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

Abstract

Pregnant women appear to be at increased risk for severe outcomes associated with COVID-19, but the pathophysiology underlying this increased morbidity and its potential impact on the developing fetus is not well understood. In this study of pregnant women with and without COVID-19, we assessed viral and immune dynamics at the placenta during maternal SARS-CoV-2 infection. Amongst uninfected women, ACE2 was detected by immunohistochemistry in syncytiotrophoblast cells of the normal placenta during early pregnancy but was rarely seen in healthy placentas at full term. Term placentas from women infected with SARS-CoV-2, however, displayed a significant increase in ACE2 levels. Using immortalized cell lines and primary isolated placental cells, we determined the vulnerability of various placental cell types to direct infection by SARS-CoV-2 in vitro. Yet, despite the susceptibility of placental cells to SARS-CoV-2 infection, viral RNA was detected in the placentas of only a subset (~13%) of women in this cohort. Through single cell transcriptomic analyses, we found that the maternal-fetal interface of SARS-CoV-2-infected women exhibited markers associated with pregnancy complications, such as preeclampsia, and robust immune responses, including increased activation of placental NK and T cells and increased expression of interferon-related genes. Overall, this study suggests that SARS-CoV-2 is associated with immune activation at the maternal-fetal interface even in the absence of detectable local viral invasion. While this likely represents a protective mechanism shielding the placenta from infection, inflammatory changes in the placenta may also contribute to poor pregnancy outcomes and thus warrant further investigation.

Figure 1.

Histopathology of representative COVID-19 (A) and matched control (B) placentas. (A) COVID-19 placenta at low magnification revealed extensive intervillous fibrin deposition, with only occasional areas of open intervillous (I) spaces. (A1) High magnification at edge of blood filled intervillous space (I) and the earliest fibrin deposition (asterisks). Trapped chorionic villi (V) have become avascular and fibrotic. Initial fibrillar fibrin (arrow heads) can be seen at the blood-fibrin interface. (A2) Older area of intervillous fibrin (asterisks) and trapped villi (V) revealing migration of trophoblasts (arrow heads) into the fibrin matrix. (A3) Oldest area of intervillous fibrin became calcified (green asterisks), encasing villous remnants (V). (B) In sharp contrast, the control placenta revealed virtually no fibrin in the intervillous space (I). (B1 and B2) Representative magnified areas revealed normal villi (V) and open, maternal blood containing intervillous space (I), with only occasional foci of fibrin formation (arrow heads). Bars represents 200 μM for images A and B and 50 μM for images A1-B2.

Figure 2.

ACE2 protein expression in the placenta varies with gestational age. (A) Human kidney used as a positive control revealed strong apical staining of the proximal tubules (P). The distal tubules (D) and glomerulus (G) were negative. Inset shows a serial section of the same kidney stained with non-immune rabbit sera resulting in no staining. (B-D) Placentas derived from normal pregnancies between 7 and 15 weeks of gestation demonstrated strong, uniform, apical microvillus syncytiotrophoblast staining (arrow heads), and patchy strong basolateral staining at the cytotrophoblast–syncytiotrophoblast contact zone (arrows). Intervillous space (I) and villous core (V). (E) A normal 21-week placenta still exhibited syncytiotrophoblast surface staining (arrow head), but to a lesser extent than the earlier samples. Cytotrophoblast– syncytiotrophoblast contact zone staining was still prominent (arrow). (F) A representative normal placenta at 39 weeks revealed almost no ACE2 staining. Occasionally, staining at the cytotrophoblast–syncytiotrophoblast contact zone was noted (arrow) (G) Normal extravillous invasive trophoblasts from a 39-week placenta demonstrated strong surface expression of ACE2, with variable cytoplasmic staining. (H) Representative image of ACE2 expression in a 38-week placenta derived from a case of symptomatic maternal COVID-19. Reappearance of strong apical microvillus syncytiotrophoblast (arrow heads) and cytotrophoblast–syncytiotrophoblast contact zone staining (arrows) was observed. All sections were cut at 5 μM, except panel (E), which was cut at 10 μM. Bar represents 50 μM for images A-H. (I) ACE2 H-score demonstrated steady loss of placental ACE2 with increasing gestational age in healthy pregnancies (p<0.001). Linear regression (blue line) was fit to data from healthy controls (circles). 95% confidence interval is shown with dashed lines. Placentas derived from COVID-19 cases are depicted as red squares. (J) ACE2 H-score was significantly increased in term placentas from COVID-19 cases (squares) compared to uninfected, matched controls (circles).

Figure 3.

SARS-CoV-2 infection of placental cells in vitro. (A) Representative images of icSARS-CoV-2-mNG infection of primary placental cells, immortalized placental cell lines, and Vero E6 cells as measured by mNeonGreen expression and immunofluorescence staining of SARS-CoV-2 nucleocapsid (NP). Images are displayed as maximum intensity projections of z-stacks and grayscale bars indicate measured fluorescence intensity in arbitrary digital units. (B) Fold-change quantification of SARS-CoV-2 N1 by RT-qPCR at 24 hours post-infection. Cells were infected at an MOI of 5 for one hour and washed three times with PBS before the addition of fresh media. Cells were washed and collected at 24 hours post-infection. Data presented are representative results from one of three replicates.

Figure 4.

HSPA1A is significantly upregulated in maternal COVID. (A) Hierarchal clustering and heatmap of differentially expressed genes (p<0.05). Bulk RNA-seq was performed on placental villi isolated from control and maternal COVID cases. (B) Gene ontology of differentially expressed genes (p<0.05) in bulk RNA-seq. (C) Volcano plot indicating differentially expressed genes between control and maternal COVID groups from bulk RNA-seq. Significant hits are depicted in red (p_adj<0.05) and black (p<0.05). Non-significant genes are shown in gray.

Figure 5.

(A) UMAP projection of 83378 single placenta cells from COVID-19 cases (n=2 decidual and n=2 villous samples) and uninfected controls (n= 2 decidual and n = 3 villous samples). Cell type annotations based on correlation with reference datasets(–28) followed by manual examination of marker genes. (B) Dotplot of the top 5 genes that are upregulated between COVID-19 and uninfected control samples for each annotated cell type based on fold-difference. Size of dots represents percent of cells in cluster expressing gene of interest; intensity of color reflects average scaled expression. Significantly altered expression between COVID-19 cases and controls (Bonferroni-adjusted, two-tailed, Wilcoxon rank-sum test P < 0.05) is marked by a solid black line. (C) Interferome analysis demonstrating the fraction of differentially expressed genes in each cell type that are interferon-responsive, in COVID-19 cases compared to controls; with p values for enrichment (observed over expected fraction) calculated using hypergeometric distribution. (D) Clustered heatmap showing the top enriched functional terms according to Metascape(76) among differentially expressed genes between COVID-19 and control samples in the annotated placental cell types. bars are colored to encode p-values of increasing statistical significance. (E) Heat map depicting the log-transformed ratio (COVID-19 cases over controls) of number of ligand-receptor interactions between all placental cell type pairs, inferred using the CellphoneDB repository of ligands, receptors and their interactions(33). Red indicates cell type pairs with more interactions in COVID-19 cases compared to control; blue indicates the opposite. (F) Violin plots of HSPA1A expression at the placental villi and maternal decidua obtained by scRNA-seq.