Discussion
Viral infections have been associated with male infertility as they can disrupt crucial processes involved in sperm production and function. Testicular inflammation caused by viruses can harm the seminiferous tubules, resulting in reduced sperm count, motility, and morphology. Sexually trasmitted viruses like Zika, HIV, and hepatitis B and C have also been linked to impaired male fertility. Therefore, it is crucial to prioritize the prevention and management of viral infections to safeguard male reproductive health .
Since the outbreak of SARS-CoV-2 in 2002, previous studies have indicated that orchitis, inflammation of the testicles, can disrupt spermatogenesis and germ cell apoptosis, impacting semen quality . In this study, semen samples were collected approximately 3 months after SARS-CoV-2 infection from 20 patients. Interestinlgy, most patients (16 out of 20) exhibited normal ranges of total motile sperm counts and sperm morphology (Table 1 )10,29–32.
Histopathological investigations from other SARS-CoV-2 studies have revealed the presence of inflammatory infiltrates primarly in the seminiferous tubules, suggesting the involvement of inflammatory and immunologic reactions in viral-induced testicular damage . Similarly, studies on
human SARS-CoV-2 infection and animal models have associated hypogonadism with increased levels of pro-inflammatory cytokines , particulalry IL-1β, IL-6, and TNF-α, which are significant inflammatory mediators in SARS-CoV-2 pathogenesis. Therefore, the study examined the levels of inflammatory cytokines and chemokines in the seminal plasma fraction of SARS-CoV-2 exposed individuals. Remarkably, high levels of IL-6, IFN-γ and IL-8 were observed (Figure 2A ). Furthermore, positive correlations were found between IL-6 and IL-8 levels and lower sperm vitality (Figure 3B ). Additionally, several inflammatory cytokines (IL-6 and IL-8, GMCSF level and IL-6, GMCSF and IFN-γ, TNF and IFN-γ) exhibited positive correlations with each other (Figure 4A, B, and C ). These results suggest that SARS-CoV-2 increases cytokines level in semen, leading to an inflammatory condition that negatively impacts sperm vitality.
Considering the increased inflammatory cytokine levels in semen and the crucial role of inflammatory and immunologic reactions in viral-mediated testicular damage , the immune cell profile of human semen samples was investigated. As reported in the literature, all the detected immune population (monocytes, NK, and T-cells, except for naïve T cells) play a critical role in clearing long- and short- term viral infection (Figure 5A ). T cells, in particular, have a significant role in adaptive immunity by recognizing antigenic peptides and differentiating into effector T cells during primary infection. After antigenic clearance, a small fraction of the effector T cells differentiate into memory T cells. However, chronic viral and bacterial infections can lead to altered differentiation, resulting in T-cell exhaustion and impaired immune responses. T-cell exhaustion has been observed in various chronic viral infections and cancer, primarily affecting CD8+ T cells. Interestinlgy, a positive correlation was found between immune cell populations involved in viral clearance (exhausted-like, CD8 effector memory T cells, CD8 terminal effector T cells) and the expression of SARS-CoV-2 receptors TMPRSS2 and ACE2 (Figure 5B ). Furthermore, several immune populations exhibited positive correlations with IFN-γ, IL-6, and IL-1β (Supplementary Figure 1, 2 and 3 ). Patient 10, the only sample in which ACE2 was detected in the plasmatic fraction, displayed increased immune cell recruitment compared to the other samples (Table 2 ). These findings suggest that the inflammatory condition persists approximately 3 months after SARS-CoV-2 infection, as observed in other studies . Moreover, the higher recruitment of immune cell populations correlates with the detection of ACE2.
Recent data indicate that SARS-CoV-2 exploits ACE2 and TMPRSS2 to enter cells . Genetic aberrations in TMPRSS2 and ACE2 may contribute to the severity of orchitis and fertility outcomes. Mild orchitis can result from SARS-CoV-2, while individuals with germline TMPRSS2 losses may be more susceptible to severe orchitis and have a higher risk of sterility. It has been suggested that ACE2 activates inflammasomes upon SARS-CoV-2 binding, and pro-inflammatory cytokines regulate the expression of TMPRSS2 and ACE2. However, individuals with non-obstructive azoospermia exhibit decreased ACE2 expression in Sertoli cells, indicating that ACE2 defects could enhance the impact of TMPRSS2 hypofunction on infertility predisposition.To investigate whether these receptors were modulated after SARS-CoV-2 infections, mRNA expression in the spermatozoa and seminal fluid fractions was analyzed. Surprisingly, both the ACE2 (1 plasmatic and in 3 spermatozoa) and TMPRSS2 (10 plasmatic and in 14 spermatozoa) were detected in both fractions (Figure 6A and B ). The presence of SARS-CoV-2 viral RNA was also assessed in the plasmatic and spermatozoa fractions and found that while N or S genes were not detected in either fractions, RdRp was present in 5 out of 20 patients (Figure 7 ). These results indicate that SARS-CoV-2 internalization receptors remain up-regulated and viral RdRp can still be detected 3 months after infection.
Understanding the safety of cryopreservation techniques such as vitrification or two steps cryopreservation for preserving human semen after SARS-CoV-2 exposure is crucial for clinicians and researchers. Sperm cryopreservation is a crucial technique employed in Assisted Reproductive Technology (ART) programs to preserve male fertility. This study aimed to assess the safety of two commonly utilized methods, namely conventional two-step freezing and vitrification for preserving semen samples from patients who had been exposed to SARS-CoV-2 and required fertility preserrvation.
In conclusion, despite the persistent inflammatory consitions observed in patients recovering from SARS-CoV-2 infection, conventional freezing and vitrification remain safe procedures for preserving male fertility in ART programs. However, it is important to acknowledge certain limitations. The relatively sample size of 20 patients limits the generalizability of our findings to a larger population. The absence of a control group hinders direct comparisons and definitive conclusions regarding the observed effects. Moreover, the potential reversibility of any observed effects on male fertility remains unclear. These limitations sould be taken into consideration when interpreting our results.