A growing body of epidemiological evidence links maternal exposure to air pollution with an increased risk of adverse pregnancy outcomes, such as preterm birth and low birth weight. Cerium dioxide nanoparticles (CeO2 NPs or nanoceria) are emerging pollutants, used as additives in diesel fuels and cigarettes for their catalytic properties, and released into the environment. Due to their high surface-to-volume ratio and reactivity, CeO2 NPs develop a surface coating during combustion, which may incorporate other released fuel-borne chemicals, such as benzo[a]pyrene (BaP), a known carcinogen, mutagen and reprotoxicant, raising concerns about their combined impacts on human health. To better reflect environmental reality, we produced BaP-coated CeO2 NPs and exposed primary human trophoblasts and chorionic villi. Our findings show that BaP-coated CeO2 NPs activate the aryl hydrocarbon receptor (AhR) pathway, enhancing trophoblast differentiation and syncytium formation, with effects distinct from those of BaP or CeO₂ NPs alone, or their unbound mixture. Additionally, exposure to CeO2 NPs alone altered homeostasis of mitochondria, affecting their phenotype and function. While individual exposures or BaP-coated CeO2 NPs had no detectable impact, parallel co-exposure resulted in a slight but significant reduction in basal respiration. Finally, uncoated CeO2 NPs altered placental steroidogenesis, increasing estrone level while decreasing dehydroepiandrosterone level, with sex-specific effects. These findings suggest that CeO2 NPs can influence the biological effects of BaP in the human placenta, including modulating trophoblast differentiation, as well as disrupting mitochondria homeostasis and steroid production, with potential implications for pregnancy outcomes in polluted environments.

Preeclampsia is a common and severe pregnancy-related disease associated with failed remodeling of the uterine spiral arteries by the placenta, which can lead to maternal and fetal mortality. Currently, there are limited strategies for early intervention of preeclampsia, primarily relying on long-term use of low-dose aspirin, which cannot reverse the pathological changes in the placenta. In this study, we propose the potential of using rosiglitazone, a peroxisome proliferator-activated receptor gamma (PPARγ) agonist, for early intervention of preeclampsia. We conducted a literature review of the mechanisms of PPARγ in preeclampsia-related research over the past few decades and evaluated the toxicity and practical outcome of rosiglitazone in clinical applications, providing feasibility for conducting clinical trials of rosiglitazone in the treatment of preeclampsia.

Background: During the process of elongation, the embryo increases in size within the uterus, while the extra-embryonic tissues (EETs) develop and differentiate in preparation for implantation. As it grows, the ovoid embryo transforms into a tubular form first and then a filamentous form. This process is directed by numerous genes and pathways, the expression of which may be altered in the case of developmental irregularities such as when the conceptus is shorter than expected or when the embryo develops after splitting. In bovines, efforts to understand the molecular basis of elongation have employed trophoblastic vesicles (TVs)—short tubular EET pieces that lack an embryo—which also elongate in vivo. To date, however, we lack molecular analyses of TVs at the ovoid or filamentous stages that might shed light on the expression changes involved.Methods: Following in vivo development, we collected bovine conceptuses from the ovoid (D12) to filamentous stages (D18), sectioned them into small pieces with or without their embryonic disc (ED), and then, transferred them to a receptive bovine uterus to assess their elongation abilities. We also grew spherical blastocysts in vitro up to D8 and subjected them to the same treatment. Then, we assessed the differences in gene expression between different samples and fully elongating controls at different stages of elongation using a bovine array (10 K) and an extended qPCR array comprising 224 genes across 24 pathways.Results:In vivo, TVs elongated more or less depending on the stage at which they had been created and the time spent in utero. Their daily elongation rates differed from control EET, with the rates of TVs sometimes resembling those of earlier-stage EET. Overall, the molecular signatures of TVs followed a similar developmental trajectory as intact EET from D12–D18. However, within each stage, TVs and intact EET displayed distinct expression dynamics, some of which were shared with other short epithelial models.Conclusion: Differences between TVs and EET likely result from multiple factors, including a reduction in the length and signaling capabilities of TVs, delayed elongation from inadequate uterine signals, and modified crosstalk between the conceptus and the uterus. These findings confirm that close coordination between uterine, embryonic, and extra-embryonic tissues is required to orchestrate proper elongation and, based on the partial differentiation observed, raise questions about the presence/absence of certain developmental cues or even their asynchronies.

Phthalates are environmental contaminants commonly used as plasticizers in polyvinyl chloride (PVC) products. In the literature, phthalate exposure has been associated with preterm birth, low birth weight, and pregnancy loss, but information on possible mechanisms linking maternal phthalate exposure and placental development is limited. One hypothesis is the involvement of the peroxisome proliferator-activated receptor-γ (PPARγ), which belongs to the superfamily of nuclear receptors that regulate, in a ligand-dependent manner, the transcription of target genes. Studies of PPARγ-deficient mice have demonstrated its essential role in lipid metabolism and placental development. In the human placenta, PPARγ is expressed in the villous cytotrophoblast (VCT) and is activated during its differentiation into syncytiotrophoblast (ST). The goal of this study was to investigate the action of mono-2-ethylhexylphthalate (MEHP) on PPARγ activity during in vitro differentiation of VCTs into ST, the essential tissue of the human placenta providing hormonal and exchange functions between maternal and fetal blood. Several techniques such as immunofluorescence, PPARγ activity / hCGβ assays, western blotting, and lipidomics analyses were combined to characterize the impacts of physiologically relevant concentrations of MEHP (0.1, 1, and 10μM) on freshly isolated VCTs from human term placenta. 0.1 μM and 1 μM MEHP significantly decreased PPARγ activity and cell fusion index compared to controls, while, surprisingly, 10 μM had the opposite effect. MEHP exposure inhibited hCG secretion regardless of the concentration used and significantly altered lipid composition. this study suggests that MEHP: (i) disrupts trophoblast differentiation, (ii) alters the production of the essential pregnancy hormone (hCG), and (iii) that these effects are in part mediated by the nuclear receptor PPARγ. Thus, phthalates act as endocrine disruptors in the human placenta.

Preeclampsia (PE) is a hypertensive disease of pregnancy associated with substantial maternal and fetal morbidity and mortality. Corin is a transmembrane type II serine protease expressed in the cardiomyocytes that converts pro-atrial natriuretic peptide (pro-ANP) into ANP, a cardiac hormone that regulates blood pressure. High levels of soluble corin (sCorin) have been reported in preeclampsia and are supposed to have cardiac origin. The hypothesis of our study was that during pregnancy sCorin is released by the syncytiotrophoblast, and that increased levels of sCorin in preeclampsia are of placental origin. Soluble corin levels were measured in plasma of 375 patients from ECLAXIR study (181 PE and 194 controls) using an ELISA kit (R&D Systems). Primary cytotrophoblasts (VCT) were isolated from human term placenta (n=3), and cultured up to 72h. Cell supernatants and cells were collected and stored at -80°C. Placental villi from PE patients (n=3) and controls (n=3) were either cultured for 24h where the explant supernatants were collected and stored at -80°C, or snap-frozen and stored at -80°C, or PFA-fixed and embedded in paraffin. mRNA and protein levels of Corin were quantified by RT-qPCR, Western Blot and immunohistochemistry. On 395 patients (181 PE and 194 controls), the high levels of sCorin in maternal blood were confirmed from preeclamptic pregnancies compared to controls. Next, experiments on differentiated primary cytotrophoblasts (VCT) showed that Corin was expressed (mRNA and protein levels) and secreted by trophoblastic cells, mostly by the syncytiotrophoblast (ST). Finally, using placental explants, a significant increase in Corin production and secretion was observed in PE cases compared to controls. This study demonstrates that Corin is secreted by trophoblastic cells and that high levels of sCorin in preeclampsia are of a placental origin.

TORCH - Toxoplasma gondii, Other agents (such as treponema pallidum, varicella zoster virus, parvovirus B19, human immunodeficiency virus), Rubella Cytomegalovirus and Herpes simplex virus - infections are responsible for 2 to 3% of all congenital disorders, or disorders present at birth. These infections can cause a variety of complications, including preterm birth, foetal growth restriction, physical malformations and sometimes, loss of pregnancy. Interferon-induced transmembrane proteins (IFITMs) are a family of restriction factors blocking the entry step of many viruses including TORCH. Elevated levels of type I interferon (IFN) during pregnancy are associated with foetal growth restriction, preterm birth, and foetal demise through mechanisms that are not well understood. We have recently published that the IFN inducer polyinosinic:polycytidylic acid promoted foetal resorption and placental abnormalities in wild-type but not in Ifitm-deleted mice. Using cultures of human trophoblasts and mouse cells, we showed that IFITMs impaired formation of the syncytiotrophoblast by inhibiting the syncytin-mediated cell fusion process (Buchrieser et al, Science, 2019). The objective of the present study was to determine whether IFITMs modify human trophoblast invasion, another critical step of placental development. Primary extravillous trophoblasts (EVTs), HTR8/SVneo cell line and placental explants (7-12 WG) were treated with IFN-ß (10, 100 or 1000 IU/mL) for 48 hours or transduced for IFITM1, IFITM2 and IFITM3. IFITMs expressions were evaluated by immunofluorescence and western blotting. Invasion of HTR8/SVneo trophoblasts was monitoring for 24h by a Matrigel wound healing assays using a live-cell imager (IncuCyte). EVTs invasion was quantified by culturing placental explants on Matrigel-coated dishes. Treatment of HTR8/SVneo and primary EVTs with IFN-ß strongly up-regulated IFITM1 and IFITM2/3. HTR8/SVneo trophoblasts treated with 100 and 1000 IU/mL IFN-ß invaded significantly less than the vehicle-treated cells. This inhibition was confirmed on IFN-ß-treated placental explants. To directly address the role of IFITMs in trophoblast invasion, we transduced cells with IFITM expression vectors. Results showed that only HTR8/SVneo expressing IFITM1 migrated significantly less than empty-, IFITM2- or IFITM3-transduced cells. This IFITM1-induced decrease in EVT migration was confirmed on IFITMs-transduced placental explants. Finally, analysis of murine placentas revealed that the distance of migration of trophoblast giant cells was inferior in Poly-IC conditions compared to controls. These results together with our previous work demonstrate that excessive levels of IFITMs inhibit both trophoblast invasion and fusion, and may mediate the pregnancy complications observed during congenital infections and other IFN-induced pathologies.

Human placenta is a multifunctional interface between maternal and fetal blood. Studying the impact of pollutants on this organ is crucial because many xenobiotics in maternal blood can accumulate in placental cells or pass into the fetal circulation. Benzo(a)pyrene (BaP) and cerium dioxide nanoparticles (CeO2 NP), which share the same emission sources, are found in ambient air pollution and also in maternal blood. The aim of the study was to depict the main signaling pathways modulated after exposure to BaP or CeO2 NP vs. co-exposure on both chorionic villi explants and villous cytotrophoblasts isolated from human term placenta. At nontoxic doses of pollutants, BaP is bioactivated by AhR xenobiotic metabolizing enzymes, leading to DNA damage with an increase in γ-H2AX, the stabilization of stress transcription factor p53, and the induction of its target p21. These effects are reproduced in co-exposure with CeO2 NP, except for the increase in γ-H2AX, which suggests a modulation of the genotoxic effect of BaP by CeO2 NP. Moreover, CeO2 NP in individual and co-exposure lead to a decrease in Prx-SO3, suggesting an antioxidant effect. This study is the first to identify the signaling pathways modulated after co-exposure to these two pollutants, which are common in the environment.

Objective: The adipogenic PPARG-encoded PPAR gamma nuclear receptor also displays essential placental functions. We evaluated the metabolic, reproductive, and perinatal features of patients with PPARG-related lipodystrophy. Methods: Current and retrospective data were collected in patients referred to a National Rare Diseases Reference Centre. Results: 26 patients from 15 unrelated families were studied (18 women, median age 43 years). They carried monoallelic PPARG variants except a homozygous patient with congenital generalized lipodystrophy. Among heterozygous patients aged 16 or more (n = 24), 92% had diabetes, 96% partial lipodystrophy (median age at diagnosis 24 and 37 years), 78% hypertriglyceridaemia, 71% liver steatosis, and 58% hypertension. The mean BMI was 26 +/- 5.0 kg/m(2). Women (n = 16) were frequently affected by acute pancreatitis (n = 6) and/or polycystic ovary syndrome (n = 12). Eleven women obtained one or several pregnancies, all complicated by diabetes (n = 8), hypertension (n = 4), and/or hypertriglyceridaemia (n = 10). We analysed perinatal data of patients according to the presence (n = 8) or absence (n = 9) of a maternal dysmetabolic environment. The median gestational age at birth was low in both groups (37 and 36 weeks of amenorrhea, respectively). As expected, the birth weight was higher in patients exposed to a foetal dysmetabolic environment of maternal origin. In contrast, 85.7% of non-exposed patients, in whom the variant is, or is very likely to be, paternally-inherited, were small for gestational age. Conclusions: Lipodystrophy-related PPARG variants induce early metabolic complications. Our results suggest that placental expression of PPARG pathogenic variants carried by affected foetuses could impair prenatal growth and parturition. This justifies careful pregnancy monitoring in affected families.

The human placenta is a transitory organ essential for fetal development, whose functions can be disrupted by xenobiotics in maternal blood. Benzo-[a]-pyrene (B[a]P) is a carcinogenic, mutagenic and reprotoxic pollutant as well as an endocrine disruptor that can be internalized in the human body by respiratory exposure. Cerium dioxide nanoparticles (CeO2 NP) have been introduced into our environment, mainly for their catalytic properties and share the same emission sources as B[a]P (cigarette smoke and diesel engine exhaust). The aim of our study is to determine the impact of these two atmospheric pollutants on the human placenta in concomitant exposure, in order to get closer to the environmental reality. Chorionic villi and villous cytotrophoblasts (VCT) from human placentas at term of pregnancy were exposed in vitro to the pollutants. The internalization of the pollutants was observed by confocal and Raman microscopy. Cytotoxicity was assessed using WST-1 test and extracellular LDH assay. Placental endocrine activity was assessed by ELISA. The signaling pathways impacted have been studied by Western Blot, RT-qPCR and IHC. CeO2 NP and B[a]P can be internalized within the chorionic villi. B[a]P alone or in co-exposure with CeO2 NP activate the metabolic pathway of the aryl hydrocarbon receptor (AhR), causing DNA damage. B[a]P also stabilizes the stress transcription factor p53 and its transcriptional target p21. Although these two pollutants do not cause major toxicity on term human trophoblasts after in vitro exposures, cellular stress markers are induced.

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 in vitro/ex vivo models of EVCT, mice treated in vivo 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.

BackgroundPreeclampsia is a pregnancy-specific disorder that always causes maternal and fetal serious adverse outcome. Disturbances in maternal immune tolerance to embryo at the maternal-fetal interface (MFI) may be associated with preeclampsia onset. Recent studies have revealed the reduced expression pattern of HLA-F at the MFI in preeclampsia, while the mechanism of it mediating maternal fetal immune tolerance has not been revealed.MethodsSingle-cell RNA sequencing on placental decidua was performed to reveal the immune disturbances landscape at the MFI in preeclampsia. Human Jar cells and NK-92MI cells were employed to study the role of HLA-F in trophoblasts and lymphocyte.ResultsA total of 101,250 cells were classified into 22 cell clusters. Disease-related IGFBP1+SPP1+ extracellular villus trophoblast (EVT) was identified in the preeclamptic placental decidua, accompanied by newly discovered immune cellular dysfunction such as reduced ribosomal functions of NK populations and abnormal expression of antigen-presenting molecules in most cell clusters. Certain genes that are characteristic of the intermediate stage of myeloid or EVT cell differentiation were found to have unexplored but important functions in the pathogenesis of preeclampsia; specifically, we detected enhanced cell cross-talk between IGFBP1+SPP1+ EVT2 or SPP1+M1 cells and their receptor cell populations at the MFI of PE patients compared to controls. With respect to HLA-F, mIF staining confirmed its reduced expression in PE samples compared to controls. Over-expression of HLA-F in Jar cells promoted cell proliferation, invasion, and migration while under-expression had the opposite effect. In NK-92MI cells, over-expression of HLA-F increased the secretion of immunoregulation cytokines such as CSF1 and CCL22, and promoted adaptive NKG2C+NK cell transformation.ConclusionsWe revealed the immune disturbance landscape at the MFI in preeclampsia. Our findings regarding cellular heterogeneity and immune cellular dysfunction, as revealed by scRNA-seq, and the function of HLA-F in cells provide new perspectives for further investigation of their roles in the pathogenesis of preeclampsia, and then provide potential new therapeutic target.

The placenta is a multifunctional organ essential for fetal development and growth, but also represents a barrier for the fetus against many xenobiotics. Benzo-(a)-pyrene (BaP) is a pollutant already known as a mutagenic and reprotoxic carcinogen as well as an endocrine disruptor, which can bypass the placental barrier. Cerium dioxide nanoparticles (CeO2 NPs) are new pollutants that currently share the same emission sources as BaP (cigarette smoke, diesel engine exhaust…). In order to understand their impact on human health, in 2010 the OECD included CeO₂ NPs in the priority list of nanomaterials requiring urgent assessment. The aim of our study is to identify the cellular effects and the mechanisms of action of these two atmospheric pollutants on the human placental barrier when they are in concomitant exposure, in order to get closer to the environmental reality. The chosen concentrations ratio is of 10 µg/cm2 of NPs CeO2 for 1 µM of BaP, which corresponds to the quantity of BaP necessary to cover the surface of the NPs when they are encountered together. Cytotoxicity was assessed using a metabolic activity test of the mitochondrial dehydrogenase (WST-1 test) and the release of LDH on villous cytotrophoblasts isolated from term human placentas. A Cell Stress Array protein-on-chip study was performed to identify the modulated signaling pathways after exposure to these two pollutants. The results obtained were confirmed by Western Blot, IHC and RTq-PCR. The endocrine function was assessed by hCG dosage in the culture medium after exposure to these pollutants. We have shown that the two pollutants are not toxic for cytotrophoblasts at realistic exposure doses. However, BaP alone or in co-exposure to CeO2 NPs activates the pathway of xenobiotic metabolism through the aryl hydrocarbon receptor (AhR), stabilizes the stress transcription factor p53 and its transcriptional target p21, without modifying the antioxidant enzyme levels. On the contrary, CeO2 NPs seem rather to have an antioxidant effect and decrease the levels of factors such as HIF-1α, HIF-2α, p38 and NFκB. These diminishing effects are potentiated after co-exposure to both pollutants. The Cell Stress Array highlights a large number of actors, whose protein levels decrease during co-exposure compared to individual exposures, in particular those of the p53 pathway and HIF. These results open up new perspectives for describing the mechanisms by which these two atmospheric pollutants alter the functioning of the placenta of exposed pregnant women

Phthalates are environmental contaminants commonly used as plasticizers in polyvinyl chloride (PVC) products. In the literature, phthalate exposure has been associated with preterm birth, low birth weight, and pregnancy loss, but information on possible mechanisms linking maternal phthalate exposure and placental development is limited. One hypothesis is the involvement of the peroxisome proliferator-activated receptor-γ (PPARγ), which belongs to the superfamily of nuclear receptors that regulate, in a ligand-dependent manner, the transcription of target genes. Studies of PPARγ-deficient mice have demonstrated its essential role in lipid metabolism and placental development. In the human placenta, PPARγ is expressed in the villous cytotrophoblast (VCT) and is activated during its differentiation into syncytiotrophoblast (ST). The goal of this study was to investigate the action of mono-2-ethylhexylphthalate (MEHP) on PPARγ activity during in vitro differentiation of VCTs into ST, the essential tissue of the human placenta providing hormonal and exchange functions between maternal and fetal blood. Several techniques such as immunofluorescence, PPARγ activity / hCGβ assays, western blotting, and lipidomics analyses were combined to characterize the impacts of physiologically relevant concentrations of MEHP (0.1, 1, and 10μM) on freshly isolated VCTs from human term placenta. 0.1 μM and 1 μM MEHP significantly decreased PPARγ activity and cell fusion index compared to controls, while, surprisingly, 10 μM had the opposite effect. MEHP exposure inhibited hCG secretion regardless of the concentration used and significantly altered lipid composition. This study suggests that MEHP: (i) disrupts trophoblast differentiation, (ii) alters the production of the essential pregnancy hormone (hCG), and (iii) that these effects are in part mediated by the nuclear receptor PPARγ. Thus, phthalates act as endocrine disruptors in the human placenta.

Aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that plays a critical role in diverse biological processes, including xenobiotic metabolism, carcinogenesis, and physiological functions such as regulation of the immune system and cell differentiation. To improve studies of AHR activity, we constructed two new reporter genes: a fluorescent GFP-tagged histone 2B (XRE-H2B-eGFP) and a secreted nanoluciferase (XRE-pNL1.3[secNluc]). Here, we demonstrate how these reporters can be used to monitor AHR activity in different types of cells, including human primary trophoblasts and cell lines, following incubation with a strong AHR ligand, benzo[a]pyrene (B[a]P), or an AHR inhibitor (CH223191). Compared to vehicle control cells, a significant increase in AHR activity was observed in cells treated with 0.5 and/or 2 µM B[a]P and a significant decrease was detected in response to treatment with 3 µM CH223191. These new plasmids have great potential for use in a variety of applications, such as screening for endogenous or exogenous ligands of AHR.