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Objective: SARS-CoV-2 entry in host cells requires the presence of angiotensin-converting enzyme 2 (ACE2) as the extracellular receptor, and the serine protease TMPRSS2 to cleave the viral spike protein for incorporation into the host cell. Basigin (BSG/CD147) may also act as an ACE2 independent receptor mechanism. The cysteine protease cathepsin-L (CTSL) may also cleave the viral spike proteins and facilitate cell entry. The objective of this study was to characterize the mRNA and protein expression of these cellular entry receptors and proteases in female reproductive cells to determine their susceptibility to SARS-CoV-2 infection. Design: Prospective Research Study. Materials and Methods: Materials and Methods: Oocytes (GV, MII), follicular cells (cumulus, CC;granulosa, GC) and embryos (1 cell, 1C;blastocyst, BL) were collected from a minimum of three different patients per sample type, with consent. Samples were analyzed for mRNA expression of ACE2, CD147, TMPRSS2, and CTSL genes relative to GAPDH using RT-qPCR. Primers were validated using human mixed tissue cDNA. Protein quantification was performed by immunoblotting using the Jess system (ProteinSimple) optimized to detect over 10 proteins/5-10 oocytes or embryos. Antibodies for ACE2, CD147, TMPRSS2, and CTSL proteins were validated and then used to determine protein abundance relative to total protein. Data were obtained from three independent biological replicates and analyzed using one-way ANOVA. Results: Results from q-PCR analysis revealed high (p

Objective: In the early stages of the COVID-19 pandemic, most IVF clinics stopped the majority of patient treatment cycles to minimize the risk of disease transmission When ASRM and other professional societies recommended resumption of treatments, procedures were put into place to ensure patient and staff safety However, the risk of SARS-CoV-2 viral exposure and potential cross contamination within the IVF laboratory remains largely unclear The objective of this study was to assess the true risk of exposure to SARS-CoV-2 in an active IVF laboratory when strict patient screening procedures are in place Design: Prospective analysis Materials and Methods: Prior to restarting IVF treatments, a COVID-19 safety protocol was implemented Patients and staff were required to wear masks, fill out a symptom-based questionnaire daily, have their temperature taken, and practice social distancing in patient waiting areas Each female patient undergoing transvaginal oocyte retrieval (TVOR) was required to have a negative SARS-CoV-2 RNA test 3-4 days prior to the procedure Male partners were not tested All cases examined utilized ICSI The first tube of follicular fluid aspirated during TVOR (FF), culture media drops following removal of embryos on day 5 (M), and vitrification solution (VS) after blastocyst cryopreservation were analyzed Self-inactivating replication incompetent lentivirus particles containing the single stranded viral RNA genome were immediately inoculated into each sample after collection as a positive control for viral RNA stability, prior to direct RNA isolation (M, VS) or sample concentration (FF) For FF, cell debris was removed by centrifugation and filtration (0 22 um) prior to concentration of virus particles with an Amicon filter RNA was isolated using the optimized QIAamp viral RNA minikit, RNA quantity and quality determined, and cDNA synthesized using SuperScript IV VILO master mix A multiplex TaqMan-based qPCR assay was developed for SARS-CoV-2 and lentivirus RNA (detection limit 5 SARS-CoV-2 copies/qPCR reaction and 50 viral copies/2 mL sample), and used to test all diagnostic samples SARS-Cov2 synthetic RNA and lentivirus RNA were used as an RT-qPCR positive control Samples with no amplification of lentivirus genome were removed from the analysis (false negative) Results: In total, culture medium from 30 patients, vitrification solution from 98 patients, and follicular fluid from 156 patients were analyzed All samples were negative for the presence of SARS-CoV-2 viral RNA Conclusions: With stringent safety protocols in place, including patient testing and use of ICSI, the presence of SARS-CoV-2 RNA can be avoided in the IVF laboratory Importantly, this study does not indicate that virus from an actively infected patient cannot be found in follicular fluid or make its way into the IVF lab However, it does provide reassurance that with proper patient testing and safety measures, cross-contamination of the virus between gametes and embryos (including within liquid nitrogen storage dewars), as well as exposure of embryologists, is minimal