key: cord-0891872-vq8c86un authors: Barragan, M; Guillén, J J; Martin-Palomino, N; Rodriguez, A; Vassena, R title: Undetectable viral RNA in oocytes from SARS-CoV-2 positive women date: 2020-09-30 journal: Hum Reprod DOI: 10.1093/humrep/deaa284 sha: 55c676e2a1f888b66f31e81e1fd39abb36a29319 doc_id: 891872 cord_uid: vq8c86un A central concern for the safe provision of ART during the current coronavirus disease 2019 (CODIV-19) pandemic is the possibility of vertical transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection through gametes and preimplantation embryos. Unfortunately, data on SARS-CoV-2 viral presence in oocytes of infected individuals are not available to date. We describe the case of two women who underwent controlled ovarian stimulation and tested positive to SARS-CoV-2 infection by PCR on the day of oocyte collection. The viral RNA for gene N was undetectable in all the oocytes analyzed from the two women. In the months since December 2019, when the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in our species, it has become apparent that the virus can affect several tissues and organs. A central concern for the safe provision of ART treatments during the current coronavirus disease 2019 pandemic is the possibility of vertical transmission of SARS-CoV-2 infection through gametes and preimplantation embryos. Despite the large body of literature gathered so far, the effects of SARS-CoV-2 infection on reproductive function are still mostly unknown. Specifically, it is not clear whether the virus can infect human gametes, and whether the use of oocytes from women harboring the virus can result in an infection of the developing embryo. The ability of SARS-CoV-2 to affect a tissue is determined by its capacity to infect cells and replicate, which requires expression of the SARSCoV-2 receptors angiotensin converting enzyme 2 (ACE2) and Basigin (BSG), and the proteases transmembrane protease serine 2 (TMPRSS2) and cathepsin L (CTSL) (Hoffmann et al. 2020; Wang et al. 2020 ). The mRNAs of these genes are expressed in most of the human female reproductive tract, whole ovary (Hikmet et al. 2020 ) including cumulus cells (Stanley et al. 2020 ), endometrium (Henarejos-Castillo et al. 2020 , and during the early developmental stages of the human embryo (Weatherbee, Glover, and Zernicka-Goetz 2020). Importantly, protein expression in these tissues has not been confirmed. A further limiting factor of all these analyses is their reliance on samples from healthy women, as no published data are available from women with a confirmed SARS-CoV-2 infection. We report here on the detection of SARS-CoV-2 viral RNA, and gene expression of ACE2, TMPRSS2, CTSL1 and BSG, in mature oocytes from two women who underwent The women in this study (A and B) contacted the clinic in late February 2020, wishing to donate their oocytes. While A did not report any respiratory symptoms in the preceding weeks, B did report symptoms compatible with a mild cold in early February. At the time of A and B screening for donation, regulations in Spain mandated that only individuals with active symptoms compatible with COVID-19 should be screened for SARS-CoV-2 infection, therefore they were not tested by PCR at that time. Cq<37). All donors were given oral and written information about the SARS-CoV-2 PCR test, and all of them consented to the test. Nasopharyngeal swabs were taken by the anesthesiologist during the anesthesia for oocyte retrieval. Samples were sent to a certified independent diagnostic laboratory, and PCR results were obtained 2 days later. Six oocytes from A and 10 from B were warmed according to standard procedures (Cryotop®, Kitazato®, 150 BioPharma Co., Ltd; Japan), and individually processed for whole transcriptome amplification (WTA) using the REPLI-g WTA Single Cell Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions for amplification of total RNA from single cells by using a mixture of random and oligo dT primers. Briefly, individual oocytes were placed in a PCR tube containing 7 µl RNAsefree-PBS and snap-frozen in liquid nitrogen. A positive control for viral RNA amplification was run to control for the recovery of potential viral particles and to assess potential amplification inhibition during the WTA protocol . One immature oocyte from a COVID-19 negative woman was included as positive control for viral RNA recovery: in this case, the oocyte was placed in 7 µl RNAse-free-PBS containing 2x10 5 copies of MS2 bacteriophage (MS2Φ; 10165948001; Roche-Merck; Madrid, Spain). After thawing on ice, 4 µl lysis buffer provided in the kit was added to each tube and all oocytes were processed simultaneously. WTA samples were diluted 1:100 following manufacturer's instructions and 2 µl were (Vandesompele et al. 2002) . Actin B (ACTB), ubiquitin C (UBC) and DNA methyltransferase-1 (DNMT1) were used as normalizers (Table I) . The REPLI-g WTA single-cell kit can isolate, reverse transcribe, and amplify viral particles present in a single oocyte; 2x10 5 copies of single strain RNA of MS2 bacteriophage were added to a single oocyte at the lysis step. After amplification, specific MS2 RNA was detected (mean Cq=17.42; SD=0.12). Additionally, the standard curve for the SARS-CoV-2 N gene determined that the limit of detection (LOD) in our set-up is Cq<35, which corresponds to 100 copies per well (R=0.997; from 10 6 copies (mean Cq=21.1, SD=0.15) to 10 2 copies (mean Cq=34.3, SD=0.61)). The RNA for the SARS-CoV-2 gene N was undetectable (Cq>38) in the six oocytes from A and 10 from B that were analyzed. Further, we analyzed the expression of genes involved in controlling SARS-CoV-2 infection, to understand whether oocytes could get infected, regardless of current undetectable viral RNA. We detected levels of ACE2 in 2/6 oocytes from A and 3/10 from B (LOD Cq<36; range [25.51 -33.85]). Additionally, TMPRSS2 was not detected in any of the oocytes (LOD Cq<38). The putative receptor BSG (6/6 for woman A and 9/10 for woman B) (LOD Cq<38; ) and the protease protein CTSL (6/6 for woman A and 10/10 for woman B) (LOD Cq<38; were both expressed at similar levels in 50% oocytes for woman A and 60% for woman B. To our knowledge, this is the first report on the detection of the viral RNA of SARS-CoV-2 in oocytes from women who were positive by PCR. We found that the viral RNA was undetectable in all 16 oocytes tested from two asymptomatic positive women. Regardless of the detection of viral RNA in oocytes, one wonders about the possibility of the virus infecting them, perhaps when present in higher concentration in the reproductive organs. Therefore, we have analyzed the expression of two functionally related pairs of genes: ACE2 and TMPRSS2 on the one hand, and CTSL and BSG on the other. ACE2 acts as receptor for SARS viruses by interacting with the S1 domain of their S protein, while TMPRSS2 facilitates the virus entry by cleaving and activating viral envelope glycoproteins. Viruses such as human coronavirus 229E (HCoV-229E), Middle East respiratory virus coronavirus (MERS-CoV), SARS-CoV and SARS-CoV-2 do use these proteins for cell entry (Hoffmann et al. 2020) . BSG is a transmembrane glycoprotein that has been identified as a putative receptor for virus infection (Wang et al. 2020) , while CTSL could cleave the S1 subunit of the SARS-CoV-2 spike protein in the absence of functional TMPRSS2 (Hoffmann et al. 2020) . It was previously suggested that expression of ACE2 and TMRPSS2 was likely in human oocytes, based on their mRNA expression in a non-human primate oocytes (up to antral follicular stages), and in human cumulus cells (Stanley et al. 2020) . We found variable expression (less than 30% of the oocytes) of ACE2, and undetectable expression for TMPRSS2 in our cohort. These results extend the observation that neither ACE2 mRNA nor protein were consistently detected in human ovaries (Hikmet et al. 2020 ). However, as we report expression of BSG and CTSL, we cannot exclude the possibility of multiple avenues through which SARS-CoV-2 may infect human oocytes. Further, we have tested RNA presence, while a more definitive assessment would need to include protein presence in the oocytes. Although all oocytes were visually inspected under the microscope at warming for absence of cumulus cells, we cannot fully discard the possibility of contamination of RNA from cumulus cells in our samples, given the physiological continuum between cumulus cells and oocytes during oocyte maturation. Prospective analysis of follicular fluid and cumulus cells in infected women would help clarify this point. The two women included in this study were asymptomatic. Although it is not possible to determine whether symptomatic women may harbor viral particles in their oocytes, the most likely clinical situation for the provision of ART treatments is with asymptomatic patients. Our report suggests that vertical transmission in these women may not occur through their oocytes during treatment, and that handling of this material in the clinical embryology laboratory may not constitute a hazard for healthcare professionals; nevertheless, more extensive reports are needed to confirm our findings. Fully anonymized data are available upon request. Quality control implementation for universal characterization of DNA and RNA viruses in clinical respiratory samples using single metagenomic next-generation sequencing workflow The transcriptome of human oocytes is related to age and ovarian reserve CDC. CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel SARS-CoV-2 infection risk assessment in the endometrium: viral infectionrelated gene expression across the menstrual cycle The protein expression profile of ACE2 in human tissues SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Expression of SARS-CoV-2 receptor ACE2 and TMPRSS2 in human primary conjunctival and pterygium cell lines and in mouse cornea Increased expression of cathepsin L: a novel independent prognostic marker of worse outcome in hepatocellular carcinoma patients Coronavirus disease-19 and fertility: viral host entry protein expression in male and female reproductive tissues Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes Quality control implementation for universal characterization of DNA and RNA viruses in clinical respiratory samples using single metagenomic nextgeneration sequencing workflow The transcriptome of human oocytes is related to age and ovarian reserve CDC. CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel Expression of SARS-CoV-2 receptor ACE2 and TMPRSS2 in human primary conjunctival and pterygium cell lines and in mouse cornea Increased expression of cathepsin L: a novel independent prognostic marker of worse outcome in hepatocellular carcinoma patients The authors wish to thank the staff of the Eudona donation program and the clinical embryology lab at Clinica EUGIN for their support.