Cryopreservation of seminal fluid or spermatozoa from epididymary and testicularretrieval represents a crucial tool in managing conditions of permanent or temporaryinfertility, as well as in cases of secretory and excretory azoospermia. Thesepathologies often affect young men and children, making it essential to maintainfertility through cryopreservation methods such as vitrification or two-stepcryopreservation of human semen.The burden of the SARS-CoV-2 virus has rapidly evolved. SARS-CoV-2 causes respiratory,cardiovascular, digestive, and urinary infections, yet no studies have explored itseffects on the reproductive system. Recent findings confirm the expression of the virusreceptor (ACE2) on certain testicular cells, including Sertoli cells, Leydig cells, andspermatogonia, which could significantly impact fertility and cryopreservation practices.This study aims to utilize semen samples from individuals exposed to SARS-CoV-2 infectionto perform two different cryopreservation procedures, evaluating virus behavior andeffects. The objective is to determine whether it is safer to cryopreserve the entirevolume of the sample or perform gradient separation to isolate the virus fromspermatozoa, potentially establishing a new cryopreservation protocol specific toSARS-CoV-2.
The pandemic caused by SARS-CoV-2 ranks among the most devastating events in recent
history. Limited knowledge exists regarding the characteristics and behavior of this beta
coronavirus, particularly its potential impact on reproductive medicine. Questions remain
about the presence of the virus in seminal fluid and whether male semen could serve as a
transmission route. Additionally, concerns exist about the virus impairing male
reproductive potential through fever or the "cytokine storm" triggered by the immune
response.
No current evidence confirms whether SARS-CoV-2 can survive semen cryopreservation,
though other viruses, such as influenza, have demonstrated survival in liquid nitrogen
freezing. Semen cryopreservation is a critical fertility preservation strategy for
conditions like cancers, autoimmune diseases, or cryptorchidism. The contamination of
cryopreserved semen by SARS-CoV-2 could turn such samples into hazardous viral
reservoirs.
The study aims to investigate the presence of the virus in semen, identify cells
expressing viral receptors ACE2 and TMPRSS2, and evaluate the virus's direct and indirect
effects on male reproductive capacity. Objectives include determining if SARS-CoV-2
survives cryopreservation procedures and identifying optimal methods to ensure virus-free
cryopreserved semen while separating infected material from male gametes.
The study will recruit 20 men exposed to SARS-CoV-2 and 20 control subjects, all treated
at the Humanitas Fertility Center, Department of Gynecology, at the Humanitas Research
Hospital. Each participant will provide a semen sample for evaluation. This observational
study will not involve clinical interventions or alter regular diagnostic procedures.
Participants undergoing routine tests for medically assisted reproduction and deemed
eligible after completing an anamnesis questionnaire will be included. Samples will be
processed in the Mucosal Immunology and Microbiota Unit at the Humanitas Clinical
Institute.
Using cell biology, molecular biology, and flow cytometry techniques, the study will
assess viral contamination, receptor expression in spermatozoa, and inflammatory cytokine
levels in seminal fluid after two cryopreservation methods: slow two-step freezing and
vitrification. Analyses will cover unprocessed semen and samples exposed in vitro to
SARS-CoV-2 under BSL3 conditions.
Additionally, the cellular fraction of semen, including granulocytes, lymphocytes, round
cells, and spermatozoa, will undergo phenotype and activation state characterization. The
study will evaluate viral contamination in cryopreserved semen and assess the
effectiveness of protocols designed to separate infected material from male gametes.
Inclusion Criteria:
Patient population inclusion criteria:
- • Male subjects, aged 18-50 requiring a semen fluid evaluation.
- Positive IgG serological test result performed not more than 3 months before
recruitment
- Signed informed consent
- Anamnestic test compilation
Control subject inclusion criteria:
- Male subjects, aged 18-50 requiring a semen fluid evaluation.
- Negative IgG serological test result performed within the last month
- Signed informed consent
- Anamnestic test compilation
Exclusion Criteria:
- Patient population exclusion criteria:
- Severe male Factor (azoospermia and <200,00 sperm per ejaculate)
- Fever (>38.5 C) in the 60 days before enrollment
- Treated with antibiotic at the time of enrolment or two months before enrolment
- Treated with cortisone at the time of enrolment or two months before enrolment
Control subject population exclusion criteria:
- Severe male Factor (azoospermia and <200,00 sperm per ejaculate)
- Fever (>38.5 C) in the 60 days before enrolment
- Known history of SARS-CoV-2 infection
- Treated with antibiotic at the time of enrolment or two months before enrolment
- Treated with cortisone at the time of enrolment or two months before enrolment
Istituto Clinico Humanitas
Rozzano 3168837, Milan, Italy
Not Provided