Identification and characterization of pharmacologically active small molecules modulating spheroid attachment and embryo implantation

Abstract

Infertility affects millions of couples worldwide, with embryo implantation failure being one of the biggest challenges. This research aims to identify and characterize small molecules that can enhance endometrial receptivity and embryo implantation. A multi-step approach was used, including high-throughput screening of 1280 small molecules from the Library of Pharmacologically Active Compounds (LOPAC), bioinformatics analysis, and in vitro co-culture assays with non-receptive endometrial cells to identify promising compounds. RNA sequencing and quantitative PCR were applied to assess differential gene expression, focusing on pathways relevant to implantation. In vivo validation was conducted using a transcervical delivery system in a mouse model, with implantation sites marked by Chicago blue dye at specific time points to precisely evaluate implantation outcomes.

Through screening the LOPAC containing 1280 small molecules, several targets, including loxapine succinate, perphenazine, and N-oleoylethanolamine, were identified that significantly improved spheroid attachment to endometrial epithelial cells. Detailed investigations including RNA sequencing, confirmed the effectiveness of these compounds in modulating key pathways involved in immune regulation, cell adhesion, and extracellular matrix remodeling in the endometrial epithelial cells. Additionally, in vivo studies demonstrated the potential of these molecules to enhance endometrial receptivity and improve implantation outcomes in mouse, further supporting their therapeutic potential of these drugs.

Further investigations focused on JS-K, a nitric oxide (NO)-releasing prodrug, as a key candidate for enhancing endometrial receptivity. JS-K was found to release NO selectively in response to cellular glutathione levels, which improved spheroid attachment in non-receptive endometrial epithelial cells. In vitro studies showed that JS-K modulates key pathways involved in immune regulation, cell adhesion, and extracellular matrix remodeling. RNA sequencing and quantitative PCR analyses provided insights into the molecular mechanisms by which JS-K enhances endometrial receptivity. Additionally, in vivo experiments using a mouse model supported JS-K's efficacy in increasing implantation rates.

These findings suggest that these compounds, including JS-K, could be potential therapeutic agents to enhance endometrial receptivity and improve implantation outcomes. However, challenges remain in translating these findings from in vitro and animal models to human applications. Future research should focus on validating these results in human models, optimizing dosing strategies, and exploring potential synergistic effects of drugs on fertility treatments. In sum, this work provides a foundation for developing new therapeutic strategies to improve endometrial receptivity and implantation success, ultimately contributing to better fertility outcomes for patients undergoing assisted reproductive treatments.