Prioritization of potential causative genes for schizophrenia in placenta.

Our earlier work has shown that genomic risk for schizophrenia converges with early life complications in affecting risk for the disorder and sex-biased neurodevelopmental trajectories. Here, we identify specific genes and potential mechanisms that, in placenta, may mediate such outcomes. We performed TWAS in healthy term placentae (N = 147) to derive candidate placental causal genes that we confirmed with SMR; to search for placenta and schizophrenia-specific associations, we performed an analogous analysis in fetal brain (N = 166) and additional placenta TWAS for other disorders/traits. The analyses in the whole sample and stratifying by sex ultimately highlight 139 placenta and schizophrenia-specific risk genes, many being sex-biased; the candidate molecular mechanisms converge on the nutrient-sensing capabilities of placenta and trophoblast invasiveness. These genes also implicate the Coronavirus-pathogenesis pathway and showed increased expression in placentae from a small sample of SARS-CoV-2-positive pregnancies. Investigating placental risk genes for schizophrenia and candidate mechanisms may lead to opportunities for prevention that would not be suggested by study of the brain alone.

Dissecting Rubella Placental Infection in an In Vitro Trophoblast Model.

Vertical transmission of rubella virus (RuV) occurs at a high rate during the first trimester of pregnancy. The modes of vertical transmission including the response of trophoblasts to RuV are not well understood. Here, RuV-trophoblast interaction was studied in the BeWo trophoblast cell line. Analysis included early and late time-point kinetics of virus infection rate and the antiviral innate immune response at mRNA and protein level. BeWo characteristics were addressed through metabolic activity by extracellular flux analysis and syncytiotrophoblast formation through incubation with forskolin. We found that RuV infection of BeWo led to profuse type III interferon (IFN) production. Transfecting trophoblast cells with dsRNA analog induced an increase in the production of type I IFN-ß and type III IFNs; however, this did not occur in RuV-infected BeWo trophoblasts. IFN-ß and to a lesser extent type III IFN-λ1 were inhibitory to RuV. While no significant metabolic alteration was detected, RuV infection reduced the cell number in the monolayer culture in comparison to the mock control and resulted in detached and floating cells. Syncytia formation restricted RuV infection. The use of BeWo as a relevant cell culture model for infection of trophoblasts highlights cytopathogenicity in the absence of a type I IFN response as a pathogenic alteration by RuV.

Activation of the Interferon Pathway in Trophoblast Cells Productively Infected with SARS-CoV-2.

SARS-CoV-2 infection during pregnancy has been associated with poor maternal and neonatal outcomes and placental defects. The placenta, which acts as a physical and immunological barrier at the maternal-fetal interface, is not established until the end of the first trimester. Therefore, localized viral infection of the trophoblast compartment early in gestation could trigger an inflammatory response resulting in altered placental function and consequent suboptimal conditions for fetal growth and development. In this study, we investigated the effect of SARS-CoV-2 infection in early gestation placentae using placenta-derived human trophoblast stem cells (TSCs), a novel in vitro model, and their extravillous trophoblast (EVT) and syncytiotrophoblast (STB) derivatives. SARS-CoV-2 was able to productively replicate in TSC-derived STB and EVT, but not undifferentiated TSCs, which is consistent with the expression of SARS-CoV-2 entry host factors, ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane cellular serine protease) in these cells. In addition, both TSC-derived EVT and STB infected with SARS-CoV-2 elicited an interferon-mediated innate immune response. Combined, these results suggest that placenta-derived TSCs are a robust in vitro model to investigate the effect of SARS-CoV-2 infection in the trophoblast compartment of the early placenta and that SARS-CoV-2 infection in early gestation activates the innate immune response and inflammation pathways. Therefore, placental development could be adversely affected by early SARS-CoV-2 infection by directly infecting the developing differentiated trophoblast compartment, posing a higher risk for poor pregnancy outcomes.

Human early syncytiotrophoblasts are highly susceptible to SARS-CoV-2 infection.

Direct in vivo investigation of human placenta trophoblast's susceptibility to SARS-CoV-2 is challenging. Here we report that human trophoblast stem cells (hTSCs) and their derivatives are susceptible to SARS-CoV-2 infection, which reveals heterogeneity in hTSC cultures. Early syncytiotrophoblasts (eSTBs) generated from hTSCs have enriched transcriptomic features of peri-implantation trophoblasts, express high levels of angiotensin-converting enzyme 2 (ACE2), and are productively infected by SARS-CoV-2 and its Delta and Omicron variants to produce virions. Antiviral drugs suppress SARS-CoV-2 replication in eSTBs and antagonize the virus-induced blockage of STB maturation. Although less susceptible to SARS-CoV-2 infection, trophoblast organoids originating from hTSCs show detectable viral replication reminiscent of the uncommon placental infection. These findings implicate possible risk of COVID-19 infection in peri-implantation embryos, which may go unnoticed. Stem cell-derived human trophoblasts such as eSTBs can potentially provide unlimited amounts of normal and genome-edited cells and facilitate coronavirus research and antiviral discovery.

COVID-19 and preeclampsia: The unique and the mutually nonexclusive clinical manifestations.

Preeclampsia (PE) is a serious, unpredictable hypertensive disorder of pregnancy present in around 8-10% of all pregnancies resulting in high rate of maternal and fetal morbidity and mortality. With the pathophysiology partially known, delivery is the only cure for PE. The disease sets due to multiple pathologic processes involving endothelial cell activation, inflammation, multiorgan damage and syncytiotrophoblast stress.  Though the primary target organ is lungs in COVID-19, other systemic manifestations which include endothelial dysfunction, dysregulated angiogenesis, thrombosis, liver injury, thrombocytopenia, hypertension and kidney damage overlap with PE. COVID-19 patients show a higher incidence of PE as compared to their noninfected counterparts and vice versa. Similar pathophysiology and clinical features make differential diagnosis challenging. For effective and specific management, it is important to differentiate actual PE from COVID-19 with PE like features. There are contradictory reports about the accuracy of diagnostic tools in distinguishing PE from severe COVID-19 with PE like features. With the available data, it can only be stated that PE is a common adverse pregnancy event, which may be exacerbated by, or may exacerbate, COVID-19. Future research should focus on cohesive understanding of the pathophysiology of the clinical manifestations, and preventive strategies during pregnancy.

Coronavirus disease 2019 and the placenta: A literature review.

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has been implicated in the clinical pathology of multiple organs and organ systems. Due to the novelty of the disease, there is a need to review emerging literature to understand the profile of SARS-CoV-2 in the placenta. This review sought to evaluate the literature on the mediators, mechanism of entry, pathogenesis, detection, and pathology of SARS-CoV-2 in the placenta. Systematic literature searches found 96 eligible studies. Our review revealed that SARS-CoV-2 canonical mediators, angiotensin-converting enzyme-2 (ACE2), and transmembrane serine protease-2 (TMPRSS2) are variably expressed in various placenta compartments, including the villous cytotrophoblasts, syncytiotrophoblasts (STBs), and extravillous trophoblasts (EVTs) throughout pregnancy. Placental SARS-CoV-2 and coronavirus-associated receptors and factors (SCARFs), including basigin (BSG/CD147), dipeptidyl peptidase-4 (DPP4/CD26), cathepsin B/L (CTL B/L), furin, interferon-induced transmembrane protein (IFITM1-3), and lymphocyte antigen 6E (LY6E) may increase or reduce the permissiveness of the placenta to SARS-CoV-2. EVTs express genes that code for proteins that may drive viral pathogenesis in the placenta. Viral RNA, proteins, and particles were detected primarily in the STBs by in situ hybridization, immunohistochemistry, electron microscopy, and polymerase chain reaction. Placental pathology in SARS-CoV-2-infected placentas included maternal and fetal vascular malperfusion and a generally nonspecific inflammatory-immune response. The localization of SARS-CoV-2 receptors, proteases, and genes involved in coding proteins that drive viral pathogenesis in the placenta predisposes the placenta to SARS-CoV-2 infection variably in all pregnancy trimesters, with antecedent placental pathology. There is a need for further studies to explicate the mechanism of entry and pathogenesis of SARS-CoV-2 in the placenta.

Gestational SARS-CoV-2 infection is associated with placental expression of immune and trophoblast genes.

INTRODUCTION: Maternal SARS-CoV-2 infection during pregnancy is associated with adverse pregnancy outcomes and can have effects on the placenta, even in the absence of severe disease or vertical transmission to the fetus. This study aimed to evaluate histopathologic and molecular effects in the placenta after SARS-CoV-2 infection during pregnancy. METHODS: We performed a study of 45 pregnant participants from the Generation C prospective cohort study at the Mount Sinai Health System in New York City. We compared histologic features and the expression of 48 immune and trophoblast genes in placentas delivered from 15 SARS-CoV-2 IgG antibody positive and 30 IgG SARS-CoV-2 antibody negative mothers. Statistical analyses were performed using Fisher's exact tests, Spearman correlations and linear regression models. RESULTS: The median gestational age at the time of SARS-CoV-2 IgG serology test was 35 weeks. Two of the IgG positive participants also had a positive RT-PCR nasal swab at delivery. 82.2% of the infants were delivered at term (≥37 weeks), and gestational age at delivery did not differ between the SARS-CoV-2 antibody positive and negative groups. No significant differences were detected between the groups in placental histopathology features. Differential expression analyses revealed decreased expression of two trophoblast genes (PSG3 and CGB3) and increased expression of three immune genes (CXCL10, TLR3 and DDX58) in placentas delivered from SARS-CoV-2 IgG positive participants. DISCUSSION: SARS-CoV-2 infection during pregnancy is associated with gene expression changes of immune and trophoblast genes in the placenta at birth which could potentially contribute to long-term health effects in the offspring.

Functional coupling between TRPV4 channel and TMEM16F modulates human trophoblast fusion.

TMEM16F, a Ca2+-activated phospholipid scramblase (CaPLSase), is critical for placental trophoblast syncytialization, HIV infection, and SARS-CoV2-mediated syncytialization, however, how TMEM16F is activated during cell fusion is unclear. Here, using trophoblasts as a model for cell fusion, we demonstrate that Ca2+ influx through the Ca2+ permeable transient receptor potential vanilloid channel TRPV4 is critical for TMEM16F activation and plays a role in subsequent human trophoblast fusion. GSK1016790A, a TRPV4 specific agonist, robustly activates TMEM16F in trophoblasts. We also show that TRPV4 and TMEM16F are functionally coupled within Ca2+ microdomains in a human trophoblast cell line using patch-clamp electrophysiology. Pharmacological inhibition or gene silencing of TRPV4 hinders TMEM16F activation and subsequent trophoblast syncytialization. Our study uncovers the functional expression of TRPV4 and one of the physiological activation mechanisms of TMEM16F in human trophoblasts, thus providing us with novel strategies to regulate CaPLSase activity as a critical checkpoint of physiologically and disease-relevant cell fusion events.

Stem-cell-derived trophoblast organoids model human placental development and susceptibility to emerging pathogens.

Trophoblast organoids derived from placental villi provide a 3D model system of human placental development, but access to first-trimester tissues is limited. Here, we report that trophoblast stem cells isolated from naive human pluripotent stem cells (hPSCs) can efficiently self-organize into 3D stem-cell-derived trophoblast organoids (SC-TOs) with a villous architecture similar to primary trophoblast organoids. Single-cell transcriptome analysis reveals the presence of distinct cytotrophoblast and syncytiotrophoblast clusters and a small cluster of extravillous trophoblasts, which closely correspond to trophoblast identities in the post-implantation embryo. These organoid cultures display clonal X chromosome inactivation patterns previously described in the human placenta. We further demonstrate that SC-TOs exhibit selective vulnerability to emerging pathogens (SARS-CoV-2 and Zika virus), which correlates with expression levels of their respective entry factors. The generation of trophoblast organoids from naive hPSCs provides an accessible 3D model system of the developing placenta and its susceptibility to emerging pathogens.

Bizarre Chorionic-type Trophoblast in Second-trimester and Third-trimester Placentas: Clinicopathologic Characterization of a Placental Pseudoneoplastic Lesion.

Bizarre (atypical/symplastic) cells have been described in various gynecologic normal tissues and benign neoplasms. This type of bizarre cytologic change is usually an incidental finding and is regarded as a benign process. We describe 17 cases of bizarre chorionic-type trophoblast in second-trimester and third-trimester placentas that created concern for an underlying/undersampled or incipient intraplacental trophoblastic neoplasm, predominantly found in intervillous trophoblastic islands (11/17), placental septae (6/17), chorionic plate (1/17), and/or the chorion layer of fetal membranes (2/17). The bizarre trophoblastic cells exhibited sheet-like or nested architecture, had a multifocal/patchy distribution, and/or were present as individual cells within hyaline stroma; they were characterized by large nuclei with smudgy chromatin and occasional intranuclear pseudoinclusions. The degree of atypia was classified as mild (0/17), moderate (3/17), or severe (14/17). Mitotic figures and necrosis were not identified. A dual immunohistochemical stain for trophoblast (hydroxyl-delta-5-steroid dehydrogenase) and a proliferation marker (Ki-67), performed in 15 cases, demonstrated 0% to very low proliferative activity within the bizarre trophoblast (0% to 2% [10/15], 3% to 8% [5/15]). Immunohistochemical stains for fumarate hydratase showed intact/retained expression in the bizarre cells in 7 of 7 cases. Clinical follow-up ranged from 1 to 45 months, and all patients were alive and well without subsequent evidence of a gestational trophoblastic or other neoplasms. We conclude that bizarre chorionic-type trophoblast in second-trimester or third-trimester placentas have the potential to mimic an intraplacental trophoblastic neoplasm but are likely a benign degenerative change. This study expands the spectrum of bizarre cells that occur in the gynecologic tract.

Intrauterine Fetal Demise After Uncomplicated COVID-19: What Can We Learn from the Case?

BACKGROUND: SARS-CoV-2 infection in pregnant women can lead to placental damage and transplacental infection transfer, and intrauterine fetal demise is an unpredictable event. CASE STUDY: A 32-year-old patient in her 38th week of pregnancy reported loss of fetal movements. She overcame mild COVID-19 with positive PCR test 22 days before. A histology of the placenta showed deposition of intervillous fibrinoid, lympho-histiocytic infiltration, scant neutrophils, clumping of villi, and extant infarctions. Immunohistochemistry identified focal SARS-CoV-2 nucleocapsid and spike protein in the syncytiotrophoblast and isolated in situ hybridization of the virus' RNA. Low ACE2 and TMPRSS2 contrasted with strong basigin/CD147 and PDL-1 positivity in the trophoblast. An autopsy of the fetus showed no morphological abnormalities except for lung interstitial infiltrate, with prevalent CD8-positive T-lymphocytes and B-lymphocytes. Immunohistochemistry and in situ hybridization proved the presence of countless dispersed SARS-CoV-2-infected epithelial and endothelial cells in the lung tissue. The potential virus-receptor protein ACE2, TMPRSS2, and CD147 expression was too low to be detected. CONCLUSION: Over three weeks' persistence of trophoblast viral infection lead to extensive intervillous fibrinoid depositions and placental infarctions. High CD147 expression might serve as the dominant receptor for the virus, and PDL-1 could limit maternal immunity in placental tissue virus clearance. The presented case indicates that the SARS-CoV-2 infection-induced changes in the placenta lead to ischemia and consecutive demise of the fetus. The infection of the fetus was without significant impact on its death. This rare complication of pregnancy can appear independently to the severity of COVID-19's clinical course in the pregnant mother.

Severe Acute Respiratory Syndrome Coronavirus 2 ACE2 and TMPRSS2 Receptor Protein Expression Patterns Throughout Gestation.

We previously demonstrated that the late gestation placental expression pattern of ACE2 (the primary severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] receptor) is localized to the villous syncytiotrophoblast (ST), usually in a polarized membranous pattern at the ST base sparing the apical surface (that directly exposed to maternal blood). We found that the late gestation placental expression pattern of TMPRSS2 (the spike proteinase required for SARS-CoV-2 cellular infection), is usually absent in the trophoblast but is rarely, weakly expressed in the placental endothelium. We now show the developmental protein expression patterns of ACE2 and TMPRSS2 by immunohistochemistry throughout gestation, from the first through third trimester. We found that TMPRSS2 expression was rarely detectable in villous endothelium and very rarely detectable in the ST across gestation. We found that ACE2 expression varied during gestation with circumferential ST expression more common in early gestations and polarized expression more common in later gestation. Although this study is small, these preliminary results suggest that earlier gestation pregnancies may be more vulnerable to infection than later gestation pregnancies.

Is SARS-CoV-2 Infection a Risk Factor for Early Pregnancy Loss? ACE2 and TMPRSS2 Coexpression and Persistent Replicative Infection in Primitive Trophoblast.

BACKGROUND: SARS-CoV-2 infection in term placenta is rare. However, growing evidence suggests that susceptibility of the human placenta to infection may vary by gestational age and pathogen. For several viral infections, susceptibility appears to be greatest during early gestation. Peri-implantation placental infections that result in pre-clinical pregnancy loss would typically go undetected. Little is known about the effects of SARS-CoV-2 on the peri-implantation human placenta since this time in pregnancy can only be modeled in vitro. METHODS: We used a human embryonic stem cell (hESC)-derived model of peri-implantation placental development to assess patterns of ACE2 and TMPRSS2 transcription and protein expression in primitive trophoblast. We then infected the same trophoblast cell model with a clinical isolate of SARS-CoV-2 and documented infection dynamics. RESULTS: ACE2 and TMPRSS2 were transcribed and translated in hESC-derived trophoblast, with preferential expression in syncytialized cells. These same cells supported replicative and persistent infection by SARS-CoV-2, while non-syncytialized trophoblast cells in the same cultures did not. CONCLUSIONS: Co-expression of ACE2 and TMPRSS2 in hESC-derived trophoblast and the robust and replicative infection limited to syncytiotrophoblast equivalents support the hypothesis that increased viral susceptibility may be a defining characteristic of primitive trophoblast.

Placental SARS-CoV-2 distribution correlates with level of tissue oxygenation in COVID-19-associated necrotizing histiocytic intervillositis/perivillous fibrin deposition.

INTRODUCTION: Recent evidence supports the - rare - occurrence of vertical transplacental SARS-CoV-2 transmission. We previously determined that placental expression of angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor, and associated viral cell entry regulators is upregulated by hypoxia. In the present study, we utilized a clinically relevant model of SARS-CoV-2-associated chronic histiocytic intervillositis/massive perivillous fibrin deposition (CHIV/MPFVD) to test the hypothesis that placental hypoxia may facilitate placental SARS-CoV-2 infection. METHODS: We performed a comparative immunohistochemical and/or RNAscope in-situ hybridization analysis of carbonic anhydrase IX (CAIX, hypoxia marker), ACE2 and SARS-CoV-2 expression in free-floating versus fibrin-encased chorionic villi in a 20-weeks' gestation placenta with SARS-CoV-2-associated CHIV/MPVFD. RESULTS: The levels of CAIX and ACE2 immunoreactivity were significantly higher in trophoblastic cells of fibrin-encased villi than in those of free-floating villi, consistent with hypoxia-induced ACE2 upregulation. SARS-CoV-2 showed a similar preferential localization to trophoblastic cells of fibrin-encased villi. DISCUSSION: The localization of SARS-CoV-2 to hypoxic, fibrin-encased villi in this placenta with CHIV/MPVFD suggests placental infection and, therefore, transplacental SARS-CoV-2 transmission may be promoted by hypoxic conditions, mediated by ACE2 and similar hypoxia-sensitive viral cell entry mechanisms. Understanding of a causative link between placental hypoxia and SARS-CoV-2 transmittability may potentially lead to the development of alternative strategies for prevention of intrauterine COVID-19 transmission.

Morphometric Parameters of Placental Villi in Parturient Women with COVID-19.

We performed a comparative morphological analysis of placental villi in parturient women with mild and moderate COVID-19 infection. The area and perimeter of terminal villi, their capillaries, and syncytiotrophoblast were assessed on immunohistochemical preparations with antibodies to CD31 using an image analysis system; the parameters of fetal vascular component in the placental villi were also assessed. Changes in the studied parameters differed in parturient women with mild and moderate COVID-19 infection. The observed increase in the total perimeter with a simultaneous decrease in the total capillary area and the degree of vascularization of the placental villi in parturient women with COVID-19 indicates impairment of circulation in the fetal compartment and the development of placental hypoxia, which can be the cause of unfavorable neonatal outcomes.

SARS-CoV-2 can infect and propagate in human placenta explants.

The ongoing SARS-CoV-2 pandemic continues to lead to high morbidity and mortality. During pregnancy, severe maternal and neonatal outcomes and placental pathological changes have been described. We evaluate SARS-CoV-2 infection at the maternal-fetal interface using precision-cut slices (PCSs) of human placenta. Remarkably, exposure of placenta PCSs to SARS-CoV-2 leads to a full replication cycle with infectious virus release. Moreover, the susceptibility of placental tissue to SARS-CoV-2 replication relates to the expression levels of ACE2. Viral proteins and/or viral RNA are detected in syncytiotrophoblasts, cytotrophoblasts, villous stroma, and possibly Hofbauer cells. While SARS-CoV-2 infection of placenta PCSs does not cause a detectable cytotoxicity or a pro-inflammatory cytokine response, an upregulation of one order of magnitude of interferon type III transcripts is measured. In conclusion, our data demonstrate the capacity of SARS-CoV-2 to infect and propagate in human placenta and constitute a basis for further investigation of SARS-CoV-2 biology at the maternal-fetal interface.

Hofbauer Cells and COVID-19 in Pregnancy.

CONTEXT.­: SARS-CoV-2 can undergo maternal-fetal transmission, heightening interest in the placental pathology findings from this infection. Transplacental SARS-CoV-2 transmission is typically accompanied by chronic histiocytic intervillositis together with necrosis and positivity of syncytiotrophoblast for SARS-CoV-2. Hofbauer cells are placental macrophages that have been involved in viral diseases, including HIV and Zika virus, but their involvement in SARS-CoV-2 is unknown. OBJECTIVE.­: To determine whether SARS-CoV-2 can extend beyond the syncytiotrophoblast to enter Hofbauer cells, endothelium, and other villous stromal cells in infected placentas of liveborn and stillborn infants. DESIGN.­: Case-based retrospective analysis by 29 perinatal and molecular pathology specialists of placental findings from a preselected cohort of 22 SARS-CoV-2-infected placentas delivered to pregnant women testing positive for SARS-CoV-2 from 7 countries. Molecular pathology methods were used to investigate viral involvement of Hofbauer cells, villous capillary endothelium, syncytiotrophoblast, and other fetal-derived cells. RESULTS.­: Chronic histiocytic intervillositis and trophoblast necrosis were present in all 22 placentas (100%). SARS-CoV-2 was identified in Hofbauer cells from 4 of 22 placentas (18.2%). Villous capillary endothelial staining was positive in 2 of 22 cases (9.1%), both of which also had viral positivity in Hofbauer cells. Syncytiotrophoblast staining occurred in 21 of 22 placentas (95.5%). Hofbauer cell hyperplasia was present in 3 of 22 placentas (13.6%). In the 7 cases having documented transplacental infection of the fetus, 2 (28.6%) occurred in placentas with Hofbauer cell staining positive for SARS-CoV-2. CONCLUSIONS.­: SARS-CoV-2 can extend beyond the trophoblast into the villous stroma, involving Hofbauer cells and capillary endothelial cells, in a small number of infected placentas. Most cases of SARS-CoV-2 transplacental fetal infection occur without Hofbauer cell involvement.