S. Assou

Gene expression profile of human endometrial receptivity: comparison between natural and stimulated cycles for the same patients

BACKGROUND The adjunction of exogenous hormones for controlled ovarian stimulation (COS) may alter endometrial receptiveness. In order to identify the genes misregulated under COS, we compared the endometrium gene expression profiles, from the same patients, in a natural cycle and in a subsequent COS cycle. METHODS For the same normal-responder patients (n = 21), endometrial biopsies (n = 84) were collected during the pre-receptive (LH + 2) and receptive stages (LH + 7) of a natural cycle and, subsequently, on oocyte retrieval day (hCG + 2) and on transfer day (hCG + 5) of a stimulated cycle. Samples were analyzed using DNA microarrays. Gene expression profiles and biological pathways involved in endometrial receptivity were analyzed. RESULTS Although endometrium transition profiles from pre-receptive to receptive phases are similar between patients, COS regimens alter endometrial receptivity in comparison with natural cycle. Under COS conditions, two endometrial profiles were identified and were associated either with a moderately altered receptivity profile for the majority of the patients or a strongly altered profile for a sub-category of patients. The receptive endometrium transcription profile under COS was defective for biological functions such as TGFbeta signaling, leukocyte transendothelial migration and the cell cycle. CONCLUSIONS Gonadotrophin treatments in COS cycles led to disruptions of the transcriptional activation of genes involved in normal endometrial receptivity. We propose that when the receptiveness of the endometrium is seriously compromised by the COS protocol, fresh embryo replacement should be cancelled, the embryo frozen and thawed embryo replacement should be performed under natural cycles.

Dynamic changes in gene expression during human early embryo development: from fundamental aspects to clinical applications

BACKGROUND The first week of human embryonic development comprises a series of events that change highly specialized germ cells into undifferentiated human embryonic stem cells (hESCs) that display an extraordinarily broad developmental potential. The understanding of these events is crucial to the improvement of the success rate of in vitro fertilization. With the emergence of new technologies such as Omics, the gene expression profiling of human oocytes, embryos and hESCs has been performed and generated a flood of data related to the molecular signature of early embryo development. METHODS In order to understand the complex genetic network that controls the first week of embryo development, we performed a systematic review and study of this issue. We performed a literature search using PubMed and EMBASE to identify all relevant studies published as original articles in English up to March 2010 (n = 165). We also analyzed the transcriptome of human oocytes, embryos and hESCs. RESULTS Distinct sets of genes were revealed by comparing the expression profiles of oocytes, embryos on Day 3 and hESCs, which are associated with totipotency, pluripotency and reprogramming properties, respectively. Known components of two signaling pathways (WNT and transforming growth factor-β) were linked to oocyte maturation and early embryonic development. CONCLUSIONS Omics analysis provides tools for understanding the molecular mechanisms and signaling pathways controlling early embryonic development. Furthermore, we discuss the clinical relevance of using a non-invasive molecular approach to embryo selection for the single-embryo transfer program.

Slow freezing and vitrification differentially modify the gene expression profile of human metaphase II oocytes.

BACKGROUND Cryopreservation is now considered as an efficient way to store human oocytes to preserve fertility. However, little is known about the effects of this technology on oocyte gene expression. The aim of this study was to examine the effect of the two cryopreservation procedures, slow freezing and vitrification, on the gene expression profile of human metaphase II (MII) oocytes. METHODS Unfertilized MII oocytes following ICSI failure were cryopreserved either by slow freezing or by the Cryotip method for vitrification. After thawing, total RNA was extracted and analyzed using Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays. The gene expression profiles and associated biological pathways in slowly frozen/thawed and vitrified MII oocytes were determined and compared with those of non-cryopreserved MII oocytes used as controls. RESULTS Both cryopreservation procedures negatively affected the gene expression profile of human MII oocytes in comparison with controls. However, slowly frozen and vitrified MI oocytes displayed specific gene expression signatures. Slow freezing was associated with down-regulation of genes involved in chromosomal structure maintenance (KIF2C and KIF3A) and cell cycle regulation (CHEK2 and CDKN1B) that may lead to a reduction in the oocyte developmental competence. In vitrified oocytes, many genes of the ubiquitination pathway were down-regulated, including members of the ubiquitin-specific peptidase family and subunits of the 26S proteasome. Such inhibition of the degradation machinery might stabilize the maternal protein content that is necessary for oocyte developmental competence. CONCLUSIONS The low pregnancy rates commonly observed when using human MII oocytes after slow freezing-thawing may be explained by the alterations of the oocyte gene expression profile.

MicroRNAs: new candidates for the regulation of the human cumulus–oocyte complex

STUDY QUESTION What is the expression pattern of microRNAs (miRNAs) in human cumulus-oocyte complexes (COCs)? SUMMARY ANSWER Several miRNAs are enriched in cumulus cells (CCs) or oocytes, and are predicted to target genes involved in biological functions of the COC. WHAT IS KNOWN ALREADY The transcriptional profiles of human MII oocytes and the surrounding CCs are known. However, very limited data are available about post-transcriptional regulators, such as miRNAs. This is the first study focussing on the identification and quantification of small RNAs, including miRNAs, in human oocytes and CCs using a deep-sequencing approach. STUDY DESIGN, SIZE, DURATION MII oocytes and CCs were collected from women who underwent IVF. PARTICIPANTS/MATERIALS, SETTING, METHODS Using the Illumina/deep-sequencing technology, we analyzed the small RNAome of pooled MII oocytes (n = 24) and CC samples (n = 20). The mRNA targets of CC and MII oocyte miRNAs were identified using in silico prediction algorithms. Using oligonucleotide microarrays, genome-wide gene expression was studied in oocytes (10 pools of 19 ± 3 oocytes/each) and 10 individual CC samples. TaqMan miRNA assays were used to confirm the sequencing results in independent pools of MII oocytes (3 pools of 8 ± 3 oocytes/each) and CC samples (3 pools of 7 ± 3 CCs/each). The functional role of one miRNA, MIR23a, was assessed in primary cultures of human CCs. MAIN RESULTS AND THE ROLE OF CHANCE Deep sequencing of small RNAs yielded more than 1 million raw reads. By mapping reads with a single location to the human genome, known miRNAs that were abundant in MII oocytes (MIR184, MIR100 and MIR10A) or CCs (MIR29a, MIR30d, MIR21, MIR93, MIR320a, MIR125a and the LET7 family) were identified. Predicted target genes of the oocyte miRNAs were associated with the regulation of transcription and cell cycle, whereas genes targeted by CC miRNAs were involved in extracellular matrix and apoptosis. Comparison of the predicted miRNA target genes and mRNA microarray data resulted in a list of 224 target genes that were differentially expressed in MII oocytes and CCs, including PTGS2, CTGF and BMPR1B that are important for cumulus-oocyte communication. Functional analysis using primary CC cultures revealed that BCL2 and CYP19A1 mRNA levels were decreased upon MIR23a overexpression. LIMITATIONS, REASONS FOR CAUTION Only known miRNAs were investigated in the present study on COCs. Moreover, the source of the material is MII oocytes that failed to fertilize. WIDER IMPLICATIONS OF THE FINDINGS The present findings suggest that miRNA could play a role in the regulation of the oocyte and CC crosstalk. STUDY FUNDING/COMPETING INTEREST(S) This work was partially supported by a grant from Ferring Pharmaceuticals. The authors of the study have no conflict of interest to report. TRIAL REGISTRATION NUMBER Not applicable.

Human Cumulus Cells Molecular Signature in Relation to Oocyte Nuclear Maturity Stage

The bi-directional communication between the oocyte and the surrounding cumulus cells (CCs) is crucial for the acquisition of oocyte competence. We investigated the transcriptomic profile of human CCs isolated from mature and immature oocytes under stimulated cycle. We used human Genome U133 Plus 2.0 microarrays to perform an extensive analysis of the genes expressed in human CCs obtained from patients undergoing intra-cytoplasmic sperm injection. CC samples were isolated from oocyte at germinal vesicle, stage metaphase I and stage metaphase II. For microarray analysis, we used eight chips for each CC category. Significance analysis of microarray multiclass was used to analyze the microarray data. Validation was performed by RT-qPCR using an independent cohort of CC samples. We identified differentially over-expressed genes between the three CC categories. This study revealed a specific signature of gene expression in CCs issued from MII oocyte compared with germinal vesicle and metaphase I. The CC gene expression profile, which is specific of MII mature oocyte, can be useful as predictors of oocyte quality.

Transcriptome analysis reveals dialogues between human trophectoderm and endometrial cells during the implantation period

BACKGROUND Crosstalk between human trophectoderm (TE) and endometrial cells during the implantation window is a complex and not well-understood process. The aims of this study were (i) to evaluate the global gene expression profile in TE cells from Day 5 human blastocysts issued from IVF, (ii) to compare these data with the transcriptomic profile of endometrial cells in stimulated cycles for IVF and (iii) to identify potential early dialogues between maternal and embryonic cells during the implantation window. METHODS Endometrial biopsies (n = 18) from normal responder patients were performed on the day of embryo transfer (Day 5 after human chorionic gonadotrophin administration). TE biopsies from five blastocysts donated for research purposes were mechanically extracted. DNA microarray analysis was carried out to identify the specific gene expression profiles and the biological pathways activated during the implantation window in endometrial and TE cells. RESULTS Several cytokines (such as PDGFA, placenta growth factor, IGF2BP1 and IGF2BP3) were up-regulated in human TE cells, whereas some of the corresponding receptors (PDGFRA and KDR) were over-expressed in the receptive endometrium, suggesting that these molecules are involved in the early dialogue between blastocyst and maternal endometrial cells. In addition, several adhesion molecules and extracellular matrix proteins (MCAM, ITGAE and LAMA1) were also over-expressed in the TE, while others (ALCAM, CEACAM1, PECAM1, ITGB8 and LAMA2) were restricted to the receptive endometrium. CONCLUSION The present study shows that several growth factors, cytokines, integrins and adhesion molecules are expressed in the TE and endometrium at the time of implantation. These results could contribute to the understanding of the mechanisms involved in the early dialogue between blastocyst and endometrium during implantation. Such results should be confirmed by further studies.

Insights into human endometrial receptivity from transcriptomic and proteomic data

The appreciation of endometrial receptivity is a crucial step in assisted reproductive technology as implantation failures are thought to result, in large part, from abnormal endometrial receptivity. Using emerging omics technologies, investigators have begun to define both molecular signatures and specific biomarkers of receptive endometrium. The aim of this review was to analyse the new perspectives brought to the appreciation of endometrial receptivity by transcriptomic and proteomic technologies, involving the analysis of gene- or protein-expression-profile shifts between the pre-receptive and receptive secretory stages and how they might lead to new strategies for endometrial receptivity assessments. The use of omics as molecular tools to determine the effects of stimulation protocols on endometrial gene expression and clinical outcomes has also been investigated.

Altered gene expression profile in cumulus cells of mature MII oocytes from patients with polycystic ovary syndrome

STUDY QUESTION Oocyte developmental competence is altered in patients with polycystic ovary syndrome (PCOS); is gene expression in cumulus cells (CCs) from mature metaphase II oocytes of patients with PCOS altered as well? SUMMARY ANSWER Compared with CCs from non-PCOS patients, the gene expression profile of CCs isolated from mature oocytes of patients with PCOS present alterations that could explain the abnormal folliculogenesis and reduced oocyte competence in such patients. WHAT IS KNOWN ALREADY Abnormal mRNA expression of several members of the insulin-like growth factor (IGF) family in CCs from PCOS patients was previously reported. Moreover, the whole transcriptome has been investigated in cultured CCs from PCOS patients. STUDY DESIGN, SIZE AND DURATION This retrospective study included six PCOS patients diagnosed following the Rotterdam Criteria and six non-PCOS patients who all underwent ICSI for male infertility in the assisted reproduction technique (ART) Department of Montpellier University Hospital, between 2009 and 2011. PARTICIPANTS/MATERIALS, SETTING AND METHODS CCs from PCOS and non-PCOS patients who underwent controlled ovarian stimulation (COS) were isolated mechanically before ICSI. Gene expression profiles were analysed using the microarray technology and the Significance Analysis of Microarray was applied to compare the expression profiles of CCs from PCOS and non-PCOS patients. MAIN RESULTS The gene expression profile of CCs from patients with PCOS was significantly different from that of CCs from non-PCOS patients. Specifically, CCs from women with PCOS were characterized by abnormal expression of many growth factors, including members of the epidermal growth factor-like (EGFR, EREG and AREG) and IGF-like families (IGF1R, IGF2R, IGF2BP2 and IGFBP2), that are known to play a role in oocyte competence. In addition, mRNA transcripts of factors involved in steroid metabolism, such as CYP11A1, CYP1B1, CYP19A1 and CYP2B7P1, were deregulated in PCOS CCs, and this could explain the abnormal steroidogenesis observed in these women. Functional annotation of the differentially expressed genes suggests that defects in the transforming growth factor β and estrogen receptors signalling cascades may contribute to the reduced oocyte developmental competence in patients with PCOS. LIMITATIONS AND REASONS FOR CAUTION Owing to the strict selection criteria (similar age, weight and reasons for ART), this study included a small sample size (six cases and six controls), and thus, further investigations using a large cohort of patients are needed to confirm these results. WIDER IMPLICATIONS OF THE FINDINGS This study opens a new perspective for understanding the pathogenesis of PCOS. STUDY FUNDING/COMPETING INTERESTS This work was partially supported by a grant from the Ferring Pharmaceutical. The authors of the study have no competing interests to report. TRIAL REGISTRATION NUMBER Not applicable.

Cell-free nucleic acids as non-invasive biomarkers of gynecological cancers, ovarian, endometrial and obstetric disorders and fetal aneuploidy

BACKGROUND Proper folliculogenesis is fundamental to obtain a competent oocyte that, once fertilized, can support the acquisition of embryo developmental competence and pregnancy. MicroRNAs (miRNAs) are crucial regulators of folliculogenesis, which are expressed in the cumulus-oocyte complex and in granulosa cells and some can also be found in the bloodstream. These circulating miRNAs are intensively studied and used as diagnostic/prognostic markers of many diseases, including gynecological and pregnancy disorders. In addition, serum contains small amounts of cell-free DNA (cfDNA), presumably resulting from the release of genetic material from apoptotic/necrotic cells. The quantification of nucleic acids in serum samples could be used as a diagnostic tool for female infertility. METHODS An overview of the published literature on miRNAs, and particularly on the use of circulating miRNAs and cfDNA as non-invasive biomarkers of gynecological diseases, was performed (up to January 2014). RESULTS In the past decade, cell-free nucleic acids have been studied for potential use as biomarkers in many diseases, particularly in gynecological cancers, ovarian and endometrial disorders, as well as in pregnancy-related pathologies and fetal aneuploidy. The data strongly suggest that the concentration of cell-free nucleic acids in serum from IVF patients or in embryo culture medium could be related to the ovarian hormone status and embryo quality, respectively, and be used as a non-invasive biomarker of IVF outcome. CONCLUSIONS The profiling of circulating nucleic acids, such as miRNAs and cfDNA, opens new perspectives for the diagnosis/prognosis of ovarian disorders and for the prediction of IVF outcomes, namely (embryo quality and pregnancy).

Transcriptome analysis during human trophectoderm specification suggests new roles of metabolic and epigenetic genes.

In humans, successful pregnancy depends on a cascade of dynamic events during early embryonic development. Unfortunately, molecular data on these critical events is scarce. To improve our understanding of the molecular mechanisms that govern the specification/development of the trophoblast cell lineage, the transcriptome of human trophectoderm (TE) cells from day 5 blastocysts was compared to that of single day 3 embryos from our in vitro fertilization program by using Human Genome U133 Plus 2.0 microarrays. Some of the microarray data were validated by quantitative RT-PCR. The TE molecular signature included 2,196 transcripts, among which were genes already known to be TE-specific (GATA2, GATA3 and GCM1) but also genes involved in trophoblast invasion (MUC15), chromatin remodeling (specifically the DNA methyltransferase DNMT3L) and steroid metabolism (HSD3B1, HSD17B1 and FDX1). In day 3 human embryos 1,714 transcripts were specifically up-regulated. Besides stemness genes such as NANOG and DPPA2, this signature included genes belonging to the NLR family (NALP4, 5, 9, 11 and 13), Ret finger protein-like family (RFPL1, 2 and 3), Melanoma Antigen family (MAGEA1, 2, 3, 5, 6 and 12) and previously unreported transcripts, such as MBD3L2 and ZSCAN4. This study provides a comprehensive outlook of the genes that are expressed during the initial embryo-trophectoderm transition in humans. Further understanding of the biological functions of the key genes involved in steroidogenesis and epigenetic regulation of transcription that are up-regulated in TE cells may clarify their contribution to TE specification and might also provide new biomarkers for the selection of viable and competent blastocysts.