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Inflammatory responses in the placenta upon SARS-CoV-2 infection late in pregnancy

Summary The effect of SARS-CoV-2 infection on placental function is not well understood. Analysis of placentas from women who tested positive at delivery showed SARS-CoV-2 genomic and subgenomic RNA in 22 out of 52 placentas. Placentas from two mothers with symptomatic COVID-19 whose pregnancies resulted in adverse outcomes for the fetuses contained high levels of viral Alpha variant RNA. The RNA was localized to the trophoblasts that cover the fetal chorionic villi in direct contact with maternal blood. The intervillous spaces and villi were infiltrated with maternal macrophages and T cells. Transcriptome analysis showed an increased expression of chemokines and pathways associated with viral infection and inflammation. Infection of placental cultures with live SARS-CoV-2 and spike protein-pseudotyped lentivirus showed infection of syncytiotrophoblast and, in rare cases, endothelial cells mediated by ACE2 and Neuropilin-1. Viruses with Alpha, Beta, and Delta variant spikes infected the placental cultures at significantly greater levels.


INTRODUCTION
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has already taken a devastating toll with the global virus caseload surpassing 490 million infections and more than 6.1 million deaths worldwide. In severe cases, SARS-CoV-2 causes a respiratory illness, whose defining features are an imbalanced inflammatory host response, reduced innate antiviral defenses and an inflammatory ''cytokine storm,'' endothelial damage, coagulopathies, and thrombosis in several tissues from infected patients (Blanco-Melo et al., 2020).
The effect of SARS-CoV-2 infection during pregnancy on the health of the mother and baby is not fully understood. In the large majority of pregnancies, babies are delivered uninfected when tested by nasopharyngeal swabs and without apparent effect. However, pregnant women with symptomatic SARS-CoV-2 infections are more likely to be admitted to intensive care and the maternal death rates are statistically higher when compared with non-pregnant infected women (Zambrano et al., 2020). Whereas preterm deliveries occur more often in women with suspected or confirmed SARS-CoV-2 infection, no increase in stillbirths or early neonatal deaths was found (Mullins et al., 2021). Prospective and retrospective studies showed that pregnant women infected with SARS-CoV-2 are at an increased risk of adverse events, including higher rates of cesarean section and increased postpartum complications (Marín Gabriel et al., 2020;Prabhu et al., 2020;Woodworth et al., 2020). Vertical transmission from mother to fetus has been reported in a few cases (Alamar et al., 2020;Hecht et al., 2020;Facchetti et al., 2020;Taglauer et al., 2020;Vivanti et al., 2020;Woodworth et al., 2020), although most studies have not detected viral transmission to the fetus (Baergen and Heller, 2020;Cribiù et al., 2021;Della Gatta et al., 2020;Edlow et al., 2020;Kimberlin and Stagno, 2020;Mourad et al., 2021;Penfield et al., 2020;Prabhu et al., 2020;Salvatore et al., 2020;Schwartz, 2020).
The effect of SARS-CoV-2 on placental function is not well understood. Several studies have detected infection of placentas in women who tested positive for the virus at or before delivery. In some cases, the placenta displayed signs of inflammation with increased vascular malperfusion indicative of thrombi within fetal vessels (Baergen and Heller, 2020;Prabhu et al., 2020;Shanes et al., 2020;Vivanti et al., 2020) and infiltration of maternal immune cells (Debelenko et al., 2021;Facchetti et al., 2020;Garrido-Pontnou et al., 2021;Hosier et al., 2020;Lu-Culligan et al., 2021;Morotti et al., 2021). Whether the inflammation resulted from the infection of maternal tissue or the placenta itself is unclear and may depend on the gestational age of the fetus at the time of maternal infection. Several viruses are known to affect placental function and virus-associated inflammation during pregnancy, resulting in chronic cardiovascular disease, diabetes, and obesity later in life (Burton et al., 2016).
SARS-CoV-2 variants that have emerged over the past year are defined by the World Health Organization (WHO) as variants of concern or interest. Four variants of concern have been identified (Alpha (B.1.1.7), Beta (B1.351), Gamma (P.1), and Delta (B.1.617.2)) that contain mutations in the spike protein receptor binding domain and result in increased rates of transmission and lethality (World Health Organization, 2021) (https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/). Whether the variants pose an increased risk of the infection of the placenta and adverse effects on placentation and pregnancies is unknown.
In this study, we analyzed placentas from women who tested positive for SARS-CoV-2 at the time of delivery to determine the extent of infection and impact on the inflammatory state of the placental tissues. Of the 22 analyzed placentas, 2 containing high levels of SARS-CoV-2 RNA were both infected with the Alpha (B.1.1.7) variant. These contained the highest levels of viral RNA and both resulted in adverse outcomes for the fetus. The placentas showed infiltration of maternal immune cells and an active immune response at the maternal-fetal interface. Ex vivo infection of placental cultures showed an infection of syncytiotrophoblast. Viruses pseudotyped by the variants of concern spike protein infected placental cells with a greater efficiency than the parental, non-variant spike protein. These results suggest that a direct infection of placental cells can occur and the variants may have increased propensity to infect this critical tissue during pregnancy.

RESULTS
Clinical presentations of SARS-CoV-2 positive mothers, fetal outcomes, and placental pathologies To understand the effects of SARS-CoV-2 infection during pregnancy, we assembled a panel of placental tissues from 52 women who had been identified as positive for SARS-CoV-2 at the time of admission for delivery at NY Presbyterian Hospital-Weill Cornell (H_1-H_2, P_1-P_20, N1-N30; Table 1). All placental samples were obtained from the Department of Pathology and most of them displayed placental pathologies or were obtained from mothers with clinical presentations. The infection status was determined by qRT-PCR from nasopharyngeal swabs. As controls, placental tissues were obtained from four women who tested negative for SARS-CoV-2 (C_1-C_4), and four SARS-CoV-2 negative women with placental inflammatory pathologies (I_1-I_4) that included acute chorioamnionitis (ACA) and chronic villitis of unknown etiology (CVUE) ( Table 1).
The pregnant women ranged in age from 16 to 51 years, with the majority in their 20s and 30s. Two pregnancies resulted in intra-uterine fetal demise (IUFD) (H_2, P_10). One fetus that was delivered preterm at 25 weeks of gestation was admitted to the neonatal intensive care unit (NICU) where the infant remained for several months (H_1). All neonates tested negative for SARS-CoV-2 by nasopharyngeal swabs at 24-h post-delivery. Among the placentas delivered from mothers who tested positive for SARS-CoV-2, 33% (17 cases) presented with fetal vascular malperfusion (FVM), 23% (12 cases) displayed maternal vascular

SARS-CoV-2 viral RNA presence in the placenta
To determine the extent of SARS-CoV-2 infection in the placenta of infected mothers, we isolated RNA from formalin-fixed paraffin embedded (FFPE) sections from all placental samples and measured genomic and replicating viral RNA by qRT-PCR using primers against SARS-CoV-2-N. The presence of a PCR amplicon of the correct size on melting curves and agarose gels confirmed the positive and negative results (Figure 1A and data not shown). Genomic and subgenomic viral RNA was detected in 22 of the 52 placentas from SARS-CoV-2 positive mothers (42%) ( Table 1). The presence of SARS-CoV-2 RNA in the placenta was not correlated with the observed FVM (10 displayed FVM out of 22 positive placentas; Table 1).
Strikingly, the three pregnancies from SARS-CoV-2 positive mothers that resulted in IUFD or admission of the neonate to the NICU-delivered placentas that were highly positive (H_1 and H_2) or positive (P_10) for SARS-CoV-2 (Table 1, grey shaded rows).
Placental syncytiotrophoblast are the primary target for SARS-CoV-2 infection of pregnant females at term We next investigated placental pathologies and identified the cells in the placental chorionic villi that were infected by SARS-CoV-2. Adjacent placental sample sections (10 microns apart) were stained with hematoxylin and eosin (H&E) and examined for the presence of replicating viral RNA by in situ hybridization and for the presence of SARS-CoV-2 nucleocapsid protein (SARS-CoV-2 N) by immunohistochemistry. Hofbauer cells (HBC) of the placenta and maternal monocytes/macrophages were visualized by immunohistochemistry with an anti-CD163 antibody. Subgenomic viral RNA was readily detected by in situ hybridization in the high positive samples H_1 and H_2 and sporadically in positive samples but not in negative controls ( Figures 1B and S1, and not shown). The presence of viral RNA was restricted to the Keratin-7 (KRT7)-positive trophoblast layers that anatomically cover the chorionic villi structures. These consist of an outer layer of syncytiotrophoblast that faces the maternal blood (MB) space and a cytotrophoblast layer underneath ( Figures 1B and S1). Similarly, the expression of the SARS-CoV-2 N protein was detected in adjacent sections within the same villi. The N protein was localized to the syncytiotrophoblast layers in the high positive placental samples ( Figures 1B and S1).

Immune cell infiltration into intervillous spaces and invasion of chorionic villi
To determine the localization of maternal immune cell populations in SARS-CoV-2-infected placentas, monocytes/macrophages, T cells, and natural killer cells (NK) in the FFPE tissue slides from high positive and negative control samples were stained for CD163, CD3, CD4, and CD56, respectively. Intravilllous HBC and intervillous maternal macrophages were not infected with SARS-CoV-2 as evidenced by the absence of SARS-CoV-2 RNA and N protein in adjacent placental tissue sections ( Figure 1B). In the high positive placental samples H_1 and H_2, immunostaining for CD163 showed massive infiltration of CD163-positive monocytes/macrophages into the intervillous space as well as increased numbers of HBC within the chorionic fetal villi compared with the uninfected negative controls ( Figures 2D, 2H, and 2I). In addition, a significantly higher number of CD163-positive HBC was detected in the chorionic plate (CP) and a slight increase in the number of macrophages in the maternal decidua ( Figure 2I). The increase in infiltrating macrophages and T cells into the fetal regions of the placenta may be an indicator for the observed adverse outcome of pregnancy (Table 1).

Significantly increased levels of
Taken together, our results show that the syncytiotrophoblast is the primary target for SARS-CoV-2 infection of the placenta. In response to infection in the high positive placentas, there was a massive migration of maternal monocyte/macrophages and T cells and a retention of CD56-positive NK cells within the maternal decidua, caused either by SARS-CoV-2 infection of the mother, the placenta, or both.

Placental explants and cell clusters are permissive for pseudotype virus
The susceptibility of placental cells to support SARS-CoV-2 replication is not clear. Virus entry requires cell surface ACE2 on the target cell and TMPRSS2 and cathepsins that serve to process the spike protein (Hoffmann et al., 2020). In addition, NRP1 is thought to play a role as a co-receptor for virus entry in some celltypes (Cantuti-Castelvetri et al., 2020;Daly et al., 2020). Spike protein pseudotyped lentiviruses provide an accurate and sensitive means to determine the susceptibility of cells to SARS-CoV-2 entry (Tada et al., 2020). We used this approach to determine the susceptibility of the placental cells to support SARS-CoV-2 entry. Fresh placental isolates from SARS-CoV-2 negative mothers were obtained shortly following delivery. The fetal CP and maternal decidua were then removed and samples containing terminal, intermediate and stem chorionic villi were used to establish placental villi explant cultures ( Figure 3C). To allow the passage of small cell clusters, additional placental cell clusters were prepared by enzymatic digestion of the chorionic villi followed by filtration ( Figure 3D). The cultures were then infected with a dual green fluorescent protein/nanoluciferase (GFP.Nluc) reporter lentiviral vector pseudotyped by the D614G SARS-CoV-2 spike (S) (Tada et al., 2020). Vesicular stomatitis virus G protein (VSV-G) pseudotyped lentivirus, which infects cells with high infectivity using a ubiquitous cell surface receptor, was used as a control. The results showed that the placental cultures were susceptible to infection by the D614G pseudotype. The infectivity was 2.7-fold lower on average than of the VSV-G pseudotype, indicative of a relatively efficient entry mediated by the spike protein ( Figures 3A, 3B, and 3E). The explant cultures were approximately five-fold less infectable as compared with the clusters and single cells, likely owing to the reduced surface accessibility in the explants. The addition of the HIV reverse transcriptase inhibitor nevirapine (NVP) confirmed that the luciferase activity was primarily owing to virus entry and not carry-over from residual virions ( Figure 3A).  Table S1. Values represent the percentage of DAB positive nuclei out of total number of cells based on nuclear detection by a hematoxylin signal. The grey bar in the boxplots represents the median, and the inner, colored boxes represent the interquartile range (25th and 75th percentiles). Statistical analysis was performed using a two-tailed Mann-Whitney U-test and adjusted for multiple testing with the Benjamini-Hochberg FDR method. *p < 0.05, **p < 0.01. See also Table S1 and Figure S2. iScience Article NRP1 (Arad et al., 2017;Baston-Buest et al., 2011;Cantuti-Castelvetri et al., 2020;Daly et al., 2020). To determine the relative roles of ACE2 and NRP1 in SARS-CoV-2 entry in the placenta, placental cell clusters were treated with anti-ACE2 or anti-NRP1 blocking antibodies before infection with the pseudotyped virus. Both antibodies decreased the infectivity of the D614G spike protein pseudotyped lentivirus (anti-ACE2, 3.5-fold; anti-NRP1, 3.1-fold). The combination of the two antibodies further decreased infectivity by two-fold, resulting in a 7.3-fold reduction compared with untreated controls ( Figure 3B, left panel; Figure S3A). For comparison, we tested the lung cell-line A549-ACE2. Whereas the overall infectability of the cells was much higher than the primary placental cells, the antibodies decreased the infection to a similar extent (anti-ACE2, 2.6-fold; anti-NRP1: 2.9-fold) consistent with an earlier report (Tada et al., 2020), and the combination resulted in no further decrease ( Figure 3B, right panel). The antibodies did not block the infection by the VSV-G pseudotype demonstrating specificity of the antibodies ( Figure 3B, left and right panels; Figure S3A). The results suggest that SARS-CoV-2 viral entry of the placental tissue is dependent on both ACE2 and NRP1, and that infectivity is increased in the presence of both receptors simultaneously.
To determine the cell types targeted for infection, placental explant cultures were infected with the spike protein pseudotypes and then visualized by fluorescence microscopy for the GFP reporter. Live GFP fluorescence could be visualized in the infected explant cultures and was more robust for the VSV-G pseudotype-infected cultures ( Figure 3C). To determine the identity of the infected cells, the explant cultures were processed for immunofluorescence by staining with an antibody against the trophoblast marker KRT7/ Cytokeratin and the endothelial marker CD31. The results showed GFP-positive cells in small patches of the syncytiotrophoblast on the outer perimeter of the chorionic villi but not in endothelial cells ( Figure 3D). The VSV-G pseudotype showed a similar pattern, typically with larger clusters of GFP-positive cells by live cell fluorescence microscopy of the infected explant cultures. No GFP-positive cells were found in mockinfected explant cultures ( Figure 3D).

Increased infection of placental cell clusters by viruses with variant spike proteins
SARS-CoV-2 variants containing mutated spike proteins have been shown to spread with increased transmissibility and replicate at higher virus loads in vivo. This raises the possibility that the variants might pose a greater risk to the pregnant mother and the developing fetus. To test the ability of the variants to infect placental cells, we infected placental cultures with lentiviruses pseudotyped by Alpha (B1.1.7), Beta (B1.351), and Delta (B.1.617.2) variants of concern spike proteins ( Tada et al., 2020Tada et al., , 2021aTada et al., , 2021bTada et al., , 2022. The results showed that the variant pseudotypes infected the placental clusters with increased infectivity relative to the earlier D614G pseudotype. Infection by the Alpha pseudotype was increased 2.7-fold, on average, whereas Beta and Delta were increased 2.2-fold and 2.1-fold, respectively ( Figure 3E, left panel; Figure S3B). The variants did not show the same increase upon infection of A549-ACE2 cells demonstrating that the increases were specific to placental cells and not caused by a general increased infectivity of the variants ( Figure 3E, right panel). To further determine the susceptibility of placental cells to SARS-CoV-2, we infected primary human placental cell clusters ex vivo. Placentas isolated from healthy term deliveries were digested into cell clusters of approximately 50-100 cells, plated on Matrigel-coated plates, and infected with live SARS-CoV-2 (MOI = 1). The cells were collected 24-h post-infection (hpi) and analyzed by qRT-PCR and immunofluorescence staining.
qRT-PCR quantification using primers targeting subgenomic N transcripts showed the presence of SARS-CoV-2 viral RNA in the placental clusters, demonstrating the presence of active virus infection ( Figure 4A).
To determine the infected cell types in the placental cell clusters, we performed immunostaining for the SARS-CoV-2 N protein and confocal imaging. This analysis showed infected KRT7-positive trophoblast cells ( Figures 4B and 4C; Video S1) and, in addition, the presence of a small number of infected CD31 + endothelial cells ( Figure 4C).
To determine the transcriptional changes induced by SARS-CoV-2 infection, we analyzed the transcriptome of ex vivo infected placental cell clusters at 24 hpi by RNA-seq. Alignment to the viral genome detected SARS-CoV-2 viral RNA levels in SARS-CoV-2-infected samples ( Figure 5A). Principal component analysis (PCA) demonstrated that the infected samples occupy a distinct transcriptional space compared with mock-infected control samples ( Figure 5B). Strong similarity among SARS-CoV-2-infected samples with distinct separation from mock-infected samples was observed in the heatmap of sample-to-sample distances ( Figure 5C).
To determine the effects of SARS-CoV-2 infection on the expression of placental cell-type specific markers genes, differential expression analysis was performed on the RNA-seq data. The analysis showed decreased expression of trophoblast cell markers KRT7, GATA3, CDKN1C and endothelial cell markers VWF, CD36, CD44, and increased the expression of the Hofbauer cell marker CD14 ( Figure 5D). In addition, a strong inflammatory response was seen in infected samples with dramatic increases in the expression of chemokine, proinflammatory cytokines ( Figure 5E) and other inflammation-related genes ( Figure 5F), a finding consistent with inflammatory responses to SARS-CoV-2 infection in other tissues Tang et al., 2021;Yang et al., 2020Yang et al., , 2021. In addition, a trend for increased expression of cell death-related genes is observed in SARS-CoV-2-infected placental cell clusters, suggesting an acute apoptotic response to infection ( Figure 5G).
Ingenuity pathway analysis (IPA) of the genes enriched in SARS-CoV-2-infected samples compared with mock-infected controls highlighted canonical biological pathways associated with viral infection and immune response, including the Th1 and Th2 activation pathways, Coronavirus pathogenesis pathway, and IL-10 and IL-6 signaling ( Figure 5H). The pathway with the most prominent alteration in the infected placental cell clusters was EIF2 signaling, a pathway previously identified as the top pathway changed in SARS-CoV-2-infected human pancreatic islets .

SARS-CoV-2 infection has been shown to induce major transcriptional alterations in infected cells.
To determine the host response of the human placenta to SARS-CoV-2 infection, we analyzed the transcriptome of placentas from COVID-19 positive and COVID-19 negative mothers shown in Table 1 and Figure 1A by RNA-seq. Of the COVID-19 positive mothers, two placentas were high positive (high positive samples, H_1, H_2) and 15 were positive (positive samples, P_1-P_15) for SARS-CoV-2 RNA by qRT-PCR ( Figure 1A, Table S2). Of the COVID-19 negative mothers, four were healthy controls (control samples, C_1-C_4) and four were placentas with non-COVID related inflammatory pathologies (inflammatory samples, I_1-I_4) ( Figure 1A, Table S2). Alignment to the viral genome detected SARS-CoV-2 viral RNA levels in the two high positive samples analyzed (11.1%; H_1 and H_2), and both samples were identified as the Alpha (B.1.1.7) variant ( Figure 6A). Furthermore, in both high positive samples, SARS-CoV-2 viral RNA reads iScience Article can be detected across the whole genome ( Figure S4A). PCA demonstrated that the high positive samples H_1 and H_2 occupy a distinct transcriptional space compared with the negative controls (C) and the inflammatory samples (I), as well from the positive samples (P) ( Figure 6B). Clustering of an expression heatmap of sample-to-sample distances demonstrated a high degree of similarity between the two high positive samples (H_1 and H_2) and a broad separation from all other samples, whereas two of the inflammatory samples (I_1 and I-4) and two positive samples (P_5 and P_10) displayed a moderate degree of similarity ( Figure 6C).
To determine the transcriptional changes in the placenta in response to virus infection, differential expression analysis was performed on the four groups based on their category and number of viral reads detected by RNA-seq (Table S2), using three comparisons: (1) high positive versus control samples, to determine the  Expression and cluster analysis of placental cell type specific markers showed an increase in the expression of HBC macrophage marker genes CD68, CD163, CD14 and a decrease in the expression of trophoblast markers KRT7, TFAP2C, HLA-G, PHLDA2 and endothelial specific markers CDH5, VWF, CD36, CD44 in the high positive samples compared with all other samples ( Figure 6D), similar to what was found in ex vivo infected human placenta cell clusters ( Figure 5D). No major differences were observed in expression levels of apoptosis-or necrosis-related genes ( Figure S4B), suggesting that the downregulation of placenta cell specific markers is owing to the loss of cell identity rather than cell death. The loss of cellular identity has also been observed in several SARS-CoV-2-infected tissues (Omer et al., 2021;Tang et al., 2021). High positive placental samples show a strong upregulation of chemokines and cytokines that is more robust than that found in inflammatory control samples ( Figure 6E) and is consistent with lung autopsy samples from COVID-19 patients . Similar to ex vivo infected placental cell clusters, the high positive placental samples also show an increased expression of inflammatory-related genes ( Figures 5F and 6F). Significantly decreased expression of several placental trophoblast and endothelial cell markers (TFAP2C, VWF), increased the expression of Hofbauer cell markers (CD68, CD163), and of chemokine and cytokine genes (CXCL10, CCL20, IL15) and inflammation markers (NURP1, TNFRSF1B) was validated by qRT-PCR of the high positive samples ( Figure S5).
IPA of the genes enriched in the high positive placental samples compared with controls from uninfected mothers highlighted canonical biological pathways associated with viral infection and immune response, including antigen presentation, Th1 and Th2 immune cell activation, and natural killer cell signaling ( Figure 6G).
The top canonical pathways altered in the inflammatory samples as compared with healthy controls included EIF2 signaling and mitochondrial dysfunction ( Figure S4C). Interferon signaling and hypercytokinemia/hyperchemokinemia that are known to play a critical role in the pathogenesis of influenza were the major pathways enriched in the positive samples compared with controls ( Figure S4D). Importantly, the IPA analysis of ex vivo infected placental cell clusters ( Figure 5H) and of the high positive placental samples ( Figure 6G) showed significant changes in Th1 and Th2 signaling pathways as well as pathways involving natural killer cells, consistent with the quantitative analysis of T cells and NK cells by immunohistochemistry ( Figure 2I). These changes were specific to the infected placentas as they were not detected in placentas with other inflammatory pathologies ( Figures 6G and S4C).
To estimate the abundances of inflammatory cell types within the placental samples, we performed cellular deconvolution by CIBERSORTx (Newman et al., 2019) using a cell-type reference specific to term placentas (Pique-Regi et al., 2020). The analysis showed that control and positive samples displayed a similar pattern of inferred relative cell proportions, whereas the inflammatory and high positive samples were more dissimilar ( Figure 6H). Specifically, the high positive samples showed an increase in the relative abundance of T cells, monocytes and macrophages, B cells, and fibroblasts in comparison with control samples. Interestingly, there was also a decrease in the pattern of signatures related to specialized placental cell types such as cytotrophoblasts (CTB), extravillous trophoblast (EVT), endothelial cells, and decidual cells. These data are consistent with the immune cell infiltration we found in the placenta (Figure 2). Furthermore, the data suggest that SARS-CoV-2 infection of placental tissue may have a profound effect on the cellular identity and tissue integrity of the placental tissue. Taken together, our findings identify an inflammatory phenotype in SARS-CoV-2 positive placentas, most pronounced in the high positive samples, that is complemented by a loss of chorionic villi cell identity.

DISCUSSION
The placenta is a vital organ that provides the gestational interface between mother and fetus. Viral infections can result in placental dysfunction, leading to pregnancy complications with increased morbidity and mortality for the mother and fetus (John and Hemberger, 2012; Maltepe et al., 2010;Rossant and Cross, 2001) and can developmentally program the fetus for a chronic disease later in life (Burton et al., 2016). iScience Article Such effects are notable for Zika virus infection where trophoblasts serve as a route of mother-to-fetus infection (Tan et al., 2019).
Although the large majority of infants born to SARS-CoV-2-infected mothers do not become infected, our results show that SARS-CoV-2 RNA can frequently be detected in the placenta. In placental tissues that were highly infected, the virus was primarily localized to the syncytiotrophoblast and the placenta was infiltrated with maternal immune cells with the transcriptional hallmarks of inflammation. Our findings do not directly show the presence of live virus in the placenta although virus is detected at least in some pregnancies. In support of this, the two highly infected placentas showed high levels of viral RNA; placental cells were infected by the pseudotyped and live SARS-CoV-2; placental samples stained for nucleocapsid protein; and RNA-seq showed high copy numbers of viral RNA across the entire virus genome.
Within a cohort of placental samples from mothers who tested positive for SARS-CoV-2 RNA at delivery, 22 of 52 (42%) had detectable genomic and replicating viral RNA, a frequency higher than that found in previous studies (Debelenko et al., 2021;Hecht et al., 2020;Facchetti et al., 2020;Lu-Culligan et al., 2021). The increased frequency may be because most of the placental samples we studied were from deliveries in which the mother was infected late in pregnancy and the mothers or newborns presented with clinical pathologies.
The two placental samples that we identified with high levels of viral RNA were both infected with the SARS-CoV-2 Alpha variant, one of which resulted in fetal demise and the other was a preterm delivery that required extended care in the NICU. Both mothers presented with symptomatic COVID-19, and tested positive for SARS-CoV-2 RNA and antiviral IgM and IgG antibody, indicating a recent infection (Kubiak et al., 2021;Prabhu et al., 2021). In contrast, only one out of 20 pregnancies of SARS-CoV-2 positive mothers with medium virus content in the placenta (P_10) and one out of 30 pregnancies of SARS-CoV-2 positive mothers with no virus detected in the placenta (N15) resulted in IUFD. This may have been triggered by poorly controlled maternal type 2 diabetes (P_10) or by anencephaly of the fetus (N15). Infants born from COVID-19 positive mothers with placentas high positive or positive for the virus tested negative for SARS-CoV-2 by PCR on nasal swabs, demonstrating the protective role of the placenta in preventing vertical transmission (Cribiù et al., 2021). Despite the protection of the newborn from infection, it remains possible that infection of the placenta could have long-term effects on the infant, suggesting the importance of continued monitoring of the child for developmental defects.
In placentas with high virus content, viral RNA and antigen were detected in a large fraction of the syncytiotrophoblast, the outer cell layer covering the fetal chorionic villi situated at the interphase to MB. In those placentas, virus was not detected in the fetal HBC or stromal and endothelial cells within the chorionic villi or outside the villi. Other studies have also provided evidence for SARS-CoV-2 infection in the placental syncytiotrophoblast (Alamar et al., 2020;Facchetti et al., 2020;Hecht et al., 2020;Hosier et al., 2020;Mulvey et al., 2020;Penfield et al., 2020;Taglauer et al., 2020;Vivanti et al., 2020). However, two reports on a preterm placenta and a placenta from a newborn with vertically transmitted SARS-CoV-2 detected SARS-CoV-2 RNA in HBC and stromal cells inside the villi and in maternal macrophages and epithelial cells at the maternal-fetal interface (Facchetti et al., 2020;Verma et al., 2021). In those cases, it is possible that infection occurred at an earlier gestational stage that allowed for viral spread beyond the syncytiotrophoblast layer.
Infection of the placenta was followed by extensive infiltration of maternal immune cells. In placentas with a high virus load, the maternal space and fetal chorionic villi contained large numbers of macrophages, T lymphocytes, increased numbers of CD3-positive and CD4-positive T cells in the fetal CP, as well as higher numbers of CD56-positive NK cells restricted to the maternal decidua. The source of the NK cells was not clear. They could have resulted from the proliferation of uterine NK cells residing at the maternal-fetal interface or infiltration of NK cells from the maternal circulation. No viral RNA or protein was detected in immune cells after careful and rigorous analysis, suggesting immune cells are not a viral source in the placenta. Other studies have reported on infiltration of maternal immune cells into the placenta in the case of live-borne and stillborn neonates that tested positive for SARS-CoV-2 (Facchetti et al., 2020;Garrido-Pontnou et al., 2021;Kirtsman et al., 2020;Vivanti et al., 2020) iScience Article infiltration by inflammatory immune cells, chronic histiocytic intervillositis with trophoblast necrosis, intervillous space collapse, and increased perivillous fibrin deposition (Debelenko et al., 2021;Facchetti et al., 2020;Garrido-Pontnou et al., 2021;Linehan et al., 2021;Lu-Culligan et al., 2021;Marton et al., 2021;Verma et al., 2021). The two heavily infected placentas H_1 and H_2 in our study were both infected with the Alpha variant. These placentas were delivered in the early Spring 2021, whereas the other infected placentas were delivered before December 2020, before the widespread presence of the Alpha variant in the US (Alpert et al., 2021).
Transcriptome analysis of placental tissue and of acutely infected placental cell clusters revealed the increased expression chemokines, cytokines, and inflammatory-related genes. Overall loss of placental cell lineages identities was detected in the high positive samples, similar to what has been reported for several SARS-CoV-2-infected tissues (Omer et al., 2021;Tang et al., 2021). Moreover, a previous study reported an increase in immunopathology gene expression in one severe SARS-CoV-2 positive placenta but not in placental tissue with a low virus load (Cribiù et al., 2021). In our study, placental samples with low virus content displayed altered interferon signaling pathways as detected by IPA; however, they showed only minor differences in their gene expression patterns compared with negative control samples. A recent single-cell RNA-seq study by Lu-Culligan et al. on two virus-negative placentas from women with severe COVID-19 revealed robust immune activation in the placenta, including the upregulation of interferonstimulated genes and of genes indicative of mediating innate-to-adaptive immune cell communication in response to maternal COVID-19 (Lu-Culligan et al., 2021). In contrast, the high virus levels detected in our study of two placentas infected with the Alpha variant, in combination with the observed up-regulation of immune response genes and infiltration of immune cells into maternal and fetal placental compartments support the hypothesis that these are in response to the infection of the mother and the presence of SARS-COV-2 in the placenta.
To determine the ability of placental cells to support SARS-CoV-2 infection, we tested the infectability of ex vivo placental explant and cell cluster culture models both with live SARS-CoV-2 virus and with spike protein pseudotyped lentiviruses. Term placentas express low levels of ACE2 and TMPRSS2 (Ouyang et al., 2021;Pique-Regi et al., 2020;Singh et al., 2020;), as well as the entry cofactor NRP1 (Cantuti-Castelvetri et al., Daly et al., 2020). NRP1 is expressed at the maternal-fetal interface in decidual cells and syncytiotrophoblast where it acts as a co-receptor for vascular endothelial growth factor (VEGF) on endothelial cells and plays an important role both in pregnancy and the immune system (Arad et al., 2017;Baston-Buest et al., 2011). In addition, SARS-CoV-2 infection has been reported to increase (Lu-Culligan et al., 2021) or decrease placental ACE2 expression, causing dysregulation of the renin-angiotensin system (Verma et al., 2021). We found that live and pseudotyped virus targeted the syncytiotrophoblast and, in rare cases, endothelial cells. The infection was partially blocked by anti-ACE2 and anti-NRP1 antibodies, suggesting the role of both proteins in virus entry into placental cells. The antibody treatment did not completely prevent infection of the placental cells suggesting the possibility of a yet unidentified entry cofactor, although we could not rule-out the possibility that the antibodies were not completely efficient at blocking virus entry. Alpha, Beta, and Delta variant pseudotypes infected the placental cell clusters with increased efficiency, raising concern that the variants may present an increased risk to the developing fetus. Such a concern was further supported by our finding that the two placentas that contained high levels of virus were both infected by the Alpha variant and were associated with a strong inflammatory response and adverse outcome of pregnancy.
This study focused on the effect of maternal infection late in pregnancy on the placenta. It will be important, in addition, to study the effect of infections during the earlier stages of pregnancy where the impact on placental health could be greater. Such a possibility is of particular importance with the high prevalence of the Delta variant that replicates with increased virus loads in vivo (Lopez Bernal et al., 2021;Luo et al., 2021) and has an increased ability to infect placental cells. The increased frequency with which variants of concern appear to infect the placenta should be considered by clinicians advising their patients on vaccination during pregnancy. In addition, the increased expression of SARS-CoV-2 entry cofactors in the first and second trimester may increase the susceptibility of the placenta to infection. An infection at these earlier stages could affect placental development and morphogenesis resulting in an increased risk of placental pathologies and clinical outcomes for mother and fetus. Our study highlights the importance of monitoring the health of COVID-19 mothers and their infants in order to identify possible long-term clinical impacts of SARS-CoV-2 infection during pregnancy. iScience Article

Limitations of the study
The present study used a limited cohort of women who tested positive for SARS-CoV-2 at delivery, and all placental samples were obtained from Pathology. It will be important to investigate the impact of SARS-CoV-2 maternal infection during the first and second trimester on placental development and on the health of the newborns. With the exception of two placentas from mothers infected with the Alpha variant, whose pregnancies resulted in adverse outcomes for the fetuses and contained high virus levels, all placentas in this study were from mothers infected with the original SARS-CoV-2. Given that viruses with variant spike proteins infected placental cultures at significantly greater levels, it will be important to study the effect of the recent SARS-CoV-2 variants Delta and Omicron on the placenta and its long-term effects on the health of mothers and their newborns.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: Data and code availability d RNA-seq data of patient placentas and ex vivo placental clusters were deposited in the GEO repository database and will be available as of the date of publication. The accession number is listed in the key resources table.
d Immunohistochemistry (IHC) analysis data were deposited in the zenodo repository database and will be available as of the date of publication. The accession number is listed in the key resources table.
d The source code for the analysis of IHC data will be available as of the date of publication. The link is listed in the key resources table.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Human placental samples
Placental samples from SARS-CoV-2 positive women and controls were obtained at delivery by the Department of Pathology and Laboratory Medicine at Weill Cornell Medicine (WCM)/NY Presbyterian Hospital. Maternal and gestational age of the patients are provided in Table 1.

Placental explant and cell cluster cultures
Fresh, de-identified placentas from SARS-CoV-2-negative mothers were collected within 0.5-2 h post-delivery from Labor & Delivery at WCM/NYP under an approved IRB exempt protocol (#20-07022453, WCM). Tissue samples were dissected by removing the fetal chorionic plate and any remaining maternal decidual tissue. Primary explant cultures and cell clusters were cultured in DMEM/F12 medium supplemented with 10% FBS and 100 U/mL penicillin, 100 mg/mL streptomycin and 0.25 mg/mL amphotericin B .

Placental samples
All placental samples from SARS-CoV-2 positive women and controls were obtained at delivery by the Department of Pathology and Laboratory Medicine at Weill Cornell Medicine. All women admitted for delivery were tested by nasal swabs for acute SARS-CoV-2 infection by qRT-PCR, and serologically for previous infection at Weill Cornell Medicine Department of Pathology and Laboratory Medicine. Infants were tested for SARS-CoV-2 at birth and 1 week of age by nasal swabs and RT-PCR. Placental samples were fixed for 48 h in formalin and then processed and embedded into formalin fixed paraffin embedded (FFPE) blocks by the pathology department. FFPE placental samples from 4 healthy women who tested negative for SARS-CoV-2 as controls and an additional 4 samples with inflammation pathologies obtained from SARS-CoV-2 negative patients were also included in the study. Unstained sections and H&E sections of the FFPE blocks were performed at the Weill Cornell Clinical & Translational Science Center (CTSC) core facility. Additional H&E staining was performed by the Weill Cornell Histology core facility.

SARS-CoV-2 detection in RNA from FFPE placental sections by qRT-PCR
Total RNA samples were prepared from FFPE placental tissue sections, followed by DNaseI treatment using a RNeasy FFPE kit (Qiagen). To quantify for replicating virus, subgenomic viral nucleocapsid RNA copy numbers were determined by qRT-PCR. cDNA was generated by using a RT SuperMix Kit (LunaScriptâ) and SARS-CoV-2 N and cellular glyceraldehyde phosphate dehydrogenase (GAPDH)