SARS-CoV2 vertical transmission with adverse effects on the newborn revealed through integrated immunohistochemical, electron microscopy and molecular analyses of Placenta

Abstract

Background . The occurrence of trans-placental transmission of severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) infection remains highly debated. Placental positivity for SARS-CoV-2 has been reported in selected cases, but infection or virus-associated disease of fetal tissues or newborns remains to be demonstrated. Methods We screened for SARS-CoV-2 spike (S) protein expression placentas from 101 women who delivered between February 7 and May 15, 2020, including 15 tested positive for SARS-CoV-2 RNA, 34 tested negative, and 52 not evaluated as they did not meet testing criteria (32), or delivered before COVID-19 pandemic declaration (20). Immunostain for SARS-CoV-2 nucleocapsid (N) was performed in the placentas of all COVID-19 positive women. One placenta resulted positive for the SARS-CoV-2 S and N proteins, which was further studied by RNA-in situ hybridization and RT-PCR for S transcripts, and by electron microscopy. A comprehensive immunohistochemical and immunofluorescence analysis of the placental inflammatory infiltrate completed the investigations. Findings SARS-CoV-2 S and N proteins were strongly expressed in the placenta of a COVID-19 pregnant woman whose newborn tested positive for viral RNA and developed COVID-19 pneumonia soon after birth. SARS-CoV-2 antigens, RNA and/or particles morphologically consistent with coronavirus were identified in villous syncytiotrophoblast, endothelial cells, fibroblasts, in maternal macrophages, and in Hofbauer cells and fetal intravascular mononuclear cells. The placenta intervillous inflammatory infiltrate consisted of neutrophils and monocyte-macrophages expressing activation markers. Absence of villitis was associated with an increase in the number of Hofbauer cells, which expressed PD-L1. Scattered neutrophil extracellular traps (NETs) were identified by immunofluorescence. Interpretation We provide first-time evidence for maternal-fetal transmission of SARS-CoV-2, likely propagated by circulating virus-infected fetal mononuclear cells. Placenta infection was associated with recruitment of maternal inflammatory cells in the intervillous space, without villitis. PD-L1 expression in syncytiotrophoblast and Hofbaeur cells, together with limited production of NETs, may have prevented immune cell-driven placental damage, ensuring sufficient maternal-fetus nutrient exchanges.

Placental Pathology of COVID-19 with and without Fetal and Neonatal Infection: Trophoblast Necrosis and Chronic Histiocytic Intervillositis as Risk Factors for Transplacental Transmission of SARS-CoV-2

“…These criteria include the use of immunohistochemistry and RNA in situ hybridization/RNAscope to identify the SARS-CoV-2 virus in cells on the fetal side of the placenta from maternal/neonatal dyads that test positive for COVID-19. In addition, some authors have also used ultrastructural analysis to identify the virus in fetal-derived placental tissues [24,41,44]. Fortunately, transplacental vertical infection appears to be uncommon.…”

“…In Italy, Fachetti et al screened placentas from 15 pregnant women with COVID-19 using immunohistochemistry for SARS-CoV-2 nucleocapsid antigen to identify infected placentas [24]. One placenta tested positive for the virus and underwent analysis in greater detail.…”

“…These criteria are based upon the molecular identification of the virus on the fetal side of the placenta, such as in cells of the trophoblast or chorionic villous stroma, by demonstrating either viral antigens by immunohistochemistry or through detection of viral nucleic acid using RNA in situ hybridization or RNAscope methods. Based upon these criteria, there have been at least several neonates who appear to have acquired their COVID-19 infection in utero by transplacental transmission of the virus from an infected mother [23][24][25][26].…”

“…Trophoblast necrosis can be seen on the surface of some chorionic villi. This placenta is described in[24].Hematoxylin & eosin, ×20 magnification. Photograph courtesy of Fabio Facchetti, MD, Ph.D. of the University of Brescia, Italy.…”

A model for COVID-19 with isolation, quarantine and testing as control measures

“…In particular, newborns of susceptible individuals shall enter the corresponding susceptible compartment. Regarding descendants of infected mothers, there is still not enough evidence neither to confirm the possibility nor to estimate the probability of trans-placental transmission ( Facchetti et al, 2020 , Fenizia et al, 2020 ). On the other hand, the natural mortality rate shall act on each compartment of system (1) , as well as on in system (2) .…”