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Non-invasive prenatal testing (NIPT) investigates placental DNA and may detect confined placental mosaicism (CPM). The aim of this study was to confirm CPM in the term placenta in cases with abnormal NIPT but normal follow-up cytogenetic studies of fetus and mother. Additionally we examined the distribution of abnormal cells over the placenta. Four chorionic villus (CV) biopsies from four placental quadrants were requested in cases where CPM was assumed. Both cell lineages of the CV, cytotrophoblast (CTB) and mesenchymal core (MC), were analyzed separately with SNP array. The chromosome aberration was confirmed in 67 % of the placentas. Three quarters of the CTB and MC biopsies from these mosaic placentas were uniformly normal (57 %) or abnormal (20 %), and a minority showed mosaicism. Among 16 cases of CPM where first trimester CV were examined as well, 11 had chromosomally normal results during pregnancy. Cytogenetic investigations of term placental biopsies suspected to be affected with CPM did not reveal the chromosome aberration in one third of the placentas. This is caused by the patchy pattern in which chromosomally abnormal cells are distributed over the placenta with the majority of the biopsies being uniformly normal. Further CPM research, including its clinical impact, requires the analysis of more than four biopsies to get insight into the extent of the affected part. Moreover, a subset of CPM type 1 and 3 seems to be only detectable with NIPT and not with first trimester CVS. • Confirming confined placental mosaicism (CPM) in term placentas is challenging. • CPM exhibits a patchy pattern over the placenta with most biopsies uniformly normal. • A subset of CPM type 1 and 3 seems to be only detectable with NIPT and not with CVS. [ABSTRACT FROM AUTHOR]

Malaria during pregnancy has been associated with significant risks to both the mother and the fetus, leading to complications such as anemia, low birth weight, and increased infant mortality. The trophoblast cells, a key component of the placenta, are crucial for nutrient and oxygen exchange between mother and fetus. The differentiation of cytotrophoblasts (CTBs) into syncytiotrophoblasts (STBs) is critical for proper pregnancy development. These cells form the bi-stratified epithelium surrounding the placental villi. While previous studies have described an inflammatory activation of STB cells exposed to Plasmodium falciparum-infected erythrocytes (P. falciparum-IE) or components such as hemozoin (HZ), little is known about the direct effect this parasite may have on the epithelial turnover and function of trophoblast cells. This study aims to contribute to understanding mechanisms leading to placental damage during placental malaria using a BeWo cell line as a differentiation model. It was found that P. falciparum-IE interferes with the fusion of BeWo cells, affecting the differentiation process of trophoblast. A reduction in syncytialization could be associated with the adverse effects of infection in fetal health, altering the remodeling of the trophoblast epithelial barrier and reducing their capacity to exchange substances. However, further studies are necessary to assess alterations in the functionality of this epithelium. [ABSTRACT FROM AUTHOR]

AbstractIntroduction: We investigated the role of E-cadherin and Ber-EP4 in tubal pregnancy by comparing their expressions in epithelial and trophoblastic cells both in ectopic tubal and intrauterine pregnancies. Methods: The Formalin-fixed paraffin embedded blocks of 17 intrauterine and 17 tubal pregnancies were immunohistochemically stained with E-cadherin and Ber-EP4. Results: E-cadherin was expressed in the epithelium, villous and extravillous trophoblast in tubal and intrauterine pregnancies but not in the syncytiotrophoblast. The staining intensity was lower in the extra-villous trophoblast in tubal ectopic pregnancies compared with intrauterine pregnancies. Ber-EP4 was expressed in the epithelium of tubal and intrauterine pregnancies and only in villous cytotrophoblast. The intensity of staining in tubal pregnancy was higher than in intrauterine pregnancy. Discussion: The loss of E-cadherin expression in extra-villous trophoblast and increased expression of Ber-EP4 in the villous cytotrophoblast may play a role in the formation of tubal pregnancy by allowing the blastocyst to attach to the tubal epithelium. [ABSTRACT FROM AUTHOR]

CYP19A1 encodes aromatase, which converts testosterone to estrogen, and is induced during placental maturation. To elucidate the molecular mechanism underlying this function, histone methylation was analyzed using the placental cytotrophoblast cell line, JEG3. Treatment of JEG3 cells with 3-deazaneplanocin A, an inhibitor of several methyltransferases, resulted in increased CYP19A1 expression, accompanied by removal of the repressive mark H3K27me3 from the CYP19A1 promoter. However, this increase was not observed in cells treated with GSK126, another specific inhibitor for H3K27me3 methylation. Expression of TFAP2C , which encodes AP-2γ, a transcription factor that regulates CYP19A1 , was also elevated on 3-deazaneplanocin A treatment. Interestingly, TFAP2C messenger RNA (mRNA) was readily degraded in JEG3 cells but protected from degradation in the presence of 3-deazaneplanocin A. TFAP2C mRNA contained N6-methyladenosines, which were reduced on drug treatment. These observations indicate that the TFAP2C mRNA undergoes adenosine methylation and rapid degradation, whereas 3-deazaneplanocin A suppresses methylation, resulting in an increase in AP-2γ levels. We conclude that the increase in AP-2γ expression via stabilization of the TFAP2C mRNA is likely to underlie the increased CYP19A1 expression. [ABSTRACT FROM AUTHOR]

The trophoblast cells are responsible for the transfer of nutrients between the mother and the foetus and play a major role in placental endocrine function by producing and releasing large amounts of hormones and growth factors. Syncytiotrophoblast cells (STB), formed by the fusion of mononuclear cytotrophoblasts (CTB), constitute the interface between the foetus and the mother and are essential for all of these functions. We performed transcriptome analysis of human placental samples from two control groups—live births (LB), and stillbirths (SB) with a clinically recognised cause—and from our study group, idiopathic stillbirths (iSB). We identified 1172 DEGs in iSB, when comparing with the LB group; however, when we compared iSB with the SB group, only 15 and 12 genes were down- and upregulated in iSB, respectively. An assessment of these DEGs identified 15 commonly downregulated genes in iSB. Among these, several syncytiotrophoblast markers, like genes from the PSG and CSH families, as well as ALPP, KISS1, and CRH, were significantly downregulated in placental samples from iSB. The transcriptome analysis revealed underlying differences at a molecular level involving the syncytiotrophoblast. This suggests that defects in the syncytial layer may underlie unexplained stillbirths, therefore offering insights to improve clinical obstetrics practice. [ABSTRACT FROM AUTHOR]

Objectives: Necroptosis, a form of programmed cell death, can occur in the placenta of patients with preeclampsia (PE). Hydrogen sulfide (H2S) can inhibit necroptosis of human umbilical vein endothelial cells under the high glucose-induced injury. Whether H2S can protect trophoblasts against necroptosis underlying PE has not been elucidated. This study aimed to explore the protective role of H2S in trophoblast cells against necroptosis underlying PE. Design: This is an in vitro experimental study. Participants: A total of 10 pregnant women with severe PE and 10 matched control normotensive pregnant women were included. The placenta tissues were extracted from participators. The human JEG-3 trophoblasts were commercially available. Methods: The expression and localization of necrotic proteins were assayed in human placenta samples, and the effect of necrotic cell death on the proliferation and apoptosis of human JEG-3 trophoblasts was evaluated. The component expressions of inflammatory cytokine and p38MAPK signaling pathway were measured in samples pretreated with or without NaHS (H2S donor) and SB203580 (p38 inhibitor). Results: RIPA1, RIPA3, and p-p38 levels were significantly higher in PE placental tissue, whereas cystathionine β-synthase expression was decreased. In JEG-3 trophoblasts, necroptosis increased apoptotic cell numbers, suppressed cell proliferation, increased inflammatory cytokine expression, and increased p38MAPK activation, which can be prevented by NaHS. Limitations: In the present study, we did not provide sufficient evidence that necroptosis was a part of the pathogenesis of PE. Conclusions: We proposed the putative role of necroptosis in early-onset PE, reflected by the blockage of caspase-8/3 and increased expression of RIPA1 and RIPA3 in PE placenta tissues. Furthermore, we demonstrated that exogenous H2S protected cytotrophoblasts against ceramide-induced necroptosis via the p38MAPK pathway. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) is a heterogeneous disease for which the current clinical classification system is based on the presence or absence of specific clinical features. PE-associated placentas also show heterogeneous findings on pathologic examination, suggesting that further subclassification is possible. We combined clinical, pathologic, immunohistochemical, and transcriptomic profiling of placentas to develop integrated signatures for multiple subclasses of PE. In total, 303 PE and 1388 nonhypertensive control placentas were included. We found that maternal vascular malperfusion (MVM) in the placenta was associated with preterm PE with severe features and with small-for-gestational-age neonates. Interestingly, PE placentas with either MVM or no histologic pattern of injury showed a linear decrease in proliferative (p63+) cytotrophoblast per villous area with increasing gestational age, similar to placentas obtained from the nonhypertensive patient cohort; however, PE placentas with fetal vascular malperfusion or villitis of unknown etiology lost this phenotype. This is mainly because of cases of fetal vascular malperfusion in placentas of patients with preterm PE and villitis of unknown etiology in placentas of patients with term PE, which are associated with a decrease or increase, respectively, in the cytotrophoblast per villous area. Finally, a transcriptomic analysis identified pathways associated with hypoxia, inflammation, and reduced cell proliferation in PE-MVM placentas and further subclassified this group into extravillous trophoblast-high and extravillous trophoblast-low PE, confirmed using an immunohistochemical analysis of trophoblast lineage-specific markers. Our findings suggest that within specific histopathologic patterns of placental injury, PE can be subclassified based on specific cellular and molecular defects, allowing the identification of pathways that may be targeted for diagnostic and therapeutic purposes.

[This corrects the article DOI: 10.3389/fcell.2021.702046.].

Trophoblast differentiation is a crucial process in the formation of the placenta where cytotrophoblasts (CTs) differentiate and fuse to form the syncytiotrophoblast (ST). The bioactive components of cannabis, such as Δ9-THC, are known to disrupt trophoblast differentiation and fusion, as well as mitochondrial dynamics and respiration. However, less is known about the impact of cannabidiol (CBD) on trophoblast differentiation. Due to the central role of mitochondria in stem cell differentiation, we evaluated the impact of CBD on trophoblast mitochondrial function and differentiation. Using BeWo b30 cells, we observed decreased levels of mRNA for markers of syncytialization (GCM1, ERVW1, hCG) following 20 µM CBD treatment during differentiation. In CTs, CBD elevated transcript levels for the mitochondrial and cellular stress markers HSP60 and HSP70, respectively. Furthermore, CBD treatment also increased the lipid peroxidation and oxidative damage marker 4-hydroxynonenal. Mitochondrial membrane potential, basal respiration and ATP production were diminished with the 20 µM CBD treatment in both sub-lineages. mRNA levels for endocannabinoid system (ECS) components (FAAH, NAPEPLD, TRPV1, CB1, CB2, PPARγ) were altered differentially by CBD in CTs and STs. Overall, we demonstrate that CBD impairs trophoblast differentiation and fusion, as well as mitochondrial bioenergetics and redox homeostasis. [ABSTRACT FROM AUTHOR]

ABSTRACT The placenta is a critical barrier against viral, bacterial, and eukaryotic pathogens. For most teratogenic pathogens, the precise molecular mechanisms of placental resistance are still being unraveled. Given the importance of understanding these mechanisms and challenges in replicating trophoblast-pathogen interactions using in vitro models, we tested an existing stem-cell-derived model of trophoblast development for its relevance to infection with Toxoplasma gondii. We grew human trophoblast stem cells (TSCT) under conditions leading to either syncytiotrophoblast (TSSYN) or cytotrophoblast (TSCYT) and infected them with T. gondii. We evaluated T. gondii proliferation and invasion, cell ultrastructure, as well as for transcriptome changes after infection. TSSYNs cells showed similar ultrastructure compared to primary cells and villous explants when analyzed by transmission electron microscopy and scanning electron microscopy (SEM), a resistance to T. gondii adhesion could be visualized on the SEM level. Furthermore, TSSYNs were highly refractory to parasite adhesion and replication, while TSCYTs were not. RNA-seq data on mock-treated and infected cells identified differences between cell types as well as how they responded to T. gondii infection. We also evaluated if TSSC-derived SYNs and CYTs had distinct resistance profiles to another vertically transmitted facultative intracellular pathogen, Listeria monocytogenes. We demonstrate that TSSYNs are highly resistant to L. monocytogenes, while TSCYTs are not. Like T. gondii, TSSYN resistance to L. monocytogenes was at the level of bacterial adhesion. Altogether, our data indicate that stem-cell-derived trophoblasts recapitulate resistance profiles of primary cells to T. gondii and highlight the critical importance of the placental surface in cell-autonomous resistance to teratogens.IMPORTANCECongenital toxoplasmosis can cause a devastating consequence to the fetus. To reach the fetus’s tissues, Toxoplasma gondii must cross the placenta barrier. However, how this parasite crosses the placenta and the precise molecular mechanisms of placental resistance to this parasite are still unknown. In this study, we aimed to characterize a new cellular model of human trophoblast stem cells to determine their resistance, susceptibility, and response to T. gondii. Syncytiotrophoblast derived from trophoblast stem cells recapitulate the resistance profile similarly to placenta cells. We also showed that these cells are highly resistant to Listeria monocytogenes, at the level of bacterial adhesion. Our results suggest that resisting pathogen adhesion/attachment may be a generalized mechanism of syncytiotrophoblast resistance, and trophoblast stem cells represent a promising model to investigate cell-intrinsic mechanisms of resistance to pathogen adhesion and replication.

The Bone Morphogenetic Protein (BMP) signaling pathway has established roles in early embryonic morphogenesis, particularly in the epiblast. More recently, however, it has also been implicated in development of extraembryonic lineages, including trophectoderm (TE), in both mouse and human. In this review, we will provide an overview of this signaling pathway, with a focus on BMP4, and its role in emergence and development of TE in both early mouse and human embryogenesis. Subsequently, we will build on these in vivo data and discuss the utility of BMP4-based protocols for in vitro conversion of primed vs. naïve pluripotent stem cells (PSC) into trophoblast, and specifically into trophoblast stem cells (TSC). PSC-derived TSC could provide an abundant, reproducible, and ethically acceptable source of cells for modeling placental development.

Malaria during pregnancy has been associated with significant risks to both the mother and the fetus, leading to complications such as anemia, low birth weight, and increased infant mortality. The trophoblast cells, a key component of the placenta, are crucial for nutrient and oxygen exchange between mother and fetus. The differentiation of cytotrophoblasts (CTBs) into syncytiotrophoblasts (STBs) is critical for proper pregnancy development. These cells form the bi-stratified epithelium surrounding the placental villi. While previous studies have described an inflammatory activation of STB cells exposed to Plasmodium falciparum-infected erythrocytes (P. falciparum-IE) or components such as hemozoin (HZ), little is known about the direct effect this parasite may have on the epithelial turnover and function of trophoblast cells. This study aims to contribute to understanding mechanisms leading to placental damage during placental malaria using a BeWo cell line as a differentiation model. It was found that P. falciparum-IE interferes with the fusion of BeWo cells, affecting the differentiation process of trophoblast. A reduction in syncytialization could be associated with the adverse effects of infection in fetal health, altering the remodeling of the trophoblast epithelial barrier and reducing their capacity to exchange substances. However, further studies are necessary to assess alterations in the functionality of this epithelium.

Trophoblast stem cells (TSCs) have recently been derived from human embryos and early-first-trimester placenta; however, aside from ethical challenges, the unknown disease potential of these cells limits their scientific utility. We have previously established a bone morphogetic protein 4 (BMP4)-based two-step protocol for differentiation of primed human pluripotent stem cells (hPSCs) into functional trophoblasts; however, those trophoblasts could not be maintained in a self-renewing TSC-like state. Here, we use the first step from this protocol, followed by a switch to newly developed TSC medium, to derive bona fide TSCs. We show that these cells resemble placenta- and naive hPSC-derived TSCs, based on their transcriptome as well as their in vitro and in vivo differentiation potential. We conclude that primed hPSCs can be used to generate functional TSCs through a simple protocol, which can be applied to a widely available set of existing hPSCs, including induced pluripotent stem cells, derived from patients with known birth outcomes.

Poly- and perfluroroalkylated substances (PFAS) are a major class of surfactants used in industry applications and consumer products. Despite efforts to reduce the usage of PFAS due to their environmental persistence, compounds such as perfluorooctanoic acid (PFOA) are widely detected in human blood and tissue. Although growing evidence supports that prenatal exposures to PFOA and other PFAS are linked to adverse pregnancy outcomes, the target organs and pathways remain unclear. Recent investigations in mouse and human cell lines suggest that PFAS may impact the placenta and impair trophoblast function. In this study, we investigated the effects of PFOA on cytotoxicity and the transcriptome in cultured second trimester human cytotrophoblasts (CTBs). We show that PFOA significantly reduces viability and induces cell death at 24 h, in a concentration-dependent manner. At subcytotoxic concentrations, PFOA impacted expression of hundreds of genes, including several molecules (CRH , IFIT1 , and TNFSF10) linked with lipid metabolism and innate immune response pathways. Furthermore, in silico analyses suggested that regulatory factors such as peroxisome proliferator-activated receptor-mediated pathways may be especially important in response to PFOA. In summary, this study provides evidence that PFOA alters primary human CTB viability and gene pathways that could contribute to placental dysfunction and disease. [ABSTRACT FROM AUTHOR]

The trophoblast cells are responsible for the transfer of nutrients between the mother and the foetus and play a major role in placental endocrine function by producing and releasing large amounts of hormones and growth factors. Syncytiotrophoblast cells (STB), formed by the fusion of mononuclear cytotrophoblasts (CTB), constitute the interface between the foetus and the mother and are essential for all of these functions. We performed transcriptome analysis of human placental samples from two control groups—live births (LB), and stillbirths (SB) with a clinically recognised cause—and from our study group, idiopathic stillbirths (iSB). We identified 1172 DEGs in iSB, when comparing with the LB group; however, when we compared iSB with the SB group, only 15 and 12 genes were down- and upregulated in iSB, respectively. An assessment of these DEGs identified 15 commonly downregulated genes in iSB. Among these, several syncytiotrophoblast markers, like genes from the PSG and CSH families, as well as ALPP, KISS1, and CRH, were significantly downregulated in placental samples from iSB. The transcriptome analysis revealed underlying differences at a molecular level involving the syncytiotrophoblast. This suggests that defects in the syncytial layer may underlie unexplained stillbirths, therefore offering insights to improve clinical obstetrics practice.

The HtrA serine peptidase 1 (HTRA1) is a multidomain secretory protein with serine–protease activity involved in the regulation of many cellular processes in both physiological and pathological conditions. HTRA1 is normally expressed in the human placenta, and its expression is higher in the first trimester compared to the third trimester, suggesting an important role of this serine protease in the early phases of human placenta development. The aim of this study was to evaluate the functional role of HTRA1 in in vitro models of human placenta in order to define the role of this serine protease in preeclampsia (PE). BeWo and HTR8/SVneo cells expressing HTRA1 were used as syncytiotrophoblast and cytotrophoblast models, respectively. Oxidative stress was induced by treating BeWo and HTR8/SVneo cells with H2O2 to mimic PE conditions in order to evaluate its effect on HTRA1 expression. In addition, HTRA1 overexpression and silencing experiments were performed to evaluate the effects on syncytialization, cell mobility, and invasion processes. Our main data showed that oxidative stress significantly increased HTRA1 expression in both BeWo and HTR8/SVneo cells. In addition, we demonstrated that HTRA1 has a pivotal role in cell motility and invasion processes. In particular, HTRA1 overexpression increased while HTRA1 silencing decreased cell motility and invasion in HTR8/SVneo cell model. In conclusion, our results suggest an important role of HTRA1 in regulating extravillous cytotrophoblast invasion and motility during the early stage of placentation in the first trimester of gestation, suggesting a key role of this serine protease in PE onset.

Background: Preeclampsia is a severe complication of pregnancy which is attributed to placental dysfunction. The retrotransposon, Paternal Expressed Gene 10 (PEG10) harbours critical placental functions pertaining to placental trophoblast cells. Limited evidence exists on whether PEG10 is involved in preeclampsia pathogenesis. This study characterised the expression and regulation of PEG10 in placentas from patients with early-onset preeclampsia compared to gestation-matched controls. Methods: PEG10 expression was measured in plasma and placentas collected from patients with early-onset preeclampsia (< 34 weeks') and gestation-matched controls using ELISA (protein) and RT-qPCR (mRNA). First-trimester human trophoblast stem cells (hTSCs) were used for in vitro studies. PEG10 expression was measured during hTSC differentiation and hTSC exposure to hypoxia (1% O2) and inflammatory cytokines (IL-6 and TNFα) using RT-qPCR. Functional studies used PEG10 siRNA to measure the effect of reduced PEG10 on canonical TGF- β signalling and proliferation using luciferase and xCELLigence assays, respectively. Results: PEG10 mRNA expression was significantly reduced in placentas from patients with early-onset preeclampsia (< 34 weeks' gestation) relative to controls (p = 0.04, n = 78 vs n = 18 controls). PEG10 protein expression was also reduced in preeclamptic placentas (p = 0.03, n = 5 vs n = 5 controls, blinded assessment of immunohistochemical staining), but neither PEG10 mRNA nor protein could be detected in maternal circulation. PEG10 was most highly expressed in hTSCs, and its expression was reduced as hTSCs differentiated into syncytiotrophoblasts (p < 0.0001) and extravillous trophoblasts (p < 0.001). Trophoblast differentiation was not altered when hTSCs were treated with PEG10 siRNA (n = 5 vs n = 5 controls). PEG10 was significantly reduced in hTSCs exposed to hypoxia (p < 0.01). PEG10 was also reduced in hTSCs treated with the inflammatory cytokine TNF α (p < 0.01), but not IL-6. PEG10 knocked down (siRNA) in hTSCs showed reduced activation of the canonical TGF-β signalling effector, the SMAD binding element (p < 0.05) relative to controls. PEG10 knockdown in hTSCs however was not associated with any significant alterations in proliferation. Conclusions: Placental PEG10 is reduced in patients with early-onset preeclampsia. In vitro studies suggest that hypoxia and inflammation may contribute to PEG10 downregulation. Reduced PEG10 alters canonical TGF- β signalling, and thus may be involved in trophoblast dysfunction associated with this pathway. [ABSTRACT FROM AUTHOR]

The human placenta and its specialized cytotrophoblasts rapidly develop, have a compressed lifespan, govern pregnancy outcomes, and program the offspring's health. Understanding the molecular underpinnings of these behaviors informs development and disease. Profiling the extraembryonic epigenome and transcriptome during the 2nd and 3rd trimesters revealed H3K9 trimethylation overlapping deeply DNA hypomethylated domains with reduced gene expression and compartment-specific patterns that illuminated their functions. Cytotrophoblast DNA methylation increased, and several key histone modifications decreased across the genome as pregnancy advanced. Cytotrophoblasts from severe preeclampsia had substantially increased H3K27 acetylation globally and at genes that are normally downregulated at term but upregulated in this syndrome. In addition, some cases had an immature pattern of H3K27ac peaks, and others showed evidence of accelerated aging, suggesting subtype-specific alterations in severe preeclampsia. Thus, the cytotrophoblast epigenome dramatically reprograms during pregnancy, placental disease is associated with failures in this process, and H3K27 hyperacetylation is a feature of severe preeclampsia.

During pregnancy, conceptus-derived extravillous trophoblast (EVT) invades the endomyometrium, anchors the placenta to the maternal uterus, and remodels the spiral arteries in order to establish maternal blood supply to the fetoplacental unit. Recent reports have described early gestation EVT as polyploid and senescent. Here, we extend these reports by performing comprehensive profiling of both the genomic organization and transcriptome of first trimester and term EVT. We define pathways and gene regulatory networks involved in both initial differentiation and maturation of this important trophoblast lineage at the maternal-fetal interface. Our results suggest that like first trimester EVT, term EVT undergoes senescence and endoreduplication, is primarily tetraploid, and lacks high rates of copy number variations. Additionally, we have highlighted senescence and polyploidy-related genes, pathways, networks, and transcription factors that appeared to be important in normal EVT differentiation and maturation and validated a key role for the unfolded protein response in this context.

The human placenta is a poorly-understood organ, but one that is critical for proper development and growth of the fetus in-utero. The epithelial cell type that contributes to primary placental functions is called "trophoblast," including two main subtypes, villous and extravillous trophoblast. Cytotrophoblast and syncytiotrophoblast comprise the villous compartment and contribute to gas and nutrient exchange, while extravillous trophoblast invade and remodel the uterine wall and vessels, in order to supply maternal blood to the growing fetus. Abnormal differentiation of trophoblast contributes to placental dysfunction and is associated with complications of pregnancy, including preeclampsia (PE) and fetal growth restriction (FGR). This review describes what is known about the cellular organization of the placenta during both normal development and in the setting of PE/FGR. It also explains known trophoblast lineage-specific markers and pathways regulating their differentiation, and how these are altered in the setting of PE/FGR, focusing on studies which have used human placental tissues. Finally, it also highlights remaining questions and needed resources to advance this field.

Argininosuccinate synthase (ASS1) is involved in nitric oxide production, which has a key role in placental development improving pregnancy outcomes. Syncytiotrophoblast and extravillous trophoblast differentiations are milestones of placental development and their impairment can cause pathologies, such as preeclampsia (PE) and fetal growth restriction (FGR). Immunohistochemistry and Western blotting were used to localize and quantify ASS1 in first trimester (8.2 ± 1.8 weeks), third trimester (38.6 ± 1.1 weeks), and PE (36.3 ± 1.5 weeks) placentas. In addition, cell cultures were used to evaluate ASS1 expression under hypoxic conditions and the syncytialization process. Our data showed that ASS1 is localized in the villous cytotrophoblast of first trimester, third trimester, and PE placentas, while the villous cytotrophoblast adjacent to the extravillous trophoblast of cell columns as well as the extravillous trophoblast were negative for ASS1 in first trimester placentas. In addition, ASS1 was decreased in third trimester compared to the first trimester placentas (p = 0.003) and no differences were detected between third trimester and PE placentas. Moreover, ASS1 expression was decreased in hypoxic conditions and syncytialized cells compared to those not syncytialized. In conclusion, we suggest that the expression of ASS1 in villous cytotrophoblast is related to maintaining proliferative phenotype, while ASS1 absence may be involved in promoting the differentiation of villous cytotrophoblast in extravillous cytotrophoblast of cell columns in first trimester placentas. [ABSTRACT FROM AUTHOR]

In humans, a subset of placental cytotrophoblasts (CTBs) invades the uterus and its vasculature, anchoring the pregnancy and ensuring adequate blood flow to the fetus. Appropriate depth is critical. Shallow invasion increases the risk of pregnancy complications, e.g., severe preeclampsia. Overly deep invasion, the hallmark of placenta accreta spectrum (PAS), increases the risk of preterm delivery, hemorrhage, and death. Previously a rare condition, the incidence of PAS has increased to 1:731 pregnancies, likely due to the rise in uterine surgeries (e.g., Cesarean sections). CTBs track along scars deep into the myometrium and beyond. Here we compared the global gene expression patterns of CTBs from PAS cases to gestational age-matched control cells that invaded to the normal depth from preterm birth (PTB) deliveries. The messenger RNA (mRNA) encoding the guanine nucleotide exchange factor, DOCK4, mutations of which promote cancer cell invasion and angiogenesis, was the most highly up-regulated molecule in PAS samples. Overexpression of DOCK4 increased CTB invasiveness, consistent with the PAS phenotype. Also, this analysis identified other genes with significantly altered expression in this disorder, potential biomarkers. These data suggest that CTBs from PAS cases up-regulate a cancer-like proinvasion mechanism, suggesting molecular as well as phenotypic similarities in the two pathologies.

BackgroundDuring pregnancy, the Zika flavivirus (ZIKV) infects human placentas, inducing defects in the developing fetus. The flavivirus nonstructural protein 1 (NS1) alters glycosaminoglycans on the endothelium, causing hyperpermeability in vitro and vascular leakage in vivo in a tissue-dependent manner. The contribution of ZIKV NS1 to placental dysfunction during ZIKV infection remains unknown.MethodsWe examined the effect of ZIKV NS1 on expression and release of heparan sulfate (HS), hyaluronic acid (HA), and sialic acid on human trophoblast cell lines and anchoring villous explants from first-trimester placentas infected with ZIKV ex vivo. We measured changes in permeability in trophoblasts and stromal cores using a dextran-based fluorescence assay and changes in HA receptor expression using immunofluorescent microscopy.ResultsZIKV NS1 in the presence and absence of ZIKV increased the permeability of anchoring villous explants. ZIKV NS1 induced shedding of HA and HS and altered expression of CD44 and lymphatic endothelial cell HA receptor-1, HA receptors on stromal fibroblasts and Hofbauer macrophages in villous cores. Hyaluronidase was also stimulated in NS1-treated trophoblasts.ConclusionsThese findings suggest that ZIKV NS1 contributes to placental dysfunction via modulation of glycosaminoglycans on trophoblasts and chorionic villi, resulting in increased permeability of human placentas.

ABSTRACT The Zika virus (ZIKV) is teratogenic and considered a TORCH pathogen (toxoplasmosis [Toxoplasma gondii], rubella, cytomegalovirus, herpes simplex virus [HSV], and other microorganisms capable of crossing the blood-placenta barrier). In contrast, the related flavivirus dengue virus (DENV) and the attenuated yellow fever virus vaccine strain (YFV-17D) are not. Understanding the mechanisms used by ZIKV to cross the placenta is necessary. In this work, parallel infections with ZIKV of African and Asian lineages, DENV, and YFV-17D were compared for kinetics and growth efficiency, activation of mTOR pathways, and cytokine secretion profile using cytotrophoblast-derived HTR8 cells and monocytic U937 cells differentiated to M2 macrophages. In HTR8 cells, ZIKV replication, especially the African strain, was significantly more efficient and faster than DENV or YFV-17D. In macrophages, ZIKV replication was also more efficient, although differences between strains were reduced. Greater activation of the mTORC1 and mTORC2 pathways in HTR8 cells infected with ZIKV than with DENV or YFV-17D was observed. HTR8 cells treated with mTOR inhibitors showed a 20-fold reduction in ZIKV yield, versus 5- and 3.5-fold reductions for DENV and YFV-17D, respectively. Finally, infection with ZIKV, but not DENV or YFV-17D, efficiently inhibited the interferon (IFN) and chemoattractant responses in both cell lines. These results suggest a gating role for the cytotrophoblast cells in favoring entry of ZIKV, but not DENV and YFV-17D, into the placental stroma. IMPORTANCE Zika virus acquisition during pregnancy is associated with severe fetal damage. The Zika virus is related to dengue virus and yellow fever virus, yet fetal damage has not been related to dengue or inadvertent vaccination for yellow fever during pregnancy. Mechanisms used by the Zika virus to cross the placenta need to be deciphered. By comparing parallel infections of Zika virus strains belonging to the African and Asian lineages, dengue virus, and the yellow fever vaccine virus strain YFV-17D in placenta-derived cytotrophoblast cells and differentiated macrophages, evidence was found that Zika virus infections, especially by the African strains, were more efficient in cytotrophoblast cells than dengue virus or yellow fever vaccine virus strain infections. Meanwhile, no significant differences were observed in macrophages. Robust activation of the mTOR signaling pathways and inhibition of the IFN and chemoattractant response appear to be related to the better growth capacity of the Zika viruses in the cytotrophoblast-derived cells.

Metformin is a widely prescribed medication whose mechanism of action is not completely defined and whose role in gestational diabetes management remains controversial. In addition to increasing the risk of fetal growth abnormalities and preeclampsia, gestational diabetes is associated with abnormalities in placental development including impairments in trophoblast differentiation. Given that metformin impacts cellular differentiation events in other systems, we assessed metformin’s impact on trophoblast metabolism and differentiation. Using established cell culture models of trophoblast differentiation, oxygen consumption rates and relative metabolite abundance were determined following 200 µM (therapeutic range) and 2000 µM (supra-therapeutic range) metformin treatment using Seahorse and mass-spectrometry approaches. While no differences in oxygen consumption rates or relative metabolite abundance were detected between vehicle and 200 µM metformin-treated cells, 2000 µM metformin impaired oxidative metabolism and increased the abundance of lactate and TCA cycle intermediates, α-ketoglutarate, succinate, and malate. Examining differentiation, treatment with 2000 μM, but not 200 µM metformin, impaired HCG production and expression of multiple trophoblast differentiation markers. Overall, this work suggests that supra-therapeutic concentrations of metformin impair trophoblast metabolism and differentiation whereas metformin concentrations in the therapeutic range do not strongly impact these processes.

We previously established a two-step protocol for differentiation of human pluripotent stem cells (hPSCs) into trophoblasts, using a StemPro-based minimal medium (EMIM) with bone morphogenetic protein-4 (BMP4). This protocol was suboptimal, resulting in induction of mixed mesoderm and trophoblast markers. Furthermore, adapting hPSCs to StemPro has proven difficult, and prolonged culture in this medium has been shown to promote genomic instability. Therefore, we moved on to the use of new media, including E8, and most recently, StemFlex, for rapid adaptation from feeder to non-feeder conditions. In the new protocol, we have incorporated the WNT inhibitor IWP2 into the first step, resulting in uniform differentiation of hPSCs into cytotrophoblast (CTB)-like cells, without induction of the mesoderm lineage. We also show that, at the end of the second step, there are distinct populations of terminally differentiated multinucleated human chorionic gonadotropin (hCG)-producing syncytiotrophoblast (STB) and HLAG+ extravillous trophoblast (EVT)-like cells. © 2019 by John Wiley & Sons, Inc.

Despite gradual legislative efforts to phase out flame retardants (FRs) from the marketplace, polybrominated diphenyl ethers (PBDEs) are still widely detected in human maternal and fetal tissues, eg, placenta, due to their continued global application in consumer goods and inherent biological persistence. Recent studies in rodents and human placental cell lines suggest that PBDEs directly cause placental toxicity. During pregnancy, trophoblasts play key roles in uterine invasion, vascular remodeling, and anchoring of the placenta-fetal unit to the mother. Thus, to study the potential consequences of PBDE exposures on human placental development, we used an in vitro model: primary villous cytotrophoblasts (CTBs). Following exposures, the endpoints that were evaluated included cytotoxicity, function (migration, invasion), the transcriptome, and the methylome. In a concentration-dependent manner, common PBDE congeners, BDE-47 and -99, significantly reduced cell viability and increased death. Upon exposures to sub-cytotoxic concentrations (≤ 5 µM), we observed BDE-47 accumulation in CTBs with limited evidence of metabolism. At a functional level, BDE-47 hindered the ability of CTBs to migrate and invade. Transcriptomic analyses of BDE-47 effects suggested concentration-dependent changes in gene expression, involving stress pathways, eg, inflammation and lipid/cholesterol metabolism as well as processes underlying trophoblast fate, eg, differentiation, migration, and vascular morphogenesis. In parallel assessments, BDE-47 induced low-level global increases in methylation of CpG islands, including a subset that were proximal to genes with roles in cell adhesion/migration. Thus, using a primary human CTB model, we showed that PBDEs induced alterations at cellular and molecular levels, which could adversely impact placental development.

An increasing body of evidence points to significant spatio-temporal differences in early placental development between mouse and human, but a detailed comparison of placentae in these two species is missing. We set out to compare placentae from both species across gestation, with a focus on trophoblast progenitor markers. We found that CDX2 and ELF5, but not EOMES, are expressed in early post-implantation trophoblast subpopulations in both species. Genome-wide expression profiling of mouse and human placentae revealed clusters of genes with distinct co-expression patterns across gestation. Overall, there was a closer fit between patterns observed in the placentae when the inter-species comparison was restricted to human placentae through gestational week 16 (thus, excluding full-term samples), suggesting that the developmental timeline in mouse runs parallel to the first half of human placental development. In addition, we identified VGLL1 as a human-specific marker of proliferative cytotrophoblast, where it is co-expressed with the transcription factor TEAD4. As TEAD4 is involved in trophectoderm specification in the mouse, we posit a regulatory role for VGLL1 in early events during human placental development.

Development of the early embryo takes place under low oxygen tension. Under such conditions, the embryo implants and the trophectoderm, the outer layer of blastocyst, proliferate, forming the cytotrophoblastic shell, the early placenta. The cytotrophoblasts (CTBs) are the so-called epithelial 'stem cells' of the placenta, which, depending on the signals they receive, can differentiate into either extravillous trophoblast (EVT) or syncytiotrophoblast (STB). EVTs anchor the placenta to the uterine wall and remodel maternal spiral arterioles in order to provide ample blood supply to the growing fetus. STBs arise through CTB fusion, secrete hormones necessary for pregnancy maintenance and form a barrier across which nutrient and gas exchange can take place. The bulk of EVT differentiation occurs during the first trimester, before the onset of maternal arterial blood flow into the intervillous space of the placenta, and thus under low oxygen tension. These conditions affect numerous signaling pathways, including those acting through hypoxia-inducible factor, the nutrient sensor mTOR and the endoplasmic reticulum stress-induced unfolded protein response pathway. These pathways are known to be involved in placental development and disease, and specific components have even been identified as directly involved in lineage-specific trophoblast differentiation. Nevertheless, much controversy surrounds the role of hypoxia in trophoblast differentiation, particularly with EVT. This review summarizes previous studies on this topic, with the intent of integrating these results and synthesizing conclusions that resolve some of the controversy, but then also pointing to remaining areas, which require further investigation.

Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during viral congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of human cytomegalovirus (hCMV) infection on sEVs production, composition, and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy, and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a potential proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from an ex vivo model of infected histocultures from early placenta. Based on these findings, we propose that placental sEVs could be important actors favoring viral dissemination to the fetal brain during hCMV congenital infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: To explore the effect of anti‐nuclear antibodies (ANAs) on the outcome of in vitro fertilization‐embryo transplantation (IVF‐ET) and to study the effect of ANAs in follicular fluid (FF) on embryonic development. Methods: The expression of ANAs in FF of patients treated with IVF‐ET and healthy group. The patients were divided into ANAs‐positive group and ANA‐negative group. The age, duration of infertility, body mass index (BMI), basic follicle stimulating hormone (bFSH), anti‐Mullerian hormone (AMH), number of retrieved oocytes, portion of metaphase II oocytes (MII), number of embryos in the cleavage stage, bipronuclear (2PN), number of embryos, number of high‐quality embryos and the outcome of IVF‐ET were compared between the two groups. In vitro, HTR8/SVneo trophoblast cells were cultivated and divided into ANAs‐negative group (control group) and ANAs‐positive group (ANAs‐positive FF was added to cytotrophoblasts). The ANAs titer in the serum and FF of patients who treated with IVF‐ET was detected using ELSIA method. CCK‐8 assay and flow cytometry (at 24 h and at 48 h) were used to detect the cell proliferation and apoptosis frequency of the two groups, respectively. Results: Among those who underwent IVF‐ET treatment, the number of retrieved oocytes, the number of fertilization and the portion of MII oocytes in the FF‐positive group were significantly lower than those in the FF‐negative group. Furthermore, the implantation rate and the clinical pregnancy rate were decreased, and early miscarriage rate was increased in the FF‐positive patients than those in the FF‐negative patients. In vitro, the cytotrophoblasts proliferation activity in the ANAs group was significantly lower than that in the control group. Moreover, the cytotrophoblasts apoptosis rate in the ANAs group was significantly higher than that in the control group. Conclusions: Our data suggested that ANAs in FF might become an obstacle to embryonic development through promoting trophoblast apoptosis and inhibiting trophoblast proliferation. ANAs in FF might be an unfavorable factor for the outcome of those who undergo IVF‐ET treatment. [ABSTRACT FROM AUTHOR]

In preeclampsia (PE), cytotrophoblast (CTB) invasion of the uterus and spiral arteries is often shallow. Thus, the placenta's role has been a focus. In this study, we tested the hypothesis that decidual defects are an important determinant of the placental phenotype. We isolated human endometrial stromal cells from nonpregnant donors with a previous pregnancy that was complicated by severe PE (sPE). Compared with control cells, they failed to decidualize in vitro as demonstrated by morphological criteria and the analysis of stage-specific antigens (i.e., IGFBP1, PRL). These results were bolstered by global transcriptional profiling data that showed they were transcriptionally inert. Additionally, we used laser microdissection to isolate the decidua from tissue sections of the maternal-fetal interface in sPE. Global transcriptional profiling revealed defects in gene expression. Also, decidual cells from patients with sPE, which dedifferentiated in vitro, failed to redecidualize in culture. Conditioned medium from these cells failed to support CTB invasion. To mimic aspects of the uterine environment in normal pregnancy, we added PRL and IGFBP1, which enhanced invasion. These data suggested that failed decidualization is an important contributor to down-regulated CTB invasion in sPE. Future studies will be aimed at determining whether this discovery has translational potential with regard to assessing a woman's risk of developing this pregnancy complication.

Preeclampsia (PE) is a prevalent obstetric complication affecting approximately 3-5% of pregnancies worldwide and is a major cause of maternal and perinatal morbidity and mortality. Preeclampsia is considered a disease of the endothelial system that can progress to eclampsia, characterized by seizures. Early diagnosis and appropriate management are crucial to improving maternal and fetal outcomes, as preeclampsia can lead to severe complications such as placental abruption, fetal growth restriction, and stroke. The pathophysiology of PE is complex, involving a combination of genetic, acquired, and immunological factors. A central feature of the condition is inadequate placentation and impaired uteroplacental perfusion, leading to local hypoxia, endothelial dysfunction, vasoconstriction, and immunological dysregulation. Recent evidence suggests that dysregulation of ion transporters may play a significant role in the adaptation of uterine circulation during placentation. These transporters are essential for maintaining maternal-fetal homeostasis, influencing processes such as nutrient exchange, hormone synthesis, trophoblast cell migration, and the function of smooth muscle cells in blood vessels. In preeclampsia, adverse conditions like hypoxia and oxidative stress result in the downregulation of ion, solute, and water transporters, impairing their function. This review focuses on membrane transporters involved in PE, discussing functional alterations and their physiological implications. The goal of this investigation is to enhance understanding of how dysregulation of ion and small molecule transporters contributes to the development and progression of preeclampsia, underscoring the importance of exploring these signaling pathways for potential therapeutic interventions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Recent advances in the use of trophoblast stem cells and organoid models have markedly enhanced our understanding of placental development and function. These models offer significant improvements over previous systems due to their extended viability in culture and capacity to replicate various trophoblast functions, such as extravillous trophoblast invasion, syncytialisation and 3D architecture. Initially, the generation of trophoblast organoids was confined to first trimester placental tissue; however, it was recently reported that term placentae can also serve as a source of trophoblast stem cells. Here, we report that both 2D proliferative cytotrophoblasts and 3D trophoblast organoids can be effectively derived from cryopreserved term cytotrophoblasts isolated by the 'Kliman' method of sequential trypsin digestion and Percoll density gradient centrifugation, when cultured in specialised medium. This was confirmed by the expression of characteristic trophoblast markers including cytokeratin-7, E-cadherin, and human chorionic gonadotropin beta (β-hCG). The proliferative cytotrophoblasts were induced to differentiate to syncytiotrophoblasts, marked by elevated β-hCG expression, reduced Ki67-positive nuclei, and a fused syncytial phenotype. The protocol described here enables the application of organoid models and in vitro functional studies to stored cytotrophoblast samples for the study of placental function from unique patient cohorts. Moreover, the utilization of term placental sources may alleviate ethical concerns with using cells from pregnancy terminations, thus expanding access for a broader research community., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier Ltd.)

The placenta plays a fundamental role in fetal growth and maintenance of pregnancy. Its cellular components include a large multinucleated syncytiotrophoblast (STB) and its progenitor, cytotrophoblasts (CTBs), both of which perform vital functions in the human placenta. Primary cytotrophoblasts isolated from term human placentas that spontaneously fuse and differentiate into syncytiotrophoblast-like cells in vitro have been utilized to investigate the functions of the syncytiotrophoblast and placenta with multiple modifications. Although recent advances have enabled the use of trophoblast stem cell-derived organoids as a model for villous trophoblasts, primary CTBs offer several advantages, including spontaneous differentiation, easy access to materials (from term-delivered human placentas), and simple methodology. Here, we present a precise step-by-step process for isolating pure CTBs from term human placenta based on previously reported placenta digestion, density centrifugation, and CTB purification using anti-HLA-A, B, C antibody. Subsequently, we provide a method to improve CTB viability and differentiation into STB-like cells using epidermal growth factor (EGF) and a ROCK inhibitor (Y-27632) that ensures long-term and stable cultures without altering their proliferation. Because these cells can grow on standard tissue culture plates, this model can be easily utilized for various placental investigations, including innate immune responses, drug resistance, and STB metabolism. Employing this approach considerably enhances our understanding of placental functions, which are key to maternal and offspring health., Competing Interests: Declaration of competing interest The authors declare that there are no potential conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The mechanism by which human cytotrophoblast cells (CTB) differentiate into extravillous trophoblast cells (EVT) is an epithelial-mesenchymal transition (EMT). Polarized CTB, anchored in an epithelial layer, are transformed into motile, non-polar EVT which invade the uterus. Our previous research has shown that over gestation, invasive first trimester EVT are converted to a non-invasive phenotype showing a reduced degree of EMT. We hypothesized that in an under-invasion pathology, such as early onset preeclampsia, third trimester EVT would display a less advanced EMT profile than controls. The goal of this study was to determine whether expression of EMT-associated genes in the EVT of early onset preeclamptics shows a less mesenchymal, more epithelial phenotype compared to control pregnancies. Measures of preeclamptic CTB and EVT gene expression, using highly purified cells from third trimester, early onset preeclamptics and gestational-age matched controls, showed clear evidence of a phenotypic pattern characteristic of an EMT. Comparison of preeclamptic EVT to gestational-age matched, control EVT demonstrated multiple changes in gene expression, including changes in well-known EMT gene markers, indicative of a more limited EMT. These changes are not explained by differences in the preeclamptic CTB precursors. In this first study of purified third trimester EVT, we show that the pattern of gene expression corresponding to EMT-associated differentiation is diminished in early onset preeclampsia. This provides a mechanistic framework for many of the molecular changes observed in preeclampsia and presents an opportunity for detailed studies of the pathways regulating the aberrant EMT and for potential biomarkers of the process. [ABSTRACT FROM AUTHOR]

In humans, the hemochorial placenta is a unique temporary organ that forms during pregnancy to support fetal development, gaseous exchange, delivery of nutrition, removal of waste products, and provides immune protection, while maintaining tolerance to the HLA-haploidentical fetus. In this review, we characterize decidual and placental immunity duringmaternal viral (co)-infectionwith HIV-1, human cytomegalovirus (HCMV), and Zika virus. We discuss placental immunology, clinical presentation, and epidemiology, before characterizing host susceptibility and cellular tropism, and how the three viruses gain access into specific placental target cells. We describe current knowledge on host-viral interactions with decidual and stromal human placental macrophages or Hofbauer cells, trophoblasts including extra villous trophoblasts, T cells, and decidual natural killer (dNK) cells. These clinically significant viral infections elicit both innate and adaptive immune responses to control replication. However, the three viruses either during mono- or co-infection (HIV-1 and HCMV) escape detection to initiate placental inflammation associated with viral transmission to the developing fetus. Aside from congenital or perinatal infection, other adverse pregnancy outcomes include preterm labor and spontaneous abortion. In addition, maternal HIV-1 and HCMV co-infection are associated with impaired fetal and infant immunity in postnatal life and poor clinical outcomes during childhood in exposed infants, even in the absence of vertical transmission of HIV-1. Given the rapidly expanding numbers of HIV-1-exposed uninfected infants and children globally, further research is urgently needed on neonatal immune programming during maternal mono-and co-infection. This review therefore includes sections on current knowledge gaps thatmay prompt future research directions. These gaps reflect an emerging but poorly characterized field. Their significance and potential investigation is underscored by the fact that although viral infections result in adverse consequences in both mother and developing fetus/newborn, antiviral and immunomodulatory therapies can improve clinical outcomes in the dyad. [ABSTRACT FROM AUTHOR]

Preeclampsia is a major obstetrical complication with short- and long-term life-threatening consequences for both mother and child. Shallow cytotrophoblast invasion through the uterine decidua into the spiral arteries is implicated in the pathogenesis of preeclampsia, although the cause of deficient arterial invasion remains unknown. Research that is focused on the "soil"-the maternal decidua-highlights the importance of this poorly understood but influential uterine layer. Decidualization of endometrial cells regulates embryo invasion, which is essential for spiral artery remodeling and establishing the maternal-fetal interface. Exploration of the association between impaired decidualization and preeclampsia revealed suboptimal endometrial maturation and uterine natural killer cells present in the decidua before preeclampsia development. Furthermore, decidualization defects in the endometrium of women with severe preeclampsia, characterized by impaired cytotrophoblast invasion, were detected at the time of delivery and persisted 5 years after the affected pregnancy. Recently, a maternal deficiency of annexin A2 expression was found to influence aberrant decidualization and shallow cytotrophoblast invasion, suggesting that decidualization resistance, which is a defective endometrial cell differentiation during the menstrual cycle, could underlie shallow trophoblast invasion and the poor establishment of the maternal-fetal interface. Based on these findings, the transcriptional signature in the endometrium that promotes decidualization deficiency could be detected before (or after) conception. This would serve to identify women at risk of developing severe preeclampsia and aid the development of therapies focused on improving decidualization, perhaps also preventing severe preeclampsia. Here, we discuss decidualization deficiency as a contributor to the pathogenesis of pregnancy disorders with particular attention to severe preeclampsia. We also review current diagnostic strategies and discuss future directions in diagnostic methods based on decidualization. [ABSTRACT FROM AUTHOR]

Introduction: Placental dysfunction triggers fetal growth restriction in congenital human cytomegalovirus (HCMV) infection. Studies suggest that HCMV infection interferes with the differentiation of human trophoblasts. However, the underlying mechanisms have not been clarified. This study investigated the impact of HCMV infection on gene transcriptomes in cytotrophoblasts (CTBs) associated with placental dysfunction.Methods: CTBs were isolated from human term placentas, and spontaneous syncytialization was observed in vitro. The transcriptome profiles were compared between CTB groups with and without HCMV infection by cap analysis gene expression sequencing. The effect of HCMV infection on trophoblast differentiation was evaluated by examining cell fusion status using immunocytochemical staining for desmoplakin and assessing the production of cell differentiation markers, including hCG, PlGF, and soluble Flt-1, using ELISA.Results: The expression of the genes categorized in the signaling pathways related to the cell cycle was significantly enhanced in CTBs with HCMV infection compared with uninfected CTBs. HCMV infection hindered the alteration of the gene expression profile associated with syncytialization. This suppressive effect under HCMV infection was concurrent with the reduction in hCG and PlGF secretion. Immunostaining for desmoplakin revealed that HCMV infection reduced the cell fusion of cultured CTBs. These findings imply that HCMV infection has a negative impact on syncytialization, which is indispensable for the maintenance of villous function.Discussion: HCMV infection interferes with gene expression profiles and functional differentiation of trophoblasts. Suppression of syncytialization may be a survival strategy for HCMV to expand infection and could be associated with placental dysfunction. [ABSTRACT FROM AUTHOR]

In humans, the hemochorial placenta is a unique temporary organ that forms during pregnancy to support fetal development, gaseous exchange, delivery of nutrition, removal of waste products, and provides immune protection, while maintaining tolerance to the HLA-haploidentical fetus. In this review, we characterize decidual and placental immunity during maternal viral (co)-infection with HIV-1, human cytomegalovirus (HCMV), and Zika virus. We discuss placental immunology, clinical presentation, and epidemiology, before characterizing host susceptibility and cellular tropism, and how the three viruses gain access into specific placental target cells. We describe current knowledge on host-viral interactions with decidual and stromal human placental macrophages or Hofbauer cells, trophoblasts including extra villous trophoblasts, T cells, and decidual natural killer (dNK) cells. These clinically significant viral infections elicit both innate and adaptive immune responses to control replication. However, the three viruses either during mono- or co-infection (HIV-1 and HCMV) escape detection to initiate placental inflammation associated with viral transmission to the developing fetus. Aside from congenital or perinatal infection, other adverse pregnancy outcomes include preterm labor and spontaneous abortion. In addition, maternal HIV-1 and HCMV co-infection are associated with impaired fetal and infant immunity in postnatal life and poor clinical outcomes during childhood in exposed infants, even in the absence of vertical transmission of HIV-1. Given the rapidly expanding numbers of HIV-1-exposed uninfected infants and children globally, further research is urgently needed on neonatal immune programming during maternal mono-and co-infection. This review therefore includes sections on current knowledge gaps that may prompt future research directions. These gaps reflect an emerging but poorly characterized field. Their significance and potential investigation is underscored by the fact that although viral infections result in adverse consequences in both mother and developing fetus/newborn, antiviral and immunomodulatory therapies can improve clinical outcomes in the dyad.

Trophoblast is the primary epithelial cell type in the placenta, a transient organ required for proper fetal growth and development. Different trophoblast subtypes are responsible for gas/nutrient exchange (syncytiotrophoblasts, STBs) and invasion and maternal vascular remodeling (extravillous trophoblasts, EVTs). Studies of early human placental development are severely hampered by the lack of a representative trophoblast stem cell (TSC) model with the capacity for self-renewal and the ability to differentiate into both STBs and EVTs. Primary cytotrophoblasts (CTBs) isolated from early-gestation (6-8 wk) human placentas are bipotential, a phenotype that is lost with increasing gestational age. We have identified a CDX2(+)/p63(+) CTB subpopulation in the early postimplantation human placenta that is significantly reduced later in gestation. We describe a reproducible protocol, using defined medium containing bone morphogenetic protein 4 by which human pluripotent stem cells (hPSCs) can be differentiated into CDX2(+)/p63(+) CTB stem-like cells. These cells can be replated and further differentiated into STB- and EVT-like cells, based on marker expression, hormone secretion, and invasive ability. As in primary CTBs, differentiation of hPSC-derived CTBs in low oxygen leads to reduced human chorionic gonadotropin secretion and STB-associated gene expression, instead promoting differentiation into HLA-G(+) EVTs in an hypoxia-inducible, factor-dependent manner. To validate further the utility of hPSC-derived CTBs, we demonstrated that differentiation of trisomy 21 (T21) hPSCs recapitulates the delayed CTB maturation and blunted STB differentiation seen in T21 placentae. Collectively, our data suggest that hPSCs are a valuable model of human placental development, enabling us to recapitulate processes that result in both normal and diseased pregnancies.

The placenta is a critical barrier against viral, bacterial, and eukaryotic pathogens. For most teratogenic pathogens, the precise molecular mechanisms of placental resistance are still being unraveled. Given the importance of understanding these mechanisms and challenges in replicating trophoblast-pathogen interactions using in vitro models, we tested an existing stem-cell-derived model of trophoblast development for its relevance to infection with Toxoplasma gondii . We grew human trophoblast stem cells (TS CT ) under conditions leading to either syncytiotrophoblast (TS SYN ) or cytotrophoblast (TS CYT ) and infected them with T. gondii . We evaluated T. gondii proliferation and invasion, cell ultrastructure, as well as for transcriptome changes after infection. TS SYNs cells showed similar ultrastructure compared to primary cells and villous explants when analyzed by transmission electron microscopy and scanning electron microscopy (SEM), a resistance to T. gondii adhesion could be visualized on the SEM level. Furthermore, TS SYNs were highly refractory to parasite adhesion and replication, while TS CYTs were not. RNA-seq data on mock-treated and infected cells identified differences between cell types as well as how they responded to T. gondii infection. We also evaluated if TS SC -derived SYNs and CYTs had distinct resistance profiles to another vertically transmitted facultative intracellular pathogen, Listeria monocytogenes . We demonstrate that TS SYNs are highly resistant to L. monocytogenes , while TS CYTs are not. Like T. gondii , TS SYN resistance to L. monocytogenes was at the level of bacterial adhesion. Altogether, our data indicate that stem-cell-derived trophoblasts recapitulate resistance profiles of primary cells to T. gondii and highlight the critical importance of the placental surface in cell-autonomous resistance to teratogens.IMPORTANCECongenital toxoplasmosis can cause a devastating consequence to the fetus. To reach the fetus's tissues, Toxoplasma gondii must cross the placenta barrier. However, how this parasite crosses the placenta and the precise molecular mechanisms of placental resistance to this parasite are still unknown. In this study, we aimed to characterize a new cellular model of human trophoblast stem cells to determine their resistance, susceptibility, and response to T. gondii . Syncytiotrophoblast derived from trophoblast stem cells recapitulate the resistance profile similarly to placenta cells. We also showed that these cells are highly resistant to Listeria monocytogenes , at the level of bacterial adhesion. Our results suggest that resisting pathogen adhesion/attachment may be a generalized mechanism of syncytiotrophoblast resistance, and trophoblast stem cells represent a promising model to investigate cell-intrinsic mechanisms of resistance to pathogen adhesion and replication., Competing Interests: The authors declare no conflict of interest.

Aim: To determine which genes are important in placenta by network analysis. Methods: Placenta expressing genes were screened from RNA‐Seq data. Protein–protein interaction data were downloaded from STRING (v11.0) database. Google PageRank (PR) algorithm was used to identify important placental genes from protein interaction network. Six placental disease‐related datasets were downloaded from NCBI GEO database, and the differential expression of the 99 genes was identified. Results: We calculated PR for each placenta expressing gene and defined the top 99 genes with high PR as important genes. GAPDH has the highest PR. The 99 genes had different expression pattern in placental cell types. FN1 is up‐regulated in 8 w EVT compared to 8 w CTB and 24 w EVT compared to 8 w EVT. HSPA4 is down‐regulated in 8 w EVT compared to 8 w CTB and 24 w EVT compared to 8 w EVT. MIB2, TLR4, and UBB are consistently changed in preeclampsia (PE). UBB and ACTG1 were identified to be down‐regulated in fetal growth restriction (FGR). SOD1 is down‐regulated in preterm birth placenta. Conclusion: Our findings confirmed that the importance of these genes in placenta‐related diseases, and provide new candidates (MIB2, UBB, ACTG1, and SOD1) for placenta‐related disease diagnosis and treatment. [ABSTRACT FROM AUTHOR]