Rimesh Pal1, Urmila Yadav2
1 Department of Endocrinology, Post Graduate Institute of Medical Education and Research, Chandigarh, India-160012
2 National Institute of Nursing Education, Post Graduate Institute of Medical Education and Research, Chandigarh, India-160012
Ever since its outbreak in Wuhan, China in December 2019, the novel coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected over 4.7 million individuals, claiming more than 316,000 lives in over 200 countries worldwide. SARS-CoV-2 primarily affects the lungs, resulting in a viral pneumonia that is often complicated by acute respiratory distress syndrome and sepsis. The coronavirus enters the pneumocyte using the host angiotensin-converting enzyme 2 (ACE2) as a receptor. Apart from the lungs, the enzyme is widely expressed in other human organs including the testis. Single-cell transcriptome analysis using adult human testis single-cell RNA-seq datasets has shown high expression of ACE2 in testis (1–3). ACE2 expression has been demonstrated in the germ cells (spermatogonial stem cells, spermatogonia, early spermatocyte, late spermatocyte, round spermatid and elongated spermatid) as well the somatic cells (Sertoli cells, Leydig cells, testicular macrophages) (2,3). The highest expression of ACE2 is exhibited by the Sertoli cells; more then 90% of the Sertoli cells express ACE2 (3). In addition, TMPRSS2, a transmembrane serine protease that is required for priming of SARS-CoV-2 spike (S) protein is also expressed in the testis, predominantly in the spermatogonial stem cells and spermatogonia (2,3). Since SARS-CoV-2 RNA has been detected in the plasma of COVID-19 patients (4), it is expected that the virus might interact with ACE2 and TMPRSS2 in testicular tissue and exert its deleterious effects.
Viral orchitis is commonly reported with mumps virus and human immunodeficiency virus (HIV) and rarely with Hepatitis B and C viruses, Epstein-Barr virus and Papilloma virus. Orchitis has also been reported on autopsy studies of SARS patients (original outbreak in 2003 caused by SARS-CoV, the ‘cousin’ of SARS-CoV-2). Xu et al. had demonstrated extensive germ cell destruction, thickening of basement membrane, peritubular fibrosis, interstitial leukocyte infiltration and vascular congestions in testicular tissues of six SARS cases. Immunohistochemistry had shown CD3+ T-cells and CD-68+ macrophages in the seminiferous tubules (5). SARS-CoV was also detected in testicular epithelial cells and Leydig cells by electron microscopy combined with in-situ hybridization (6). Data on semen analysis, serum testosterone and fertility in SARS survivors are however not available. Nevertheless, SARS-CoV infection has been shown to significantly reduce serum testosterone in male mice (7). This observation needs to be interpreted cautiously as any acute critical illness can lead to a decrease in serum testosterone secondary to cytokine-mediated suppression of hypothalamic-pituitary-testicular axis, biochemically manifesting as low luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone (T) (hypogonadotropic hypogonadism). Nevertheless, considering the fact that the novel SARS-CoV-2 shares 76% amino acid sequence homology with the original SARS-CoV combined with hardcore evidence of expression of ACE2/TMPRSS2 in human testis, it is likely that COVID-19 may have its repercussions on male reproductive function.
Effect of COVID-19 on Leydig cells
The interstitial cells of Leydig represent the endocrine cells of the human testis and are concerned with production of testosterone. Leydig cells express ACE2 and are likely targets of SARS-CoV-2 (2,8). A recent study by Ma et al. from China compared sex-related hormones of 81 reproductive-aged men with COVID-19 with 100 age-matched healthy men. Men with COVID-19 had lower serum total testosterone (T) (although not statistically significant) and significantly higher serum luteinizing hormone (LH). Serum T:LH ratio was also significantly lower in cases compared to controls and was negatively associated with disease severity. No significant difference was observed in either serum estradiol (E2) or T: E2 ratio (9). High LH and low T points to a primary testicular defect that could have been the result of direct action of SARS-CoV-2 on Leydig cells (10). Besides, low serum testosterone, as seen in patients with hypogonadism, is associated with increased pro-inflammatory cytokines that may promote progression of COVID-19 infection due to the cytokine storm (11).
Effect of COVID-19 on cells of seminiferous tubules
Seminiferous tubules constitute upto 90% of the human testis. They are lined by sustentacular cells called Sertoli cells surrounded by germ cells and spermatozoa at different stages of development and maturation. As has already been described, the Sertoli cells and the germ cells express ACE2 and can be potential sites of involvement in COVID-19. This may affect spermatogenesis that would be reflected in semen analysis. Permanent damage to the germ cells may also manifest clinically as infertility. However, data on infertility or semen quality in COVID-19 are not available. Available studies have concentrated only on demonstrating SARS-CoV-2 in semen of COVID-19 patients. Among 12 men in the recovery phase of COVID-19, none tested positive for SARS-CoV-2 in their semen specimens (12). A study in 34 adult Chinese men who had recovered from COVID-19 reported that 6 patients (19%) had demonstrated scrotal discomfort around the time of disease confirmation that might suggest underlying viral orchitis. However, semen analysis could not identify SARS-CoV-2 in the specimen. Semen quality was not assessed (13). In another report, semen and urine samples were tested for SARS-CoV-2 in a 31-year-old man eight days after confirmation of COVID-19. Both were negative for the virus (14). In another study involving 38 male patients with COVID-19 aged 15 years and above, 6 patients (15.8%) were found to have SARS-CoV-2 in their semen specimen (15). The discordant results between the two aforementioned studies can possibly be explained on the basis of stage of infection when the semen samples were collected (13,15). In the study by Pan et al, semen samples were collected from patients recovering from COVID-19 after a median of 31 days (IQR: 29-36 days) from COVID-19 diagnosis (13). On the contrary, the study by Li et al. had included patients who were in the acute stage of disease as well as those who had achieved clinical recovery. Twenty-seven percent of patients in acute stage and only 9% of patients who had recovered tested positive for SARS-CoV-2 in semen. Besides, the two patients with clinical recovery who were found to harbor SARS-CoV-2 in semen were tested early in the course of their convalescence (15). Thus, it is possible that the SARS-CoV-2, interacting with ACE2 in the cells of the seminiferous tubules, may seed the male reproductive tract during the acute stage of infection. This might be aided by systemic inflammatory milieu, especially interleukin-6 (IL-6) that disrupts blood-testis barrier (16). Even if it cannot replicate in the male reproductive system, it may persist for a few days (but not for long), possibly because testes are immune-privileged sites (15). The hitherto available studies on COVID-19 and male reproductive function have been shown in table 1.
A probable explanation offered by Pan et al. for non-isolation of SARS-CoV-2 in semen was the sparse expression of ACE2 and TMPRSS2 in the human testis as identified on single-cell transcriptome analysis. This is in stark contrast to most of the available studies (1–3). In addition, Pan et al. found a non-overlapping pattern of expression of ACE2 and TMPRSS2, which would theoretically make it difficult for the SARS-CoV-2 to invade the host cell, as both ACE2 and TMPRSS2 are required for viral invasion (13). Prior studies have shown a similar finding with TMPRSS2 expression being highest in the spermatogonial stem cells and lowest in the Sertoli cells, reciprocal to that of ACE2 expression (3). However, it has been hypothesized that apart from the ACE2-TMPRSS2, there exists other viral invasion pathways as well. Notably, CD147 can bind to the S protein and mediate viral invasion. CD147 is expressed in all types of testicular cells with the highest expression seen in differentiating spermatogonia. Moreover, endosomal cysteine proteases, cathepsin B and L are associated with S protein priming in cell lines. Interestingly, CTSB and CTSL are also expressed in human testicular tissues and thereby may obviate the need for S protein priming by TMPRSS2 (3).
Apart from direct invasion of the Sertoli cells/germ cells, certain indirect mechanisms have been implicated in COVID-19-mediated involvement of the male reproductive system. Like influenza virus, SARS-CoV-2 can induce oxidative stress at the cellular level. Increased oxidative stress, in turn, has been implicated in promoting sperm DNA fragmentation and in reducing sperm motility (17,18). In addition, Ang (1–7) produced by ACE2 in the testicular tissues acts through Mas receptor, promotes activation of sperm motility via PI3K/AKT signaling pathway (19). In COVID-19, the host expression of ACE2 is downregulated following viral intrusion (20), thereby abrogating the Ang (1-7)/Mas/PI3K/AKT3 pathway in the testis (21). This might have repercussions on sperm motility and male fertility.
In conclusion, data on male reproductive function in patients with COVID-19 are limited and mostly conjectural at this point of time. Most of the available data are based on small-scale studies and long-term follow-up data is not available. Nevertheless, possibility of testicular involvement in COVID-19 has to be kept in mind. Considering the magnitude of the pandemic and the fact that a large proportion of patients are young and are presently in their reproductive age (22), testicular function in young men recovering from COVID-19 perhaps needs to be monitored periodically. A morning sample of serum T, LH and follicle-stimulating hormone (FSH) would be adequate with seminal analysis being ordered only when hormonal profile is deranged. Certain questions like how frequently and how long one needs to monitor is still unanswered. Large-scale studies in young men recovering from COVID-19 need to be carried to resolve these queries.
- Li M-Y, Li L, Zhang Y, Wang X-S. Expression of the SARS-CoV-2 cell receptor gene ACE2 in a wide variety of human tissues. Infect Dis Poverty [Internet]. 2020 [cited 2020 Apr 30];9. Available from: https://idpjournal.biomedcentral.com/articles/10.1186/s40249-020-00662-x
- Wang Z, Xu X. scRNA-seq Profiling of Human Testes Reveals the Presence of the ACE2 Receptor, A Target for SARS-CoV-2 Infection in Spermatogonia, Leydig and Sertoli Cells. Cells. 2020;9:920.
- Liu X, Chen Y, Tang W, Zhang L, Chen W, Yan Z, et al. Single-cell transcriptome analysis of the novel coronavirus (SARS-CoV-2) associated gene ACE2 expression in normal and non-obstructive azoospermia (NOA) human male testes. Sci China Life Sci [Internet]. 2020 Apr 30 [cited 2020 May 20]; Available from: http://link.springer.com/10.1007/s11427-020-1705-0
- Chang L, Yan Y, Wang L. Coronavirus Disease 2019: Coronaviruses and Blood Safety. Transfus Med Rev [Internet]. 2020 [cited 2020 Apr 1]; Available from: https://linkinghub.elsevier.com/retrieve/pii/S0887796320300146
- Xu J, Qi L, Chi X, Yang J, Wei X, Gong E, et al. Orchitis: A Complication of Severe Acute Respiratory Syndrome (SARS)1. Biol Reprod. 2006;74:410–6.
- Zhao J, Zhou G, Sun Y, Wang S, Yang J, Meng E, et al. Clinical pathology and pathogenesis of severe acute respiratory syndrome. Zhonghua Shi Yan He Lin Chuang Bing Xue Za Zhi Zhonghua Shiyan He Linchuang Bingduxue Zazhi Chin J Exp Clin Virol. 2003;17:217–21.
- Channappanavar R, Fett C, Mack M, Ten Eyck PP, Meyerholz DK, Perlman S. Sex-Based Differences in Susceptibility to Severe Acute Respiratory Syndrome Coronavirus Infection. J Immunol. 2017;198:4046–53.
- Douglas GC, O’Bryan MK, Hedger MP, Lee DKL, Yarski MA, Smith AI, et al. The Novel Angiotensin-Converting Enzyme (ACE) Homolog, ACE2, Is Selectively Expressed by Adult Leydig Cells of the Testis. Endocrinology. 2004;145:4703–11.
- Ma L, Xie W, Li D, Shi L, Mao Y, Xiong Y, et al. Effect of SARS-CoV-2 infection upon male gonadal function: A single center-based study [Internet]. Sexual and Reproductive Health; 2020 [cited 2020 Apr 2]. Available from: http://medrxiv.org/lookup/doi/10.1101/2020.03.21.20037267
- Wang S, Zhou X, Zhang T, Wang Z. The need for urogenital tract monitoring in COVID-19. Nat Rev Urol [Internet]. 2020 [cited 2020 Apr 30]; Available from: http://www.nature.com/articles/s41585-020-0319-7
- Pozzilli P, Lenzi A. Commentary: Testosterone, a key hormone in the context of COVID-19 pandemic. Metabolism. 2020;108:154252.
- Song C, Wang Y, Li W, Hu B, Chen G, Xia P, et al. Absence of 2019 novel coronavirus in semen and testes of COVID-19 patients†. Biol Reprod [Internet]. 2020 [cited 2020 May 20]; Available from: https://academic.oup.com/biolreprod/advance-article/doi/10.1093/biolre/ioaa050/5820830
- Pan F, Xiao X, Guo J, Song Y, Li H, Patel DP, et al. No evidence of SARS-CoV-2 in semen of males recovering from COVID-19. Fertil Steril [Internet]. 2020 [cited 2020 Apr 30]; Available from: https://linkinghub.elsevier.com/retrieve/pii/S0015028220303848
- Paoli D, Pallotti F, Colangelo S, Basilico F, Mazzuti L, Turriziani O, et al. Study of SARS-CoV-2 in semen and urine samples of a volunteer with positive naso-pharyngeal swab. J Endocrinol Invest [Internet]. 2020 [cited 2020 Apr 30]; Available from: http://link.springer.com/10.1007/s40618-020-01261-1
- Li D, Jin M, Bao P, Zhao W, Zhang S. Clinical Characteristics and Results of Semen Tests Among Men With Coronavirus Disease 2019. JAMA Netw Open. 2020;3:e208292.
- Zhang H, Yin Y, Wang G, Liu Z, Liu L, Sun F. Interleukin-6 disrupts blood-testis barrier through inhibiting protein degradation or activating phosphorylated ERK in Sertoli cells. Sci Rep [Internet]. 2015 [cited 2020 May 20];4. Available from: http://www.nature.com/articles/srep04260
- Anifandis G, Messini CI, Daponte A, Messinis IE. COVID-19 and fertility: A virtual reality. Reprod Biomed Online [Internet]. 2020 [cited 2020 May 20]; Available from: https://linkinghub.elsevier.com/retrieve/pii/S1472648320302637
- Homa S, Vassiliou A, Stone J, Killeen A, Dawkins A, Xie J, et al. A Comparison Between Two Assays for Measuring Seminal Oxidative Stress and their Relationship with Sperm DNA Fragmentation and Semen Parameters. Genes. 2019;10:236.
- Valdivia A, Cortés L, Beitia M, Totorikaguena L, Agirregoitia N, Corcostegui B, et al. Role of Angiotensin-(1–7) via MAS receptor in human sperm motility and acrosome reaction. Reproduction. 2020;159:241–9.
- Pal R, Bhansali A. COVID-19, Diabetes Mellitus and ACE2: The conundrum. Diabetes Res Clin Pract. 2020;162:108132.
- Tsuji A, Ikeda Y, Murakami M, Matsuda S. COVID-19, an infertility risk? Clin Obstet Gynecol Reprod Med. 2020;
- Wu Z, McGoogan JM. Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72 314 Cases From the Chinese Center for Disease Control and Prevention. JAMA [Internet]. 2020 [cited 2020 Mar 21]; Available from: https://jamanetwork.com/journals/jama/fullarticle/2762130