Endometrial expression profiling in women with recurrent early pregnancy loss
ABSTRACT: Pregnancy loss is the most common complication of human pregnancy. Recurrent early pregnancy loss (REPL) has multiple etiologies, including endometrial dysregulation leading to “suboptimal” implantation. Although the implantation process is tightly regulated in Eutherian (placental) mammals, the molecular factors contributing to dysregulated endometrial gene expression patterns in women with REPL are largely unknown. We hypothesized that genes that gained novel expression in the endometria of mammals that evolved in the Eutherian stemlineage, coincident with the evolution of pregnancy, are likely essential for establishment and maintenance of normal pregnancy and are, therefore, good candidates for genes whose expression may be dysregulated in disorders such as REPL. To test this hypothesis, we took an evolutionary forward genomics approach to characterize gene expression profiles of midsecretory endometria from women with REPL associated with abnormal endometria based either on histology or molecular expression of cyclin E. We identified 58 genes that were differentially expressed (P<0.001) between women with out-of-phase histological dating vs normal histology, and 81 genes that were differentially expressed (P<0.001) between women with abnormally elevated cyclin E levels vs normal cyclin E. Remarkably, genes that were recruited into endometrial expression during the evolution of pregnancy in Eutherian mammals were significantly enriched for dysregulated genes (P=0.002 for histology, P=0.021 for cyclin E), as well as for immune and signaling pathways with essential roles in endometrial biology. Thus, our novel evolutionary-based forward genomics approach identified genes whose dysregulation during the mid-secretory phase likely contributes to the molecular etiologies of recurrent early pregnancy loss. Total RNA obtained from mid luteal phase endometrium (two replicates per biopsy) from women with recurrent early pregnancy loss (REPL). Endometrial gene expression levels were compared 1) between women with out-of-phase (n=10) and normal histological dating (n=22), 2) between women with abnormally elevated (n=9) and normal (n=23) cyclin E levels. For 5 additional women with abnormally high cyclin E levels, biopsy samples were collected before and after progesterone treatment to investigate the gene expression profiles in response to progesterone.
Project description:In GnRH-antagonist/rec-FSH stimulated cycles, advanced endometrial maturation on the day of oocyte retrieval correlates with altered gene expression Keywords: gene expression analysis, advanced endometrial maturation Overall design: Endometrial biopsies taken on the day of oocyte retrieval in stimulated IVF cycles with 1 or 2 embryos replaced on day 3 in the same cycle. Endometrial dating was performed according to Noyes' criteria. All endometria taken on the day of oocyte retrieval showed an advanced maturation, ranging from +d2 to +d4. The patients with a subsequent clinical pregnancy all showed a histological dating corresponding to +d2 or +d3. Gene expression of histologically more advanced endometria, as assessed by Noyes' criteria, (n=3, +d4) was compared with matched less advanced endometria (n=4,+d2-3).
Project description:In GnRH-antagonist/rec-FSH stimulated cycles, advanced endometrial maturation on the day of oocyte retrieval correlates with altered gene expression Experiment Overall Design: Endometrial biopsies taken on the day of oocyte retrieval in stimulated IVF cycles with 1 or 2 embryos replaced on day 3 in the same cycle. Endometrial dating was performed according to Noyes' criteria. All endometria taken on the day of oocyte retrieval showed an advanced maturation, ranging from +d2 to +d4. The patients with a subsequent clinical pregnancy all showed a histological dating corresponding to +d2 or +d3. Gene expression of histologically more advanced endometria, as assessed by Noyes' criteria, (n=3, +d4) was compared with matched less advanced endometria (n=4,+d2-3).
Project description:Endometrial receptivity is imperative to achieving pregnancy in humans. A disruption in the development of endometrial receptivity is responsible for recurrent implantation failures (RIF) of endometrial origin. To further understand the molecular mechanisms behind the endometrial receptivity process, we used the 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) method to compare and quantify the proteomes from endometrial biopsies of three different endometrial statuses (fertile women, IUD carriers and RIF patients). Overall, iTRAQ allowed to identify 1,889 non-redundant proteins. Of these, 188 were differentially expressed proteins (DEP) (p-value < 0.05) among the three endometrial groups. Pairwise comparisons revealed 133 significant DEP in fertile vs. IUD carriers and 158 DEP in RIF vs. IUD carriers. However, no DEP were identified between fertile and RIF patients. Western blot validation of three DEP involved in endometrial receptivity (Plastin 2, Lactotrasferrin, and Lysozyme) confirmed our iTRAQ results. Moreover, functional KEGG enrichment revealed that complement and coagulation cascades and peroxisome were the two most significant pathways for the RIF vs. IUD comparison and ribosome and spliceosome for the fertile vs. IUD comparison, as possible important pathways involved in the endometrial receptivity acquisition. Our findings confirm that an IUD introduces numerous changes in the endometrial protein profile when compared to fertile and RIF endometria, revealing some key proteins involved in endometrial receptivity. The lack of DEP between fertile and RIF patient endometria suggest either that idiopathic RIF may not have an endometrial origin, with other as-yet-unknown factors involved.
Project description:Use of long-acting progestin-only contraceptives (LAPCs) offers a discrete and highly effective family planning method. Abnormal uterine bleeding (AUB) is the major side effect of, and cause for, discontinuation of LAPCs. The endometria of LAPC-treated women display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow and oxidative stress. To understand the mechanisms underlying AUB, we propose to identify LAPC-modulated unique gene cluster(s) in human endometrial stromal cells (HESCs). Protein and RNA isolated from cultured HESCs treated 7 days with estradiol (E2) or E2+medroxyprogesterone acetate (MPA) or E2+ etonogestrel (ETO) or E2+progesterone (P4) were analyzed by q-PCR and immunoblotting. HSCORES were determined for immunostained-paired endometria of pre-and 3 months post-Depo-Provera (Depo) treated women (n=6) and ovariectomized guinea pigs (GPs; n=12) treated with placebo or E2 or MPA or E2+MPA for 21 days. In HESCs, whole genome analysis identified a 67-gene group regulated by all three progestins, whereas a 235-gene group was regulated by E2+ETO and E2+MPA, but not E2+P4. Ingenuity pathway analysis identified glucocorticoid receptor (GR) activation as an upstream regulator of the 235 LAPCMPA and ETO-specific genes. Among these, microarray results demonstrated significant enhancement of FKBP51, a repressor of PR/GR transcriptional activity, by both LAPCsMPA and ETO. q-PCR and immunoblot analysis confirmed the microarray results. In endometria of post-Depo versus pre-Depo administered women, FKBP51 expression was significantly increased in endometrial stromal and glandular cells. In GPs, E2+MPA or MPA significantly increased FKBP51 immunoreactivity in endometrial stromal and glandular cells versus placebo- and E2-administered groups. LAPC MPA or ETO administration activates GR signaling and increases endometrial FKBP51 expression, which could be one of the mechanisms causing AUB by inhibiting feeds back to inhibit PR- and GR-mediated transcription. The resultant PR- and/or GR-mediated functional withdrawal may contribute to associated endometrial inflammation, aberrant angiogenesis, and bleeding. Overall design: Total RNA (n=3) obtained from cultured HESCs treated with vehicle only (CON) or ETO or M or P for 6h.
Project description:In order to try and identify characteristics of gene expression in the endometrium of women suffering infertility or recurrenty miscarriage, we performed RNAseq on endometrial pipelle biopsies from 20 women. The endometrial transcriptome in the mid-luteal phase of the cycle (window of implantation) is highly divergent in women suffering infertility or miscarriages. 20 mid-luteal endometrial biopsies were analysed from infertile women and patients suffering recurrent pregnancy loss.
Project description:Molecular mechanisms responsible for abnormal endometrial vasculature in women receiving long-acting progestin-only contraceptives (LAPCs) are unknown. We hypothesize that LAPCs impairs vascular smooth muscle cell (VSMC) and pericyte proliferation and/or migration producing thin-walled hyperdilated fragile microvessels prone to bleeding. Proliferating cell nuclear antigen (PCNA) and alpha smooth muscle actin (aSMA) double-immunohistochemistry assessed VSMC differentiation and proliferation in endometria from women pre- and postDepoProvera (Depo) treatment and from oophorectomized guinea pigs (OVX-GPs) treated with vehicle, estradiol (E2), medroxyprogesterone acetate (MPA) or E2+MPA pellets. After treating cultured VSMCs with MPA or etonogestrel (ETO), whole genome profiling, proliferation and migration assays were performed. Endometrial vasculature of Depo-administered women displayed reduced ĮSMA immunoreactivity and fewer PCNA (+) nuclei among aSMA (+) cells (P<0.008). Microarray analysis of VSMCs identified several MPA and ETO-altered transcripts regulated by STAT1 signaling (p<2.22x10-6), including chemokine (C-C motif) ligand 2 (CCL2). Both MPA and ETO reduced VSMC proliferation and migration (p<0.001), recombinant CCL2 reversed this progestin inhibition, and, in turn, a STAT1 inhibitor abolished these CCL2 effects. Similarly, the endometria of MPA treated OVX-GPs displayed decreased aSMA staining and fewer PCNA (+) nuclei in VSMC (p<0.005). In conclusion, LAPCs promote abnormal endometrial vessel formation by inhibiting VSMC proliferation and migration. Total RNA (n=3) obtained from cultured TDCs incubated 6h with estradiol or estradio + medroxyprogesterone acetate with or without 1 ng/ml IL-1β for 6h.
Project description:the aim of this present study was to characterize the complex transcriptome changes in different endometrial cell types during the preimplantation period for improving our understanding of localization of endometrial gene expression regulation in context of recognition of pregnancy. To do this analysis in a transcriptome-wide manner, RNA-seq was performed for samples derived from luminal epithelium (LE), glandular epithelium (GE), and stroma (S) isolated by the use of LCM Overall design: three types of endometrial cells, luminal epithelial (LE), glandular epithelial (GE), and stromal cells (S) were isolated by laser capture microdissection from endometria of Day 12 pregnant and cyclic control gilts (each group n=4), respectively, and then subjected to RNA-sequencing (RNA-Seq). The obtained datasets were compared to a RNA-Seq dataset from complete endometrial tissue samples collected in the same experiment.
Project description:An increase in circulating progesterone (P4) concentrations is associated with increased pregnancy success in beef and dairy cattle. Our objective was to ascertain differential effects of elevated P4 concentrations following conception on endometrial gene expression in beef heifers on Days 5, 7, 13 and 16 of pregnancy, corresponding to the morula, blastocyst, elongation and maternal recognition of pregnancy stages, respectively. Estrus was synchronized in beef heifers (N=263). Two-thirds (N=140) were inseminated (Day 0), and all animals were randomly assigned to one of the following treatments: (i) pregnant, high P4; (ii) pregnant, normal P4; (iii) cycling, high P4; (iv) and cycling, normal P4. All high P4 groups received a P4 release intravaginal device (PRID) on Day 3 post-estrus/mating. Tissue was collected on Days 5, 7, 13 or 16 of the cycle or pregnancy, and pregnancy was confirmed by the presence of an appropriately developed embryo/conceptus. PRID insertion elevated (P<0.05) P4 concentrations from Day 3.5 to 8 compared with untreated animals and conceptus size was larger (P<0.05) in animals with elevated P4 on Days 13 and 16 compared with normal P4. Total RNA was extracted from predominantly intercaruncular endometria from the ipsilateral uterine horn. Samples from individual heifers were selected on the basis of their P4 profiles and gene expression was analyzed using bovine Affymetrix microarrays (N=5 per treatment per time point). Microarray data from analyses using Bioconductor GCRMA and Limma packages were subjected to a modified t-test and P-values were adjusted for multiple testing using the Benjamin and Hochberg false discovery rate method. Differentially expressed genes were selected on the basis of an adjusted P-value of <0.01. There were no detectable differences in gene expression in endometria from pregnant and cyclic heifers on Days 5, 7 and 13 post-estrus, but, the expression of 764 genes was altered due to the presence of the conceptus at maternal recognition of pregnancy (Day 16). On Days 5 and 7, elevated P4 in pregnant heifers, altered the expression of 36 and 124 genes respectively but on Days 13 and 16 there were relatively few DEG between high and normal P4 heifers (15 and 25). Of the genes that were differentially regulated by P4, the majority were unique to a specific day of the estrous cycle/early pregnancy. In conclusion, gene expression in endometria did not differ between pregnant and cycling heifers until Day 16 of pregnancy (i.e. the time of maternal recognition of pregnancy and production of interferon tau by conceptus trophectoderm); however, elevating P4 in early pregnancy programmed changes in gene expression in endometria that are hypothesized to impact early conceptus growth and development. Thus, on Days 5, 7 and 13 differential gene expression was affected by P4, but on Day 16 the conceptus primarily influenced gene expression in uterine endometria of heifers. Endometiral samples were taken from estrous synchronized beef heifers that were either cyclic or confirmed pregnant with either normal or high concentrations of progesterone on Day 5, 7, 13 and 16 of the estrous cycle/early pregnancy.
Project description:Interferon tau (IFNT), a Type I IFN similar to alpha IFNs (IFNA), is the pregnancy recognition signal, produced by the ruminant conceptus. To elucidate specific effects of bovine IFNT and of other conceptus-derived factors, endometrial gene expression changes during early pregnancy were compared to gene expression changes after intrauterine application of human IFNA2. In study one, endometrial tissue samples were obtained on days (D) 12, 15, and 18 post-mating from nonpregnant or pregnant heifers. In study two, heifers were treated from D14 to D16 of the estrous cycle with an intrauterine device releasing IFNA2 or placebo lipid extrudates or PBS only as controls. Endometrial biopsies were collected after flushing the uterus. All samples from both experiments were analyzed with an Affymetrix Bovine Genome Array. Study one revealed differential gene expression between pregnant and nonpregnant endometria on D15 and D18. In study two, IFNA2 treatment resulted in differential gene expression in the bovine endometrium. Comparison of the datasets from both studies identified genes that were differentially expressed in response to IFNA2 but not in response to pregnancy on D15 or D18. Vice versa, genes were found as differentially expressed during pregnancy but not after IFNA2 treatment. In study three, spatiotemporal alterations in expression of selected genes were determined in uteri from nonpregnant and early pregnant heifers using in situ hybridization. The findings of this study suggest differential effects of bovine IFNT compared to human IFNA2 and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT. Study I: Early pregnancy; day 12 of pregnancy (n=5 heifers), day 15 of pregnancy (n=3), day 18 of pregnancy (n=4), day 12 cyclic controls (n=5), day 15 cyclic controls (n=3), day 18 cyclic controls (n=4). Study II: Treatment with human interferon alpha (IFNA); IFNA treatment group (IFNA, n=3 heifers), placebo group (PLAC, n=3 heifers), control group (CONT, n=3 heifers).
Project description:Endometriosis is a common gynecological disease that affects 10-20% of women in child-bearing age. It is defined by the presence of endometrial tissues outside the uterus. The causes remain elusive, but the endometrium of these patients differs profoundly from that of disease-free subjects. During pregnancy, the maternal-fetal interface is built onto decidualized endometrium. We investigated whether this interface is normal when the subjects suffer of endometriosis and showed remarkly properties of eutopic endometrium from endometriosis-affected women to spontaneously develop endometriotic-like lesions within direct contact to non-uterine tissues. Overall design: Methylation analysis was conducted on three distinct pools of three choriodecidua DNAs from women suffering of severe endometrosis and three distinct pools of healthy choriodecidua from eighteen pregnant women in total. These women were delivered at term by cesarean section for feto-pelvic disproportion or scarred uterus, after an uncomplicated pregnancy.