Single-nucleus transcriptional profiling of the placenta reveals the syncytiotrophoblast stress response to COVID-19

Abstract

Background: COVID-19 in pregnancy is associated with placental immune activation, inflammation, and vascular malperfusion, but its impact on syncytiotrophoblast biology and function is unclear.

Objective: This study aimed to determine the effects of maternal COVID-19 on placental syncytiotrophoblasts using single-nucleus transcriptional profiling and to compare placental stress responses in COVID-19 and preeclampsia.

Study design: For transcriptional characterization of syncytiotrophoblasts, we used the single-nucleus RNA sequencing platform, single-cell combinatorial indexing RNA sequencing (sci-RNA-seq3), to profile placental villi and fetal membranes from unvaccinated patients with symptomatic COVID-19 at birth (n = 4), gestational age-matched controls (n = 4), and a case of critical COVID-19 in the second trimester with delivery at term (n = 1). Clustering of nuclei and differential gene expression analysis was performed in Seurat. Gene ontology analysis was conducted using Enrichr. High-confidence transcriptional target analysis was used to identify key transcription factor nodes governing the syncytiotrophoblast response to maternal SARS-CoV-2 infection. Bioinformatic approaches were further used to compare the COVID-19 dataset to published preeclampsia gene signatures. Tissue analysis, including immunofluorescence, was conducted to validate the transcriptional data and to compare COVID-19 and preeclampsia placental histology for an expanded cohort of placentas: controls (n = 6), asymptomatic COVID-19 (n = 3), symptomatic COVID-19 (n = 5), and preeclampsia with severe features (n = 7).

Results: The analyzed dataset comprised 15 cell clusters and 47,889 nuclei. We identified 3 clusters of syncytiotrophoblasts representing fusing and mature nuclei with overlapping but distinct transcriptional responses to COVID-19. Bioinformatic analyses indicated that COVID-19 is associated with the following alterations in syncytiotrophoblasts: (1) endoplasmic reticulum stress and activation of stress signaling pathways, including the unfolded protein response and integrated stress response; (2) regulation of gene expression by CCAAT/enhancer-binding protein beta (CEBPB), a master transcription factor of the syncytiotrophoblast lineage; and (3) upregulation of preeclampsia-associated genes. Using complementary methods, we confirmed increased levels of stress response proteins (eg, BiP, G3BP1) in syncytiotrophoblasts, unfolded protein response signaling (spliced XBP1 mRNA), and CEBPB activation (phosphorylation) in COVID-19. Increased cytotrophoblast proliferation (Ki-67) was also detected in COVID-19, consistent with a trophoblast response to injury. Markers of stress detected in preeclampsia demonstrated similarities in the placental stress phenotype of COVID-19 and preeclampsia.

Conclusion: Maternal COVID-19 is associated with syncytiotrophoblast endoplasmic reticulum stress and activation of the syncytiotrophoblast lineage transcription factor, CEBPB. Similarities between syncytiotrophoblast stress in COVID-19 and preeclampsia provide insights into their clinical association.

Keywords: CEBPB; COVID-19; SARS-CoV-2; endoplasmic reticulum stress; placenta; preeclampsia; pregnancy; single-nucleus RNA sequencing; syncytiotrophoblast; transcriptomics; trophoblast; unfolded protein response.