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Implantation of the human embryo requires Rac1-dependent endometrial stromal cell migration.

ABSTRACT: Failure of the human embryo to implant into the uterine wall during the early stages of pregnancy is a major cause of infertility. Implantation involves embryo apposition and adhesion to the endometrial epithelium followed by penetration through the epithelium and invasion of the embryonic trophoblast through the endometrial stroma. Although gene-knockdown studies have highlighted several molecules that are important for implantation in the mouse, the molecular mechanisms controlling implantation in the human are unknown. Here, we demonstrate in an in vitro model for human implantation that the Rho GTPases Rac1 and RhoA in human endometrial stromal cells modulate invasion of the human embryo through the endometrial stroma. We show that knockdown of Rac1 expression in human endometrial stromal cells inhibits human embryonic trophoblast invasion into stromal cell monolayers, whereas inhibition of RhoA activity promotes embryo invasion. Furthermore, we demonstrate that Rac1 is required for human endometrial stromal cell migration and that the motility of the stromal cells increases at implantation sites. This increased motility correlates with a localized increase in Rac1 activation and a reciprocal decrease in RacGAP1 levels. These results reveal embryo-induced and localized endometrial responses that may govern implantation of the human embryo.

SUBMITTER: Grewal S

PROVIDER: S-EPMC2562412 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PURPOSEThe purpose of this paper is to study whether human preimplantation embryos regulate endometrial stromal cell (hESC) migration.METHODSPrimary hESCs were isolated from fertile patients undergoing hysterectomy for benign conditions (uterine scar niche n?=?3, dysmenorrhea n?=?2; no hormonal treatment). Migration and proliferation assays were performed by culturing decidualized or non-decidualized hESCs in the presence of embryo conditioned medium (ECM) from high-quality embryos (fragmentation ??20%) or from low-quality embryos (fragmentation >?20%) or in non-conditioned medium from the same dishes (control). ECM samples from 425 individually cultured human embryos were used in this study.RESULTSECM from high-quality embryos, i.e., with a low percentage of fragmentation, actively stimulated decidualized hESC migration (p?<?0.001). This effect was consistent throughout embryonic development from cleavage stage embryos with 2-7 cells (high quality vs. control; p?=?0.036), 8-18 cells (high quality vs. control; p?<?0.001) to morulae (high quality vs. control; p?=?0.003). Additionally, linear regression analysis showed that hESC migration was influenced by embryo quality (fragmentation, ? -?0.299; p?=?0.025) and not developmental stage (cell number, ? 0.177; p?=?0.176) or maternal age (? -?0.036; p?=?0.78). Opposite to decidualized hESCs, the migration response of non-decidualized hESCs was inhibited by ECM from high-quality embryos (p?=?0.019). ECM from low-quality embryos, i.e., with a high percentage of fragmentation, did not cause an altered migration response in decidualized hESCs (p?=?0.860) or non-decidualized hESCs (p?=?0.986). Furthermore, ECM of both high- and low-quality human embryos did not influence the number of proliferating cells (p?=?0.375) and the cell cycle time (p?=?0.297) of non-decidualized or decidualized hESCs.CONCLUSIONThis study reveals a mechanism by which high-quality human preimplantation embryos actively interact with the endometrium to increase their chances of successful implantation.

Project description:PurposeDuring early embryo invasion (48 h after embryo attachment), what functional changes accompany dynamic gene expression alterations in human endometrial stromal cells?MethodIn the present study, primary human endometrial stromal cells (phESCs) were cultured. After in vitro decidualization, primary human endometrial stromal cells (phESCs) were cultured with blastocysts for 48 h. During this process, blastocysts attached and invaded the phESCs (embryo-invaded primary human endometrial stromal cells, ehESCs). We performed comprehensive transcriptomic profiling of phESCs (two replicates) and ehESCs (five replicates) and analyzed the differentially expressed gene (DEGs) sets for gene ontology (GO) terms and Kyoto encyclopaedia of genes and genomes (KEGG) pathway enrichment. To analyse potential connectivity patterns between the transcripts in these DEG sets, a protein-protein interaction (PPI) network was constructed using the STRING database.ResultsA total of 592 DEGs were identified between phESCs and ehESCs after embryo invasion. Primary human endometrial stromal cells underwent significant transcriptomic changes that occur in a stepwise fashion. Oxidative phosphorylation, mitochondrial organization, and P53 signalling pathways were significantly altered in phESCs after embryo invasion. EP300 may play a key role in regulating transcription via chromatin remodelling to facilitate the adaptive gene expression changes that occur during embryo invasion.ConclusionsOur data identify dynamic transcriptome changes that occur in endometrial stromal cells within 48 h after embryo invasion. The pathways that we found to be enriched in phESCs after embryo invasion (oxidative phosphorylation, mitochondrial organization, and P53 signalling) may represent novel mechanisms underlying embryo implantation, and may illuminate the reasons that some women experience reproductive failure.Key messagesHuman endometrial stromal cells have undergone changes in gene expression regulation and signalling pathways during the embryo invasion.Mitochondrial-oxidative phosphorylation changes in human stromal cells manifested as down-regulation of gene expression in the electron transport chain.TP53 signalling pathway and transcriptional regulator EP300 assist stromal cells to get adaptive changes during embryo invasion phase.

Project description:Objective:This experimental study aimed to evaluate the effects of 17?-estradiol (E2) and progesterone (P4) on the interaction between mouse embryo and human endometrial mesenchymal stromal cells, and gene expressions related to implantation [?V and ?3 integrins, interleukin-1 receptor (IL-1R), and leukemia inhibitory factor receptor (LIFR)] using an in vitro twodimensional model. Materials and Methods:In this experimental study, the endometrial stromal cells were isolated enzymatically and mechanically, and cultured to the fourth passage. Next, their immunophenotype was confirmed by flow cytometric analysis as mesenchymal stromal cells. The cells were cultured as either the experimental group in the presence of E2 (0.3 nmol) and P4 (63.5 nmol) or control group without any hormone treatment. Mouse blastocysts were co-cultured with endometrial mesenchymal stromal cells in both groups for 48 hours. Their interaction was assessed under an inverted microscope and scanning electron microscopy (SEM). Expressions of ?V and ?3 integrins, LIFR, and IL-1R genes were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR). Results:Similar observations were seen in both groups by light microscopy and SEM. We observed the presence of pinopode-like structures and cell secretions on the apical surfaces of endometrial mesenchymal stromal cells in both groups. The trophoblastic cells expanded and interacted with the mesenchymal monolayer cells. At the molecular level, expression of IL-1R significantly increased in the hormonal treated group compared to the control (P?0.05). Expressions of the other genes did not differ. Conclusion:This study has shown that co-culture of endometrial mesenchymal stromal cells with mouse embryo in media that contained E2 (0.3 nmol) and P4 (63.5 nmol) could effectively increase the expression of IL-1R, which is involved in embryo implantation. However, there were no significant effects on expressions of ?V and ?3 integrins, LIFR, and on the morphology and ultrastructure of endometrial mesenchymal stromal cells.

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Implantation of the human embryo requires Rac1-dependent endometrial stromal cell migration.

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