J. De Vos

A non-invasive test for assessing embryo potential by gene expression profiles of human cumulus cells: a proof of concept study

Identification of new criteria for embryo quality is required to improve the clinical outcome of in vitro fertilization. The aim of this study was to determine the gene expression profile of cumulus cells (CC) surrounding the oocyte as biomarkers for embryo potential and to identify genes to be used as prognostic indicators of successful pregnancy. CC from single oocytes were analysed using DNA microarrays. Gene expression profiles of CC surrounding the oocyte associated with good embryonic quality and pregnancy outcome were computed. We observed that CC issued from oocytes that developed into embryos with a good morphology had differing gene expression profile according to the pregnancy outcome of the embryo. We demonstrated that the expression of BCL2L11, PCK1 and NFIB in CC is significantly correlated with embryo potential and successful pregnancy. These results were confirmed by quantitative RT-PCR. The gene expression profiling of human CC correlates with embryo potential and pregnancy outcome. BCL2L11, PCK1 and NFIB genes are proposed as biomarkers for predicting pregnancy. Our findings suggest a non-invasive approach, offering a new potential strategy for competent embryo selection. This approach should be validated in single-embryo transfer programmes.

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.

Identification of new biomarkers of human endometrial receptivity in the natural cycle

BACKGROUND Identification of new markers assessing endometrial receptivity may help in improving the clinical outcome of IVF. This study aimed at identifying genes expressed in human endometrium during the implantation window that could be used as such markers. METHODS A series of normoresponder patients (n = 31) underwent endometrial biopsies (n = 62, 2 per patient) during the early secretory phase, 2 days after the LH surge (LH + 2) and the mid-secretory phase (LH + 7) of the same natural cycle that preceded a new ICSI attempt for male infertility factor. Samples were analyzed using DNA microarrays and gene expression profiles at the time of the implantation window were computed. Systems biology analysis allowed the identification of biological pathways that were over-represented in this signature. A new approach for class prediction applied to microarray experiments was then used to identify biomarkers putatively involved in endometrial receptiveness. RESULTS Five genes expressed during the implantation window were all up-regulated in the LH + 7 samples compared with LH + 2 [laminin beta3 (P = 0.002), microfibril-associated protein 5 (P = 0.009), angiopoietin-like 1 (P = 0.005), endocrine gland-derived vascular endothelial growth factor (P = 0.049) and nuclear localized factor 2 (P = 0.007)]. Increased expression was validated by quantitative RT-PCR. CONCLUSIONS Five genes have been identified for the first time as being up-regulated during the implantation window and are proposed as new biomarkers for exploration of endometrial receptiveness. As the endometrial biopsy procedure can be performed during a natural cycle, it would be worth testing this approach as a novel strategy in patients with poor implantation after IVF or ICSI.

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.

LH/hCGR gene expression in human cumulus cells is linked to the expression of the extracellular matrix modifying gene TNFAIP6 and to serum estradiol levels on day of hCG administration

BACKGROUND Recent studies suggest a role for luteinizing hormone and human chorionic gonadotrophin receptor (LH/hCGR) signalling in the regulation of the oocyte-cumulus oophorus cell interplay. The present study aimed at assessing the LH/hCGR gene expression in cumulus cells (CCs) surrounding oocytes in patients undergoing controlled ovarian hyperstimulation (COS) before ICSI and to relate the LH/hCGR expression to other COS quality parameters. METHODS CCs from single oocytes of normal responder patients were analysed by DNA microarrays. Concomitantly, estradiol levels on the day of hCG administration, CC morphology, total collected oocyte and metaphase II oocyte number were assessed in relation to LH/hCGR gene expression in CC. RESULTS The transcriptome analysis of CC indicated a variable expression of LH/hCGR among the patients and intra-patients. LH/hCGR mRNA expression was negatively correlated with serum estradiol level on the day of hCG administration. Eighty-five genes were significantly modulated between CCs from patients with a high and a low LH/hCGR expression. These genes are involved principally in steroid metabolism and in the ovulation process and include TNFAIP6, a gene expressed during CC-oocyte complex (COC) expansion. There were no significant differences in LH/hCGR gene expression profile between COS protocols. CONCLUSIONS LH/hCGR is expressed in CC under COS conditions. LH/hCGR expression level is associated with TNFAIP6 gene expression and negatively correlated with serum estradiol level on the day of hCG administration.

Les cellules souches embryonnaires humaines: de la transgression de l'embryon humain à la médecine régénératrice de demain

Human embryonic stem cells (hESC) are derived from the inner cell mass (ICM) of the human blastocyst at day 5 or 6 of the early embryo development. These cells display two cardinal features: they are able to differentiate into cell types from many if not all human tissue (pluripotency) and they proliferate strongly and indefinitely without senescence in vitro. Therefore, hESC are a source of choice for stem cells for regenerative medicine and are a reference model to study the biology of pluripotency. Since 2004, the French law (loi de Bioéthique) authorizes hESC research under certain conditions.

Les marqueurs apoptotiques ont-ils leur place comme marqueurs potentiels de l'exploration de l'infertilité masculine ?

Apoptosis is a cell death program involved in different steps of spermatogenesis, first at puberty, at the beginning of spermatogenesis, then in adult testicles by controlling normal spermatogenesis. As a result, apoptosis deregulation can affect spermatogenesis. Many studies have provided evidence that apoptosis deregulation in germinal cells resulted in male infertility. In addition, apoptosis detection in ejaculated spermatozoa arouses a growing interest in research as a reliable marker of spermatozoon quality. The aim of this review is to summarize our knowledge on physiological apoptosis during spermatogenesis, and then analyse the possibility of using apoptotic markers as selective markers of spermatozoon quality to optimize the rate of success of in vitro fertilization.

Des cellules souches au gamète mâle : science-fiction ou avenir proche ?

In mammalian species, gametes are issued from primordial germ cells (PGC). PGC represent a small cell population, appearing during early embryo development. Gametes production is essential for fertilization process and therefore the establishment of the next generation. The gametes are different from somatic cells because they have a haploid number of chromosomes that are normally generated during the cellular division known as meiosis. The embryonic stem cells that appear at the fifth or sixth day within the blastocyst inner cellular mass are pluripotent cells. Therefore, they can differentiate into the three cellular lineages, somatic as well as germinal. It is well established that the mouse embryonic stem cells can be differentiated in vitro into primordial germinal cells. These cells enter into meiosis forming male or female gametes. In vitro production of gametes by induction from embryonic stem cells has become a very important aspect of the actual research constituting an alternative tool for infertility treatment.