In Situ Analyses of Placental Inflammatory Response to SARS-CoV-2 Infection in Cases of Mother-Fetus Vertical Transmission

Abstract

It has been shown that vertical transmission of the SARS-CoV-2 strain is relatively rare, and there is still limited information on the specific impact of maternal SARS-CoV-2 infection on vertical transmission. The current study focuses on a transcriptomics analysis aimed at examining differences in gene expression between placentas from mother-newborn pairs affected by COVID-19 and those from unaffected controls. Additionally, it investigates the in situ expression of molecules involved in placental inflammation. The Papa Giovanni XXIII Hospital in Bergamo, Italy, has recorded three instances of intrauterine transmission of SARS-CoV-2. The first two cases occurred early in the pandemic and involved pregnant women in their third trimester who were diagnosed with SARS-CoV-2. The third case involved an asymptomatic woman in her second trimester with a twin pregnancy, who unfortunately delivered two stillborn fetuses due to the premature rupture of membranes. Transcriptomic analysis revealed significant differences in gene expression between the placentae of COVID-19-affected mother/newborn pairs and two matched controls. The infected and control placentae were matched for gestational age. According to the Benjamani-Hochberg method, 305 genes met the criterion of an adjusted p-value of less than 0.05, and 219 genes met the criterion of less than 0.01. Up-regulated genes involved in cell signaling (e.g., CCL20, C3, MARCO) and immune response (e.g., LILRA3, CXCL10, CD48, CD86, IL1RN, IL-18R1) suggest their potential role in the inflammatory response to SARS-CoV-2. RNAscope^® technology, coupled with image analysis, was utilized to quantify the surface area covered by SARS-CoV-2, ACE2, IL-1β, IL-6, IL-8, IL-10, and TNF-α on both the maternal and fetal sides of the placenta. A non-statistically significant gradient for SARS-CoV-2 was observed, with a higher surface coverage on the fetal side (2.42 ± 3.71%) compared to the maternal side (0.74 ± 1.19%) of the placenta. Although not statistically significant, the surface area covered by ACE2 mRNA was higher on the maternal side (0.02 ± 0.04%) compared to the fetal side (0.01 ± 0.01%) of the placenta. IL-6 and IL-8 were more prevalent on the fetal side (0.03 ± 0.04% and 0.06 ± 0.08%, respectively) compared to the maternal side (0.02 ± 0.01% and 0.02 ± 0.02%, respectively). The mean surface areas of IL-1β and IL-10 were found to be equal on both the fetal (0.04 ± 0.04% and 0.01 ± 0.01%, respectively) and maternal sides of the placenta (0.04 ± 0.05% and 0.01 ± 0.01%, respectively). The mean surface area of TNF-α was found to be equal on both the fetal and maternal sides of the placenta (0.02 ± 0.02% and 0.02 ± 0.02%, respectively). On the maternal side, ACE-2 and all examined interleukins, but not TNF-α, exhibited an inverse mRNA amount compared to SARS-CoV-2. On the fetal side, ACE-2, IL-6 and IL-8 were inversely correlated with SARS-CoV-2 (r = -0.3, r = -0.1 and r = -0.4, respectively), while IL-1β and IL-10 showed positive correlations (r = 0.9, p = 0.005 and r = 0.5, respectively). TNF-α exhibited a positive correlation with SARS-CoV-2 on both maternal (r = 0.4) and fetal sides (r = 0.9) of the placenta. Further research is needed to evaluate the correlation between cell signaling and immune response genes in the placenta and the vertical transmission of SARS-CoV-2. Nonetheless, the current study extends our comprehension of the molecular and immunological factors involved in SARS-CoV-2 placental infection underlying maternal-fetal transmission.

Keywords: COVID-19; SARS-CoV-2 vertical transmission; placenta; pregnancy; twins.

Figure 1

The volcano plot illustrates differential gene expression between SARS-CoV-2-infected placentae and control placentae. Up-regulated genes are shown as green dots, down-regulated genes as blue dots, and SARS-CoV-2-specific genes as red dots. Grey dots represent statistically non-significant genes. Thresholds are set at −log10 adjusted p-values greater than 1.3 (red dashed line) and 2 (yellow line), corresponding to statistical significance levels of 0.05 and 0.01, respectively, with|log2 fold changes|greater than 1 (green dashed line).

Figure 2

Analysis of the top 25 up-regulated genes with the most significant differential expressions (Set 2) between SARS-CoV-2-affected placentae and controls analyzed through the STRING database.

Figure 3

Placental tissue showing SARS-CoV-2 RNA distribution in fetal side (upper third) (A), intermediate zone (middle third) (B) and maternal side (lower third of the placental disk thickness) (C). In all images, the positive signal is confined to the syncytiotrophoblast of the chorionic villi. Scale bar: 500 μm.

Figure 4

(A) Visual representations of “gradients” from the maternal side, through the intermediate zone, to the fetal side of the placenta. The color black indicates a “high” RNA-covered surface for the specific probe, while lighter shades of gray indicate progressively “lower” RNA-covered surfaces. (B) Gradients based on the minimum and maximum RNA-covered surfaces of single probes observed across all four cases.

Figure 5

A heatmap showing the two-dimensional distribution of SARS-CoV-2 RNA, displaying different amounts from the fetal zone (F) of the placenta towards the maternal side (M) in Case 1. The values within the subregions represent the percentage of mRNA-covered surface. SARS-CoV-2 RNA was found to be heterogeneously distributed with a coefficient of variability of 128%.