key: cord-0853224-54hv57hv authors: Boudry, Liese; Essahib, Wafaa; Mateizel, Ileana; Van de Velde, Hilde; De Geyter, Deborah; Piérard, Denis; Waelput, Wim; Uvin, Valerie; Tournaye, Herman; De Vos, Michel; De Brucker, Michael title: Undetectable viral RNA in follicular fluid, cumulus cells and endometrial tissue samples in SARS-CoV-2 positive women date: 2022-01-01 journal: Fertil Steril DOI: 10.1016/j.fertnstert.2021.12.032 sha: d9bcbb20611bd17abde05452d53ac503e635e30b doc_id: 853224 cord_uid: 54hv57hv Objectives To study the presence of viral RNA in follicular fluid, cumulus cells and endometrial tissue samples in SARS-CoV-2 positive women undergoing assisted reproductive technologies (ART). Design Prospective, single-center, observational study. Setting Tertiary hospital, University Hospital UZ Brussel, Belgium. Patients Sixteen patients undergoing transvaginal oocyte retrieval who had a positive SARS-CoV-2 RNA test less than 48 hours before the procedure. All cases were done between September 2020 and June 2021 and used in-vitro fertilization or intracytoplasmic sperm injection. All embryos were vitrified to avoid conception during SARS-CoV-2 infection. Intervention Follicular fluid aspirated during oocyte retrieval, cumulus cells and endometrial samples were analyzed for SARS-CoV-2 RNA using RealStar® SARS-CoV-2 RT-PCR-Kit1.0 (Altona-Diagnostics). Main outcome measures The primary outcome parameter was the detection of viral RNA in follicular fluid, cumulus cells and endometrial cells. Fertilization rate, embryo developmental potential and clinical outcome after frozen embryo transfer were secondary outcome parameters. Results Samples from sixteen patients were analyzed. Cycle threshold (Ct) values < 40 were considered positive. All samples were negative for SARS-CoV-2 viral RNA. No inflammatory lesions of the endometrium were identified histologically. Fertilization rate, embryo development and clinical outcomes after embryo transfer were reassuring. Conclusions In women infected with SARS-CoV-2 who underwent ART, viral RNA was undetectable in follicular fluid, cumulus cells and endometrium. Caution is warranted in view of the small sample size, and the risk of SARS-CoV-2 affecting the embryo via ART cannot be ruled out. Adequate counselling of women and couples undergoing ART is crucial in parallel with further research on the effect of exposure of the early human embryo to SARS-CoV-2. Introduction before oocyte retrieval. Secondary outcomes included fertilization rate, embryo developmental potential and the clinical outcome after frozen embryo transfer (FET). women with a positive PCR test after nasopharyngeal swab screening less than 48 hours before 114 oocyte retrieval. All women who were scheduled for ovarian stimulation for ART between September During the months corresponding to the peak of the COVID-19 pandemic, routine nasopharyngeal 123 PCR testing of all patients including asymptomatic ones who were scheduled for an ART cycle was 124 performed less than 48 hours before oocyte retrieval; in case of a positive PCR test on the day of 125 ovulation triggering or on the following day, the patient or couple was counselled about the un-126 known effect of viral infection on the outcome of ART treatment and the unknown risk of vertical 127 transmission. If the patient or couple decided not to cancel the oocyte retrieval but to proceed, the 128 procedure was performed as planned, considering all necessary protective safety measures accord-retrieval. Of those, one patient was an anonymous oocyte donor and was therefore not considered eligible for inclusion and another had a negative PCR confirmation test. A third patient refused to Hormone antagonist or long Gonadotropin-Release Hormone agonist protocol, or in a modified natcarried out under vaginal ultrasound-guided aspiration 34-36 hours after the above trigger. All pro- All FET procedures were performed in a natural or artificial cycle. Cycle monitoring, endometrial 237 preparation and vitrified-warmed embryo transfer were conducted as previously described (14, 15) . Embryo development after IVF (one cycle only) was also normal. In total, thirty-four embryos were vitrified in twelve cycles, of which fifteen embryos on day 3 and 267 nineteen embryos on day 5 or day 6 (including eight blastocysts following trophectoderm biopsy for 268 PGT). Four patients had no embryos available for vitrification due to insufficient embryo quality on 269 day 5 and/or day 6 or, in one single case, due to absence of fertilization. (TMPRSS2) on host cells for S-protein priming (17, 18) . Co-expression of ACE2 and TMPRSS2 proteins 301 in human metaphase II (MII) oocytes, zygotes and blastocysts marks a theoretical opportunity for 302 invasion by SARS-CoV-2 (19, 20) . A second ACE2-independent mechanism has recently been de-303 scribed, with CD147 -also known as Basigin (BSG) -as cellular receptor and Cathepsin L (CTSL) as 304 the protease (21, 22) . The presence of CD147 protein has also been shown on human oocytes and 305 pre-and peri-implantation embryos (19) . Montano In this study we investigated the susceptibility of follicular fluid, cumulus cells, and endometrium to 320 SARS-CoV-2 inoculation in sixteen patients who tested positive less than 48 hours before oocyte of viral RNA in follicular fluid (24) . In another recent study SARS-CoV-2 mRNA was not identified in our knowledge, this is the first report on the absence of viral RNA in cumulus cells and endometrium 326 of SARS-CoV-2 patients. Although cumulus cells may be susceptible to SARS-CoV-2 infection based 327 on the available machinery conducive to SARS-CoV-2 invasion (19) , cumulus cells in our patients 328 were found not to be infected by the virus. We suggest that this observation could be explained by 329 the existence of multiple transcript variants of ACE2 (19) . Different ACE2 isoforms resulting from Our results support that a positive SARS-CoV-2 test on a nasopharyngeal swab before oocyte retrieval 346 should lead to cycle cancellation. This information is highly relevant to couples embarking on ART 347 treatment during the long-haul COVID-19 pandemic. We advocate shared decision making whether 348 to proceed with oocyte retrieval; stringent safety protocols should be followed, involves collection 349 and handling of gametes in a way that mitigates the risk of infection and embryo culture in a labor-350 atory designed and equipped to treat IVF/ICSI patients with infectious diseases. A freeze-all ap-chances (27) . There are some important limitations on the validity of conclusions from these observational data. 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