key: cord-0933499-8pp3q0wg
authors: Abdel-Moneim, Adel
title: COVID-19 Pandemic and Male Fertility: Clinical Manifestations and Pathogenic Mechanisms
date: 2021-03-19
journal: Biochemistry (Mosc)
DOI: 10.1134/s0006297921040015
sha: 296999df497658eb428a8508520dd63879c6c7e3
doc_id: 933499
cord_uid: 8pp3q0wg

The novel coronavirus disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been a major public health emergency worldwide with over 118.27-million confirmed COVID-19 cases and 2.62-million deaths recorded, as of March 12, 2021. Although this disease primarily targets lungs, damages in other organs, such as heart, kidney, liver, and testis, may occur. Testis is the cornerstone of male reproduction, while reproductive health is the most valuable resource for continuity of the human race. Given the unique nature of SARS-CoV-2, the mechanisms of its impact on the testes have yet to be fully explored. Notably, coronaviruses have been found to invade target cells through the angiotensin-converting enzyme 2 receptor, which can be found in the respiratory, gastrointestinal, cardiovascular, urinary tract, and reproductive organs, such as testes. Coronavirus studies have suggested that testes might be a potential target for SARS-CoV-2 infection. The first etiopathogenic concept proposed by current hypotheses indicates that the virus can invade testes through the angiotensin-converting enzyme 2 receptor. Next, the activated inflammatory response in the testes, disease-associated fever, and COVID-19 medications might be implicated in testicular alterations. Although evidence regarding the presence of SARS-CoV-2 mRNA in semen remains controversial, this emphasizes the need for researchers to pay closer attention to sexually transmitted diseases and male fertility after recovering from COVID-19. In this review the latest updates regarding COVID-19-associated testicular dysfunction are summarized and possible pathogenic mechanisms are discussed.

BIOCHEMISTRY (Moscow) more attention should be given to the potential risk of SARS CoV 2 infection on the male fertility [8] . Testes are the cornerstone of male reproduction, while repro ductive health is the most valuable resource for continu ity of the human race. However, little is known about the potential short and long term impact of COVID 19 on the male reproductive system. This review provides insights into clinical manifestations and possible patho genicity of the SARS CoV 2 infection in relation to tes ticular injury based on the latest studies.

Testis orchitis. Previous studies have reported that a number of viruses could infect testes, including mumps virus, human immunodeficiency virus (HIV), and SARS CoV [5, 9] . Regarding the novel SARS CoV 2, which shares 76% amino acid sequence homology with the SARS CoV, one can hypothesize that SARS CoV 2 may have the ability to invade testes. Indeed, Gagliardi et al. reported the case of orchi epididymitis associated with SARS CoV 2 infection [10] . Moreover, six patients with SARS CoV showed signs of orchitis and testicular injury including reduced numbers of germ cells and apoptotic death with interstitial leukocyte infiltration. Additionally, histopathological findings showed inflammatory infiltra tions and accumulation of immunoglobulin G (IgG), particularly in seminiferous epithelium, interstitium, degenerated germ cells, and Sertoli cells [5] . This finding is consistent with the results presented in the study of Pan et al., where 6 out of 34 men who recovered from SARS CoV 2 infection developed signs of scrotal dis comfort [11] . Additionally, the study of serious COVID 19 cases in Brazil identified fibrin microthrombi orchitis in two of the investigated testes [12] . Similarly, post mortem examination of 12 COVID 19 male patients in China showed marked seminiferous cellular damage, reduced Leydig cell number, and moderate lymphocytic inflammation [13] . One study suggested that the orchitis may have resulted from vasculitis considering correlation between the COVID 19 and coagulation disorders, and that the segmental vascularization of the testes could cause an orchitis like syndrome [6] . The aforementioned findings could suggest that SARS CoV 2 infection may contribute to testicular ultrastructural lesions and orchitis in the severely infected males.

Sex hormone abnormalities. Regarding androgen secretion, the decreased total testosterone and calculated free testosterone with elevated luteinizing hormone (LH) levels recorded in the severe cases of COVID 19, have been significantly correlated with the increased plasma lactate dehydrogenase and ferritin levels and as well as with high er neutrophil counts and reduced lymphocyte counts [14] . These results are in agreement with the findings of another study that showed substantial reduction in the serum testosterone levels in 113 patients (51.1%) with severe COVID 19, suggesting this parameter as a negative predic tor of COVID 19 progression [15] . In addition, the recent study on 119 men with COVID 19 observed that the infected men had slightly lower overall serum testosterone levels, markedly higher serum LH, and lower testosterone: LH and follicle stimulating hormone (FSH): LH ratios compared to 273 age matched healthy men [16] . Similarly, the German study revealed that the critically ill male COVID 19 patients exhibited elevated LH and FSH levels with decreased testosterone and dihydrotestosterone levels [17] . Interestingly, the serum LH elevation in male patients with COVID 19 could possibly inhibit the hypo thalamic-hypophyseal-testicular axis and perhaps explain the primary Leydig cell injury [18] . Moreover, low testosterone level had been the most important predictive hormonal factor for in hospital mortality in the group of aged male patients [19] . Accordingly, COVID 19 may induce acute male hypogonadism that is clinically mani fested as a reduction in testosterone levels (table) .

Virus detection in seminal fluid. To date, no records on the sexual transmission of SARS CoV 2 exist, while evi dence regarding the presence of SARS CoV 2 in semen remains limited. Recently, the contradictory findings have been reported regarding the presence of SARS CoV 2 in the semen of patients diagnosed with COVID 19. Meanwhile, individuals infected with SARS CoV 2 should take all possible precautions to minimize the possi ble risk of transmitting the infection via sexual intercourse [20] . Although actual fertility situation in the patients diagnosed with COVID 19 is still to be explored, the American Society for Reproductive Medicine (ASRM) and the Society for Assisted Reproductive Technology (SART) had already published warnings regarding the SARS CoV 2 transmission via sexual intercourse [21] . Importantly, among the 38 patients with COVID 19 who provided semen specimens, 6 patients (15.8%) had posi tive results for SARS CoV 2 RNA, including 4 of 15 patients (26.7%) who were at the acute stage of infection and 2 of 23 patients (8.7%) who were recovering [22] .

In contrast, Pan et al. reported no virus in patients' semen 29 36 days after recovery, although viral orchitis symptoms were observed in 19% of the patients with COVID 19 and six men displayed mild scrotal discomfort at the time of disease [11] . Furthermore, Song et al. [23] collected 12 semen samples from the COVID 19 patients and one postmortem testicular biopsy. SARS CoV 2 RNA was not detected in the semen samples and testicu lar biopsy. The authors concluded, based on the fact that SARS CoV 2 was not identified in the semen and testis samples, that the probability of testicular infection during the early and symptomatic phase of the infection is low but cannot be ruled out, when certain organs such as lung, heart, and gut are infected. In line with this, Holtmann et al. [24] found no SARS CoV 2 RNA in the semen of 34 recovered or acutely infected males with SARS CoV 2 43 days after diagnosis on average. Furthermore, among the 12 deceased patients with COVID 19, 11 were negative for SARS CoV 2 RNA in the testicular tissue, while one was positive for the virus. However, the patients with COVID 19 had been found to display seriously damaged seminiferous tubules, decreased Sertoli cells, and mild inflammatory infiltrates in the interstitium [13] (table). Therefore, evidence avail able from 132 cumulative patients over 7 studies revealed that the virus was present in the semen of 7 (5%) patients with COVID 19 [11, 13, 22 26] .

Moreover, Ma et al. [16] reported that 4 patients with COVID 19 (33.3%, 4/12) had low sperm motility. Duration between the semen collection and the disease onset ranged from 56 days to 109 days (with a median of 78.5 days). However, a marked negative effect on the semen parameters such as sperm concentration, total sperm count, and the total number of progressive motili ty was observed in the recovered participants with mild symptoms and need for hospitalization compared to the control group. This finding indicates that SARS CoV 2 infection has short term effects on spermatogenesis in patients with mild symptoms associated with COVID 19 [24] . Notably, Yang et al. [13] stated that COVID 19 can induce testicular tissue injury and affect fertility, particu larly in young men, which highlights the need for exam ining testicular function after recovery. several organs such as heart, gut, lungs, kidneys, testes, and brain [7] , directly influence invasion of the three coronavirus strains into human cells: SARS CoV, NL63, and SARS CoV 2. Seminiferous tubules represent up to 90% of the human testis tissues, the Sertoli cells and the germ cells express ACE2 making them a potential site for SARS CoV 2 infection that subsequently impacts sper matogenesis ( figure) . Interestingly, testicular cells showed the highest level of ACE2 mRNA in the seminiferous duct cells, spermatogonia, Leydig cells, primordial germ cells, and Sertoli cells. Considerable seminiferous tubular dam age, decreased number of Leydig cells, and moderate lymphocyte inflammation was reported in the studies of patients with COVID 19 [7, 27] . The physiological roles of ACE2 in Leydig cells include control of steroidogene sis and spermatogenesis, modulation of testosterone syn thesis, and regulation of the local vascular regulatory sys tem to balance the volume of interstitial fluid by control ling conversion of Angiotensin II to Angiotensin I [14, 28] . Additionally, histopathological studies of biop sies from COVID 19 male patients showed impaired spermatogenesis in three out of six samples. Also, histo logical staining of one sample from the patient with COVID 19 demonstrated interstitial macrophage and leukocyte infiltration. Furthermore, immunofluores cence analysis of six biopsies derived from the COVID 19 patients demonstrated direct correlation between the increased ACE2 levels and impaired spermatogenesis indicating possible mechanism of infection of testis by SARS CoV 2 [29] . Since the expression of ACE2 is high in testes, Li et al. [30] concluded that SARS CoV 2 enters the testicular interstitium via the circulation route during peak viremia and that Leydig cells could be one of the initial targets. Moreover, the testicular ACE2 expres sion has been related to age, the largest ACE2 expression was observed in 30 years old patients, while 60 year old patients showed the lowest expression in testes [31] sug gesting that younger males with COVID 19 were at sig nificantly higher risk for testicular damage compared to older males.

Efficient entry of SARS CoV 2 into the host cells depends not only on the presence of ACE2 receptors but also on the transmembrane protease serine 2 (TMPRSS2), which cleaves S protein of the human coro naviruses on the membrane priming them for viral entry into the cells [32] . In addition to ACE2, TMPRSS2 is also expressed in spermatogonia, Leydig cells, and Sertoli cells, providing potential route of entry for SARS CoV 2 into these cells [33] . The virus is expected to bind to ACE2 and TMPRSS2 in the testicular tissue to induce its adverse effects. Importantly, Abobaker and Raba [34] highlighted the possibility for testicular injury and subse quent male infertility after SARS CoV 2 infection, which might be caused by either direct viral invasion through ACE2 receptors or indirectly via the inflammatory immune response.

While cytokines play a vital role in the testicular function, they also participate in pathological processes [35] . Elevated concentrations of cytokines following viral infection can influence spermatogenesis and steroidogenesis, thereby seriously impacting fertility [36] (figure) . Notably, testic ular inflammation causes upregulation of interleukin (IL) 1β, IL 1α, IL 6, and tumor necrosis factor α, which produce harmful effects on the germ cells and inflammatory conditions in testes that interfere with the process of spermatogenesis [37] . Moreover, ACE2 present on Leydig cells may also influence local microvascular flows and permeability and promote inflammation that interferes with the role of Leydig cells, thereby inhibiting testosterone production and damaging seminiferous tubular cells [5] . The SARS CoV 2 could invade the male reproductive tract during acute infection through ACE2 present on the cells of seminiferous tubules. The virus could probably stay for only a few days due to the privi leged immune status of testes [22] . In particular, the immunosuppressive characteristics of Sertoli cells and testicular macrophages are known to play a vital role in suppressing inflammation and reducing testicular damage associated with viruses. Nevertheless, the COVID 19 associated inflammation may temporarily affect integrity of the blood testis barrier (BTB), which could negatively impact spermatogenesis [33] . SARS CoV 2 could cause an increase of ACE2 expression and promote typical inflammatory response that could interfere with the func tion of Leydig and Sertoli cells. Proinflammatory cytokines released by Leydig and Sertoli cells may acti vate the autoimmune response and damage the seminifer ous epithelium, leading to autoimmune orchitis [5] . Accordingly, despite their privileged immune status, testes cannot be protected from the general immune response. Infiltration of leukocyte as well as CD 3+ T lym phocytes and CD 68+ macrophages into the interstitial tis sue of testis can generate interferons that may also decrease testosterone production [36] . Moreover, testos terone depletion has also been associated with autoim mune diseases and elevation of the inflammatory bio markers such as C reactive protein (CRP), IL 6, and TNF α [38] .

COVID 19 associated fever. The hyperactivated immune responses along with cytokine storms following SARS CoV 2 infection affect many organs, including heart, liver, kidney, and testes. Interestingly, fever, is an additional risk of the COVID 19 that may affect male fer tility. More importantly, hyperinflammatory condition with persistent fever, fulminant, and fatal hypercytokine mia have been associated with multi organ failure [39] . High inflammatory response associated with fever, immune cell activation, and inflammatory mediators such as interferons and cytokines can affect testicular function [40, 41] (figure). The hypothesis that fever and elevation of testicular temperature contribute to sperm deficiency has been generally accepted. Given that sper matogenesis could be affected by COVID 19 associated fever, semen parameters such as sperm concentration and motility could be decreased for 72 to 90 days after the viral infection [42] . Moreover, participants with verified COVID 19 associated fever tended to have lower motile sperm count, sperm concentration, and total sperm count [43] . COVID 19 and gonadal toxic drugs. Interferon α and ribavirin (in combination with interferon or lopinavir/ritonavir), as well as chloroquine phosphate, have been recommended for COVID 19 treatment [44] . Animal studies have revealed that ribavirin administration reduced testosterone levels and inhibited spermatogenesis [45] , while lopinavir/ritonavir has also been observed to inhibit spermatogenesis in rats probably due to oxidative stress and inflammation [46] . Unfortunately, testicular dysfunction could be also caused by glucocorticoids and infectious diseases related stress due to psychological crises induced by COVID 19 [22] (figure). Additionally, chloroquine phosphate has been found to affect spermato genesis and epididymal function in male rats, which indi cates that certain medication can affect testicular function in the male patients with COVID 19 [47] (table) .

Unfortunately, the aforementioned primary studies on male fertility have some limitations related to sample size, research methodology, and disease course. Further comprehensive investigations at all levels are therefore required to improve the levels of evidence and under standing of the impact of SARS CoV 2 on male repro duction and testicular health. Fortunately, many researchers have recently launched a highly promising multidimensional andrological research project in men called the PROTEGGIMI study (prospective multidi mensional andrological translational research project) to develop international collaboration for the data registry in hormonal and genomic studies in hopes of filling the gaps in the current knowledge on association between SARS CoV 2 and male frailty [48] THE IMPACT OF LONG TERM VIRAL INFECTION ON REPRODUCTIVE FUNCTION Given the novelty of the COVID 19 pandemic, long term studies on its influence on male fertility have been unavailable. Nonetheless, several studies have addressed the effects of long term viral infection on male reproduc tive function. Several viruses, such as human papillo maviruses, hepatitis B (HBV), hepatitis C viruses (HCV), human herpes viruses, influenza viruses, cytomegalo viruses, HIV, mumps virus, Zika viruses, and Ebola virus es have been known to cause orchitis and influence male fertility [49] . Interestingly, the study on 298 patients with mumps orchitis found that 24% of adults and 38% of ado lescents exhibited seminal abnormalities for up to three years after recovery. At least 24% of adults and 38% of young individuals had abnormal ejaculation even three years after orchitis [50] . Ultrasonography of eight patients with mumps orchitis revealed atrophic testes with an oblong shape, heterogeneous low echogenicity, and decreased vascularity 40 to 230 days after the initial diag nosis [51. Histological characteristics of testis from 57 autopsied patients with chronic HIV, suggested a slightly lower degree of spermatogenesis as well as increased thickening of the cell membrane and interstitial fibrosis [52] . Moreover, the male patients with chronic HCV infection showed lower total serum testosterone, sperm count, and progressive sperm motility, and abnormal sperm morphology compared to the healthy controls [53] . Altered sperms morphology with decreased sperms motility, viability, and concentration were observed in the semen from patients with chronic HBV infection com pared to the healthy individuals [54] .

Notably, Ebola RNA may be found in seminal fluid for periods exceeding 13 months after infection [55] . Moreover, the study on virus persistence following the 2014 2016 Ebola virus outbreak had reported the presence of Ebola RNA in seminal fluid up to 565 days after infec tion [56] . Additionally, among a cohort of 135 male patients with Ebola who were followed up from 2015 to 2017, 8% reported erectile dysfunction, whereas 12% showed decreased libido [57] . Regarding the Zika virus, Counotte et al. [58] reported the presence of Zika RNA in semen for a median duration of 40 to 370 days. However, Avelino Silva et al. [59] found normal levels of the serum sex hormone and absence of the Zika RNA in the semen samples from six patients 1 year post Zika infection, although impaired motility was observed in three samples and low sperm count was reported in one sample.

The current review suggests that the recent coron avirus pandemic COVID 19 could have various effects on male reproductive health. Although, the data on the male reproductive system in patients with COVID 19 have been scarce with most relevant evidence coming from the small scale studies without available long term follow up data. Taken together, several etiopathogenic hypotheses have been suggested implying that the SARS CoV 2 infection might be responsible for impairment of testicu lar function. ACE2 is primarily expressed in spermatogo nia and Leydig and Sertoli cells of the human testes, which may lead to a testicular dysfunction in patients infected with SARS CoV 2. Moreover, testicular damage may be attributed to the inflammatory responses and fever associated inflammation, as well as medications used in the severe cases. Thus, clinical and translational (short /long term) investigations in larger cohorts of cur rently infected subjects are needed to evaluate the impact of COVID 19 on human spermatogenesis, determine protective and curative approaches against testicular injuries, and establish clear conclusions.

Ethics declarations. The author declares no conflicts of interest. This article does not contain any studies with human participants or animals performed by the author.

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