SARS-CoV-2 ACE-receptor detection in the placenta throughout pregnancy

Published:October 03, 2020DOI:https://doi.org/10.1016/j.cmi.2020.09.049
      Fetal risks for women exposed to SARS-CoV-2 and subsequent pregnancy outcomes remain uncertain [
      • Bahadur G.
      • Homburg R.
      • Yoong W.
      • Singh C.
      • Bhat M.
      • Kotabagi P.
      • et al.
      Adverse outcomes in SAR-CoV-2 (COVID-19) and SARS virus related pregnancies with probable vertical transmission.
      ]. Recent reports have raised controversial concerns regarding the potential for vertical transmission by transplacental infection. It has been proposed that the occurrence of intrauterine transplacental SARS-CoV-2 among infected mother–infant dyads be based upon identification of SARS-CoV-2 in chorionic villous cells using immunohistochemistry or nucleic acid methods such as in situ hybridization [
      • Schwartz D.A.
      • Morotti D.
      • Beigi B.
      • Moshfegh F.
      • Zafaranloo N.
      • Patane L.
      Confirming vertical fetal infection with COVID-19: neonatal and pathology criteria for early onset and transplacental transmission of SARS-CoV-2 from infected pregnant mothers.
      ]. Hypothetically, two conditions are necessary for transplacental transmission to be possible: (a) the virus must reach the placenta; and (b) the receptor for the virus, angiotensin-converting enzyme 2 (ACE2), must be present in the placenta.
      Regarding the first condition, we and others recently reported RT-PCR or RNAscope data supporting the presence of SARS-CoV-2 in placental tissue [
      • Baud D.
      • Nielsen-Saines K.
      • Qi X.
      • Musso D.
      • Pomar L.
      • Favre G.
      Authors' reply.
      ,
      • Kotlyar A.
      • Grechukhina O.
      • Chen A.
      • Popkhadze S.
      • Grimshaw A.
      • Tal O.
      • et al.
      Vertical transmission of COVID-19: a systematic review and meta-analysis.
      ,
      • Vivanti A.J.
      • Vauloup-Fellous C.
      • Prevot S.
      • Zupan V.
      • Suffee C.
      • Do Cao J.
      • et al.
      Transplacental transmission of SARS-CoV-2 infection.
      ,
      • Patane L.
      • Morotti D.
      • Giunta M.R.
      • Sigismondi C.
      • Piccoli M.G.
      • Frigerio L.
      • et al.
      Vertical transmission of coronavirus disease 2019: severe acute respiratory syndrome coronavirus 2 RNA on the fetal side of the placenta in pregnancies with coronavirus disease 2019-positive mothers and neonates at birth.
      ]. To date, conflicting data exists regarding the second condition [
      • Kotlyar A.
      • Grechukhina O.
      • Chen A.
      • Popkhadze S.
      • Grimshaw A.
      • Tal O.
      • et al.
      Vertical transmission of COVID-19: a systematic review and meta-analysis.
      ,
      • Li M.
      • Chen L.
      • Zhang J.
      • Xiong C.
      • Li X.
      The SARS-CoV-2 receptor ACE2 expression of maternal–fetal interface and fetal organs by single-cell transcriptome study.
      ,
      • Pique-Regi R.
      • Romero R.
      • Tarca A.L.
      • Luca F.
      • Xu Y.
      • Alazizi A.
      • et al.
      Does the human placenta express the canonical cell entry mediators for SARS-CoV-2?.
      ].
      In order to investigate ACE2 expression in the placenta throughout pregnancy, we selected formalin-fixed placental tissues between 14 and 40 weeks gestation from 28 patients who delivered before the current COVID-19 outbreak (two different cases for each 2 weeks of gestation, i.e. 14, 16, 18, … 38, 40 weeks; please see supplementary material), as well as placental tissues at 19 weeks' gestation from a COVID-19-positive patient (Fig. 1). Kidney was used as positive (brush border of proximal renal tubules and podocytes of glomeruli) and negative (distal renal tubules and renal medulla) controls. Using a monoclonal anti-ACE2 antibody (Atlas antibodies, clone CL4035, dilution 1/1000), we demonstrated in situ expression of ACE2 at the maternal–fetal interface (Fig. 1 and supplementary material), a prerequisite for transplacental transmission. We observed a strong and diffuse membranous staining of cytotrophoblast and syncytiotrophoblast cells of placental villi, as well as a membranous expression in extravillous trophoblast. By testing placental tissues at various gestational ages in both COVID-19-positive and -negative mothers, we confirmed that ACE expression is present consistently throughout pregnancy regardless of COVID-19 status.
      Fig. 1
      Fig. 1Diffuse membranous staining of villous cytotrophoblast and syncytiotrophoblast cells (arrows) with monoclonal Anti-ACE2 antibody (clone CL4035), dilution 1/1000 in a COVID-19 positive mother, 19 weeks of amenorrhea.
      Our in situ analyses by specific immunohistochemistry and SARS-CoV-2 detection by RT-PCR indicate a possible placental infection by SARS-CoV2. Trophoblastic cells, which are in direct contact with the maternal blood in the intervillous space, show strong expression of ACE2 throughout pregnancy, supporting that SARS-CoV2 is able to infect the placenta via a receptor-mediated mechanism. SARS-CoV-2 can cross the placental barrier, as it has been demonstrated in a neonate born to a mother infected in the last trimester and presenting with neurological compromise [
      • Vivanti A.J.
      • Vauloup-Fellous C.
      • Prevot S.
      • Zupan V.
      • Suffee C.
      • Do Cao J.
      • et al.
      Transplacental transmission of SARS-CoV-2 infection.
      ]. However, the rate of vertical transmission to the fetus warrants further investigation. By analogy to other pathogens (i.e. cytomegalovirus or toxoplasmosis), this may occur once sufficient time has elapsed for the virus to breach the placental barrier (6–8 weeks after infection).
      SARS-CoV-2 infection of the maternal placental surface may induce acute or chronic placental insufficiency [
      • Baud D.
      • Nielsen-Saines K.
      • Qi X.
      • Musso D.
      • Pomar L.
      • Favre G.
      Authors' reply.
      ,
      • Kotlyar A.
      • Grechukhina O.
      • Chen A.
      • Popkhadze S.
      • Grimshaw A.
      • Tal O.
      • et al.
      Vertical transmission of COVID-19: a systematic review and meta-analysis.
      ,
      • Vivanti A.J.
      • Vauloup-Fellous C.
      • Prevot S.
      • Zupan V.
      • Suffee C.
      • Do Cao J.
      • et al.
      Transplacental transmission of SARS-CoV-2 infection.
      ] or be responsible for subsequent miscarriage or fetal growth restriction as reported in 40% of MERS or SARS-1 maternal infections. In consequence, these findings could lead to increased protective measures for pregnant women, as well as a heightened level of concern/monitoring of pregnancies exposed to SARS-CoV-2.

      Transparency declaration

      G.G. reported having ongoing research agreements with Resistell and Becton-Dickinson and being medical advisor for Resistell; he also reported being developer of a card game on microbes and funding JeuPro, a start-up company that distributes the game Krobs. No other disclosures were reported by any of the other authors. No external funding was received.

      Author contributions

      D.B. and C.G. had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Conceptualization: C.G., E.D., D.B.
      Acquisition, analysis, or interpretation of data: all authors. Writing – original draft: C.G., E.D., D.B. Writing – review and editing: C.G., G.G., G.F., L.L., D.B. Administrative, technical, or material support: all authors. Supervision and validation: C.G., D.B.

      Appendix A. Supplementary data

      The following is/are the supplementary data to this article:

      References

        • Bahadur G.
        • Homburg R.
        • Yoong W.
        • Singh C.
        • Bhat M.
        • Kotabagi P.
        • et al.
        Adverse outcomes in SAR-CoV-2 (COVID-19) and SARS virus related pregnancies with probable vertical transmission.
        JBRA Assist Reprod. 2020; 24: 351-357
        • Schwartz D.A.
        • Morotti D.
        • Beigi B.
        • Moshfegh F.
        • Zafaranloo N.
        • Patane L.
        Confirming vertical fetal infection with COVID-19: neonatal and pathology criteria for early onset and transplacental transmission of SARS-CoV-2 from infected pregnant mothers.
        Arch Pathol Lab Med. 2020; https://doi.org/10.5858/arpa.2020-0442-SA
        • Baud D.
        • Nielsen-Saines K.
        • Qi X.
        • Musso D.
        • Pomar L.
        • Favre G.
        Authors' reply.
        Lancet Infect Dis. 2020; 20: 775-776
        • Kotlyar A.
        • Grechukhina O.
        • Chen A.
        • Popkhadze S.
        • Grimshaw A.
        • Tal O.
        • et al.
        Vertical transmission of COVID-19: a systematic review and meta-analysis.
        Am J Obstet Gynecol. 2020;
        • Vivanti A.J.
        • Vauloup-Fellous C.
        • Prevot S.
        • Zupan V.
        • Suffee C.
        • Do Cao J.
        • et al.
        Transplacental transmission of SARS-CoV-2 infection.
        Nat Commun. 2020; 11: 3572
        • Patane L.
        • Morotti D.
        • Giunta M.R.
        • Sigismondi C.
        • Piccoli M.G.
        • Frigerio L.
        • et al.
        Vertical transmission of coronavirus disease 2019: severe acute respiratory syndrome coronavirus 2 RNA on the fetal side of the placenta in pregnancies with coronavirus disease 2019-positive mothers and neonates at birth.
        Am J Obstet Gynecol MFM. 2020; 2: 100145
        • Li M.
        • Chen L.
        • Zhang J.
        • Xiong C.
        • Li X.
        The SARS-CoV-2 receptor ACE2 expression of maternal–fetal interface and fetal organs by single-cell transcriptome study.
        PloS One. 2020; 15e0230295
        • Pique-Regi R.
        • Romero R.
        • Tarca A.L.
        • Luca F.
        • Xu Y.
        • Alazizi A.
        • et al.
        Does the human placenta express the canonical cell entry mediators for SARS-CoV-2?.
        Elife. 2020; 9