Project description:Gene expression microarrays investigating the transcriptome of matched populations of human side-population trophoblasts and cytotrophoblasts isolated from first and third trimester placentae
Project description:To investigate the role of miRNA in the placental development, we analyzed miRNA expression profiles in the first and third trimester human placentas. Total RNA was isolated from 12 placentas (6 from first trimester and 6 from third trimester). miRNA expression profiles were determined by Affymetrix GeneChip 2.0 miRNA Microarray. Using miRNA microarray to determine miRNA expression profiles in the human placenta between first and third trimester pregnancies.
Project description:Human placenta bulk small RNA-seq from healthy pregnancies without infertility, from n=113 first trimester (58 female, 55 male) and n=47 third trimester (19 female, 28 male) tissue samples. Tissue was collected at Cedars-Sinai Medical Center in Los Angeles, California, USA. First trimester placenta was collected at 70-102 days of gestation from leftover chorionic villus sampling for prenatal genetic diagnosis. Third trimester placenta was collected after delivery at 254-290 days gestation from the fetal side near the umbilical cord insertion site beneath the amnion. Mothers with pre-existing diabetes or hypertension were excluded. All pregnancies were conceived without fertility treatments, were normal karyotype, and resulted in live singleton births. The average parental age was advanced (over 35 years old) but PCA analysis did not show clustering by either maternal or paternal age. Gonzalez et al 2021 [PMID: 34030457] analyzes similarities and differences between first and third trimester miRNA expression overall. Flowers et al 2021 focuses on the effect of fetal sex on miRNA expression across gestation.
Project description:In this work, we have isolated a Hoescht side-population of trophoblasts from first trimester human placentae that cluster separately from more differentiated trophoblast populations, and have a transcriptomic profile indicative of a stem cell population. Hoescht side-population cells were compared in quintuplicate with extravillous trophoblasts and cytotrophoblasts extracted from the same placentae, giving a total of 15 samples.
Project description:Human placenta bulk mRNA-sequencing from n=124 first trimester (59 female, 65 male) and n=43 third trimester (18 female, 25 male) tissue samples. A subset of 23 pregnancies has matched placenta tissue collected at both first and third trimester. Tissue was collected at Cedars-Sinai Medical Center in Los Angeles, California, USA. First trimester placenta was collected at gestational ages of 70-100 days from leftover tissue from chorionic villus sampling for prenatal genetic diagnosis, after cleaning to remove maternal decidua. Third trimester placenta was collected after delivery at gestational ages 254-290 days from the fetal side near the umbilical cord insertion site beneath the amnion. Tissue was stored in RNAlater RNA Stabilization Solution (Invitrogen) at -80C until further processing. All pregnancies were conceived without fertility treatments, were normal karyotype, and resulted in live singleton births. Mothers with pre-existing diabetes or hypertension were excluded from the study, but not excluded if complications developed during pregnancy, though most pregnancies were uncomplicated. The average parental age was advanced (over 35 years old) but principal components analysis did not show clustering by either maternal or paternal age. RNA extraction was performed with physical homogenization of tissue followed by the AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN), then 1 ug of the total RNA elution was used for library construction using the Illumina TruSeq Stranded mRNA library preparation kit (Illumina), with polyA mRNA selection then cDNA synthesis using SuperScript II reverse transcriptase (Invitrogen) and random primers. The cDNA was converted into double stranded DNA and PCR-amplified, then purified with Agencourt AMPure XP beads (Beckman Coulter). Sample libraries were multiplexed and sequenced on a NovaSeq 6000 platform (Illumina) using 75bp single-end mRNA-sequencing, with average 30 million reads per sample. Differential expression analysis was performed with DEseq2 to compare first versus third trimester placenta, adjusted for fetal sex [PubMed ID: 38271627]. Subanalyses were also performed to identify sex differences.
Project description:In this work, we have isolated a Hoescht side-population of trophoblasts from first trimester human placentae that cluster separately from more differentiated trophoblast populations, and have a transcriptomic profile indicative of a stem cell population.
Project description:To investigate the role of miRNA in the placental development, we analyzed miRNA expression profiles in the first and third trimester human placentas. Total RNA was isolated from 12 placentas (6 from first trimester and 6 from third trimester). miRNA expression profiles were determined by Affymetrix GeneChip 2.0 miRNA Microarray.
Project description:To better understand how DNA methylation influences placentation, DNA from first trimester primary trophoblast populations (side-population trophoblasts, cytotrophoblasts and extravillous trophoblasts) isolated using FACS underwent reduced representation bisulfite sequencing and were compared to publicly available data of blastocyst-derived and somatic cell populations.
Project description:Seven patient paired primary human HLA-G+ extravillous trophoblasts (EVT) and Villous trophoblasts (VT) obtained from 1st trimester (7-9 weeks) villous tissue were obtained. RNA was isolated directly after isolation and purification using FACS sort for CD45-HLA-G+ (EVT) and CD45-HLA-G-EGFR1+ (VT) fractions. Expression profiles were compared to two samples of the choriocarcinoma cell line JEG-3 and four samples of decidual stromal cells (DSC) at passage 2 after cell culture.
Project description:Oxidative stress plays a crucial role in preeclampsia (PE) pathogenesis. Evidence indicates altered microRNAs (miRNAs) expression in PE, however, the relationship between oxidative stress and miRNA changes in placenta as a potential mechanism involved in PE is not fully elucidated. In the present study, we investigate the impact of increased oxidative stress on miRNA and mRNA expression profiles of genes known to be associated with PE in villous 3A first trimester trophoblast cells. Cells were exposed to H2O2 at 10 different concentrations (0-1 mM) for 0.5, 4, 24, and 48 h. Cytotoxicity was determined using the SRB assay and data was used to calculate the IC50 of H2O2. Total RNA was extracted after short-term exposure (4 h) to H2O2 for miRNA expression profiling. H2O2 exerted a concentration and time-dependent cytotoxicity on 3A trophoblast cells. Short-term exposure of 3A cells to low concentration of H2O2 (5% of IC50) significantly altered miRNA profile as evidenced by significant changes in 195 out of 595 evaluatable miRNAs. Short-term exposure of villous first trimester trophoblasts to low concentrations of H2O2 significantly alters miRNA profile. Oxidative stress plays a crucial role in preeclampsia (PE) pathogenesis. Evidence indicates altered microRNAs (miRNAs) expression in PE, however, the relationship between oxidative stress and miRNA changes in placenta as a potential mechanism involved in PE is not fully elucidated. In the present study, we investigate the impact of increased oxidative stress on miRNA and mRNA expression profiles of genes known to be associated with PE in villous 3A first trimester trophoblast cells. Cells were exposed to H2O2 at 10 different concentrations (0-1 mM) for 0.5, 4, 24, and 48 h. Cytotoxicity was determined using the SRB assay and data was used to calculate the IC50 of H2O2. Total RNA was extracted after short-term exposure (4 h) to H2O2 for miRNA expression profiling. H2O2 exerted a concentration and time-dependent cytotoxicity on 3A trophoblast cells. Short-term exposure of 3A cells to low concentration of H2O2 (5% of IC50) significantly altered miRNA profile as evidenced by significant changes in 195 out of 595 evaluatable miRNAs. Short-term exposure of villous first trimester trophoblasts to low concentrations of H2O2 significantly alters miRNA profile.