Trophoblast Function Research Articles

Microbiome-producing SCFAs are associated with preterm birth via trophoblast function modulation

ABSTRACT Although preterm birth (PTB) is one of the major causes of perinatal mortality and neonatal morbidity, little is known about its complex etiology. An abnormal cervicovaginal microbiome during pregnancy is associated with an increased risk of PTB. The cervicovaginal microbiota and its active metabolites, such as short-chain fatty acids (SCFAs), might be effectively used to predict and diagnose PTB. However, the roles of these proteins and the underlying mechanisms involved remain elusive. We conducted 16S rRNA gene sequencing and used a targeted metabolomics approach to study cervicovaginal swabs obtained from 51 singleton pregnancies and 52 twin pregnancies in the second trimester. Next, functional in vitro experiments were performed to investigate the roles and mechanisms of SCFAs in placental trophoblast cells (HTR8/SVneo cells). Significant cervicovaginal microbiome dysbiosis, characterized by a substantial reduction in the abundance of lactobacilli and overgrowth of anaerobes, was revealed in the second trimester and was strongly associated with subsequent PTB ( P = 0.036). Among the paired samples ( n = 103), acetic acid was significantly greater in the preterm group than in the term group ( P = 0.047). Data obtained from integrated gas chromatography‒mass spectrometry and 16S RNA studies revealed metabolites that were distinctly associated with particular microbial communities. Gardnerella vaginalis was the species most positively associated with acetic acid content. In addition, we identified a marker set consisting of the pregnancy type, acetic acid concentration, and community state type to accurately diagnose PTB. Acetate was associated with increased interleukin (IL)-8 and IL-6 levels and extravillous trophoblast cell migration and invasion through the activation of the extracellular signal-regulated kinase 1/2 signaling pathway in HTR8/SVneo cells. Cervicovaginal microbiota dysbiosis is an important etiological factor of PTB. The cervicovaginal microbiota and its active metabolites can be efficiently used to predict and diagnose PTB. Our findings enrich the microbiota–placenta axis theory and contribute to the development of microecological products for pregnancy. IMPORTANCE Preterm birth (PTB) is a leading cause of infant mortality and long-term health issues, affecting millions of families worldwide. Despite its prevalence, the exact causes of PTB remain unclear. Our study reveals that certain bacteria and their metabolic byproducts in the cervicovaginal environment, specifically short-chain fatty acids (SCFAs), are linked to the risk of preterm birth. By analyzing samples from pregnant women, we found that an imbalance in the vaginal microbiota and increased levels of SCFAs are associated with changes in cells that can lead to early labor. This research provides new insights into how the microbiome influences pregnancy outcomes and highlights potential biomarkers for predicting preterm birth. Understanding these microbial influences could lead to innovative strategies for early diagnosis and prevention, ultimately improving maternal and infant health.

BMP5 promotes trophoblast functions upon N-glycosylation via the BMP5-SMAD1/5 signaling pathway in preeclampsia

IntroductionPreeclampsia (PE) is one of the most common pregnancy-related complications worldwide and currently lacks an effective treatment. While trophoblast cell dysfunction has been identified as the fundamental cause of PE, the underlying mechanisms remain unclear. Bone morphogenetic protein 5 (BMP5) is a secreted glycoprotein highly expressed in the placenta that is involved in cell proliferation, migration, and invasion. However, the role and mechanism of BMP5 glycosylation of trophoblast cell function remain unclear. MethodsThe expression of BMP5 and N-glycosylation in preeclamptic placental tissues was investigated. We predicted and validated the N-glycosylation sites of BMP5. Additionally, we evaluated the effect of BMP5 N-glycosylation on the proliferation, migration, invasion, and angiogenesis of human immortalized trophoblastic HTR-8/SVneo cells. Furthermore, the role of N-glycosylated BMP5 in activating the BMP5-SMAD1/5 signaling pathway and regulating trophoblastic cell functions was explored. ResultsOur study reveals that PHA-E+L (recognizing branching N-glycans) reactive N-glycans and BMP5 expression levels are lower in preeclamptic villous tissues compared to normal placental tissues. Additionally, we demonstrated that BMP5 is an N-glycosylation-modified protein. Furthermore, N-glycosylated BMP5 promoted the functional trophoblastic cells (HTR-8/SVneo). We also revealed that N-glycosylation of BMP5 regulates multiple cell functions through the BMP5-SMAD1/5 signaling pathway. ConclusionN-glycosylated BMP5 promotes trophoblast cell proliferation, migration, invasion, and angiogenesis. This study provides mechanistic insight as to how N-glycosylation of BMP5 in trophoblast cells can contribute to the pathogenesis of preeclampsia and provides a new basis for its diagnosis and treatment.

Simulated ischaemia/reperfusion impairs trophoblast function through divergent oxidative stress- and MMP-9-dependent mechanisms.

Early-onset pre-eclampsia is believed to arise from defective placentation in the 1st trimester, leading to placental ischaemia/reperfusion (I/R) and oxidative stress. However, our current understanding of the effects of I/R and oxidative stress on trophoblast function is ambiguous in part due to studies exposing trophoblasts to hypoxia instead of I/R, and which report conflicting results. Here we present a model of simulated ischaemia/reperfusion (SI/R) to recapitulate the pathophysiological events of early-onset PE, by exposing 1st trimester cytotrophoblast HTR-8/SVneo cells to a simulated ischaemia buffer followed by reperfusion. We examined different ischaemia and reperfusion times and observed that 1h ischaemia and 24h reperfusion induced an increase in reactive oxygen species (ROS) production (p < 0.0001) and oxygen consumption rate (p < 0.01). SI/R-exposed trophoblast cells exhibited deficits in migration, proliferation and invasion (p < 0.01). While the deficits in migration and proliferation were rescued by antioxidants, suggesting a ROS-dependent mechanism, the loss of invasion was not affected by antioxidants, which suggests a divergent ROS-independent pathway. In line with this, we observed a decrease in MMP-9, the key regulatory enzyme necessary for trophoblast invasion (p < 0.01), which was similarly unaffected by antioxidants, and pharmacological inhibition of MMP-9 replicated the phenotype of deficient invasion (p < 0.01). Collectively, these data demonstrate that I/R impairs trophoblast migration and proliferation via a ROS-dependent mechanism, and invasion via a ROS-independent loss of MMP-9, disambiguating the role of oxidative stress and providing insights into the response of trophoblasts to I/R in the context of early-onset PE.

ITGA3 participates in the pathogenesis of recurrent spontaneous abortion by downregulating ULK1-mediated autophagy to inhibiting trophoblast function.

Recurrent spontaneous abortion (RSA) is a significant challenge encountered by couples of reproductive ages, with inadequate trophoblast invasion identified as a primary factor in RSA pathogenesis. However, the precise molecular mechanisms through which trophoblast cells dysfunction leads to RSA remain incompletely understood. Research has highlighted the critical role of integrins in embryo implantation and development. While integrin α-3 (ITGA3) is recognized for its promotion of invasion in cancer cells, its involvement in miscarriage remains poorly characterized. This investigation initially assessed ITGA3 expression in villous tissues obtained from RSA patients and induced abortion patients. The findings demonstrated a notable reduction in ITGA3 levels in the villous tissues of RSA patients compared control group. Subsequent in vitro analyses indicated that ITGA3 knockdown inhibited the migration, invasion, and proliferation of trophoblast cells. Through RNA sequencing and subsequent experimentation, it was revealed that ITGA3 regulated ULK1-mediated autophagy to influence trophoblast cells invasion, migration, and proliferation. Furthermore, utilizing a miscarriage animal model, the diminished expression of ITGA3 and ULK1 in the placentas of RSA mice was confirmed. In conclusion, the study findings suggest that the downregulation of ITGA3 suppresses ULK1 expression, consequently impeding autophagy to initiation and impeding trophoblast cells invasion and migration, thereby contributing to the pathological progression of RSA.

Cleistocalyx nervosum var. paniala mitigates oxidative stress and inflammation induced by PM10 soluble extract in trophoblast cells via miR-146a-5p

Air pollution poses a significant global concern, notably impacting pregnancy outcomes through mechanisms such as DNA damage, oxidative stress, inflammation, and altered miRNA expression, all of which can adversely affect trophoblast functions. Cleistocalyx nervosum var. paniala, known for its abundance of anthocyanins with diverse biological activities including anti-mutagenic, antioxidant, and anti-inflammatory properties, is the focus of this study examining its effect on Particulate Matter 10 (PM10) soluble extract-induced trophoblast cell dysfunction via miRNA expression. The study involved the extraction of C. nervosum fruit using 70% ethanol, followed by fractionation with hexane, dichloromethane, and ethyl acetate. Subsequent testing for total phenolics, flavonoids, anthocyanins, and antioxidant activity revealed the ethyl acetate fraction (CN-EtOAcF) as possessing the highest phenolic and anthocyanin content along with potent antioxidant activity, prompting its selection for further investigation. In vitro studies on HTR-8/SVneo cells demonstrated that 5–10 µg/mL PM10 soluble extract exposure inhibited cell proliferation, migration, invasion, and induced apoptosis. However, pretreatment with 20–80 µg/mL CN-EtOAcF followed by 5 µg/mL PM10 soluble extract exposure exhibited protective effects against PM10 soluble extract-induced damage, including inflammation inhibition and intracellular ROS suppression. Notably, CN-EtOAcF down-regulated PM10-induced miR-146a-5p expression, with SOX5 identified as a potential target. Overall, CN-EtOAcF demonstrated the potential to protect against PM10-induced harm in trophoblast cells, suggesting its possible application in future therapeutic approaches.

The Deubiquitinating Enzyme USP4 Promotes Trophoblast Dysfunction by Stabilizing RYBP.

Previous studies have suggested that impaired spiral artery remodeling, placental dysfunction, and insufficient trophoblast infiltration are the etiology and pathogenesis of Preeclampsia (PE). Ring 1 and YY1 binding protein (RYBP) has been reported to be associated with trophoblast dysfunction. However, the molecular mechanism of RYBP involved in trophoblasts in the pathogenesis of PE is poorly defined. RYBP and Ubiquitin-specific peptidase 4 (USP4) mRNA levels were determined using real-time quantitative polymerase chain reaction (RT-qPCR). RYBP, USP4, p-PI3K, PI3K, p-AKT, and AKT protein levels were measured using western blot assay. Cell viability, proliferation, apoptosis, invasion, and migration were assessed using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, and wound healing assays. After ubibrowser database analysis, the interaction between USP4 and RYBP was verified using Co-immunoprecipitation (CoIP) assay. RYBP and USP4 expression were upregulated in placental tissues from PE patients. By using JEG-3 and HTR-8/SVneo trophoblast cells, RYBP overexpression or USP4 upregulation could hinder cell viability, proliferation, invasion, migration, and promote apoptosis. Mechanistically, USP4 could trigger the deubiquitination of RYBP and prevent its degradation. In addition, USP4 repressed the PI3K/AKT signaling pathway by regulating RYBP. In total, Decreased USP4-mediated ubiquitination results in an adverse impact on trophoblast function by enhancing RYBP expression, providing a novel therapeutic target for PE.

KLF4 regulates trophoblast function and associates with unexplained recurrent spontaneous abortion

BackgroundRecurrent spontaneous abortion (RSA) is defined as two or more consecutive spontaneous abortions before 20 weeks with the same spouse [1]. However, approximately 50% of RSA cases of unknown cause are classified as unexplained recurrent spontaneous abortion (URSA). Potential factors include decreased trophoblast cell migration and invasion, leading to impaired placental implantation and maintenance of the normal maternal-fetal interface. However, the mechanism of this pathogenesis remains unknown. In this study, we investigated the potential role and mechanism of KLF4 in regulating URSA by influencing the invasion and migration ability of trophoblast cells.MethodsWe firstly identified 817 differentially expressed genes by performing a difference analysis of the dataset GSE121950 [2] related to recurrent abortion, and intersected the top 10 genes obtained respectively by the three algorithms: DMNC, MNC, and EPC using Venn Diagram.To detect the expression levels of core genes, villi samples were obtained from normal pregnant women and patients with URSA. RT-qPCR analysis revealed a significant difference in KLF4 mRNA expression and KLF4 was then analyzed. Trophoblast cell lines HTR8 and JEG3 were used to investigate the effect of KLF4 on trophoblastic function. Wound healing and transwell assays was performed to detect the invasion and migration of trophoblast cells. The expression of epithelial-mesenchymal transition(EMT) molecules were detected by RT-qPCR and western blot. Promoter detection and epigenetic modification were detected by chromatin immunoprecipitation (ChIP) assay. Molecular nuclear localization was detected by immunofluorescence and subcellular fractionation. Miscarried mice model was used to study the effects of KLF4 on URSA induced by reduced trophoblast invasion and migration.ResultsKLF4 is highly expressed in the villi of patients with URSA. KLF4 inhibits the expression level of H3R2ME2a in trophoblast cells by regulating the transcriptional level and nuclear translocation of PRMT6, thereby inhibiting the possible regulatory mechanism of trophoblastic invasion and providing a potential treatment strategy for URSA in vivo.ConclusionsThe KLF4/PRMT6/H3R2ME2a axis regulates mechanisms associated with unexplained recurrent spontaneous abortion by regulating trophoblast function.

DOCK1 deficiency drives placental trophoblast cell dysfunction by influencing inflammation and oxidative stress, hallmarks of preeclampsia.

Preeclampsia (PE) is a globally prevalent obstetric disorder, pathologically characterized by abnormal placental development. Dysfunctions of angiogenesis, vasculogenesis and spiral artery remodeling are demonstrated to be involved in PE pathogenesis; however, the underlying mechanisms remain largely unknown. Here, we investigated the role of the dedicator of cytokinesis 1 (DOCK1), crucial molecule in various cellular processes, in PE progression using HTR-8 cells derived from first-trimester placental extravillous trophoblasts. Our analysis revealed an aberrant DOCK1 expression in the placental villi of PE patients and its impact on essential cellular functions for vascular network formation. A deficiency of DOCK1 in HTR-8 cells impaired the vascular network formation, exacerbated the expression of anti-angiogenic factor ENG, and reduced VEGF levels. Moreover, DOCK1 knockout amplified apoptosis, as indicated by an altered BCL2: BAX ratio and enhanced levels of cleaved PARP. DOCK1 depletion also boosted NF-κB activation and pro-inflammatory cytokine production (IL-6 and TNF-α). Furthermore, the mice treated with DOCK1 inhibitor, TBOPP, exhibited PE-like symptoms. These findings highlight the multifaceted roles of DOCK1 in the pathophysiology of PE, demonstrating that its deficiency can lead to placental dysfunction by orchestrating inflammatory responses and oxidative stress. These insights emphasize the pathogenic role of DOCK1 in PE development and suggest potential treatment strategies that require further exploration. In the graphical abstract, a split image of placental villi contrasts the effects of normal and reduced DOCK1 expression on preeclampsia. The left side illustrates adequate DOCK1 levels supporting healthy trophoblast function and effective spiral artery remodeling. The right side highlights the consequences of DOCK1 deficiency, leading to trophoblast dysfunction and impaired spiral artery remodeling, accompanied by angiogenic imbalance, increased inflammation, oxidative stress, and apoptosis, contributing to placental dysfunction and the development of preeclampsia.

A systematic review and risk of bias analysis of in vitro studies on trophoblast response to immunological triggers

An increasing amount of evidence suggests that immune responses may affect trophoblast functioning, which in turn may play a role in gestational disorders and fetal development. This systematic review offers the first summary of in vitro studies on the trophoblast response to immunological triggers, in conjunction with a risk of bias analysis.A search in Pubmed and Embase yielded 110 relevant studies. Primary trophoblasts were the most commonly used cell type, but trophoblast subtypes were not always defined. Similarly, the exact natures of trophoblast cell lines were sometimes unclear. Cytokines and Toll-like receptor agonists were often used as interventions, but most studies focused on a select few substances such as tumor necrosis factor-α and lipopolysaccharide. In regard to the outcome parameters, some important trophoblast functions, such as hormone production and barrier formation were underrepresented.Whether or not risk of bias was high varied strongly between types of bias. Risk of selection bias, for example, was usually low. However, none of the included studies mentioned blinding or plate randomization. Only a select few studies mentioned passage numbers, use of vehicle control or conflict of interest.In conclusion, better characterization of trophoblast subtypes and a broader range of studied interventions and outcome parameters would contribute to a more complete understanding of trophoblast responses to immune stimuli. Additionally, researchers are encouraged to replicate experiments and pay close attention when setting up and writing down methodologies, in order to improve the reproducibility and translatability of their work.

A heterozygous SPRY4 variant identified in female infertility characterized by reduced oocyte potential and early embryonic arrest.

Can novel genetic factors contributing to early embryonic arrest in infertile patients be identified, along with the underlying mechanisms of the pathogenic variant? We identified a heterozygous variant in the SPRY4 (sprouty RTK signaling antagonist 4) in infertile patients and conducted in vitro and in vivo studies to investigate the effects of the variant/deletion, highlighting its critical role in female reproductive health. SPRY4 acts as a negative regulator of receptor tyrosine kinases (RTKs) and functions as a tumor suppressor. Its abnormal expression can lead to recurrent miscarriage by affecting trophoblast function. In mice, Spry4 knockout (KO) leads to craniofacial anomalies and growth defects. A human study links the SPRY4 variant to a male patient with isolated hypogonadotropic hypogonadism (IHH), hypothetically impacting gonadotropin-releasing hormone (GnRH) neurons, and causing reproductive dysfunctions. SPRY4 is thus potentially integral in regulating endocrine homeostasis and reproductive function. To date, no study has reported SPRY4 variants associated with female fertility, and a causal relationship has not been established with functional evidence. Whole-exome sequencing (WES) was performed in 392 infertile women who suffered from primary infertility of unknown reason, and the heterozygous SPRY4 variant were identified in one independent family. The infertile patients presenting were recruited from July 2017 to November 2023. Women diagnosed with primary infertility were recruited from the Reproduction Center of Zhongshan Hospital, Fudan University. Genomic DNA was extracted from peripheral blood for WES analysis. The SPRY4 variant were identified through WES, in silico analysis, and variant screening. All variants were confirmed by Sanger sequencing. The effects of the variants were investigated in human embryonic kidney (HEK) 293T (HEK293T) cells via western blotting, and in mouse oocytes and embryos through complementary RNA (cRNA) injection, RNA sequencing, fluorescence, absorbance, and RT-qPCR assays. Gene function was further examined in Spry4 KO mice via histology, western blotting, ELISA, and RT-qPCR assays. We identified a missense heterozygous pathogenic variant in SPRY4 (GRCh38, GenBank: NM_030964.5, c.157C>T p.(Arg53Trp), rs200531302) that reduces SPRY4 protein levels in HEK293T cells and disrupts the redox system and mitochondrial function in mouse oocyte, and perturbs developmental potential in mouse embryos. These phenotypes could be partially reversed by the exogenous addition of Nrf1 cRNA. Additionally, Spry4-/- mice exhibit ovarian oxidative stress and decreased ovarian function. Due to the limited WES data and population, we identified only one family with a SPRY4 mutation. The deeper mechanism and therapeutic strategy should be further investigated through mutant mice and recovery experiment. Our study has identified a pathogenic variant in SPRY4 associated with early embryonic arrest in humans. These findings enhance our understanding of the role of SPRY4 in early embryonic development and present a new genetic marker for female infertility. This work was supported by the National Natural Science Foundation of China (82071643 and 82171655) and Natural Science Foundation of Shanghai (22ZR1456200). None of the authors have any competing interests. N/A.

Altered microRNA composition in the uterine lumen fluid in cattle (Bos taurus) pregnancies initiated by artificial insemination or transfer of an in vitro produced embryo

BackgroundMicroRNAs (miRNAs) are presented in the uterine lumen of many mammals, and in vitro experiments have determined that several miRNAs are important for the regulation of endometrial and trophoblast functions. Our aim was to identify and contrast the miRNAs present in extracellular vesicles (EVs) in the uterine lumen fluid (ULF) at the onset of attachment in cattle pregnancies (gestation d 18) initiated by artificial insemination (AI) or by the transfer of an in vitro-produced blastocyst (IVP-ET). A third group had no conceptus after the transfer of an IVP embryo.ResultsThe abundance of 263 annotated miRNAs was quantified in the EVs collected from ULF. There was an increase in the transcript abundance of 20 miRNAs in the ULF EVs from the AI pregnant group, while 4 miRNAs had a lower abundance relative to the group not containing a conceptus. Additionally, 4 miRNAs were more abundant in ULF EVs in the AI pregnant group relative to IVP-ET group (bta-mir-17, bta-mir-7-3, MIR7-1, MIR18A). Specific miRNAs in the ULF EVs were co-expressed with messenger RNAs expressed in extra-embryonic tissues and endometrium, including genes that are known to be their targets.ConclusionsThe results provide biological insights into the participation of miRNAs in the regulation of trophoblast proliferation and differentiation, as well as in endometrium receptivity. The knowledge that in vitro cultured embryos can contribute to the altered abundance of specific miRNAs in the uterine lumen can lead to the development of corrective approaches to reduce conceptus losses during the first month of pregnancy in cattle.

Down-regulation of CORO1C mediated by lncMALAT1/miR-133a-3p axis contributes to trophoblast dysfunction and preeclampsia

IntroductionPlacental trophoblast dysfunction has been proved to be closely related to the pathogenesis of preeclampsia. Coronaryxin-like actin-binding protein 1C (CORO1C) plays an important role in cell proliferation, apoptosis, invasion, and signal transduction, but its involvement in trophoblast dysfunction and preeclampsia remains uncertain. MethodsThe expression of CORO1C in placental tissues of preeclampsia (PE) pregnant women and pregnant mice PE model were detected by real-time quantitative polymerase chain reaction (RT-qPCR), western blotting (WB) and immunohistochemical (IHC) staining. Next, the proliferation, invasion, migration and apoptosis were performed to explore the functions of CORO1C in HTR8/SVneo cell. Furthermore, the expression of CORO1C were detected in lncMALAT1 knockdown and overexpression HTR-8/SVneo cell. And then we investigated the possible regulatory mechanism of lncMALAT1 on CORO1C through bioinformatics analysis, FISH assays, RIP assays, RNA pull down and dual luciferase reporter assays. Finally, we further validated that lncMALAT1 regulate the function of placental trophoblast cells through CORO1C. ResultsThe expression of CORO1C was significantly decreased in the placenta of PE patients and mice model, and positively associated with neonatal birth weight. And we found that CORO1C inhibited trophoblast proliferation, migration and invasion. Furthermore, reduced expression of lncMALAT1 impaired CORO1C level, thereby resulting in trophoblast dysfunction. Mechanistically, the dysregulation of lncMALAT1 promoted the expression of miR-133a-3p, strongly enhancing its binding to the 3′UTR region of CORO1C mRNA for degradation. DiscussionThis study demonstrated that the dysregulation of CORO1C via lncMALAT1/miR-133a-3p axis impairs trophoblast function and contributes to preeclampsia pathogenesis, providing novel insights in PE therapy through modulating CORO1C level.

Association between Kisspeptin and Foxa2 in regulation of trophoblast function and placentation: possible role in pregnancy viability

Association between Kisspeptin and Foxa2 in regulation of trophoblast function and placentation: possible role in pregnancy viability

Analysis of Non-Coding RNAs in Placental Tissue and Extracellular Vesicles: Implications for Trophoblast Function

Analysis of Non-Coding RNAs in Placental Tissue and Extracellular Vesicles: Implications for Trophoblast Function

EFFECTS OF ARYL HYDROCARBON RECEPTOR LIGAND TCDD ON HUMAN TROPHOBLAST CELL DEVELOPMENT.

The primary interface between mother and fetus, the placenta, serves two critical functions: extraction of nutrients from the maternal compartment and facilitation of nutrient delivery to the developing fetus. This delivery system also serves as a barrier to environmental exposures. The aryl hydrocarbon receptor (AHR) is an important component of the barrier. AHR signaling is activated by environmental pollutants and toxicants that can potentially affect cellular and molecular processes, including those controlling trophoblast cell development and function. In this study, we investigated the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an effective AHR ligand, exposure on human trophoblast cells. Human trophoblast stem (TS) cells were used as in vitro model system for investigating the downstream consequences of AHR activation. The actions TCDD were investigated in human TS cells maintained in the stem state or in differentiating TS cells. TCDD exposure stimulated the expression of CYP1A1 and CYP1B1 in human TS cells. TCDD was effective in stimulating CYP1A1 and CYP1B1 expression and altering gene expression profiles in human TS cells maintained in the stem cell state or induced to differentiate into extravillous trophoblast cells (EVT) or syncytiotrophoblast (ST). These actions were dependent upon the presence of AHR. TCDD exposure did not adversely affect maintenance of the TS cell stem state or the ability of TS cells to differentiate into EVT cells or ST. However, TCDD exposure did promote the biosynthesis of 2 methoxy estradiol (2ME), a biologically active catechol estrogen, with the potential to modify the maternal-fetal interface. Human trophoblast cell responses to TCDD were dependent upon AHR signaling and possessed the potential to shape development and function of the human placentation site.

High expression of CX3CL1/CX3CR1 at the mother-fetus interface of preeclampsia inhibits trophoblast invasion and migration

IntroductionPreeclampsia is associated with maternal inflammatory overreaction and imbalanced immunity at the mother-fetus interface. The pro-inflammatory chemokine fractalkine (CX3CL1) is recently recognized apart from imbalanced immunity. In this study, CX3CL1- CX3C chemokine receptor 1(CX3CR1) regulation of decidual macrophage function and trophoblast invasion ability in preeclampsia was initially explored. MethodsThe study comprised 60 women allocated to NP group (normotensive pregnant woman, n = 30) and sPE group (woman with severe preeclampsia, n = 30). After the delivery, the expression of CX3CL1 in placental tissues of the two groups was detected by immunohistochemical analysis. The protein level of CX3CL1 in placental tissue and CX3CR1 in decidua tissue was detected by Western Blot and the localization of CX3CR1 expression in decidua was detected by immunofluorescence. Macrophages were polarized into classically activated (M1) macrophages. M1 were treat with PBS (control group), recombinant human CX3CL1 (CX3CL1 group), recombinant human CX3CL1+ selective CX3CR1 antagonist-JMS-17-2 (CX3CL1+anti-CX3CR1 group) and recombinant human CX3CL1 + selective CX3CR1 antagonist-JMS-17-2 + VS-6063 (CX3CL1+anti-CX3CR1+ FAK inhibitor group). M1 and HTR8/SVneo cells were co-cultured as described previously to assess invasion and migration capacity by transwell assays and Wound-healing assay. ResultsIn this study, CX3CL1 expression is high in the placental tissues of severe preeclampsia (sPE) patients than in normotensive pregnancies (NP). CX3CR1 expression is high in the decidual tissues of severe preeclampsia patients and mainly expressed in macrophages of decidual tissues. CX3CL1/CX3CR1 decreased VEGF expression in M1 macrophages and reduced the invasion and migration function of HTR-8/SVneo through the FAK signaling pathway. DiscussionThese findings revealed that CX3CL1-CX3CR1 regulate the trophoblast function by FAK and provided new insights into the pathogenesis of preeclampsia.

Impact of the Immunomodulatory Factor Soluble B7-H4 in the Progress of Preeclampsia by Inhibiting Essential Functions of Extravillous Trophoblast Cells.

A key aspect of preeclampsia pathophysiology is the reduced invasiveness of trophoblasts and the impairment of spiral artery remodelling. Understanding the causes of altered trophoblast function is critical to understand the development of preeclampsia. B7-H4, a checkpoint molecule, controls a wide range of processes, including T-cell activation, cytokine release, and tumour progression. Our previous findings indicated that B7-H4 levels are elevated in both maternal blood and placental villous tissue during the early stages of preeclampsia. Here, we investigated the function of B7-H4 in trophoblast physiology. Recombinant B7-H4 protein was used to treat human SGHPL-5 extravillous trophoblast cells. Biological functions were investigated using MTT, wound healing, and transwell assays. Signalling pathways were analysed by immunoblotting and immunofluorescence. The functionality of B7-H4 was further confirmed by immunoblotting and immunohistochemical analysis in placental tissues from control and preeclamptic patients following therapeutic plasma exchange (TPE) or standard of care treatment. This study showed that B7-H4 inhibited the proliferation, migration, and invasion capacities of SGHPL-5 extravillous cells while promoting apoptosis by downregulating the PI3K/Akt/STAT3 signalling pathway. These results were consistently confirmed in placental tissues from preterm controls compared to early-onset preeclamptic placental tissues from patients treated with standard of care or TPE treatment. B7-H4 may play a role in the development of preeclampsia by inhibiting essential functions of extravillous trophoblast cells during placental development. One possible mechanism by which TPE improves pregnancy outcomes in preeclampsia is through the elimination of B7-H4 amongst other factors.

Hydroquinone impairs trophoblast migration and invasion via AHR-twist-IFITM1 axis

IntroductionEmbryo implantation is a tightly regulated process, critical for a successful pregnancy. After attachment of the blastocyst to the surface epithelium of the endometrium trophoblast migrate from the trophectoderm and invade into the stromal component of endometrium. Alterations on either process will lead to implantation failure or miscarriage. Volatile organic compounds (VOCs) such as benzene induce pregnancy complications, including preterm birth and miscarriages. The mechanism of this effect is unknown. The objective of this study was to elucidate the impact of benzene metabolite, Hydroquinone, on trophoblast function. We tested the hypothesis that Hydroquinone activates the Aryl hydrocarbon receptor (AhR) pathway modulating trophoblast migration and invasion. MethodsFirst-trimester trophoblast cells (Sw.71) were treated with hydroquinone (6 and 25 μM). Trophoblast migration and invasion was evaluated using a 3D invasion/migration model. Gene expression was quantified by q-PCR and Western blot analysis. ResultsHydroquinone impairs trophoblast migration and invasion. This loss is associated with the activation of the AhR pathway which reduced the expression of Twist1and IFITM1. IFITM1 overexpression can rescue impaired trophoblast migration. DiscussionOur study highlights that hydroquinone treatment induces the activation of the AhR pathway in trophoblast cells, which impairs trophoblast invasion and migration. We postulate that activation of the AhR pathway in trophoblast suppress Twist1 and a subsequent IFITM1. Thus, the AhR-Twist1-IFITM1 axis represent a critical pathway involved in the regulation of trophoblast migration and it is sensitive to benzene exposure. These findings provide crucial insights into the molecular mechanisms underlying pregnancy complications induced by air pollution.

Transcriptome comparisons of trophoblasts from regenerative cell models with peri-implantation human embryos.

Mechanisms controlling trophoblast proliferation and differentiation during embryo implantation are poorly understood. Human trophoblast stem cells (TSC) and BMP4/A83-01/PD173074-treated pluripotent stem cell-derived trophoblast cells (BAP) are two widely employed, contemporary models to study trophoblast development and function, but how faithfully they mimic early trophoblast cells has not been fully examined. We evaluated the transcriptomes of trophoblast cells from BAP and TSC and directly compared them with those from peri-implantation human embryos during extended embryo culture (EEC) between embryonic day 8 to 12. The BAP and TSC grouped closely with trophoblast cells from EEC within each trophoblast sublineage following dimensional analysis and unsupervised hierarchical clustering. However, subtle differences in transcriptional programs existed within each trophoblast sublineage. We also validated the presence of six genes in peri-implantation human embryos by immunolocalization. Our analysis reveals that both BAP and TSC models have features of peri-implantation trophoblasts, while maintaining minor transcriptomic differences, and thus serve as valuable tools for studying implantation in lieu of human embryos.

Dysregulated lncSNHG12 suppresses the invasion and migration of trophoblasts by regulating Dio2/Snail axis to involve in recurrent spontaneous abortion

Recurrent spontaneous abortion (RSA) is a complex pathological process involving diverse factors, in which the dysregulated functions of trophoblasts cannot be ignored. Long noncoding RNA (lncRNA) has been reported to play a significant role in regulating the functions of trophoblasts in RSA. However, the impact and potential mechanism of lncRNA small nucleolar RNA host gene 12 (lncSNHG12) remain unclear. The role of lncSNHG12 in RSA was investigated through in vivo experiments and clinical samples. Co-IP and RNA pull down were conducted to explore the molecular mechanisms in trophoblasts. Our results showed that lncSNHG12 promoted the migration and invasion of trophoblasts by interacting with Iodothyronine deiodinase 2 (Dio2), which regulating the EMT process of trophoblasts by interacting with Snail. Moreover, in vivo experiments confirmed that lncSNHG12 could improve the fetal absorption rate of the abortion mice. The clinical samples revealed that lncSNHG12, Dio2 and Snail were down-regulated in the villous tissues of RSA patients, and positive correlations were confirmed between lncSNHG12 and Dio2, as well as Dio2 and Snail. In summary, the lncSNHG12/Dio2/Snail axis might be involved in the development of RSA by regulating the invasion and migration of trophoblasts.Abbreviations: RSA, recurrent spontaneous abortion; EVTs, extravillous trophoblasts; EMT, epithelial-to-mesenchymal transition; lncRNA, long non-coding RNA; Dio2, iodothyronine deiodinase 2; SNHGs, small nuclear RNA host genes; snoRNAs, small nuclear cell RNAs; LPS, lipopolysaccharide; De, derived decidua; Jz, junctional zone; Lz, labyrinth zones; RIP, RNA Binding Protein Immunoprecipitation; Co-IP, Co-Immunoprecipitation; RPISeq, RNA-Protein Interaction Prediction.