(121) Impact of Human Relevant Di-n-Butyl Phthalate Exposure on Ovulation, Fertilization, and Early Embryo Development in a Mouse Model

Phthalates are used in beauty and personal care products, food packaging, medical devices, and the coating of some medications. Many epidemiological studies have reported associations between phthalate burden and human reproductive health outcomes. Women of reproductive age are considered a high exposure/high risk population based on biomonitoring data showing higher phthalate burden, greater use of cosmetics and personal care products, and higher exposures in the occupational setting. Phthalate exposure in women has been associated with reduced egg yield and early pregnancy loss during medically assisted reproduction. Therefore, it is critical to elucidate the direct impacts of phthalates on ovulation, fertilization, and early embryo development in an animal model. We developed a mouse model of phthalate exposure using dibutyl phthalate (DBP) in which CD-1 female mice are orally exposed to the parent compound DBP at human relevant doses and its active metabolite, mono-n-butyl phthalate (MBP), is distributed to the ovary. This study was designed to determine whether DBP exposure negatively influences ovulatory function, fertilization, and early embryo development in adult CD-1 mice. The impact of DBP on ovulation was assessed using cycling adult CD-1 mice that were exposed to tocopherol-stripped corn oil (vehicle), two human relevant DBP exposures (10 and 100 µg/kg/day), or a classical high dose of DBP (1000 mg/kg/day) for 9-12 days. Mice were euthanized as they reached the stage of estrus, their oviducts dissected, and the number of released ova quantified. Relative to vehicle control natural ovulation incidence (90% of mice), ovulation was lower in the DBP10µg group (50% of mice) and significantly reduced in the DBP100µg and DBP1000mg groups (30% of mice). Among mice who successfully ovulated, DBP-treatment did not impact ova yield or fertilization rate. Hormone-primed ovariectomized and superovulated mice were used to determine whether DBP100µg interfered with ovulation via neuroendocrine and/or ovarian sensitivity mechanisms. Ovariectomized DBP-treated mice responded to hormone priming (17β-estradiol and progesterone) by releasing luteinizing hormone (LH) and producing peak serum LH levels like vehicle-treated controls. Interestingly, the onset of LH surging in DBP-treated mice seemed delayed by two hours and with a greater area under the curve, strongly suggesting a broadening of the surge window. Finally, while superovulation resulted in ovulation, fertilization, and cleavage rates comparable between treatments, DBP-treated mice produced less blastocysts and more fragmented embryos than vehicle controls. Our results provide evidence that pituitary hormone release, ovarian sensitivity to gonadotropins, and preimplantation embryo development are impaired following daily oral exposure to DBP.  These findings add essential information to help understand the mechanisms responsible for the poor ovulatory and pregnancy outcomes observed in women with high phthalate burden.

This work was funded by NIEHS Grants K99/R00ES021467, R01ES026998, and R01ES034690 to ZRC.