Syncytiotrophoblast Formation Research Articles

Sodium-dependent phosphate transporter PiT1/SLC20A1 as the receptor for the endogenous retroviral envelope syncytin-B involved in mouse placenta formation.

Syncytins are envelope genes of retroviral origin that play a critical role in the formation of a syncytial structure at the fetomaternal interface via their fusogenic activity. The mouse placenta is unique among placental mammals since the fetomaternal interface comprises two syncytiotrophoblast layers (ST-I and ST-II) instead of one observed in all other hemochorial placentae. Each layer specifically expresses a distinct mouse syncytin, namely syncytin-A (SynA) for ST-I and syncytin-B (SynB) for ST-II, which have been shown to be essential to placentogenesis and embryonic development. The cellular receptor for SynA has been identified as the membrane protein LY6E and is not the receptor for SynB. Here, by combining a cell-cell fusion assay with the screening of a human ORFeome-derived expression library, we identified the transmembrane multipass sodium-dependent phosphate transporter 1 PiT1/SLC20A1 as the receptor for SynB. Transfection of cells with the cloned receptor, but not the closely related PiT2/SLC20A2, leads to their fusion with cells expressing SynB, with no cross-reactive fusion activity with SynA. The interaction between the two partners was further demonstrated by immunoprecipitation. PiT1/PiT2 chimera and truncation experiments identified the PiT1 N-terminus as the major determinant for SynB-mediated fusion. RT-qPCR analysis of PiT1 expression on a panel of mouse adult and fetal tissues revealed a concomitant increase of PiT1 and SynB specifically in the developing placenta. Finally, electron microscopy analysis of the placenta of PiT1 null embryo before they die (E11.5) disclosed default of ST-II formation with lack of syncytialization, as previously observed in cognate SynB null placenta, and consistent with the present identification of PiT1 as the SynB partner.IMPORTANCESyncytins are envelope genes of endogenous retroviruses, coopted for a physiological function in placentation. They are fusogenic proteins that mediate cell-cell fusion by interacting with receptors present on the partner cells. Here, by devising an in vitro fusion assay that enables the screening of an ORFeome-derived expression library, we identified the long-sought receptor for syncytin-B (SynB), a mouse syncytin responsible for syncytiotrophoblast formation at the fetomaternal interface of the mouse placenta. This protein - PiT1/SLC20A1 - is a multipass transmembrane protein, also known as the receptor for a series of infectious retroviruses. Its profile of expression is consistent with a role in both ancestral endogenization of a SynB founder retrovirus and present-day mouse placenta formation, with evidence-in PiT1 knockout mice-of unfused cells at the level of the cognate placental syncytiotrophoblast layer.

Hypoxia and loss of GCM1 expression prevents differentiation and contact inhibition in human trophoblast stem cells.

The placenta develops alongside the embryo and nurtures fetal development to term. During the first stages of embryonic development, due to low blood circulation, the blood and ambient oxygen supply is very low (∼1-2% O 2 ) and gradually increases upon placental invasion. While a hypoxic environment is associated with stem cell self-renewal and proliferation, persistent hypoxia may have severe effects on differentiating cells and could be the underlying cause of placental disorders. We find that human trophoblast stem cells (hTSC) thrive in low oxygen, whereas differentiation of hTSC to trophoblast to syncytiotrophoblast (STB) and extravillous trophoblast (EVT) is negatively affected by hypoxic conditions. The pro-differentiation factor GCM1 (human Glial Cell Missing-1) is downregulated in low oxygen, and concordantly there is substantial reduction of GCM1-regulated genes in hypoxic conditions. Knockout of GCM1 in hTSC caused impaired EVT and STB formation and function, reduced expression of differentiation-responsive genes, and resulted in maintenance of self-renewal genes. Treatment with a PI3K inhibitor reported to reduce GCM1 protein levels likewise counteracts spontaneous or directed differentiation. Additionally, chromatin immunoprecipitation of GCM1 showed enrichment of GCM1-specific binding near key transcription factors upregulated upon differentiation including the contact inhibition factor CDKN1C. Loss of GCM1 resulted in downregulation of CDKN1C and corresponding loss of contact inhibition, implicating GCM1 in regulation of this critical process.

TFEB controls syncytiotrophoblast formation and hormone production in placenta.

TFEB, a bHLH-leucine zipper transcription factor belonging to the MiT/TFE family, globally modulates cell metabolism by regulating autophagy and lysosomal functions. Remarkably, loss of TFEB in mice causes embryonic lethality due to severe defects in placentation associated with aberrant vascularization and resulting hypoxia. However, the molecular mechanism underlying this phenotype has remained elusive. By integrating in vivo analyses with multi-omics approaches and functional assays, we have uncovered an unprecedented function for TFEB in promoting the formation of a functional syncytiotrophoblast in the placenta. Our findings demonstrate that constitutive loss of TFEB in knock-out mice is associated with defective formation of the syncytiotrophoblast layer. Indeed, using in vitro models of syncytialization, we demonstrated that TFEB translocates into the nucleus during syncytiotrophoblast formation and binds to the promoters of crucial placental genes, including genes encoding fusogenic proteins (Syncytin-1 and Syncytin-2) and enzymes involved in steroidogenic pathways, such as CYP19A1, the rate-limiting enzyme for the synthesis of 17β-Estradiol (E2). Conversely, TFEB depletion impairs both syncytial fusion and endocrine properties of syncytiotrophoblast, as demonstrated by a significant decrease in the secretion of placental hormones and E2 production. Notably, restoration of TFEB expression resets syncytiotrophoblast identity. Our findings identify that TFEB controls placental development and function by orchestrating both the transcriptional program underlying trophoblast fusion and the acquisition of endocrine function, which are crucial for the bioenergetic requirements of embryonic development.

Comparative analysis of the syncytiotrophoblast in placenta tissue and trophoblast organoids using snRNA sequencing.

The outer surface of chorionic villi in the human placenta consists of a single multinucleated cell called the syncytiotrophoblast (STB). The unique cellular ultrastructure of the STB presents challenges in deciphering its gene expression signature at the single-cell level, as the STB contains billions of nuclei in a single cell. There are many gaps in understanding the molecular mechanisms and developmental trajectories involved in STB formation and differentiation. To identify the underlying control of the STB, we performed comparative single nucleus (SN) and single cell (SC) RNA sequencing on placental tissue and tissue-derived trophoblast organoids (TOs). We found that SN was essential to capture the STB population from both tissue and TOs. Differential gene expression and pseudotime analysis of TO-derived STB identified three distinct nuclear subtypes reminiscent of those recently identified in vivo . These included a juvenile nuclear population that exhibited both CTB and STB marker expression, a population enriched in genes involved in oxygen sensing, and a fully differentiated subtype. Notably, suspension culture conditions of TOs that restore the native orientation of the STB (STB out ) showed elevated expression of canonical STB markers and pregnancy hormones, along with a greater proportion of the terminally differentiated mature STB subtype, compared to those cultivated with an inverted STB polarity (STB in ). Gene regulatory analysis identified novel markers of STB differentiation conserved in tissue and TOs, including the chromatin remodeler RYBP, that exhibited STB-specific RNA and protein expression. Finally, we compared STB gene expression signatures amongst first trimester tissue, full-term tissue, and TOs, identifying many commonalities but also notable variability across each sample type. This indicates that STB gene expression is responsive to its environmental context. Our findings emphasize the utility of TOs to accurately model STB differentiation and the distinct nuclear subtypes observed in vivo , offering a versatile platform for unraveling the molecular mechanisms governing STB functions in placental biology and disease.

Impact of COVID-19 Vaccination on In Vitro Fertilization Outcomes: A Systematic Review.

COVID-19 vaccination, especially vaccines that mimic the structure of the COVID-19 virus (mRNA vaccines), may be wrongly assumed to be disrupting factors affecting in vitro fertilization (IVF) outcome. This study aims to evaluate any significant impact of COVID-19 vaccination in women undergoing IVF to improve vaccine compliance and promote COVID-19 eradication. This was a systematic review study. We searched studies published between 2020 and 2022 using databases such as PubMed, Cochrane, PMC, and CINAHL. Selected studies were carefully analyzed to review the impact of the COVID-19 vaccine on IVF outcomes. Seven retrospective and prospective cohort studies, which involved 3232 female patients undergoing IVF, who also received full doses of COVID-19 vaccinations (mRNA), were included. All studies in the present review showed that despite presenting anti-SARS-CoV-2 antibodies after vaccination, there were no significant differences in IVF outcomes, implantation rates, and pregnancy rates. Contrary to the theory that presumed cross-reactivity between anti- SARS-CoV-2 antibodies and the human syncytin-1 protein could affect syncytiotrophoblast formation and embryo implantation. The present review concluded that COVID-19 vaccination does not result in any detrimental effects on IVF outcomes and is safe for women undergoing IVF treatment. The results of our study are important to tackle misinformation regarding COVID-19 vaccination and infertility that may cause vaccine hesitancy in women of reproductive age.

Epigenetic drug screening for trophoblast syncytialization reveals a novel role for MLL1 in regulating fetoplacental growth.

Abnormal placental development is a significant factor contributing to perinatal morbidity and mortality, affecting approximately 5-7% of pregnant women. Trophoblast syncytialization plays a pivotal role in the establishment and maturation of the placenta, and its dysregulation is closely associated with several pregnancy-related disorders, including preeclampsia and intrauterine growth restriction. However, the underlying mechanisms and genetic determinants of syncytialization are largely unknown. We conducted a systematic drug screen using an epigenetic compound library to systematically investigate the epigenetic mechanism essential for syncytialization, and identified mixed lineage leukemia 1 (MLL1), a histone 3 lysine 4 methyltransferase, as a crucial regulator of trophoblast syncytialization. BeWo cells were utilized to investigate the role of MLL1 during trophoblast syncytialization. RNA sequencing and CUT&Tag were further performed to search for potential target genes and the molecular pathways involved. Human placenta tissue was used to investigate the role of MLL1 in TEA domain transcription factor 4 (TEAD4) expression and the upstream signaling during syncytialization. A mouse model was used to examine whether inhibition of MLL1-mediated H3K4me3 regulated placental TEAD4 expression and fetoplacental growth. Genetic knockdown of MLL1 or pharmacological inhibition of the MLL1 methyltransferase complex (by MI-3454) markedly enhanced syncytialization, while overexpression of MLL1 inhibited forskolin (FSK)-induced syncytiotrophoblast formation. In human placental villous tissue, MLL1 was predominantly localized in the nuclei of cytotrophoblasts. Moreover, a notable upregulation in MLL1 expression was observed in the villus tissue of patients with preeclampsia compared with that in the control group. Based on RNA sequencing and CUT&Tag analyses, depletion of MLL1 inhibited the Hippo signaling pathway by suppressing TEAD4 expression by modulating H3K4me3 levels on the TEAD4 promoter region. TEAD4 overexpression significantly reversed the FSK-induced or MLL1 silencing-mediated trophoblast syncytialization. Additionally, decreased hypoxia-inducible factor 1A (HIF1A) enrichment at the MLL1 promoter was observed during syncytialization. Under hypoxic conditions, HIF1A could bind to and upregulate MLL1, leading to the activation of the MLL1/TEAD4 axis. In vivo studies demonstrated that the administration of MI-3454 significantly enhanced fetal vessel development and increased the thickness of the syncytial layer, thereby supporting fetoplacental growth. These results revealed a novel epigenetic mechanism underlying the progression of syncytialization with MLL1, and suggest potential avenues for identifying new therapeutic targets for pregnancy-related disorders.

Galectin-1 Modulates the Fusogenic Activity of Placental Endogenous Retroviral Envelopes.

Syncytin-1 and -2 are glycoproteins encoded by human endogenous retrovirus (hERV) that, through their fusogenic properties, are needed for the formation of the placental syncytiotrophoblast. Previous studies suggested that these proteins, in addition to the EnvP(b) envelope protein, are also involved in other cell fusion events. Since galectin-1 is a β-galactoside-binding protein associated with cytotrophoblast fusion during placental development, we previously tested its effect on Syncytin-mediated cell fusion and showed that this protein differently modulates the fusogenic potential of Syncytin-1 and -2. Herein, we were interested in comparing the impact of galectin-1 on hERV envelope proteins in different cellular contexts. Using a syncytium assay, we first demonstrated that galectin-1 increased the fusion of Syncytin-2- and EnvP(b)-expressing cells. We then tested the infectivity of Syncytin-1 and -2 vs. VSV-G-pseudotyped viruses toward Cos-7 and various human cell lines. In the presence of galectin-1, infection of Syncytin-2-pseudotyped viruses augmented for all cell lines. In contrast, the impact of galectin-1 on the infectivity of Syncytin-1-pseudotyped viruses varied, being cell- and dose-dependent. In this study, we report the functional associations between three hERV envelope proteins and galectin-1, which should provide information on the fusogenic activity of these proteins in the placenta and other biological and pathological processes.

Analyzing Trophoblast Fusion Using Immunofluorescence and Split Protein Complementation Assays.

The fusion of cytotrophoblasts into a multinucleatedsyncytiotrophoblast is essential for placental development. For studies investigating syncytiotrophoblast formation, various methods are available to analyze the fusion efficiency of trophoblast cells in vitro. Here, we describe protocols for measuring trophoblast fusion using immunofluorescence and an assay employing complementary parts of a split green fluorescent protein that self-reassociates and generates a fluorescent signal following cell fusion. Together, these approaches allow for a comprehensive and robust analysis of the fusion index in trophoblast cells and can strengthen the accuracy and throughput of investigations into factors that may regulate syncytiotrophoblast development.

Development of a Cre-recombination-based color-switching reporter system for cell fusion detection

Cell fusion plays a key role in the development and formation of tissues and organs in several organisms. Skeletal myogenesis is assessed in vitro by cell shape and gene and protein expression using immunofluorescence and immunoblotting assays. However, these conventional methods are complex and do not allow for easy time-course observation in living cells. Therefore, this study aimed to develop a Cre recombination-based fluorescent reporter system to monitor cell-cell fusion. We combined green and red fluorescent proteins with a Cre-loxP system to detect syncytium formation using a fluorescent binary switch. This allowed us to visualize mononucleated cells with green fluorescence before fusion and multinucleated syncytia with red fluorescence by conditional expression after cell fusion. The formation of multinuclear myotubes during myogenic differentiation was detected by the change in fluorescence from green to red after Cre-mediated recombination. The distribution of the fluorescence signal correlated with the expression of myogenic differentiation markers. Moreover, red reporter fluorescence intensity was correlated with the number of nuclei contained in the red fluorescent-positive myotubes. We also successfully demonstrated that our fusion monitoring system is applicable to the formation of skeletal muscle myotube and placental syncytiotrophoblast. These results suggest that the color-switching fluorescent reporter system, using Cre-mediated recombination, could be a robust tool used to facilitate the study of cell-to-cell fusion.

Activation of human endogenous retroviruses and its physiological consequences.

Human endogenous retroviruses (HERVs) are abundant sequences that persist within the human genome as remnants of ancient retroviral infections. These sequences became fixed and accumulate mutations or deletions over time. HERVs have affected human evolution and physiology by providing a unique repertoire of coding and non-coding sequences to the genome. In healthy individuals, HERVs participate in immune responses, formation of syncytiotrophoblasts and cell-fate specification. In this Review, we discuss how endogenized retroviral motifs and regulatory sequences have been co-opted into human physiology and how they are tightly regulated. Infections and mutations can derail this regulation, leading to differential HERV expression, which may contribute to pathologies including neurodegeneration, pathological inflammation and oncogenesis. Emerging evidence demonstrates that HERVs are crucial to human health and represent an understudied facet of many diseases, and we therefore argue that investigating their fundamental properties could improve existing therapies and help develop novel therapeutic strategies.

IFITM1 inhibits trophoblast invasion and is induced in placentas associated with IFN-mediated pregnancy diseases

Interferon-induced transmembrane proteins (IFITMs) are restriction factors that block many viruses from entering cells. High levels of type I interferon (IFN) are associated with adverse pregnancy outcomes, and IFITMs have been shown to impair the formation of syncytiotrophoblast. Here, we examine whether IFITMs affect another critical step of placental development, extravillous cytotrophoblast (EVCT) invasion. We conducted experiments using invitro/exvivo models of EVCT, mice treated invivo with the IFN-inducer poly (I:C), and human pathological placental sections. Cells treated with IFN-β demonstrated upregulation of IFITMs and reduced invasive abilities. Transduction experiments confirmed that IFITM1 contributed to the decreased cell invasion. Similarly, migration of trophoblast giant cells, the mouse equivalent of human EVCTs, was significantly reduced in poly (I:C)-treated mice. Finally, analysis of CMV- and bacterial-infected human placentas revealed upregulated IFITM1 expression. These data demonstrate that high levels of IFITM1 impair trophoblast invasion and could explain the placental dysfunctions associated with IFN-mediated disorders.

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-β and type III IFNs; however, this did not occur in RuV-infected BeWo trophoblasts. IFN-β 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.

Genome-Wide Analysis of Hypoxia-Inducible Factor Binding Reveals Targets Implicated in Impaired Human Placental Syncytiotrophoblast Formation under Low Oxygen

Preeclampsia (PE) is a common and serious complication of pregnancy with no cure except premature delivery. The root cause of PE is improper development of the placenta-the temporary organ supporting fetal growth and development. Continuous formation of the multinucleated syncytiotrophoblast (STB) layer via differentiation and fusion of cytotrophoblasts (CTBs) is vital for healthy placentation and is impaired in preeclamptic pregnancies. In PE, there is reduced/intermittent placental perfusion, likely resulting in a persistently low O2 environment. Low O2 inhibits differentiation and fusion of CTBs into STB and may thus contribute to PE pathogenesis; however, the underlying mechanisms are unknown. Because low O2 activates a transcription factor complex in cells known as the hypoxia-inducible factor (HIF), the objective of this study was to investigate whether HIF signaling inhibits STB formation by regulating genes required for this process. Culture of primary CTBs, the CTB-like cell line BeWo, and human trophoblast stem cells under low O2 reduced cell fusion and differentiation into STB. Knockdown of aryl hydrocarbon receptor nuclear translocator (a key component of the HIF complex) in BeWo cells restored syncytialization and expression of STB-associated genes under different O2 levels. Chromatin immunoprecipitation sequencing facilitated the identification of global aryl hydrocarbon receptor nuclear translocator/HIF binding sites, including several near genes implicated in STB development, such as ERVH48-1 and BHLHE40, providing new insights into mechanisms underlying pregnancy diseases linked to poor placental O2 supply.

Laminin switches terminal differentiation fate of human trophoblast stem cells under chemically defined culture conditions

Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development invitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we neither utilize forskolin for STB formation nor transforming growth factor-beta (TGFβ) inhibitors or a passage step for EVT differentiation. Strikingly, the presence of a single additional extracellular cue-laminin-111-switched the terminal differentiation of hTSCs from STB to the EVT lineage under these conditions. In the absence of laminin-111, STB formation occurred, with cell fusion comparable to that obtained with differentiation mediated by forskolin; however, in the presence of laminin-111, hTSCs differentiated to the EVT lineage. Protein expression of nuclear hypoxia-inducible factors (HIF1α and HIF2α) was upregulated during EVT differentiation mediated by laminin-111 exposure. A heterogeneous mixture of Notch1+ EVTs in colonies and HLA-G+ single-cell EVTs were obtained without a passage step, reminiscent of heterogeneity invivo. Further analysis showed that inhibition of TGFβ signaling affected both STB and EVT differentiation mediated by laminin-111 exposure. TGFβ inhibition during EVT differentiation resulted in decreased HLA-G expression and increased Notch1 expression. On the other hand, TGFβ inhibition prevented STB formation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation and will enable mechanistic studies invitro.

Endogenous retrovirus-derived enhancers confer the transcriptional regulation of human trophoblast syncytialization.

Endogenous retroviruses (ERVs) have been proposed as a driving force for the evolution of the mammalian placenta, however, the contribution of ERVs to placental development and the underlying regulatory mechanism remain largely elusive. A key process of placental development is the formation of multinucleated syncytiotrophoblasts (STBs) in direct contact with maternal blood, through which constitutes the maternal-fetal interface critical for nutrient allocation, hormone production and immunological modulation during pregnancy. We delineate that ERVs profoundly rewire the transcriptional program of trophoblast syncytialization. Here, we first determined the dynamic landscape of bivalent ERV-derived enhancers with dual occupancy of H3K27ac and H3K9me3 in human trophoblast stem cells (hTSCs). We further demonstrated that enhancers overlapping several ERV families tend to exhibit increased H3K27ac and reduced H3K9me3 occupancy in STBs relative to hTSCs. Particularly, bivalent enhancers derived from the Simiiformes-specific MER50 transposons were linked to a cluster of genes important for STB formation. Importantly, deletions of MER50 elements adjacent to several STB genes, including MFSD2A and TNFAIP2, significantly attenuated their expression concomitant to compromised syncytium formation. Together, we propose that ERV-derived enhancers, MER50 specifically, fine-tune the transcriptional networks accounting for human trophoblast syncytialization, which sheds light on a novel ERV-mediated regulatory mechanism underlying placental development.

Directed Differentiation of Human Pluripotent Stem Cells to Cytotrophoblast and Syncytiotrophoblast.

Human pluripotent stem cells (hPSCs) form an ideal system to study the formation of placental cells, from an undifferentiated human embryonic stem cell state. The conventional human in vitro model systems to study the human placenta cannot be employed for understanding placental dysfunctions or the development of specialized placental cell types. Hence, human PSCs make an ideal model system to study human placental development and disorders. Here, we describe an efficient and validated protocol to reproducibly study the formation of human cytotrophoblasts (CTBs) and syncytiotrophoblast (STBs) from undifferentiated hPSCs. CTBs are the trophoblast stem cells that can differentiate into specialized placental cell types such as STBs. The multinucleated STB plays vital role in the exchange of nutrients and gases across the placenta and secretes several hormones during pregnancy, such as human chorionic gonadotropin β (hCGβ). Here we describe two methods of seeding the hPSCs: chemical (clumps method) and enzymatic methods (single cells) to differentiate them to CTB and STB, activating BMP (B) signaling and inhibiting ACTIVIN/NODAL and FGF signaling pathways (2i), thus naming our protocol as "B2i" (Sudheer et al., Stem Cells Dev 21:2987-3000, 2012). This protocol forms the perfect model system for understanding in vitro placentation, modeling diseases arising from abnormal placentation that cause complications such as miscarriage, preeclampsia or intrauterine growth restriction (IUGR), and drug discovery for placental disorders.

Decreased junctional adhesion molecule 3 expression induces reactive oxygen species production and apoptosis in trophoblasts†.

Junctional adhesion molecule 3 (JAM3) is involved in epithelial cell junction, cell polarity, and motility. The molecular mechanisms underlying the role of JAM3 in placental dysfunction remain unclear. We hypothesized that JAM3 expression regulates trophoblast fusion, differentiation, proliferation, and apoptosis. Our results revealed that JAM3 was expressed in the cytotrophoblasts and syncytiotrophoblasts of first-trimester and term placental villi. JAM3 expression in cell-cell junctions decreased with the formation of syncytiotrophoblasts. Using trophoblasts as an in vitro model, we observed that forskolin and JAM3 knockdown significantly reduced JAM3 expression and increased syncytium formation. JAM3 knockdown additionally inhibited trophoblast proliferation and increased the number of trophoblasts in the sub-G1 and G2/M phases, indicating cell-cycle disturbance and apoptosis. Cell-cycle arrest was associated with the engagement of checkpoint kinase 2-cell division cycle 25C-cyclin-dependent kinase 1/cyclin B1 signaling. Increased expression of BIM, NOXA, XAF1, cytochrome c, and cleaved caspase-3 further indicated trophoblast apoptosis. Overexpression of JAM3 or recombinant JAM3 protein enhanced trophoblast adhesion and migration, which were inhibited by JAM3 knockdown. JAM3 knockdown induced reactive oxygen species and syncytin 2 expression in trophoblasts. Furthermore, H2O2-induced oxidative stress reduced JAM3 expression in trophoblasts and cell culture supernatants. H2O2 simultaneously induced trophoblast apoptosis. JAM3 expression was significantly decreased in the plasmas and placentas of patients with early-onset severe preeclampsia. Thus, our results show that JAM3 may not only be a structural component of trophoblast cell junctions but also regulates trophoblast fusion, differentiation, proliferation, apoptosis, and motility. Dysregulated trophoblast JAM3 expression is crucial in preeclampsia development.

How trophoblasts fuse: an in-depth look into placental syncytiotrophoblast formation.

In humans, cell fusion is restricted to only a few cell types under normal conditions. In the placenta, cell fusion is a critical process for generating syncytiotrophoblast: the giant multinucleated trophoblast lineage containing billions of nuclei within an interconnected cytoplasm that forms the primary interface separating maternal blood from fetal tissue. The unique morphology of syncytiotrophoblast ensures that nutrients and gases can be efficiently transferred between maternal and fetal tissue while simultaneously restricting entry of potentially damaging substances and maternal immune cells through intercellular junctions. To maintain integrity of the syncytiotrophoblast layer, underlying cytotrophoblast progenitor cells terminate their capability for self-renewal, upregulate expression of genes needed for differentiation, and then fuse into the overlying syncytium. These processes are disrupted in a variety of obstetric complications, underscoring the importance of proper syncytiotrophoblast formation for pregnancy health. Herein, an overview of key mechanisms underlying human trophoblast fusion and syncytiotrophoblast development is discussed.

Elucidating the Role of Hypoxia Signaling in Placental Trophoblast Differentiation

IntroductionPreeclampsia is a common and severe disease of pregnancy presenting as hypertension and blood vessel damage in mothers that can lead to organ failure, stroke, and death of the mother and/or baby. There is no cure except early delivery of the baby, which poses immediate and long‐term health risks for the prematurely delivered infant. The root cause of preeclampsia is improper development of the placenta – the temporary organ supporting growth of the baby in the womb. Continuous formation of the placental syncytiotrophoblast (STB) via differentiation and fusion of progenitor cytotrophoblast (CTB) cells is vital for healthy placentation and is impaired in preeclampsia. Preeclamptic placentas are also associated with chronic hypoxia, or insufficient O2, which activates the hypoxia‐inducible factor (HIF) transcriptional complex to elicit an adaptive cellular response. Past studies suggest that hypoxia inhibits CTB differentiation into STB, yet the underlying mechanisms are not well understood.ObjectiveThe goal of our study is to assess whether HIF activity inhibits CTB differentiation and to identify genes regulated by HIF that are involved. Therefore, we hypothesize that hypoxia‐HIF signaling impairs STB formation by interfering with transcription of genes needed for CTB differentiation.MethodsCells of the CTB‐like cell line, BeWo, were transduced with lentivirus carrying shRNA specific to ARNT (a key component of the HIF complex) or control shRNA. Cells were induced to differentiate into STB by treating with 8‐Bromo‐cAMP for 48 h under hypoxic (1% O2) or standard culture (20% O2) conditions. The capacity to form STB was evaluated using qRT‐PCR to assess transcript levels of CTB and STB markers as well as immunofluorescence to quantify multinucleated, hormone‐producing cells. Data were analyzed using two‐way ANOVA followed by Holm‐Sidak’s multiple comparisons test, with P<0.05 considered significant.ResultsBeWo cells induced to differentiate under hypoxic conditions showed decreased STB‐associated transcripts (75%, 86%, and 99% decreased ERVFRD‐1, OVOL1, and CGB expression respectively) as well as an 82% reduction in percent fusion compared to cells differentiated under standard conditions (N=3, P<0.05). Knockdown of ARNT restored the capacity of cells to form STB as evidenced by an induction of STB‐associated transcripts (18, 20, and 17‐fold increased ERVFRD‐1, OVOL1, and CGBexpression respectively) as well as a 5.6‐fold increase in percent fusion for ARNT‐knockdown versus control cells induced to differentiate under hypoxic conditions (N=3, P<0.05).ConclusionHypoxia represses expression of genes vital for CTB differentiation, impairing STB formation. ARNT knockdown prevents this inhibitory effect, suggesting HIF plays a role.SignificanceThis study provides insight into mechanisms underlying disrupted STB formation during hypoxia and in preeclampsia pathogenesis. Future work will identify genes regulated by HIF that are involved, which will open doors to develop novel therapeutics that could restore impaired STB formation and improve disease prevention and management.

Inefficient development of syncytiotrophoblasts in the Atp11a-deficient mouse placenta

The P4-ATPases ATP11A and ATP11C function as flippases at the plasma membrane to translocate phosphatidylserine from the outer to the inner leaflet. We herein demonstrated that Atp11a-deficient mouse embryos died at approximately E14.5 with thin-walled heart ventricles. However, the cardiomyocyte- or epiblast-specific Atp11a deletion did not affect mouse development or mortality. ATP11C may have compensated for the function of ATP11A in most of the cell types in the embryo. On the other hand, Atp11a, but not Atp11c, was expressed in the mouse placenta, and the Atp11a-null mutation caused poor development of the labyrinthine layer with an increased number of TUNEL-positive foci. Immunohistochemistry and electron microscopy revealed a disorganized labyrinthine layer with unfused trophoblasts in the Atp11a-null placenta. Human placenta-derived choriocarcinoma BeWo cells expressed the ATP11A and ATP11C genes. A lack of ATP11A and ATP11C eliminated the ability of BeWo cells to flip phosphatidylserine and fuse when treated with forskolin. These results indicate that flippases at the plasma membrane play an important role in the formation of syncytiotrophoblasts in placental development.