Covid-19: Is the placenta a safe space

Problem: Several large studies have demonstrated that COVID-19 pregnant individuals are at a significant risk for severe disease and adverse pregnancy outcomes. The mechanisms underlying these phenomena remain to be elucidated and are the focus of our project. Although fetal and placental infection is rare, placental abnormalities and adverse pregnancy outcomes associated with placental dysfunction in COVID-19 cases have been widely reported. In particular, placental thrombosis and lesions consistent with maternal vascular malperfusion (MVM) of the placenta are common in individuals with COVID-19. Since thrombotic complications have been associated with COVID-19, it is not surprising that pregnant individuals with COVID- 19 are at risk for placental thrombosis. Method of Study: Placentas were evaluated histologically. Extracellular vesicles were isolated by serial centrifugation. Result(s): Adverse pregnancy outcomes associated with these placental lesions, including hypertensive disorders of pregnancy (gestational hypertension and preeclampsia), small for gestational age (SGA, birthweight < 10th percentile for gestational age), and preterm birth (PTB, < 37 weeks) are significantly increased among pregnant individuals with COVID-19. Placental infection with SARSCoV- 2 is uncommon, but multiple inflammatory and metabolic factors are likely to affect the placenta, including circulating extracellular vesicles (EVs) derived from various organs that have been associated with COVID-19 pathology and disease severity.We have analyzed over 500 placentas from COVID-19 pregnancies and found marked changes in placental morphology, characterized by abnormal maternal and fetal vessels, intervillous thrombi, and fibrin deposition, even in the face of mild or asymptomatic disease. We detected increased levels of small EVs in maternal serum from COVID-19 cases compared to controls and increased levels of mitochondrial DNA in EVs from COVID-19 cases. In in vitro experiments, we found increased oxidative stress in uterine endothelial cells and primary trophoblasts. Syncytialization of trophoblast cells following exposure to EVs from pregnant COVID-19 patients was markedly reduced. RNAseq of trophoblast cells exposed to EVs from pregnant COVID-19 patients revealed disruption of multiple pathways related to mitochondria function, oxidative stress, coagulation defects, and inflammation. Timing of infection during pregnancy (first, second, and third trimester) altered EV size distribution, cargo content, and functional consequences of trophoblast EV exposure. Conclusion(s): Our studies show that COVID-19 infection during pregnancy has profound effects on placenta morphology and function. It remains to be determined what the long-term consequences are on the offspring.

The immunopathology of COVID-19 placentitis: A multi-omic spatial approach

Problem: COVID-19 placentitis is a rare complication of maternal SARS-CoV-2 respiratory infection associated with serious adverse obstetric outcomes, including intra-uterine death. The precise role of SARS-CoV-2 in COVID-19 placentitis is uncertain, as trophoblast infection is only observed in around one-half of the affected placenta. Method of Study: Through multi-omic spatial profiling, including Nanostring GeoMX digital spatial profiling and Lunaphore COMET multiplex IHC, we provide a deep characterization of the immunopathology of placentitis from obstetrically complicated maternal COVID-19 infection. Result(s):We show that SARS-CoV-2 infection of placental trophoblasts is associated with a distinct innate and adaptive immune cell infiltrate, florid cytokine expression and upregulation of viral restriction factors. Quantitative spatial analyses reveal a unique microenvironment surrounding virus-infected trophoblasts characterizedd by multiple immune evasion mechanisms, including immune checkpoint expression, cytotoxic T-cell exclusion, and interferon blunting. Placental viral loads inversely correlated with the duration of maternal infection consistent with progressive virus clearance, potentially explaining the absence of virus in some cases. Conclusion(s): Our results demonstrate a central role for placental SARS-CoV-2 infection in driving the unique immunopathology of COVID-19 placentitis.

SARS-CoV-2 infection disrupts trophoblast function and placental syncytial barrier integrity via its accessory protein, ORF3a

Problem: Although it is rare for a SARS-CoV-2 infection to transmit vertically to the fetus during pregnancy, there is a significantly increased risk of adverse pregnancy outcomes due to maternalCOVID- 19. However, there is a poor understanding of such risks because mechanistic studies on how SARS-CoV-2 infection disrupts placental homeostasis are significantly lacking. The SARS-CoV-2 proteome includes multiple structural and non-structural proteins, including the non-structural accessory proteinORF3a. The roles of these proteins in mediating placental infection remain undefined. We and others have shown that autophagy activity in placental syncytium is essential for barrier function and integrity. Here, we have used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placenta exposed to SARS-CoV-2. The objective of our study was to investigate potential mechanisms through which SARS-CoV-2 impairs placental homeostasis and causes adverse pregnancy outcomes. We tested the central hypothesis that an essential SARS-CoV-2 non-structural and accessory protein, ORF3a, uniquely (amongst multiple viral proteins tested) with a novel three-dimensional structure andwith no homology to any other proteins is a key modulator of placental trophoblast cell dynamics via autophagy and intracellular trafficking of a tight junction protein (TJP), ZO-1. Method(s): We used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placentas exposed to SARS-CoV- 2. Autophagic flux was measured in placental villous biopsies from SARS-CoV-2-exposed and unexposed pregnant women by quantifying the expression of autophagy markers, LC3 and P62. Trophoblast cells (JEG-3, Forskolin-treated JEG-3, HTR8/SVneo, or primary human trophoblasts (PHTs)) were transfected with expression plasmids encoding SARS-CoV-2 proteins including ORF3a. Using western blotting, multi-label immunofluorescence, and confocal imaging, we analyzed the effect of ORF3a on the autophagy, differentiation, invasion, and intracellular trafficking of ZO-1 in trophoblasts. Using coimmunoprecipitation assays, we tested ORF3a interactions with host proteins. t-tests and one-way analyses of variance (ANOVAs) with post hoc tests were used to assess the data, with significance set at P < .05. Result(s): We discovered :1) increased activation of autophagy, but incomplete processing of autophagosome-lysosomal degradation;2) accumulation of protein aggregates in placentas exposed to SARS-CoV- 2. Mechanistically, we showed that the SARS-CoV-2 ORF3a protein, uniquely 3) blocks the autophagy-lysosomal degradation process;4) inhibits maturation of cytotrophoblasts into syncytiotrophoblasts (STBs);5) reduces production ofHCG-beta, a key pregnancy hormone that is also essential for STB maturation;and 6) inhibits trophoblast invasive capacity. Furthermore, ORF3a harbors an intrinsically disordered C-terminus withPDZ-bindingmotifs.We show for the first time that, 7) ORF3a binds to and co-localizes with the PDZ domain of ZO-1, a junctional protein that is essential for STB maturation and the integrity of the placental barrier. Conclusion(s): Our work outlines a new molecular and cellular mechanism involving the SARS-CoV-2 accessory protein ORF3a that may drive the virus's ability to infect the placenta and damage placental syncytial integrity. This implies that the mechanisms facilitating viral maturation, such as the interaction of ORF3a with host factors, can be investigated for additional functionality and even targeted for developing new intervention strategies for treatment or prevention of SARS-CoV-2 infection at the maternal-fetal interface.

Placental pathology in sudden intrauterine death (SIUD) in SARS-CoV-2-positive oligosymptomatic women.

BACKGROUND: Pregnant women are also susceptible to SARS-CoV-2. Although an infection of the placenta may be rare, pregnancy may occasionally be affected by intrauterine failure. The knowledge of placental morphology on sudden intrauterine demise is still limited. METHODS: Fetal and placental tissue of two cases of sudden intrauterine death in the second trimester were analysed morphologically and by immunohistochemistry. One case was evaluated by RT-PCR. RESULTS: Both mothers were tested positive for the Alpha variant of SARS-CoV-2 but were oligosymptomatic for COVID-19. Unexpected sudden intrauterine death (SIUD) occurred at 15 + 2 and 27 + 3 weeks of gestation. One fetus demonstrated an intrauterine growth restriction. No malformations nor inflammatory changes were observed in either fetus on autopsy. In contrast to the placentas, the fetal tissue was negative for SARS-CoV-2 on immunohistochemical and RT-PCR analyses. Macroscopically, the placentas showed an increased consistency with a white, reticular cutting surface covering about 95% of the whole placenta. Only very focal histiocytic chronic intervillositis was noted histologically. Massive perivillous fibrin deposits with extensive necroses of the villous trophoblast were present in more than 90% of the placental tissue. Immunohistochemical staining was strong and diffusely positive for SARS-CoV-2 in the villous trophoblast and rarely within the villous stromal cells. Placental SARS-CoV-2 infection was confirmed by RT-PCR. CONCLUSION: Sudden intrauterine death may occur in mothers who are oligosymptomatic for COVID-19. Acute placental failure is responsible for SIUD, demonstrated by massive perivillous fibrin deposits and extensive necroses of the villous trophoblast with SARS-CoV-2-positivity based on immunohistochemical staining and RT-PCR. Detailed histopathological examination of placental and fetal tissue is mandatory to verify SARS-CoV-2 and to evaluate the pathogenesis and functionality of this disease.

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.

Pathological involvement of placenta in COVID-19: a systematic review.

The mammalian placenta, which is responsible for bonding between the mother and the fetus, is one of the first organs to develop. Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection has caused a great threat to public health and affected almost all the organs including the placenta. Owing to limited available data on vertical transmission and pathological changes in the placenta of SARS-CoV-2 positive patients, we aim to review and summarize histopathological and ultrastructural changes in the placental tissue following SARS-CoV-2 infection. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2009 guidelines were used for review writing. Multiple studies have reported significant pathological changes in the placental tissue of SARS-CoV-2 positive mothers. On the other hand, some studies have demonstrated either no or very little involvement of the placental tissue. The most common pathological changes reported are fetal and maternal vascular malformation, villitis of unknown etiology, thrombus formation in the intervillous space and sub-chorionic space, and chorangiosis. Reports on vertical transmission are less in number. The observations of this review present a strong base for the pathological involvement of the placenta in SARS-CoV-2 infected mothers. However, a smaller number of original studies have been done until now, and most of them have small sample sizes and lack matched control groups, which are the big limitations for drawing an effective conclusion at this stage. Antenatal care can be improved by a better understanding of the correlation between maternal SARS-CoV-2 infection and placental pathology in COVID-19.

Stem cell-derived trophoblast organoids model human placental development, X chromosome inactivation, and susceptibility to emerging pathogens

Problem: Trophoblast organoids derived from human placental villi provide a powerful 3D model system of placental development, but access to first-trimester tissues is limited due to ethical and legal restrictions. Here we sought to establish a methodology for establishing 3D trophoblast organoids from naïve human pluripotent stem cells (hPSCs), which have an expanded potential for extraembryonic differentiation. Method of Study: We previously demonstrated that naïve hPSCs readily give rise to self-renewing human trophoblast cells (hTSCs) that resemble post-implantation cytotrophoblast (CTB) progenitors and can further differentiate into specialized trophoblast lineages. Here we examined whether hTSCs derived from three distinct sources (naïve hPSCs, human blastocysts, and first-trimester placental tissues) have the potential to self-organize into 3D trophoblast organoids by transfer to Matrigel droplets in the presence of trophoblast organoid medium. The expression of protein markers in the resulting stem cellderived trophoblast organoids (SC-TOs) was examined by immunofluorescence and light-sheet microscopy, while their single cell transcriptome was analyzed using the 10X Genomics platform. We also investigated the X chromosome inactivation (XCI) status of organoids derived from female naïve hPSCs and their ability to differentiate into invasive extravillous trophoblast (EVT) organoids. Finally, we evaluated whether SC-TOs are susceptible to infection by various emerging pathogens (SARS-CoV-2 and Zika virus), as a basis for establishing a stem cell-based model system of placental infections during the first trimester. Results: Trophoblast organoids generated from naïve and primary hTSCs displayed comparable tissue architecture, placental hormone secretion, microRNA expression, and capacity for long-term selfrenewal. In-depth single cell transcriptome profiling revealed that SCTOs encompass a variety of trophoblast identities that closely correspond to CTB progenitor, syncytiotrophoblast (STB) and EVT cell types found in human post-implantation embryos. Interestingly, the cellular composition in trophoblast organoids derived from naïve and primary hTSCs was highly similar, which suggests that trophoblast organoid culture represents a powerful attractor state in which the influence of subtle epigenetic differences between naïve and primary hTSCs is mitigated. These organoid cultures displayed clonal XCI patterns previously described in the human placenta.Upon differentiation into specialized EVT organoids, extensive trophoblast invasion was observed in co-culture assays with human endometrial cells. We further demonstrated that SC-TOs display selective vulnerability to infection by SARS-CoV-2 and Zika virus, which correlated with the expression levels of their respective entry factors. Conclusions: The generation of trophoblast organoids from naïve hPSCs provides an accessible and patient-specific 3D model system of the developing placenta and its susceptibility to emerging pathogens. The ability to genetically manipulate naïve hPSCs prior to differentiation into SC-TOs enables functional interrogation of regulatory factors implicated in placental organogenesis.

My Journeys with Three Microbes in the Reproductive Tract

Problem: The placenta performs various functions of the lung/GI/GU tract for the developing fetus, while also moderating host defenses of the fetus against infections in utero, and likely educates the developing fetal immune system. It thus has long-term impacts on the health of both the woman and the child. Knowledge is limited about the underlying mechanisms that enable the placenta to serve as a protective barrier for the fetus against infection. The long-term goals of my research program are to, 1) elucidate the normal barriers to infection in the placenta and show how dysfunction in barrier function can lead to adverse maternal-fetal outcomes, 2) define how viral infections impact placental biology, and 3) characterize possible functional roles for the newly described microbiota at the maternal-fetal interface. Method of Study: To address the above questions, our research includes the use human placentas, primary human trophoblasts and immune cells derived from term placentas, cultured placental cells, trophoblast organoids, and mousemodels. Results: We found that placentas from women who gave birth prematurely exhibit reduced autophagy activity. Prematurity and reduced autophagy levels were also strongly associated with maternal infection. In a mouse model of pregnancy, we showed that placentas from mice deficient for Atg16L1 were significantly less able to withstand infection, and the deficient mice gave birth prematurely upon an inflammatory stimulus. We have also shown that the autophagy pathway plays a key role in ZIKV vertical transmission from mother to fetus. We demonstrated that hydroxychloroquine (HCQ), an autophagy inhibitor approved for use in pregnant women, can attenuate placental and fetal ZIKV infection and ameliorate adverse placental and fetal outcomes. More recently, we have identified a small molecule inhibitor that targets the NS2B-NS3 protease of ZIKV and inhibits viral replication. It has recently become evident that SARS-CoV-2 infection is also associated with adverse outcomes for pregnant women, including preterm birth, preeclampsia, and fetal growth restriction. We localized SARS-CoV-2 to the placenta and showed that infection alters the Renin Angiotensin System (RAS) that regulates blood pressure, thereby increasing risk for preeclampsia. In new work, we are showing that SARS-CoV-2 non-structural proteins affect autophagy in different ways than in Zika virus. Finally, we have discovered that the maternal fetal interface of the placenta harbors intracellular resident microbes, and functionally demonstrated that they do not induce any inflammatory response or cell death but may promote immune tolerance and support normal pregnancy outcomes. Conclusions: For the past 10 years of my career, I have been working on host microbial interactions at the maternal fetal interface. Our work has led to new insights into viral infections, showing how they co-opt host defenses, and that tolerance may have microbial drivers. We have shown how cellular pathways in the placenta such as autophagy and RAS mechanistically regulate host defenses against pathogens, including ZIKV and SARS-CoV-2. Additionally, our studies provide a foundation for understanding possible 'commensal' microbial- placental interactions and hint at the functional importance of microbes at the fetal maternal interface in maintaining placental health and supporting fetal development.

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.

EFFECTS OF COVID-19 VECTOR VACCINE ON AUTOANTIBODY PROFILE IN REPRODUCTIVE AGE WOMEN

Autoimmune mechanisms have been implicated in the negative effects of vaccines on female reproductive health. This study evaluates the endogenous levels of self-reactive antibodies and ovarian reserve-associated hormones before and after immunization with the domestically developed Gam-COVID-Vac combined vector vaccine to check for possible reproductive sequelae. The prospective study enrolled 120 women aged 18–49, subject to vaccination with Gam-COVID-Vac. Ovarian reserve was assessed prior to vaccination and 90 days after the first component injection. Profiles of specific antibodies to self-antigens, including phospholipids, nuclear antigens, FSH, progesterone, and also thyroid, ovarian, trophoblast, and zona pellucida antigens, were assessed at the same time points by enzyme immunoassay. Overall, the vaccination had no effect on the levels of ovarian reserve-associated hormones and autoantibodies, apart from a transient increase in positivity for antiphosphatidylethanolamine IgM and anti-dsDNA IgG. Seroprevalence of elevated serum autoantibodies constituted 70.8% before and 75% after vaccination. According to the results, immunization with Gam-COVID-Vac does not affect ovarian reserve or autoimmune status, thus being safe for the female reproductive potential.

Healthy Baby Delivery of a COVID-19 Infected Mother in Iran: Process and Protection

Background: In late December 2019, a kind of pneumonia caused by a novel coronavirus (SARS-CoV-2) emerged in Wuhan, Hubei Province, China. This virus rapidly spread worldwide and infected 195 countries and territories, including Iran. By March 22, 2020, the virus had affected more than 40000 people worldwide and caused more than 19000 deaths. Pregnant women are a vulnerable group to viral infections because partial immune suppression occurs during pregnancy. Therefore, the COVID-19 epidemic may cause a rising global concern about its consequences for pregnant women and fetuses. Objectives: In this case study, we report the delivery of a pregnant woman after her COVID-19 confirmation. Methods: We report a 44-year-old pregnant woman (32 weeks gestation) with COVID-19 who gave birth to a healthy baby with no evidence of COVID-19. We did not observe any worse clinical outcomes, such as maternal mortality, stillbirth, spontaneous abortion, and preterm delivery. Results: A preterm baby girl with 2500 g weight and Apgar scores at 5 minutes and 10 minutes were 9 and 10 was delivered. The preterm baby was normocephalic, had no icteric sclera, and the heart sounded normal without murmurs, Lung ventilation was normal. Conclusion: Viral pneumonia may severely be presented in pregnancy because of physiological and immunological changes and shift from cell-mediated to humoral-mediated immunity during the pregnancy period. Vertical transmission of COVID-19 from mother to child, short-term and long-term adverse effects on mother and newborn are still unclear and controversial.

Characteristic Histological and Molecular Findings in SARS-CoV-2-infected Placentas and its Impact on the Fetus. Study of 279 Cases from Pregnant Women Affected by COVID-19 during the Pandemic

Background: Placental pathology in pregnant women infected by SARS-CoV-2 has generated many reports with non-specific or contradictory results. The objective is to identify histological and molecular findings of infected placentas and its impact on the course of pregnancy. Design: Placentas from 279 women infected with SARS-CoV-2 during pregnancy were reviewed, 137 of them with active infection within 10 days prior to delivery and 142 with past infection beyond this period. Four fetal autopsies were submitted for pathological study. Placental and fetal infection was investigated by immunohistochemistry (IHC) in 279 cases, RT-PCR in 27 cases and in situ hybridization (ISH) in 5 cases. Significance of association between qualitative variables was tested by Fisher's exact test. Results: SARS-CoV-2 infection of placental tissues was demonstrated in 16/279 (5,7%) by IHQ (granular cytoplasmic villous trophoblast staining) and confirmed by ISH and RT-PCR (carried on 5 IHQ positive cases). All these 16 infected placentas showed diffuse (6/16) or focal (10/16) Trophoblast Damage (DTD/FTD) characterized by trophoblast necrosis with preserved villous stroma, collapse of the intervillous space, intervillous fibrinoid deposits, and variable polymorphous inflammatory infiltrates. Cases with DTD (>80% of the parenchyma affected) resulted in 5 stillbirth and 1 premature alive baby (27w) born after induction of labor for pathologic fetal non-stress test. In 4/5 deaths an autopsy was performed and infection of fetal tissues was not demonstrated by IHQ. The cases with FTD (<35% of the parenchyma affected) hadn't negative impact on the fetus. Correlation between placental infection and DTD/FTD had statistical significance (p<0,001). 15/16 placentas belonged to the group of pregnant women with active infection within 10 days prior delivery. Correlation between DTD/FTD and maternal active infection had statistical significance (p<0,002). Conclusions: Placentas infected by SARS-CoV2 has a characteristic histological pattern that we have defined as DTD or FTD, characterized by a predominant villous trophoblast necrosis, intervillous space collapse and variable degrees of mixed inflammation and intervillous fibrinoid deposition. DTD/FTD is an infrequent lesion observed in 5,7% of pregnant women affected by COVID-19 in our series. DTD can be the cause of fetal death due to placental insufficiency. In this study, vertical transmission of the virus to the fetus has not been demonstrated.

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.

Regulation of Proinflammatory Molecules and Tissue Factor by SARS-CoV-2 Spike Protein in Human Placental Cells: Implications for SARS-CoV-2 Pathogenesis in Pregnant Women.

SARS-CoV-2 infects cells via binding to ACE2 and TMPRSS2, which allows the virus to fuse with host cells. The viral RNA is detected in the placenta of SARS-CoV-2-infected pregnant women and infection is associated with adverse pregnancy complications. Therefore, we hypothesize that SARS-CoV-2 infection of placental cells induces pro-inflammatory cytokine release to contribute to placental dysfunction and impaired pregnancy outcomes. First, expression of ACE2 and TMPRSS2 was measured by qPCR in human primary cultured term cytotrophoblasts (CTBs), syncytiotrophoblast (STBs), term and first trimester decidual cells (TDCs and FTDCs, respectively), endometrial stromal cells (HESCs) as well as trophoblast cell lines HTR8, JEG3, placental microvascular endothelial cells (PMVECs) and endometrial endothelial cells (HEECs). Later, cultured HTR8, JEG3, PMVECs and HEECs were treated with 10, 100, 1000 ng/ml of recombinant (rh-) SARS-CoV-2 S-protein ± 10 ng/ml rh-IFNγ. Pro-inflammatory cytokines IL-1ß, 6 and 8, chemokines CCL2, CCL5, CXCL9 and CXCL10 as well as tissue factor (F3), the primary initiator of the extrinsic coagulation cascade, were measured by qPCR as well as secreted IL-6 and IL-8 levels were measured by ELISA. Immunohistochemical staining for SARS-CoV-2 spike protein was performed in placental specimens from SARS-CoV-2-positive and normal pregnancies. ACE2 levels were significantly higher in CTBs and STBs vs. TDCs, FTDCs and HESCs, while TMPRSS2 levels were not detected in TDCs, FTDCs and HESCs. HTR8 and JEG3 express ACE2 and TMPRSS2, while PMVECs and HEECs express only ACE2, but not TMPRSS2. rh-S-protein increased proinflammatory cytokines and chemokines levels in both trophoblast and endothelial cells, whereas rh-S-protein only elevated F3 levels in endothelial cells. rh-IFNγ ± rh-S-protein augments expression of cytokines and chemokines in trophoblast and endothelial cells. Elevated F3 expression by rh-IFNγ ± S-protein was observed only in PMVECs. In placental specimens from SARS-CoV-2-infected mothers, endothelial cells displayed higher immunoreactivity against spike protein. These findings indicated that SARS-CoV-2 infection in placental cells: 1) induces pro-inflammatory cytokine and chemokine release, which may contribute to the cytokine storm observed in severely infected pregnant women and related placental dysfunction; and 2) elevates F3 expression that may trigger systemic or placental thrombosis.

Loss of imprinting of the Igf2-H19 ICR1 enhances placental endocrine capacity via sex-specific alterations in signalling pathways in the mouse.

Imprinting control region (ICR1) controls the expression of the Igf2 and H19 genes in a parent-of-origin specific manner. Appropriate expression of the Igf2-H19 locus is fundamental for normal fetal development, yet the importance of ICR1 in the placental production of hormones that promote maternal nutrient allocation to the fetus is unknown. To address this, we used a novel mouse model to selectively delete ICR1 in the endocrine junctional zone (Jz) of the mouse placenta (Jz-ΔICR1). The Jz-ΔICR1 mice exhibit increased Igf2 and decreased H19 expression specifically in the Jz. This was accompanied by an expansion of Jz endocrine cell types due to enhanced rates of proliferation and increased expression of pregnancy-specific glycoprotein 23 in the placenta of both fetal sexes. However, changes in the endocrine phenotype of the placenta were related to sexually-dimorphic alterations to the abundance of Igf2 receptors and downstream signalling pathways (Pi3k-Akt and Mapk). There was no effect of Jz-ΔICR1 on the expression of targets of the H19-embedded miR-675 or on fetal weight. Our results demonstrate that ICR1 controls placental endocrine capacity via sex-dependent changes in signalling.

Stillbirth after COVID-19 in Unvaccinated Mothers Can Result from SARS-CoV-2 Placentitis, Placental Insufficiency, and Hypoxic Ischemic Fetal Demise, Not Direct Fetal Infection: Potential Role of Maternal Vaccination in Pregnancy.

Stillbirth is a recently recognized complication of COVID-19 in pregnant women. Other congenitally transmitted infections from viruses, bacteria and parasites can cause stillbirth by infecting fetal organs following transplacental transmission of the agent from the maternal bloodstream. However, recent research on pregnant women with COVID-19 having stillbirths indicates that there is another mechanism of stillbirth that can occur in placentas infected with SARS-CoV-2. In these cases, viral infection of the placenta results in SARS-CoV-2 placentitis, a combination of concurrent destructive findings that include increased fibrin deposition which typically reaches the level of massive perivillous fibrin deposition, chronic histiocytic intervillositis and trophoblast necrosis. These three pathological lesions, in some cases together with placental hemorrhage, thrombohematomas and villitis, result in severe and diffuse placental parenchymal destruction. This pathology can involve greater than one-half of the placental volume, averaging 77% in the largest study of 68 cases, effectively rendering the placenta incapable of performing its function of oxygenating the fetus. This destructive placental process can lead to stillbirth and neonatal death via malperfusion and placental insufficiency which is independent of fetal infection. Fetal autopsies show no evidence that direct infection of fetal organs is contributory. Because all mothers examined have been unvaccinated, maternal vaccination may prevent viremia and consequent placental infection.

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.

Human placental biology at single-cell resolution: a contemporaneous review.

Single-cell technologies capture cellular heterogeneity to focus on previously poorly described subpopulations of cells. Work by our laboratory and many others has metagenomically characterised a low biomass intrauterine microbial community, alongside microbial transcripts, antigens and metabolites, but the functional importance of low biomass microbial communities in placental immuno-microenvironments is still being elucidated. Given their hypothesised role in modulating inflammation and immune ontogeny to enable tolerance of beneficial microbes while warding off pathogens, there is a need for single-cell resolution. Herein, we summarise the potential for mechanistic understanding of these and other key fundamental early developmental processes by applying single-cell approaches.