Villus Tissue Research Articles

The circRNA Landscape in Recurrent Pregnacy Loss (RPL): A Comparison of Four Reproductive Tissues

Recurrent Pregnancy Loss (RPL), also named Recurrent Spontaneous Abortion (RSA), is a common fertility problem that refers to at least two consecutive pregnancy losses and affects 1–2% of couples all over the world. Despite common causes such as genetic abnormalities, uterine anomalies or hormonal and metabolic disorders, there is still a huge challenge in identifying the causes of about 40–60% of RPL patients. Circular RNAs (circRNAs) are endogenous ncRNAs with a unique closed-loop and single-stranded structure. Accumulated evidence indicates the role of circRNAs in embryonic development and implantation, which may help decipher the mechanisms and causes underlying RSA. Four works were selected in the SRA public repository that used RNAseq analysis in control and RPL samples in four tissues: endometrium, chorionic villus tissue, decidua and decidua immune cells. Two programs were selected for circRNA detection: DCC and CIRI2. A total of 1715 candidate circRNAs were detected after filtering the results. In the differential expression analysis, decidual tissue showed the highest percentage of circRNA with differential expression between cases and controls. CircRNAs originating from OGA, FNDC3B, RAB11FIP1, SIPA1L2 and GREB1L showed the highest expression in women suffering from pregnancy losses, in decidual tissue or endometrium. In the GO term enrichment analysis, multiple terms related to embryonic development and immunological response were consistently enriched in villus and decidual tissues. Although some differentially expressed circRNAs were shared between tissues, decidua seems the tissue of choice for analyzing the role of circRNAs in RPL.

Glutamine metabolism promotes human trophoblast cell invasion via COL1A1 mediated by PI3K-AKT pathway

Abnormal trophoblast invasion function is an important cause of recurrent spontaneous abortion (RSA). Recent research has revealed a connection between glutamine metabolism and RSA. However, the interplay between these three factors and their related mechanisms remains unclear. To address this issue, we collected villus tissues from 10 healthy women with induced abortion and from 10 women with RSA to detect glutamine metabolism. Then, the trophoblast cell line HTR-8/SVneo was used in vitro to explore the effect of glutamine metabolism on trophoblast cells invasion, which was tested by transwell assay. We found that the concentration of glutamine in the villi of the normal pregnancy group was significantly higher than that in the RSA group. Correspondingly, the expression levels of key enzymes involved in glutamine synthesis and catabolism, including glutamine synthetase and glutaminase, were significantly higher in the villi of the normal pregnancy group. Regarding trophoblast cells, glutamine markedly enhanced the proliferative and invasive abilities of HTR-8/SVneo cells. Additionally, collagen type I alpha 1 (COL1A1) was confirmed to be a downstream target of glutamine, and glutamine also activated the PI3K-AKT pathway in HTR-8/SVneo cells. These findings indicate that glutamine metabolism facilitates the invasion of trophoblasts by up-regulating COL1A1 expression through the activation of the PI3K-AKT pathway, but the specific mechanism of COL1A1 requires further study.

Copaiba oil’s bactericidal activity and its effects on health and zootechnical performance for Nile tilapia after oral supplementation

Tilapia is one of the most important farmed fish in the world and the most cultivated in Brazil. The increase of this farming favors the appearance of diseases, including bacterial diseases. Therefore, the aim of this study was to evaluate the bactericidal activity of copaiba oil, Copaifera duckei, against Streptococcus agalactiae and Flavobacterium columnare and the dietary effect of copaiba oil on zootechnical performance, hematological, biochemical, immunological, and histological analysis before and after an intraperitoneal infection (body cavity) with S. agalactiae in Nile tilapia. For this, fish were randomly distributed into 15 fiber tanks in five treatments (0, 0.25, 0.50, 0.75, and 1.0%) and fed with a commercial diet supplemented with copaiba oil for 30 days. After this period, the fish were randomly redistributed for the experimental challenge with S. agalactiae into six treatments (T0, T1, T2, T3, T4, and T5), the fish were anesthetized, and blood samples were collected to assess hematological, biochemical, immunological, and histological parameters. Copaiba oil showed bactericidal activity against Streptococcus spp. and Flavobacterium spp. in vitro. In addition, concentrations of 0.75 and 1.0% of copaiba oil have an anti-inflammatory effect and improve hematological and immunological parameters, increasing leukocyte numbers, albumin, and serum lytic activity. Furthermore, there is an increase in the intestinal villus length and tissue damage in groups at concentrations of 0.75 and 1.0% of copaiba oil. In conclusion, copaiba oil presented bactericidal activity against Streptococcus spp. and Flavobacterium spp. in vitro, and oral supplementation at concentrations of 0.75 and 1.0% compared to the control group enhanced non-specific immune parameters and digestibility in Nile Tilapia.

Leveraging chorionic villus biopsies for the derivation of patient-specific trophoblast stem cells.

Human trophoblast stem (TS) cells are an informative in vitro model for the generation and testing of biologically meaningful hypotheses. The goal of this project was to derive patient-specific TS cell lines from clinically available chorionic villus sampling biopsies. Cell outgrowths were captured from human chorionic villus tissue specimens cultured in modified human TS cell medium. Cell colonies emerged early during the culture and cell lines were established and passaged for several generations. Karyotypes of the newly established chorionic villus-derived trophoblast stem (TS CV ) cell lines were determined and compared to initial genetic diagnoses from freshly isolated chorionic villi. Phenotypes of TSCV cells in the stem state and following differentiation were compared to cytotrophoblast-derived TS (TS CT ) cells. TSCV and TSCT cells uniformly exhibited similarities in the stem state and following differentiation into syncytiotrophoblast and extravillous trophoblast cells. Chorionic villus tissue specimens provide a valuable source for TS cell derivation. They expand the genetic diversity of available TS cells and are associated with defined clinical outcomes. TSCV cell lines provide a new set of experimental tools for investigating trophoblast cell lineage development.

The relationship between Sjögren's syndrome and recurrent pregnancy loss: a bioinformatics analysis

Research QuestionSjögren's syndrome (SS) is an autoimmune disease and also an essential factor in recurrent pregnancy loss (RPL). What are the gene-related relationships between SS and RPL pathogenesis? DesignWe obtained the SS- and RPL-related gene datasets from the Gene Expression Omnibus database and identified the co-expression modules and shared differentially expressed genes (DEGs) in diseases through the weighted gene co-expression network analysis (WGCNA) and limma analysis based on sample size. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were applied to reveal the hidden biological pathways. Additionally, shared hub gene identification, gene set enrichment analysis, hub gene's association with ferroptosis and immunity, drug sensitivity analysis, single-cell RNA sequencing analysis, and construction of ceRNA network were conducted. ResultsBy intersecting the genes from the WGCNA and limma analysis, one shared hub gene (KCNN3) was derived, exhibiting upregulation in SS and RPL. There was a positive relationship between KCNN3 and the immune-related gene TLR2. The ceRNA network revealed that XIST was the most shared lncRNA and could competitively bind with 8 miRNAs to regulate the expression of KCNN3. Forty-eight drugs were found to be strongly associated with KCNN3 expression, such as Estramustine and cyclosporine. Moreover, KCNN3 exhibited high expression in RPL endothelial cells of villus tissue. ConclusionsThis research is one of the initial studies to reveal that SS shares common biological pathways with RPL. KCNN3 was identified as the one hub gene correlated with SS and RPL, and it may be a new target for mechanistic studies on SS and RPL.

P-420 ALG3 is a new novel marker in preeclampsia

Abstract Study question To investigate the potential function of ALG3 (Asparagine-Linked Glycosylation 3, Alpha-1,3-Mannosyltransferase Homolog) in the pathogenesis of preeclampsia(PE). Summary answer ALG3 plays an important role in the pathogenesis of PE by influencing the function of the trophoblast cells. What is known already PE is the leading cause of maternal and perinatal mortality and morbidity. The underlying mechanism is still unclear. Disorder migration and invasion function of trophoblasts are one of the mechanisms in PE. ALG3 regulates the Dol-P-Man-dependent mannosylation, leading to the formation of a polyterpene alcohol-oligosaccharide precursor and subsequent N-glycan synthesis. The expression of ALG3 in villus tissue during the early stages of preeclampsia is significantly reduced, as indicated by public databases (GSE12767). The investigation into the potential role of ALG3 in early trophoblast development and its contribution to the pathogenesis of PE holds significant scientific interest. Study design, size, duration The placental samples were acquired quickly (<30 mins) from a group of normal pregnant women (without PE or other complications) (n = 15) and PE patients (n = 20) to investigate the expression of ALG3. Secondly, lentiviral systems were employed to establish loss and gain of function models in the trophoblast cell lines JEG3 and HTR8/Sneov for elucidating the role of ALG3 in the pathogenesis of PE. Participants/materials, setting, methods We examined the expression of ALG3 both in placental tissues from PE and normal pregnant. Meanwhile, we generated ALG3 loss and gain of function models in JEG3 and HTR8/Sneov cells by lentivirus systems. Transwell assays, scratch-wound assays, EDU and plate clone formation assays, and villous explant culture were used to examine the function of ALG3 in JEG3 and HTR8/Sneov cells model. The effect of ALG3 on N-glycosylation in trophoblasts was analyzed using label-free quantitative proteomics. Main results and the role of chance In this study, ALG3 was significantly decreased in PE placentas by immunohistochemistry. And further confirmed the expression of ALG3 in placental tissues by qRT-PCR and Western blot. We demonstrated that the knockdown of ALG3 in trophoblasts dramatically decreased cell proliferation, migration, and invasion. Extravillous explants transfected with siALG3 inhibited the expansion of the EVT outgrowth across and into the Matrigel. While overexpression of ALG3 in trophoblasts significantly increased the ability of cell proliferation, migration, and invasion. Further, we demonstrated that ALG3 regulated the N-glycosylation of EGFR in trophoblasts and regulated the EGFR/ERK/MMP9 signal pathway. Therefore, the functional axis, ALG3/EGFR/ERK axis, plays an important role in PE initiation and development. These findings uncover a novel N-glycosylation mechanism of the ALG3 in PE pathogenesis. Limitations, reasons for caution Although we show that ALG3 played an important role in the function of trophoblasts, the study of the working mechanism of ALG3 in PE is still limited. The crucial role it plays in early placentation has yet to be fully elucidated. More studies will be performed to explore underline mechanisms. Wider implications of the findings This study was the first time to explore the role of ALG3 and N-glycosylation in trophoblast. It will help us to better understand the pathogenesis of PE, which might be helpful in the future application of novel therapeutic targets in PE. Trial registration number not applicable

Impact of PEDV infection on the biological characteristics of porcine intestinal exosomes.

Porcine epidemic diarrhea (PED) is a highly contagious intestinal infection primarily affecting pigs. It is caused by the porcine epidemic diarrhea virus (PEDV). PEDV targets the villus tissue cells in the small intestine and mesenteric lymph nodes, resulting in shortened intestinal villi and, in extreme cases, causing necrosis of the intestinal lining. Moreover, PEDV infection can disrupt the balance of the intestinal microflora, leading to an overgrowth of harmful bacteria like Escherichia coli. Exosomes, tiny membrane vesicles ranging from 30 to 150 nm in size, contain a complex mixture of RNA and proteins. MicroRNA (miRNA) regulates various cell signaling, development, and disease progression processes. This study extracted exosomes from both groups and performed high-throughput miRNA sequencing and bioinformatics techniques to investigate differences in miRNA expression within exosomes isolated from PEDV-infected porcine small intestine tissue compared to healthy controls. Notably, two miRNA types displayed upregulation in infected exosomes, while 12 exhibited downregulation. These findings unveil abnormal miRNA regulation patterns in PEDV-infected intestinal exosomes, shedding light on the intricate interplay between PEDV and its host. This will enable further exploration of the relationship between these miRNA changes and signaling pathways, enlightening PEDV pathogenesis and potential therapeutic targets.

Association between ACE1 and missed abortion: ACE1 promotes H2O2-induced trophoblast cell injury in vitro†.

The aim of this study was to evaluate the role of angiotensin-converting enzyme 1 (ACE1) in H2O2-induced trophoblast cell injury and the potential molecular mechanisms. Oxidative stress was modeled by exposing HTR-8/SVneo cells to 200μM H2O2. Western blot and real-time quantitative PCR methods were used to detect protein and mRNA expression level of ACE1 in chorionic villus tissue and trophoblast HTR-8/SVneo cell. Inhibition of ACE1 expression was achieved by transfection with small interfering RNA. Then flow cytometry, Cell Counting Kit-8, and Transwell assay was used to assess apoptosis, viability, and migration ability of the cells. Reactive oxygen species (ROS) were detected by fluorescent probes, and malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) activities were determined by corresponding detection kits. Angiotensin-converting enzyme 1 expression was upregulated in chorionic villus tissue of patients with missed abortion (MA) compared with individuals with normal early pregnancy abortion. H2O2 induced elevated ACE1 expression in HTR-8/SVneo cells, promoted apoptosis, and inhibited cell viability and migration. Knockdown of ACE1 expression inhibited H2O2-induced effects to enhance cell viability and migration and suppress apoptosis. Additionally, H2O2 stimulation caused increased levels of ROS and MDA and decreased SOD and GSH activity in the cells, whereas knockdown of ACE1 expression led to opposite changes of these oxidative stress indicators. Moreover, knockdown of ACE1 attenuated the inhibitory effect of H2O2 on the Nrf2/HO-1 pathway. Angiotensin-converting enzyme 1 was associated with MA, and it promoted H2O2-induced injury of trophoblast cells through inhibiting the Nrf2 pathway. Therefore, ACE1 may serve as a potential therapeutic target for MA.

Pyroptosis is involved in the immune microenvironment regulation of unexplained recurrent miscarriage.

Unexplained recurrent miscarriage (URM) is a common pregnancy complication with few effective therapies. Moreover, little is known regarding the role of pyroptosis in the regulation of the URM immune microenvironment. To address this issue, gene expression profiles of publicly available placental datasets GSE22490 and GSE76862 were downloaded from the Gene Expression Omnibus database. Pyroptosis-related differentially expressed genes were identified and a total of 16 differentially expressed genes associated with pyroptosis were detected, among which 1 was upregulated and 15 were downregulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that the functionally enriched modules and pathways of these genes are closely related to immune and inflammatory responses. Four hub genes were identified: BTK, TLR8, NLRC4, and TNFSF13B. BTK, TLR8, and TNFSF13B were highly connected with immune cells, according to the correlation analysis of four hub genes and 20 different types of immune cells (p < 0.05). The four hub genes were used as research objects to construct the interaction networks. Chorionic villus tissue was used for quantitative real-time polymerase chain reaction and western blot to confirm the expression levels of hub genes, and the results showed that the expression of the four hub genes was significantly decreased in the chorionic villus tissue in the URM group. Collectively, the present study indicates that perhaps pyroptosis is essential to the diversity and complexity of the URM immune microenvironment, and provides a theoretical basis and research ideas for subsequent target gene verification and mechanism research.

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

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

FTO protein regulates the TGF-β signalling pathway through RNA N6-methyladenosine modification to induce unexplained recurrent spontaneous abortion.

Recent studies have revealed the involvement of RNA m6A modification in embryonic development; however, the relationship between aberrant RNA m6A modification and unexplained recurrent spontaneous abortion (URSA) remains unclear. In this study, we analysed the level of RNA m6A modification in trophoblasts using dot blot, RNA m6A quantification, and MeRIP assays. By integrating data from the GEO database, RNA-Seq, and MeRIP-Seq, we examined the aberrant expression of m6A methyltransferases and their downstream molecules in chorionic villus (placental) tissues. RNA pull-down, RIP, and electrophoretic mobility shift assay were used to analyse the binding relationship between the YTHDC1 protein and MEG3. Additionally, RNA stability and BrU immunoprecipitation chase assays were utilised to elucidate the regulation of MEG3 stability by YTHDC1. ChIP and DNA pull-down RNA experiments were performed to elucidate the mechanism by which MEG3 targets EZH2 to the TGF-β1 promoter. The results showed that the expression of the m6A demethylase FTO protein was significantly increased in URSA trophoblasts, leading to inhibition of the MEG3 m6A modification and weakening of the stabilising effect of the m6A binding protein YTHDC1 on MEG3. Furthermore, MEG3 was found to bind simultaneously with the EZH2 protein and the TGF-β1 gene promoter, enabling the localisation of EZH2 protein to the TGF-β1 gene promoter and subsequent inhibition of TGF-β1 gene expression. In summary, our findings elucidate the mechanism by which FTO protein regulates the MEG3-TGF-β signalling pathway, thereby suppressing trophoblast invasion and proliferation in URSA trophoblast cells. These findings provide new insights for the treatment of URSA.

KISS-1 knockdown inhibits cell growth, migration, and invasion in HTR-8/SVneo cells by regulating the GRP54-mediated PI3K/AKT signaling pathway

Recurrent spontaneous abortions (RSA) affect reproductive health and increase the risk of subsequent abortions. To investigate the role of KISS-1/GPR-54 signaling in RSA progression. Villus tissue was collected from RSA patients, and human trophoblastic HTR-8/SVneo cells were used. KISS-1 and GRP54 levels were detected using RT-qPCR and immunohistochemistry. Western blotting was performed to analyze ZO-1 and ZEB1 levels. Cell proliferation was determined via CCK-8 and cell clone formation assays. Transwell assays were performed to assess cell migration and invasion abilities. KISS-1 was down-regulated in the villus tissues of RSA patients. KISS-1 overexpression dramatically inhibited trophoblast proliferation, migration, and invasion. Mechanistically, ZEB1 expression was down-regulated, whereas ZO-1 expression was up-regulated, after KISS-1 overexpression. GPR54 silencing neutralized the effect of KISS-1 in HTR-8/SVneo cells. Additionally, KISS-1 overexpression inactivated the PI3K/AKT signaling pathway through GRP54. The KISS-1/GPR-54 signaling axis regulates RSA progression by regulating the PI3K/AKT signaling pathway.

Genetic analysis of chorionic villus tissues in early missed abortions

Chromosomal abnormalities are the most common etiology of early spontaneous miscarriage. However, traditional karyotyping of chorionic villus samples (CVSs) is limited by cell culture and its low resolution. The objective of our study was to investigate the efficiency of molecular karyotyping technology for genetic diagnosis of early missed abortion tissues. Chromosome analysis of 1191 abortion CVSs in early pregnancy was conducted from August 2016 to June 2021; 463 cases were conducted via copy-number variations sequencing (CNV-seq)/quantitative fluorescent-polymerase chain reaction (QF-PCR) and 728 cases were conducted using SNP array. Clinically significant CNVs of CVSs were identified to clarify the cause of miscarriage and to guide the couples’ subsequent pregnancies. Among these, 31 cases with significant maternal cell contamination were removed from the study. Among the remaining 1160 samples, 751 cases (64.7%) with genetic abnormalities were identified, of which, 531 (45.8%) were single aneuploidies, 31 (2.7%) were multiple aneuploidies, 50 (4.3%) were polyploidies, 54 (4.7%) were partial aneuploidies, 77 (6.6%) had submicroscopic CNVs (including 25 with clinically significant CNVs and 52 had variants of uncertain significance), and 8 cases (0.7%) were uniparental disomies. Our study suggests that both SNP array and CNV-seq/QF-PCR are reliable, robust, and high-resolution technologies for genetic diagnosis of miscarriage.

Clinical Significance of Application of Chromosomal Karyotyping of Villus Tissues.

Karyotype analysis of villus chromosomes in spontaneous abortion by high-throughput ligation-dependent probe amplification (HLPA) was performed to explore relationship between spontaneous abortion and chromosomal abnormalities in spontaneous abortion tissues. The karyotypes of chromosomes of villus tissues from 516 patients with spontaneous abortion who were admitted to our hospital between 2014 and 2019 were analyzed. The data were grouped by the age (ie, ≤29, 30-35, and ≥36 years old) in which embryonic chromosomal abnormality rate, and abnormal distribution of chromosome number in abortion tissue of 294 patients. The examination of 516 samples of spontaneous abortion tissues showed that the chromosomes of 294 samples were abnormal, and the abnormality rate was 60.97%. Among 294 samples with chromosomal abnormalities, 136 (89.47%) samples had numerical chromosomal abnormalities, 15 (9.87%) samples had structural chromosomal abnormalities, and 1 (0.07%) sample had uniparental disomy. A total of 308 couples voluntarily underwent peripheral blood chromosome analysis. The results showed that 56 couples had abnormality in one or both spouses, and rate of embryonic chromosomal abnormality in these 26 cases with chromosomal abnormalities was 100%. Numerical and structural chromosomal abnormalities are among the important causes of embryonic arrest, while the increase in age could also be a high-risk factor for embryonic chromosomal abnormality. Therefore, examination of the karyotypes of embryo chorionic villus may help understand the reasons of embryonic arrest, which could provide important guidance for more genetic counseling for patients with early spontaneous abortion, as well as improving the preconception preparation.

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.

H19 Promoter DNA Methylation is Lower Among Early Abortion Patients Undergoing IVF Embryo Transfer.

H19 is the first long noncoding RNA (lncRNA) found to be associated with gene imprinting. It is highly expressed in the embryonic stage and may have important regulatory effects on human embryonic development. We investigated the differences between the levels of H19 promoter DNA methylation in the chorionic villi of patients who experienced spontaneous abortion (SA) following in vitro fertilization embryo transfer (IVF-ET) and those of patients with a normal early pregnancy (NEP). We also analyzed the associated DNA methyltransferase (DNMT) activity. Chorionic villus tissue from patients with SA and NEP were collected. The DNA methylation levels of two CpG islands in the promoter region of the H19 gene in the two groups were detected by bisulfite sequencing, and the mRNA expression of DNMTs was analyzed by real-time polymerase chain reaction. The sample size of each group was 32, and there were no significant differences in baseline data, including age, parity, and body mass index, between the two groups. Among the 7 CpG islands measured, the methylation rates of 3 CpG islands (CpG 1, 6, and 7) were significantly lower in the SA group than in the NEP group (P < 0.01). The methylation levels of the other 4 CpG islands were not significantly different between the two groups. There were no differences in the expression of DNMT1 between the two groups (P > 0.05), but DNMT3a and DNMT3b RNA levels were significantly lower in SA group than in the NEP group (P < 0.01). The lower H19 promoter DNA methylation levels found in the chorionic villi of patients with SA patients following IVF-ET may be explained by decreased expression of DNMT3a and DNMT3b.

GPR65 inhibits human trophoblast cell adhesion through upregulation of MYLK and downregulation of fibronectin via cAMP-ERK signaling in a low pH environment

BackgroundExtravillous trophoblasts (EVTs) are essential cells during the formation of the placenta, with the major function of invading the maternal decidua, anchoring the developing placenta to the uterus, remodeling uterine arteries, and regulating immune responses to prevent rejection. During early pregnancy, the decidua undergoes a hypoxic and acidic microenvironment, which has been shown to participate in tumor cell migration, invasion, growth, and angiogenesis. Nevertheless, the mechanisms by which EVTs sense and respond to the acidic microenvironment, thereby executing their functions, remain poorly understood.MethodsThe effects of G protein-coupled receptor 65 (GPR65) on cell adhesion and other cellular functions were tested using JAR spheroids, mouse blastocysts, and HTR-8/SVneo cells. Specifically, we employed HTR-8/SVneo cells for gene overexpression and silencing to investigate the underlying mechanism of GPR65's impact on trophoblast cell function under acidic conditions. Additionally, villus tissue samples obtained from early pregnancy loss patients were utilized to explore the potential association between GPR65 and its related signaling pathway molecules with the disease.ResultsThis study identified GPR65 expression widely in trophoblasts, with the highest level in EVTs. Importantly, optimal GPR65 levels are required for maintaining normal adhesion, migration, and invasion, whereas overexpression of GPR65 inhibits these functions by activating the cAMP-ERK signaling pathway, upregulating myosin light chain kinase (MYLK) and MYLK3 expression, and subsequently downregulating fibronectin. Consistently, elevated expression of GPR65, MYLK, and MYLK3 is observed in patients suffering from early pregnancy loss.ConclusionsThis work offers insights into the suppressive effects of GPR65 on EVT function under acidic conditions and highlights a putative target for therapeutic intervention in early pregnancy complications.E9R9tt1jd9fu3QritsNs7QVideo

Expression of Mas1 receptor in human placenta and its effect on the function of trophoblast cells in pre-eclampsia patients

Objective: To investigate the expression change of the Mas1 receptor in the placenta of healthy pregnant women during different gestation periods, analyze the expression level of the Mas1 receptor in the placenta of pre-eclampsia (PE) patients, and its biological function in trophoblast cells. Methods: Placental villous tissues were collected from normal pregnant women in early, mid and late pregnancy. Human trophoblast stem cells were isolated and cultured from early pregnancy villous tissues. The expression of the Mas1 receptor was detected by fluorescence immunoassay and real-time fluorescence quantitative PCR. In a case-control study, patients with full-term PE were selected as the case group and healthy women with full-term pregnancy were selected as the control group. Placental villus tissues were collected from both groups. Immunofluorescence chemistry and immunoprotein blotting were used to study the changes in Mas1 receptor expression in PE. Mas1 receptor agonists and blockers induced HTR8/Svneo cells and BeWo cells, and the effects of the Mas1 receptor on the proliferation and migration of trophoblast cells were detected by the CCK8 proliferation test and scratch test. Results: Eight cases were included in early pregnancy, seven cases in mid-pregnancy and six cases in late pregnancy. Mas1 receptors in normal placental villi tissue were mainly expressed in human trophoblast stem cell membranes and cytoplasm, and the expression of Mas1 receptor mRNA in villi tissue was significantly higher in late pregnancy than in mid-pregnancy. There were 24 cases included in the case group and 12 cases in the control group. Mas1 receptor expression in placental villi was significantly lower in the case group compared to the control group; Activation/inhibition of the Mas1 receptor had no significant effect on the proliferation of HTR8/Svneo cells and BeWo cells. Activated Mas1 receptor had no significant effect on the migration ability of HTR8/Svneo cells. Conclusion: Mas1 receptors are expressed in placental villous tissue and their expression varies with gestation. Mas1 receptor expression is reduced in PE patients, but it does not affect the value-added or migratory function of trophoblast cells.

Dysregulation of Histone Deacetylases Inhibits Trophoblast Growth during Early Placental Development Partially through TFEB-Dependent Autophagy-Lysosomal Pathway.

Dysregulated biological behaviors of trophoblast cells can result in recurrent spontaneous abortion (RSA)-whose underlying etiology still remains insufficient. Autophagy, a conserved intracellular physiological process, is precisely monitored throughout whole pregnancy. Although the exact mechanism or role remains elusive, epigenetic modification has emerged as an important process. Herein, we found that a proportion of RSA patients exhibited higher levels of autophagy in villus tissues compared to controls, accompanied with impaired histone deacetylase (HDAC) expression. The purpose of this study is to explore the connection between HDACs and autophagy in the pathological course of RSA. Mechanistically, using human trophoblast cell models, treatment with HDAC inhibitor (HDACI)-trichostatin A (TSA) can induce autophagy by promoting nuclear translocation and transcriptional activity of the central autophagic regulator transcription factor EB (TFEB). Specifically, overactivated autophagy is involved in the TSA-driven growth inhibition of trophoblast, which can be partially reversed by the autophagy inhibitor chloroquine (CQ) or RNA interference of TFEB. In summary, our results reveal that abnormal acetylation and autophagy levels during early gestation may be associated with RSA and suggest the potential novel molecular target TFEB for RSA treatment.

Four Decades of Carrier Detection and Prenatal Diagnosis in Hemophilia A: Historical Overview, State of the Art and Future Directions.

Hemophilia A (HA), a rare recessive X-linked bleeding disorder, is caused by either deficiency or dysfunction of coagulation factor VIII (FVIII) resulting from deleterious mutations in the F8 gene encoding FVIII. Over the last 4 decades, the methods aimed at determining the HA carrier status in female relatives of HA patients have evolved from phenotypic studies based on coagulation tests providing merely probabilistic results, via genetic linkage studies based on polymorphic markers providing more accurate results, to next generation sequencing studies enabling highly precise identification of the causative F8 mutation. In parallel, the options for prenatal diagnosis of HA have progressed from examination of FVIII levels in fetal blood samples at weeks 20-22 of pregnancy to genetic analysis of fetal DNA extracted from chorionic villus tissue at weeks 11-14 of pregnancy. In some countries, in vitro fertilization (IVF) combined with preimplantation genetic diagnosis (PGD) has gradually become the procedure of choice for HA carriers who wish to prevent further transmission of HA without the need to undergo termination of pregnancies diagnosed with affected fetuses. In rare cases, genetic analysis of a HA carrier might be complicated by skewed X chromosome inactivation (XCI) of her non-hemophilic X chromosome, thus leading to the phenotypic manifestation of moderate to severe HA. Such skewed XCI may be associated with deleterious mutations in X-linked genes located on the non-hemophilic X chromosome, which should be considered in the process of genetic counseling and PGD planning for the symptomatic HA carrier. Therefore, whole exome sequencing, combined with X-chromosome targeted bioinformatic analysis, is highly recommended for symptomatic HA carriers diagnosed with skewed XCI in order to identify additional deleterious mutations potentially involved in XCI skewing. Identification of such mutations, which may profoundly impact the reproductive choices of HA carriers with skewed XCI, is extremely important.