SARS-CoV-2 infection leads to sustained testicular injury and functional impairments in K18 hACE2 mice (preprint)

Compromised male reproductive health is one of the symptoms of long COVID with a decrease in male fertility markers including testosterone levels and sperm count for months in recovering patients. However, the long-term impact of SARS-CoV-2 infection on testicular injury and underlying mechanisms remains unknown. We previously demonstrated a disrupted tissue architecture with no evidence of virus replication in the testis during the acute stage of the disease in K18-hACE2 mice. Here, we systematically delineate the consequences of SARS-CoV-2 infection on the testis injury and function both during the acute stage of the disease and up to 4 weeks after infection in survivor K18-hACE2 mice. The gross morphological defects included sloughing of healthy spermatids and spermatocytes into the lumen, lack of lumen, and increase in apoptotic cells that sustained for at least 2 weeks after infection. Testis injury correlated with systemic and testicular inflammation, and infiltration of immune cells in the interstitial space and seminiferous tubules. Transcriptomic analysis identified dysregulation of key pathways of testicular immune homeostasis, spermatogenesis, and cell death at the symptomatic and short-term recovery stages. Further, a significant reduction in testosterone levels was associated with transient reduction in sperm count and mouse fertility. Most of the testicular impairments except testosterone levels were resolved within 4 weeks, which is almost one spermatogenesis cycle in mice. These findings provide much-needed mechanistic insights beyond our current understanding of testicular pathogenesis, suggesting that recovering COVID-19 patients should be closely monitored to rescue the pathophysiological effects on male reproductive health.

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury. (preprint)

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ failure including testicular injury and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury can likely result either from direct virus infection of resident cells or by exposure to systemic inflammatory mediators or virus antigens. We here characterized SARS-CoV-2 infection in different human testicular 2D and 3D models including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not establish a productive infection in any testicular cell types. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma depicted a significant decrease in cell viability and death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 envelope protein, but not Spike or nucleocapsid proteins led to cytopathic effects on testicular cells that was dependent on the TLR2 receptor. A similar trend was observed in the K18h-ACE2 mouse model which revealed gross pathology in the absence of virus replication in the testis. Collectively, data strongly indicates that the testicular injury is not due to direct infection of SARS-CoV-2 but more likely an indirect effect of exposure to systemic inflammation or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.