The Human Embryo-Endometrial Dialogue: Impact of a Single Blastocyst in the Gene Expression Pattern of Endometrial Epithelial Cells

Abstract

Functional genomics of human endometrial receptivity is the next step for the understanding of the molecular mechanisms involved in the cross-talk between embryo and endometrial epithelium. In the present work, we have investigated the gene expression profile induced in the human endometrial epithelium by the presence of a human blastocyst using a wide genome approach. Primary human endometrial epithelial cells (hEEC) cultures were isolated and cultured from endometrial samples according to our published protocol (Simón et al., 1994). HEEC were used in our co-culture system as previously reported (Mercader et al., 1993). HEEC were analyzed in two different conditions: cells that have been cultured in the presence or in the absence of single blastocysts during the last 24 hours before the transfer. Only co-cultures with high score blastocyst (A/A) were selected for this study. Genomic analysis of endometrial epithelial cells was performed using the GSp_HMN1 microarray from the University of Cambridge containing more than 9,000 genes. After the embryo was transferred, conditioned media was removed, cells were collected from a total of 30 wells (15 without blastocyst and 15 with blastocyst) from 8 different patients. They were washed with cold PBS1X and collected with 300μl of TRIzol per well (Life Technologies, Inc., USA) for RNA isolation. The generation of the amplified labelled cDNA targets and the chip hybridization was performed as described (Petadilis et al., 2003). Results were validated by Taqman and Quantitative-PCR. From the 15 comparisons performed (with/without single blastocyst) 6 were not considered for the final analysis for the following reasons: RNA was degraded (n=2), not response to the embryos (n=4). The remaining 9 samples responded consistently to the presence of a blastocyst. The presence of the blastocyst induces significant changes in the gene expression pattern of the endometrial epithelial cells in culture. We found almost one hundred genes up-regulated 1.75 fold-change or more in the presence of a single blastocyst and it was statistically significant by t test p≤0.05. Furthermore, most of these genes never have been identified in studies of endometrial receptivity and they do not belong to the classic window of implantation genes. The molecular cross-communication between blastocyst and endometrial epithelial cells is one of the less-known aspects in endometrial receptivity and embryonic implantation. This work reveals, for the first time, that a single blastocyst is able to induce significant changes in the gene expression profile of the endometrial epithelial cells in the co-culture system. Furthermore, this new approach identified a cluster of genes that are directly involved in the response of the epithelial cells to the embryonic signals giving us information about the local effect that the embryo can induces in the implantation area.