SARS-CoV-2 niches in human placenta revealed by spatial transcriptomics

Abstract

Background: Functional placental niches are presumed to spatially separate maternal-fetal antigens and restrict the vertical transmission of pathogens. We hypothesized a high-resolution map of placental transcription could provide direct evidence for niche microenvironments with unique functions and transcription profiles.

Methods: We utilized Visium Spatial Transcriptomics paired with H&E staining to generate 17,927 spatial transcriptomes. By integrating these spatial transcriptomes with 273,944 placental single-cell and single-nuclei transcriptomes, we generated an atlas composed of at least 22 subpopulations in the maternal decidua, fetal chorionic villi, and chorioamniotic membranes.

Findings: Comparisons of placentae from uninfected healthy controls (n = 4) with COVID-19 asymptomatic (n = 4) and symptomatic (n = 5) infected participants demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in syncytiotrophoblasts occurred in both the presence and the absence of maternal clinical disease. With spatial transcriptomics, we found that the limit of detection for SARS-CoV-2 was 1/7,000 cells, and placental niches without detectable viral transcripts were unperturbed. In contrast, niches with high SARS-CoV-2 transcript levels were associated with significant upregulation in pro-inflammatory cytokines and interferon-stimulated genes, altered metallopeptidase signaling (TIMP1), with coordinated shifts in macrophage polarization, histiocytic intervillositis, and perivillous fibrin deposition. Fetal sex differences in gene expression responses to SARS-CoV-2 were limited, with confirmed mapping limited to the maternal decidua in males.

Conclusions: High-resolution placental transcriptomics with spatial resolution revealed dynamic responses to SARS-CoV-2 in coordinate microenvironments in the absence and presence of clinically evident disease.

Funding: This work was supported by the NIH (R01HD091731 and T32-HD098069), NSF (2208903), the Burroughs Welcome Fund and the March of Dimes Preterm Birth Research Initiatives, and a Career Development Award from the American Society of Gene and Cell Therapy.

Keywords: COVID-19; Translation to patients; maternal-fetal; microenvironment; perinatal; visium.

Figure 1.. Detection of SARS-CoV-2 in placentae by histology and bulk RT-qPCR.

(A-C) Fresh-frozen in optimal cutting temperature serum (FF-OCT) tissue blocks from spatial transcriptomics samples were cryosectioned and subject to (A) RNA in situ probing for SARS-CoV-2 Spike (S), (B) immunohistochemistry (IHC) for S, and (C) IHC staining for SARS-CoV-2 Nucleocapsid (N). Images were taken at 20x magnification and each row represents images obtained from an individual participant. (D) FF-OCT and formalin-fixed and paraffin-embedded (FFPE) blocks were subject to reverse transcription and quantitative polymerase chain reactions (RT-qPCR) probing for S, N, or ORF1ab SARS-CoV-2 transcripts. Based on these results, placentae were grouped for analysis into negative controls (NC), maternal positive but SARS-CoV-2 was not detected in the placenta (ND), sparse positive (SP) if SARS-CoV-2 was detected by RT-qPCR where ct values <27 were observed (limit of detection = 1/7,000 cells) or ≥1 SARS-CoV-2 transcripts per spot were observed spatial transcriptomics (limit of detection = 1/661 cells), and high positive (HP) where RT-qPCR ct values <15 and ≥2–1,554 SARS-CoV-2 transcripts per spot were observed. Abbreviations: CV= chorionic villi, IVS= intervillous space, and SYT= syncytiotrophoblast.

Figure 2.. A term single-cell and spatial transcriptomics atlas predicts cell-type niches with or without SARS-CoV-2.

(A) With 273,944 placenta single-cell and single-nuclei transcriptomes, a term placenta single-cell transcriptomics atlas was generated and used to (B) predict the cell-type profiles of the spatial transcriptomics niches. (C) Dimension reduction Unique Manifold Approximation and Projection (UMAP) of the 17,927 spatial transcriptomics labeled by analysis cohort or niche annotation. (D) Spatial locations of each transcriptomics niche for each sample. Abbreviations: DC=dendritic cell, EVT= extravillous trophoblast, NK= natural killer cell, PVC= perivillous cell, RBC= red blood cell, SYT= syncytiotrophoblast, VCT= villous cytotrophoblast, and VEC= vascular endothelial cell

Figure 3.. Transcriptomic niches of sparse or high SARS-CoV-2 levels in placentae.

(A) H&E stain of sparsely positive SARS-CoV-2 placentae with the H&E, spatial transcriptome overlay, and zooming in on areas of SARS-CoV-2 transcript detection. The pie charts reveal how many spatial transcriptome spots were positive for SARS-CoV-2, and the annotation of those spots. The intervillous space (IVS) and chorionic villi (CV) are labeled. Each row represents images from a separate participant. The niches in the pie charts refer to the spatial transcriptome annotation. (B) H&E stain of highly positive SARS-CoV-2 placenta samples annotating areas with perivillous fibrinoid (PVF) deposition or SARS-CoV-2 transcripts. Each row represents images in separate sections from participant HP13.

Figure 4.. Unique spatial transcription markers in placentae depending on SARS-CoV-2 detection levels.

(A) Differential expression between spatial transcriptomes in each analysis cohort relative to the negative controls identified 54 significantly differentially expressed transcripts (q<0.05, Log₂(fold-change)>2) unique or shared between analysis cohorts. (B) The 37 transcripts unique to highly positive SARS-CoV-2 placentae were subject to EnrichR pathway analysis with the Reactome 2022 database, revealing the top 90^th-quartile of significant (q<0.05) pathways. (C-D) Violin plots with the expression levels of KISS1 and TIMP1, which were markers for placentae where SARS-CoV-2 was not detected or highly positive, respectively. (E-F) Spatial gene expression of KISS1 and TIMP1 representative of each analysis cohort.

Figure 5.. Analysis of infected spatial transcriptomes identifies distinct phases of inefficient and coordinated SARS-CoV-2 gene expression.

(A) The 752 spatial transcriptomes with detectable SARS-CoV-2 transcripts were subset, clustered, and further analyzed. For continuity, the trajectories are plotted on A and D, where the white circle represents the starting point, black circles represent branchpoints, and grey represents endpoints. (B) Expression of viral transcripts revealed patterns in distinct clusters. (C) The mean counts of viral RNAs per cluster revealed viral RNA levels were highest in clusters 1>4>2>0>3. (D) Pseudotime trajectory analysis starting at cluster 3, which had the fewest viral transcripts, identified distinct endpoints at clusters 2, 0, and 1. (E) Pearson’s correlation analysis of each cluster revealed significant correlations in viral RNAs only in cluster 1

Figure 6.. Tracing placental macrophage polarization trajectories identifies depletion of anti-inflammatory M2 macrophages and histiocytic intervillositis in highly positive SARS-CoV-2 placentae.

(A) Schematic of macrophage polarization from naïve monocytes (M0) to pro-inflammatory M1, and anti-inflammatory M2. (B) Canonical markers for each subpopulation. ^#Note: caveats exist including potential differences by gestational age and between single-cell RNA and protein levels. (C) The 3,180 placental macrophages were analyzed by Monocle3, revealing pseudotime trajectories starting at M0 monocytes and trajectories going to M1 or M2 polarized subpopulations. (D) Using the pseudotime trajectory results, subpopulations were annotated based on predicted polarization states including intermediates (e.g. M0 to M1 is M0.M1).(E) Proportions of macrophages according to predicted polarization states. (F-G) IHC staining for CD163, a classical macrophage marker. Images were taken at 40x magnification. (G) Proportions of all spatial transcriptomes (see Figure 2 and Figure S2c) separated based on virus detection grouping from Figure 2 and the cluster analysis of SARS-CoV-2 positive transcriptomes in Figure 5. Significance of P<0.05 (**P<0.001, ***P<0.0001, ns= P>0.05) was determined by two-way ANOVA with Tukey’s multiple comparisons test. Error bars represent the standard error of the mean

Figure 7.. Caution in assigning fetal sex differences in gene expression associated with placental SARS-CoV-2 without spatial resolution.

(A) The 208 significantly (q<0.05) differentially expressed genes in the SARS-CoV-2 scRNA-seq (147,906 transcriptomes; n=15 female and 30 male and spatial transcriptomics (9,446 transcriptomes; n=4 male and 5 female) data based on fetal sex were uploaded to EnrichR for BioPlanet pathway analysis. The top 90^th-quartile of most significant (q<0.05) pathways were plotted. (B) The 208 genes with sex differences in expression from the SARS-CoV-2 scRNA-seq and spatial transcriptomics datasets were compared and plotted as a Venn diagram, revealing 4 male and 1 female cross-validated genes. (C-D) Spatial gene expression of cross-validated genes (C) TIMP1 and (D) PRG2 were upregulated in the villous space in females and maternal decidua regions of male SARS-CoV-2 placentae, respectively.