Preimplantation Factor Effect on Endometrial and Decidual Cells
Ontology highlight
ABSTRACT: Embryos secrete preimplantation factor (PIF), a peptide present in maternal circulation during viable pregnancy. We compared downstream synthetic PIF effect on gene expression in non-pregnant Human Endometrial Stromal Cell (HESC) and First Trimester Decidual cell (FTDC) culture to mimic the maternal intrauterine environment during embryo implantation and trophoblast invasion. Cells were cultured in triplicate with and without synthetic PIF for 24 hours. Cultured cells were then pooled for chip analysis. Altered gene expression relative to controls was assessed by Affymetrix microarray
Project description:Pregnancy is an immune paradox: where the mother accepts a semi-allograft/allograft (donor embryo), while avoiding maternal immune suppression. Indeed, impaired development of maternal immune tolerance towards the conceptus may be an important cause of implantation failure and pregnancy loss. Successful implantation rates for allogenic donor embryos are unexpectedly high, while abnormal embryos may be unable to initiate maternal tolerance, suggesting that embryo-specific compounds can play a vital role in this process. Both a tolerant T-suppressor (TH2), and inflammatory, T-helper 1 (TH1) cytokine profiles are an integral part of pregnancy and the viable embryo/fetus plays a critical role in creating this delicate balance. Maternal recognition of pregnancy occurs prior to implantation. There were several embryo derived factors reported. However, we previously found that preimplantation factor (PIF) is distinct from the other non-pregnancy-specific embryo-derived factors. Herein we aimed to characterize PIF and examine its role in early pregnancy events by observing the isolated novel peptide's effect on human Peripheral Blood Mononuclear Cell (PBMC). We did affymetrix, GeneChip Human Genome U133 plus 2.0 array on RNA isolated from normal human PBMC cultured for 24hrs in the presence or absence of PIF to study the genes modulated by PIF. Experiment Overall Design: Blood was collected from a male volunteer and PBMC were separated using the Ficoll Hypaque method. Cells were cultured in RPMI culture media for 24 hours in four different groups in duplicate viz., Media alone, Media + 50 nM PIF, Media + 50nM PIF +CD3 (10µg/ml)+ CD28 (10µg/ml) and Media + CD3 (10µg/ml) +CD28 (10µg/ml). At the end of incubation cells were removed from the media, washed and collected in RNAlater (Qiagen) and transferred to Biocodon Sciences (Tx, USA) for Gene array analysis on dry ice. Samples were analyzed using the Agilent Bioanalyzer Process found to be adequate. Each 100 µl contained 2.7-5.4 µg RNA (Nanodrop analyzer). Each sample was tested integrity. RNA from duplicate samples was combined and array was performed on an Affymetrix chip (Human Genome U 133 Plus 2.0 Array). Subsequently, arrays were processed, scanned, data was extracted and statistical evaluation was carried out comparing the various groups. Data generated were categorized as robust changes or mild changes based on differences in expression.
Project description:To understand the regulation of cytotoxicity by decidual CD8+ T cells (CD8+ dT) at the maternal-fetal interface, gene expression analysis of CCR7-CD45RA- effector-memory CD8+ T cells isolated from peripheral blood of unrelated healthy controls and decidual tissue from first trimester (6-12 weeks) and term (>37 weeks) pregnancy was performed. Furthermore, first trimester decidual effector-memory CD8+ T cells were stimulated with anti-CD3/CD28 in the presence of IL-2 for 12 hours or 72 hours. RNA was isolated and gene expression profiles were generated by employing Affymetrix HG_U133_Plus 2 arrays on the Affymetrix Geneatlas system.
Project description:An allorecognition system depending on interactions between uterine Natural Killer (uNK) cells and placental extravillous trophoblast (EVT) during early pregnancy influences reproductive outcomes in humans. Our previous immunogenetic studies show that particular combinations of maternal Killer Immunoglobulin-like Receptors (KIR) in uNK cells with fetal HLA-C variants on EVT are associated with disorders of pregnancy, especially pre-eclampsia. To identify responses stimulated in uNK cells specifically when activating KIR (KIR2DS1) binds to C2+HLA-C, a combination protective against pre-eclampsia, we modelled KIR-HLA interactions by co-culturing uNK cells with the 721.221 HLA-null cell line transfected to express either C1+ or C2+HLA-C molecules, followed by transcriptome profiling by RNA sequencing. We detect the secretion of key cytokines by uNK cells under the protective combination between KIR2DS1 and C2+HLA-C.
Project description:Embryos secrete preimplantation factor (PIF), a peptide present in maternal circulation during viable pregnancy. We compared downstream synthetic PIF effect on gene expression in non-pregnant Human Endometrial Stromal Cell (HESC) and First Trimester Decidual cell (FTDC) culture to mimic the maternal intrauterine environment during embryo implantation and trophoblast invasion.
Project description:Gene expressions were measured in 8 normal breast duct samples and 8 normal breast lobule samples from premenopausal patients using Agilent-028004 SurePrint G3 Human GE 8x60K Microarray. A total of 426 transcripts were identified as being significantly differentially expressed. Supervised hierarchical clustering based on the 426 differentially expressed genes showed distinct clustering of the duct and lobule samples.The significant enrichment analysis of GO terms for the differentially expressed genes using the R language package software demonstrated that these 426 differentially expressed genes were involved in many important biological processes, including negative regulation of low-density lipoprotein receptor biosynthetic process, response to steroid hormone stimulus and oxidation reduction. 17 differential expressed genes identified by microarray were randomly chosen for further validation using quantitative RT-PCR. As results, fold changes measured by quantitative RT-PCR were closely correlated with those measured by microarray. 8 normal breast duct samples vs 8 normal breast lobule samples from premenopausal patients, unpaired
Project description:Gestation-matched endometrium was collected from women with ectopic pregnancy (EP) (n=11) and intrauterine pregnancies (IUP) (n=13). RNA was extracted from the tissue. In addition, tissues were prepared for histological analysis for degree of decidualization. We compared a) the samples from EP that were decidualized (n=6) with non-decidualized samples (n=5), and b) the decidualized EP (n=6) with decidualization-matched IUP (n=6) samples.
Project description:The aim of the study was to compare gene expression profiles in decidua basalis from cases with complicated pregnancies to those from healthy pregnant controls. Cases included pregnant women with preeclampsia (PE) and/or fetal growth restriction (FGR). Decidual tissue was obtained by vacuum suction of the placental bed after the placenta was delivered. Women with PE and/or FGR were included as cases. PE was defined as persistent hypertension (blood pressure (BP) ¡Ý 140/90 mm Hg), plus proteinuria (¡Ý0.3g/24 h or ¡Ý2+ according to a dipstick test), developing after 20 weeks of pregnancy. FGR implied birth weight under 2 standard deviations (SD) below the expected birth weight, as related to gestational age (GA) and sex. Due to tissue sampling procedures, only cases delivered by cesarean section (CS) were included. Healthy women with normal pregnancies, undergoing CS for various reasons considered irrelevant to the aim of this study (e. g. breech presentation and previous CS), served as controls. Tissue was immediately submerged in a RNA stabilisation solution (RNAlater, Ambion, Huntingdon, U.K.), incubated at 4¡ãC overnight and stored at -80¡ãC
Project description:Analysis of gene expression in uterine natural killer (NK) cells purified from either lean (control) or obese pregnanct women. The studys' aim is to identify differentially expressed genes and pathways in natural killer cells that are affected by maternal obesity. Results provide mechanitic insight into gene pathways altered in the condition of obeisty. Total RNA obtained from immuno-magnetic bead purified natural killer cells from uterine decidual mucosa from 7 to 10 week old pregnancies from 13 lean (BMI 20-24.9) and 11 obese (BMI >30) women. All women were between 19 and 35 years of age. In addition to BMI, blood serum CRP (hsCRP), a measure of inflammation, was measured via ELISA.
Project description:This project consisted of three HDX-MS experiments. First, we compared the dimeric PDK1(SKD-PIF) to monomeric PDK1(SKD) and mapped the differences in deuterium incorporation onto the dimer model. We then compared the deuterium incorporation kinetics for the kinase (PDK1(SKD)) and PH (PDK1(PH) domains of PDK1 with full-length PDK1 (PDK1(FL)) in pairwise experiments.