key: cord-0721016-eixymqdy
authors: Colson, Arthur; Depoix, Christophe L.; Dessilly, Géraldine; Baldin, Pamela; Danhaive, Olivier; Hubinont, Corinne; Sonveaux, Pierre; Debiève, Frédéric
title: COVID-19 during Pregnancy: Clinical and In Vitro Evidence against Placenta Infection at Term by SARS-CoV-2
date: 2021-06-08
journal: Am J Pathol
DOI: 10.1016/j.ajpath.2021.05.009
sha: 230ca73b9e17443f0d54401da4c5fedb4aed7e2d
doc_id: 721016
cord_uid: eixymqdy

Despite occasional reports of SARS-CoV-2 vertical transmission during pregnancy, the question of placental infection and its consequences for the newborn remain questionable. Here, we analyzed the placentas of 31 COVID-19-positive mothers by RT-PCR, immunohistochemistry and in situ hybridization. We only detected one case of placental infection, which was associated with intrauterine demise of the fetus. We then isolated and differentiated primary trophoblasts from non-pathological human placentas at term, and exposed them to SARS-CoV-2 virions. Unlike for positive control cells Vero E6, we were not able to detect the virus inside cytotrophoblasts and syncytiotrophoblasts or in the supernatant four days after infection. As a mechanism of defense, we hypothesized that trophoblasts at term do not express ACE2 and TMPRSS, the two main host membrane receptors for SARS-CoV-2 entry. The quantification of these proteins in the placenta during pregnancy confirmed the absence of TMPRSS2 at the surface of the syncytium. Surprisingly, a transiently induced experimental expression of TMPRSS2 did not allow the entry or replication of the virus in differentiated trophoblasts. Altogether, these results underline that trophoblasts are not likely to be infected by SARS-CoV-2 at term, but the reported case raises concern about preterm infection.

Over a year, the COVID-19 pandemic has become a worldwide health and social crisis, deeply affecting human lives and questioning the future of human kind. Our knowledge has exponentially increased, but many questions remain to be answered.

In this context, pregnant women are particularly vulnerable to respiratory infectious diseases because of the remodeling of their immune and cardiovascular systems. 1 Initially, it has been shown that pregnant women infected by SARS-CoV-2 were more prone to require intensive care treatment, and their pregnancies have been associated with increased incidence of miscarriage, preterm birth, preeclampsia, cesarean delivery, and perinatal deaths. 2 The last meta-analyses are less clear, as maternal COVID-19 was not necessarily associated with adverse pregnancy and neonatal complications. [3] [4] [5] [6] Maternofetal transmission of SARS-CoV-2 remains a matter of investigation since very few neonates have shown evidence of contamination following maternal infection. 7 Direct involvement of the placenta is a rare event, and robust virological evidence, whether by RT-PCR, immunohistochemistry (IHC), or in situ hybridization (ISH), is most of the time missing in the reported cases. [8] [9] [10] However, some authors revived the debate by documenting intrauterine transmission and placental infection. [11] [12] [13] As SARS-CoV-2 has been detected in the blood of severely ill patients, circulating viral particles are likely to interact with the maternofetal interface. 14 Especially, the mononuclear villous cytotrophoblasts (CTB) fuse and differentiate into syncytiotrophoblasts (STB), which are in direct contact with maternal blood. 15 SARS-CoV-2 is a β-coronavirus composed of spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins encompassing a single-stranded RNA genome of 29,891 nucleotides. 16 Its infectivity mainly depends on the expression, localization, and structure of the transmembrane proteins angiotensin-converting enzyme 2 (ACE2) and the transmembrane protease serine 2 (TMPRSS2). 17 To infect human cells, SARS-CoV-2 engages ACE2 as the entry receptor and employs TMPRSS2 for S protein priming, allowing the fusion of viral and cellular membranes. ACE2 and TMPRSS2 expression at the surface of cells is therefore considered as a biological indicator of their susceptibility for SARS-CoV-2 infection. ACE2 and TMPRSS2 were detected in the human placenta throughout pregnancy, but their co-expression by syncytiotrophoblasts remains subject to controversy. [18] [19] [20] J o u r n a l P r e -p r o o f [3' ]BHQ-1) was performed with Takyon One-

Step ROX Probe 5X MasterMix dTTP blue (Eurogentec, Seraing, Belgium) and

amplified on a StepOne Real-time PCR System (Applied Biosystems, Thermo Fisher Scientific). In addition to unknown samples, each assay includes one negative control (H 2 O) and five positive controls consisting in in vitro synthesized RNA transcripts (10 7 , 10 6 , 10 5 , 10 4 , and 10 3 copies genome equivalent).

The presence of antibodies in maternal and fetal plasmas was investigated by using Exposure to SARS-CoV-2

The SARS-Cov-2 strains were isolated from patient samples (clade 20E/EU1).

Twenty-four hours or 96 h after isolation, the cells were exposed to three concentrations of viral particles (10 9 , 10 8 , 10 7 copies/mL). Two hours after, the cells were washed three times with PBS, and fresh medium was added. A negative control Fluorescence was examined on an AxioImager microscope combined with ApoTome (Zeiss, Oberkochen, Germany) or on an LSM 800 confocal microscope (Zeiss).

Acquisition parameters and color histograms were kept similar between conditions.

Expression of ACE2 and TMPRSS2 during pregnancy was quantified by IHC in 

Thirty-one pregnant women who delivered after 22 weeks and who were tested positive for SARS-CoV-2 during their pregnancy were included in the study. Maternal and fetal characteristics are described in Table 3 . Gestational age was estimated from the first day of the last menstrual period and confirmed by first-trimester ultrasound examination. Twenty-seven patients were tested positive during their third trimester, mainly the week before childbirth. Of these patients, 18 were asymptomatic, five had mild symptoms (fatigue, anosmia, ageusia), and four had moderate symptoms, including fever and dyspnea. In most cases, the pregnancy ended without complications and the mothers gave birth to healthy neonates.

However, four patients were tested positive before 30 weeks of gestation. The first one has fever associated to dyspnea. She was diagnosed at 26 weeks and 3 days and gave birth prematurely by C-section because of severe preeclampsia. The baby was growth-restricted and was tested positive for SARS-CoV-2 by RT-PCR, but seven days after birth. 29 The second patient was tested at 28 weeks and 6 days. She was hospitalized in the Intensive Care Unit (ICU) for 14 days for severe respiratory distress, but she was able to give birth at term without complication. The third patient was tested positive at 23 weeks and 3 days of gestation because of dyspnea. She Maternal SARS-CoV-2 antibodies IgG are transferred to the fetus For the first twelve patients of our cohort, additional samples were available ( Table   4 ). The virus was only detected in the maternal plasma of the ICU patient during her hospitalization. All patient gave birth to healthy babies at term, and none of these babies was tested positive for SARS-CoV-2 by RT-PCR on a nasopharyngeal swab performed just after delivery. In our cohort, several mothers had SARS-CoV-2 IgM and/or IgG, but their newborns only had IgG, suggesting passive antibody transfer across the placenta.

For all included patients, a piece of placenta and the amniotic membrane were available for the detection of SARS-CoV-2 by RT-PCR ( Table 5) IHC and ISH in lung, kidney, heart, intestinal tract, thyroid, spleen, liver, pancreas, testes, cerebellum or eye tissues (Supplemental Figure S6) .

Trophoblasts are not susceptible to infection by SARS-CoV-2

As placenta at term seemed to constitute a natural barrier against the maternal pathogens, our hypothesis was that the trophoblasts were not susceptible to SARS-CoV-2 infection, regardless their state of differentiation. To test this hypothesis, we first isolated mononuclear CTBs from human placentas at term and differentiated them in vitro. Forty-eight hours after isolation, the cells started expressing (CGB mRNA) (Figure 2A) and secreting a high amount of free β hCG in the supernatant ( Figure 2B ). In addition, plurinuclear cells expressing cytokeratin 7 were visualized,

suggesting an adequate differentiation of the cells (Figure 2C) . 31 To assess their vulnerability to SARS-CoV-2 infection, undifferentiated (CTB -48 h after isolation) and differentiated (STB -96 h after isolation) trophoblasts were exposed to increasing amounts of SARS-CoV-2 virions in the medium. Four days after, we were not able to detect viral antigens or RNA in culture media. On the opposite, Vero E6 cells, known to be vulnerable to SARS-CoV-2, 32 were infected and released a high number of viruses in the supernatant. This finding was further confirmed by detecting the nucleocapsid of the virus in the cytoplasm of Vero E6 but not in primary trophoblasts by IF ( Figure 2D ). As expected, the cytopathic effects (CPE) were obvious in the infected Vero E6 while there was no difference were between the infected trophoblast cultures and controls (Supplemental Figure S7) .

Trophoblasts poorly co-expressed ACE2 and TMPRSS2 at term

As already mentioned, combined ACE2 and TMPRSS2 expression at the cell surface could be considered as a biological indicator of susceptibility to SARS-CoV-2 infection. Compared with lung and kidney tissues, we showed that the placenta at term expressed ACE2 moderately (Supplemental Figure S8A ) but not TMPRSS2 (Supplemental Figure S8B) . Computer-assisted quantification demonstrated that ACE2 expression in placentas from different gestational ages decreased from the second trimester to term (Figure 3, A-D) , while TMPRSS2 expression almost disappeared after 15 weeks of gestation (Figure 3, E-H) . High magnification analysis of the placentas at term showed that ACE2 was expressed at the apical and the J o u r n a l P r e -p r o o f basolateral poles of STBs (Figure 4A) , while TMPRSS2 was only expressed by CTBs ( Figure 4B ). This finding was further confirmed by RT-qPCR and western blot in our primary cell cultures: CTBs expressed TMPRSS2, while the STBs highly expressed ACE2 (Figure 4, C and D) .

Inducing TMPRSS2 expression by syncytiotrophoblasts is not sufficient to allow

Given expressional observations, we envisioned that the syncytium at term was protected from SARS-CoV-2 infection because of the absence of TMPRSS2 at its surface. To confirm this hypothesis, we transfected differentiated STBs with an expression vector of TMPRSS2 48 hours before exposure to SARS-CoV-2 virions.

TMPRSS2 expression was verified by RT-qPCR ( Figure 5A ) and western blot ( Figure 5B) , while its localization was specified by IF ( Figure 5C ) before adding viral particles to the culture medium. Surprisingly, we were not able to detect SARS-CoV-2 in the supernatant by RT-PCR nor in the cells by IF (Figure 5D) , suggesting that transient expression of TMPRSS2 is not sufficient to allow the entry of the virus or its replication in differentiated trophoblasts.

Our data confirmed that the vertical transmission of SARS-CoV-2 is a rare event, which does not usually occur around the term of a pregnancy. Analysis of the clinical samples showed that maternal infection does not easily spread to the fetus, but the consequences can be dramatic. Isolated cases of vertical transmission are regularly published, but their validity must be taken with caution since a pertinent diagnostic classification is rarely used. 33 antibodies developed for immunostaining lack specificity in practice. 36 For instance, the anti-spike antibodies had to be excluded of the study as they showed specificity problems (Supplemental Figures S1 and S2 ). Yet, the anti-nucleocapsid antibody showed a higher specificity (Supplemental Figure S3) . It is therefore difficult to trust findings based on one antibody alone. In this context, ISH appears to be the solution of choice but its implementation is limited because it requires a specific expertise, a substantial budget, and good-quality samples. Finally, a few papers localized viral particles in the syncytium by electron microscopy. 37, 38 Although interesting, these observations have been controversial because of the difficulty in distinguishing viral particles from cellular components. 39, 40 Like immunostaining, electron microscopy should be used in combination with other methods, including the histological diagnosis. Like us, others reported isolated cases of positive placentas identified by IHC and/or ISH. 13, 41 All the infected placentas were characterized by the prominent positivity of STBs for SARS-CoV-2, an unusual chronic histiocytic intervillositis, and syncytial necrosis. 42 Conversely, the uninfected placentas showed significant variability in the spectrum of pathology findings, even if it has been shown that MVM features are preferentially associated with adverse outcomes. 43 As observed in our cohort, direct interactions between SARS-CoV-2 and STBs remain possible in vivo. For this reason, we directly exposed undifferentiated and differentiated trophoblasts to different concentrations of SARS-CoV-2 virions. These concentrations were much higher than what we could find in the blood of the hospitalized patient (< 10 5 copies/mL). This set of data is unprecedented, as primary cells isolated from human placentas at term were cultured with real viral particles for the first time. Only one publication showed that SARS-CoV-2 pseudovirus could eventually enter CTBs, but neither the localization nor the replication of the virus has been studied. 44 By contrast, our experiments clearly demonstrated that term trophoblasts, whatever their state of differentiation, are resistant to SARS-CoV-2

infection.

Others and we have speculated that resistance was conferred by the low coexpression of ACE2 and TMPRSS2 by STBs. 45 We confirmed the findings retrieved from scRNA-seq data, and reliably quantified the expression of ACE2 and TMPRSS2 in the syncytial area over the weeks of gestation. 20, 46 In addition to a very low J o u r n a l P r e -p r o o f TMPRSS2 expression by the syncytium, a polarized pattern of ACE2 with the highest expression on the stromal side of the STBs has been suggested. 47 However, our immunostaining was both localized on the basolateral and the apical sides of the syncytium. Complementarily, we also looked for an abnormal expression of SARS-CoV-2 receptors in the infected placenta. Yet, immunostaining of ACE2 and TMPRSS2 did not appear stronger than in a placenta of similar gestational age (Supplemental Figure S9) .

To assess the essential role of TMPRSS2 for viral entry, we induced its transient expression in fully differentiated trophoblasts, and exposed them to SARS-CoV-2.

Surprisingly, expressing the isoform 1 of TMPRSS2 was not sufficient to allow the infection of the cells. We acknowledge that even if the expression was confirmed by different techniques, it is difficult to prove that the produced protein was fully functional and localized at the membrane. For instance, alternative splicing could explain the inability of the expressed TMPRSS2 to prime the spike protein.

Interestingly, TMPRSS2 transcription is under androgen regulation, and androgen receptor inhibitors were found to be protective against SARS-CoV-2 infection. 48, 49 As progesterone has anti-androgenic effects, the high production of progesterone by the syncytium could also explain the natural protection of the placenta against the virus. 50

It is important to acknowledge that the role of TMPRSS2 has been highlighted by knockdown or overexpression techniques in already infectable cells, including enterocytes or Vero E6 cells. 51, 52 Besides, recent studies tend to prove the existence of other proteases and host factors required to permit SARS-CoV-2 entry. [53] [54] [55] Therefore, it is important to admit that the entry processes by which SARS-CoV-2 infects human cells are far more complex than initially hypothesized, and do not only Immunostaining of TMPRSS2 is visualized in the villi at term at high magnification.

STBs are negative (full arrow) while CTBs are weakly positive (discontinuous arrow). Table 2 

Gene Forward Reverse CGB 5'-GCTACTGCCCCACCATGACC-3' 5'-ATGGACTCGAAGCGCACATC-3' ACE2 5'-CGAGTGGCTAATTTGAAACCAAGAA-3' 5'-ATTGATACGGCTCCGGGACA-3' TMPRSS2 5'-CCTGTGTGCCAAGACGACTG-3' 5'-TTATAGCCCATGTCCCTGCAG-3' SDHA 5'-TGGGAACAAGAGGGCATCTG-3' 5'-CCACCACTGCATCAAATTCATG-3' TBP 5'-GAACATCATGGATCAGAACAACA-3' 5'-ATAGGGATTCCGGGAGTCAT-3'

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Vero E6 cells (nuclei in blue and viral nucleocapsid in red) (bottom left picture)

4408S IF: secondary antibody

Alexa Fluor 555 anti-rabbit ¶ 4413 IF: secondary antibody

Human ACE2 ∥ AMAb91268 IHC: 1/200, citrate buffer pH 5

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need for further explorations of the vertical transmission of SARS-CoV-2 during pregnancy and the systematic screening for the virus in case of neonatal infection.