Over the course of the last four decades, IVF has allowed an increasing number of infertile couples the chance to conceive. Considering the extensive research and advances in ART, too many IVF attempts still do not result in a successful pregnancy [1, 2]. Embryo implantation is a crucial event in the establishment of a pregnancy. It is now clear that embryo implantation relies upon cross-talk and synchronicity between the implanting embryo and a receptive endometrium [3]. This embryo-maternal cross-talk involves an elaborate and coordinated network of communication via timely released embryonic, maternal-derived signals, and well-targeted actions [4]. If the clinical and culture conditions to obtain a “good quality” embryo are well advanced today, ER remains the last barrier in ART. When a high-quality embryo is transferred, impaired uterine receptivity is believed to be one of the major reasons behind failure of the establishment of pregnancy [5, 6]. It has been suggested in a few studies that up to two-thirds of implantation failures are due to defects in ER whereas the quality of the embryo itself is responsible for only one-third of failures [7, 8]. An endometrium is receptive to an embryo in a spatially and temporally restricted period called the window of implantation (WOI). In natural cycles, this period, occurring during the mid-secretory phase, is limited to approximately 48 h, starting around the seventh day after the LH (luteinizing hormone) surge [9, 10]. The dynamic transition from a non-receptive to a receptive endometrium is still poorly understood. Several reports have shown that ER is defined by specific changes in factors involved in adhesion, invasion, survival, growth, differentiation, decidualization and immuno-modulation. The correct spatio-temporal synthesis and balance of these various factors are thought to play an important role in human uterine preparation for implantation [4, 11]. Extensive efforts have been made to understand and characterize a receptive endometrium, from the first histological dating methods to the ‘omics’ technologies [3, 7, 12]. Several endometrial dating criteria have been commonly used in clinical practice. One or more panels of biomarkers, predictive of optimal ER, have been analyzed in blood and uterine fluid. Leukaemia inhibitor factor (LIF) is an example of a potential biomarker of the WOI [13]. However, this approach has been judged unsatisfactory by several studies because LIF measurements in serum do not reflect fertility status and similar conclusions were reported for other biomarkers [3, 14]. Cervical mucus has also been used to date ER by analyzing cytokines and growth factors produced by a receptive endometrium and their transport to the cervical mucus [15]. However, other studies have been unable to detect these growth factors in cervical secretions throughout the menstrual cycle and in these studies no correlation was observed between cytokine levels in cervico-vaginal secretions and serum and between the cytokines gene expression level in the secretory endometrium and the concentrations in serum [3, 16]. Another group evaluated ER during IVF cycles using three-dimensional power Doppler ultrasound [17]. However, Sterzik et al. concluded that ultrasonography is an inadequate method to predict ER in IVF cycles since neither the endometrial thickness nor the echogenic pattern correlate with histological findings [18]. As demonstrated, endometrial dating criteria have been questioned in various randomized studies. This has encouraged further investigation and application of new technologies to try to objectively diagnose ER. Omics technologies, such as transcriptomics, have been used to identify biomarkers of human endometrium [7, 19]. Based on the transcriptomic signature identified in these studies, only two ER diagnostic tools have been commercialized in order to personalize the frozen embryo transfer (FET): the ERA test (Endometrial Receptivity Array) [20] and the Win-Test (Window Implantation Test) [21]. In this review, we screened publications of the transcriptomic profiles of fertile and infertile women during the secretory phase of natural and stimulated cycles in order to understand lessons learned from endometrial gene profiling.

In assisted reproduction, about 30% of embryo implantation failures are related to inadequate endometrial receptivity. To identify molecules involved in endometrial receptivity acquisition, we investigated, using a SELDI-TOF approach, the protein expression profile of early-secretory and mid-secretory endometrium samples. Among the proteins upregulated in mid-secretory endometrium, we investigated the function of S100A10 in endometrial receptivity and implantation process. S100A10 was expressed in epithelial and stromal cells of the endometrium of fertile patients during the implantation windows. Conversely, it was downregulated in the mid-secretory endometrium of infertile patients diagnosed as non-receptive. Transcriptome analysis of human endometrial epithelial and stromal cells where S100A10 was silenced by shRNA revealed the deregulation of 37 and 256 genes, respectively, related to components of the extracellular matrix and intercellular connections. Functional annotations of these deregulated genes highlighted alterations of the leukocyte extravasation signaling and angiogenesis pathways that play a crucial role during implantation. S100A10 silencing also affected the migration of primary endometrial epithelial and stromal cells, decidualization and secretory transformation of primary endometrial stromal cells and epithelial cells respectively, and promoted apoptosis in serum-starved endometrial epithelial cells. Our findings identify S100A10 as a player in endometrial receptivity acquisition.

ObjectiveThe aim of this study was to identify key actors of human endometrial receptivity by performing complete study from Omics pre-screenings with both proteome and transcriptome until validation in independent patients and function determination.DesignUsing Omics technologies, we identified biomarkers of human endometrial receptivity by comparing pre-receptive (LH+2) with receptive (LH+7) secretory endometrial samples. Then, potential candidates have been selected for validation in independent cohort of fertile patients by western blot and immunofluorescent staining of endometrial tissues. One candidate was selected for function(s) investigations in purified primary endometrial cells using the shRNAs.Materials and MethodsRNA and proteins content were extracted from each endometrial biopsy. Gene and protein expression profiles at the LH+2 and LH+7 were analyzed by DNA microarray chips (n=62 samples, 31 patients) and SELDI TOF (18 samples, 9 patients) respectively. One biomarker over-expressed during the implantation window (at mRNA and protein level) was selected for validation (western blot and immunofluorescent staining of endometrial tissue). Primary endometrial cell cultures of epithelial and stromal cells were performed to target the extinction of the candidate using shRNAs in each endometrial cell type. The obtained phenotype was analyzed in regard of physiological processes involved in early implantation.ResultsTranscriptome of shRNA S100 epithelial cells revealed the over-expression of 34 genes related to components of the extracellular matrix or intercellular connections (COL4A5, GJA5). In stromal cells, 256 genes were differentially expressed in infected cells compared to control cells (174 up-regulated, 82 down-regulated). Functional annotation revealed alteration of leukocyte transendothelial migration (VAV3, MMP16) and TGFβ signaling (ACVR1C, BMPER). Western blot and immunofluorescence analyses of the selected biomarker, belonging of the S100 family, confirmed the over-expression of the candidate during the implantation window and its localization in both endometrial cell types. Candidate extinction significantly reduced cells migration and affected decidualization in both cell types. In addition, candidate extinction sensitized apoptosis induced by serum withdrawal in epithelial cells.ConclusionsBy performing a complete study from pre-screenings to functional analyses, we identify crucial actor of the human endometrial receptive phenotype, opening new perspectives in the understanding of mechanisms regulating human endometrial receptivity of infertile patients. ObjectiveThe aim of this study was to identify key actors of human endometrial receptivity by performing complete study from Omics pre-screenings with both proteome and transcriptome until validation in independent patients and function determination. The aim of this study was to identify key actors of human endometrial receptivity by performing complete study from Omics pre-screenings with both proteome and transcriptome until validation in independent patients and function determination. DesignUsing Omics technologies, we identified biomarkers of human endometrial receptivity by comparing pre-receptive (LH+2) with receptive (LH+7) secretory endometrial samples. Then, potential candidates have been selected for validation in independent cohort of fertile patients by western blot and immunofluorescent staining of endometrial tissues. One candidate was selected for function(s) investigations in purified primary endometrial cells using the shRNAs. Using Omics technologies, we identified biomarkers of human endometrial receptivity by comparing pre-receptive (LH+2) with receptive (LH+7) secretory endometrial samples. Then, potential candidates have been selected for validation in independent cohort of fertile patients by western blot and immunofluorescent staining of endometrial tissues. One candidate was selected for function(s) investigations in purified primary endometrial cells using the shRNAs. Materials and MethodsRNA and proteins content were extracted from each endometrial biopsy. Gene and protein expression profiles at the LH+2 and LH+7 were analyzed by DNA microarray chips (n=62 samples, 31 patients) and SELDI TOF (18 samples, 9 patients) respectively. One biomarker over-expressed during the implantation window (at mRNA and protein level) was selected for validation (western blot and immunofluorescent staining of endometrial tissue). Primary endometrial cell cultures of epithelial and stromal cells were performed to target the extinction of the candidate using shRNAs in each endometrial cell type. The obtained phenotype was analyzed in regard of physiological processes involved in early implantation. RNA and proteins content were extracted from each endometrial biopsy. Gene and protein expression profiles at the LH+2 and LH+7 were analyzed by DNA microarray chips (n=62 samples, 31 patients) and SELDI TOF (18 samples, 9 patients) respectively. One biomarker over-expressed during the implantation window (at mRNA and protein level) was selected for validation (western blot and immunofluorescent staining of endometrial tissue). Primary endometrial cell cultures of epithelial and stromal cells were performed to target the extinction of the candidate using shRNAs in each endometrial cell type. The obtained phenotype was analyzed in regard of physiological processes involved in early implantation. ResultsTranscriptome of shRNA S100 epithelial cells revealed the over-expression of 34 genes related to components of the extracellular matrix or intercellular connections (COL4A5, GJA5). In stromal cells, 256 genes were differentially expressed in infected cells compared to control cells (174 up-regulated, 82 down-regulated). Functional annotation revealed alteration of leukocyte transendothelial migration (VAV3, MMP16) and TGFβ signaling (ACVR1C, BMPER). Western blot and immunofluorescence analyses of the selected biomarker, belonging of the S100 family, confirmed the over-expression of the candidate during the implantation window and its localization in both endometrial cell types. Candidate extinction significantly reduced cells migration and affected decidualization in both cell types. In addition, candidate extinction sensitized apoptosis induced by serum withdrawal in epithelial cells. Transcriptome of shRNA S100 epithelial cells revealed the over-expression of 34 genes related to components of the extracellular matrix or intercellular connections (COL4A5, GJA5). In stromal cells, 256 genes were differentially expressed in infected cells compared to control cells (174 up-regulated, 82 down-regulated). Functional annotation revealed alteration of leukocyte transendothelial migration (VAV3, MMP16) and TGFβ signaling (ACVR1C, BMPER). Western blot and immunofluorescence analyses of the selected biomarker, belonging of the S100 family, confirmed the over-expression of the candidate during the implantation window and its localization in both endometrial cell types. Candidate extinction significantly reduced cells migration and affected decidualization in both cell types. In addition, candidate extinction sensitized apoptosis induced by serum withdrawal in epithelial cells. ConclusionsBy performing a complete study from pre-screenings to functional analyses, we identify crucial actor of the human endometrial receptive phenotype, opening new perspectives in the understanding of mechanisms regulating human endometrial receptivity of infertile patients. By performing a complete study from pre-screenings to functional analyses, we identify crucial actor of the human endometrial receptive phenotype, opening new perspectives in the understanding of mechanisms regulating human endometrial receptivity of infertile patients.

L'acquisition du phenotype de la receptivite endometriale est une etape cle de l'implantation embryonnaire. Dans ce contexte, ce projet vise a mieux cerner les mecanismes moleculaires de la receptivite endometriale. Des approches globales (transcriptome et proteome), ont ete utilisees pour nos activites de recherche et le developpement d'outils diagnostiques en AMP. Nous avons determine les fonctions d'un de nos bio-marqueurs de la receptivite endometriale par shARNs, S100A10. L'extinction de S100A10 dans les cellules primaires endometriales affecte la migration, la decidualisation et l'apoptose des cellules endometriales, des fonctions biologiques majeures impliquees dans le processus d'implantation. D'autre part, nous avons implante notre test d'appreciation de la receptivite endometriale dans une clinique de fertilite a Montreal et initie une etude prospective clinique de l'evaluation de la receptivite endometriale en cycle naturel de patientes en attente d'une procedure de FIV/ICSI. De plus, nous avons demontre un effet deletere d'une progesteronemie elevee le jour du declenchement de l'ovulation sous traitement de stimulation ovarienne sur la transition des profils transcriptomiques des endometres pre-receptif et receptif, suggerant une acceleration de la maturation endometriale pendant la phase peri-ovulatoire, sans pour autant affecter la receptivite endometriale. Enfin, nous avons montre que la receptivite endometriale des patientes sous traitements hormonaux substitutifs differait de celles en cycle naturel, caracterisee par une alteration des voies de signalisation mediee par les recepteurs aux œstrogenes, des membres de la famille VEGF et des integrines.

The impact of a premature elevation of serum progesterone level, the day of hCG administration in patients under controlled ovarian stimulation during IVF procedure, on human endometrial receptivity is still debated. In the present study, we investigated the endometrial gene expression profile shifts during the prereceptive and receptive secretory stage in patients with normal and elevated serum progesterone level on the day of hCG administration in fifteen patients under stimulated cycles. Then, specific biomarkers of endometrial receptivity in these two groups of patients were tested. Endometrial biopsies were performed on oocyte retrieval day and on day 3 of embryo transfer, respectively, for each patient. Samples were analysed using DNA microarrays and qRT-PCR. The endometrial gene expression shift from the prereceptive to the receptive stage was altered in patients with high serum progesterone level (>1.5 ng/mL) on hCG day, suggesting accelerated endometrial maturation during the periovulation period. This was confirmed by the functional annotation of the differentially expressed genes as it showed downregulation of cell cycle-related genes. Conversely, the profile of endometrial receptivity was comparable in both groups. Premature progesterone rise alters the endometrial gene expression shift between the prereceptive and the receptive stage but does not affect endometrial receptivity.

We previously identified and validated the over-expression of S100 protein family member during the receptive phase as biomarker of endometrial receptivity, using omics (transcriptome and proteome). This study aims to identify this candidate roles in receptive endometrial phenotype acquisition of fertile woman. Primary stromal and epithelial cells cultures were purified from endometrial biopsies of fertile woman. Protein extinction was completed by loss of function (shRNA) for stable gene silencing. We investigated the impact of candidate knockdown on transcriptome, migration and decidualization. Extinction of candidate was performed using 3 shRNAs (pLKO.1-puro-CMV-tGFP) in purified primary endometrial cells. Transcriptome of infected and control cells were studied using DNA microarray. We performed migration assay by wound healing and decidualization assay using 8-Br-cAMP with quantification by qRT-PCR of decidualization biomarkers. Transcriptome analysis demonstrated, in stromal cells, that 256 genes were differentially expressed in infected cells compared to control cells (174 up-regulated, 82 down-regulated). Functional annotation revealed alteration of leukocyte transendothelial migration (VAV3, MMP16) and TGFβ signaling (ACVR1C, BMPER). In epithelial cells, 34 genes were up-regulated in shRNA cells compared with controls. Several of these genes were components of the extracellular matrix or intercellular connections (COL4A5, GJA5). Candidate extinction significantly reduced cells migration over 24hrs. As well, 9 days after the start of cAMP treatment, we observed in infected cells, a reduction of cellular differentiation/decidualization that was confirmed by the down-regulation of connexin 43. Interestingly, candidate extinction induced a significant reduction of ARNm expression of the decidualization biomarker prolactin in stromal cells (44%) while a strong increase (61%) was observed in epithelial cells compared to controls. Extinction of our candidate affected decidualization, migration and transcriptome of endometrial cells demonstrating its essential role in the gradual acquisition of the receptive endometrial phenotype.

This study aims to identify specific biomarkers of human endometrial receptivity and to determine the function(s) of these new biomarkers using transcriptomic and proteomic approaches. We performed the gene and protein expression profiles between LH+2 and LH+7 from the same patients (n=9) during natural cycles. Two biomarkers over-expressed during the implantation window (mRNA and protein level) have been selected for functional analyses using primary endometrial cells. The obtained phenotypes were analyzed in regard of the morphology, survival/death, proliferation, adhesion, migration and decidualization. RNA and proteins content were extracted from each endometrial biopsy. Gene and protein expression profiles during the LH+2 and LH+7 transitions were analyzed by DNA microarray chips and SELDI TOF. Two biomarkers were selected and their over-expression were validated in independent cohort of fertile patients (n=4) by western blot. Then, functional analyses in the target cellular type (primary epithelial and/or stromal cells) were investigated by shRNAs. Western blot confirmed the over-expression of our candidates, belonging of the S100 family of proteins, during the implantation window. Immunofluorescence staining of sections from paraffin-embedded endometrium, as well as western blot analyses of purified endometrial cells, revealed a localization in both cellular types with a stronger expression in epithelial cells for the first candidate, and exclusively epithelial for the second candidate. An approach by loss of function via shRNA was used in the target cellular type. Obtained phenotypes revealed that the two candidates don't play a central role in cellular morphology, proliferation and survival/death. Impact on adhesion, migration and decidualization are in the course of analyses. This study provides tools for clinical application for exploring human endometrial receptivity.