Flow Cytometry Analysis of Semen Samples Recovered from COVID-19 Patients.
In order to gain insight of how the immune cell populations in semen may impact the clereance of SARS-CoV-2, we utilized flow cytometry to characterize the immune cell profile of human semen samples. Due to limited sample recovery after cell isolation, data from 5 out of 16 patients are presented (Supplementary Table 1 ). The scheme presented in Supplementary Table 2 was used to characterize the CD45+ population in monocytes, B cells, memory B cells, cytokine producing NK cells, cytotoxic NK cells, dendritic cells, CD8 central memory T cells, CD8 effector memory T cells, CD8 naïve T cells, CD8 terminal effector T cells, TPEX (exhausted cells progenitors), and exhausted-like T cells (Figure 5A ). Notably, significant positive correlations were assessed between exhausted-like T cells, CD8 effector memory T cells, CD8 terminal effector T cells and the expression of TMPRSS2 and ACE2 (Figure 5B ). Furthermore, to elucidate the potential relationship between immune populations and the cytokines released in the seminal fluid, we conducted correlation analyses. Notably, IFN-γ showed positive correlation with monocytes, exhausted-like T cells, CD8 effector memory T cells, and CD8 terminal effector T cells. Positive correlations were also found between cytotoxic NK, dendritic cells, and IL-6. Lastly, a positive correlation was found between IL-1β and monocytes, B cells, and memory B cells (Supplementary Figure 1, 2, and 3 ).
Detection of ACE2 and TMPRSS2 in Plasma and Sperm of Recovered SARS-CoV-2 Patients.
SARS-CoV-2 utilizes the glycoprotein ACE2 to bind to host cells, including those in the testicular tissue. Upon binding to ACE2, the virus enters the cell through endocytosis facilitated by the transmembrane serine protease 2 (TMPRSS2) . In light of this, we aimed to investigate the gene expression of the SARS-CoV-2 receptor ACE2 and the co-receptor TMPRSS2 in spermatozoa and seminal fluid. Among the 20 patients analyzed, ACE2 expression was detected only in one plasmatic fraction (patient 10) and in 3 spermatozoa fraction (patient 1, 13 and 17) (Figure 6A ). On the other hand, TMPRSS2 gene expression was identified in 10 plasmatic fractions and 14 spermatozoa fractions (Figure 6B ). Notably, all the spermatozoa that tested positive for ACE2 expression also exhibited TMPRSS2 expression, and there was a positive correlation between ACE2 and TMPRSS2 expression levels (Figure 6C ).