key: cord-0707705-27c2murw
authors: Dubucs, C.; Groussolles, M.; Ousselin, J.; Sartor, A.; Van Acker, N.; Vayssière, C.; Pasquier, C.; Reyre, J.; Batlle, L.; Favarel, S.; Duchanois, D.; Jauffret, V.; Courtade-Saïdi, M.; Aziza, J.
title: Severe placental lesions due to maternal SARS-CoV-2 infection associated to intrauterine fetal death
date: 2022-01-05
journal: Hum Pathol
DOI: 10.1016/j.humpath.2021.12.012
sha: 6b9f39dc506ddf6a8b39d67f777140ab422131db
doc_id: 707705
cord_uid: 27c2murw

SARS-CoV-2 infection can cause severe placental lesions leading rapidly to intra uterine fetal death (IUFD). From August 2020 to September 2021, in the pathology Department of Toulouse Oncopole, we analyzed 50 placentas from COVID-19 positive unvaccinated mothers. The purpose of our study is to describe the clinicopathological characteristics of these placental damages and to understand the pathophysiology. Ten of them (20%) showed placental lesions with positive immunohistochemistry for SARS-CoV-2 in villous trophoblast. In five cases (10%), we observed massive placental damage associating trophoblastic necrosis, fibrinous deposits, intervillositis as well as extensive hemorrhagic changes due to SARS-CoV-2 infection probably responsible of IUFD by functional placental insufficiency. In five other cases, we found similar placental lesions but with a focal distribution that did not lead to IUFD but live birth. These lesions are independent of maternal clinical severity of COVID-19 infection since they occur despite mild maternal symptoms and are therefore difficult to predict. In our cases, they occurred 1 to 3 weeks after positive SARS-CoV-2 maternal RT-PCR testing and were observed in 2nd and 3rd trimesters of pregnancies. When these lesions are focal, they do not lead to IUFD and can be involved in intra-uterine growth restriction. Our findings, together with recent observations, suggest that future pregnancy guidance should include stricter pandemic precautions such as screening for a wider array of COVID-19 symptoms, enhanced ultrasound monitoring as well as newborn medical surveillance.

Vertical transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and pregnancy complications induction are serious concerns for pregnant individuals with COVID-19. Based on early RNA detection of SARS-CoV-2 after birth, vertical transmission's incidence of SARS-CoV-2 has been estimated to occur in around 2 to 3% of neonates [1, 2] . The short and long-term consequences of this infection on the newborn are still poorly understood. However, even if a neonate tests negative for SARS-CoV-2, different abnormal placental findings have been reported in cases of COVID-19-positive mothers [3] . Since the beginning of the pandemic, many nonspecific placental lesions have been described, including fetal and maternal vascular malperfusion, or chorioamnionitis, all negative in immunohistochemistry with the anti SARS-CoV-2 antibody [4] . On the other hand, lesions characterized by trophoblastic necrosis, fibrinous deposits, and intervillositis are more suggestive of viral involvement and can lead to IUFD [5] .

The purpose of our study is to describe the clinicopathological characteristics of placental damages associated with SARS-CoV-2 maternal infection in 50 placentas from COVID-19 positive unvaccinated mothers and to understand the pathophysiology. Histopathological findings are heterogeneous. Among positive SARS-CoV-2 placentas, specific histological findings are extremely variable in intensity from diffuse placental hemorrhagic lesions to focal perivillous fibrin deposition lesions possibly sequellar. In 10% of cases lesions are diffuse and associated to an increase of IUFD in the 2 nd and 3rd trimester of pregnancy.

From August 2020 to September 2021, in the pathology Department of Toulouse Oncopole, (attached to the biggest maternity unit in Occitania (south of France) with more than 5000 births a year), we analyzed fifty placentas from COVID-19 positive unvaccinated mothers. Forty of them were normal or non-specific at histological examination, while 10 of them showed particular abnormal histological signs reported here.

Automated classical immunohistochemical (IHC) stains were performed using the Benchmark ULTRA (Roche, Ventana Medical Systems, Innovation Park Drive Tucson, Arizona 85755 USA) on FFPE tissue sections (3µm). After dewaxing, tissue slides were heat pre-treated using a CC1 (pH8) buffer (05424569001, Roche) for 64 minutes at 98°C. The slides were blocked for endogenous peroxidase activity and incubated with primary anti-2019-nCOV Spike rabbit polyclonal (A20022, ABclonal, Diagomics, Blagnac, France) (1/100 in Envision FLEX antibody diluent, K800621-2, Agilent, Santa Clara, CA 95051, USA) and anti-SARS-CoV-2 nucleocapsid mouse monoclonal (Ready to Use, clone BSB-134, BioSB, Diagomics, Blagnac, France) antibodies for 64 and 92 minutes respectively. Targets were then visualized using the OptiView DAB detection kit (06396500001, Roche). The tissue slides were counterstained using hematoxylin (05277965001, Roche) for 8 minutes followed by post-coloration using Bluing reagent for 4 minutes at room temperature (05266769001, Roche). The slides were then dehydrated (ethanol and xylene) and mounted using xylene-based mounting (TissueTek Prisma®, Sakura Finetek Europe B.V., The Netherlands).

SARS-CoV-2 genomic RNA was detected on specimen taken by nasopharyngal swabs using Hologic Aptima SARs-CoV-2 transcription mediated amplification on Panther instrument as recommended by manufacturer. For SARS-CoV-2 genomic RNA detection on placenta, samples were crushed and nucleic acids extracted using Magnapure 96 small volume protocol (Roche Diagnostics). The targets were localized on the RNA-dependent polymerase gene (IP2 and IP4, Institut Pasteur, Paris, France). Amplification and real-time detection were performed using qScript XLT 1-step RT-qPCR ToughMix (Quantabio, VWR International SAS, France) on Light Cycler 480 (Roche Diagnostics).

In our study, 40 of the 50 placentas analyzed showed histological findings either normal (n=16/40, 40%) or with various nonspecific lesions as previously described in the literature [4, 6, 7] such as fetal vascular malperfusion (n=1/40) and maternal vascular malperfusion (n=17/40, 42,5%), chorioamnionitis (n=8/40, 20%) or focal intervillositis (n=1/40) ( Table 1) according to the Amsterdam Placental Consensus classification [8] . All these cases were negative with anti SARS-CoV-2 antibody and RT-PCR in the placenta. Thirty-eight of them were associated with live births; the two other cases were late miscarriages, one associated with a monochorial biamniotic twin pregnancy with transfused transfusion syndrome and one in a context of septate uterus with history of multiple miscarriages.

Among the 10 cases with significant placental lesions, five were associated with IUFD while the other five were associated with live birth.

Five out of the ten patients (Cases n°1-5) were hospitalized in our department for IUFD discovered between 19 WoG (Weeks of Gestation) and 36+6 WoG. None of them had a medical history. The pregnancy was marked by maternal COVID-19 infection between 18+4 WoG and 34 WoG, therefore between 1 to 3 weeks before IUFD occurred. Maternal symptoms were unspecific and mild, mainly hyperthermia (detailed in Table 1 ). Interestingly, three of them were tested for SARS-CoV-2 by nasopharyngeal swabs not because of symptoms suggestive of COVID but because they were considered contact cases. All five patients presented in our emergency unit for decreased fetal movements. Ultrasound exam discovered IUFD with a variable estimated fetal weight from <3 rd percentile to 50 th percentile. The standard IUFD exams (CGH array, CMV and Parvovirus B19 research in amniotic fluid, as well as maternal autoimmune tests excluding other causes of IUFD) were negative in the five cases. Placental bacteriology came back negative as well. All five placentas weighed within 10 th -25 th percentile for reference ranges. Cross-placental examination revealed unusual diffuse macroscopic lesions, characterized by several intraplacental and subchorial hematoma, dissociating the placenta tissue (Figure 1 , A3). These lesions involved at least 80% of the parenchyma.

Histologically, lesions were characterized by massive hemorrhagic inundation of intervillous space and extensive villous trophoblastic necrosis ( Figure 1 , B1, D1-2, E). Surrounding the villi, chronic intervillositis and fibrin deposits were present (Figure 1 , B2, E2). Polymorphonuclears were observed, due to hemorrhagic lesions. At high magnification, villous trophoblast showed signs of cellular injury including karyorrhexis, fragmentation of trophoblast nuclei and clearing of cytoplasm (Figure 1, D3) . Intravillous inflammation was not detected nor vascular thrombosis in villous capillaries. Associated chronic intervillous inflammatory infiltrate was characterized by abundant presence of T lymphocytes (CD3 +, with no predominance of neither CD4+ nor CD8+ T cell subsets), and CD68+ monocytic cells. Few mononuclear cells circulating in maternal vascular space also showed strong positivity with anti-SARS-CoV-2 nucleocapsid (N) antibody. Among these five cases, two fetuses were autopsied and none had visceral malformation nor visceral (lung, kidney, heart) positive SARS-CoV-2 immunostaining.

The five other cases (cases n°6-10) showed similar lesions associating trophoblastic necrosis, fibrinoid deposits and intervillositis but focally located with less than 30% of the parenchyma involved ( Figure 2 , A1). Immunostaining with anti-SARS-CoV-2 nucleocapsid (N) antibody showed positive cytoplasmic staining only located in pathological villous trophoblast ( Figure  2 , A2, A3). Likewise, the lympho-histiocytic infiltrate demonstrated by anti-CD3 and CD68 antibodies was limited to the lesion areas.

One case (n°10) was particularly interesting since placental histological examination showed pathological foci characterized by perivillous fibrin deposition surrounding necrotic villi. These lesions were less inflammatory with more fibrin deposits, in the process of organization. Immunohistochemical anti-SARS-CoV-2 staining was not diffusely detected in trophoblast as observed in other cases, but was only very focally present (Figure 2, B1, B2, B3) . Clinically, the time from maternal infection to childbirth was more than 2 months.

J o u r n a l P r e -p r o o f

In cases of maternal SARS-CoV-2 infection, most of the placental histopathological lesions largely reported were non-specific such as fetal vascular malperfusion, maternal vascular malperfusion, chorangiosis, chorioamnionitis and villositis [4, 6, 7] . Indeed 40 of the 50 placentas analyzed in our study (80%) showed histological findings either normal or with various nonspecific lesions as previously described in the literature and all these cases were negative with anti SARS-CoV-2 antibody and RT-PCR.

However, 10 out of the 50 placentas analyzed showed specific placental lesions directly linked to SARS-CoV-2 infection, since all were positive with anti SARS-CoV-2 antibody and SARS-CoV-2 RT-PCR. Depending on their intensity, these lesions had variable consequences on pregnancy outcome ranging from intra-uterine growth restriction (IUGR) to IUFD by functional placental insufficiency.

In five cases (cases n°1-5), placental damages were diffuse, involving at least 80% of parenchyma. Lesions were characterized by unusual massive hemorrhagic inundation of intervillous space and extensive villous trophoblastic necrosis associated with chronic intervillositis and fibrin deposits. Placental lesions observed in our cases, were in agreement with the few IUFD cases described in literature but were additionally characterized by multiple intraplacental hemorrhagic lesions associated with extensive trophoblastic necrosis and chronic intervillositis [5, [9] [10] [11] . The trophoblastic necrosis showed disappearance of trophoblastic cytoplasmic membranes, enlarged nuclei often vesicular with diffuse fine chromatin or fragmentation of trophoblast nuclei possibly suggestive of viral toxicity of SARS-CoV-2 infection (Figure 1, D3) . This was supported by immunohistochemical studies showing positivity of villous trophoblast with SARS-CoV-2 antibodies. Otherwise, decidua arteriopathy was not seen but rather hemorrhagic changes dissociating the decidua, which is in agreement with literature [12] . Unlike other authors, we did not find fetal vascular thrombi [13] .

The fact that these lesions were unusually intense and largely diffuse, both inflammatory and hemorrhagic, seems sufficient to explain functional placental insufficiency and its involvement in the fetal death due to placental destruction. Especially since the IUFD assessment was negative.

On the clinical level, these lesions were observed between the 7 th day and the 20 th day after maternal infection occurred which is in agreement with observation in the literature [12] .

It is interesting to point out that hemorrhagic changes were especially marked in the diffuse placental damage associated to poor fetal outcome. These hematomas because of their intensity are unusual and cannot be explained by caesarian section. Interestingly, Flores et al., analyzed the impact of COVID-19 infection on placental endothelium, by analyzing expression of Von Willebrand Factor (vWf), claudin-5 and vascular endothelial cadherin (VEC) in placentas of women with severe COVID-19 [14] . Their results showed an increase in the expression of vWf, suggesting the existence of a vascular thrombotic condition, and a decreased expression of both Claudine-5 and VEC. Thus, enhanced vessel permeability is possibly responsible for a possible breach in the maternal-fetal interface leading to hemorrhages in the decidua and the intervillous space. In the setting of adults SARS-CoV-2 infections, hemorrhagic lesions affecting other different organs have also been described such as hemorrhagic colitis [15] , hemorrhagic pericardial effusion with tamponade [16] , and hemorrhagic encephalopathy related to blood-brain barrier breakdown [17, 18] . Some authors have suggested that these lesions are the result of a cytokine storm syndrome responsible for widespread microvascular injury [19] .

In five other cases (cases n°6-10), lesions were focal or plurifocal mainly associated with IUGR and involved less than 30% of the parenchyma. On microscopic examination, several focal lesions were observed, still characterized by trophoblastic necrosis, chronic histiocytic intervillositis and perivillous fibrin deposition, contrasting with non-lesional areas. Hemorrhagic changes were absent or rare. Immunohistochemistry with anti SARS-CoV-2 antibody was in agreement with the histological aspect and showed diffuse trophoblastic positivity in the lesion foci, whereas no positivity was noted elsewhere.

Interestingly, in case 10, the time from maternal infection to childbirth was more than 2 months, which is much longer than the 3 weeks observed in IUFD cases. This could suggest that lesions induced by SARS-CoV-2 infection evolve over time with only focal or even negative immunostaining for SARS-CoV-2, making the histological diagnosis of SARS-CoV-2 infection difficult.

Among placentas and neonates tested for SARS-CoV-2 infection, a minority of neonates (2 to 3%) and placental samples (around 20%) tested positive for SARS-CoV-2 infection from infected mother. This is what we have as well in our study since 10 of the 50 placentas tested positive for SARS-CoV-2 (20%) [20] .

On reminder, SARS-CoV-2 infects tissues via its receptor Angiotensin-converting enzyme 2 (ACE2), and its entry into cells requires spike protein cleavage by the serine protease TMPRSS2. ACE2 is highly expressed in maternal-fetal interface cells, such as syncytiotrophoblasts and cytotrophoblasts from 7 weeks onwards and across gestation with similar intensity and distribution of ACE2 staining [21, 22] . It is believed that high cellular coexpression of ACE2 (allowing virus attachment in cells) and TMPRSS2 (allowing virus penetration in cells) is required for viral transmission [23] . However, this rare coexpression in placenta could explain the high heterogeneity of placental lesions and thus variable and low occurrence of SARS-CoV-2 transplacental infection [14] , as we expose it in case n°10 were lesions were highly heterogeneous (Figure 1, D) . Schwartz et al., summarized the spectrum of pathology findings from pregnant women with COVID-19 based upon the infection status of their infants. Authors concluded that cooccurrence of chronic histiocytic intervillositis and trophoblastic necrosis appears to be a risk factor for placental infection with SARS-CoV-2 as well as for maternal-fetal viral transmission, and suggests a potential mechanism by which the coronavirus can breach the maternal-fetal interface [5] . However, the intensity and acute onset of the placental lesions could explain the rare positive fetal cases described to date, since the death occurs rapidly by placental functional insufficiency before a possible fetal viral diffusion.

In our study, placental lesions did not appear to be correlated with maternal infection severity. Indeed, third trimester's pregnant women placentas received in our laboratory showed subnormal placental examination while they presented severe symptoms with respiratory distress requiring emergency fetal extraction. On the other hand, second trimester's or third trimester's maternal infections have been reported several times with IUFD despite mild maternal COVID-19 symptoms [13, [24] [25] [26] [27] . This makes clinical monitoring difficult and raises the question of ultrasound monitoring. Retrospectively, we did not find any ultrasound finding in the five cases with IUFD and suggest a possible rapid and fulminant onset of placental damage within a period of 1 to 3 weeks.

SARS-CoV-2 infection can cause severe placental lesions associating chronic inflammation with trophoblastic necrosis and massive hemorrhage leading rapidly to placental destruction and intra uterine fetal death as observed in our laboratory. These lesions are independent of maternal symptoms since they occur despite mild maternal symptoms and are therefore difficult to predict. In our cases, they occurred 1 to 3 weeks after positive SARS-CoV-2 maternal RT-PCR testing and were observed in 2nd and 3rd trimesters of pregnancy. IUFD associated to SARS-CoV-2 placental lesions are not so rare since in our study they occurred in 5 out of 50 cases (10%). These lesions can also appear focally in the placenta and lead to livebirth with sometimes IUGR. The discovery of such lesions in the placenta should initiate a medical monitoring of the newborns for which the long-term effects are not known to date. Moreover, focal SARS-CoV-2 lesions seem to evolve over time with more fibrin deposits, becoming difficult to diagnose since SARS-CoV-2 immunostaining could become negative. This study highlights the need for systematic placental and fetal gross and microscopic evaluation, which can help elucidate the pathophysiology of COVID-19. case n°3 E Microscopic aspects comparable to cases 1 and 2 (H&E). F IHC with anti-SARS-CoV-2 nucleocapsid antibody is highly positive in necrosis trophoblast. Intervillous inflammatory infiltrate G1 with both T lymphocytes (IHC with CD3 antibody) and G2 monocytic cells (IHC with CD68 antibody).

Case n°9 A1 (H&E) multifocal lesions occupying less than 30% of the placental parenchyma. A2 and A3 IHC with anti-SARS-CoV-2 nucleocapsid antibody is highly positive. Some areas show strong diffuse staining of the trophoblast (facing necrosis trophoblast) contrasting with negative non-affected villi (facing preserved trophoblast).

B1 (H&E) massive perivillous fibrin deposition with villous infarctions and without significant inflammatory infiltrate B2 and B3 IHC with anti-SARS-CoV-2 nucleocapsid antibody. Note residual and focal immunostaining on this more fibrinous lesion.

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-SARS-CoV-2 placental lesions can be involved in intrauterine fetal death (10% of cases) by functional placental insufficiency when placental damage is massive associating trophoblastic necrosis, perivillous fibrin deposition, chronic intervillositis as well as extensive hemorrhagic changes.-Intrauterine fetal death occurred 1 to 3 weeks after positive SARS-CoV-2 maternal RT-PCR testing and are observed in 2nd and 3rd trimesters of pregnancies.-10% of cases show focal lesions, that over time become less inflammatory, richer in fibrin deposits with only focal or even negative immunostaining for SARS-CoV-2, making the histological diagnosis of SARS-CoV-2 infection difficult.-Severe SARS-CoV-2 placenta lesions occur despite mild maternal COVID-19 symptoms.