Dissecting the First Transcriptional Divergence During Human Embryonic Development

The trophoblast cell lineage is specified early at the blastocyst stage, leading to the emergence of the trophectoderm and the pluripotent cells of the inner cell mass. Using a double mRNA amplification technique and a comparison with transcriptome data on pluripotent stem cells, placenta, germinal and adult tissues, we report here some essential molecular features of the human mural trophectoderm. In addition to genes known for their role in placenta (CGA, PGF, ALPPL2 and ABCG2), human trophectoderm also strongly expressed Laminins, such as LAMA1, and the GAGE Cancer/Testis genes. The very high level of ABCG2 expression in trophectoderm, 7.9-fold higher than in placenta, suggests a major role of this gene in shielding the very early embryo from xenobiotics. Several genes, including CCKBR and DNMT3L, were specifically up-regulated only in trophectoderm, indicating that the trophoblast cell lineage shares with the germinal lineage a transient burst of DNMT3L expression. A trophectoderm core transcriptional regulatory circuitry formed by 13 tightly interconnected transcription factors (CEBPA, GATA2, GATA3, GCM1, KLF5, MAFK, MSX2, MXD1, PPARD, PPARG, PPP1R13L, TFAP2C and TP63), was found to be induced in trophectoderm and maintained in placenta. The induction of this network could be recapitulated in an in vitro trophoblast differentiation model

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

Pertinence of Apoptosis Markers for the Improvement of In Vitro Fertilization (IVF).: Apoptotic markers in ART

International audienceIn assisted reproductive technology (ART), the pregnancy and birth rates following in vitro fertilization (IVF) attempts are still low. Recently, apoptotic markers have been suggested as new criteria for oocyte and embryo quality selection. Many studies have provided evidence that poor oocyte and embryo quality can be associated with apoptosis. The aim of this review is to summarize our current knowledge on the apoptotic process in oocytes and embryos, and focus on the possibility for using apoptotic markers as a reliable and predictive marker to select competent oocytes and embryos during IVF. Moreover, it is currently accepted that IVF failures, linked to poor embryo quality, are, in part, associated with suboptimal in vitro culture conditions. Here, we also review the current state of knowledge concerning how the genetic control of apoptosis during folliculogenesis and pre-implantation embryonic development is affected by in vitro culture conditions during IVF. In the future, identification of apoptotic markers in ART for oocyte and embryo selection should result in the development of new agonistic or antagonistic molecules of apoptosis by medicinal chemistry

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

International audienceBACKGROUND: 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

Endometrial miRNome profile according to the receptivity status and implantation failure

International audienceThis is a retrospective study to evaluate if the miRNome profile of endometrium samples collected during the implantation window predicts Assisted Reproduction Technology (ART) outcomes. We first investigated the endometrial miRNome profile according to the receptivity status in 20 patients with repeated implantation failures (RIF) (discovery cohort). After customized embryo transfer, the miRNome profiles of receptive patients with a positive or negative β-hCG, and with early miscarriage or live birth were analysed. Some differentially expressed miRNAs were selected for validation by RT-qPCR in endometrial samples from 103 RIF patients (validation cohort). Analysis of the different miRNome profiles identified endometrial receptivity, implantation failure, and early miscarriage-associated miRNA signatures that included 11, 261, and 76 miRNAs, respectively. However, only four miRNAs associated with the endometrial receptivity status (miR-455-3p and miR-4423-3p) and implantation failure (miR-152-3p and miR-155-5p) were significantly validated in endometrial samples. The miRNome profile of endometrial tissues during the implantation window can predict the pregnancy outcome. These data are crucial for opening new perspectives to predict implantation failure and consequently, to increase ART success

Comparative Gene Expression Profiling in Human Cumulus Cells according to Ovarian Gonadotropin Treatments

In in vitro fertilization cycles, both HP-hMG and rFSH gonadotropin treatments are widely used to control human follicle development. The objectives of this study are (i) to characterize and compare gene expression profiles in cumulus cells (CCs) of periovulatory follicles obtained from patients stimulated with HP-hMG or rFSH in a GnRH antagonist cycle and (ii) to examine their relationship with in vitro embryo development, using Human Genome U133 Plus 2.0 microarrays. Genes that were upregulated in HP-hMG-treated CCs are involved in lipid metabolism (GM2A) and cell-to-cell interactions (GJA5). Conversely, genes upregulated in rFSH-treated CCs are implicated in cell assembly and organization (COL1A1 and COL3A1). Interestingly, some genes specific to each gonadotropin treatment (NPY1R and GM2A for HP-hMG; GREM1 and OSBPL6 for rFSH) were associated with day 3 embryo quality and blastocyst grade at day 5, while others (STC2 and PTX3) were related to in vitro embryo quality in both gonadotropin treatments. These genes may prove valuable as biomarkers of in vitro embryo quality

Coprs inactivation leads to a derepression of LINE1 transposons in spermatocytes

International audienceRepression of retrotransposons is essential for genome integrity during germ cell development and is tightly controlled through epigenetic mechanisms. In primordial germ cells, protein arginine N-methyltransferase (Prmt5) is involved in retrotransposon repression by methylating Piwi proteins , which is part of the piRNA pathway. Here, we show that in mice, genetic inactivation of coprs (which is highly expressed in testis and encodes a histone-binding protein required for the targeting of Prmt5 activity) affects the maturation of spermatogonia to spermatids. Mass spectrom-etry analysis revealed the presence of Miwi in testis protein lysates immunoprecipitated with an anti-Coprs antibody. The observed deregulation of Miwi and pachytene pre-piRNAs levels and the derepression of LINE1 repetitive sequences observed in coprs-/-mice suggest that Coprs is implicated in genome surveillance mechanisms. Spermatogenesis is a multistep process that takes place in the seminiferous tubules whereby mature spermato-zoa are continuously produced during adulthood reproduction lifetime. Specifically, primordial germ cells (PGCs) generate spermatogonia that either self-renew to maintain a pool of stem cells or undergo differentiation. After two meiotic divisions, secondary spermato-cytes are produced. Then, during spermiogenesis, they differentiate into elongating spermatids and finally spermatozoa [1-5]. Importantly, these changes are associated with chromatin compaction, transcription arrest, and specific epigenetic modifications that contribute to the production of mature sperm. Concomi-tantly, to maintain genome integrity, PGCs need to repress repetitive DNA elements because their spread can lead to inherited diseases. These repetitive sequences [for instance, long interspersed nuclear elements 1 (LINEs), short interspersed nuclear element (SINEs), and intracisternal A-particle (IAP) sequences] derive mostly from transposable elements and represent Abbreviations Coprs, coordinator of Prmt5 and differentiation stimulator; IAP, intracisternal A-particle; LINE1, long interspersed nuclear element 1; piRNA, Piwi-interacting RNA; Prmt5, protein arginine N-methyltransferase; SINE, short interspersed nuclear element. 159 FEBS Open Bio 9 (2019) 159-168

Controlled Ovarian Hyperstimulation for In Vitro Fertilization Alters Endometrial Receptivity in Humans: Protocol Effects1

International audienceThe impact of gonadotropin-releasing hormone (GnRH) agonist long compared with GnRH antagonist protocols, under in vitro fertilization conditions on endometrial receptivity, is still debated. Therefore, we compared the effect of both GnRH antagonist and agonist long protocols on the endometrial receptivity by analyzing, to our knowledge for the first time, the global gene expression profile shift during the prereceptive and receptive stages of stimulated cycles under the two GnRH analogue protocols compared with natural cycles in the same patients. For the same normal-responder patients, endometrial biopsies were collected on the day of oocyte retrieval and on the day of embryo transfer after human chorionic gonadotropin administration of a stimulated cycle with either GnRH agonist long or GnRH antagonist protocols and compared with the prereceptive and receptive stages of a natural cycle. Samples were analyzed using DNA microarrays. Gene expression profiles and biological pathways involved during the prereceptive stage to the receptive endometrial transition of stimulated and natural cycles were analyzed and compared for each patient. Both protocols affect endometrial receptivity in comparison with their natural cycle in the same patients. Major differences in endometrial chemokines and growth factors under stimulated cycles in comparison with natural cycles were observed. Such an effect has been associated with gene expression alterations of endometrial receptivity. However, the endometrial receptivity under the GnRH antagonist protocol was more similar to the natural cycle receptivity than that under the GnRH agonist protocol

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

International audienceBACKGROUND: 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