Prenatal exposure to a phthalate mixture leads to multigenerational and transgenerational effects on uterine morphology and function in mice

Phthalates are commonly used plasticizers and additives that are found in plastic containers, children's toys and medical equipment. Phthalates are classified as endocrine-disrupting chemicals and exposure to phthalates has been associated with several human health risks including reproductive defects. Most studies focus on a single phthalate; however, humans are exposed to a mixture of phthalates daily. We hypothesized that prenatal exposure to an environmentally relevant phthalate mixture would lead to changes in uterine morphology and function in mice in a multi-generational manner. To test this hypothesis, pregnant CD-1 dams were orally dosed with vehicle or a phthalate mixture (20 μg/kg/day, 200 μg/kg/day, 200 mg/kg/day, and 500 mg/kg/day) from gestational day 10.5 to parturition. The mixture contained 35 % diethyl phthalate, 21 % di-(2-ethylhexyl) phthalate, 15 % dibutyl phthalate, 15 % diisononyl phthalate, 8% diisobutyl phthalate, and 5% benzylbutyl phthalate. The F1 pups were maintained and mated to produce two more generations (F2 and F3). At the age of 13 months, all females were euthanized and tissue samples were collected in diestrus. Our results showed that exposure to a phthalate mixture caused a decrease in progesterone levels in the treated groups in the F2 generation. The 200 mg/kg/day treatment group showed a decreased and increased luminal epithelial cell proliferation in the F1 and F2 generations respectively. In addition, these mice in the F2 generation had reduced Hand2 expression in the sub-epithelial stroma compared to the controls. A higher incidence of multilayered luminal epithelium and large dilated endometrial glands were observed in the phthalate mixture exposed groups in all generations. The mixture also caused a higher incidence of smooth muscle actin expression and collagen deposition in the endometrium compared to controls. Collectively, our results demonstrate that prenatal exposure to an environmentally relevant phthalate mixture can have adverse effects on female reproductive functions.