(115) Co-Exposure to Mono(2-ethylhexyl) Phthalate and Elevated Temperature Inhibits Mouse Antral Follicle Growth and Estradiol Synthesis In Vitro

Abstract Authors: Sundus Ghuneim¹; Gretchen Ruschman²; Caroline Harper²; Madison Wilson²; Patrick Hannon^1,2

1. Department of Pharmacology and Nutritional Sciences, University of Kentucky, Lexington, KY, United States
2. Department of Obstetrics and Gynecology, University of Kentucky, Lexington, KY, United States

Abstract Text: Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer used in common consumer goods that is rapidly metabolized upon ingestion to its bioactive metabolite, mono(2-ethylhexyl) phthalate (MEHP). DEHP and MEHP are endocrine-disrupting chemicals that have been shown to target the ovary and disrupt follicle development and steroidogenesis. Due to the extensive use of DEHP in consumer goods, humans are ubiquitously exposed and therefore vulnerable to endocrine and reproductive dysfunction by MEHP. Simultaneously, humans are ubiquitously exposed to non-chemical stressors like rising global temperatures, and studies have shown that exposure to elevated temperatures can impair female reproductive health. Given the progression of climate change and the unavoidable exposure to both increasing heat and DEHP, this study used an in vitro follicle culture system to test the hypothesis that exposure to high temperature will exacerbate the negative effects of MEHP exposure on antral follicle function. Antral follicles were isolated from 4-5-week-old CD-1 mice and cultured for 24-96hr in media containing follicle-stimulating hormone (FSH) at a control temperature (CT; 37°C) or high temperature (HT; 42°C). Follicles were treated with vehicle control (dimethyl sulfoxide; DMSO) or MEHP (0.2-20µg/ml) in both temperature groups. Separate incubators were used for the CT and HT treatment groups, in which all HT treatments occurred for 8hr per 24hr period to model daytime, occupational exposure to high temperatures. Follicular growth was measured every 24hr. Follicles and media were collected at 24, 72, and 96hr for gene expression analyses and sex steroid hormone measurements, respectively (n=3-7 with 5-10 follicles/group/replicate; p≤0.05). Antral follicle growth was decreased at 72hr and 96hr by HT+MEHP compared to CT+DMSO. At 96hr, there was an interaction between HT and MEHP where estradiol levels were decreased by CT+MEHP, HT+DMSO, and HT+MEHP compared to CT+DMSO, demonstrating decreased estradiol levels with exposure to MEHP alone, HT alone, and HT+MEHP co-exposure. The mRNA levels of Cyp11a1 (72hr) and Hsd17b1 (24hr) were also decreased by HT+MEHP compared to CT+DMSO. Further, the mRNA levels of Cyp19a1 were decreased by HT+DMSO (72hr) and HT+MEHP (24hr, 72hr) compared to CT+DMSO and when compared to certain doses’ CT+MEHP equivalents, indicating decreased Cyp19a1 levels with exposure to HT alone and HT+MEHP co-exposure. At 96hr, the Bax/Bcl2 ratio was increased by HT+MEHP compared to CT+MEHP. At 24hr, there was an interaction between HT and MEHP where mRNA levels of Fshr were increased by HT+DMSO and HT+MEHP compared to CT+DMSO and when compared to certain doses’ CT+MEHP equivalents, indicating increases in Fshr levels with exposure to HT alone and HT+MEHP co-exposure. These results show that HT+MEHP co-exposure inhibits antral follicle growth and steroidogenesis in vitro, where observed decreases in steroidogenic mRNA levels correlate to decreases in estradiol levels. Further, the decreases in follicle growth, Cyp11a1, and Hsd17b1 by HT+MEHP co-exposure, but not by exposure to HT alone or MEHP alone, demonstrate for the first time that HT and MEHP may exert additive/synergistic effects. The increased Bax/Bcl2 ratio suggests apoptosis as a mechanism by which follicle growth and estradiol levels are decreased by HT+MEHP exposure. In addition, the early increase in Fshr levels suggests a compensatory mechanism and/or that HT alone and HT+MEHP co-exposure may dysregulate the intricate balance in FSH/FSHR signaling. Thus, exposure to chemical and non-chemical stressors like MEHP and HT disrupts ovarian function and may potentiate dysfunction when combined. Supported by R01ES033767 and pilot funding from UL1TR001998 and P30ES026529.