Screening and characterization of molecules modulating embryo implantation using a high throughput in vitro co-culture model

Abstract

Embryo attachment is critical to the establishment of pregnancy. Whether small molecules could be used to enhance pregnancy outcome remains largely unexplored. The use of in-vitro trophoblastic spheroids (blastocyst surrogate) and Ishikawa cell (endometrial surrogate) co-culture model could enhance the screening of drugs in a laboratory setting. Here, we report the use of a high-throughput in-vitro spheroid-endometrial cells co-culture model to screen a library of pharmacologically active compound (LOPAC) containing 1280 small molecules in order to identify molecules that either enhance or suppress spheroid attachment. In this study, we used AggreWell to produce spheroids with homogenous size from trophoblast cells (BeWo) and cocultured with the Ishikawa monolayer. The spheroids were labeled with the fluorescent dye Calcein-AM. After 1 h co-culture, a linear florescent signal could be produced when 0 to 100 spheroids were seeded on Ishikawa monolayer in 96-well plates. Then we screened the LOPAC by treating the Ishikawa monolayer in duplicates at 10µM for 24h, and the attachment rates of spheroids were determined in repeated experiments. Each screening of assay contained negative control and vehicle control (0.1% DMSO). A significant different at p<0.05, one-way ANOVA was used. Until now, 320 LOPAC compounds have been screened and 6 primary hits have been found can suppress the attachment rate. In summary, this result indicates that some constituents of the LOPAC panel can modulate the implantation process. Our study will shed light on identifying potential small molecules that can enhance or suppress embryo implantation which may have great potential in the enhancement of IVF treatment or development of emergency contraception.