Cytotrophoblast Cells Research Articles

ALKBH5 modulation of ferroptosis in recurrent miscarriage: implications in cytotrophoblast dysfunction.

As one of the most common and abundant internal modifications of eukaryotic mRNA, N6-methyladenosine (m6A) modifications are closely related to placental development. Ferroptosis is a newly discovered form of programmed cell death. During placental development, placental trophoblasts are susceptible to ferroptosis. However, the interactions of m6A and ferroptosis in trophoblast physiology and injury are unclear. Recurrent miscarriage (RM) was selected as the main gestational disease in this study. Published data (GSE76862) were used to analyze the gene expression profiles in patients with RM. The extent of m6A modification in total RNA of villous tissues between patients with RM and healthy controls (HC) was compared. ALKBH5 (encoding AlkB homolog 5, RNA demethylase) was selected as the candidate gene for further research. Quantitative real-time reverse transcription PCR, western blotting, and immunohistochemistry (IHC) confirmed the elevated expression of ALKBH5 in the cytotrophoblasts of patients with RM. Then, cell counting kit-8 assays, glutathione disulfide/glutathione quantification, 2',7'-dichlorfluorescein-diacetate staining, and malonaldehyde assays were used to explore the alterations of ferroptosis-related characteristics following RAS-selective lethal (RSL3) stimulation after overexpression of ALKBH5. Thereafter, we re-analyzed the published RNA sequencing data upon knockdown of ALKBH5, combined with published tissue RNA-seq data, and FTL (encoding ferritin light chain) was identified as the ferroptosis-related gene in cytotrophoblasts of patients with RM that is regulated by ALKBH5. Finally, western blotting and IHC confirmed the increased expression of FTL in the cytotrophoblasts from patients with RM. Total m6A levels were decreased in patients with RM. The most significant differentially m6A-related gene was ALKBH5, which was increased in patients with RM. In vitro cell experiments showed that treatment with RSL3 resulted in increased cell death and upregulated ALKBH5 expression. Overexpression of ALKBH5 alleviated RSL3-induced HTR8 cell death and caused decreased levels of intracellular oxidation products. Published transcriptome sequencing revealed that FTL was the major ferroptosis-related gene regulated by ALKBH5 in the villous tissues of patients with RM. Consistent with the expression of ALKBH5, FTL was increased by RSL3-induction and increased in patients with RM. Elevated ALKBH5 alleviated RSL3-induced cytotrophoblast cell death by promoting the expression of FTL in patients with RM. Our results supported the view that ALKBH5 is an important regulator of the ferroptosis-related etiology of RM and suggested that ALKBH5 could be responsible for epigenetic aberrations in RM pathogenesis.

MCM proteins are up-regulated in placentas of women with reduced insulin sensitivity.

In the first trimester of pregnancy the human placenta grows rapidly, making it sensitive to changes in the intrauterine environment. To test whether exposure to an environment in utero often associated with obesity modifies placental proteome and function, we performed untargeted proteomics (LC-MS/MS) in placentas from 19 women (gestational age 35-48 days, i.e. 5+0-6+6 weeks). Maternal clinical traits (body mass index, leptin, glucose, C-peptide and insulin sensitivity) and gestational age were recorded. DNA replication and cell cycle pathways were enriched in the proteome of placentas of women with low maternal insulin sensitivity. Driving these pathways were the minichromosome maintenance (MCM) proteins MCM2, MCM3, MCM4, MCM5, MCM6 and MCM7 (MCM-complex). These proteins are part of the pre-replicative complex and participate in DNA damage repair. Indeed, MCM6 and γH2AX (DNA-damage marker) protein levels correlated in first trimester placental tissue (r = 0.514, P<0.01). MCM6 and γH2AX co-localized to nuclei of villous cytotrophoblast cells, the proliferative cell type of the placenta, suggesting increased DNA damage in this cell type. To mimic key features of the intrauterine obesogenic environment, a first trimester trophoblast cell line, i.e., ACH-3P, was exposed to high insulin (10 nM) or low oxygen tension (2.5% O2). There was a significant correlation between MCM6 and γH2AX protein levels, but these were independent of insulin or oxygen exposure. These findings show that chronic exposure in utero to reduced maternal insulin sensitivity during early pregnancy induces changes in the early first trimester placental proteome. Pathways related to DNA replication, cell cycle and DNA damage repair appear especially sensitive to such an in utero environment.

Galectin-8 Contributes to Human Trophoblast Cell Invasion.

Galectins are a class of lectins that are extensively expressed in all organisms. Galectins are involved in a range of functions, including early development, tissue regeneration, cancer and inflammation. It has been shown that galectin-8 is expressed in the villous and extravillous trophoblast (EVT) cells of the human placenta; however, its physiological role in pregnancy establishment has not been elucidated. Taking these factors into account, we investigated the functional role of galectin-8 in HTR-8/SVneo cells-a human EVT cell line-and human primary cytotrophoblast cells isolated from a first-trimester placenta. We analyzed the effects of recombinant human galectin-8 (rh galectin-8) on the adhesion, migration and invasion of HTR-8/SVneo cells. We used qPCR, cell-based ELISA (cELISA) and gelatin zymography to study the effects of galectin-8 on mediators of these processes, such as integrin subunits alpha-1 and beta-1 and matrix metalloproteinases (MMPs)-2 and -9, on the mRNA and protein levels. Further, we studied the effects of galectin-8 on primary cytotrophoblast cells' invasion. Galectin-8 stimulated the adhesion, migration and invasion of HTR-8/SVneo cells, as well as the invasion of primary cytotrophoblasts. In addition, the MMP-2 and -9 levels were increased, while the expression of integrins alpha-1 and beta-1 was not affected. Galectin-8 has the ability to positively affect EVTs' invasion, so it can be considered a significant factor in the trophoblast cell invasion process.

Reduced bioenergetics and mitochondrial fragmentation in human primary cytotrophoblasts induced by an EGFR-targeting chemical mixture

Exposures to complex environmental chemical mixtures during pregnancy reach and target the feto-placental unit. This study investigates the influence of environmental chemical mixtures on placental bioenergetics. Recognizing the essential role of the epidermal growth factor receptor (EGFR) in placental development and its role in stimulating glycolysis and mitochondrial respiration in trophoblast cells, we explored the effects of chemicals known to disrupt EGFR signaling on cellular energy production. Human primary cytotrophoblasts (hCTBs) and a first-trimester extravillous trophoblast cell line (HTR-8/SVneo) were exposed to a mixture of EGFR-interfering chemicals, including atrazine, bisphenol S, niclosamide, PCB-126, PCB-153, and trans-nonachlor. An RNA sequencing approach revealed that the mixture altered the transcriptional signature of genes involved in cellular energetics. Next, the impact of the mixture on cellular bioenergetics was evaluated using a combination of mitochondrial and glycolytic stress tests, ATP production, glucose consumption, lactate synthesis, and super-resolution imaging. The chemical mixture did not alter basal oxygen consumption but diminished the maximum respiratory capacity in a dose-dependent manner, indicating a disruption of mitochondrial function. The respiratory capacity and ATP production were increased by EGF, while the Chem-Mix reduced both EGF- and non-EGF-mediated oxygen consumption rate in hCTBs. A similar pattern was observed in the glycolytic medium acidification, with EGF increasing the acidification, and the Chem-Mix blocking EGF-induced glycolytic acidification. Furthermore, direct stochastic optical reconstruction microscopy (dSTORM) imaging demonstrated that the Chem-Mix led to a reduction of the mitochondrial network architecture, with findings supported by a decrease in the abundance of OPA1, a mitochondrial membrane GTPase involved in mitochondrial fusion. In conclusion, we demonstrated that a mixture of EGFR-disrupting chemicals alters mitochondrial remodeling, resulting in disturbed cellular bioenergetics, reducing the capacity of human cytotrophoblast cells to generate energy. Future studies should investigate the mechanism by which mitochondrial dynamics are disrupted and the pathological significance of these findings.

Gestational Trophoblastic Disease: Complete versus Partial Hydatidiform Moles.

Hydatidiform moles, including both complete and partial moles, constitute a subset of gestational trophoblastic diseases characterized by abnormal fertilization resulting in villous hydrops and trophoblastic hyperplasia with or without embryonic development. This involves chromosomal abnormalities, where one or two sperms fertilize an empty oocyte (complete hydatidiform mole (CHM); mostly 46,XX) or two sperms fertilize one oocyte (partial hydatidiform mole (PHM); mostly 69,XXY). Notably, recurrent occurrences are associated with abnormal genomic imprinting of maternal effect genes such as NLRP7 (chromosome 19q13.4) and KHDC3L (chromosome 6q1). Ongoing efforts to enhance identification methods have led to the identification of growth-specific markers, including p57 (cyclin-dependent kinase inhibitor 1C; CDKN1C), which shows intact nuclear expression in the villous cytotrophoblast and villous stromal cells in PHMs and loss of expression in CHMs. Treatment of hydatidiform moles includes dilation and curettage for uterine evacuation of the molar pregnancy followed by surveillance of human chorionic gonadotropin (HCG) levels to confirm disease resolution and rule out the development of any gestational trophoblastic neoplasia. In this review, we provide a synopsis of the existing literature on hydatidiform moles, their diagnosis, histopathologic features, and management.

The role of placental kisspeptin in trophoblast invasion and migration: an assessment in Kiss1r knockout mice, BeWo cell lines and human term placenta.

Context There is mounting evidence implicating kisspeptin signalling in placental development and function. Aims This study aimed to elucidate kisspeptin's role in trophoblast invasion and migration using three experimental models. Methods First, we examined the mouse fetus and placenta in a kisspeptin receptor (Kiss1r) knockout (KO) model. Fetal/placental weights and gene expression (quantitative polymerase chain reaction) were assessed. Second, we determined kisspeptin effects on a human trophoblast (BeWo) cell line in vitro . Third, we examined KISS1 and KISS1R gene expression in human placenta from term and pre-term pregnancies. Key results No difference was found in fetal or placental weight between Kiss1r KO and wildtype mice. However, expression of the trophoblast invasion marker, Mmp2 mRNA, was greater in the placental labyrinth zone of Kiss1r KO mice. BeWo cell models of villus cytotrophoblast and syncytiotrophoblast cells exhibited kisspeptin protein expression, with greater expression in syncytiotrophoblast, consistent with KISS1 mRNA. Kisspeptin treatment inhibited the migratory potential of cytotrophoblast-like cells. Finally, while no difference was seen in KISS1 and KISS1R mRNA between term and pre-term placentas, we saw a difference in the relative expression of each gene pre-term. We also observed a positive correlation between KISS1 expression and maternal body mass index. Conclusions Our results indicate that kisspeptin may inhibit trophoblast invasion. Implications Further investigation is required to clarify specific regulatory mechanisms.

Epigenetic changes regulating the epithelial-mesenchymal transition in human trophoblast differentiation.

The phenotype of human placental extravillous trophoblast (EVT) at the end of pregnancy reflects both first trimester differentiation from villous cytotrophoblast (CTB) and later gestational changes, including loss of proliferative and invasive capacity. Invasion abnormalities are central to two major placental pathologies, preeclampsia and placenta accreta spectrum, so characterization of the corresponding normal processes is crucial. In this report, our gene expression analysis, using purified human CTB and EVT cells, highlights an epithelial-mesenchymal transition (EMT) mechanism underlying CTB-EVT differentiation and provides a trophoblast-specific EMT signature. In parallel, DNA methylation profiling shows that CTB cells, already hypomethylated relative to non-trophoblast cell lineages, show further genome-wide hypomethylation in the transition to EVT. However, a small subgroup of genes undergoes gains of methylation (GOM) in their regulatory regions or gene bodies, associated with differential mRNA expression (DE). Prominent in this GOM-DE group are genes involved in the EMT, including multiple canonical EMT markers and the EMT-linked transcription factor RUNX1, for which we demonstrate a functional role in modulating the migratory and invasive capacities of JEG3 trophoblast cells. This analysis of DE associated with locus-specific GOM, together with functional studies of an important GOM-DE gene, highlights epigenetically regulated genes and pathways acting in human EVT differentiation and invasion, with implications for obstetric disorders in which these processes are dysregulated.

P-800 Best matrix match: Laminin-511 improves human trophoblast stem cell identity in vitro

Abstract Study question Can alternative basement membranes support human trophoblast stem cell (hTSC) culture and recapitulate the in vivo environment more effectively than Collagen-IV (Col-IV)? Summary answer Laminin-511 (Lam-511) is the optimal alternative xeno-free matrix that enhances the stem cell state and more closely recapitulates the in vivo hTSC niche. What is known already Our knowledge on early placentation is limited due to a historical lack of robust in vitro models. Recently, hTSCs isolated from human blastocysts and first trimester placentas were successfully cultured on Col-IV, recapitulating cytotrophoblast cell self-renewal and differentiation. Even though Col-IV has been detected in the cytotrophoblast basement membrane, previously published RNA-seq analysis does not detect transcripts encoding Col-IV in the human trophectoderm. Attachment of adhesion molecules to the ECM can have an effect on cell proliferation, differentiation, polarity and gene expression. Using the correct matrix ensures a stable cell growth, as it can influence cell behaviour and identity. Study design, size, duration This was a prospective study carried out over a 6-month period. Patient-derived hTSC lines, BTS5 and CT27, obtained from RIKEN BRC Cell Bank were cultured according to the previously published protocol (1). hTSCs were maintained on culture plates coated with Col-IV, Lam-511, laminin-521 and fibronectin. Experiments were performed with biological replicates of both cell lines (n = 4), grown in every condition. Cells grown on Col-IV were used as a positive control. Participants/materials, setting, methods hTSCs were maintained on the candidate matrices for minimum of 5 passages and then were used for subsequent experiments. hTSC identity was determined by immunofluorescence analysis for known hTSC markers, GATA2 and KRT18. qRT-PCR analyses were also performed to detect additional markers EGFR, ITGA6, TEAD4, TP63, and expression from the chromosome 19 miRNA cluster (C19MC). The self-renewal capacity was assessed by cell counting and Ki67-staining. The differentiation potential was examined by performing previously published protocols. Main results and the role of chance RNA-seq analysis identified expression of ECM ligand and receptor transcripts encoding Lam-511, laminin-521 and fibronectin, which were used as candidate matrices to test. hTSC identity was maintained in all conditions as confirmed by morphological assessment, expression of known hTSC markers at the transcript and protein level, and detection of expression of C19MC miRNAs. The highest hTSC marker expression was found in cells grown on laminins. However, interestingly it was observed that only Lam-511 was capable of supporting hTSCs for more than 15 passages. Compared to Col-IV, hTSCs grown on Lam-511 appear to be more proliferative as there was a higher cell count over a 6-day period and a higher proportion of Ki-67-positive proliferative cells (p = 0.03). Additionally, preliminary data suggests that differentiation ability of hTSCs grown on Lam-511 into syncytiotrophoblasts and extravillous cytotrophoblasts is retained. Therefore, Lam-511 is an improvement to the conventional model as it enhances the stem-cell identity and closely recapitulates the in vivo niche. It can also be used as an alternative xeno-free matrix in hTSC culture in order to overcome current supply chain issues and reduce batch-to-bath variability associated with Col-IV. Limitations, reasons for caution During the 6-month period of this study, two contaminations occurred that led to slower progression of the experiments and smaller sample size. Also, there was low efficiency of the extravillous cytotrophoblast differentiation which was due to the fact that the previously published protocol performed has not yet been optimised. Wider implications of the findings Recapitulating the in vivo niche more closely will allow a deeper understanding of early placentation and help create more accurate 3D models to study the pathomechanisms of various pregnancy complications. Furthermore, utilising the xeno-free Lam-511 as an alternative matrix is a step closer to building a GMP-compliant hTSC culture. Trial registration number Not applicable

The Effects of Low Concentrations of Pravastatin on Placental Cells

Pravastatin is a promising medication to treat preeclampsia. However, the appropriate dose of pravastatin for managing preeclampsia has not been established. In this in vitro study, we examined the effects of low concentrations of pravastatin (0.01 to 10 µM) under hypoxic conditions on two types of placental cells and found that pravastatin decreased sFlt-1 levels up to 34% in cytotrophoblast cells isolated from human term placentas. Furthermore, we showed that sFlt-1 levels in HTR-8/SVneo cells, a cell line derived from first trimester trophoblast cells, decreased after exposure to very low concentrations of pravastatin (0.01, 0.1 µM). We also examined the effects of pravastatin on uterine spiral artery remodeling-related events and showed in wound healing and tube formation assays that low concentrations of pravastatin upregulated cell migration and invasion in HTR-8/SVneo cells. These results demonstrated that a low dose of pravastatin has in vitro effects that suggest a potential for anti-preeclamptic effects in vivo.

CYP19A1 Expression Is Controlled by mRNA Stability of the Upstream Transcription Factor AP-2γ in Placental JEG3 Cells.

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.

Placental Vimentin Expression in Preeclampsia and Gestational Diabetes Mellitus

OBJECTIVE: This study investigated vimentin expression in placentas of patients with preeclampsia and gestational diabetes mellitus (GDM). STUDY DESIGN: Placentas of preeclamptic women (n=25), women with GDM (n=25), and control cases (n=25) were enrolled in this study. Placental samples were fixed in zinc-formalin and further processed for paraffin wax tissue embedding. Demographic and laboratory parameters of patients were recorded. Vimentin immune activity was analyzed in the placental sections with immunohistochemistry. Sections were imaged and analyzed under a light microscope. A semiquantitative measurement was done between groups by comparing the Vimentin signal and significance was calculated. Network construction and pathway enrichment analysis were conducted using Cytoscape (v3.10.1) and ShinyGO, respectively. RESULTS: Vimentin expression was high in the placental sections of the control group. The preeclampsia group showed positive Vimentin expression in cytotrophoblast and syncytiotrophoblast cells and connective tissue of placental villi in the preeclampsia group. Vimentin expression was generally recorded as negative in placental villi, fibrinoid substances, and connective tissue cells in the GDM group. Bioinformatic analysis showed that the AGE-RAGE signaling pathway and cancer-related pathways were mainly observed in Vimentin-associated pathways, which finally activate inflammatory pathways in both preeclampsia and GDM. CONCLUSION: Vimentin expression patterns in placental tissue sections reveal nuanced regulatory mechanisms, emphasizing the need for further exploration into the functional roles of vimentin in placental physiology and pathology.

SP6 controls human cytotrophoblast fate decisions and trophoblast stem cell establishment by targeting MSX2 regulatory elements

The commitment and differentiation of human placental progenitor cytotrophoblast (CT) cells are crucial for a successful pregnancy, but the underlying mechanism remains poorly understood. Here, we identified the transcription factor (TF), specificity protein 6 (SP6), as a human species-specific trophoblast lineage TF expressed in human placental CT cells. Using pluripotent stem cells as a model, we demonstrated that SP6 controls CT generation and the establishment of trophoblast stem cells (TSCs) and identified msh homeobox 2 (MSX2) as the downstream effector in these events. Mechanistically, we showed that SP6 interacts with histone acetyltransferase P300 to alter the landscape of H3K27ac at targeted regulatory elements, thereby favoring transcriptional activation and facilitating CT cell fate decisions and TSC maintenance. Our results established SP6 as a regulator of the human trophoblast lineage and implied its role in placental development and the pathogenies of placental diseases.

Maternal SARS-CoV-2 infection in pregnancy disrupts gene expression in Hofbauer cells with limited impact on cytotrophoblasts.

Hofbauer cells (HBCs) and cytotrophoblasts (CTBs) are major cell populations in placenta. The indirect impact of maternal SARS-CoV-2 disease on these cells that are not directly infected has not been extensively studied. Herein, we profiled gene expression in HBCs and CTBs isolated from placentae of recovered pregnant subjects infected with SARS-CoV-2 during all trimesters of pregnancy, placentae from subjects with active infection, SARS-CoV-2 vaccinated subjects, and those who were unexposed to the virus. Placentae were collected within 4 h post-delivery and membrane-free tissues were enzymatically digested for the isolation of HBCs and CTBs. RNA extracted from HBCs and CTBs were sequenced using 150bp paired-end reads. Differentially expressed genes (DEGs) were identified by DESeq2 package in R and enriched in GO Biological Processes, KEGG Pathway, Reactome Gene Sets, Hallmark Gene Sets, and Canonical Pathways. Protein-protein interactions among the DEGs were modelled using STRING and BioGrid. Pregnant subjects (n = 30) were recruited and categorized into six groups: infected with SARS-CoV-2 in i) the first (1T, n = 4), ii) second (2T, n = 5), iii) third (3T, n = 5) trimester, iv) tested positive at delivery (Delivery, n = 5), v) never infected (Control, n = 6), and vi) fully mRNA-vaccinated by delivery (Vaccinated, n = 5). Compared to the Control group, gene expression analysis showed that HBCs from infected subjects had significantly altered gene expression profiles, with the 2T group having the highest number of DEGs (1,696), followed by 3T and 1T groups (1,656 and 958 DEGs, respectively). These DEGs were enriched for pathways involved in immune regulation for host defense, including production of cytokines, chemokines, antimicrobial proteins, ribosomal assembly, neutrophil degranulation inflammation, morphogenesis, and cell migration/adhesion. Protein-protein interaction analysis mapped these DEGs with oxidative phosphorylation, translation, extracellular matrix organization, and type I interferon signaling. Only 95, 23, and 8 DEGs were identified in CTBs of 1T, 2T, and 3T groups, respectively. Similarly, 11 and 3 DEGs were identified in CTBs and HBCs of vaccinated subjects, respectively. Reassuringly, mRNA vaccination did not induce an inflammatory response in placental cells. Our studies demonstrate a significant impact of indirect SARS-CoV-2 infection on gene expression of inner mesenchymal HBCs, with limited effect on lining CTB cells isolated from pregnant subjects infected and recovered from SARS-CoV-2. The pathways associated with these DEGs identify potential targets for therapeutic intervention.

Diagnostic Role of Cell-Free miRNAs in Identifying Placenta Accreta Spectrum during First-Trimester Screening.

Placenta accreta spectrum (PAS) is a severe complication of pregnancy associated with excessive invasion of cytotrophoblast cells at the sites of the endometrial-myometrial interface and the myometrium itself in cases of adherent (creta) and invasive (increta and percreta) forms, respectively. This leads to a high risk of massive blood loss, maternal hysterectomy, and preterm birth. Despite advancements in ultrasound protocols and found associations of alpha-fetoprotein, PAPP-A, hCG, PLGF, sFlt-1, IL-8, and IL-33 peripheral blood levels with PAS, there is a high need for an additional non-invasive test to improve the diagnostic accuracy and to select the real PAS from the suspected ones in the first-trimester screening. miRNA signatures of placental tissue, myometrium, and blood plasma from women with PAS in the third trimester of pregnancy, as well as miRNA profiles in exosomes from the blood serum of women in the first trimester with physiologically progressing pregnancy, complicated by PAS or pre-eclampsia, were obtained using deep sequencing. Two logistic regression models were constructed, both featuring statistically significant parameters related to the levels of miR-26a-5p, miR-17-5p, and miR-101-3p, quantified by real-time PCR in native blood serum. These models demonstrated 100% sensitivity in detecting PAS during the first pregnancy screening. These miRNAs were identified as specific markers for PAS, showing significant differences in their blood serum levels during the first trimester in the PAS group compared to those in physiological pregnancies, early- or late-onset pre-eclampsia groups. Furthermore, these miRNAs exhibited differential expression in the PAS placenta and/or myometrium in the third trimester and, according to data from the literature, control angiogenesis. Significant correlations were found between extracellular hsa-miR-101-3p and nuchal translucency thickness, hsa-miR-17-5p and uterine artery pulsatility index, and hsa-miR-26a-5p and hsa-miR-17-5p with PLGF. The developed test system for early non-invasive PAS diagnosis based on the blood serum level of extracellular miR-26a-5p, miR-17-5p, and miR-101-3p can serve as an auxiliary method for first-trimester screening of pregnant women, subject to validation with independent test samples.

CPY27B1-related vitamin D deficiency contributes to the impaired function of CD4+T cells in Recurrent spontaneous miscarriage

The study aimed to explore the relationship between the expression of cytochrome P450 family 27 subfamily B member 1 (CYP27B1), vitamin D, and impaired T cell subsets in recurrent spontaneous miscarriage (RSM). A Total of 779 healthy women of childbearing age and 1031 women with a history of RSM were involved in this study. The results of flow cytometry showed that the proportion of Tregs was higher in healthy women than in the women with RSM. For cytokines, the levels of interleukin-17 (IL-17) and interferon-gamma (IFN-γ) were significantly higher in RSM patients than in healthy women, while IL-10 was notably lower in RSM patients. Furthermore, compared to healthy individuals, RSM patients had lower levels of serum 25(OH)D detected by chemiluminescence. The frequency of Tregs was negatively correlated with 25(OH)D. Specifically, for every 10 ng/ml increase in 25(OH)D, the percentage of Tregs increased by 0.58 as calculated. IL-17 and IFN-γ were inversely correlated with 25(OH)D, while the serum interleukin-10 (IL-10) level was positively correlated with 25(OH)D. CYP27B1 was found to be expressed in both cytotrophoblast and extracellular villi trophoblast cells. However, reduced expression of CYP27B1 was observed in the placenta with RSM. Notably, the level of 25(OH)D increased in the supernatant of CYP27B1 knockdown BeWo compared to normal cells, while human chorionic gonadotropin (hCG) was significantly reduced. The hCG secretion of CYP27B1 KO BeWo cells was partially restored after 1,25(OH)2D3 supplementation. In addition, 1,25(OH)2D3 treatment could induce more CD4+ T cells to convert to Foxp3+iTreg, which in turn inhibited the secretion of IL-17, IFN-γ. In summary, this research unveiled a connection between reduced CYP27B1 and vitamin D deficiency in RSM. Our study underscores the potential benefits of vitamin D treatment supplementation in the context of RSM. However, it is important to note that further research is imperative to validate these observations.

Isolation of Human Villous Cytotrophoblastic Cells from Term Placenta for Transfection.

Isolation of villous cytotrophoblastic cells (vCTBs) from placental tissue provides the possibility of studying differentiation of these cells as well as their function. We describe herein a protocol, in which term placentas are used to isolate a population of vCTB in high quantities. These cells are subsequently used to transfect siRNA through microporation and thereby to achieve efficient silencing of targeted genes.

EGFR expression in COVID19 placentas

Introduction: Coronavirus disease 2019 (COVID-19) pandemic is a global health problem that has claimed many lives worldwide. The disease is affecting almost all populations, but it poses a particular risk for groups such as the elderly and those with chronic diseases. Although the number of studies on clinical situations involving pregnant women and their infants infected with COVID-19 is increasing, the factors supporting the negative association between COVID-19 and pregnancy remain unclear. In this study, we aimed to examine the placental histopathologic structure using EGFR (Epidermal Growth Factor) antibody in placentas of COVID-19 pregnant patients.&#x0D; Materials and methods: In our study, placentas of a total of 40 pregnant women (regardless of age), including 20 pregnant women with C0VID-19 and 20 normotensive pregnant women, were used, and placentas were compared histopathologically and immunohistochemically.&#x0D; Results: In the hematoxylin and eosin stained sections of the control group placentas, tissue integrity was preserved and cellular structures were normal. In the placentas of Covid -19 group patients, it was observed that degenerations increased and cellular deformations developed. In sections of control group placentas, EGFR expression was negative in decidual cells and in areas with syncytial nodes. EGFR expression was positive in cytotrophoblast cells. In placenta sections of Covid-19 patients, EGFR expression was positive in vascular endothelium and Hofbauer cells.&#x0D; Conclusion: In our study, it was observed that EGFR expression, an important biomarker in tissue regeneration, was inhibited. We also concluded that Covid-19 caused degeneration and inflammation in placental tissue.

α1,3-fucosylation of MEST promotes invasion potential of cytotrophoblast cells by activating translation initiation

Embryo implantation into the uterus is the gateway for successful pregnancy. Proper migration and invasion of embryonic trophoblast cells are the key for embryo implantation, and dysfunction causes pregnancy failure. Protein glycosylation plays crucial roles in reproduction. However, it remains unclear whether the glycosylation of trophoblasts is involved in trophoblast migration and invasion processes during embryo implantation failure. By Lectin array, we discovered the decreased α1,3-fucosylation, especially difucosylated Lewis Y (LeY) glycan, in the villus tissues of miscarriage patients when compared with normal pregnancy women. Downregulating LeY biosynthesis by silencing the key enzyme fucosyltransferase IV (FUT4) inhibited migration and invasion ability of trophoblast cells. Using proteomics and translatomics, the specific LeY scaffolding glycoprotein of mesoderm-specific transcript (MEST) with glycosylation site at Asn163 was identified, and its expression enhanced migration and invasion ability of trophoblast cells. The results also provided novel evidence showing that decreased LeY modification on MEST hampered the binding of MEST with translation factor eIF4E2, and inhibited implantation-related gene translation initiation, which caused pregnancy failure. The α1,3-fucosylation of MEST by FUT4 may serve as a new biomarker for evaluating the functional state of pregnancy, and a target for infertility treatment.

OR09-06 The Cholesterol Oxidation Product 7-Ketosterol Reduces Progesterone Production By Human Cytotrophoblast Cells In A Sex Dimorphic Manner

Abstract Disclosure: E. Ticiani: None. N. Pascuali: None. P. Danos: None. C. Murga-Zamalloa: None. I. Buhimschi: None. A. Veiga-Lopez: None. Many pregnancy complications including recurrent miscarriage and preterm birth are strongly associated with placental dysfunction and heightened inflammation. Diet-derived cholesterol, especially via consumption of fast foods that have undergone thermal processing or long-term storage, is highly susceptible to oxidation which results in cholesterol oxidation products, known as oxysterols. Oxysterols are known drivers of inflammation and have been implicated in chronic diseases, including atherosclerosis and neurodegeneration. We hypothesized that the prototype oxysterol 7-ketosterol (7-KT) induces reduced steroid synthesis and/or secretion in human placental cells associated with a pro-inflammatory response. To test this hypothesis, we isolated human primary cytotrophoblast cells (hCTB) from healthy term pregnancies with singleton female fetuses (n=4) and exposed them to 7-KT (2.5 µg/ml) or vehicle control (C; 0.1% DMSO) for 96h. Cells were subjected to RNA sequencing using Illumina TruSeq (p&amp;lt;0.05, FDR &amp;lt;0.05 and log2 fold change &amp;gt;1). Pathway analysis discovered that 10 of the most down-regulated genes (CYP51A1, MSMO1, IDI1, HMGCS1, HMGCR, ACAT2, DDX31, DHCR7, SREBF2, NLRP7) were associated with lipid metabolism. Among them, sterol regulatory element binding transcription factor 2 (SREBF2), is a known activator of cholesterol synthesis. Using female (n=4) and male hCTB (n=4), we further evaluated sex specific effects of 7-KT after 96h of exposure using RT-qPCR. We confirmed that 7-KT reduced the expression of SREBF2 in female and in male hCTB, as well as of genes responsible for cholesterol influx (LDLR) and synthesis (HMGCR and MVK), but not of the cholesterol efflux gene ABCA1. Similar gene expression patterns were observed in JEG-3 cells exposed to 7-KT, a choriocarcinoma cell line that retains the ability to synthetize progesterone. This was coupled with a ∼50% reduction in intracellular cholesterol in JEG-3 cells exposed to 7-KT. Considering that cholesterol is the precursor for progesterone, we assessed progesterone secretion in JEG-3 and in male and female-derived hCTBs cells by ELISA. 7-KT reduced progesterone secretion by JEG-3 and female hCTB by ∼40% while male hCTB were not affected. Additionally, a short exposure (24h) to 7-KT increased the expression of pro-inflammatory genes (IL1α, IL6 and TNFα) in female, but not male hCTB. Altogether, our results demonstrate that the oxysterol 7-KT induces early pro-inflammatory gene expression, followed by a reduction in cholesterol metabolism, lower intracellular cholesterol, and progesterone secretion. Future studies should further investigate the mechanism by which 7-KT drives sex-specific changes in placental cells, and the impact of excess oxysterols on pregnancy outcomes. Presentation: Friday, June 16, 2023

Zika virus targets human trophoblast stem cells and prevents syncytialization in placental trophoblast organoids

Zika virus (ZIKV) infection during pregnancy threatens pregnancy and fetal health. However, the infectivity and pathological effects of ZIKV on placental trophoblast progenitor cells in early human embryos remain largely unknown. Here, using human trophoblast stem cells (hTSCs), we demonstrated that hTSCs were permissive to ZIKV infection, and resistance to ZIKV increased with hTSC differentiation. Combining gene knockout and transcriptome analysis, we demonstrated that the intrinsic expression of AXL and TIM-1, and the absence of potent interferon (IFN)-stimulated genes (ISGs) and IFNs contributed to the high sensitivity of hTSCs to ZIKV. Furthermore, using our newly developed hTSC-derived trophoblast organoid (hTSC-organoid), we demonstrated that ZIKV infection disrupted the structure of mature hTSC-organoids and inhibited syncytialization. Single-cell RNA sequencing (scRNA-seq) further demonstrated that ZIKV infection of hTSC-organoids disrupted the stemness of hTSCs and the proliferation of cytotrophoblast cells (CTBs) and probably led to a preeclampsia (PE) phenotype. Overall, our results clearly demonstrate that hTSCs represent the major target cells of ZIKV, and a reduced syncytialization may result from ZIKV infection of early developing placenta. These findings deepen our understanding of the characteristics and consequences of ZIKV infection of hTSCs in early human embryos.