Endometrial RNA Research Articles

Endometrioid Endometrial RNA Index Predicts Recurrence in Stage I Patients.

Risk prediction with genomic and transcriptomic data has the potential to improve patient outcomes by enabling clinicians to identify patients requiring adjuvant treatment approaches, while sparing low-risk patients from unnecessary interventions. Endometrioid endometrial carcinoma (EEC) is the most common cancer in women in developed countries, and rates of endometrial cancer are increasing. We collected a 105-patient case-control cohort of stage I EEC comprised of 45 patients who experienced recurrence less than 6 years after excision, and 60 FIGO grade matched controls without recurrence. We first utilized two RNA based, previously validated machine learning approaches, namely EcoTyper and Complexity Index in Sarcoma (CINSARC). We developed Endometrioid Endometrial RNA Index (EERI) which uses RNA expression data from 46 genes to generate a personalized risk score for each patient. EERI was trained on our 105-patient cohort and tested on a publicly available cohort of 263 stage I EEC patients. EERI was able to predict recurrences with 94% accuracy in the training set and 81% accuracy in the test set. In the test set, patients assigned as EERI high-risk were significantly more likely to experience recurrence (30%) than the EERI low-risk group (1%) with a hazard ratio of 9.9 (95% CI 4.1-23.8, P <0.001). Tumors with high-risk genetic features may require additional treatment or closer monitoring and are not readily identified using traditional clinicopathologic and molecular features. EERI performs with high sensitivity and modest specificity, which may benefit from further optimization and validation in larger independent cohorts.

Integrated analysis of endometrial stromal cell long noncoding RNA and mRNA expression profiles associated with TGF-β1-induced fibrosis.

Integrated analysis of endometrial stromal cell long noncoding RNA and mRNA expression profiles associated with TGF-β1-induced fibrosis.

Bioinformatics Analysis Identifies Key Genes in Recurrent Implantation Failure Based on Immune Infiltration.

Recurrent implantation failure (RIF) is a thorny problem often encountered in the field of assisted reproduction. Existing evidences suggest that immune dysregulation may be involved in the pathogenesis of RIF. The purpose of this study is to explore immune-related genes contributing to RIF through data mining. The endometrial expression profiles of 24 RIF and 24 controls were obtained from the GEO database. The immune infiltration in bulk tissue was estimated by single sample gene set enrichment analysis (ssGSEA) method based on marker gene sets for immune cells generated from endometrial single-cell RNA sequencing data. The results showed that the infiltration levels of B cells and regulatory T cells (Tregs) were significantly reduced in the RIF group. Four hub genes (GJA1, PRKAG2, CPT1A, and ICA1) were identified by integrated analysis of weighted gene co-expression network analysis (WGCNA), random forest and LASSO regression. Moreover, these hub genes were significantly correlated with certain immune-related factors, especially CXCL12, CEACAM1, and XCR1. Single-gene GSEA indicated that the pathways associated with hub genes included the regulation of cell cycle, the process of epithelial-mesenchymal transition and transplant rejection, etc. A predictive model for RIF was constructed based on hub genes and performed well in the training dataset and the other two external datasets. Thus, this study identified immune-related key genes in RIF and provided new biomarkers for early diagnosis.

O-142 COVID19-free endometrium: Undetectable viral RNA in endometrial biopsies from positive symptomatic SARS-CoV-2 women

Abstract Study question Does SARS-CoV-2 infect the endometrial tissue in women with coronavirus disease 2019 (COVID-19)? Summary answer Symptomatic women with COVID-19 report no presence, in the short term, of viral RNA from SARS-CoV-2 in the endometrium. What is known already The recent emergence of COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has not allowed yet to establish putative relations between this disorder and other existing pathologies. It is the case with fertility problems and the reproductive organs, including a possible endometrial disorder caused by the virus. Thus, an important step is to elucidate the viral presence in different human tissues to improve diagnostics, prevention and/or treatment. The hypothesis of the possible infection of the endometrial tissue comes from the described expression of ACE2 protein in the human endometrium, mainly in stromal cells during the secretory phase. Study design, size, duration 15 endometrial biopsies from symptomatic and hospitalized women with COVID-19 were collected. Endometrial samples were obtained from August to November 2020 at the Hospital Universitari i Politècnic La Fe (Valencia, Spain); the project received the approval of the hospital’s medical ethics committee (registration number: 2020-268-1). The main objective was to study by real-time PCR (RT-PCR) the presence of viral RNA from SARS-CoV-2 as well as the expression of ACE2 receptor on the endometrial tissue. Participants/materials, setting, methods 15 women in the reproductive age (24-46 years) accepted to participate in the study and signed the informed consent. All these patients tested positive for SARS-CoV-2 by RT-PCR of nasopharyngeal swabs (1-17 days before the biopsy collection) and were hospitalized due to health complications (pneumonia) derived from COVID-19. Endometrial biopsies were taken by aspiration and preserved in RNA-later until -80ºC cryopreservation in a biobank; RNA was extracted for RT-PCR for N1, N2, and ACE2 genes. Main results and the role of chance The 15 recruited patients represented the different phases of the menstrual cycle: proliferative (n = 3) and secretory (n = 10); 2 patients had amenorrhea. The viral RNA for SARS-CoV-2, measured by the detection of N1 and N2 gene targets (fragments of N gene, from the viral nucleocapsid) by RT-PCR methodology, was undetectable in all the endometrial biopsies analyzed (n = 15). In all the cases the housekeeping gene RPP30 was used as positive control and to check RNA integrity. To correlate the presence or absence of SARS-CoV-2 with the organ-specific expression of ACE2 (angiotensin-converting enzyme 2), the main postulated entry receptor of SARS-CoV-2, the endometrial RNA was also analyzed by RT-PCR for the ACE2 receptor gene. This gene was only detectable in 10 of the 15 biopsies, and the levels ranged from 28.65 to 36.19 Ct values, revealing a very low expression of ACE2 in the tissue. Moreover, ACE2 results did not report any correlation with the phase of the menstrual cycle. Limitations, reasons for caution These results imply endometrium is safe from SARS-CoV-2 infection, at least in the short term. All the endometrial samples were taken at maximum of 17 days after a positive test by RT-PCR of nasopharyngeal swabs (to note that all were hospitalized during the early stages of the disease). Wider implications of the findings In conclusion, the SARS-CoV-2 RNA is not present in the human endometrial tissue of positive patients. This hypothesis was reinforced by the low ACE2 receptor levels. However, an in-depth genetic analysis comparing to a negative control group could elucidate a systemic affectation of the endometrium, despite the negative RT-PCR results. Trial registration number not applicable

P–555 Recurrent pregnancy loss is associated with changes in the pre-pregnant endometrial gland transcriptome

Abstract Study question Do endometrial gland factors influence recurrent pregnancy loss? Summary answer The endometrial gland transcriptome during the window of implantation is altered in women with recurrent pregnancy loss compared to controls. What is known already Secretions from endometrial glands contribute to the uterine environment that supports the attachment and implantation of the embryo in early pregnancy. Studies have attempted to identify an endometrial gene expression pattern associated with recurrent pregnancy loss however, the cellular heterogeneity within the endometrium may obscure important differences in specific cell populations. Study design, size, duration An observational study comparing controls and women with recurrent pregnancy loss. Participants/materials, setting, methods Endometrial samples were collected during the implantation period of the menstrual cycle from five matched participant egg donor controls and women with recurrent pregnancy loss. Endometrial glands were isolated from fresh endometrial biopsies and RNA sequencing was performed. A differential gene expression analysis and a gene ontology enrichment analysis was performed between egg donor controls and women with recurrent pregnancy loss. Main results and the role of chance This study reports a glandular epithelium specific gene expression profile and demonstrates differential gene expression of endometrial glands from women with recurrent pregnancy loss compared to controls. 18 genes were upregulated and 1 gene was downregulated in the endometrial glands from women with recurrent pregnancy loss compared to controls (5% false discovery rate). Biological processes which contain genes that were differentially expressed in women with recurrent pregnancy loss compared to controls include epithelial cell migration and regulation of secretion by the cell. Limitations, reasons for caution This is an observational study with a relatively small sample size. Wider implications of the findings: This study identified differences in gene expression in women with recurrent pregnancy loss that are specifically associated with endometrial glands rather than endometrium as a whole. These differences could be used to identify a perturbed endometrium, isolate causes of recurrent pregnancy loss and develop targeted therapies. Trial registration number Not applicable

Species-specific and collection method-dependent differences in endometrial susceptibility to seminal plasma-induced RNA degradation

This study aimed to determine the effect of bull seminal plasma (SP) and sperm on endometrial function. Bovine endometrial explants were incubated with: ejaculated sperm with or without SP, epididymal sperm, or SP alone. Neither ejaculated nor epididymal sperm induced differential expression of IL1A, IL1B, IL6, IL8, PTGES2, TNFA, and LIF. Interestingly, SP had a detrimental effect on endometrial RNA integrity. Addition of an RNase inactivation reagent to SP blocked this effect, evidencing a role for a SP-RNase. Because bulls deposit the ejaculate in the vagina, we hypothesized that the bovine endometrium is more sensitive to SP-RNase than vaginal and cervical tissues (which come into contact with SP during mating), or to endometrium from intrauterine ejaculators (such as the horse). In addition, due to differences in SP-RNase abundance depending on SP collection method (i.e., with an artificial vagina, AV, or by electroejaculation, EE), this effect was also tested. Bull SP, collected by AV, degrades RNA of mare endometrium, and bovine vagina, cervix and endometrium. However, stallion SP or bull SP collected by EE did not elicit this effect. Thus, results do not support a role for SP in modulating endometrial function to establish pregnancy in cattle.

Up-regulation of genes involved in the insulin signalling pathway (IGF1, PTEN and IGFBP1) in the endometrium may link polycystic ovarian syndrome and endometrial cancer

BackgroundEndometrial cancer (EC) is the most common gynaecological cancer amongst women in the UK. Although previous studies have found that women with polycystic ovary syndrome (PCOS) have at least a three-fold increase in endometrial cancer (EC) risk compared to women without PCOS, the precise molecular mechanisms which link between PCOS and EC remain unclear. It has been suggested that insulin resistance may contribute to the increased risk of EC in PCOS. The specific expression of genes related to the insulin-signalling pathway including the IGF system in the endometrium of women with PCOS has however never been measured and compared to that in women with EC without PCOS and control women without EC or PCOS. . ObjectivesTo test the hypothesis that insulin signalling plays a key role in the development of EC in women with PCOS by measuring and comparing the expression of three key genes involved in the insulin signalling pathway (IGF1, PTEN and IGFBP1) in endometrial tissue obtained from three groups of women; PCOS without EC, women with EC without PCOS and non-PCOS women without EC (controls). We also aimed to determine the correlation between the gene expressions to various clinical variables among participants. MethodsThis was a cross-sectional study of 102 women in 3 groups (PCOS, EC and controls) at a University teaching hospital in the United Kingdom. Clinical assessment (blood pressure, body mass index (BMI) and waist-hip-circumference ratio), venepuntures (fasting blood sugar, insulin, lipid profile, hormones) and endometrial tissue biopsies were taken in all participants. Endometrial tissue RNA extraction was performed before real time polymerase-chain-reaction for the genes of interest (IGF1, IGFBP1 and PTEN) was carried out. To compare the baseline characteristics of the study population, One-Way-ANOVA test or the Independent t-test was used. For variables that were not normally distributed, the Spearman correlation test was used to calculate the r value. A “p” value of <0.05 was considered statistically significant. ResultsIGF1, IGFBP1 and PTEN gene expression were significantly up-regulated in the endometrium of PCOS and EC women compared to controls. However there was no significant difference in the expression of these genes in PCOS compared to EC endometrium. The BMI of women with PCOS and controls, were not significantly different (29.28 (±2.91) vs 28.58 (±2.62) kg/m2) respectively, women with EC however had a higher mean BMI (32.22 (±5.70) kg/m2). PCOS women were younger (31.8 (±5.97) years) than women with EC (63.44 (±10.07) years) and controls (43.68 (±13.12) years). The changes in gene expression were independent of BMI, waist hip ratio, estradiol and androgen levels. Protein validation test in the serum samples in the three groups were consistent with the gene findings. ConclusionWomen with PCOS and EC have an increased endometrial expression of genes (IGF1, IGFBP1 and PTEN) involved in the insulin signalling pathway compared with control women. This may explain the increased risk of EC in PCOS women. This study provides a strong basis for clinical trials aiming to prevent EC in women with PCOS by investigating drugs targeting the insulin signalling pathway. This panel of genes may also serve as clinically useful early biomarkers which predict which women with PCOS will go on to develop EC.

Expression of Endometrial Receptivity Genes Increase After Myomectomy of Intramural Leiomyomas not Distorting the Endometrial Cavity.

This study was designed to investigate whether endometrial receptivity genes are altered in infertile patients with intramural leiomyomas (IM) not distorting the endometrial cavity undergoing myomectomy. We measured endometrial HOXA-10, HOXA-11, LIF, ITGB3, and ITGAV messenger RNA (mRNA) expressions levels before and after myomectomy/metroplasty during mid-luteal phase in participants with IM, submucosal leiomyomas (SM), and septate uterus and fertile participants without fibroids. Initial endometrial sampling was obtained at the time of surgery, and second sampling was obtained 3 months after myomectomy/metroplasty. Expressions of each gene were evaluated using real-time reverse transcriptase polymerase chain reaction (RT-PCR). A trend toward decreased endometrial HOXA-10, HOXA-11, and ITGAV mRNA expression was detected in both SM and IM groups before myomectomy when compared to both fertile group and septate uterus. However, the differences failed to show statistical significance. After myomectomy of IM, we have detected 12.8-fold increase in endometrial HOXA-10 mRNA expression and 9.0-fold increase in endometrial HOXA-11 mRNA expression. This increase in endometrial HOXA-10 and 11 mRNA expression was significant. Accordingly, 2 patients having intramural fibroids greater than 5 cm were able to remain pregnant after myomectomy. Conversely, submucosal myomectomy did not cause any significant effect on endometrial receptivity markers. Likewise, all markers of endometrial receptivity remained unchanged after metroplasty. Myomectomy of IM have favorable effect on endometrial HOXA-10 and 11 mRNA expression.

Effects of intrauterine infusion of Trueperella pyogenes on endometrial mRNA expression of proinflammatory cytokines and luteolytic cascade genes and their association with luteal life span in dairy cows

Objectives were to determine the effects of intrauterine infusion (IUI) of Trueperella pyogenes on endometrial expression of proinflammatory cytokines and luteal life span. Holstein cows (n = 32) were allocated randomly, in two replicates (15 then 17 cows), to receive one of three treatments on Day 5 of the estrous cycle: TP (n = 13), IUI containing 109 colony-forming units/mL of T pyogenes; tumor necrosis factor (TNF; n = 9), IUI containing 1 μg of TNFα; and control (n = 10), IUI of saline solution. Five cows per treatment had uterine biopsies collected at 6, 12, and 24 hours after treatment to evaluate the endometrial messenger RNA expression of TNFα (TNF), interleukin-1β (IL1B), IL6, IL8, prostaglandin E synthase (PGES), prostaglandin F synthase (PGFS), and oxytocin receptor (OXR), and histologic evidence of inflammation. Messenger RNA expression was measured using quantitative reverse transcription polymerase chain reaction. The remaining cows had ovaries scanned and blood collected for progesterone evaluation; however, only seven, four, and three cows in the TP, TNF, and control groups were used for comparison in replicate 2. The GLIMMIX procedure of SAS was used for statistical analysis. All TP and TNF cows had moderate to severe endometrial inflammation, whereas only one control had mild inflammation. Premature luteolysis occurred in three, one, and zero cows in the TP, TNF and control groups, respectively. Delayed luteolysis occurred in one TP and one TNF cow. Interleukin-1β expression was greater in the TP cows than in the TNF cows at 24 hours after IUI. Moreover, IL6 expression tended to be greater for the TP cows than for the control cows at 12 hours after IUI. Interleukin 8 expression was greater in the TP cows than in the control and TNF cows at 24 hours after IUI. Oxytocin receptor expression tended to be greater for the TP cows and was greater for the TNF cows than for the control cows at 12 hours. The messenger RNA expressions of TNF, PGES, and PGFS were not affected by treatment, time, or their interaction. In conclusion, IUI of T pyogenes or TNFα led to histologic evidence of inflammation and early luteolysis in some cows, which may have been caused by increased endometrial expression of proinflammatory cytokines (i.e., IL1B, IL6), chemokines (i.e., IL8), and luteolytic cascade factors (i.e., OXR).

Possible Human Papillomavirus 38 Contamination of Endometrial Cancer RNA Sequencing Samples in The Cancer Genome Atlas Database.

Viruses are causally associated with a number of human malignancies. In this study, we sought to identify new virus-cancer associations by searching RNA sequencing data sets from >2,000 patients, encompassing 21 cancers from The Cancer Genome Atlas (TCGA), for the presence of viral sequences. In agreement with previous studies, we found human papillomavirus 16 (HPV16) and HPV18 in oropharyngeal cancer and hepatitis B and C viruses in liver cancer. Unexpectedly, however, we found HPV38, a cutaneous form of HPV associated with skin cancer, in 32 of 168 samples from endometrial cancer. In 12 of the HPV38-positive (HPV38(+)) samples, we observed at least one paired read that mapped to both human and HPV38 genomes, indicative of viral integration into the host DNA, something not previously demonstrated for HPV38. The expression levels of HPV38 transcripts were relatively low, and all 32 HPV38(+) samples belonged to the same experimental batch of 40 samples, whereas none of the other 128 endometrial carcinoma samples were HPV38(+), raising doubts about the significance of the HPV38 association. Moreover, the HPV38(+) samples contained the same 10 novel single nucleotide variations (SNVs), leading us to hypothesize that one patient was infected with this new isolate of HPV38, which was integrated into his/her genome and may have cross-contaminated other TCGA samples within batch 228. Based on our analysis, we propose guidelines to examine the batch effect, virus expression level, and SNVs as part of next-generation sequencing (NGS) data analysis for evaluating the significance of viral/pathogen sequences in clinical samples. High-throughput RNA sequencing (RNA-Seq), followed by computational analysis, has vastly accelerated the identification of viral and other pathogenic sequences in clinical samples, but cross-contamination during the processing of the samples remain a major problem that can lead to erroneous conclusions. We found HPV38 sequences specifically present in RNA-Seq samples from endometrial cancer patients from TCGA, a virus not previously associated with this type of cancer. However, multiple lines of evidence suggest possible cross-contamination in these samples, which were processed together in the same batch. Despite this potential cross-contamination, our data indicate that we have detected a new isolate of HPV38 that appears to be integrated into the human genome. We also provide general guidelines for computational detection and interpretation of pathogen-disease associations.

A Murine Uterine Transcriptome, Responsive to Steroid Receptor Coactivator-2, Reveals Transcription Factor 23 as Essential for Decidualization of Human Endometrial Stromal Cells1

Recent data from human and mouse studies strongly support an indispensable role for steroid receptor coactivator-2 (SRC-2)-a member of the p160/SRC family of coregulators-in progesterone-dependent endometrial stromal cell decidualization, an essential cellular transformation process that regulates invasion of the developing embryo into the maternal compartment. To identify the key progesterone-induced transcriptional changes that are dependent on SRC-2 and required for endometrial decidualization, we performed comparative genome-wide transcriptional profiling of endometrial tissue RNA from ovariectomized SRC-2(flox/flox) (SRC-2(f/f) [control]) and PR(cre/+)/SRC-2(flox/flox) (SRC-2(d/d) [SRC-2-depleted]) mice, acutely treated with vehicle or progesterone. Although data mining revealed that only a small subset of the total progesterone-dependent transcriptional changes is dependent on SRC-2 (∼13%), key genes previously reported to mediate progesterone-driven endometrial stromal cell decidualization are present within this subset. Along with providing a more detailed molecular portrait of the decidual transcriptional program governed by SRC-2, the degree of functional diversity of these progesterone mediators underscores the pleiotropic regulatory role of SRC-2 in this tissue. To showcase the utility of this powerful informational resource to uncover novel signaling paradigms, we stratified the total SRC-2-dependent subset of progesterone-induced transcriptional changes in terms of novel gene expression and identified transcription factor 23 (Tcf23), a basic-helix-loop-helix transcription factor, as a new progesterone-induced target gene that requires SRC-2 for full induction. Importantly, using primary human endometrial stromal cells in culture, we demonstrate that TCF23 function is essential for progesterone-dependent decidualization, providing crucial translational support for this transcription factor as a new decidual mediator of progesterone action.

Endometrial RNA expressions of interleukin 7, interleukin 17 and their receptors during the implantation window, in patients with repeated implantation failures, repeated miscarriages and fertile women

Endometrial RNA expressions of interleukin 7, interleukin 17 and their receptors during the implantation window, in patients with repeated implantation failures, repeated miscarriages and fertile women

Transcriptome Changes in the Porcine Endometrium During the Preattachment Phase1

The porcine conceptus undergoes rapid differentiation and expansion of its trophoblastic membranes between Days 11 and 12 of gestation. Concomitant with trophoblast elongation, production of conceptus estrogen, the porcine embryonic pregnancy recognition signal, increases. Conceptus attachment to the uterine surface epithelium starts after Day 13, initiating epitheliochorial placentation. To analyze the transcriptome changes in the endometrium in the course of maternal recognition of pregnancy, deep sequencing of endometrial RNA samples of Day 12 pregnant animals (n = 4) and corresponding nonpregnant controls (n = 4) was performed using RNA sequencing (RNA-Seq). Between 30 000 000 and 35 000 000 sequence reads per sample were produced and mapped to the porcine genome (Sscrofa10.2). Analysis of read counts revealed 2593 differentially expressed genes (DEGs). Expression of selected genes was validated by the use of quantitative real-time RT-PCR. Bioinformatics analysis identified several functional terms specifically overrepresented for up-regulated or down-regulated genes. Comparison of the RNA-Seq data from Days 12 and 14 of pregnancy was performed at the level of all expressed genes, the level of the DEG, and the level of functional categories. This revealed specific gene expression patterns reflecting the different functions of the endometrium during these stages (i.e., recognition of pregnancy and preparation for conceptus attachment). Genes related to mitosis, immune response, epithelial cell differentiation and development, proteolysis, and prostaglandin signaling and metabolism are discussed in detail. This study identified comprehensive transcriptome changes in porcine endometrium associated with establishment of pregnancy and could be a resource for targeted studies of genes and pathways potentially involved in regulation of this process.

Deep Sequencing of the Porcine Endometrial Transcriptome on Day 14 of Pregnancy1

In pigs, conceptus attachment to the uterine surface epithelium starts around Day 14 of pregnancy preceded by a pronounced vascularization at the implantation zones, initiating the epitheliochorial placentation. To characterize the complex transcriptome changes in the endometrium in the course of initial conceptus attachment, deep sequencing of endometrial RNA samples of pregnant animals (n = 4) and corresponding cyclic controls (n = 4) was performed using Illumina RNA-Seq. The obtained sequence reads were mapped to the porcine genome, and relative expression values were calculated for the analysis of differential gene expression. Statistical analysis revealed 1933 differentially expressed genes (false discovery rate 1%), 1229 with higher and 704 with lower mRNA concentration, in the samples from pregnant animals. Expression of selected genes was validated by the use of quantitative real-time RT-PCR. The RNA-Seq data were compared to results of a microarray study of bovine endometrium on Day 18 of pregnancy and additional related data sets. Bioinformatics analysis revealed for the genes with higher mRNA concentration in pregnant samples strong overrepresentation, particularly for immune-related functional terms but also for apoptosis and cell adhesion. Overrepresented terms for the genes with lower mRNA concentration in pregnant samples were related to extracellular region, ion transport, cell adhesion, and lipid and steroid metabolic process. In conclusion, RNA-Seq analysis revealed comprehensive transcriptome differences in porcine endometrium between Day 14 of pregnancy and corresponding cyclic endometrium and highlighted new processes and pathways probably involved in regulation of noninvasive implantation in the pig.

118 DEEP SEQUENCING OF THE PORCINE ENDOMETRIAL TRANSCRIPTOME ON DAYS 12 AND 14 OF PREGNANCY

Establishment of pregnancy in mammals requires prolongation of luteal life span for sustained progesterone (P4) production. P4 stimulates secretory functions of the endometrium required for conceptus growth and development and is essential for endometrial receptivity. The porcine conceptus undergoes rapid differentiation and expansion of its trophoblastic membranes between Day 11 and Day 12 of gestation. With trophoblast elongation production of estrogen, the porcine embryonic pregnancy recognition signal, by the conceptus increases. A complex interplay of estrogen signaling and prostaglandin (PG) metabolism in the endometrium finally results in prevention of luteolysis. Conceptus attachment to the uterine surface epithelium starts around Day 14 of pregnancy, preceded by a pronounced vascularization at the implantation zones, initiating the epitheliochorial placentation. To characterize the complex transcriptome changes in the endometrium in the course of maternal recognition of pregnancy and initial placentation, deep sequencing of endometrial RNA samples of pregnant animals (n = 4) and corresponding non-pregnant controls (n = 4) was performed on Day 12 and Day 14 after ovulation using Illumina RNA-Seq (Illumina Inc., San Diego, CA, USA). Between 21 and 36 million 76-bp single-end reads were produced per sample. Reads were mapped with TopHat to the porcine genome assembly Sscrofa10.2 and relative expression values were calculated based on the National Center for Biotechnology Information’s gene annotation for the analysis of differential gene expression. Statistical analysis revealed more than 3500 differentially expressed genes (DEG) for Day 12 and almost 2500 DEG for Day 14 of pregnancy (DESeq, FDR 1%). Differential expression of selected genes was validated by the use of quantitative real-time PCR (RT-PCR). Comparison of the RNA-Seq data from Day 12 and Day 14 of pregnancy based on a comparison of the DEGs and of overrepresented functional categories revealed distinct differences in gene regulation, reflecting the different functions of the endometrium during these stages; that is, recognition of pregnancy on Day 12 and preparation for conceptus implantation on Day 14. The data from Day 14 was also compared to the published microarray data set from Day 14 of pregnancy (Østrup et al. 2010 Biol. Reprod.) and other sets of DEG by the use of gene set enrichment analysis (GSEA). Overall, a very good agreement with the results of the previous microarray study was found. Furthermore, a significant overlap with genes upregulated in bovine endometrium on Day 18 of pregnancy was found for the genes upregulated in porcine endometrium on Day 14. More than half of these genes were known interferon-regulated genes. In conclusion, RNA-Seq revealed distinct transcriptome changes on Day 12 and Day 14 of pregnancy in porcine endometrium associated with MRP and initiation of implantation.

Global Gene Expression Profiling of Proliferative Phase Endometrium Reveals Distinct Functional Subdivisions

The human endometrium follows a predictable pattern of development during the proliferative phase. Endometrial thickness increases after day 3 and then plateaus at days 9 to 10 of the menstrual cycle despite continued high serum levels of estrogen. We hypothesized that proliferative phase endometrium undergoes more than simple estrogen responsive growth, rather it is characterized by complex time-dependent functional activities reflected in differential gene expression. Nine endometrial RNA samples from healthy participants were subjected to microarray analysis and 15 samples were used for quantitative real-time polymerase chain reaction. The samples were divided into early, mid, or late proliferative phase. The early proliferative phase showed higher expression of genes including transforming growth factor β2, chemokine (C-C motif) ligand 18 (CCL18), and metallothionein 2A. The mid-proliferative phase was characterized by higher expression of heat shock proteins and implantation-associated genes including Indian hedgehog, secreted frizzled protein 4, and progesterone receptor. In the late proliferative phase, we identified increased angiotensin II receptor, type 2 and large decrease in expression of genes related to natural killer (NK) cell function. We demonstrate a unique gene expression signature at distinct time points within the proliferative phase. The early proliferative phase is characterized by tissue remodeling, angiogenesis, and modulation of inflammation; the mid-proliferative phase is characterized not only by proliferation in response to estrogens but also marks the onset of expression of genes required for endometrial receptivity and a dampening of estrogen responsiveness. In the late proliferative phase, changes in immune function and NK cells predominate. The proliferative phase is not simply a uniform period of estrogen responsive endometrial growth that can be considered as a single experimental time point when evaluating endometrial development; rather the proliferative phase is complex with differing functions and patterns of gene expression.

Expression of Akt Isoforms in the Rat Endometrium During Pregnancy and Pseudopregnancy.

Rat gestation can be divided into various parts that would include fertilization, implantation, decidualization, decidual regression and parturition. However, little is known about the intracellular and molecular mechanisms involved in the endometrium regulating these different steps of pregnancy. Akt isoforms (Akt1, Akt2 and Akt3) are expressed and differentially regulated in normal tissues but their specific roles remain to be clarified in the endometrium. Our hypothesis is that the Akt signaling pathway and the three Akt isoforms are playing important and different roles in the rat endometrium during pregnancy. This pathway is critically involved in cell proliferation and survival but its regulation during pregnancy remains to be elucidated. It has been shown that X-linked Inhibitor of Apoptosis Protein (XIAP) might be involved in cell survival through regulation of Akt phosphorylation in different systems. XIAP is inhibited by second mitochondria-derived activator of caspase/Direct IAP Binding Protein with Low pI (Smac/DIABLO). Transcription factor NF-kappaB plays a pivotal role in inflammatory and immune responses. It can activate the transcription of many genes involved in the pregnancy process and is also known to be activated by Akt. Objective of the present study was to investigate the regulation of Akt pathway in the rat endometrium during pregnancy. Pseudopregnant rats were killed, from day 1 to 9, and during normal pregnancy, every two days from day 1 to 22. Uteri were removed to collect endometrial RNA, protein and tissues sections were prepared for immunochemistry. In the early stages of pregnancy, an increase of pAkt (active form) at day 5 was observed and only Akt3 was the active isoform. An increase of pAkt at day 3 was observed in pseudopregnant rats. It was possible to observe a translocation of NF-kappaB to the nuclus in pregnant but not in pseudopregnant rats. The difference observed between pregnancy and pseudopregnancy suggests that the presence of an embryo has an impact on the regulation of the Akt pathway. During the late state of pregnancy, we found that the active form of Akt was reduced during decidual regression and was increased at day 1 post-partum and that Akt3 was the active isoform. XIAP expression showed a peak on day 14 and then decreased thereafter. Smac/Diablo also showed a peak of expression at day 14. In vivo, PI3-K inhibitor wortmannin blocked Akt phosphorylation and reduced XIAP expression in the endometrium. In vitro, PI3-K inhibition decrease both pAkt and XIAP expression in decidual cells. These results suggest that Akt3 may be an important signalling protein in the rat endometrium to control cell survival.

Progesterone concentration requirements for normal mid-secretory endometrial structure and function

OBJECTIVE: Determine the threshold P concentration necessary for normal endometrial structure and/or function. DESIGN: Endometrial biopsy was performed in normal volunteers (21 - 37y, n=25) during the mid-secretory phase of a natural cycle or during randomized treatment with one of four P doses. MATERIALS AND METHODS: Endometrial cycles were modeled using leuprolide down-regulation, transdermal E (0.2 mg/d × 20 days), and i.m. P at a randomly assigned dose (40, 10, 5, and 2.5 mg/d) during the last 10 days of E treatment. Endometrial biopsy specimens were collected on the 10th day of P exposure. Blinded histological dating was performed on H&E stained sections using Noyes criteria. Function was assessed by gene expression profiling of whole endometrial RNA using an Agilent 44k microarray. Differential expression of selected genes was confirmed using real-time RT-PCR. RESULTS: Women exposed to 40, 10, 5, and 2.5 mg/d P had serum P levels of about 20, 5, 2.5, and 1 ng/mL, respectively. No difference in histologically assigned dates was seen at any P dose, except 2.5 mg/d, which resulted in a mean histologic date 4.5 d earlier than expected. Gene expression differences were seen only at 2.5 mg/d. At adjusted p ≤ 0.05, 337 genes are differentially regulated between the control and 2.5 mg groups. Using a cutoff of 1.5 or 2-fold change, 221 and 54 genes, respectively, were identified. Among the most differentially regulated genes were SPP1, RPRM, CFD, and CXCL13 (increased in controls); and MMP11, DIO2, SFRP4, and NOTCH1 (increased in the 2.5 mg P group). CONCLUSION: Serum P concentrations ≤ 1 ng/mL are insufficient, while those ≥ 2.5 ng/mL are sufficient for normal mid-secretory endometrial structure and function in young, healthy women. Since women in ART cycles without P supplementation have luteal serum P >> 2.5 ng/mL, these findings suggest that benefits of P supplementation are either extra-endometrial or due to an increased P requirement for some women with infertility.

Role of PTGS2 and PTGES in Receptivity and Initiation of Decidualization in Rat Endometrium.

Implantation is a complex process involving many physiological events including uterine receptivity follow by blastocyst attachment, apoptosis of endometrial luminal epithelium, an increase in endometrial vascular permeability, and finally decidualization. Previous studies conducted in this laboratory suggest that prostaglandin E2 (PGE2) but not prostaglandin I2 (PGI2) has a major role in the early events of implantation in the rat endometrium (Gillio-Meina et al 2007, 53rd Annual Meeting of the Canadian Fertility and Andrology Society, poster FP21). To further elucidate if PGE2 is involved in uterine receptivity and initiation of decidualization, we determined the temporal and spatial pattern of endometrial expression of genes encoding enzymes involved in PGE2 synthesis (prostaglandin-endoperoxide synthase 1 and 2 [PTGS1 and PTGS2], and microsomal and cytosolic prostaglandin E synthases [PTGES, PTGES2 and PTGES3]) in ovariectomized rats treated on the afternoon of the equivalent of day 4 of pseudopregnancy with estradiol (E2) and progesterone (P4), equivalent to nidatory E2 to induce receptivity), or P4 alone (to maintain the neutral state). Endometrium was separated from myometrium at 0, 5 or 15 h after the hormone injections and RNA and protein were isolated. Western blot analysis showed a significant increase in PTGES2 protein levels at 5 h in the presence of both E2 and P4, and at 15 h in the presence of P4 alone or in combination with E2. Moreover, PTGS2 protein levels were significantly higher 15 h after the treatment with both E2 and P4. To further study the role of PGE2 during initiation of decidualization, ovariectomized rats were treated with E2 and P4 to mimic pseudopregnancy and on day 5 of pseudopregnancy a bilateral intrauterine injection of sesame oil was given as a deciduogenic stimulus. Endometrial RNA and protein were isolated and uterine sections were prepared at 0 and at 2, 4, 8, 16 or 32 h following the stimulus. We found that Ptgs2 mRNA also significantly increased at 2 and 4 h after the deciduogenic stimulus, whereas the protein was upregulated at 8, 16 and 32 h. In addition, PTGES2 protein was upregulated at 2 and 4 h, whereas PTGES3 protein was significantly higher at 2, 4 and 8 h. By immunohistochemistry, PTGS1, PTGES2 and PTGES3 were co-localized in glands and luminal epithelium at all times, but the signals appeared to be higher at 2 and 4 h after the stimulus. By contrast, PTGS2 was localized in the stromal cells and the signal increased at 16 and 32 h. Because PTGS2 protein levels are increased by nidatory E2, it is possible that a product of that increase, PGE2, could be necessary for initiation of decidualization. Moreover, the upregulation of PTGES2 and PTGES3 proteins in the early stages of decidualization and an upregulation of PTGS2 in the later stages of decidualization, provides additional evidence that PGE2 is the PG most likely involved in decidualization in rat endometrium. [This work was supported by the Canadian Institutes of Health Research.]

Galectin 15 (LGALS15): A Gene Uniquely Expressed in the Uteri of Sheep and Goats that Functions in Trophoblast Attachment1

Galectins are a family of secreted animal lectins with biological roles in cell adhesion and migration. In sheep, galectin 15 (LGALS15) is expressed specifically in the endometrial luminal (LE) and superficial glandular (sGE) epithelia of the uterus in concert with blastocyst elongation during the peri-implantation period. The present study examined LGALS15 expression in the uterus of cattle, goats, and pigs. Although the bovine genome contains an LGALS15-like gene, expressed sequence tags encoding LGALS15 mRNA were found only for sheep, and full-length LGALS15 cDNAs were cloned only from endometrial total RNA isolated from pregnant sheep and goats, but not pregnant cattle or pigs. Ovine and caprine LGALS15 were highly homologous at the mRNA (95%) and protein (91%) levels, and all contained a conserved carbohydrate recognition domain and RGD recognition sequence for integrin binding. Endometrial LGALS15 mRNA levels increased after Day 11 of both the estrous cycle and pregnancy, and were considerably increased after Day 15 of pregnancy in goats. In situ hybridization detected abundant LGALS15 mRNA in endometrial LE and sGE of early pregnant goats, but not in cattle or pigs. Immunoreactive LGALS15 protein was present in endometrial epithelia and conceptus trophectoderm of goat uteri and detected within intracellular crystal structures in trophectoderm and LE. Recombinant ovine and caprine LGALS15 proteins elicited a dose-dependent increase in ovine trophectoderm cell attachment in vitro that was comparable to bovine fibronectin. These results support the hypothesis that LGALS15 is uniquely expressed in Caprinae endometria and functions as an attachment factor important for peri-implantation blastocyst elongation.