Project description:<p>Chorionic villus sampling (CVS) is routinely used for prenatal diagnosis of cytogenetic disorders, but also possesses great potential for studying placentation. To better understand villi biology, human placentation, and how these relate to pregnancy outcomes, we examined the morphology and transcriptomes of villi obtained via CVS from 10-14 weeks of pregnancy and correlated these with pregnancy attributes and clinical outcomes.</p>
Project description:The maternal signs of preeclampsia, principally the new onset of high blood pressure, are thought to occur secondary to faulty placentation. Previous studies profiled the gene expression patterns of chorionic villi, the maternal-fetal interface or isolated cytotrophoblasts in this pregnancy complication. We theorized that transcriptomic analyses of trophoblast subpopulations in situ would give us new insights into the role of these cells in preeclampsia pathogenesis.
Project description:Purpose: During early placentation, extravillous trophoblast (EVT) cell invasion into maternal decidua plays a crucial role in establishment of the successful pregnancy. However, little is known about the characteristic features of EVT-associated ncRNAs. The purpose of this study is to elucidate gene expression profile for both coding and non-coding genes (i.e., mRNAs, lncRNAs, and miRNAs) expressed in EVT cells isolated from the first-trimester human placenta by RNA sequencing analysis. Methods: Human first trimester placental tissues (at gestational week 7, n=3) were obtained after legal abortions. EVT cells growing from explanted human chorionic villi were isolated. Minced chorionic villous tips were defined as first-trimester chorionic villous trophoblast (CVT) cells. Using total RNAs of these samples, mRNA/lncRNA-sequencing analysis was carried out on an Illumina MiSeq (San Diego, California, USA) using MiSeq v2 Reagent Kit 300cycle (cat. no. MS-102-2002; Illumina) and MiSeq v2 Reagent Kit 50cycle (cat. no. MS-102-2001; Illumina), respectively. The paired-end reads of 151 bp length for mRNA/lncRNA-sequencing and single reads of 26 bp length for miRNA-sequencing were obtained as fastq files which were imported into CLC genomics workbench (version 8.0.1; Qiagen, Venlo, Netherlands). Clean reads were aligned to the human genome sequence retrieved from the Ensembl database (Assembly GRCh38.p13, database version 82.38 and 101.38) and microRNA database miRbase (release 21), using CLC Genomics Workbench Map Reads to Reference tool. Normalization of gene expression data was performed by the TMM method of edgeR package. Statistically differentially expressed transcripts were conducted using edgeR. Genes that had a false discovery rate (FDR)-corrected p-value (q-value) of < 0.05 and an absolute log2 fold change (log FC) ≥ 1 were judged to be significantly differentially expressed (i.e., differentially expressed genes, DEGs). Results: Five thousand ninty-two mRNAs , 422 lncRNAs and 400 miRNAs were found to be differentially expressed. One hundred differentially expressed genes (i.e., 10 mRNAs, 7 lncRNAs, and 83 miRNAs) that are located in the chromosome 14q32.2 region containing the eutherian-specific imprinted DLK1-DIO3 domain were significantly down-regulated in EVT cells. In terms of imprinted genes (http://www.geneimprint.com/site/genes-by-species), 68 genes were found to be differentially expressed; 71% of these genes were down-regulated in EVT cells. lncRNA H19 was a DEG (q = 1.29.E-09, log FC = 2.98) and the most highly expressed lncRNA (70.47%) in EVT cells. Conclusions: The present research should be valuable for a resource for future ncRNA research in EVT cells.
Project description:Preeclampsia (PE) is the leading cause of prenatal morbidity and mortality. It is associated with defective trophoblast functions at implantation, but manifestation of its phenotypes is in late pregnancy. There is no reliable method for early prediction and treatment of PE. Adrenomedullin (ADM) is an abundant placental peptide in early pregnancy. Here, integrated single-cell sequencing and spatial transcriptomics confirm a high ADM expression in the human villous cytotrophoblast. The levels of ADM in chorionic villi and serum were lower in first-trimester pregnant women who later developed PE than those with normotensive pregnancy. ADM stimulates differentiation of trophoblast stem cells and trophoblast organoids in vitro. In pregnant mice, placenta-specific ADM suppression led to PE-like phenotypes. The PE-like phenotypes in a mouse PE model were reduced by a novel placenta-specific nanoparticle-based forced expression of ADM. Our study reveals the roles of trophoblastic ADM in placental development, PE pathogenesis and its potential clinical uses.
Project description:In the chorionic villi of placenta, trophoblasts and endothelial cells are present, and moreover mesenchymal cells (stromal cells) can be obtained. We generated cells with the mesenchymal phenotype from the chorionic mesoderm, and showed that: a) physiologically functioning cardiomyocytes were transdifferentiated from human placenta-derived chorionic villi cells, but these cells did not induce to osteoblasts and adipocytes ; b) the cardiomyogenic induction rate obtained using our system was relatively high compared to that obtained using the previously described method ; c) co-cultivation with fetal murine cardiomyocytes alone without transdifferentiation factors such as 5-azaC or oxytocin is sufficient for cardiomyogenesis in our system; d) Chorionic villi cells have the electrophysiological properties of 'working' cardiomyocytes. The chorionic mesoderm contained a large number of cells with a cardiomyogenic potential. Experiment Overall Design: To isolate chorionic villi cells, we used the explant culture method, in which the cells were outgrown from pieces of chorionic villi attached to dishes. Chorionic villi cells were harvested with 0.25% trypsin and 1 mM EDTA, and overlaid onto the cultured fetal cardiomyocytes at 7 x 103/cm2. Every 2 days, the culture medium was replaced with fresh culture medium that was supplemented with 10% FBS and 1 ug/ml Amphotericin B (GIBCO). The morphology of the beating chorionic villi cells was evaluated under a fluorescent microscope.
Project description:Miscarriage occurs in 15-20% of clinical pregnancies. While chromosomal errors are observed in over 50%, causes of karyotypically normal losses are poorly understood. DNA methylation undergoes reprogramming during development and must be appropriately set to maintain a healthy pregnancy. We hypothesize that aberrant DNA methylation may cause karyotypically normal miscarriage, particularly among women experiencing recurrent miscarriage (RM). DNA methylation in first trimester chorionic villi was assessed in chromosomally normal miscarriages from women with RM (N=33) or isolated miscarriage (M, N=21), and elective terminations (TA, N=16). Differentially methylated candidate loci were identified using the Illumina Infinium HumanMethylation27 BeadChip array by comparing 10 RM to 10 TA samples. Follow up showed a significant increase in methylation in RM and M compared to TA placentae at the CYP1A2 (p=0.002) and AXL (p=0.02) promoters and decrease at the DEFB1 (p=0.008) promoter. Gene function analysis showed an enrichment of imprinted genes (p=9.53E-10) and genes previously associated with RM (p=9.51E-06). DNA methylation was evaluated at 7 imprinted loci using bisulfite pyrosequencing. An increase of outliers at these loci was observed in RM (3.9%) compared to M (0%) and TA (0.9%) (p=0.02), with increased average methylation at the H19/IGF2 ICR1 in M samples (p<0.0001). Altered DNA methylation in the placenta at specific loci, as well as global dysregulation in specific cases, may contribute to or be a consequence of placental insufficiency in karyotypically normal miscarriage. First-trimester placental villi samples from karyotypically normal miscarriages from recurrent miscarriage patients (N, N=10) and chromosomally normal elective terminations (PZET, N=10).
Project description:Pregnancy loss is often caused by chromosomal abnormalities of the conceptus. The prevalence of these abnormalities and the allocation of (ab)normal cells in embryonic and placental lineages during intrauterine development remain elusive. We analyzed 1,745 spontaneous pregnancy losses and found that roughly half (50.4%) of the products of conception (POC) were karyotypically abnormal, with maternal and paternal age independently contributing to the increased genomic aberration rate in pregnancy loss. We applied genome haplarithmisis to a subset of 94 pregnancy losses with normal parental and POC karyotypes. Genotyping of parental DNA as well as POC extraembryonic mesoderm and chorionic villi DNA, representing embryonic and trophoblastic tissues, enabled characterization of the genomic landscape of both lineages. Of these pregnancy losses, 35.1% had chromosomal aberrations not previously detected by karyotyping, increasing the rate of aberrations of pregnancy losses to 67.8% by extrapolation. In contrast to viable pregnancies where mosaic chromosomal abnormalities are often restricted to chorionic villi, such as confined placental mosaicism, we found a higher degree of mosaic chromosomal imbalances in extraembryonic mesoderm rather than chorionic villi in pregnancy losses. Our results stress the importance of scrutinizing the full allelic architecture of genomic abnormalities in pregnancy loss to improve clinical management and basic research of this devastating condition.