Validation of a SARS-CoV-2 RT-PCR assay: a requirement to evaluate viral contamination in human semen

• Published in: Reproductive biomedicine online 2022-12, Vol.45 (6), p.1247-1254
• Main Authors: Chabrolles, Hélène, Pons-Rejraji, Hanae, Chaput, Laure, Brebion, Amélie, Fiot, Mélanie, Pereira, Bruno, Brugnon, Florence, Henquell, Cécile
• Format: Article
• Language: English
• Subjects: Assisted reproductive technology; COVID-19 - diagnosis; Fertility preservation; Human spermatozoa; Humans; Life Sciences; Male; Real-time RT-PCR; Reproductive Biology; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral - analysis; SARS-CoV-2; SARS-CoV-2 - genetics; Semen; Semen Analysis
• ISSN: 1472-6483; 1472-6491
• DOI: 10.1016/j.rbmo.2022.09.004

Is it possible to validate an accurate and reliable method for direct detection of SARS-CoV-2 by reverse transcription polymerase chain reaction (RT-PCR) in human semen fractions? This qualitative improvement study aimed to provide a prospective validation of SARS-CoV-2 detection in male semen. The SARS-CoV-2 genome was detected by multiplex real-time RT-PCR on patient samples that underwent routine semen analyses for infertility at the Center for Reproductive Medicine at the University Hospital of Clermont-Ferrand. Samples comprised surplus semen collected for treatment with assisted reproductive technology. Seminal fluid and spermatozoa fractions were isolated with density gradient centrifugation and cryopreserved. Positive samples were prepared with a standard of inactivated SARS-CoV-2 particles. The analytical method was validated in both seminal fluid and spermatozoa fractions. In both semen fractions, the assay was repeatable, reproducible and showed high sensitivity with a limit of detection of 0.33 SARS-CoV-2 genome copies/µl. The limit of quantification was 1 copy of the SARS-CoV-2 genome/µl. The method was effective regardless of semen quality (normal and altered sperm parameters), number of spermatozoa or the cryoprotectant media used to freeze spermatozoa. This validated RT-PCR assay provided accurate and reliable screening of SARS-CoV-2 in seminal fluid and spermatozoa fractions. This method is essential to ensure protection against viral contamination in the cryobanking process.