A single-cell RNA expression map of coronavirus receptors and associated factors in developing human embryos
Stacy Colaco, Karisma Chhabria, Domdatt Singh, Anshul Bhide, Neha Singh, Abhishek Singh, Atahar Husein, Anuradha Mishra, Richa Sharma, Nancy Ashary, Deepak Modi
11 Expression of SARS-CoV-2 receptor ACE2 and the spike protein processing enzymes in developing human embryos
Stacy Colaco† , Karisma Chhabria† , Neha Singh , Anshul Bhide , Domdatt Singh , Abhishek Singh , Atahar Husein , Anuradha Mishra , Richa Sharma , Nancy Ashary , Deepak Modi* Molecular and Cellular Biology Laboratory, ICMR-National Institute for Research in Reproductive Health, Indian Council of Medical Research (ICMR), JM Street, Parel, Mumbai 400012, India MGM Institute of Health Sciences, MGM Educational Campus, Sector 1, Kamothe, Navi Mumbai-410209, India Corresponding author: [email protected], [email protected] † equal contribution
Abstract
The novel coronavirus disease, COVID-19 caused by Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2), has resulted in thousands of critically ill patients and poses a serious threat globally. To investigate if SARS-CoV-2 can affect early human embryos, we analysed scRNA-seq datasets of human embryos for the SARS-CoV-2 receptors ACE2 and BSG (CD147), the serine protease TMPRSS2 for viral spike protein priming, and the endosomal protease CTSL for spike protein activation via the endosomal route. The results reveal that
ACE2 and
TMPRSS2 are co-expressed in a proportion of epiblast cells, and both
BSG and
CTSL are expressed in all the stages of embryonic development. The cells of the blastocysts express genes encoding for other coronavirus receptors such as
DPP4 and
ANPEP as well. Interestingly, the cells of the epiblast also express genes involved in viral endocytosis and replication. We further identified 194 genes that are differentially expressed in
ACE2- and
TMPRSS2 -positive cells as compared to
ACE2- and
TMPRSS2 -negative cells of the epiblast. Our results show that developing human embryos express the receptors for SARS-CoV-2 and other coronaviruses; embryos also harbour the necessary machinery for viral internalization and replication. We suggest that couples be advised to avoid conceiving during the pandemic and that IVF procedures be kept to a minimum to prevent any possible hazard to the developing embryos.
Keywords : SARS-CoV-2, COVID-19, ACE2, BSG, TMPRSS2, CTSL, Embryo, coronaviruses, viruses
Introduction
In December 2019, a cluster of atypical pneumonia associated with a novel coronavirus was first reported from Wuhan, Hubei province, in China (Zhu et al. , 2020). The disease, termed COVID-19, is caused by coronavirus Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2), previously termed 2019-nCoV (Singhal, 2020). The virus has since spread worldwide and is a serious global health concern as it has high human-to-human transmission (Ralph et al. 2020). SARS-CoV-2 has sequence similarity to the bat-SL-CoVZC45 and bat-SL-CoVZXC21; the external subdomain of the SARS-CoV-2 receptor binding domain is similar to that of the SARS-CoV identified in 2002 (Yadav et al. , 2020; Zhu et al. , 2020). Like most viruses, the coronaviruses enter the host cell by receptor binding followed by endocytosis, genome replication, exocytosis, and budding (Fehr et al. 2015). Recent studies have shown that the human Angiotensin-Converting Enzyme II (ACE2) is the host receptor for SARS-CoV-2 (Letko, Marzi and Munster, 2020). Furthermore, the SARS-CoV-2 does not use other coronavirus receptors viz; alanine aminopeptidase N (ANPEP) and dipeptidyl peptidase 4 (DPP4), which are used by CoV-229E and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), respectively, for entry into host cells (Li, 2015; Letko, Marzi and Munster, 2020). ACE2 is necessary for entry of SARS-CoV-2 into host cells, and studies have shown that the spike (S) protein of SARS-CoV-2 is processed by transmembrane serine protease 2 (TMPRSS2) (Matsuyama et al. 2020, Hoffmann and Pöhlmann 2020) for enhanced viral entry. Another endosomal protease, cathepsin L (CTSL), is also involved in processing of the SARS-CoV-2 S protein via the endosomal route (Ou et al. , 2020). In addition, the SARS-CoV-2 utilizes Basigin (BSG), also known as CD147, as a receptor for viral entry (Wang et al. , 2020). These findings imply that, in a given tissue, the cells co-expressing these molecules would be the key determinants of susceptibility to SARS-CoV-2 infection. Respiratory distress is one of the common clinical presentations of SARS-CoV-2 infection, and the lung airway epithelium is a primary target for SARS-CoV-2 action. Indeed,
ACE2 and
TMPRSS2 co-expressing cells are reported amongst human airway alveolar epithelial cells (Lukassen et al. , 2020; Qi et al. , 2020). Beyond the lung,
ACE2 is expressed in other human organs such as the oral and nasal mucosa, nasopharynx, stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow, spleen, liver, and kidney (Hamming et al. , 2004; Qi et al. , 2020; Seow et al. , 2020; Xu et al. , 2020), suggesting that SARS-CoV-2 may have more widespread effects than presently thought based on clinical presentations. Beyond the adult tissues, certain viruses can also infect the gametes and the developing embryos of the host (Racicot and Mor 2017). However, the main focus of the studies on SARS-CoV-2 and host interactions is on adult tissues, little is known about the effects of the virus on embryos. Studies on preimplantation mammalian embryos are important because maternal viral infections during early pregnancy can induce embryonic death or abnormal embryonic development (Hardy, 1974; Silasi et al. , 2015; Racicot and Mor, 2017). With the high population estimates of SARS-CoV-2 spread (Li et al. , 2020) and the observation that a large proportion of individuals with SARS-CoV-2 infection may be asymptomatic, (Ling et al. 2020, Pan et al. 2020, Wu et al. 2020) it is likely that many women may have conceived during the course of subclinical infection. Secondly, many assisted reproduction clinics may have handled human gametes and embryos from subclinically infected couples or inadvertently exposed them to infected laboratory personnel. Given the fact that SARS-CoV-2 has a long shelf life on various surfaces (Ong et al. , 2020; van Doremalen et al. , 2020), there are many potential routes by which gametes/embryos could be exposed to SARS-CoV-2. However, whether the gametes or early human embryos can be affected by SARS-CoV-2 or other coronaviruses remains unknown. To explore this possibility, in the present study, we analysed single-cell RNA-Seq datasets of human embryos (Yan et al. , 2013; Stirparo et al. , 2018) to determine the expression of the SARS-CoV-2 receptors
ACE2 and
BSG as well as S protein primers
TMPRSS2 and
CTSL in zygote to late-blastocyst-stage human embryos. In addition, we determined whether the cells of early embryos also express endocytotic and viral replication machinery.
Methods
Single-cell RNA-Seq data were obtained (Yan et al. , 2013; Stirparo et al. , 2018) and levels of
ACE2 , BSG , TMPRSS2 , CTSL , DPP4 , and
ANPEP (in terms of fragments per kilobase of transcript per million mapped reads, FPKM) were obtained for each cell at each stage. The numbers and percentage of cells co-expressing
ACE2 and
BSG , ACE2 and
TMPRSS2 , ACE2 and
CTSL , and
BSG and
CTSL were also calculated. Supplementary Table 1 gives the number of embryos and total number of cells tested at each time point of embryonic development. The list of genes involved in the Endosomal Sorting Complex Required for Transport (ESCRT I, II, and III) and the list of genes involved in SARS virus replication in host cells were obtained (de Wilde et al. , 2018; Ahmed et al. , 2019). A recent study identified 332 human proteins that physically interact with SARS-CoV-2 (Gordon et al. , 2020). Mean FPKM values for these genes were extracted and datasets were visualized using Morpheus (https://software.broadinstitute.org/morpheus.) and R Studio version 3.6.2. To determine the phenotypes of
ACE2 in epiblast cells, the numbers of
ACE2- and
TMPRSS2- positive and
ACE2- and
TMPRSS2 -negative cells were identified, and the mean FPKM values of all the genes expressed in both the datasets were computed. The data were filtered for genes whose mean values were >0.5 FPKM. This was followed by obtaining the ratio of the mean value in
ACE2- and
TMPRSS2 -positive cells /
ACE2- and
TMPRSS2 -negative cells. Only those genes that had a ratio of >1.5 or < 0.5 were filtered. Student’s t test was applied. Only those genes that had significantly different mean values (p<0.05) were filtered. The biological processes associated with the differentially expressed genes in
ACE2- and
TMPRSS2 -positive cells were obtained using the DAVID Knowledgebase version 6.8 (https://david.ncifcrf.gov) using the GOTERM_BP_FAT filters with highest stringency. Categories with enrichment score >0.5 were considered for analysis.
Results mRNA levels of coronavirus-associated genes
ACE2 , TMPRSS2 , BSG , CTSL , DPP4 , and
ANPEP in early stages of human embryonic development
Table.1. summarizes the receptors used by the various coronaviruses known to infect humans. We explored publicly available online datasets of human preimplantation embryos at different stages of development to determine the mRNA level of these proteins. As evident from Figure.1., high amounts of
ACE2 mRNA was detected in the zygotic stage of development followed by a decrease until morula stage with a subsequent sudden increment in the inner cell mass (ICM), epiblast, and the primitive endoderm.
BSG mRNA was consistently detected in all stages of embryonic development with an increase in levels from zygotic stage to blastocyst stage.
TMPRSS2 was not detected in the zygotes, but a low abundance of transcripts was detected at early stages of embryonic development that further increased during later stages. The highest mRNA levels of
TMPRSS2 were observed in epiblast cells.
DPP4 and
ANPEP mRNA was not detected in the zygote or 4-cell embryos; however, abundant
DPP4 and
ANPEP transcripts were abundantly detected at all stages of embryonic development (Figure.1.). The pattern of expression for all the genes remained roughly consistent in another independent dataset of single-cell RNA-Seq of human embryos (Supplementary Fig.1.).
Virus
Human host receptor
SARS-CoV-2 ACE2, CD147 (BSG) SARS-CoV ACE2, CD147 (BSG) MERS-CoV DPP4 HCoV-OC43 9-O-acetylsialic acids HCoV-HKU1 9-O-acetylsialic acids HCoV-229E ANPEP CoV-NL63 ACE2
Co-expression of transcripts of SARS-CoV-2 receptor
ACE2 and
TMPRSS2 , BSG , and
CTSL in early stages of human embryonic development Table 1: Summary of host receptors of coronaviruses that infect humans
We next determined the numbers of cells at different stages of embryonic development that co-express transcripts of
ACE2 and
TMPRSS2 . In addition, we determined the co-expression of
ACE2 and
CTSL , ACE2 and
BSG , and
BSG and
CTSL . The results reveal that at the zygote and 4-cell stage, all cells co-express
ACE2 and
BSG (Figure.2.). Nearly 86% (37/43) of cells co-expressed
ACE2 and
BSG in the ICM, 39% (25/64) in the epiblast, and 39% (11/28) in the primitive endoderm (Supplementary Table 1). Maximum numbers of
ACE2 and
TMPRSS2 co-expressing cells were detected in the epiblast (Figure.2.). All the
ACE2- and
TMPRSS2- positive cells in the epiblast also co-expressed
BSG and
CTSL . In general,
BSG and
CTSL were abundantly detected in almost all cells of the developing embryos from the morula stage to the blastocyst stage (Figure.2.).
Fig.1. mRNA expression of coronavirus receptors and spike protein processing enzymes in early human embryos.
Data was extracted from single-cell RNA-Seq of developing human embryos (Stirparo et al., 2018) . The X axis represents various stages of human embryonic development and Y axis represents FPKM values. Each dot represents data of a single cell at that developmental stage. ACE2 and BSG are the receptors for SARS-CoV-2 and SARS-CoV, DPP4 is the receptor for MERS-CoV, and ANPEP is the receptor for HCoV-229E. The viral spike protein is processed by host enzymes TMPRSS2 or CTSL.
Fig.2. Co-expression of mRNA of SARS-CoV-2 receptors and spike protein processing enzymes in early human embryos.
Co-expression of
ACE2 and
TMPRSS2 , ACE2 and
BSG, ACE2 and
CTSL , and
BSG and
CTSL in single cells of human embryos at zygote, 4-cell, 8-cell, compact morula, early inner cell mass (ICM) epiblast and primitive endoderm.
Each dot represents data of a single cell; double positive co-expressing cells are shown in blue, single positive cells are shown in red and purple. Data was extracted from single-cell RNA-Seq of developing human embryos (
Stirparo et al., 2018 ). mRNA levels of genes involved in viral endocytosis and replication in human embryos at different stages of development. We next hypothesized that if the virus can bind to the embryonic cells, the cells may contain the necessary machinery for viral endocytosis and viral replication. Towards this, we analysed the average FPKM values of the 33 genes involved in human ESCRT of viruses. We observed that from zygote to morula stage, most of the ESCRT genes are absent or present in low abundance. However, the transcripts of almost 90% of these genes dramatically increased in the ICM, epiblast, and primitive endoderm (Figure.3A.). SARS-CoV viruses enter the host cells and utilize the host cell machinery for replication. We analysed the mRNA levels for host genes involved in SARS-CoV replication and observed that the genes involved in viral replication were absent or present at low abundance from zygote to morula stages of embryonic development. However, the mRNA levels of most of these genes surged in the ICM, epiblast, and the primitive endoderm (Figure.3B.). Analysis of individual cells of the epiblast revealed that almost all the genes whose protein products play a role in ESCRT were expressed in most cells of the
ACE2- and
TMPRSS2- positive and negative cells with minimal cell-to-cell heterogeneity in level of expression (Figure.3C.). Almost all the host genes involved in viral replication are expressed in most of the cells with little heterogeneity (Figure.3D.).
Fig.3. Heat maps of the genes involved in viral endocytosis and replication in human embryos at different stages of development.
All data were extracted from single-cell RNA-Seq of developing human embryos (
Stirparo et al., 2018 ). In all heat maps, each row depicts a gene and each column depicts a stage of embryonic development (in A and B) or each cell in the epiblast (in C and D). A) Genes involved in endosomal sorting complexes required for transport (ESCRT) in different stages of embryonic development. B) Genes involved in coronavirus replication in different stages of embryonic development. C) Genes involved in ESCRT in
ACE2- and
TMPRSS2- negative (
ACE2–TMPRSS2– ) and
ACE2- and
TMPRSS2- positive (
ACE2+TMPRSS2+ ) cells of the epiblast D) Genes involved in coronavirus replication in in
ACE2- and
TMPRSS2- negative (
ACE2–TMPRSS2– ) and
ACE2- and
TMPRSS2- positive (
ACE2+TMPRSS2+ ) cells of the epiblast.
SARS-CoV-2 interacts with 332 proteins in human cells. In the epiblast of human embryos, mRNA for 320/332 SARS-CoV-2 interacting proteins was detected. However, some of the mRNA transcripts were highly abundant and others were weakly expressed (Figure.4.). Although the heterogeneity in mRNA levels of SARS-CoV-2 interacting proteins between cells was not high, 54/320 genes were overexpressed (ratio>1.5) in
ACE2- and
TMPRSS2- positive cells as compared to
ACE2- and
TMPRSS2- negative cells of the epiblast.
Fig.4. mRNA levels of genes in the epiblast of human embryos whose protein products are known to interact with SARS-CoV-2.
Information for 320 genes whose protein products are reported to interact with SARS-CoV-2 was extracted from single-cell RNA-Seq of the epiblast of developing human embryos (
Stirparo et al., 2018 ). Each row is a gene and each column is a single cell of the epiblast. The data is a relative color scheme in which the minimum and maximum values in each row are used to convert values to colors.
Differentially expressed genes and the biological processes enriched in
ACE2 and
TMPRSS2 co-expressing cells of the epiblast
In the epiblast, 19 cells expressed
ACE2 and
TMPRSS2 while 23 cells did not express both
ACE2 and
TMPRSS2 (Figure.2.). We identified 194 genes that were differentially expressed (fold change >1.5 or <0.5) with statistical significance (p<0.05) between
ACE2- and
TMPRSS2- positive cells and
ACE2- and
TMPRSS2- negative cells. Figure.5A. depicts the distribution of these 194 differentially expressed genes in each cell of the epiblast. As evident from Figure.5A., as compared to
ACE2- and
TMPRSS2- negative cells, most (189/194) of these genes were overexpressed in the
ACE2- and
TMPRSS2- positive cells of the epiblast; only 5 genes were downregulated. While there was extensive cell-to-cell heterogeneity in expression of the 194 differentially abundant genes in the
ACE2- and
TMPRSS2- negative cells, most of these genes were uniformly upregulated in most
ACE2- and
TMPRSS2- positive cells. Analysis of the differentially expressed genes revealed that they belong to diverse biological processes including genes involved in various metabolic processes, development, and viral entry into host cells (Figure.5B.). Fig.5 . Differentially expressed genes and the biological processes enriched in
ACE2- and
TMPRSS2- positive cells in epiblast of developing human blastocysts.
Data were extracted from single-cell RNA-Seq of the epiblast of developing human embryos (Stirparo et al 2018). A)
Distribution of 194 differentially expressed genes in
ACE2- and
TMPRSS2- negative (
ACE2–TMPRSS2– ) and
ACE2- and
TMPRSS2- positive (
ACE2+TMPRSS2+ ) cells of the epiblast. Rows represent genes and columns represent individual cells, presented on a relative colour scale. B) Biological processes enriched in the
ACE2+TMPRSS2 + cells of the epiblast showing differential expression of 194 genes. The Y axis indicates the enriched biological processes and X axis is the enrichment score. Discussion
The results of the present study demonstrate that early human embryos express coronavirus entry receptors and S protein proteases. Embryonic cells also express the genes for proteins that are involved in viral endocytosis and replication. Considering the scale at which the SARS-CoV-2 virus has spread globally and the fact that a proportion of individuals harbouring the virus are initially asymptomatic, it is likely that some of the infected individuals may have conceived or are trying to conceive during the duration of the pandemic. Further, with nearly 1 in 6 couples worldwide facing infertility, many couples resort to in vitro fertilization (IVF) techniques for achieving biological parenthood. In both these scenarios, it is imperative to understand whether the developing embryo is at risk of SARS-CoV-2 infection. To address this question, we analysed single-cell RNA-Seq datasets of developing human embryos for SARS-CoV-2 receptors
ACE2 and
BSG along with the spike protein processing enzymes
TMPRSS2 and
CTSL in zygotes to hatched blastocysts. Our results reveal that gametes, zygotes, and 4-cell embryos express abundant amounts of
ACE2 and
BSG along with
CTSL but not
TMPRSS2 . While the levels of
ACE2 decline in the compact morula, the expression of
ACE2 and the processing enzymes increases in the ICM and epiblast. While ACE2 is essential, the processing of the spike protein by the membrane-bound serine protease TMPRSS2 promotes viral infectivity. We observed that although more than 80% of cells of the ICM express
ACE2, none of these cells express
TMPRSS2 , indicating that the early ICM may not be highly susceptible to viral infection. However, in the epiblast (but not the primitive endoderm) a significant proportion of cells co-express ACE2 and
TMPRSS2 suggesting that these cells would be targets for viral entry. Beyond ACE2, a study has shown that the extracellular matrix metalloproteinase enhancer CD147 encoded by the gene
BSG binds to both SARS-CoV-2 and SARS-CoV and promotes viral entry independent of ACE2 and TMPRSS2 (Chen et al. , 2005; Wang et al. , 2020). We observed that
BSG transcripts were abundantly expressed in all the cells of the developing embryos from zygote stage to blastocyst stage. With regard to S protein priming, cathepsins are a class of endosomal proteases, and of these, cathepsin L encoded by
CTSL is required for endosomal cleavage of SARS-CoV and SARS-CoV-2 spike proteins (Ou et al. , 2020). Interestingly, along with
BSG , CTSL was co-expressed in most of the cells of the developing embryo. Presently it is unclear if CD147-mediated viral entry requires cathepsin L; the fact that both these proteins are co-expressed in most embryonic cells implies that developing embryos may be susceptible to coronavirus infections by the ACE2-independent mode of action. Two other members of the coronavirus family that are highly infectious and cause significant mortality are MERS and HCoV-NL63. Unlike SARS-CoV and SARS-CoV-2, MERS and HCoV-229E utilize DPP4 and ANPEP as receptors to infect the human host cells (Li, 2015). Interestingly, we found that
DPP4 and
ANPEP transcripts are not expressed in the zygotes until the morula stage, but both receptors are expressed in almost all the cells of the ICM, epiblast, and early endoderm. Together our data for the first time suggest that early human embryos could be susceptible to infection by coronaviruses in general. Once the virus binds to the receptor and the spike protein undergoes cleavage, SARS-CoV-2 releases its content into the host cells and initiates replication. In this course, most enveloped viruses recruit the ESCRT machinery in which the viral structural protein engages the tumour susceptibility gene protein (TSG101) in ESCRT-I, which ultimately delivers ESCRT-III to sites of viral budding where membrane scission releases the viral particles (Votteler and Sundquist, 2013). Several proteins are involved in the ESCRT pathway, and the blocking of any of these enzymes can prevent viral endocytosis including that of the coronavirus family (Wang et al. , 2017; Mazzon and Marsh, 2019). For SARS-CoV-2 to be pathogenic to human embryos, the early embryonic cell must possess the components of the ESCRT machinery for viral endocytosis and release. Interestingly, we observed that genes for many members of the ESCRT machinery are not expressed in zygote and morula stages, but a surge in the expression of these select genes occurs in the cells of the blastocyst including the ICM, epiblast, and primitive endoderm. These results indicate that the human blastocysts expressing SARS-CoV-2 receptors not only have the necessary machinery for the virus to bind, but also facilitate endocytosis and viral budding. Once the virus enters the cells, the next step in the infection cycle is viral replication via the interaction of cellular host proteins with the viral proteins (de Wilde et al. , 2018). Our results reveal that as compared to the zygote, 4- to 8-cell stages, and morula stage embryos, the cells of the blastocyst show a surge in mRNA expression of most host proteins involved in viral replication. Almost all of the proteins involved in viral replication were abundantly expressed in the ICM, epiblast, and primitive endoderm. Together our data show that early developing human embryos not only have the necessary machinery to bind the virus and permit its entry and exit, but also facilitate viral replication. Our analysis, so far, suggests that several genes involved in endocytosis and viral replication are expressed in the cells of developing embryos. However, it remains imperative to determine if all or a subset of the cells express these genes. Since most ACE2- and
TMPRSS2- positive cells were present in the epiblast, we analysed the data of individual cells of the epiblast and observed that irrespective of the expression of
ACE2 and
TMPRSS2 , cells of the epiblast express transcripts of genes involved in viral endocytosis and replication, albeit with varying degrees of expression. These genes were also uniformly expressed in cells of the early ICM and the primitive endoderm (not shown). These results imply that the epiblast and plausibly other cells of the blastocyst are susceptible to SARS-CoV-2 infection. Since the developing embryos lack a functional immune system, the viruses from these embryonic cells may not be cleared, allowing the viral replication cycle to proceed soon after infection. Once the viral RNA enters the host cells, it translates several of its non-structural proteins, which in turn physically interact with various host proteins to regulate host cell machinery. A recent study has identified 332 human proteins that physically interact with SARS-CoV-2 in HEK293T cells (Gordon et al. , 2020). The proteins that interact with SARS-CoV-2 have a wide range of functional roles including DNA replication, vesicle trafficking, lipid modification, RNA processing and regulation, nuclear transport machinery, cytoskeletal organization, mitochondrial functions, and extracellular matrix modelling (Gordon et al. , 2020). We observed that mRNA encoding for 320 of the 332 SARS-CoV-2 interacting proteins are expressed in the epiblast of the developing embryo. Almost all of these 320 genes were uniformly expressed in all the cells of the epiblast and also in the ICM and primitive endoderm (not shown). Interestingly, as compared to
ACE2- and
TMPRSS2- negative cells, 54/320 genes whose protein products physically interact with SARS-CoV-2 are overexpressed in
ACE2- and
TMPRSS2- positive cells of the epiblast. Together our observations imply that in human embryos, a proportion of cells in the epiblast abundantly expresses the mRNA for proteins that interact with SARS-CoV-2 to regulate host cellular processes. In developing embryos, the health of the cells of the epiblast is crucial, since these cells subsequently undergo organogenesis. Any damage to these cells, physically or functionally, may lead to embryo lethality or dysfunctions in the organs of the foetus/adults. To understand the potential damage that may occur to the embryos by SARS-CoV-2 infection, we analysed the gene signature of
ACE2- and
TMPRSS2- positive cells of the epiblast. We identified mRNA for 194 genes that were differentially expressed between the
ACE2- and
TMPRSS2- positive cells and
ACE2- and
TMPRSS2- negative cells of the epiblast. Unique gene signatures of
ACE2- positive cells have also been reported for other tissues (Seow et al. , 2020; Xu et al. , 2020). Interestingly, as compared to
ACE2- and
TMPRSS2- negative cells, 189/194 genes were overexpressed in the
ACE2- and
TMPRSS2- positive cells, and only 5/194 genes were present in lower abundance in the
ACE2- and
TMPRSS2- positive cells. Amongst the overabundant genes were cathepsins (including CTSL), prostaglandin synthetase, and glutamyl aminopeptidase, which have been associated with SARS-CoV-2 infection or enriched with ACE2 in different tissues (Ou et al. , 2020; Qi et al. , 2020). In the adult human liver, the highest expression of
ACE2 and
TMPRSS2 is detected in
TROP2 -positive cells (Seow et al. , 2020). In the present study, high expression of
TROP2 (also known as
TACSTD2 ) was also detected in the cells of the epiblast. These results imply that the
ACE2- and
TMPRSS2- positive cells may have unique functions in developing embryos. Interestingly, functional annotation revealed that the 194 differentially abundant genes play important roles in many aspects of development such as cell migration and metabolism. Furthermore, the gene signature also included genes facilitating viral entry into host cells and vacuole acidification. In this context it is imperative to note that pH reduction inside endosomes is essential for CTSL activation, which in turn is required to cleave the spike protein of SARS-CoV and SARS-CoV-2 (Ou et al. , 2020). Thus, our analyses reveal that the
ACE2- and
TMPRSS2- positive cells in the epiblast have key roles in embryonic development and may be particularly susceptible to SARS-CoV infection. Damage to these cells may cause embryo lethality at early stages of development or damage the process of tissue specification. Beyond ACE2 for SARS-CoV, DPP4 and ANPEP are other known coronavirus receptors (Li, 2015). We herein show the expression profiles of both
DPP4 and
ANPEP at the blastocyst stage, indicating that different coronaviruses can target similar cell types in the human host. Future studies should investigate these proteins and their genetic networks in embryonic cells to experimentally confirm the effects of coronaviruses on early human development. With regard to other viral infections in pregnancy, miscarriages and stillbirths are more common with influenza, and the mosquito-borne Zika virus adversely affects pregnancy and foetal development (Adams Waldorf et al. , 2018). Currently, little is known about the impact of COVID-19 on pregnancy. While coronaviruses are unrelated to the influenza and Zika viruses, our data imply potential embryo-toxic effects of SARS-CoV-2 and other coronaviruses. Presently, the American Society of Reproductive Medicine recommends that individuals with confirmed or presumed COVID-19 should avoid pregnancy. However, there is no clarity on the course of action for women/couples seeking IVF treatment, and several clinics continue to service patients seeking IVF, excluding only those who are symptomatic or who have recent travel history. The European Society of Human Reproduction and Embryology (ESHRE) has released its report of the ESHRE COVID-19 working group to monitor scientific reports relevant to reproductive medicine. The committee recommends that assisted reproduction treatments should not be started during the COVID-19 pandemic mainly for social reasons, as the report indicates that there is no specific data available; instead, it states an assumption that sperm, oocytes, and embryos do not have receptors for SARS-CoV-2 and are unlikely to be infected. Contradicting this assumption, we herein show that the receptors for coronaviruses are expressed in developing embryos. While we believe that the zona pellucida will act as a barrier to viral entry in the zygotes until the morula stages, receptors for coronaviruses are abundantly expressed in the cells of hatched blastocysts that can potentially get exposed to contaminated media and surfaces. We believe that our data will aid clinicians and societies in decision making regarding the management of patients seeking IVF or reporting miscarriages during the pandemic. In the light of our data and the possibility that the SARS-CoV-2 infected patients/clinic and laboratory personnel may be asymptomatic, we do not suggest performing IVF procedures during the COVID-19 pandemic. Procedures, if any, carried out during the pandemic should be executed with utmost precaution; patients, embryologists, and doctors should be aware of the potential risks; and couples must be appropriately counselled. Conclusion
Our single-cell RNA-Seq analysis shows that many cells of developing human embryos express the receptors for coronaviruses and also contain the necessary machinery for viral internalization and replication. We suggest that couples should be advised to avoid conception and pregnancy during the pandemic and IVF procedures should be kept to a minimum. Asymptomatic patients and laboratory/clinical personnel should be frequently screened for SARS-CoV-2 and other coronaviruses to prevent potential threats to the developing embryos.
Acknowledgements
DM lab is funded by grants from Indian Council of Medical Research (ICMR), Govt. of India. SC is thankful to Department of Health Research (DHR), Govt. of India for the Young Scientist fellowship. KC is thankful to Nehru-Fulbright Scholarship. AH and NS are thankful to ICMR-RA and ICMR-SRF respectively. RS is thankful to DST-Inspire fellowship, AM and DS are thankful to UGC for research fellowship. NA and AB are thankful to DST-SERB project fellowship. The manuscript bears the NIRRH ID RA/894/04-2020. References
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ACE2+TMPRSS2 +, ACE2+BSG+ , ACE2+CTSL+ , BSG+CTSL+ in human embryos at different stages. Data was extracted from single-cell RNA-Seq of the epiblast of developing human embryos (Stirparo et al 2018). The total cells and the numbers of embryos analysed are also given. Supplementary Fig.1. mRNA expression of coronavirus receptors and spike protein processing enzymes in early human embryos.
Data was extracted from single-cell RNA-Seq of developing human embryos (Yan et al., 2013)(Yan et al., 2013)