Dibutyl phthalate impairs steroidogenesis and a subset of LH-dependent genes in cultured human mural granulosa cell in vitro

doi:10.1016/j.reprotox.2016.12.007

Reproductive Toxicology

Volume 69, April 2017, Pages 13-18

https://doi.org/10.1016/j.reprotox.2016.12.007 Get rights and content

Highlights

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Exposure of human granulosa cells to a high concentration of di-butyl phthalate (DBP) in vitro impairs progesterone and estradiol secretion.
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Ovarian steroidogenesis is altered by exposure to high doses of DBP.
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EGF factors (AREG, EREG and BTC) secreted in response to LH surge during ovulation are sensitive to DBP.

Abstract

Exposure to di-butyl phthalate (DBP) exerts negative effects on female fertility in animal models, but human studies remain limited. Here, the effects of DBP exposure on mural granulosa cell function were investigated in primary cultures from women undergoing in vitro fertilization. Cultured cells treated with various doses of DBP (0, 0.01 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL, or 100 μg/mL) for 48 h were assessed using enzyme-linked immunosorbent assay and qRT-PCR. Treatment with 100 μg/mL DBP resulted in significantly lower 17β-estradiol and progesterone production (p < 0.01). It also resulted in altered mRNA expression of steroidogenic, angiogenic, and epidermal growth factor-like growth factor genes: CYP11A1 (p < 0.001), CYP19A1 (aromatase) (p < 0.001), VEGF-A (p < 0.02), BTC (p = 0.009), and EREG (p = 0.04). StAR expression was impaired after exposure to both 10 and 100 μg/mL (p < 0.03 and p < 0.001, respectively). Our results indicate that in vitro exposure of granulosa cells to high doses of DBP alters cell functions.

Introduction

Phthalates are man-made chemicals widely used in a variety of daily merchandise including personal care products, cosmetics, plastic packaging, inflatable toys, medical tubing, and polyvinyl chloride plastics [1]. They can leach and evaporate into the environment during use or disposal of the products [2]. The diester forms of phthalate metabolites are processed into toxic monoester forms that exert anti-estrogenic, anti-androgenic, or anti-thyroid activity and are therefore known as endocrine-disrupting chemicals (EDCs) [3], [4]. Exposure to phthalates is ubiquitous via inhalation, ingestion, or direct contact [2], [5], [6], and phthalate metabolites have been detected in various body fluids including blood, urine, and follicular fluid [1], [7]. Clinical studies have reported an inverse correlation between urinary phthalate levels, antral follicular count, and chances of conception among women undergoing infertility treatments [8], [9]. Moreover, increased urinary phthalate levels have been associated with an increased incidence of biochemical pregnancies (i.e., elevation of beta human chorionic gonadotropin but no gestational sac visible on ultrasound scan) and of pregnancy loss among women undergoing medically assisted reproduction [10].

Dibutyl phthalate (DBP), a phthalate commonly incorporated in consumer and personal care products [2], [11], is widely used among women of reproductive age. According to the Centers for Disease Control and Prevention, exposure to DBP is ubiquitous, and higher levels are measured in women compared with men [12]. Interestingly, the DBP metabolite Mono-n-butyl phthalate (MnBP) was one of the few phthalate metabolites detected in human follicular fluid [13].

Mural granulosa cells surround the oocyte in the ovarian follicle. Together with the theca cells, they play a critical role in follicular growth, oocyte development, and steroidogenesis. Mid-cycle (ovulatory) luteinizing hormone (LH) secretion triggers a cascade of activations in the follicle, leading to secretion of epidermal growth factors in mural granulosa cells and further to resumption of oocyte meiosis, cumulus expansion, ovulation, vascular endothelial growth factor A (VEGF-A) secretion, steroidogenesis and luteinization, all of which are crucial for fertilization, embryo development, and successful implantation [14], [15], [16], [17]. Alterations of these processes may lead to infertility. Animal studies have shown that DBP decreases estradiol (E₂) production, alters the expression of apoptotic factors, reduces cell proliferation, and impairs follicle stimulating hormone (FSH)-dependent signaling in mural granulosa cells [18], [19], [20]. The effects of DBP in human cells currently remain unknown. Prompted by a lack of understanding of the effects of this phthalate on female fertility, the aim of this study was to investigate the effects of DBP (using primary cultures of human mural granulosa cells) on the production of steroids and the mRNA expression of a subset of LH-dependent genes.

Section snippets

Participants

The study was approved by Sheba Medical Center's Institutional Review Board (IRB), and all participants provided written informed consent. The study included women who were <38 years old with BMI <35 kg/m² and were: 1) undergoing IVF due to male factor infertility, mechanical factor, or unexplained infertility, or 2) fertile and undergoing IVF for pre-gestational diagnosis (PGD) of autosomal recessive diseases. All women underwent controlled ovarian stimulation. We excluded anovulatory women,

Patients

We collected mural granulosa cells from 44 women during oocyte retrieval. Patients' age (mean +SD) was 31.9 ± 3.3 (range 24.6–37.8 years old). Patient BMI ranged from 16.3 to 33.2 kg/m², with a mean ± SD of 23.6 ± 4 kg/m². Etiologies for fertility treatment included preimplantation genetic diagnosis (n = 17), male factor (n = 16), unexplained (n = 6), and mechanical infertility (n = 5).

Production of 17β-estradiol and progesterone

Levels of 17β-estradiol and progesterone were measured in the culture supernatant samples. Production of both hormones was

Discussion

In the present study, we used an in vitro model of primary cultures of human mural granulosa cells to evaluate the effects of DBP exposure on the reproduction cascades induced by mid-cycle LH surge. Short-term exposure to DBP led to altered steroidogenesis, upregulated VEGF-A expression, and reduced expression of EGF-like factors in human mural granulosa cells. However, in this study, these effects were significant at high DBP levels.

We found that exposure to DBP at high doses impaired both

Conclusions

Short-term exposure to supra-physiological doses of DBP led to altered steroidogenesis and dysregulation of angiogenesis and EGF-like factors in human mural granulosa cells. Further in vitro and clinical studies are needed to clarify potential downstream effects on reproductive function, including expanding the research to evaluate the effects of DBP on apoptosis and cell cycle control in human granulosa cells. The current findings, along with previous publications, can help focus on clinical

Funding

This work was supported by the Israeli Science Foundation (grant number 1936/12).

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Dibutyl phthalate impairs steroidogenesis and a subset of LH-dependent genes in cultured human mural granulosa cell in vitro

Highlights

Abstract

Introduction

Section snippets

Participants

Patients

Production of 17β-estradiol and progesterone

Discussion

Conclusions

Funding

Environ. Int.

Environ. Res.

Environ. Int.

Reprod. Toxicol.

Fertil. Steril.

Mol. Cell. Endocrinol.

Mol. Cell. Endocrinol.

Arch. Med. Res.

Reprod. Toxicol.

Biol. Reprod.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Pharmacol.

Fertil. Steril.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Phthalates and human health

Occup. Environ. Med.