In a recent study posted to the bioRxiv* preprint server, a team of researchers demonstrated that the testicular injury in humans is not due to direct severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection but more likely is an indirect effect of exposure to systemic inflammation or SARS-CoV-2 antigens.
SARS-CoV-2 infects susceptible human cells by binding to angiotensin-converting enzyme 2 (ACE2) receptors which can progress to severe coronavirus disease 2019 (COVID-19). Apart from acute lung injury, other hallmarks of severe COVID-19 are multi-organ injury, cardiac complications, and kidney failure.
Epidemiological studies suggest that males present a higher case-to-fatality ratio than females, and some also experience COVID-19-related inflammation of the testis associated with pain and discomfort termed mild orchitis. Furthermore, postmortem studies have revealed signs of severe testicular pathology as well. Overall, the ability of SARS-CoV-2 to gain access to testis might be a rare event, but testicular injury is one of the complications of COVID-19.
Abundant ACE2 receptors in resident testicular cells might be responsible for testicular injury during SARS-CoV-2 infection. In testis, ACE2 regulates testosterone production and interstitial fluid volume via modulating the conversion of Angiotensin 2 to Angiotensin 1. Other causes of testicular injury could be direct virus infection of resident cells or exposure to systemic inflammatory infection mediators (e.g., cytokine storm) or virus antigens. However, the mechanisms governing testicular injury, one of the hallmarks of severe COVID-19, are not entirely understood.
Moreover, there is a lack of data to back up the observed SARS-CoV-2 replication and pathogenesis in extra-pulmonary organs.
About the study
In the present study, researchers used different models of human testicular cells, including primary human Sertoli cells (SC), Leydig cells (LC), mixed two-dimensional (2D) cultures of seminiferous tubule cells (STC), and three-dimensional (3D) human testicular organoids (HTO) to study SARS-CoV-2 infection dynamics in testes. In addition, they validated the effects of SARS-CoV-2 infection on testicular injury using K18-hACE2 mice.
First, the team infected well differentiated 2D cultures of human airway epithelial cells (HAE) grown on transwell inserts at a multiplicity of infection (MOI) equals one and measured infectious virions on the apical and basal sides of the inserts.
The team exposed STC to UV-inactivated supernatant from HAE and COVID-19 plasma to determine the cytopathic effects (CPE) of SARS-CoV-2 infection-derived inflammatory mediators on testicular cells.
The team measured the messenger ribonucleic acid (mRNA) levels of critical cytokines associated with COVID-19 in SC, LC, and HTO 48 hours post-infection (hpi). The production of key cytokines in the HAE supernatant was further confirmed and compared to plasma from COVID-19 patients during the acute infection phase, i.e., four to six days of symptom onset using enzyme-linked immunosorbent assay (ELISA). They also conducted a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay on STC exposed to both UV-inactivated HAE supernatant and COVID-19 plasma for 24 hours. Finally, they conducted a histopathological assessment of Periodic acid–Schiff (PAS) stained testes sections.
The researchers detected SARS-CoV-2 RNA in the range of log 2 to 3 genome copies in all cell models, but the virus copies did not increase between 24 and 96 hpi. SARS-CoV-2 infection of SC and LC at a higher MOI of 10 also did not trigger SARS-CoV-2 virions release in the HAE supernatant over 96 hours. Likewise, the immunofluorescence assay could not detect the SARS-CoV-2 spike (S) protein in infected SC and LC.
Overall, SARS-CoV-2 could not establish a productive infection in these primary cell cultures of any testicular cell type. However, peak SARS-CoV-2 titers at four dpi correlated with induction of key inflammatory cytokine genes like interleukin 6 (IL6) and tumor necrosis factor-alpha (TNFα) and antiviral genes, including interferon-induced protein with tetratricopeptide repeat one (IFIT1). All these findings reinstated that SARS-CoV-2 did not establish a productive infection in resident human testicular cells. Exposure of testicular cells to inflammatory media from SARS-CoV-2 infected HAE cells led to apoptotic death of undifferentiated spermatogonia. Exposure to SARS-CoV-2 envelope (E) protein but not S1 and nucleocapsid (N) protein induced a pro-inflammatory response associated with cytotoxic effects of severe COVID-19.
SARS-CoV-2 infection-induced CPEs also compromised the integrity of the air-liquid barrier. Accordingly, transepithelial electrical resistance (TEER) readings declined at three dpi with significantly lower values at five and six dpi. Despite no active replication, SARS-CoV-2 infection resulted in interstitial and tubular abnormalities in the testis of hACE2 mice, similar to humans. Similarly, supernatant from SARS-CoV-2 infected HAE mimicked the profile of select cytokines observed in COVID-19 patients and could help evaluate the indirect effect of infection on 2D and 3D human testicular models.
Postmortem studies have characterized testicular injuries in humans to identify its markers. The current study pursued evidence of the association of these injury markers with SARS-CoV-2 infection and showed that the testicular damage is triggered by systemic inflammation caused due to SARS-CoV-2 infection.
There is a need for long-term follow-up of male reproductive health markers, such as testosterone levels, in male patients following moderate to severe COVID-19. Studies should also evaluate specific pathways associated with pathological events following testicular injury, as cytokine storm and viral antigen exposure induced apoptotic death of germ cells that could cause orchitis symptoms.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.