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\begin{document}

\title{Impact of SARS-CoV-2 on the Male Reproductive Tract: Insights from Semen
Analysis and Cryopreservation}



\author[1]{Silvia Giugliano}%
\author[1]{Alessandro Mozzarelli M}%
\author[2]{Annalisa Navarra}%
\author[2]{Gabriele De Simone}%
\author[1]{Maria  Rescigno}%
\author[1]{Paolo Levi Setti E}%
\author[2]{Elena Albani}%
\affil[1]{Humanitas University}%
\affil[2]{IRCCS Humanitas Research Hospital}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
SARS-CoV-2, the virus behind the COVID-19 pandemic, affects multiple
organs, including the male reproductive system. The male reproductive
tract expresses ACE-2, the primary viral entry receptor, in
spermatogonia, Leydig, and Sertoli cells. While viral infections can
harm male fertility through cytokine storms, the effects of SARS-CoV-2
on fertility are still unclear. Thus, this study aimed to examine the
impact of COVID-19 on the male reproductive tract. Semen samples from 20
patients, collected 3 months post-SARS-CoV-2 infection, revealed the
presence of ACE2 and TMPRSS2 receptors in both plasmatic and spermatozoa
fractions. Five patients showed viral RNA-dependent RNA polymerase
(RdRp), indicating potential viral persistence. Elevated levels of
inflammatory cytokines and chemokines were observed in the plasmatic
fraction, suggesting a persistent inflammatory condition affecting sperm
vitality. Immune cell populations associated with viral clearance were
identified in the semen fraction, correlating with receptor expression
and inflammatory cytokines. To address SARS-CoV-2 uncertainties in
reproductive medicine, we assessed the virus's presence in seminal fluid
before and after cryopreservation using slow freezing and vitrification.
Our study confirms the safety of both techniques in preserving male
fertility for assisted reproductive technology programs. In conclusion,
our study highlights the impact of SARS-CoV-2 on male reproductive
health, emphasizing the persistence of viral entry receptors, potential
viral RNA presence, the inflammatory environment, and the involvement of
immune populations in the male reproductive tract post-infection.
Importantly, we confirm the safety of conventional freezing and
vitrification techniques for preserving male fertility in assisted
reproductive technology programs amidst the COVID-19 pandemic.%
\end{abstract}%



\sloppy


Silvia Giugliano ORCHID ID: 0000-0003-1819-0642

\textbf{Impact of SARS-CoV-2 on the Male Reproductive Tract: Insights
from Semen Analysis and Cryopreservation}

Authors: Silvia Giugliano\textsuperscript{1,4}*\selectlanguage{ngerman}\textsuperscript{§},
Alessandro M Mozzarelli\textsuperscript{1§}, Annalisa
Navarra\textsuperscript{2}, Gabriele De Simone\textsuperscript{3}, Maria
Rescigno\textsuperscript{4}, Paolo E Levi Setti\textsuperscript{1,2},
Elena Albani\textsuperscript{2}*

\textbf{Silvia Giugliano:} \textsuperscript{1}Department of Biomedical
Sciences, Humanitas University, Pieve Emanuele, MI, 20072,
Italy;\textsuperscript{4}Lead Contact; \textsuperscript{*}Corresponding
Author\textsuperscript{; §}Shared first-authorship; email
:\emph{silvia.giugliano@hunimed.eu}; Orchid: Silvia Giugliano:
0000-0003-1819-0642

\textbf{Alessandro M Mozzarelli:} \textsuperscript{1}Department of
Biomedical Sciences, Humanitas University, Pieve Emanuele, MI, 20072,
Italy; \textsuperscript{§}Shared first-authorship; email
:Alessandro.Mozzarelli@nyulangone.org

\textbf{Annalisa Navarra:} \textsuperscript{2}Department of Gynecology,
Division of Gynecology and Reproductive Medicine, Fertility Center,
Humanitas Research Hospital, IRCCS, Rozzano Milan 20089,
Italy;\emph{annanavarra@hotmail.it}

\textbf{Gabriele De Simone:} \textsuperscript{3}Flow Cytometry Core,
IRCCS Humanitas Research Hospital, 20089 Rozzano, Milan, Italy; email:
Gabriele.De\_Simone@humanitasresearch.it

\textbf{Maria Rescigno:} \textsuperscript{1}Department of Biomedical
Sciences, Humanitas University, Pieve Emanuele, MI, 20072,
Italy;\textsuperscript{4}IRCCS Humanitas Research Hospital, 20089
Rozzano, Milan, Italy; email:\emph{maria.rescigno@hunimed.eu}

\textbf{Paolo E Levi Setti:} \textsuperscript{1}Department of Biomedical
Sciences, Humanitas University, Pieve Emanuele, MI, 20072,
Italy;\textsuperscript{2}Department of Gynecology, Division of
Gynecology and Reproductive Medicine, Fertility Center, Humanitas
Research Hospital, IRCCS, Rozzano Milan 20089, Italy;
email:\emph{paolo.levi\_setti@hunimed.eu}

\textbf{Elena Albani:} \textsuperscript{2}Department of Gynecology,
Division of Gynecology and Reproductive Medicine, Fertility Center,
Humanitas Research Hospital, IRCCS, Rozzano Milan 20089,
Italy;\textsuperscript{*}Corresponding Author; email
elena.albani@humanitas.it

\textbf{Running Head:} ''SARS-CoV-2 and Male Reproductive Health''

\textbf{Conflict of Interest:} The authors declair no competing
interests

\textbf{Funding:} This investigation was financially supported by the
FERRING COVID-19 INVESTIGATIONAL GRANTS in Reproductive Medicine and
Maternal Health (RMMH) 2020.

\textbf{Abstract}

SARS-CoV-2, the virus behind the COVID-19 pandemic, affects multiple
organs, including the male reproductive system. The male reproductive
tract expresses ACE-2, the primary viral entry receptor, in
spermatogonia, Leydig, and Sertoli cells. While viral infections can
harm male fertility through cytokine storms, the effects of SARS-CoV-2
on fertility are still unclear. Thus, this study aimed to examine the
impact of COVID-19 on the male reproductive tract.

Semen samples from 20 patients, collected 3 months post-SARS-CoV-2
infection, revealed the presence of ACE2 and TMPRSS2 receptors in both
plasmatic and spermatozoa fractions. Five patients showed viral
RNA-dependent RNA polymerase (RdRp), indicating potential viral
persistence. Elevated levels of inflammatory cytokines and chemokines
were observed in the plasmatic fraction, suggesting a persistent
inflammatory condition affecting sperm vitality. Immune cell populations
associated with viral clearance were identified in the semen fraction,
correlating with receptor expression and inflammatory cytokines.

To address SARS-CoV-2 uncertainties in reproductive medicine, we
assessed the virus's presence in seminal fluid before and after
cryopreservation using slow freezing and vitrification. Our study
confirms the safety of both techniques in preserving male fertility for
assisted reproductive technology programs.

In conclusion, our study highlights the impact of SARS-CoV-2 on male
reproductive health, emphasizing the persistence of viral entry
receptors, potential viral RNA presence, the inflammatory environment,
and the involvement of immune populations in the male reproductive tract
post-infection. Importantly, we confirm the safety of conventional
freezing and vitrification techniques for preserving male fertility in
assisted reproductive technology programs amidst the COVID-19 pandemic.

\textbf{Keywords}

SARS-CoV-2, male reproductive sytem, inflammation, viral clearance,
cryopreservation

\textbf{Introduction}

In late 2019, an outbreak of pneumonia caused by a newly discovered
coronavirus called Severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) originated in Wuhan, China . This virus spread quickly
worldwide and let to a severe acute respiratory syndrome known as
''COVID-19'' as designated by the World Health Organization (WHO). The
rapid transmission of this novel virus resulted in the WHO declaring it
a pandemic, causing significant changes in social behavior. COVID-19 has
had a profound impact on the health of affected individuals and has also
greatly affected healthcare delivery for all patients, not just those
with COVID-19.

Coronaviruses have a spherical shape awith spike proteins on their
envelope, giving them a club-like appearance. The virus contains a
single-stranded RNA genome with a positive sense, which is enclosed
within a helical nucleocapsid. The entry of SARS-CoV-2 into cells is
mediated by the viral glycoprotein Spike binding to the enzymatic domain
of angiotensin-converting enzyme 2 (ACE-2) on the cell surface . The
virus is then internalized through endocytosis by another membrane bound
protease called transmembrane serine protease 2 (TMPRSS2) . Several
testicular cells, including Sertoli, Leydig cells and spermatogonia
cells, express the ACE-2 receptor on their surface along with proteins
belonging to the angiotensin pathway (ATR1, ATR2, ACE1, and TMPRSS2) .
There receptors and proteins are crucial for SARS-CoV-2 infection. Given
that other RNA viruses such as Ebola, Marburg, and Zika, have been found
to breach the blood-testis barrier and cause inflammation , it was
hypothesized that the male reproductive system could also be a target
for SARS-CoV-2. Additionally, numerous studies have reported that viral
infections significantly contribute to long-term male infertility.
Viruses like HPV (papillomavirus), HSV (herpes simplex virus), and HIV
(immunodeficiency virus) can infect the male reproductive system,
leading to severe consequences for reproductive health , possibly due to
triggering an inflammatory response that result in the release of
inflammatory cytokines and subsequent oxidative stress.

In light of these considerations, we conducted an investigation into the
inflammatory status of the testis by evaluating the presence of
inflammatory cytokines and immune cell populations in seminal fluid
after viral clearance.

To date, there has been limited research on the mechanisms responsible
for the observed effects of COVID-19 infection on semen quality.
However, a recent study has provided valuable insights, confirming that
COVID-19 infection can reduce semen volume, progressive motility,
morphology, sperm count, and DNA integrity .

Preserving male reproductive cells, including semen, epididymal, and
testicular spermatozoa, through cryopreservation, is a crucial approach
for managing permanent or temporary infertility in young men and
children. Cryopreservation technology, in particular, plays an important
role in preserving fertility for these patients . It remains uncertain
whether viral particles persist after cryopreservation, and this
question is especially relevant for cryopreserved samples processed
during the COVID outbreak. Therefore, we investigated the presence of
SARS-CoV-2 genome in seminal fluid before and after cryopreservation
using two methods: conventional slow freezing and vitrification. While
slow freezing is a commonly employed technique in assisted reproductive
technology, seminal fluid vitrification may offer a more efficient and
faster approach for sperm vitrification. However, due to the risk of
contamination from direct contact with liquid nitrogen, sperm
vitrification is considered a hazardous procedure
.\textsuperscript{13,271113--19}

Our aim was to investigate the effect of COVID-19 on the male
reproductive tract. We collected semen samples from 20 patients, with a
median interval of 3 months from SARS-CoV-2 infection. Our findings
demonstrated that both ACE2 and TMPRSS2 were detectable in both the
plasma and sperm fractions even months after the viral infection.
Additionally, we found viral RNA-dependent RNA polymerase (RdRp) in 5
out of 20 patients, indicating the potentional presence of the virus in
the body three months after the initial infection. The study also
revealed high levels of inflammatory cytokines and chemokines in the
plasma seminal fraction of individuals exposed to SARS-CoV-2-,
suggesting the persistence of an inflammatory condition that could
impact spermatozoa vitality. We also identified immune populations in
the semen fraction that are known to play a role in clearing viral
infection, and these populations positively correlate with the
expression of of SARS-CoV-2 receptors and inflammatory cytokines.

Furthermore, we investigated the potential implicaitons of SARS-CoV-2 in
reproductive medicine, particularly in medically assisted reproduction
and gamete cryopreservation. We examined the presence of SARS-CoV-2
genome in seminal fluid before and after cryopreservation using two
methods: conventional slow freezing and vitrification. Our findings
demonstrate that both conventional freezing and vitrification are safe
procedures for preserving male fertility in assisted reproductive
technology programs.

\textbf{Methods}

\textbf{Semen Sample Collection}

Semen sample were collected by masturbation with an ejaculatory
abstinence of 3 to 5 days for all patients. Semen analysis was performed
according to the WHO 2010 guidelines, evaluating sperm volume,
concentration, motility, viability, morphology. Susequently, semen
samples were treated using conventional freezing and vitrification
methods. For each patient, a semen sample was used to perform both
conventional freezing and vitrification. Additionally, a partion of the
sample was treated to separate seminal plasma from pellet for the
molecular and cellular analysys. In more detail, we recruited 20
patients from January to April 2021. The average age of 20 patients was
39 years (\textbf{Table 1} ) and none of the patients were hospitalized
due to the SARS-CoV-2 infection. The median interval from viral
infection to provide semen samples was 3 months.

\textbf{Semen Preparation}

The semen sample was divided, with 0,5 mL being used for
cryopreservation. Another 0.5 mL was centrifuged at 2000 rpm for 10
minutes, to separate seminal plasma and the pellet. Furthermore, 1 mL of
the sample was stratified using a 80\% / 40\% of a solution of Pure
sperm 100 Nidacon in Sperm Washing Medium (Irvine Scientific, Santa Ana,
CA) to separate round cells from the remaining semen. The resulting
fraction was then centrifuged at 1600 rpm for 15 minutes, and the pellet
containing spermatozoa was resuspended in TRIZOL (Thermo Scientific,
Waltham, MA) and stored at -80C. The layer between the gradients, which
contained round cells, was recoverd, centrifuged at 1800 rpm for 10
minutes, and resuspended in 500 mL of FBS10\% DMSO.

\textbf{Vitrification}

For vitrification aliquots of 30\selectlanguage{greek}µ\selectlanguage{english}l of prepared sperm mixed with a
sucrose solution were directly dropped into liquid nitrogen using
micropipettes. A stainless strainer was present in the liquid nitrogen
to capture the formed spheres. This spheres were then transferred into
pre-cooled cryovials and stored in liquid nitrogen for further storage.

The vitrification protocol followed the procedure described in ``A
simple method of human sperm vitrification'' \textbf{.} Swim up sperm
(30 microliters) combined with 0.5 M sucrose solution in a 1:1 ratio
were dropped into liquid nitrogen using a micropipette, causing them to
harden into spheres that sank into a stainless steel tea strainer. These
spheres were quickly transferred into cryovials previously cooled in
liquid nitrogen for future storage.

After vitrification, prompt transfer into cryovials in liquid nitrogen
was crucial to maintain their integrity. Larger volumes necessitated the
transfer of more spheres, increasing the risk of spontaneous thawing
during the process. This transfer procedure was vital for achieving high
post-warming survival and motility.

For devitrification/warming, the spheres were removed from storage after
a week and placed in 5 ml of prewarmed gamete handling fluid
supplemented with human serum albumin at 42 degrees Celsius for 10
seconds.

\textbf{Conventional Freezing}

The conventional freezing method was performed following the guidlines
of SpermFreeze Solution: Quinn's Advantage Sperm Freezing Medium (Cooper
Surgical Trumbull CT). SpermFreeze solution was added to the seminal
sample in cryotube vials at a 1:1 ratio. The cryovials were kept at room
temperature for10 minutes, after which they were cooled in a vapor state
for 20 minutes. They were placed horizontally, approximately 3 cm above
the liquid nitrogen surface, reaching a temperature of approximately -70
to -90\selectlanguage{ngerman}°C. Finally, the cryovials were submerged in liquid nitrogen and
stored at -196 \selectlanguage{english}0C.

\textbf{Flow-Cytometry Analysis}

Multiparameter Flow Cytometry analysis was performed on semen samples
from COVID-19 patients using a BD FACSymphony A5 instrument (BD
Biosciences) and analyzed with FlowJo (FlowJo LLC, Ashland, OR). Gates
were done on live cells and the following primary antibodies were used
for immune cell characterization: CD163 (BD Horizon, Franklin Lakes, NJ,
clone MAC2-158), CD45 (Biolegend, San Diego, CA, clone HI30), CD56 (BD
Biosciences, clone MY31), CD19 (Biolegend, clone HIB19), CD16
(Biolegend, clone 3G8), CD25 (BD Biosciences, clone 2A3), CD123 (BD
Biosciences, clone 7G3), HLA-DR (BD Biosciences, clone G46-6), PD-1
(Biolegend clone EH12.2H7), CD8 (BD Biosciences, clone SK1), CD95 (BD
Biosciences, clone DX2), CD4 (BD Biosciences, clone M-T477), CD11c
(Biolegend, clone Bu15), CD127 (eBioscience, clone eBioRDR5), CD45RO(BD
Biosciences, clone UCHL1), CD3 (BD Biosciences, clone UCHT1), CD14
(Biolegend, clone M5E2), CD68 (BD Biosciences, clone Y1/82A), CD27 (BD
Biosciences, clone M-T271), CCR7 (Bd Biosciences, clone 3D12).

\textbf{RT-qPCR Assay}

Viral RNA was extracted from plasma and sperm of recovered patients
using RNA with Quick-RNA MiniPrep (ZYMO RESEARCH, Irvine CA, catalog
\#1054) according to the manufacturer's guidelines. Genomic DNA
contamination was removed with DNase-Free DNase set (ZYMO RESEARCH,
catalog \#E1010). The total RNA extracted was stored at -80\selectlanguage{ngerman}°. Then,
500ng RNA was retro-transcribed with ImProm-II Reverse Transcriptase kit
(Promega) following manufacturer's instruction and random primers (0,5
ug/ul). qPCR assay was performed with Luna Universal qPCR Master Mix
(New England Biolabs) on 10 ng cDNA template/reaction using
exon-spanning primers (final concentration 1 \selectlanguage{greek}μ\selectlanguage{english}M). \selectlanguage{greek}ΔΔ\selectlanguage{english}CT and 2\^{}-\selectlanguage{greek}Δ\selectlanguage{english}CT
methods were used to calculate the expression levels, samples were
normalized to GAPDH.

\textbf{Cytometric Bead Array (CBA)}

Using the BD Cytometric Bead Array (CBA) Human Kit Flex Set (BD
Bioscience-Pharmingen, San Diego, CA). to measure IL-2 (catalog
\#558270), IL-6 (catalog \#558276), IFN-\selectlanguage{greek}γ \selectlanguage{english}(catalog \#558269), IL-10
(catalog \#558274), TNF (catalog \#560112), IL-1\selectlanguage{greek}β \selectlanguage{english}(catalog \#558279),
IFN-\selectlanguage{greek}α \selectlanguage{english}(catalog \#560379), GM-CSF (catalog \#558335) and IL-8 (catalog
\#558277). The kit contains three bead populations with distinct
fluorescence intensities. The beads were coated with capture antibodies
specific to IL-2, IL-6, IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-10, TNF, IL-1\selectlanguage{greek}β, \selectlanguage{english}IFN-\selectlanguage{greek}α, \selectlanguage{english}GM-CSF and IL-8
and mixed with phycoerythrin (PE) conjugated detection antibodies. These
capture beads were incubated with recombinant standards or test samples
to form sandwich complexes. The CBA was analyzed at FACSCcanto flow
cytometer (BD Bioscience-Pharmingen). Results were anayzed using graphic
and tabular formate using CBA analysis software (BD
Bioscience-Pharmingen).

\textbf{Statistical analysis}

The investigators were not blinded to allocation during experiments and
outcome assessment. Data were analyzed using Prism software (GraphPad8,
San Diego, CA). Each singel dot in every graph represents an individual
patient. Analyses were performed using Single Linear regression. A
p-value of less than 0.05 is considered statistically significant.

\textbf{Results}

\textbf{Demographics and Characteristics of Recovered COVID-19
Patients.}

We enrolled a total of 20 patients between January and April 2021. The
average age of the patients was 39 years (\textbf{Table 1} ) and none of
them required hospitalization due to the SARS-CoV-2 infection. The
median time between viral infection and collection of semen samples was
3 months. In the semen specimens, all patients tested negative for
SARS-CoV-2 RNA. However, before cryopreservation, 5 patients tested
positive for the RNA-dependent RNA polymerase (RdRp) in their
spermatozoa.

The total sperm count per ejaculate averaged 88
x10\textsuperscript{6}+/- 70,16. Progressive motility percentage was
26\% +/-7,71 and eosin percentage was 78,5\% +/-8,42. Out of the 20
patients, 16 had total motile sperm counts and sperm within normal
ranges. One patient showed mild necrozoospermia and asthenospermia, two
showed mild asthenospermia and one had a mild oligospermia
(\textbf{Table 1} ).

\textbf{Impact of Testis Inflammatory State on Sperm Vitality.}

To investigate the potential impacy of SARS-CoV-2 infection on sperm
concentration, motility and vitality, we assessed macroscopic and
microscopic parameters of seminal fluid (\textbf{Table 1} ). Our
findings, as shown in \textbf{Figure 1,} confirmed that spermatozoa with
higher motility percentages exhibited greater vitality scores
(p=0,0334;\textbf{Figure 1} ). Furthermore, we analyzed the presence of
inflammatory cytokines and chemokines in the seminal fluid of our cohort
(\textbf{Figure 2A} ) to evaluate the inflammatory state of the testis.
Specifically, we observed elevated levels of IL-6, IFN-\selectlanguage{greek}γ \selectlanguage{english}and IL-8.
Notably, the concentration of IL-6 positively correlated with
concentration of IL-8 (p\textless{}0,0001; \textbf{Figure 2B} ), and the
two samples with the highest IL-6 concentration had also exhibited the
highest IL-8 level (red circle, \textbf{Figure 2A} ).

To determine whether high levels of inflammatory cytokines could impact
spermatozoa and affect vitality and motility we conducted linear
regression analyses. Interestingly, we found that IL-6 and IL-8 level
did not significantly influence spermatozoa motility (\textbf{Figure 3A}
), but both cytokine levels were positively correlated with reduced
spermatozoa vitality (IL-6 p=0,0493 and IL-8 p=0.0350; \textbf{Figure
3B} ). Additionally, we explored the correlations among various
inflammatory cytokines and chemokines detected in the seminal plasma.
Notably, we observed a positive correlation between GMCSF level and IL-6
(p=0,0097; \textbf{Figure 4A} ), GMCSF and IFN-\selectlanguage{greek}γ
\selectlanguage{english}(p=0,0219;\textbf{Figure 4B} ), TNF and IFN-\selectlanguage{greek}γ \selectlanguage{english}(p=0,035; \textbf{Figure
4C} ).

\textbf{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 (\textbf{Supplementary Table 1} ). The scheme
presented in \textbf{Supplementary Table 2} was used to characterize the
CD45\textsuperscript{+} 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 na\selectlanguage{ngerman}ïve T
cells, CD8 terminal effector T cells, TPEX (exhausted cells
progenitors), and exhausted-like T cells (\textbf{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 (\textbf{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-\selectlanguage{greek}γ \selectlanguage{english}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-1\selectlanguage{greek}β \selectlanguage{english}and monocytes, B cells, and memory B cells
(\textbf{Supplementary Figure 1, 2, and 3} ).

\textbf{Detection of ACE2 and TMPRSS2 in Plasma and Sperm} \textbf{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) \textbf{(Figure 6A} ). On the other hand, TMPRSS2 gene expression
was identified in 10 plasmatic fractions and 14 spermatozoa fractions
(\textbf{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
(\textbf{Figure 6C} ).

\textbf{Safety of Cryopreservation for SARS-CoV-2 Samples.}

During the COVID-19 pandemic, cryopreservation techniques were employed
by Assistance Reproductive Technology (ART) clinics for precautionary
purposes, but without conducting a semen COVID-19 screening due to its
unavailability. Therefore, our objective was to investigate whether we
could identify SARS-CoV-2 viral RNA in the plasmatic fraction and
spermatozoa before and after cryopreservation. We performed viral RNA
extraction from both fractions and utilized RT-PCR to detect the nuclear
N gene, the Spike S gene and the RdRp gene. Interestingly, we were
unable to detect the N and S gene in eithr the plasmatic or spermatozoa
fraction. However, RdRp detection was succseful in 5 out of 20 patients
before cryopreservation (\textbf{Figure 7} ). Notably, when the same
samples were tested after undergoing slow conventional cryopreservation
and vitrification, the RdRp viral gene was no longer detectable. This
suggest that both freezing procedures are safe for storing samples
collected from individuals exposed to SARS-CoV-2.

\textbf{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 (\textbf{Table 1} )\textsuperscript{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-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, and TNF-\selectlanguage{greek}α, \selectlanguage{english}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-\selectlanguage{greek}γ \selectlanguage{english}and IL-8 were observed (\textbf{Figure 2A} ). Furthermore,
positive correlations were found between IL-6 and IL-8 levels and lower
sperm vitality (\textbf{Figure 3B} ). Additionally, several inflammatory
cytokines (IL-6 and IL-8, GMCSF level and IL-6, GMCSF and IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF and
IFN-\selectlanguage{greek}γ\selectlanguage{english}) exhibited positive correlations with each other (\textbf{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 na\selectlanguage{ngerman}ïve T cells) play a
critical role in clearing long- and short- term viral infection
(\textbf{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
(\textbf{Figure 5B} ). Furthermore, several immune populations exhibited
positive correlations with IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-6, and IL-1\selectlanguage{greek}β \selectlanguage{english}(\textbf{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 (\textbf{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 (\textbf{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 (\textbf{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.

\textbf{Ethic Statement}

The study complied with the Declaration of Helsinki and was approved by
the institutional ethics committee of Humanitas Research Hospital
(Protocol number 230879). Semen samples were obtained after written and
informed consent from 20 patients undergoing a seminal fluid evaluation.

\textbf{Acknowledgments}

The authors would like to thank the patients from Humanitas Research
Hospital.

\textbf{Author Contributions}

SG designed the study, conceived and performed experiments, analyzed
data, wrote the manuscript, supervised the funding of the work, and
secured the findings; AMM performed experiments, analyzed the data and
wrote the manuscript; AA performed experiments and wrote the manuscript,
GD acquired data, analyzed data and contributed to interpretation of the
results, EA performed experiments, contributed to interpretation of the
results and wrote the manuscript. TS provided expertise and feedback. MR
and PELS supervised the study. All authors discussed the results and
contributed to the final version of the manuscript.

\textbf{Data Availability Statment}

The raw data supporting the current study have been deposited to Zenodo
public repository10.5281/zenodo.8138194

\textbf{Figures and Tables}

Table 1: Semen parameters of SARS-Cov-2 recovered patients.\selectlanguage{english}
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