Extracellular Vesicles Alter Trophoblast Function in Pregnancies Complicated by COVID-19

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and resulting coronavirus disease (COVID-19) cause placental dysfunction, which increases the risk of adverse pregnancy outcomes. While abnormal placental pathology resulting from COVID-19 is common, direct infection of the placenta is rare. This suggests that pathophysiology associated with maternal COVID-19, rather than direct placental infection, is responsible for placental dysfunction. We hypothesized that maternal circulating extracellular vesicles (EVs), altered by COVID-19 during pregnancy, contribute to placental dysfunction. To examine this hypothesis, we characterized circulating EVs from pregnancies complicated by COVID-19 and tested their effects on trophoblast cell physiology in vitro. Trophoblast exposure to EVs isolated from patients with an active infection (AI), but not controls, altered key trophoblast functions including hormone production and invasion. Thus, circulating EVs from participants with an AI, both symptomatic and asymptomatic cases, can disrupt vital trophoblast functions. EV cargo differed between participants with COVID-19, depending on the gestational timing of infection, and Controls, which may contribute to the disruption of the placental transcriptome and morphology. Our findings show that COVID-19 can have effects throughout pregnancy on circulating EVs, and circulating EVs are likely to participate in placental dysfunction induced by COVID-19.

Keywords: COVID‐19; extracellular vesicle cargo; extracellular vesicle origin; placental dysfunction; pregnancy.

FIGURE 1

Differential gene expression in the placentas of patients with COVID‐19 during pregnancy is represented by volcano plots. (A–D) The number and direction of differentially expressed genes between Controls (n = 5) and resolved infections in the first trimester (R1) (A), second trimester (R2) (B) and third trimester (R3) (C), and active infection (AI) (D) is listed at the top of each graph. The gene name for those transcripts with differential expression in more than one COVID‐19 group compared to Control is listed. (n = 3–5/group).

FIGURE 2

Extracellular vesicles isolated from the plasma by serial centrifugation. (A) Representative transmission electron microscope images demonstrated EV structure including a lipid membrane. (B) Nanotracking analysis was used to determine the size of particles in three Control samples. (C) CD9 expression evaluated by immune blotting was abundant in isolated particles in three Control samples.

FIGURE 3

Circulating EVs were persistently altered in participants who experienced COVID‐19 in the second trimester. (A) The number of LEVs in the circulation at the time of delivery (n = 14–22/group). (B) The diameter of LEVs in the circulation (n = 14–22/group). (C) Relative frequency of LEVs derived from trophoblasts, endothelial cells, platelets and immune cells (n = 11–16). (D) The number of small EVs in circulation at the time of delivery (n = 14–22/group). (E) The diameter of small EVs in the circulation (n = 14–22/group). (F) The relative frequency of small EVs derived from endothelial cells, platelets, immune cells and trophoblasts (n = 13–20/group). All data are presented as mean ± SD. All analyses were performed by one‐way ANOVA or the Kruskal–Wallis test, followed by post hoc tests. Comparisons were made between Controls and resolved infection in the first trimester (R1), second trimester (R2), third trimester (R3) and active infection (AI).

FIGURE 4

Trophoblast function was disrupted by exposure to EVs isolated from patients with an active infection (AI) compared to Controls. (A) Extravillous trophoblasts (EVTs) were isolated from three placentas (circle (gestational age 6 weeks 0 day), triangle (gestational age 8 weeks 1 day) and square (gestational age 10 weeks 6 days)) and exposed to Control or AI EVs (n = 9–10/group, three experiments). Invasion was calculated and normalized to the invasion of EVTs derived from the same placenta not exposed to EVs (noEVs). All data are presented as mean ± SD. (B) Human chorionic gonadotropin (hCG) and progesterone were measured in the media of forskolin‐treated (syncytialized) BeWo cells. The ratio of hCG to progesterone was normalized to hormone production by cells not exposed to EVs (noEVs). The results of two experiments (identified by symbol shape) are reported in (B). (n = 6–7/group, two experiments). All analyses were performed by one‐way ANOVA or Kruskal–Wallis test, followed by post hoc tests. (C) Following EV exposure, BeWo cell transcriptome was measured, and the top differentially expressed genes comparing the cellular response to AI EVs to Control EVs are listed in the heat map (red represents increased and blue represents decreased expression). (D) The biological processes altered by AI EVs compared to Control EVs were determined by Gene Ontology enrichment analysis.

FIGURE 5

Large EV abundance of mtDNA is inversely correlated with gestational timing of infection. The amount of mtDNA in each large EV (A) and small EV (C) is reported for each group (n = 13–20/group). Data are presented as mean ± SD and tested by ANOVA followed by post hoc tests. Independent pairwise comparisons were made between Controls and resolved infection in the 1st trimester (R1), second trimester (R2), third trimester (R3) or active infection (AI). The Pearson correlation between gestational age at infection and mtDNA content is reported for large EV (B) and small EV (D).

FIGURE 6

Control EVs carried transcripts that were absent in COVID‐19 groups. (A, B) mRNA transcripts uniquely detected in EVs isolated from Controls but absent in EVs isolated from COVID‐19 groups (grey bars) and differential expression (log 2‐fold change) in other COVID‐19 groups are listed in the heat map (increased expression is red and decreased expression is blue). The general cellular function of each gene product is listed on the right. Large EV transcripts are reported in (A) and small EV transcripts are reported in (B) (n = 9–10/group). Independent pairwise comparisons were made between Controls and resolved infection in the first trimester (R1), second trimester (R2), third trimester (R3) or active infection (AI). Transcripts known to be highly abundant in trophoblasts are marked by (T).