Human Blastocyst is Susceptible to SARS-CoV-2 Infection based on Protein Level Examination of Key Entry Factors in Human Oocytes and Early Embryos

Objective ACE2, TMPRSS2 and NRP1 are key factors for SARS-CoV-2 infection. Here, we used immunouorescence to examine the expression patterns of ACE2, TMPRSS2 and NRP1 in human oocytes and different stages of preimplantation embryos to investigated the susceptibility to be infected by SARS-CoV-2. Methods We collected human GV oocytes and different stages of early embryos donated by patients and then performed immunouorescence followed by confocal microscopy for signals of ACE2, TMPRSS2 and NRP1 proteins in these oocytes and embryos.


Abstract
Objective ACE2, TMPRSS2 and NRP1 are key factors for SARS-CoV-2 infection. Here, we used immuno uorescence to examine the expression patterns of ACE2, TMPRSS2 and NRP1 in human oocytes and different stages of preimplantation embryos to investigated the susceptibility to be infected by SARS-CoV-2.

Methods
We collected human GV oocytes and different stages of early embryos donated by patients and then performed immuno uorescence followed by confocal microscopy for signals of ACE2, TMPRSS2 and NRP1 proteins in these oocytes and embryos.

Results
We found that ACE2 was abundant in both inner cell mass and trophectoderm at blastocyst stage, while TMPRSS2 was mainly enriched in trophectoderm. Both of the two factors had faint signal in cleavage embryos and oocytes. In contrast, NRP1 was barely detectable in oocytes or any stage of early embryos.

Conclusion
Taken together, we propose that human blastocysts, instead of human oocytes and other stages of early embryos, are susceptible to be infected by SARS-CoV-2. Therefore, speci c attention should be paid to manipulation of human blastocysts in assisted reproductive technology.

Background
Studies show that in developing countries where economies are underdeveloped, pandemics tend to bring baby booms. Attention to reproductive health issues during the COVID-19 pandemic is therefore extremely important. There are cases reporting positive results for SARS-CoV-2 in placenta and umbilical cord blood [1], and the presence of antibodies against SARS-CoV-2 in some newborns [2], indicating that there is possibility of infection before delivery by mother-to-child transmission. However, in two cases of women who tested positive for SARS-CoV-2, no viral RNA was detected in oocytes collected through ovarian stimulation [3]. Whether human oocytes and early embryos can be infected with SARS-CoV-2 remains elusive. SARS-CoV-2 was reported to enter cells through binding the cell membrane receptor ACE2, and then initiating entering through TMPRSS2 [4]. SARS-CoV-2 may be able to enter endometrial stromal cells through ACE2 receptor, and cause pathological manifestations in women with COVID-19, including increased risk of early pregnancy loss [5]. Studies have found co-expression of ACE2 and TMPRSS2 in primate ovarian cells. Moreover, ACE2 level was high in granular cells, but extremely low in TMPRSS2 level; ACE2 expression was low in endometrium, and TMPRSS2 expression was moderate [6] [7]. However, another study did not detect TMPRSS2 in oocytes by real-time PCR [3]. Single-cell transcriptome data analysis showed that ACE2 maintained a high level from the fertilized egg stage to the 4-cell embryo stage, and the expression was low from 8-cell embryo to morula stage. The expression levels of ACE2 in TE (trophectoderm) and ICM (inner cell mass) of blastocyst were close to that of fertilized eggs. On the contrary, TMPRSS2 has a continuous low expression from the fertilized egg to the morula stage, and the blastocyst has the strongest expression in TE [8]. Another SARS-CoV-2 host receptor neuropilin-1 (NRP1), which is known to bind furin-cleaved substrates could potentiate SARS-CoV-2 infectivity. NRP1 coexpression with ACE2 and TMPRSS2 markedly enhanced viral infection [9]. NRP1 is abundant in endothelial and epithelial cells of the respiratory tract [10], vascular endothelial cells and trophoblast cells in the villi [11], but the expression pro le of NRP1 in oocytes and early embryos is not clear. Few experiments have con rmed the protein level of above factors potentially mediating SARS-CoV-2 infection in human oocytes and early embryos. Exploring the potential of the vertical transmission of SARS-CoV-2 infection through gametes and preimplantation embryos has signi cance of guiding ART (assisted reproductive technology) treatment and normal pregnancy during current coronavirus pandemic.

Results
To elucidate protein levels of factors potentially mediating SARS-CoV-2 infection, we collected human GV oocytes and different stages of early embryos donated by patients. Next, we performed immuno uorescence followed by confocal microscopy for signals of ACE2, TMPRSS2 and NRP1 proteins in these oocytes and embryos ( Fig. 1 and Fig. 2).
Notably, ACE2 was present in both ICM and TE, while TMPRSS2 was mainly observed in TE. In contrast, both of the two factors had weak expression in cleavage embryos and GV oocytes. What's more, NRP1 is hardly detectable in oocytes and any stage of embryos. To verify the effectiveness of NRP1 antibody, we also performed immuno uorescence for signals of NRP1 proteins in embryo stem cells (Fig. S1).
We also analyzed the transcriptome data for human ACE2, TMPRSS2 and NRP1 genes from public database (Fig. 3). Generally, ACE2 transcript maintains a relatively high level in oocytes and from the fertilized egg to the four-cell stage embryo, but its level becomes extremely low from 8-cell embryo to morula stage. On the contrary, TMPRSS2 has a continuously low expression level in oocytes and from the fertilized egg to the morula stage. It should be noted that both ACE2 and TMPRSS2 transcripts are enriched in TE of blastocyst, and this is in agreement with our immuno uorescence result. Moreover, similar like its protein level, NRP1 transcript is not expressed in oocytes or early embryos.

Discussion
Our work focuses on documenting the protein level and localization of the three factors ACE2, TMPRSS2, NRP1 in human GV oocytes and early embryos, so as to predict the potential of SARS-CoV-2 infection. This work provides important information on how to safely perform assisted reproductive technology during the epidemic, and provide reference for asymptomatic infected and mild patients to choose the timing of pregnancy. According to the expression and distribution of ACE2, TMPRSS2 and NRP1, oocytes and cleavage stage embryos seem to have very low risk to be infected by coronavirus, and may be more suitable for embryo transfer during the epidemic. Despite of this, considering that viruses may infect cells through other receptors like HDL-scavenger receptor B type 1 [12], analyzing the protein expression of other receptors will be helpful to further explore the exact risk of SARS-CoV-2 infecting oocytes and early embryos and its impact on assisted reproduction. Our shortcomings were that the types of collected samples and tested receptors were limited. It will be also informative to test oocytes or embryos from patients who are positive for SARS-CoV-2. Taken together, exploring the potential of vertical transmission of SARS-CoV-2 infection through gametes and preimplantation embryos has guiding signi cance for the safe provision of ART treatment.

Conclusions
Taken together, both immuno uorescence result and transcriptome analysis support that TE of human blastocyst is susceptible to SARS-CoV-2 infection (Table 1).

RNA-seq Data
The RNA-seq result of human oocyte and pre-implantation embryo was downloaded from a previously published database (GSE36552) [13].

Declarations
Ethics approval and consent to participate Ethical approval for the study was obtained by the CEIC (Ethics Committee for Clinical Research) of Tongji Medical College, HUST. The committee's reference number is (S314, 2020). Informed consent was obtained from all patients in this study.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the publicly available GEO database (GSE36552).