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

Summary

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×g for 5 min. After centrifugation, the plate was kept inverted and directly examined microscopically to determine whether the blastocysts or spheroids wereaattached 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 Ca²⁺ influx with extracellular Ca²⁺ chelators (ethylenediaminetetraacetic acid or ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) or a Ca²⁺ channel blocker (verapamil) but not by interfering with Ca²⁺ 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.