limited evidence, overwhelmingly based on observational studies and case series, suggests that gonadotropin therapy for males with NOA might increase sperm retrieval success rates. In a few cases, it was associated with the return of minimal sperm numbers to the ejaculate. Further high-quality studies are warranted to confirm whether gonadotropin therapy improves sperm retrieval success rates in this population. Additionally, more data are needed to identify the NOA patients who might benefit the most from gonadotropin therapy and the optimal treatment regimen and duration.
Summary
RWD on gonadotropin stimulation for eugonadal and hypogonadal infertile males with idiopathic oligozoospermia or NOA indicates an overall positive therapeutic effect. FSH therapy in patients with idiopathic oligozoospermia, mainly using recombinant preparations, may increase sperm quantity and quality because of the FSH proliferative action on Sertoli cells, synergically complemented by the action of androgens and other growth factors. However, the effect is not universal, and patients’ specific genotypes might impact therapeutic effectiveness. Leydig cells express LHCGRs to which both LH and hCG can bind; thus, hCG preparations, mainly urine-derived, have been the drugs of choice to boost ITT production in eugonadal and hypergonadotropic hypogonadal men with NOA. Low ITT concentration is typical in men with NOA and intrinsic testicular pathology; it causes spermatogenesis disruption that hCG can revert. The increase in testosterone production by hCG might suppress the elevated endogenous gonadotrophin levels, overcoming the Sertoli cell receptor desensitization caused by chronically raised circulating FSH levels commonly seen in NOA males. Although the optimal testosterone level for stimulating spermatogenesis is unknown, RWD on hCG-based therapy in NOA males overwhelmingly based on small case series and observational studies has shown increased sperm retrieval rates (by 10-15%) than with no treatment, and in some cases, the return of small sperm numbers to the ejaculate. The most suitable NOA patients for gonadotropin therapy seem to be hypogonadal men with testicular histopathology showing maturation arrest (late stages) or hypospermatogenesis. Still, the evidence concerning the role of exogenous gonadotropins in male infertility treatment is limited and needs validation by large-scale, well-designed studies. Further research is required to identify the best candidates for treatment and optimal gonadotropin regimens.
Sandro C. Esteves, Arnold P.P. Achermann, Manuela Simoni, Daniele Santi, Livio Casarini
Abstract
Gonadotropin therapy to treat specific male infertility disorders associated with hypogonadotropic hypogonadism is evidence-based and effective in restoring spermatogenesis and fertility. In contrast, its use to improve fertility in men with idiopathic oligozoospermia or nonobstructive azoospermia remains controversial, despite being widely practiced. The existence of two major inter-related pathways for spermatogenesis, including FSH and intratesticular testosterone, provides a rationale for empiric hormone stimulation therapy in both eugonadal and hypogonadal males with idiopathic oligozoospermia or nonobstructive azoospermia. Real-world data (RWD) on gonadotropin stimulating for these patient subsets, mainly using human chorionic gonadotropin and follicle-stimulating hormone, accumulated gradually, showing a positive therapeutic effect in some patients, translated by increased sperm production, sperm quality, and sperm retrieval rates. Although more evidence is needed, current insights from RWD research indicate that selected male infertility patients might be managed more effectively using gonadotropin therapy, with potential gains for all parties involved.
Introduction
Male infertility is a disease of the male reproductive system with various non-mutually exclusive causes and contributory factors, encompassing congenital and genetic factors, anatomical dysfunctions, hormonal disorders, ejaculatory dysfunctions, genital tract infections, immunological abnormalities, chronic diseases, cancer and related treatments, gonadotoxin exposure, inadequate lifestyle, and sexual disorders incompatible with intercourse [1-9].
Global estimates indicate that male factors, alone or combined with female factors, contribute to at least 50% of reported infertility cases and that over 50 million men have infertility worldwide [10]. In many cases, the factor(s) impairing male fertility can be identified through an andrological evaluation and treated or alleviated, positively impacting the couple's pregnancy prospects [11-15]. However, only about half of the men facing infertility seek medical assistance in developed countries [16]. A well-conducted andrological evaluation is critical to achieving these goals and includes a detailed medical and reproductive history of the patient, a physical examination, and at least two routine semen analyses carried out in a specialized andrology laboratory [1,7,17]. The second-line investigations (such as hormonal assessment, sperm functional tests, genetic analysis, and imaging studies) might be necessary and are based on the clinical and semen analysis findings [2,9,15,18-22].
Hormonal therapy has been integral to male infertility treatment options [23]. The rationale of this approach relates to the critical regulatory role of the hypothalamic-pituitary-gonadal (HPG) axis on spermatogenesis and the common knowledge that hormonal abnormalities are potentially treatable causes of male infertility [7,11,23-25]. Indeed, the use of hormonal therapy to treat specific endocrine disorders (such as hypogonadotropic hypogonadism and hyperprolactinemia) is well-established and evidence-based [7,24]. The administration of exogenous human chorionic gonadotropin (hCG), alone or combined with exogenous follicle-stimulating hormone (FSH), restores spermatogenesis to varying degrees in up to 90% of patients with hypogonadotropic hypogonadism, with reported pregnancy rates of up to 65% (natural or assisted) [7,26,27]. In these patients, sperm output is usually higher when hCG is combined with FSH (vs. hCG alone), particularly in men with congenital forms of hypogonadotropic hypogonadism [7,26-28]. Despite that, data from randomized controlled trials (RCTs) comparing drugs and regimens in men with hypogonadotropic hypogonadism are lacking.
By contrast, the use of hormonal therapy to improve fertility in men with idiopathic oligozoospermia or nonobstructive azoospermia (NOA) remains controversial [23], despite being widely practiced. Surveys conducted among urologists indicate that 65-87% of practitioners routinely prescribe drugs targeting the HPG axis, including selective estrogen receptor modulators (SERMs), aromatase inhibitors, hCG, FSH, and human menopausal gonadotropin (hMG) [29,30]. Moreover, a multicenter study conducted in Italy where the National Medicines Agency approves FSH therapy for infertile men with idiopathic infertility and FSH levels <8 IU/L, independently of semen parameters showed that hormonal therapy is prescribed to 55% of eligible patients [31]. The existence of two major pathways for spermatogenesis, including the supraphysiological FSH stimulation and intratesticular testosterone (ITT) production boosting, has provided a rationale for empiric hormone stimulation therapy in both eugonadal and hypogonadal males with idiopathic oligozoospermia or NOA [28,32].
Notwithstanding these observations, large-scale RCTs on the matter concerned are lacking, and knowledge about side effects and reproductive outcomes after the treatment is scanty, precluding conclusive clinical recommendations. Accordingly, major urological/andrological guidelines do not currently advocate the routine use of empiric hormonal therapy for spermatogenesis stimulation in men with idiopathic oligozoospermia or NOA [23,33]. Despite that, real-world data (RWD) on the use of hormonal stimulation for male infertility treatment, mainly through exogenous gonadotropins, have accumulated gradually.
Data from observational studies may represent a valid study type to acquire knowledge about the potential role of hormonal therapy in specific male infertility conditions [34]. RWD may also present an opportunity for obtaining data on patients with characteristics outside those typically required for trial eligibility [35-37]. RWD allows for assessing how therapy affects a heterogeneous population, generating hypotheses for testing in RCTs. Also, it adds information for evaluating trial feasibility by examining the impact of planned inclusion/exclusion criteria in the relevant population, both within a geographical area and at a particular trial site [35]. Informing prior probability distributions and identifying prognostic indicators or patient baseline characteristics for enrichment or stratification are other advantages of RWD observational studies [38,39].
In this review, we summarize the evidence concerning the use of hormonal therapy with exogenous gonadotropins for male infertility treatment, focusing on men with idiopathic oligozoospermia and those with NOA. We also review the essential knowledge about spermatogenesis hormonal control, summarize the recent animal and human data on the molecular mechanisms of gonadotropin action, and discuss the biological plausibility of gonadotropin therapy and its potential clinical utility, focusing on semen and pregnancy data. Lastly, we appraise the knowledge gaps and suggest a path for future research.
Spermatogenesis hormonal regulation
Spermatogenesis consists of the diploid spermatogonial proliferation and differentiation into haploid spermatozoa [25,40]. The process occurs in close contact with the Sertoli cells, the male counterpart of granulosa cells, located within testicular seminiferous tubules, and is regulated primarily by FSH and luteinizing hormone (LH)-driven testosterone [41]. The pulsatile secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus stimulates the release of pituitary FSH and LH. Inhibin B and estradiol are the primary inhibitors of FSH secretion, but many other pituitary-regulatory proteins, such as activin and follistatin, have been implicated in FSH secretion and action [25,28,40].
Follicle-stimulating hormone
The FSH is a heterodimer displaying an a subunit, common to all gonadotropins and the cognate molecule thyroid-stimulating hormone (TSH), and a b subunit specific for its receptor (FSHR) [42]. The a subunit is coded by the CGA gene located in chromosome 6 [43], while the FSHB gene codes the b subunit and is in chromosome 11 [44,45]. The gene transcriptional activity is primarily modulated by the presence of a single-nucleotide variation (SNV; formerly single-nucleotide polymorphism [SNP]) falling within the promoter FSHB gene [46], 211 base pairs upstream from the transcription start site. It is characterized by the G to T nucleotide change (211 G/T; rs10835638) and is associated with reduced serum FSH levels, decreased sperm count, and reduced testicular volume; patients carrying the T allele do not adequately upregulate FSH circulating levels to achieve full spermatogenesis [47]. Other SNVs, also located in chromosome 11 and within the FSHB gene, might also affect the FSH serum level and its action on spermatogenesis in men with idiopathic or unexplained infertility [48].
The number of spermatogonia modulates pituitary FSH secretion [49]. Normal FSH levels are typically associated with adequate spermatogonial quantity. By contrast, when spermatogonia are absent or their number is markedly reduced, the endogenous FSH levels increase [50]. Furthermore, low FSH levels are typically observed in men with primary pituitary dysfunction (e.g., primary hypogonadotropic hypogonadism) or in those using testosterone replacement therapy [7,49]. Along these lines, glycosylation partially regulates FSH activity, which provides several hormone variants naturally released by the pituitary [51]. In particular, hypoglycosylated FSH molecules have a higher bioactivity than the fully glycosylated variants [52], a feature linked to different glycoproteinereceptor interactions on the cell surface [53].
The FSH provides indirect structural and metabolic support for spermatogenesis via its receptors in the Sertoli cells [28,40,41]. It regulates structural genes involved in the organization of cell-cell junctions and genes required for the metabolism and transport of regulatory and nutritive substances from Sertoli to germ cells [40]. In the testis, endothelial FSHRs mediate FSH transport across the gonadal endothelial barrier [54]. The FSH also has a regulatory role in the Sertoli cell number that is critical to maintaining spermatogenesis. Specifically, FSH regulates the mitotic proliferation of Sertoli cells, supports their growth and maturation, and prompts the release of androgen-binding protein [41].
The amplitude of the Sertoli cell response to FSH is modulated by a common FSHR SNP, characterized by the A to G nucleotide change at position 2039 of the transcription start site, resulting in the asparagine to serine change at position 680 of the protein chain [54]. In particular, it is known that homozygous women for the serine receptor phenotype undergoing medically assisted reproduction are less sensitive to ovarian stimulation with exogenous FSH [55], and some evidence is also rising in men [56]. Another FSHR SNP likely modulating the response of Sertoli cells to FSH is the G to A nucleotide change at position 29 of the FSHR gene [57]. An in vitro study suggested that this SNP influences receptor mRNA transcriptional levels [58], although some clinical observations found no association with serum FSH levels and sperm parameters [59]. Reasonably, the pharmacogenomic response to FSH should account for the combined effect of several FSHB and FSHR SNPs [60].
*In summary, the primary role of FSH is to increase sperm quantity in synergy with ITT. Although an adequate FSH level is not mandatory for the completion of spermatogenesis in humans, its deficiency markedly reduces sperm production.
Luteinizing hormone
LH is a glycoprotein released by pituitary gonadotrope cells in a pulsatile fashion upon assembly of the a and LHb subunits; the molecule carries two glycosylations in the a and one in the b subunits [42,61]. The latter originated from mRNA transcripts coded by the LHB gene located in chromosome 19q13.32. The LHB gene is embedded in a genetic cluster containing six highly similar genes and pseudogenes (CGBs) coding the b subunit of hCG in pregnant women [62]. LHb and hCGb coding genes share about 95% of their identities and differ mainly for an additional sequence of CGBs, resulting in a 28-amino acid extension of the molecule and five additional glycosylation sites [63].
LH's primary function is to stimulate testosterone production by the Leydig cells. It acts through its transmembrane receptors (LHCGR) located in the Leydig cells [25]. It is common knowledge that, upon hormone binding, the receptor undergoes a conformational change linked to Gas protein dissociation from the bg dimer and triggers the activation of multiple signaling pathways. However, several decades ago, pioneering experiments performed with hCG used as an LHCGR ligand demonstrated that treatment with hCG leads to the intracellular increase in the cyclic adenosine monophosphate (cAMP) [64]. The second messenger, cAMP, is one of the critical players triggering steroidogenesis, which occurs as an event involving upstream activation of protein kinase A (PKA), phosphorylation of the transcription factor, cAMP-responsive element-binding protein (CREB), and upregulation of target genes coding steroidogenic enzymes [65].
Furthermore, recent studies have elucidated the role of other LHCGR intracellular interactors, such as Gq and Gai proteins and b arrestins [66]. These molecules mediate relatively marked LH-induced phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2), leading to survival and mitogenic signals [67,68]. Moreover, they support steroidogenesis [69] and are involved in receptor internalization and trafficking [70], inducing the intracellular routing of the receptor and transmitting sustained cAMP signaling [71]. Therefore, LH controls its receptor, inducing the sequestration from the cell membrane and changing its mode of action. Downstream LH-mediated events account for the production of growth factors (e.g., EGF-like) by Leydig cells and play a critical role in spermatogonial proliferation [72].
Testosterone
In males, testosterone is the primary circulating androgen. Over 95% of testosterone is secreted by the Leydig cells in the testes, which produce approximately 6-7 mg of testosterone daily [73]. The remaining circulating testosterone comes from the adrenal gland. The major substrate for testosterone synthesis is cholesterol [25]. Testosterone can be converted to estradiol by aromatase or dihydrotestosterone by 5a-reductase [74]. LH stimulates the transcription of genes that encode the enzymes involved in the steroidogenic pathways.
LH-driven testosterone is critical for spermatogenesis. ITT acts via its intracellular androgen receptors, present in the Sertoli cells, which secrete testosterone-dependent paracrine stimuli for the development of germ cells [73,75,76]. The primary function of ITT relates to the post-meiotic progression of round spermatids to mature sperm (spermiogenesis) [32,40,73,75]. ITT is also needed to transition from type A to type B spermatogonia and androgen receptor up-regulation, ultimately enhancing Sertoli cell function [73,75].
Biological plausibility of gonadotropin therapy for male infertility treatment in nonobstructive azoospermia and idiopathic oligozoospermia
Imbalances of reproductive hormones are common in men with infertility. Primary testicular disorders caused by congenital (cryptorchidism and spermatogenic failure), genetic (Klinefelter syndrome and Y chromosome microdeletions), anatomical (varicocele, testicular trauma, or torsion or infection), and neoplasms (e.g., testicular cancer and related gonadotoxic treatments) are associated with oligozoospermia or azoospermia; these factors can impair testosterone synthesis [2-4,23,77]. Indeed, biochemical hypogonadism, defined by low serum testosterone levels (e.g., <300-350 ng/dL or <10-12 nmol/L) [78], affects up to 50% of males with NOA [79,80]. As a result, the pituitary gland typically responds by increasing FSH and LH secretion to stimulate sperm and androgen production. A hormonal assessment, including FSH and testosterone levels as minimum standards, is recommended for patients presenting with oligozoospermia or azoospermia, erectile dysfunction, hypospermia (ejaculate volume <1 ml), endocrinopathies (current or previous), or testicular hypotrophy [1,4,11,23,77].
*Lessons from mouse knockout models
Gonadotropins’ molecular properties, action, and preparations
Effect of FSH on Sertoli cells
In Sertoli cells, FSH binding to its receptor triggers the activation of Gas protein and, in turn, of the adenylyl cyclase enzyme. These events lead to the intracellular increase in the second messenger cAMP [91], achieving a new equilibrium between its production and the degradation rate operated by phosphodiesterases (PDEs) [92]. cAMP binds PKA, resulting in the release of PKA catalytic subunits and indirectly mediating the phosphorylation of the ERK1/2 mitogen-activated protein kinase (MAPK) and the transcription factor CREB [91] (Fig. 1). The activation of these molecules is associated with mitogenic signals at the postnatal age [93]. Thus, FSH is a central regulator of intracellular cAMP concentration and a critical player in promoting Sertoli cell proliferation, synergically complemented by the action of androgens, activin A, and insulin-like growth factor (IGF)-I [94].
Interestingly, intracellular levels of cAMP are developmentally stage-dependent, because FSH efficacy in inducing second messenger production increases from birth to puberty [93]. The age-related rise of the Sertoli cell dependence from cAMP could be a requirement to maintain the effectiveness of FSH in activating proliferative signals. These events are accompanied by the decline in sensitivity to FSH, over time, inducing the production of other molecules associated with survival signals at the prenatal stage, such as phosphatidyl-inositide-3 kinase (PI3K), AKT, mammalian target of rapamycin (mTOR), and ribosomal protein S6 kinase beta-1 (p70S6K) [95].
Besides the well-known cAMP/PKA pathway, the last two decades provided new insights into the FSH mode of action in Sertoli cells, revealing a complex network of intracellular signaling cascades activated by the hormone. The FSHR coupling to Gai protein mediates the phosphorylation of ERK1/2 (pERK1/2) independently of cAMP, further supporting the FSH-dependent mitogenic signal [93,96]. Moreover, G protein-dependent pERK1/2 activation would occur very rapidly, within 1-5 min upon hormone-receptor binding, while prolonged ERK1/2 phosphorylation is sustained by other FSHR intracellular interactors, such as b-arrestins [97]. These molecules are scaffold proteins that sequentially dictate the kinetics of the ERK1/2 phosphorylation profile, cooperate with Gas protein in triggering p70S6K activation, and are involved in the trafficking of the receptor [98]. Therefore, b-arrestins exert several actions related to FSH functioning, thus playing an essential role in supporting male fertility. These issues are demonstrated by clinical observations revealing that b-arrestin-, but not cAMP-dependent signaling, is maintained in oligozoospermic men bearing the inactivating Ala189Val FSHR mutation [99]. Notably, after binding its ligand, FSHR undergoes internalization into intracellular compartments via pathways not involving b-arrestins and with regulatory functions of cAMP signaling [100].
Additionally, recent investigations found increasing relevance of micro-RNAs (miRNAs) in regulating FSH action. Studies in rats with suppressed FSH and testosterone activity described the miRNA network at spermiation as susceptible to hormonal control [101]. Two of the transcripts identified in this model were attributable to the phosphatase and tensin homolog (PTEN) enzyme mRNA, suggesting that they would be a target of FSH and/or androgen action. Indeed, Sertoli cell treatment with FSH in vitro enhanced PTEN protein levels massively within minutes, restoring terminal cell differentiation and proliferation [102]. Moreover, FSH treatment induced the decline of miR-92a-3p levels, which could be linked to increased FSHR expression [103]. These data suggest that the miRNA network might have a crucial role in the modulation of the FSH signaling in Sertoli cells, although knowledge about the miRNA-mediated regulation of Sertoli cell functioning remains limited.
Currently, both urinary and recombinant FSH preparations are available for clinical use. Urinary FSH preparations are marketed in lyophilized vials (typically containing 75 IU), whereas recombinant FSH preparations are available in both lyophilized vials (typically containing 75 IU) and pen devices (with doses varying from 300 IU to 900 IU, for fractionated use) [42]. Both preparations are administered subcutaneously; the lyophilized products must be reconstituted using sterile water before injection, whereas the pen devices are manufactured ready-to-use. After each injection, the peak serum FSH levels are reached within 10-12 h; the FSH levels then decline until the next injection. Recently, a long-acting recombinant FSH preparation was developed for subcutaneous use by combining recombinant human FSH with the hCG C-terminal peptide (CTP) [42]; the preparation has a plasma half-life of 65 h. Menotropins (e.g., hMG) extracted from the urine of postmenopausal women also provide lyophilized FSH for daily subcutaneous or intramuscular administrations; these preparations contain FSH and LH activity (mainly derived from the addition of hCG) in a 1:1 ratio, i.e., 75 of IU FSH and 75 IU of LH activity [42].
Recombinant FSH has a better safety and quality profile than its urinary counterparts [42,104]. In general, recombinant FSH preparations are purer than urine-derived FSH, and the incorporation of vial filling by mass virtually eliminated batch-to-batch variations and enabled accurate dosing. While FSH preparations have been extensively used for ovarian stimulation in women undergoing fertility treatments [38,39,42,43], data concerning their use in the context of male infertility treatment are less robust. The label indication for FSH use in males is typically restricted to hypogonadotropic hypogonadism (HH), which must be combined with hCG. In HH males, the doses vary from 75 IU to 225 IU, administered twice or thrice a week [7,26,27]. Similar doses have been prescribed for stimulating spermatogenesis in idiopathic oligozoospermic and hypergonadotropic hypogonadal or eugonadal NOA patients [3,27-29,32,56,105], as an off-label prescription in most countries.
Effect of LH (and hCG) on Leydig cells
Given the steroidogenic fingerprint of Leydig cells, which express LHCGRs to which both LH and hCG can bind, it is understandable that hCG found clinical utility in replacing LH functions [66]. Both glycoproteins share a common a and a specific b subunit, assembled to form a noncovalently linked heterodimer acting on specific leucine-rich repeats (LRRs) and rhodopsin-like G protein-coupled receptors (GPCRs). However, hCG is easily purified in high concentrations from the urine of pregnant women. In contrast, human LH of pituitary origin is difficult to obtain and lacks full biological activity because it embeds a proteolytic site leading to internally cleaved hormones [66]. Recombinant technology allowed the production of human recombinant LH, but it has only recently become available for clinical use in ovarian stimulation, and its use in male infertility has been anecdotal [42,66]. Additionally, hCG has a longer half-life than LH, making it more patient-friendly than LH, as less frequent injections would be required [42]. The differences in the half-lives of the two molecules relate mainly to oligosaccharide structures, especially O-linked [42,66]. Notably, hCG has a long carboxy-terminal segment with 24 amino acids containing four O-linked oligosaccharide sites, whereas LH has just one [42]. After intravenous administration, hCG has a terminal half-life of 23-31 h compared to 10-12 h of recombinant LH [106,107].
It is well known that hCG binds LHCGRs and has a higher steroidogenic potential than LH [66]. In vitro data from several primary and engineered cell models demonstrated that hCG acts preferentially as an inducer of cAMP [66,67,108] and intracellular Ca2þ increase [109] rather than ERK1/ 2 or AKT phosphorylation, markedly upregulating the transcription of genes coding steroidogenic enzymes. For example, in a mouse Leydig tumor cell line, the activation of gonadal steroid synthesis is more pronounced using hCG than LH [110]. Another in vitro study confirmed the higher cAMP production after the treatment of mouse Leydig cells with hCG vs. LH, although no differences were found in testosterone production [68]. Such experiments are characterized by cell lines expressing nonhuman receptors and need a prudent interpretation. Nevertheless, these data support the use of hCG for testosterone biosynthesis and spermatogenesis.
Given the relatively low availability of human Leydig cells for in vitro studies, assessing the differential action of LH and hCG has been provided by in vivo clinical studies. A trial conducted on 19 healthy men undergoing pituitary suppression with GnRH antagonist indicated that Leydig cell stimulation might be exerted by relatively low exogenous LH doses (787.5 IU/week), which induced similar testosterone levels to those obtained by administration of 5000 IU/week of hCG [111]. This finding may lead to the speculation that LH might be used at a lower dosage than hCG to induce androgen production in vivo, even if it is non-pulsatile, indicating a differential action of the two molecules in human males.
The results of the study mentioned above may be explained by the fact that LH exerts a preferential kinase-dependent activation of mitogenic signals [67,108], which could improve the metabolic state of the cell, positively impacting steroidogenesis (Fig. 1). This effect would be detectable in cells expressing the human receptor, which may discriminate between LH and hCG binding [112]. By contrast, in vitro studies using mouse receptor-expressing cells are unable to provide the complete pattern of LH- and hCG-specific intracellular pathways [68].
Three additional studies are noteworthy as they pioneered the comparisons between hCG and LH in the human male setting. Two decades ago, a case of an 18-year-old boy with a deletion of the exon 10- coded portion of the LHCGR gene was described [113]. The patient had Leydig cell hypoplasia, delayed pubertal development, small testes, high serum LH, and low testosterone levels. Interestingly, he was unresponsive to endogenous LH, but exogenous hCG administration restored androgen biosynthesis and spermatogenesis (Fig. 2). In vitro analysis confirmed the lack of LH-induced cAMP increase, while hCG activated the signal transduction pathway upon mutant receptor binding [114]. Although the clinical scenario described above is very rare, it highlights the capability of the LHCGR to discriminate between the two natural ligands (Fig. 2), possibly reflecting a different regulation of gametogenesis and placentation in humans. Most importantly, this case report suggested that hCG may have different actions than LH in male gonads.
Subsequently, in 2008, a randomized, single-blind study including 20 healthy eugonadal men aged 18-30 evaluated hCG's and LH's effect on Leydig cell function [115]. A subgroup received either vehicle alone or urinary hCG intramuscularly in doses varying from 50 to 5000 IU, while another group received recombinant hCG intramuscularly in a dose of 250 mcg (6500 IU) or recombinant LH intravenously in doses varying from 75 to 225 IU. A serial assessment of serum testosterone and estradiol was carried out to determine the response to stimulation. There was a dose-dependent increase in testosterone and estradiol levels with urinary hCG administration, with a peak observed at 48 h and 72 h after the injection of 500 IU and 5000 IU, respectively. The peaks in testosterone and estradiol obtained with urinary hCG (5000 IU) and recombinant hCG (6500 IU) did not differ. Serum LH levels increased dose-dependently after injection of recombinant LH; the peak value was obtained 30 min after injections, progressively dropping to reach basal values 6 h later. Similarly, a dose-dependent increase in testosterone (but not estradiol) was observed after recombinant LH administration, with a peak observed 6 h after administering 225 IU. The lack of a stimulatory effect on estradiol levels by recombinant LH is attributed to its lesser steroidogenic potency than hCG, probably explained by its short plasma half-life and the reversibility of its binding to the LHCGR. In this study, the highest serum testosterone levels attained within 5 h after the injection of either 150 or 225 IU recombinant LH correlated with the 48-h peak increase in testosterone in response to the injection of 50 IU recombinant hCG [115]. The results of this study confirm the ability of both recombinant hCG and recombinant LH to promote adequate androgen production in normal men.
Along these lines, a study comparing the effects of a daily, low (75 IU) dose of recombinant LH versus 75 IU hCG in a hypogonadotropic hypogonadal hypophysectomized man found no differences between the two hormones in terms of restoring the eugonadal status after 12-14 days of treatment [116]. These findings may be explained by the known mitotic effects of testosterone demonstrated in seminal vesicles of animal models [117], and other growth factors, such as estrogens, produced in vivo, which could have covered the gonadotropin-specific signal. In any case, more data are needed to elucidate the gonadotropin-specific action in human Leydig cells.
Historically, only hCG has been used in the context of male infertility, mainly for males with hypogonadism [7,26,118]. Urinary hCG preparations are currently marketed in lyophilized vials with 5000 or 10,000 IU for intramuscular use. In contrast, recombinant hCG is available in prefilled syringes or pen devices containing 250 mcg of pure hCGdequivalent to approximately 6750 IU of urinary hCG [42]. Like recombinant FSH preparations, recombinant hCG is purer than urine-derived hCG and has a better quality and safety profile than its urinary counterparts [42,104]. Nevertheless, data concerning recombinant hCG in the context of male infertility treatment are minimal, and its use remains off-label. By contrast, urinary hCG has a label indication to treat males with hypogonadism, including those with oligozoospermia. Given the shortage of urinary hCG availability in many countries, recombinant hCG must receive approval from regulatory agencies for use in male hypogonadism.
Clinical application
The rationale for using hCG therapy (as a surrogate for LH) off-label for treating male infertility conditions associated with hypogonadism relies on the fact that low ITT concentrations cause spermatogenesis disruption. In rodents, reductions of over 75% of the ITT concentration are incompatible with sperm maturation [119-121]. Furthermore, gonadotropin action is determined by its secretory pulses' frequency, amplitude, and duration [32,122-124]. In NOA males with elevated circulating levels of endogenous FSH and LH, the relative amplitudes of FSH and LH are low, leading to a paradoxically weak stimulation of Leydig and Sertoli cells [28,32,79,122-124]. Human studies have shown that hormonal therapy with hCG increases ITT and circulating testosterone in men with nonobstructive azoospermia [122,123]. hCG treatment also promotes spermatogonial DNA synthesis, as assessed by the expression of proliferating cell nuclear antigen (PCNA) [122]. Expression of the PCNA genes is associated with cell proliferation and, thus, with DNA synthesis during genome replication in the S phase of the cell cycle [122].
Nevertheless, it remains unclear what the optimal testosterone levels are for stimulating spermatogenesis and improving sperm retrieval rates (SRR) in NOA males. The assessment of ITT levels is challenging because measurements require testicular aspiration, which is an invasive procedure [76]. Measurement of circulating testosterone levels has been used as a proxy of ITT assessment because of the high correlation (r = 0.82) in the concentrations of both hormones in normal men [125]. It should be noted that ITT concentrations are markedly higher (~100-200x) than circulating testosterone concentrations [76,125], making direct extrapolations between intratesticular and circulating testosterone levels potentially inaccurate. For example, the correlations between ITT and testosterone levels are poor in men receiving testosterone replacement therapy because of its negative feedback on the hypothalamic-pituitary-gonadal axis [126]. Serum 17-hydroxyprogesteronedan intermediate steroid produced by the testicles and adrenal glands have been proposed as an alternative biomarker for evaluating ITT levels [127].
Furthermore, human and animal data suggest pathological desensitization of the FSHR caused by high circulating FSH levels [128-131]. It has been postulated that hormone therapy using GnRH or hCG could benefit these patients by suppressing the endogenous gonadotrophin levels and thereby overcoming Sertoli cell receptor desensitization caused by chronically raised circulating FSH levels [132,133].
Lastly, as previously discussed, FSH promotes cell proliferation when acting in synergy with ITT [40]. Transgenic murine studies have suggested that spermatogenesis can be stimulated by high FSH concentrations using exogenous FSH administration, even in the presence of low ITT [40,134]. Moreover, it has been suggested that a subset of men with idiopathic oligozoospermia and low-to-normal circulating FSH levels are FSH deficient as a consequence of reduced FSH activity, which depends on the amount of circulating FSH, its glycosylation, as well as the genetically determined expression levels and function of the FSHR [56,60,134,135]. Therefore, hormonal therapy with FSH might help improve sperm parameters in some patients, as shown in carriers of the FSHR p. N680S homozygous N [56]. Although limited, these data have provided a rationale for the off-label administration of exogenous FSH to males with idiopathic oligozoospermia.
Real-world data
Idiopathic oligozoospermia
Collectively, modest evidence indicates that exogenous FSH therapy might increase sperm quantity in men with idiopathic oligozoospermia, with an apparent positive effect on pregnancy rates. The current evidence is based on studies including few participants and using different treatment protocols and follow-up periods. Further high-quality research is needed to clarify the potential clinical role of FSH therapy in overcoming oligozoospermia and improving pregnancy rates. The optimal treatment regimen and duration and the role of pharmacogenomics in identifying the best candidates for treatment have also to be determined.
Nonobstructive azoospermia
The treatment protocol used at the ANDROFERT Clinic relies primarily on recombinant hCG used off-label to boost ITT production (Fig. 4). Our experience shows that most patients' circulating testosterone levels increase after hCG treatment. The increased testosterone levels reset some patients' elevated baseline FSH levels to normal levels. This is beneficial as the FSH reset might increase FSH receptors' expression and improve Sertoli cell function [123,123,132]. Notably, some patients exhibit a remarkable decline in circulating FSH levels during hCG treatment; we add recombinant FSH to the hCG regimen when the FSH levels drop below 1.5 IU/L. Patients are followed up with a monthly hormonal assessment, and an aromatase inhibitor is added during the treatment when the testosterone-to-estradiol ratio turns less than 10 [3,77,188,193].
Collectively, limited evidence, overwhelmingly based on observational studies and case series, suggests that gonadotropin therapy for males with NOA might increase sperm retrieval success rates. In a few cases, it was associated with the return of minimal sperm numbers to the ejaculate. Further high-quality studies are warranted to confirm whether gonadotropin therapy improves sperm retrieval success rates in this population. Additionally, more data are needed to identify the NOA patients who might benefit the most from gonadotropin therapy and the optimal treatment regimen and duration.
Summary
RWD on gonadotropin stimulation for eugonadal and hypogonadal infertile males with idiopathic oligozoospermia or NOA indicates an overall positive therapeutic effect. FSH therapy in patients with idiopathic oligozoospermia, mainly using recombinant preparations, may increase sperm quantity and quality because of the FSH proliferative action on Sertoli cells, synergically complemented by the action of androgens and other growth factors. However, the effect is not universal, and patients’ specific genotypes might impact therapeutic effectiveness. Leydig cells express LHCGRs to which both LH and hCG can bind; thus, hCG preparations, mainly urine-derived, have been the drugs of choice to boost ITT production in eugonadal and hypergonadotropic hypogonadal men with NOA. Low ITT concentration is typical in men with NOA and intrinsic testicular pathology; it causes spermatogenesis disruption that hCG can revert. The increase in testosterone production by hCG might suppress the elevated endogenous gonadotrophin levels, overcoming the Sertoli cell receptor desensitization caused by chronically raised circulating FSH levels commonly seen in NOA males. Although the optimal testosterone level for stimulating spermatogenesis is unknown, RWD on hCG-based therapy in NOA males overwhelmingly based on small case series and observational studies has shown increased sperm retrieval rates (by 10-15%) than with no treatment, and in some cases, the return of small sperm numbers to the ejaculate. The most suitable NOA patients for gonadotropin therapy seem to be hypogonadal men with testicular histopathology showing maturation arrest (late stages) or hypospermatogenesis. Still, the evidence concerning the role of exogenous gonadotropins in male infertility treatment is limited and needs validation by large-scale, well-designed studies. Further research is required to identify the best candidates for treatment and optimal gonadotropin regimens.
