Maternal respiratory SARS-CoV-2 infection in pregnancy is associated with a robust inflammatory response at the maternal-fetal interface

  1. Alice Lu-Culligan
  2. Arun R. Chavan
  3. Pavithra Vijayakumar
  4. Lina Irshaid
  5. Edward M. Courchaine
  6. Kristin M. Milano
  7. Zhonghua Tang
  8. Scott D. Pope
  9. Eric Song
  10. Chantal B.F. Vogels
  11. William J. Lu-Culligan
  12. Katherine H. Campbell
  13. Arnau Casanovas-Massana
  14. Santos Bermejo
  15. Jessica M. Toothaker
  16. Hannah J. Lee
  17. Feimei Liu
  18. Wade Schulz
  19. John Fournier
  20. M. Catherine Muenker
  21. Adam J. Moore
  22. Liza Konnikova
  23. Karla M. Neugebauer
  24. Aaron Ring
  25. Nathan D. Grubaugh
  26. Albert I. Ko
  27. Raffaella Morotti
  28. Seth Guller
  29. Harvey J. Kliman
  30. Akiko Iwasaki
  31. Shelli F. Farhadian

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Abstract

No abstract available

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  1. Version published to 10.1016/j.medj.2021.04.016
  2. ScreenIT

    SciScore for 10.1101/2021.01.25.21250452: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementConsent: Women who were admitted to Yale New Haven Hospital Labor and Birth during the study period and who were positive for SARS-CoV-2 by nasopharyngeal swab RT-qPCR in the antepartum period or at the time of delivery hospitalization were approached for consent to donate additional tissue for research studies through the Yale IMPACT study (Implementing Medical and Public Health Action Against Coronavirus in CT).
    IRB: The study was approved by the Yale Institutional Review Board (protocol #2000027690 and 2000028550).
    Randomizationnot detected.
    BlindingTwo pathologists (LI and RM) blinded to patient information and diagnostic report independently scored all H&E stained placental tissue for villitis (absent, low-grade or high-grade; if low-grade, focal or multifocal and if high-grade, patchy or diffuse), intervillositis (absent or present), increased intervillous fibrin (absent or present, defined as intervillous fibrin occupying >10% of a full thickness section on 20x low-power magnification), chorioamnionitis (absent or present; if present, maternal and fetal inflammatory responses were staged and graded), fetal and/or maternal vascular malperfusion (absent or present; if present, a histologic description was recorded) and increased decidual lymphocytes (absent or present, defined as clusters of >10 lymphocytes in >3 foci in the placental disc and/or peripheral membranes).
    Power Analysisnot detected.
    Sex as a biological variableStudy participants: We searched the electronic medical record of Yale-New Haven Hospital for all women who tested positive for SARS-CoV-2 in the antepartum or peripartum period and presented for delivery hospitalization during the study period (March 27, 2020 to June 1, 2020).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    SARS-CoV-2 S1 spike protein IgM and IgG serology testing: ELISA assays for IgG and IgM antibodies towards SARS-CoV-2 were performed on plasma as described by Amanat et al (62).
    SARS-CoV-2 S1 spike protein IgM
    suggested: None
    IgM
    suggested: None
    ACE2 Immunohistochemistry: The following rabbit polyclonal antibodies were used: anti-ACE2 (Abcam, Cambridge, MA, ab10 8252, used at 1 µg IgG/ml); and, as a negative control, normal rabbit serum (Sigma-Aldrich, St. Louis, MO, R9133, used at 1 µg IgG/ml).
    R9133
    suggested: (Fitzgerald Industries International Cat# 70R-9133, RRID:AB_11150162)
    Cells migrating to the 35%/45% Percoll interface were recovered by centrifugation at 300 x g for 10 minutes at room temperature and immunopurified by negative selection using mouse anti-human CD9 antibody and mouse anti-human CD45 antibody.
    anti-human CD9
    suggested: None
    anti-human CD45
    suggested: None
    Cells from the 20%/25% to 30%/35% interfaces were combined and immunopurified by negative selection using sequential treatment with anti-EGFR and anti-CD10 antibodies conjugated to magnetic beads.
    anti-EGFR
    suggested: None
    anti-CD10
    suggested: None
    Mouse anti-SARS-CoV-2 spike antibody (clone 1A9, GeneTex GTX632604) was used at a dilution of 1:400 and rabbit anti-ACE2 (clone EPR4435(2), Abcam ab108252) was used at 1:200 dilution.
    anti-SARS-CoV-2
    suggested: None
    anti-ACE2
    suggested: None
    Each sample was incubated overnight with anti-SARS-CoV-2-NP rabbit polyclonal antibody (GeneTex # GTX135357) at a dilution of 1:200.
    anti-SARS-CoV-2-NP
    suggested: None
    After washing in PBS, coverslips were incubated for 1 hour in a 1:500 dilution of Alexa 594 anti rabbit secondary antibody (Jackson ImmunoResearch 711-585-152), washed again in PBS and treated with 1 µg/mL Hoechst 33342 for 10 min and washed a final time in PBS.
    anti rabbit
    suggested: (Jackson ImmunoResearch Labs Cat# 711-585-152, RRID:AB_2340621)
    SARS-CoV-2 S1 spike protein IgM and IgG serology testing: ELISA assays for IgG and IgM antibodies towards SARS-CoV-2 were performed on plasma as described by Amanat et al (62).
    SARS-CoV-2 S1 spike protein IgM
    suggested: None
    IgM
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    BeWo cells were maintained in F12K Kaighn’s modified media, HTR8 cells in RPMI media, and Vero E6 cells in F12:DMEM media, all with 10% FBS and antibiotic/antimycotic.
    HTR8
    suggested: RRID:CVCL_D728)
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    FASTQ files from HiSeq 2500 were analyzed using Kallisto v0.46.1(69) using the “-b 100 and -t 20” options to obtain transcript abundances in TPM and estimated counts.
    Kallisto
    suggested: (kallisto, RRID:SCR_016582)
    The kallisto index used during transcript quantification was built (31bp k-mer length) from the Homo sapiens transcriptome GRCh38 downloaded as a FASTA file from Ensembl (Ensembl.org).
    Ensembl
    suggested: (Ensembl, RRID:SCR_002344)
    Transcripts were annotated using the Bioconductor package biomaRt v2.40.5(70) in R v3.6.2.
    Bioconductor
    suggested: (Bioconductor, RRID:SCR_006442)
    biomaRt
    suggested: (biomaRt, RRID:SCR_019214)
    Differential expression analysis was performed using DESeq2 with default parameters(72), comparing all 3rd trimester samples by maternal status.
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)

    Results from OddPub: Thank you for sharing your code.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    This study is subject to several limitations. First, only 15 placentas were available for RT-qPCR analysis, and thus we had insufficient sample size to understand if specific clinical features (e.g., severe COVID-19 or duration of symptoms) correlated with the presence of local virus in the placenta. Furthermore, our analysis is limited in that we only assessed placenta from women who were infected with SARS-CoV-2 during the third trimester of pregnancy, and thus does not account for pathological and inflammatory changes at the placenta that result from infection during the first or second trimester. Indeed, our results demonstrating widespread ACE2 expression in the placenta during the first and second trimesters indicates that early pregnancy may be the most vulnerable time for SARS-CoV-2 induced placental pathology, and additional studies are needed to assess for potential placental and fetal abnormalities associated with infection during early pregnancy. By characterizing changes at the maternal-fetal interface in the context of systemic infection, our research indicated that maternal SARS-CoV-2 infection at term is associated with an inflammatory state in the placenta that may contribute to poor pregnancy outcomes in COVID-19, even in the absence of viral invasion of the placenta(57). These immune responses in the placenta may serve to protect the placenta and fetus from infection, but they also have the potential to drive pathological changes with adverse consequences f...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.01.25.21250452v1 on medRxiv