ABSTRACT
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<0.05) abundance of ACE2 transcripts in GV and MII oocytes compared to CC, GC, and BL. ACE2 protein was present in all the samples, but was relatively higher (p<0.17) in M2 oocytes, 1C, BL, and CC compared to GV oocytes and GC. TMPRSS2 protein was abundant in MII oocytes, 1C, and BL, and was present but at low levels in GV oocytes and follicular cells. Protein abundance of CD147 was five-fold higher (p<0.05) in GV and ∼1.5 fold higher in GC compared to all other samples analyzed. No CTSL protein was observed with the expected molecular weight (38 kD), although a 55 kD band (a possible isoform) was detected in GV oocytes and CC. Conclusions: Cumulus and granulosa cells are least susceptible to SARS-CoV-2 infection due to the lack of required receptors and proteases. Co-expression of the protein for ACE2 and TMPRSS2 in MII oocytes, zygotes, and blastocysts suggests that these reproductive cells are susceptible to SARS-CoV-2 infection. In conclusion, using a combined approach of mRNA and protein analysis from the same samples suggests that mature human oocytes and preimplantation embryos have the cellular machinery required for SARS-Cov2 entry, although we do not know if this occurs in vivo. The potential for viral infection in oocytes and embryos has important ramifications for ART. Care must be taken to avoid introduction of the virus to the embryo while in the ART laboratory, as well as potentially introducing the virus from an infected embryo to laboratory workspaces.
ABSTRACT
RESEARCH QUESTION: Is there a risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral exposure and potential cross-contamination from follicular fluid, culture media and vitrification solution within the IVF laboratory using strict patient screening and safety measures? DESIGN: This was a prospective clinical study. All women undergoing transvaginal oocyte retrieval were required to have a negative SARS-CoV-2 RNA test 3-5 days prior to the procedure. Male partners were not tested. All cases used intracytoplasmic sperm injection (ICSI). The first tube of follicular fluid aspirated during oocyte retrieval, drops of media following removal of the embryos on day 5, and vitrification solution after blastocyst cryopreservation were analysed for SARS-CoV-2 RNA. RESULTS: In total, medium from 61 patients, vitrification solution from 200 patients and follicular fluid from 300 patients was analysed. All samples were negative for SARS-CoV-2 viral RNA. CONCLUSIONS: With stringent safety protocols in place, including testing of women and symptom-based screening of men, the presence of SARS-CoV-2 RNA was not detected in follicular fluid, medium or vitrification solution. This work demonstrates the possibility of implementing a rapid laboratory screening assay for SARS-CoV-2 and has implications for safe laboratory operations, including cryostorage recommendations.