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.