A study of the effect of bisphenol compounds on embryo implantation : in vitro and in vivo models

Abstract

Bisphenol A (BPA) is an organic synthetic compound commonly used to make plastics and epoxy resins that are found in the coating of food and beverage cans. With a raising concern of BPA as a weak estrogenic compound to human body endocrine function, structurally similar to BPA including BPF and BPS were developed and used in manufacturing industries. The aims of this study were to use in vitro and in vivo models to (1) compare the low-dose effect of BPA, BPF and BPS on embryo implantation using in vitro spheroid-endometrial co-culture assay, (2) study the effect on reporter gene activation and the underlying molecular changes using microarray analysis, (3) evaluate the effect of bisphenols on the outgrowth and invasion of trophoblastic cells and decidualization of stromal cells, and (4) investigate the trans-generation effect on reproductive functions using mouse model. Using in-vitro spheroid-endometrial co-culture assay, chronic low-dose (100nM for 1 month) BPA but not BPF and BPS treatment suppressed spheroid attachment when compared to DMSO control. BPA, BPF and BPS at 0.1, 1 and 1μM, respectively activated the ERE-luciferase reporter gene in the transfected Ishikawa cells. BPA at 100nM induced ERa and phospho-ERK, but suppressed ERb and Axin2 expression in the Ishikawa cells. Microarray analysis compared gene expression profiles of 1 and 3 months BPA treated Ishikawa identified a few dozen differentially expressed genes that contained estrogen responsive element (ERE) sequence at the promoter regions. Furthermore, microarray analysis of acute high dose (10μM for 24 hr) BPA, BPF and BPS treated Ishikawa confirmed upregulation of PR and other transcripts that changes could be nullified by ER antagonist (ICI 182,780) treatment. BPA at 100μM suppressed the expression of ERa and ERb in Jeg-3 cells; while BPA, BPF and BPS at 1-100μM suppressed the expression of membrane receptor GPR30 in Jeg-3 cells. BPA and BPF stimulated the outgrowth and invasion of Jeg-3 spheroids, possibly through down-regulation of TIMP1 in Jeg-3 cells. BPA, BPF and BPS did not affect endometrial stromal cell decidualization in vitro, but indirect co-culture of BPA and BPF, not PBS treated decidualized stromal cells stimulated Jeg-3 spheroid invasion, probably through dys-regulation of invasion-related transcripts (e.g. TIMP4 and PAI-1). Chronic low-dose of BPA, BPF, BPS or BPA+BPF treatment for 1 month before and during the whole gestation period, neither changed the number of implantation site on day 7 of pregnancy, nor the length of estrous cycle in the offspring mice. However, the time to puberty and estrous cycle length was longer, and lower body weight at puberty was found in the BPA treated group. Crossing of BPA-treated offspring mice have significantly lower number of implantation site on day 7 when compared to other groups, suggesting the trans-generation effect of BPA in compromising fertility in mice. In summary, BPA exhibited a stronger estrogenic activity and suppression on spheroid attachment than BPF and BPS in vitro. Chronic low dose BPA treatment affected the reproductive functions of the offspring. However, the risk of BPF and BPS in other reproductive functions remains to be investigated.