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Single-cell RNA sequencing of SARS-CoV-2 cell entry factors in the preconceptional human endometrium.
Hum Reprod. 2021 09 18; 36(10):2709-2719.HR

Abstract

STUDY QUESTION

Are SARS-CoV-2 canonical cell entry machinery, consisting of ACE2, TMPRSS2, NRP1 and LY6E, or alternative potential cell entry machinery, consisting of BSG, ANPEP, CD209, CLEC4G, TMPRSS4, TMPRSS11A, FURIN, CTSB, CTSL and IFITM1, expressed in the human endometrium across the menstrual cycle?

SUMMARY ANSWER

Analysis of cell entry factors for SARS-CoV-2 by single-cell RNA-sequencing (scRNAseq) in the preconceptional human endometrium reveals low risk of infection.

WHAT IS KNOWN ALREADY

Gene expression datasets from bulk endometrial tissue show no significant expression of the SARS-CoV-2 receptor ACE2 and TMPRSS2. This is in contrast to reported expression of ACE2 at the single-cell level in the decidua and trophoblast cells at the maternal-fetal interface in early pregnancy, as well as vertical transmission of SARS-CoV-2 during pregnancy.

STUDY DESIGN, SIZE, DURATION

This analysis of SARS-CoV-2 cell entry machinery gene expression was conducted by scRNAseq in 73 181 human endometrial cells isolated from endometrial biopsies obtained from 27 donors across the menstrual cycle.

PARTICIPANTS/MATERIALS, SETTING, METHODS

ScRNAseq examined the expression of genes encoding cell entry machinery for SARS-CoV-2. The raw data were from a previously published dataset.

MAIN RESULTS AND THE ROLE OF CHANCE

ScRNAseq analysis showed no significant expression of ACE2 in stromal or unciliated epithelial cells in any phase of the menstrual cycle. TMPRSS2 was expressed in epithelial cells during the early proliferative and mid-secretory phases. Interestingly, the expression of NRP1 was observed in both stromal and epithelial cells across all phases of the menstrual cycle, and LY6E was highly expressed in stromal cells. In the mid-secretory phase, coexpression of ACE2 and TMPRSS2 was detected in 0.07% of luminal epithelial cells. No cells simultaneously expressed ACE2, NRP1 and TMPRSS2 at the time of embryo implantation. Focusing on non-canonical cell entry machinery, BSG was highly expressed in all cell types across the menstrual cycle and may interact with CTSB or CTSL proteases, but viral infection using this machinery has not yet been confirmed.

LARGE SCALE DATA

All raw data in this study can be found at NCBI's Gene Expression Omnibus (series accession code GSE111976) and Sequence Read Archive (accession code SRP135922).

LIMITATIONS, REASONS FOR CAUTION

Our findings at the single-cell level imply low efficiency of SARS-CoV-2 endometrial infection using canonical receptors in a cohort of healthy reproductive-age women; however, infection of endometrial cells can only be assessed in the presence of the virus. All samples were processed for scRNAseq, so no samples are remaining to analyze protein expression or spatial transcriptomics.

WIDER IMPLICATIONS OF THE FINDINGS

Our results offer a useful resource to guide reproductive decisions when assessing risk of endometrial infection by SARS-CoV-2 during the preconceptional period in asymptomatic COVID-19 carriers.

STUDY FUNDING/COMPETING INTEREST(S)

This study was jointly supported by the March of Dimes, Chan Zuckerberg Biohub and MINECO/FEDER (SAF-2015-67164-R, to C.S.) (Spanish Government), and the European Union's Horizon 2020 Framework Programme for Research and Innovation (Grant agreement 874867). W.W. was supported by the Stanford Bio-X Graduate Bowes Fellowship and Chan Zuckerberg Biohub. F.V. was supported by the Miguel Servet Program Type II of ISCIII (CPII18/00020) and the FIS project (PI18/00957). A patent disclosure has been filed for the study with the title 'Methods for assessing endometrial transformation' and the global patent number 'EP 3807648 A2' under the inventors S.R.Q., C.S., W.W. and F.V. C.S. is the Founder and Head of the Scientific Advisory Board of Igenomix SL. S.R.Q is the Director of Mirvie. I.M. is partially employed by Igenomix SL. B.R. has no interests to declare.

Authors+Show Affiliations

Igenomix Foundation, INCLIVA Health Research Institute, Valencia, Spain. Department of Obstetrics & Gynecology, Beth Israel Deaconess Medical Center, Harvard University, Boston, MA, USA.Department of Bioengineering, Stanford University, Stanford, CA, USA.Igenomix Foundation, INCLIVA Health Research Institute, Valencia, Spain.Igenomix Foundation, INCLIVA Health Research Institute, Valencia, Spain.Department of Bioengineering, Stanford University, Stanford, CA, USA. Department of Applied Physics, Stanford University, Stanford, CA, USA. Chan Zuckerberg Biohub, San Francisco, CA, USA.Igenomix Foundation, INCLIVA Health Research Institute, Valencia, Spain. Department of Obstetrics & Gynecology, University of Valencia, Valencia, Spain. Department of Obstetrics & Gynecology, Beth Israel Deaconess Medical Center, Harvard University, Boston, MA, USA.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

34329437

Citation

Vilella, F, et al. "Single-cell RNA Sequencing of SARS-CoV-2 Cell Entry Factors in the Preconceptional Human Endometrium." Human Reproduction (Oxford, England), vol. 36, no. 10, 2021, pp. 2709-2719.
Vilella F, Wang W, Moreno I, et al. Single-cell RNA sequencing of SARS-CoV-2 cell entry factors in the preconceptional human endometrium. Hum Reprod. 2021;36(10):2709-2719.
Vilella, F., Wang, W., Moreno, I., Roson, B., Quake, S. R., & Simon, C. (2021). Single-cell RNA sequencing of SARS-CoV-2 cell entry factors in the preconceptional human endometrium. Human Reproduction (Oxford, England), 36(10), 2709-2719. https://doi.org/10.1093/humrep/deab183
Vilella F, et al. Single-cell RNA Sequencing of SARS-CoV-2 Cell Entry Factors in the Preconceptional Human Endometrium. Hum Reprod. 2021 09 18;36(10):2709-2719. PubMed PMID: 34329437.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Single-cell RNA sequencing of SARS-CoV-2 cell entry factors in the preconceptional human endometrium. AU - Vilella,F, AU - Wang,W, AU - Moreno,I, AU - Roson,B, AU - Quake,S R, AU - Simon,C, PY - 2020/12/30/received PY - 2021/05/17/revised PY - 2021/7/31/pubmed PY - 2021/9/23/medline PY - 2021/7/30/entrez KW - ACE2 KW - COVID-19 KW - NRP1 KW - SARS-CoV-2 KW - TMPRSS2 KW - scRNAseq SP - 2709 EP - 2719 JF - Human reproduction (Oxford, England) JO - Hum Reprod VL - 36 IS - 10 N2 - STUDY QUESTION: Are SARS-CoV-2 canonical cell entry machinery, consisting of ACE2, TMPRSS2, NRP1 and LY6E, or alternative potential cell entry machinery, consisting of BSG, ANPEP, CD209, CLEC4G, TMPRSS4, TMPRSS11A, FURIN, CTSB, CTSL and IFITM1, expressed in the human endometrium across the menstrual cycle? SUMMARY ANSWER: Analysis of cell entry factors for SARS-CoV-2 by single-cell RNA-sequencing (scRNAseq) in the preconceptional human endometrium reveals low risk of infection. WHAT IS KNOWN ALREADY: Gene expression datasets from bulk endometrial tissue show no significant expression of the SARS-CoV-2 receptor ACE2 and TMPRSS2. This is in contrast to reported expression of ACE2 at the single-cell level in the decidua and trophoblast cells at the maternal-fetal interface in early pregnancy, as well as vertical transmission of SARS-CoV-2 during pregnancy. STUDY DESIGN, SIZE, DURATION: This analysis of SARS-CoV-2 cell entry machinery gene expression was conducted by scRNAseq in 73 181 human endometrial cells isolated from endometrial biopsies obtained from 27 donors across the menstrual cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS: ScRNAseq examined the expression of genes encoding cell entry machinery for SARS-CoV-2. The raw data were from a previously published dataset. MAIN RESULTS AND THE ROLE OF CHANCE: ScRNAseq analysis showed no significant expression of ACE2 in stromal or unciliated epithelial cells in any phase of the menstrual cycle. TMPRSS2 was expressed in epithelial cells during the early proliferative and mid-secretory phases. Interestingly, the expression of NRP1 was observed in both stromal and epithelial cells across all phases of the menstrual cycle, and LY6E was highly expressed in stromal cells. In the mid-secretory phase, coexpression of ACE2 and TMPRSS2 was detected in 0.07% of luminal epithelial cells. No cells simultaneously expressed ACE2, NRP1 and TMPRSS2 at the time of embryo implantation. Focusing on non-canonical cell entry machinery, BSG was highly expressed in all cell types across the menstrual cycle and may interact with CTSB or CTSL proteases, but viral infection using this machinery has not yet been confirmed. LARGE SCALE DATA: All raw data in this study can be found at NCBI's Gene Expression Omnibus (series accession code GSE111976) and Sequence Read Archive (accession code SRP135922). LIMITATIONS, REASONS FOR CAUTION: Our findings at the single-cell level imply low efficiency of SARS-CoV-2 endometrial infection using canonical receptors in a cohort of healthy reproductive-age women; however, infection of endometrial cells can only be assessed in the presence of the virus. All samples were processed for scRNAseq, so no samples are remaining to analyze protein expression or spatial transcriptomics. WIDER IMPLICATIONS OF THE FINDINGS: Our results offer a useful resource to guide reproductive decisions when assessing risk of endometrial infection by SARS-CoV-2 during the preconceptional period in asymptomatic COVID-19 carriers. STUDY FUNDING/COMPETING INTEREST(S): This study was jointly supported by the March of Dimes, Chan Zuckerberg Biohub and MINECO/FEDER (SAF-2015-67164-R, to C.S.) (Spanish Government), and the European Union's Horizon 2020 Framework Programme for Research and Innovation (Grant agreement 874867). W.W. was supported by the Stanford Bio-X Graduate Bowes Fellowship and Chan Zuckerberg Biohub. F.V. was supported by the Miguel Servet Program Type II of ISCIII (CPII18/00020) and the FIS project (PI18/00957). A patent disclosure has been filed for the study with the title 'Methods for assessing endometrial transformation' and the global patent number 'EP 3807648 A2' under the inventors S.R.Q., C.S., W.W. and F.V. C.S. is the Founder and Head of the Scientific Advisory Board of Igenomix SL. S.R.Q is the Director of Mirvie. I.M. is partially employed by Igenomix SL. B.R. has no interests to declare. SN - 1460-2350 UR - https://www.unboundmedicine.com/medline/citation/34329437/Single_cell_RNA_sequencing_of_SARS_CoV_2_cell_entry_factors_in_the_preconceptional_human_endometrium_ DB - PRIME DP - Unbound Medicine ER -