SARS-CoV-2 Infects Syncytiotrophoblast and Activates Inflammatory Responses in the Placenta

Abstract

SARS-CoV-2 infection during pregnancy leads to an increased risk of adverse pregnancy outcomes. Although the placenta itself can be a target of virus infection, most neonates are virus free and are born healthy or recover quickly. Here, we investigated the impact of SARS-CoV-2 infection on the placenta from a cohort of women who were infected late during pregnancy and had tested nasal swab positive for SARS-CoV-2 by qRT-PCR at delivery. SARS-CoV-2 genomic and subgenomic RNA was detected in 23 out of 54 placentas. Two placentas with high virus content were obtained from mothers who presented with severe COVID-19 and whose pregnancies resulted in adverse outcomes for the fetuses, including intrauterine fetal demise and a preterm delivered baby still in newborn intensive care. Examination of the placental samples with high virus content showed efficient SARS-CoV-2 infection, using RNA in situ hybridization to detect genomic and replicating viral RNA, and immunohistochemistry to detect SARS-CoV-2 nucleocapsid protein. Infection was restricted to syncytiotrophoblast cells that envelope the fetal chorionic villi and are in direct contact with maternal blood. The infected placentas displayed massive infiltration of maternal immune cells including macrophages into intervillous spaces, potentially contributing to inflammation of the tissue. Ex vivo infection of placental cultures with SARS-CoV-2 or with SARS-CoV-2 spike (S) protein pseudotyped lentivirus targeted mostly syncytiotrophoblast and in rare events endothelial cells. Infection was reduced by using blocking antibodies against ACE2 and against Neuropilin 1, suggesting that SARS-CoV-2 may utilize alternative receptors for entry into placental cells.

Figure 1.. SARS-CoV-2 virus is present in placentas from infected mothers and results in inflammatory responses.

(A) Graph showing ΔΔCT values of RNA samples isolated from FFPE patient placenta slides from the different cohorts. A student’s t-test comparing the 3 positive cohorts (High Positive, Positive, Borderline Positive) to the negative control cohort resulted in statistically significant higher viral load in the High Positive and Positive cohorts (*** = p-value < 0.001). (B) Brightfield microscopy images of a representative COVID High positive patient (P3) and a representative negative control patient sample (C1). Slides were stained by H&E, in situ PLAYR for SARS-CoV2-RNA counterstained for syncytial trophoblast marker cytokeratin (KRT7, red), and by immunohistochemistry for SARS-CoV2-N protein (brown) as well as for CD163+ Hofbauer cells (HBC). Scale bars = 100μm.

Figure 2.. Placental explant and cell clusters can be infected by SARS-CoV-2 S protein pseudotyped lentivirus and infection can be blocked by anti-ACE2 and anti-NRP1 antibodies.

(A) Graphs showing relative luminescence units (RLU) from infected explant cultures 72 hpi with the addition of reverse transcriptase inhibitor, Nevirapine (NVP). (B) Graphs showing RLU from infected isolated primary placental clusters 72 hpi with the addition of blocking antibodies against ACE2, NRP1. Statistical analysis was performed using one-way Anova (** = p-value < 0.005, *** = p-value < 0.001). (C) Brightfield and live fluorescence microscopy images of cultured placental explants Mock (left column), or 72hpi with either Lenti-SARS-CoV2-S Pseudovirus (center column) or Lenti-VSV-G (right column). (D) Fluorescence microscopy on mock (top row) and Lenti-SARS-CoV2-S infected (center row) or Lenti-VSV-G infected (bottom row) explant sections stained for the GFP reporter (green) syncytial trophoblast marker, cytokeratin (KRT7, grey), endothelial marker CD31 (red) and DAPI nuclear stain (blue). Scale bars = 500μm.

Figure 3.. Primary human placenta cells can be infected with SARS-CoV-2 ex vivo.

(A) qRT-PCR analysis of relative viral N subgenomic RNA expression in primary placental cell clusters infected with SARS-CoV-2 ex vivo (MOI=1) for 24 hours and normalized to ACTB levels. (mean+/− SD; n=12 from 4 repeated experiments; ****p<0.0001). (B) Three-dimensional reconstruction of confocal imaging of primary placental cell clusters infected with SARS-CoV-2 ex vivo (MOI=1) at 24hpi, stained for trophoblast marker KRT7 (green), SARS-N (red), endothelial marker CD31 (grey), and DAPI (blue). Scale bar = 30μm. (C) Confocal imaging of primary placental cell clusters infected with MOCK (top rows) or SARS-CoV-2 (MOI=1, bottom rows) ex vivo at 24hpi, stained for trophoblast marker KRT7 (green), SARS-N (red), endothelial marker CD31 (grey), and DAPI (blue). Arrows indicate presence of SARS-N nucleocapsid protein in trophoblast and endothelial cells. Scale bar = 20μm.