Screening and characterization of pharmacologically active compounds regulating embryo implantation in vitro and in vivo

Abstract

Abstract of the thesis entitled SCREENING AND CHARACTERIZATION OF PHARMACOLOGICALLY ACTIVE COMPOUNDS REGULATING EMBRYO IMPLANTATION IN VITRO AND IN VIVO Submitted by CHEN Xian for the degree of Doctor of Philosophy at The University of Hong Kong in March 2022 More than 25% of pregnancies are unintended. Current approaches to prevent pregnancy include physical or hormonal methods by inhibiting folliculogenesis, fertilization and embryo implantation. Hormonal contraceptive pills are widely used to avoid ovulation but are less effective once ovulation occurs. Other non-hormonal compounds that suppress embryo implantation can be potentially used for contraceptives are less explored. This study therefore aimed (1) to identify compounds that modulate spheroid attachment by screening the Library of Pharmacologically Active Compounds (LOPAC) in a high-throughput attachment assay, (2) to further identify compounds that suppress spheroid attachment on different human endometrial epithelial cells, and (3) to confirm the effects of such compounds in vivo using a mouse model. A high-throughput in-vitro BeWo spheroids-Ishikawa endometrial epithelial cells co- culture model was established by quantifying fluorescent-labeled spheroids attachment in a 96-well plate format. This model was used to screen the LOPAC and identify compounds that modulate spheroid attachment. The cytotoxicity and spheroid attachment in both endometrial Ishikawa and RL95-2 cells at different concentrations of selected compounds were studied. The number of implantation sites in pregnant ICR mice was studied after transcervical delivery of the compound into one uterine horn, and compared with the contralateral side transferred with solvent (5% DMSO) which was used as the control. Moreover, quantitative PCR was used to evaluate the expression of receptivity markers in the uterine epithelium of treated mice. Of the 1280 compounds in LOPAC, 174 were identified that significantly suppressed BeWo spheroid attachment onto endometrial Ishikawa cells. Among the top 20 of these 174 compounds, Dihydroouabain (1, 3 and 10 μM), JS-K (10 μM) and Nemadipine-A (10 μM) significantly suppressed BeWo spheroid attachment in both Ishikawa and RL95-2 cells at the concentrations that are lower than their half-lethal concentrations (LC50). Dihydroouabain and Nemadipine-A also suppressed the BeWo spheroid attachment onto human primary endometrial epithelial cells collected at LH+7/8 days. A single transcervical transfer of Dihydroouabain (150 μg/kg and 15 μg/kg) or Nemadipine-A (100 μg/kg) suppressed embryo implantation sites significantly when compared with controls, while JS-K (6 μg/kg) was surprisingly found to increase implantation sites when compared with the control. The expressions of endometrial receptivity markers integrin αV (ITGAV) and mucin-1 (MUC1), but not β-catenin (CTNNB1), were significantly decreased in the uterus of ICR mice at 2.5 days post coitum (dpc) after treatment with Dihydroouabain, JS-K and Nemadipine-A. The suppression of embryo implantation by Dihydroouabain and Nemadipine-A was likely mediated through decreasing the mRNA expressions of ITGAV and MUC1.

Taken together, Dihryoouabain and Nemadipine-A suppressed spheroid attachment in vitro and embryo implantation in vivo. Detailed molecular and functional changes in mouse endometrium after treatment need to be investigated before the two compounds are used in the clinical setting. Also, the pharmacological role and doses of JS-K in regulating embryo implantation needs to be further explored. (Word count: 462)