The effects of di(2-ethylhexyl) phthalate exposure in women with polycystic ovary syndrome undergoing in vitro fertilization

Abstract

Objectives: Di(2-ethylhexyl) phthalate (DEHP) is a common endocrine-disrupting chemical, which has potential reproductive toxicity. This study aimed to explore the effects of DEHP exposure in women with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization.

Methods: In this case-control study, DEHP levels in follicular fluid (FF) of women with PCOS (n = 56) and controls (n = 51) were measured. The in vitro effects of DEHP exposure on primary-cultured human granulosa cells (GCs) and a steroidogenic human granulosa-like tumor cell line (KGN cells) were analyzed.

Results: Concentrations of DEHP in FF were significantly higher in women with PCOS than in controls. The clinical pregnancy rate was significantly lower in women with PCOS with high levels of DEHP than in controls. The levels of androgens produced by human GCs were significantly increased following DEHP exposure. Compared with controls, DEHP-treated human GCs and KGN cells showed significantly lower viability, cell cycle arrest, higher apoptosis, and altered expression of apoptosis-related genes.

Conclusion: Women with PCOS are exposed to increased levels of DEHP in follicles, which may be associated with pregnancy loss following in vitro fertilization. DEHP may disrupt steroid production, balance in cellular proliferation, and apoptosis in human granulosa cells.

Keywords: Polycystic ovary syndrome (PCOS); apoptosis; case-control study; di(2-ethylhexyl) phthalate (DEHP); in vitro study; steroid production.

Figure 1.

Concentrations of DEHP in FF of women with PCOS and controls. The data are shown as mean ± SD. The red horizontal line represents the geometric mean. **P < 0.01 versus the control by Student’s t-test. DEHP, di(2-ethylhexyl) phthalate; FF, follicular fluid; PCOS, polycystic ovary syndrome.

Figure 2.

Effects of DEHP on steroid production of primary-cultured human GCs and KGN cells. (a) The ratios of E2 to T and E1 to A in the supernatant of primary-culture medium. (b) mRNA expression levels of CYP19A1 and CYP17A1 in primary-cultured human GCs or KGN cells. (c) Protein expression levels of CYP19A1 and CYP17A1 in primary-cultured human GCs and KGN cells. Data are shown as mean ± SD. *P < 0.05 versus control by ANOVA, ^#P < 0.05, KGN DEHP versus PC DEHP by ANOVA. PC, primary-cultured human GCs; KGN, steroidogenic human granulosa-like tumor cell line; DEHP, di(2-ethylhexyl) phthalate; T, testosterone; E1, estrone; E2, estradiol; A, androsterone.

Figure 3.

Effect of DEHP on viability of primary-cultured human GCs and KGN cells measured by MTT assay. Data are shown as mean ± SD. *P < 0.05 versus control by Student’s t-test. PC, primary-cultured human GCs; KGN, steroidogenic human granulosa-like tumor cell line; DEHP, di(2-ethylhexyl) phthalate.

Figure 4.

Effect of DEHP on cell cycle of primary-cultured human GCs and KGN cells analyzed by flow cytometry. (a) Flow cytometry results. The vertical and horizontal axis indicate cell numbers and cell division cycle, respectively. (b) Histogram. Data are shown as mean ± SD. *P < 0.05 versus control by ANOVA, ^#P < 0.05, KGN DEHP versus PC DEHP group by ANOVA. PC, primary-cultured human GCs; KGN, steroidogenic human granulosa-like tumor cell line; DEHP, di(2-ethylhexyl) phthalate.

Figure 5.

Effect of DEHP on cell of primary-cultured human GCs and KGN cells. (a) Annexin V flow cytometry assay was performed to analyze cell apoptosis. The vertical and horizontal axes indicate PI- and FITC-positive areas, respectively. Identified by flow cytometry, cells were divided into four sections: UL (annexin V-FITC− PI+) was representative of mechanical error; UR (annexin V-FITC+ PI+) was representative of late apoptosis or necrosis cells; LL (annexin V-FITC− PI−) was representative of living cells; LR (annexin V-FITC+ PI−) was representative of early apoptosis cells. (b) mRNA expression levels of caspase-3, caspase-7, caspase-8, caspase-9, and Bcl-2 in primary-cultured human GCs and KGN cells measured using real-time quantitative PCR. Data are shown as mean ± SD. *P < 0.05 versus control by ANOVA, ^#P < 0.05, KGN DEHP versus PC DEHP group by ANOVA. PI, propidium iodide; FITC, fluorescein isothiocyanate, PC, primary-cultured human GCs; KGN, steroidogenic human granulosa-like tumor cell line; DEHP, di(2-ethylhexyl) phthalate.