Establishment of an efficient method to quantify embryo attachment to endometrial epithelial cell monolayers

During implantation, complex embryo-endometrium interactions result in blastocyst adhesion. To study the mechanisms of implantation, an effective assay for monitoring adhesiveness between embryos and endometrial epithelium is essential. In this study, we describe a simple and reliable method to quantify embryo-endometrium adhesion in vitro. Murine blastocysts or BeWo trophoblast spheroids were cocultured with monolayers of RL95-2 endometrial epithelial cells (EEC) grown in 96-well plates. At the end of coculture, the wells were filled with medium, and the plate was sealed with an adhesive film, inverted, and centrifuged at 25 x g for 5 min. After centrifugation, the plate was kept inverted and directly examined microscopically to determine whether the blastocysts or spheroids were attached to EEC monolayers. Our assay demonstrated that blastocysts recovered at 1200-1400 h on d 4 were more adherent to EEC than those recovered earlier, consistent with the timing of intrauterine embryo activation. Serum also enhanced blastocyst-EEC adhesion. Spheroid-EEC adhesion was inhibited by blocking Ca(2+) influx with extracellular Ca(2+) chelators (ethylenediaminetetraacetic acid or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) or a Ca(2+) channel blocker (verapamil) but not by interfering with Ca(2+) release from intracellular stores using chelating (1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or depleting (thapsigargin) agents. Using 96-well plates for coculture, centrifugation, and examination to minimize transfer procedures, our assay system is readily applicable to investigate implantation mechanisms.