Is there a role for glucagon‐like peptide‐1 receptor agonists in the treatment of male infertility?

Abstract Glucagon‐like peptide‐1 receptor agonists (GLP1‐RAs) are novel anti‐hyperglycemic drugs efficacious on glucose control, weight loss, and cardiovascular prevention. These drugs may also be effective in modulating testicular function. In fact, they increase serum testosterone levels in diabetic and/or obese patients with functional hypogonadism on a dysmetabolic basis. Although part of this effect can be ascribed to weight loss, some evidence suggests that there is a direct effect at the testicular level. Indeed, human Leydig, Sertoli, and germ cells express GLP1 receptors. GLP1‐RAs improve sperm metabolism, motility, and insulin secretion in vitro. Likewise, GLP1‐RAs exert positive effects on the metabolism of human Sertoli cells in vitro. Finally, GLP1 is secreted by mouse Leydig cells and this suggests the presence of a paracrine mechanism by which these cells could support the metabolism of Sertoli cells. Therefore, the widespread use of GLP1‐RAs in clinical practice may reveal an important role in the management of male infertility in obese and/or diabetic patients given the negative impact of these diseases on testicular steroidogenesis and spermatogenesis. This should suggest the design of randomized controlled studies aimed at evaluating the effects of these drugs on testicular function.

increasing urinary glucose excretion and lowering blood glucose levels. 6 The glucose-lowering action of both GLP1-RAs and DPP4i is based on their capability to increase the biochemical activity of GLP1, by stimulating its receptor in the case of GLP1-RAs or by reducing its catabolism in the case of DPP4i. Under physiologic conditions, GLP1 is released from the intestine and can induce glucose-dependent

| INTRODUC TI ON
Since the first case of coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was reported in Wuhan, China, it has rapidly spread and affected more than 21 million people worldwide as of 17 August 2020. 1 SARS-CoV-2 uses angiotensin-converting enzyme II (ACE2) to enter host cells, similar to SARS-CoV, which emerged 18 years ago. 2 COVID-19 induces respiratory-predominant multiorgan dysfunction, including myocardial, renal, enteric and hepatic dysfunction, which coincides with the tissue expression of ACE2. 3 Meanwhile, several studies have shown that ACE2 is expressed in human testes (eg spermatogonia, Leydig cells and Sertoli cells), 4,5 suggesting that the testes may be another organ affected by COVID-19.
Numerous viruses have been detected in human semen. 6 Viruses may persist in semen and last longer in seminal fluid than in other body fluids due to the immune privilege of the testes and the contribution of the blood-testes barrier to resistance to therapeutic agents. 7,8 Semen may also have higher loads of viruses, such as Zika virus, than blood. 9,10 Therefore, the testes may act as a reservoir of virus, which may cause imprecise evaluation of viral clearance in patients. Viruses, including Zika virus, Ebola virus, cytomegalovirus and human immunodeficiency virus (HIV), have been isolated from semen and can be sexually transmitted. 6,11,12 Furthermore, some viruses (eg HIV, Zika virus, herpes simplex virus (HSV) and human papillomavirus) can adhere to or be internalized by spermatozoa, 7,13 which may pose a risk for embryonic infection and cause adverse reproductive outcomes.
On the other hand, many viruses, such as mumps virus, HIV and HSV, 7,14 have been found to impair semen quality, and they may directly interact with spermatozoa or affect spermatogenesis by inducing local inflammation. [15][16][17] Previous studies found that SARS, 1 of the 3 epidemic coronaviruses to emerge in the past 20 years and that shows similar clinical presentations to COVID-19, 18 could cause orchitis 19 and focal testicular atrophy. 20 Considering the tens of millions of COVID-19 cases and that men are more vulnerable to COVID-19 than women, [21][22][23] it is imperative to determine the effect of COVID-19 on male reproduction. 24 Several studies have been performed on this topic. However, the results are controversial. For example, some researchers have reported that SARS-CoV-2 was not detected in the male reproductive tract, [25][26][27][28][29][30][31][32][33][34] while others reported that SARS-CoV-2 RNA was found in the semen or testes of COVID-19 patients. 35,36 There are also unknown factors regarding COVID-19 and male reproduction.
Orchitis and broad destruction of the testes were found in deceased COVID-19 patients, 35,37 while the pathological characteristics in survivors remain unknown. In this review, we summarize the current research focusing on the effects of COVID-19 on male reproduction from the following 3 aspects: detection of SARS-CoV-2 in the male reproductive tract, determination of the impact of COVID-19 on sperm quality and exploration of pathological changes in the testes of COVID-19 patients. We further discuss the discrepancies and summarize the unknown topics, which we believe will be helpful for future research.

| ME THODS
A systematic search of published studies was conducted in the PubMed and Ovid Embase databases for studies published from December 2019 to 18 August 2020 in accordance with PRISMA. 38 All titles or abstracts of English-language studies were reviewed for eligibility. Citations and references of the retrieved studies were used as additional sources. There was no limitation on sample size, and case reports were included. A full-text review was performed by 2 independent reviewers (Y.Y. and X.Y.) on studies that reported the

| RE SULTS AND D ISCUSS I ON
After reviewing the studies retrieved from the database, citations and references were added based on a review of the title or abstract ( Figure 1). Fourteen studies were eligible and were included in this study, with 12 studies detecting SARS-CoV-2 in the male reproductive tract, 3 determining the impact of COVID-19 on sperm quality and 3 exploring pathological changes in the testes of COVID-19 patients.

| Detection of COVID-19 in the male reproductive tract
Twelve studies investigated the presence of SARS-CoV-2 in the male reproductive tract (eg semen, prostatic secretion or testicular tissue) and are shown in Table 1. Most studies were cross-sectional in design and included mainly Chinese subjects. In brief, ten of 12 studies reported that none of the participants had SARS-CoV-2 RNA 2 | CANNARELLA Et AL.
insulin secretion from pancreatic β-cells. [7][8][9] Specifically, GLP-1 derives from post-translational processing of proglucagon. Although the proglucagon gene is expressed in enteroendocrine L-cells and pancreatic α-cells, 10 GLP-1 is synthesized only by intestinal opentype L-cells, predominantly located in the ileum and colon, that are in direct contact with nutrients in the intestinal lumen. 11 Glucose and fat (unlike proteins) are able to stimulate GLP-1 secretion from L-cells, with a biphasic pattern (15-30 min after a meal, followed by a second minor peak at 90-120 min). 12,13 GLP-1 also hinders glucagon secretion and reduces gastric emptying, promoting glucose control and weight-loss. 14 GLP1-RAs and DPP4i are incretin-based drugs and positively influence risk factors for cardiovascular disease. 15 Male hypogonadism is defined by the presence of total testosterone levels lower than 9.2 nmol/L in at least two blood measurements. 16 About half (44%) of the patients with DM2 and obesity (the so-called "diabesity") have hypogonadism mainly with a central mechanism, because of the altered signaling of insulin and leptin at the level of kisspeptin neurons. 17 The possible role of the newhyperglycemic drugs on testosterone levels and sexual function in patients with DM has recently been reviewed. 18 Evidence in mice has shown that both DPP4i and SGLT2 inhibitors could be able to improve sexual function by their positive effects on the endothelium [eg, by the increase of nitric oxide levels, by the release of vascular endothelial growth factor (VEGF) which induces vasorelaxation, or by exerting anti-atherogenic effects]. [19][20][21] In line with these data, treatment for four weeks with empagliflozin in rat model of DM2 with erectile dysfunction significantly improved the erectile response in vivo that to electrical stimulation of the cavernous nerve, compared with untreated rats. 22 These findings support that empagliflozin has a favorable effect on erectile function.
No data are available relative to the impact of DPP4i and SGLT2 inhibitors on sexual function and testosterone levels in patients with DM.
In contrast, evidence on the effects of GLP1-RAs on human testicular function is available. Jensterle and colleagues reported a significant increase in testosterone and a significant reduction of body weight in male patients with obesity-associated functional hypogonadism. 23 Specifically, although one-third of the patients enrolled in this study were diabetic, the prescribed dose of liraglutide was that approved for obesity (3.0 mg/day), which is higher than that approved for diabetes (1.8 mg/day). 23 Similar conclusions were reported in a retrospective study, 24 which included diabetic obese patients with overt hypogonadism and poor response to testosterone and metformin administration. Treatment with liraglutide led to a significant increase in testosterone and a significant decrease in body weight. The increased testosterone levels can be likely ascribed to the weight-lowering effect of GLP1-RAs, which enhance the leptin signal and stimulates GnRH neuronal function in the hypothalamus. However, the magnitude of testosterone increase in patients treated with the GLP1-RA liraglutide has been claimed to be higher than expected for the amount of weight loss. 24 This suggests a possible direct effect of liraglutide in the modulation of the testicular function, which is further supported by the recent identification of GLP1 receptors (GLP1R) in human healthy (non-tumoral) Leydig cells. 25 Notably, the positive effects of GLP1-RA on the erectile function have also been ascribed to the direct action of these drugs on the endothelium. 26 Interestingly, the role of GLP1-RAs may not be confined to the management of hypogonadism in patients with DM and/or obesity, because the GLP1/GLP1R axis seems to influence sperm metabolism.
Spermatogenesis can be affected in both type 1 DM (DM1) and DM2. Accordingly, the expression of genes involved in DNA repair is altered in DM1, thus leading to a high rate of DNA fragmentation, 27 mitochondrial DNA deletions, [28][29][30][31] and alteration of the respiratory chain with a consequential decrease of sperm motility. 32,33 Furthermore, being the sperm plasma membrane and acrosome sensitive to insulin, both insulin resistance (as in DM2) or its deficiency (as in DM1) can lead to impaired spermatogenesis. 34,35 Moreover, glucose plays an important role in spermatozoon metabolism.
Indeed, spermatozoa synthesize ATP through glycolysis, mitochondrial oxidative phosphorylation, or the pentose phosphate pathway. 36 However, not only glucose but also non-hexose compounds (eg, citrate, pyruvate, lactate) are substrates that spermatozoa can use to obtain energy. 36 Notably, glucose uptake is highly deregulated in patients with DM. 36 Particularly, the impairment of glucose homeostasis leads to depletion of GLUTs, which are membrane channels devoted to glucose transport and are expressed in spermatozoa.
Their depletion decreases glucose uptake and reduced glucose intracellular availability, which in turn impair sperm metabolism and ATP production. This leads to a decreased sperm motility, increased oxidative stress (OS), and sperm DNA damage. 36 GLP1 is a potent regulator of glucose homeostasis. Recently, a study carried out in human spermatozoa showed that the GLP1/ GLP1R system can influence their metabolism. 37 In greater detail, the authors demonstrated that GLP1Rs are expressed in human spermatozoa. In β-pancreatic cells, GLP1, by interacting with its receptor, activates an intracellular signaling pathway involving 3′,5′-cyclic adenosine 5′-monophosphate (cAMP) and protein kinase A (PKA). 38,39 By incubating spermatozoa with increasing concentrations of the GLP1-RA exenatide, the authors reported a significant increase in sperm motility and cholesterol efflux at the dose of 300 pM. 37 They further demonstrated that exenatide stimulates insulin secretion from spermatozoa, and, interestingly, influences glucose metabolism because both the lactate dehydrogenase (LDH)-derived product and the glucose-6-phosphate dehydrogenase (G6PDH) activity increased after incubation with exenatide. Similar to what was described in pancreatic β cells, the PKA was involved in the intracellular signaling of exenatide. 37 This evidence strongly associates spermatozoon metabolism with that of pancreatic β cells, as both of them are insulin-sensitive and insulin-secreting cells, and they are both sensitive to the GLP1/GLP1R system. 37 According to this line of evidence, a study showed that the administration of exenatide (24 nmol/kg/day for 8 weeks) was able to improve sperm quality in terms of sperm motility, mitochondrial membrane potential, and DNA integrity, in high fat diet-induced obese mice. 40  On the other hand, many viruses, such as mumps virus, HIV and HSV, 7,14 have been found to impair semen quality, and they may directly interact with spermatozoa or affect spermatogenesis by inducing local inflammation. [15][16][17] Previous studies found that SARS, 1 of the 3 epidemic coronaviruses to emerge in the past 20 years and that shows similar clinical presentations to COVID-19, 18 could cause orchitis 19 and focal testicular atrophy. 20 Considering the tens of millions of COVID-19 cases and that men are more vulnerable to COVID-19 than women, [21][22][23] it is imperative to determine the effect of COVID-19 on male reproduction. 24 Several studies have been performed on this topic. However, the results are controversial. For example, some researchers have reported that SARS-CoV-2 was not detected in the male reproductive tract, [25][26][27][28][29][30][31][32][33][34] while others reported that SARS-CoV-2 RNA was found in the semen or testes of COVID-19 patients. 35,36 There are also unknown factors regarding COVID-19 and male reproduction.
Orchitis and broad destruction of the testes were found in deceased COVID-19 patients, 35   Other evidence supports the functional role of the GLP1/GLP1R system also in testicular somatic cells. GLP1Rs have recently been identified in human Sertoli cells. 41 The exposure to increasing concentrations of GLP1 was able to influence Sertoli cell metabolism.
At the lowest concentration, GLP1 increased the efficiency of LDH, while at the highest concentration it reduced mitochondrial membrane potential and oxidative damage. 41 This leads to speculate that GLP1-RAs may impact sperm quality and function not only directly-by triggering GLP1R expression in spermatozoa, but also indirectly-by acting via GLP1Rs present in Sertoli cells. In this context, it is interesting to highlight that mouse Leydig cells secrete GLP1. 42 Notably, GLP1 null male mice are completely infertile and show an abnormal gonadal development. 42 Thus, Leydig cell-derived GLP1 may play a role in testicular development at least in mice.
Interestingly, this evidence leads to speculate a paracrine mechanism by which Leydig cells might support Sertoli cell metabolism.
Administration of GLP1 to healthy men does not have any effect on gonadotropin levels 43 but may reduce the number of testosterone pulses. 44 So far there is no evidence of its effects on in vivo sperm production. Based on the available in vitro data, it is possible to hypothesize that GLP1/GLP1RAs may improve fertility in patients with DM. In fact, these molecules could have a positive impact on sperm metabolism by directly improving their motility and quality and, speculatively, by acting on Sertoli cells (Figure 1).
In conclusion, given the negative impact of DM and obesity on gonadal function in humans, the use of GLP1-RAs could play an important role in the management of hypotestosteronemia and infertility in patients with these dysmetabolic disorders. At the same time, the evidence reported in this comment should push toward the design of randomized and controlled clinical trials aimed at evaluating the effects of these drugs on the testicular function.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.  On the other hand, many viruses, such as mumps virus, HIV and HSV, 7,14 have been found to impair semen quality, and they may directly interact with spermatozoa or affect spermatogenesis by inducing local inflammation. [15][16][17] Previous studies found that SARS, 1 of the 3 epidemic coronaviruses to emerge in the past 20 years and that shows similar clinical presentations to COVID-19, 18 could cause orchitis 19 and focal testicular atrophy. 20 Considering the tens of millions of COVID-19 cases and that men are more vulnerable to COVID-19 than women, [21][22][23] it is imperative to determine the effect of COVID-19 on male reproduction. 24 Several studies have been performed on this topic. However, the results are controversial. For example, some researchers have reported that SARS-CoV-2 was not detected in the male reproductive tract, [25][26][27][28][29][30][31][32][33][34] while others reported that SARS-CoV-2 RNA was found in the semen or testes of COVID-19 patients. 35,36 There are also unknown factors regarding COVID-19 and male reproduction.

Conceptualization
Orchitis and broad destruction of the testes were found in deceased COVID-19 patients, 35,37 while the pathological characteristics in survivors remain unknown. In this review, we summarize the current research focusing on the effects of COVID-19 on male reproduction from the following 3 aspects: detection of SARS-CoV-2 in the male reproductive tract, determination of the impact of COVID-19 on sperm quality and exploration of pathological changes in the testes of COVID-19 patients. We further discuss the discrepancies and summarize the unknown topics, which we believe will be helpful for future research.

| RE SULTS AND D ISCUSS I ON
After reviewing the studies retrieved from the database, citations and references were added based on a review of the title or abstract ( Figure 1). Fourteen studies were eligible and were included in this study, with 12 studies detecting SARS-CoV-2 in the male reproductive tract, 3 determining the impact of COVID-19 on sperm quality and 3 exploring pathological changes in the testes of COVID-19 patients.

| Detection of COVID-19 in the male reproductive tract
Twelve studies investigated the presence of SARS-CoV-2 in the male reproductive tract (eg semen, prostatic secretion or testicular tissue) and are shown in Table 1 On the other hand, many viruses, such as mumps virus, HIV and HSV, 7,14 have been found to impair semen quality, and they may directly interact with spermatozoa or affect spermatogenesis by inducing local inflammation. [15][16][17] Previous studies found that SARS, 1 of the 3 epidemic coronaviruses to emerge in the past 20 years and that shows similar clinical presentations to COVID-19, 18 could cause orchitis 19 and focal testicular atrophy. 20 Considering the tens of millions of COVID-19 cases and that men are more vulnerable to COVID-19 than women, [21][22][23] it is imperative to determine the effect of COVID-19 on male reproduction. 24 Several studies have been performed on this topic. However, the results are controversial. For example, some researchers have reported that SARS-CoV-2 was not detected in the male reproductive tract, [25][26][27][28][29][30][31][32][33][34] while others reported that SARS-CoV-2 RNA was found in the semen or testes of COVID-19 patients. 35,36 There are also unknown factors regarding COVID-19 and male reproduction.
Orchitis and broad destruction of the testes were found in deceased COVID-19 patients, 35 on sperm quality and exploration of pathological changes in the testes of COVID-19 patients. We further discuss the discrepancies and summarize the unknown topics, which we believe will be helpful for future research.

| reprodu
Twelve st reproduc sue) and in design studies re