Materials and Methods
Study Population and Design
This prospective cohort study was conducted following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines. All male patients with SARS-CoV-2 infection, confirmed using molecular methods on respiratory specimens, were identified. Sexually active men aged 18–65 years with proven recovery from SARS-CoV2 infection (two consecutive negative NP-swabs for SARS-CoV2 RNA) were eligible for the study (World Health Organization, 2020). Men without sexual activity or with ejaculatory disorders, being treated with prostatic surgery or alpha-blockers and those with an inability to express informed consent were excluded from the study. All the partners of the enrolled patients were tested for SARS-CoV-2 positivity (NP swabs) at the time of the original diagnosis of the men and retested according to National Health Care System criteria until NP-swab double negativity, as suggested by World Health Organization (WHO) on clinical management of COVID-19 (WHO, 2020).
Moreover, if one or more specimens collected from the patients enrolled during the study were positive, partners were retested.
The study was carried out with the approval of the local Ethics Committee (Rif: CEAVC17104) and registered on clinicaltrial.gov (Rif: NCT04446169), in compliance with the Declaration of Helsinki. All enrolled men provided written informed consent.
Clinical Data and Specimen Collection
Clinical data included patient demographics, comorbidities, medications, hospitalization time and features (including intensive care need), and laboratory tests and treatments (including oxygen therapy). Moreover, patients were asked to fill out questionnaires, including International Index of Erectile Function (IIEF-5) and Male Sexual Health Questionnaire Short Form (MSHQ-SF), in order to assess a comprehensive urological, sexual and reproductive anamnesis. Data on COVID-19 status were recorded for patients' partners.
Four biological fluid samples (saliva, pre-ejaculation urine, sperm obtained with masturbation, and first fraction post-ejaculation urine) were collected in four sterile jars to be tested for SARS-CoV-2. After liquefaction of semen and assessment of volume, semen samples were divided into two aliquots, one for evaluation of the presence of SARS-CoV-2 virus by RT-PCR and one for semen analysis.
Detection of SARS-CoV-2 in Biological Specimens
All samples were processed on the same day as collection or stored at −80°C until further analysis. Nucleic acids from samples were extracted with the Microlab Nimbus IVD system (Seegene Inc, Seoul, South Korea) using the Starmag Universal Cartridge and amplified with the multiplex RT-PCR Allplex™ 2019-nCoV assay (Seegene Inc), targeting RdrP, E and N genes, according to the manufacturers' instructions.
Semen Analysis and IL-8 Evaluation
Semen analysis was carried out according to WHO guidelines (WHO, 2010). Quantification of leukocytes in semen was performed by counting the number of round cells per milliliter using an improved Neubauer hemocytometer and evaluating the percentage of leukocytes and immature germ cells after May-Grunwald staining of the sample.
For IL-8 evaluation, semen plasma aliquots were stored frozen and IL-8 levels were quantified by conventional two-step ELISA using a human IL-8 ELISA kit (BD Bioscience, San Diego, CA, USA), according to the manufacturer's instructions.
Patients were divided into different groups for comparisons according to hospitalization, intensity of treatment, and semen parameters. Differences were tested with Independent Sample Student's t-test, Mann–Whitney u-test, and univariate analysis of variance (ANOVA) for continuous variables, and with χ 2 and Fisher's Exact Test for categorical variables according to sample size. A logistic regression was carried out including significant factors to better define the risk of sexual transmission and to identify the main determinants of impairment of semen quality. A value of P < 0.05 was considered to be significant. All statistical analyses were performed using IBM SPSS version 20.0 (SPSS Inc, Chicago, IL, USA).
Hum Reprod. 2021;36(6):1520-1529. © 2021 Oxford University Press