Maternal-fetal immune responses in pregnant women infected with SARS-CoV-2

Abstract

Pregnant women represent a high-risk population for severe/critical COVID-19 and mortality. However, the maternal-fetal immune responses initiated by SARS-CoV-2 infection, and whether this virus is detectable in the placenta, are still under investigation. Here we show that SARS-CoV-2 infection during pregnancy primarily induces unique inflammatory responses at the maternal-fetal interface, which are largely governed by maternal T cells and fetal stromal cells. SARS-CoV-2 infection during pregnancy is also associated with humoral and cellular immune responses in the maternal blood, as well as with a mild cytokine response in the neonatal circulation (i.e., umbilical cord blood), without compromising the T-cell repertoire or initiating IgM responses. Importantly, SARS-CoV-2 is not detected in the placental tissues, nor is the sterility of the placenta compromised by maternal viral infection. This study provides insight into the maternal-fetal immune responses triggered by SARS-CoV-2 and emphasizes the rarity of placental infection.

Fig. 1. Serological and cytokine responses in maternal and cord blood of women with SARS-CoV-2 infection and their neonates.

a Serum concentrations of IgM and IgG in the maternal blood [n = 9 control, 11 SARS-CoV-2 (+)] (left panel) and cord blood [n = 6 control, 7 SARS-CoV-2 (+)] (right panel). Data are shown as boxplots where midlines indicate medians, boxes indicate interquartile range and whiskers indicate minimum/maximum range. Differences between groups were evaluated by two-sided Mann–Whitney U-tests. p-values < 0.05 were used to denote a significant result. Plasma concentrations of IL-8, IL-6, IL-10, IL-15, IL-16, IL-17A, IFN-γ, and TNF in b maternal blood [n = 8 control, 11 SARS-CoV-2(+)] and c cord blood [n = 7 control, 9 SARS-CoV-2(+)]. Data are shown as boxplots where midlines indicate medians, boxes indicate interquartile range and whiskers indicate minimum/maximum range. Gray dotted lines indicate the lower limit of detection. Differences in cytokine concentrations between groups were evaluated by linear mixed-effects models with adjustment for covariates. Scatter plots of two principal components (PC1 and PC3 or PC1 and PC2) from plasma cytokine concentrations in the d maternal blood [n = 8 control, 11 SARS-CoV-2(+)] and e cord blood [n = 7 control, 9 SARS-CoV-2(+)]. The association between principal components (PC1, PC2, and PC3 jointly for maternal blood and PC2 for cord blood) and SARS-CoV-2 (+) status was assessed by logistic regression. Blue dots indicate control women, light red dots indicate SARS-CoV-2 (+) women, and dark red dots indicate women with severe COVID-19. Significant differences are based on p < 0.05.

Fig. 2. Immunophenotyping of T cells in the maternal and cord blood of women with SARS-CoV-2 infection and their neonates.

a Maternal blood and cord blood were collected for immunophenotyping. b Numbers of T cells in the peripheral blood [n = 10 control, 11 SARS-CoV-2 (+)] and cord blood [n = 9 control, 8 SARS-CoV-2 (+)]. Data are shown as boxplots where midlines indicate medians, boxes indicate interquartile range and whiskers indicate minimum/maximum range. Differences between groups were evaluated by Mann–Whitney U-tests. P values < 0.05 were used to denote a significant result. Blue dots indicate control women, light red dots indicate SARS-CoV-2 (+) women, and dark red dots indicate women with severe COVID-19. c Heatmap showing the abundance (z-scores) of T-cell subsets in the maternal blood from SARS-CoV-2 (+) or control women [n = 10 control, 11 SARS-CoV-2 (+)], where cell numbers and proportions are shown. Differences between groups were assessed using two-sample t-tests. P values were adjusted for multiple comparisons using the false-discovery rate (FDR) method to obtain q values. *q < 0.1; **q < 0.05. Red and blue indicate increased and decreased abundance, respectively. d Three-dimensional scatter plot showing the distribution of flow cytometry data. Blue dots indicate control women, light red dots indicate SARS-CoV-2 (+) women, and dark red dots indicate women with severe COVID-19 [n = 10 control, 11 SARS-CoV-2(+)] based on principal component (PC)1–PC3.

Fig. 3. T-cell subsets in the maternal and cord blood of women with SARS-CoV-2 infection and their neonates.

a Representative gating strategy used to identify CD4⁺ and CD8⁺ T cells, and their respective subsets, within the total T-cell population (CD45⁺CD3⁺ cells) in the maternal blood and cord blood from SARS-CoV-2 (+) or control women. b Numbers of CD4⁺ T cells, CD4⁺ T_CM, CXCR3⁺CCR6⁺ Th1-like cells, and CXCR3⁺CCR6^- Th1-like cells (upper row); and numbers of CD8⁺ T cells, CD8⁺ T_CM, CD8⁺ T_EM, and Tc17-like cells (lower row) in the maternal blood [n = 10 control, 11 SARS-CoV-2 (+)]. c Numbers of CD4⁺ T cells, CD4⁺ T_CM, CXCR3⁺CCR6⁺ Th1-like cells, and CXCR3⁺CCR6⁻ Th1-like cells (upper row); and numbers of CD8⁺ T cells, CD8⁺ T_CM, CD8⁺ T_EM, and Tc17-like cells (lower row) in the cord blood [n = 9 control, 8 SARS-CoV-2 (+)]. Data are shown as boxplots where midlines indicate medians, boxes indicate interquartile range and whiskers indicate minimum/maximum range. Blue dots indicate control women, light red dots indicate SARS-CoV-2 (+) women, and dark red dots indicate women with severe COVID-19. Differences between groups were evaluated by two-sided Mann–Whitney U-tests, where p < 0.05 is considered significant.

Fig. 4. Single-cell transcriptomics of the placental tissues from women with SARS-CoV-2 infection.

a Uniform Manifold Approximation and Projection (UMAP) plot showing the combined cell type classifications of the chorioamniotic membranes (CAM) and placental villi and basal plate (PVBP) from SARS-CoV-2 (+) (n = 9) or control women (n = 10), where each dot represents a single cell. Abbreviations used are CTB cytotrophoblast, EVT extravillous trophoblast, LED lymphoid endothelial decidual cell, npiCTB non-proliferative interstitial cytotrophoblast, STB syncytiotrophoblast. b UMAP plots showing cell populations separated based on placental compartment (CAM and PVBP) from SARS-CoV-2 (+) or control women. c Bar plots showing the numbers of each cell type in the CAM and PVBP of SARS-CoV-2 (+) or control women. d Numbers of differentially expressed genes (DEGs) associated with SARS-CoV-2 infection from the CAM and PVBP with false discovery rate (FDR) adjusted p < 0.1. e Quantile–quantile (Q–Q) plots showing the differential expression of all tested genes in each cell type of maternal or fetal origin from the CAM and PVBP samples. Deviation above the 1:1 line (solid black line) indicates enrichment.

Fig. 5. Single-cell characterization of major cell clusters in the chorioamniotic membranes (CAM) and placental villi and basal plate (PVBP) of women with SARS-CoV-2 infection.

a Scatter plots showing the effects of SARS-CoV-2 on gene expression [log₂ fold change (FC)] in maternal T cells from the CAM and PVBP compared to a previously reported dataset (Meckiff et al., 2020). Black dots represent genes with nominal p < 0.01 in this study, which were used to calculate Spearman’s correlation. b Forest plots showing the log₂(FC) of differentially expressed genes (DEGs) associated with SARS-CoV-2 infection in Stromal-1, T cell, Macrophage-2, decidual, lymphoid endothelial decidual cell (LED), Monocyte, and extravillous trophoblast (EVT-1) cell types in the CAM and PVBP of SARS-CoV-2 (+) (n = 9) or control women (n = 10). DEGs shown are significant after false discovery rate (FDR) adjustment (q < 0.1). c Violin plots showing the distribution of single-cell gene expression levels for the top six DEGs in the maternal T cell, maternal Macrophage-2, and fetal Stromal-1 cell types in the CAM comparing SARS-CoV-2 (+) and control women. d Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched for DEGs in the maternal (M) cell types (decidual, T cells, and Macrophage-2) and fetal (F) Stromal-1 cell type from the CAM based on the over-representation analysis. A one-sided Fisher’s exact test was used. KEGG pathways with q < 0.05 were selected.

Fig. 6. Bulk transcriptomics in the maternal and cord blood of women with SARS-CoV-2 infection and their neonates.

a Maternal blood [n = 10 control, 11 SARS-CoV-2 (+)] and cord blood [n = 8 control, 9 SARS-CoV-2 (+)] were collected for RNA sequencing (RNA-seq). b Scatter plot showing the log₂ fold changes (FC) identified between SARS-CoV-2 (+) and control women that are similar or different between the maternal blood (x-axis) and cord blood (y-axis). A two-sided Spearman’s correlation test was used. Each dot represents a gene tested (in pink DEG for the maternal blood only, in green DEG for the cord blood only, in purple DEG for both compartments and same direction, gray if otherwise not significant). c Volcano plots showing the adjusted p-values (-log₁₀ thereof) on the y-axis and log₂ fold change with SARS-CoV-2 infection on the x-axis in the maternal and cord blood. The number of upregulated and downregulated genes were determined based on fold change > 1.25 and adjusted p value < 0.1. Bar plots showing the top 10 over-represented biological processes from the Gene Ontology (GO) database based on upregulated (top panel; red bars) and downregulated (bottom panel; blue bars) DEGs associated with SARS-CoV-2 infection with adjusted p value < 0.05 in the d maternal blood and e cord blood. NS not significant. The p-values were computed based on one-sided hypergeometric distribution and adjusted by false-discovery rate. f Comparative analyses were performed between bulk transcriptomic data of the maternal blood and cord blood and scRNA-seq data from the placental tissues using DEG from the bulk analysis (q < 0.1). g Scatter plots (pink boxes) showing cell-type-specific Spearman correlations between bulk RNA-seq data from the maternal blood and scRNA-seq data from the chorioamniotic membranes (CAM) for the T cell, Macrophage-2, and Monocyte populations of maternal origin. A two-sided Spearman’s correlation test was used. Scatter plot (green box) showing the cell type-specific Spearman correlation between bulk RNA-seq data from the cord blood and scRNA-seq data from the placental villi and basal plate (PVBP) for the T cell population of fetal origin.

Fig. 7. Immunohistological and molecular detection of SARS-CoV-2 proteins/RNA in placentas of women with SARS-CoV-2 infection.

a Schematic representation showing various sampling locations in the placental villi (PV), basal plate (BP), and chorioamniotic membranes (CAM) of SARS-CoV-2 (+) or control women that were evaluated for SARS-CoV-2 protein detection by immunohistochemistry, followed by RNA viral detection using RT-qPCR [n = 5 control, 10 SARS-CoV-2(+)] (see Supplementary Table 3). b Images showing a positive signal for SARS-CoV-2 spike (left panel) and nucleocapsid (right panel) proteins in the PV, BP, and CAM of spike-in positive control. c Images showing a putative positive signal for SARS-CoV-2 spike (left panel) and nucleocapsid (right panel) proteins in the PV, BP, and CAM of a SARS-CoV-2 (+) woman. Images are representative of two independent experiments. d Three representative images showing a negative signal for SARS-CoV-2 spike (left panel) and nucleocapsid (right panel) proteins in the PV, BP, and CAM of SARS-CoV-2 (+) women (n = 10). e Three representative images showing a negative signal for SARS-CoV-2 spike (left panel) and nucleocapsid (right panel) proteins in the PV, BP, and CAM of control women (n = 5). Brown staining indicates a putative positive signal. All images were taken at 200× magnification. Scale bars represent 100 µm (or 5 µm for digital zoom-in images). f SARS-CoV-2 viral RNA detection by RT-qPCR in the PV, BP, and CAM from histological slides of SARS-CoV-2 (+) (n = 10) and control women (n = 5). N1 (green dot/triangle) and N2 (purple dot/triangle) denote two SARS-CoV-2 nucleocapsid (N) genes, and RP (blue dot/triangle) denotes the human RNase P gene, which serves as a positive internal PCR control. Triangles indicate women with severe COVID-19. Spike-in positive controls were also included. Undetermined quantification cycle (C_q) values are represented below the detection limit (gray area).

Fig. 8. Bacterial DNA profiles of the placental tissues from women with SARS-CoV-2 infection.

a Schematic representation of sampling locations from the chorioamniotic membranes (CAM), amnion–chorion interface of the placenta (AC), and within the placental villous tree (VT) from SARS-CoV-2 (+) women who delivered by cesarean section [n = 3 control, 2 SARS-CoV-2(+)] or vaginally [n = 5 control, 5 SARS-CoV-2(+)]. b Quantitative real-time PCR analyses illustrating the bacterial loads (i.e., 16S rRNA gene abundance) of the CAM, AC, and VT from SARS-CoV-2 (+) or control women (cesarean section or vaginal delivery). The solid line denotes the lowest cycle of quantification (i.e., highest bacterial load) for any blank DNA extraction kit negative control. Data from three human vaginal swabs are included for perspective. c Heatmap illustrating the relative abundances of prominent (>2% average relative abundance) amplicon sequence variants (ASVs) among the 16S rRNA gene profiles of the CAM, AC, and VT from SARS-CoV-2 (+) or control women (cesarean section or vaginal delivery). Data from blank DNA extraction kit negative controls and human vaginal swabs are included for perspective. d Principal coordinates analyses (PCoA) illustrating similarity in the 16S rRNA gene profiles of the CAM, AC, and VT obtained through vaginal delivery from SARS-CoV-2 (+) or control women. Statistical analysis was performed using permutational multivariate analysis of variance (PERMANOVA) through the “adonis” function. Blue dots indicate control women, light red dots indicate SARS-CoV-2 (+) women, and dark red dots indicate women with severe COVID-19.