P-274 Development of peri-implantation embryo culture method using endometrial cells

Study question There is no culture method that can analyze embryo-endometrium interactions during peri-implantation embryo development. Summary answer We developed a culture method that can analyze the roles of endometrial cells (ECs) during peri-implantation embryo development in mice. What is known already To better understand the mechanisms of peri-implantation embryo development, several methods of culturing embryo have been developed. Embryo culture methods utilizing hydrogels or extracellular matrix components can analyze embryonic development from blastocyst to egg cylinder stage. However, these methods cannot investigate embryo-endometrium interactions. While embryo culture methods with ECs allow the study of embryo-endometrium interactions, such as attachment and invasion, no embryo culture method with ECs supports embryo development from blastocyst to egg cylinder stage. To evaluate peri-implantation embryo-endometrium interactions, a culture method capable of analyzing peri-implantation embryo development needs to be developed. Study design, size, duration First, the ECs used for co-culturing with embryo were selected. A mouse embryo collected from the uterus was cultured with spheroid derived from mouse ECs (EC spheroid) for 24–72 hours. The embryo’s developmental stage and the expression of the implantation marker Cox2 in ECs were analyzed by immunofluorescence staining. Furthermore, to analyze the effects of ECs on peri-implantation embryo development, we developed an embryo culture method combined with a gene expression regulation system. Participants/materials, setting, methods ECs were isolated from 3.5 dpc uteri or from uteri at each stage of estrous cycle. ECs were cultured and their decidualization potential was assessed by immunofluorescence staining. A 4.5 dpc embryo was co-cultured on an EC spheroid or in the hole of an EC spheroid. For the gene expression regulation system, fluorescent-labelled siRNA and several transfection reagents were used. The transfection efficiency was evaluated by flow cytometry. Main results and the role of chance To select endometrial cells for co-culture, the characteristics of ECs at each stage were compared. 3.5 dpc ECs showed superior spheroid forming capacity and decidualization characterized by nuclear enlargement and prolactin expression. An embryo cultured on 3.5 dpc EC spheroid attached to surface of the spheroid after 24 hours. The attached embryo did not grow after 48 hours. Next, prior to co-culturing, a hole was made in the 3.5 dpc EC spheroid, and an embryo was cultured in the hole. After 72 hours, the embryo was developed into egg cylinder stage in the EC spheroid. The developed embryo had Reichert’s membrane. Furthermore, expression of Cox2 (implantation marker) was observed around the implantation site. In addition, we established a method for transient gene expression regulation in ECs using siRNA and transfection reagent. The transfection efficiency was more than 90% without the loss of their spheroid-forming ability. In this study, we developed a co-culture method that can analyze the role of ECs during peri-implantation embryo development in mice. Limitations, reasons for caution In this method, an embryo was developed in EC spheroid, making it difficult to observe the developmental process using time-lapse imaging. The embryo attaches to the endometrial epithelium during implantation. The interactions between epithelial cells and embryo are not possible in this study because EC spheroid lack the epithelial layer. Wider implications of the findings Our culture method enables analysis of the effects of ECs on peri-implantation embryo development and serves as a valuable tool for elucidating the mechanisms of implantation. Furthermore, it would be a screening platform for developing therapeutic agents to treat implantation failure, thereby contributing to drug discovery. Trial registration number No