The role of SARS-CoV-2 in the pathogenesis of testicular damage


SARS-CoV-2 can cause acute testicular damage with subsequent chronic asymmetric testicular atrophy and associated hormonal changes despite a self-limiting pneumonia in hamsters.

Awareness of possible hypogonadism and subfertility is important in managing convalescent COVID-19 males.

Hamsters are now well established as a physiological small animal model for COVID-19 [12]. The animals recovered clinically by 28dpi. Here we showed that testicles were sub- clinically affected with acute decrease in sperm count and subsequent chronic asymmetric testicular atrophy associated with oligospermia and persistently low serum testosterone and inhibin B.

These testes showed degeneration, necrosis and inflammation at both the interstitium with Leydig cells, and also seminiferous tubules with disruption of orderly spermatogenesis from 4 to 120dpi. Immunohistochemical staining of biomarkers showed that both Sertoli and germ cells of seminiferous tubules were severely affected.

Viral N-protein expression was occasionally found in germ, interstitial and epididymal epithelial cells. These changes were generally more severe with higher virus inoculum. Moreover, intranasal challenge with wild-type SARS-CoV-2, Omicron or Delta variants could consistently cause testicular damage. To prove the causative role of SARS-CoV-2 in the pathogenesis of

testicular damage, we directly injected SARS-CoV-2 into the testicles transcutaneously  which demonstrated far more positive N-protein expression in the interstitium and the presence of viral sgRNA in testicular tissue than with A(H1N1)pdm09 control virus.

Blood-borne and hepatitis viruses including HIV, HBV, HCV, and HEV are associated with subfertility and decreased sperm count or quality [21]. Clinical cases of coxsackie virus associated epididymo-orchitis are reported [22], but only mumps virus is convincingly associated with human orchitis [23, 24]. However, no animal models proving the causative role of these viruses in orchitis were reported.

Filovirus with a prolonged high level of viremia [25], leading to severe haemorrhagic fever can lead to persistent infection of immunoprivileged organs including the central nervous system and the male reproductive tract in human and animal models [26]. Similarly, HEV infection can be associated with a prolonged viremia and subfertility in human [27].

Some arboviruses such as the Zika with prolonged viremia [28], is associated with sexual transmission, central nervous system infection, orchitis, and subfertility. Its causative role in epididymoorchitis is proven in an interferon α/β receptor deficient mice model.

Here we showed that SARS-CoV-2, with absent or transient viremia could cause persistent testicular damage in the clinically relevant hamster model with self-limiting pneumonia.

Involvement of the male reproductive tract by SARS-CoV-2 has previously been suspected because angiotensin converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, and/or transmembrane protease serine 2 (TMPRSS2), enzyme for proteolytic activation of spike, are abundantly expressed in spermatogonia, Sertoli, Leydig and myoid cells[29, 30].

Furthermore, case series of testicular or epididymal pain, histopathological changes of orchitis on postmortem examination, SARS-CoV-2 RNA detection in semen, reduced sperm

counts and serum testosterone level were also reported[31-34] . Besides conflicting clinical reports[8, 9], the testicular damage could be due to direct viral invasion or an indirect effect of systemic hypercytokinaemia, endotheliitis, vasculitis, thrombosis, and hypoxia due to respiratory failure or shock in severe COVID-19 [2].

Two previous hamster studies demonstrated very low viral load in testicles. However, one intranasal challenge study showed expression of testicular SARS-CoV-2 RNA without histopathological damages up to one month post-infection, despite the detection of viral replication in ex vivo hamster testicular cells[35]. Another hamster study only showed oophoritis but not orchitis despite patchy prostatitis and seminal vasculitis[36].

Here, we demonstrated that increasing infectious SARS-CoV-2 dose was associated with worsening testicular damage as evident by lower testicular weight at 120dpi and more severe and persistent histopathological changes. This may explain the high incidence of testicular changes found in deceased patients[33, 37]. The higher virus inoculum may increase the likelihood of SARS-CoV-2 crossing the blood-testicular-barrier into this immune-privileged organ.

Though virus replication and leukocyte infiltration in testicles were far lower than lungs, we speculate that acute damage at 4 to 7dpi is more related to highly susceptible germ cells to the cytokine/chemokines produced by Sertoli cells and interstitial macrophages for innate immune response against the small amount of SARS-CoV-2 going across the blood- testicular-barrier[38].

While the testicular cytokine/chemokines normalized between 42 and 120dpi, the process of immune complex deposition and immunopathological damage might start after 7dpi. As expected, the tissue mRNA expression level of proinflammatory cytokine/chemokines with acute damage, and the apoptotic markers were associated with both acute and chronic testicular damage.

This is consistent with the pathogenesis of mumps orchitis where mumps virus triggers type 1 interferons, TNF-a, IL-6, CXCL10, MCP1 which induce apoptosis, disrupt blood-testicular- barrier, inhibit testosterone synthesis in Leydig cells and recruit inflammatory cells into testes[24].

To prove the causative role of SARS-CoV-2 in pathogenesis of testicular damage, we directly injected SARS-CoV-2 into animal testes to bypass the blood-testicular-barrier. More abundant N-protein expression was found in interstitial cells. Interstitial Leydig cells may be a target of SARS-CoV-2. Leydig cells are the primary source of testosterone in males.

Its high concentration at seminiferous tubules is pivotal for maintaining testicular microvascular blood flow, Sertoli cell maturation, orderly spermatogenesis, and an intact blood-testicular- barrier. Intratesticular inoculation of mumps[39] and  Zika[40]  were used in rodent models of orchitis. Finally, we showed that vaccination given as late as 3 days before virus challenge could protect the animal testes against SARS-CoV-2 induced damage similar to the protection of their lungs[14]

Our findings here are limited to this hamster model. Few antibodies against hamster biomarkers for immunohistochemistry were available to locate infected cell types. We did not follow up beyond 120dpi due to limited availability of BSL3 facilities.

In summary, SARS- CoV-2 can cause acute and chronic testicular damage in hamsters and is consistent with the anecdotal reports of clinical orchitis and hypogonadism in recovered COVID-19 males. Long term follow-up of sperm count and sex hormone profile of convalescent COVID-19 males is warranted.

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