The immediate and long-term consequences of temporary exposure during adulthood to di(2-ethylhexyl) phthalate and diisononyl phthalate on female reproduction

The ovary is a heterogenous structure that produces a variety of sex steroid hormones and participates in several feedback loops within the hypothalamic-pituitary-ovary axis. Further, it is the site of storage and maturation of the female gamete: the oocyte. Females are born with a finite number of oocytes in the form of the primordial follicle pool. Primordial follicles are composed of an oocyte surrounded by supporting somatic cells. Activated primordial follicles will grow through the primary, preantral, and antral follicle stages before ovulating. Proper maintenance of this follicular growth, termed folliculogenesis, is imperative for maintenance of proper fertility and overall somatic health. Female fertility relies on a steady stream of maturing follicles in addition to the proper maintenance of the cyclical rise and fall of sex steroid and peptide hormones. Further, sex steroid hormones play a role in maintenance of multiple aspects of somatic health, including the cardiovascular system, bone density, and neurological health. Thus, chemical exposures that target the ovary can lead to disruptions in reproductive and overall health. The phthalate chemical family has several prominent members with endocrine disrupting capabilities. One of the most common phthalates is di(2-ethylhexyl) phthalate (DEHP), with up to 100 million pounds produced within the U.S. each year. DEHP is used as a plasticizer to impart strength and flexibility into products. DEHP can be found in products such as plastic food packaging, clothes, shower curtains, upholstery, and medical equipment. Because DEHP is noncovalently bound to products, it can leach out over time and be absorbed via inhalation, ingestion, and dermal contact. The estimated average daily intake of DEHP in humans is 3-30 µg/kg/day, and DEHP metabolites have been found in samples of human blood, urine, semen, amniotic fluid, cord blood, and ovarian follicular fluid. This is concerning because DEHP is a known reproductive toxicant and endocrine disrupting chemical. Although some studies have shown that DEHP targets the ovary, very few studies have investigated whether short-term exposure to DEHP during adulthood can have long-lasting impacts on the exposed individual. Some manufacturers have elected to start replacing DEHP with other plasticizers. Unfortunately, these replacements are often understudied, and few studies have investigated the reproductive toxicity potential of these compounds. Diisononyl phthalate (DiNP) is one such understudied DEHP substitute. Few reproductive toxicity studies using DiNP have been conducted, and the majority focus on males. Further, studies that report data on the reproductive toxicity of DiNP in females tend to only include data on the effects of DiNP on the weights of reproductive organs. Although reproductive organ weights are an important and useful metric for assessing the reproductive toxicity for a particular chemical, these data do not provide a complete understanding of the potential reproductive toxicity of the chemical. Further, the present literature concerning the effects of DiNP on female reproduction often use doses well above the range of environmental relevance. It is imperative to investigate these chemicals at relevant doses because of the non-monotonic dose responses that some endocrine disrupting chemicals can elicit. Together, information is lacking on the effects of DEHP and DiNP on female reproduction, especially as it concerns exposure during adulthood. Therefore, the purpose of my doctoral dissertation work was to investigate the effects of DEHP alongside a common DEHP replacement chemical, DiNP, on the reproductive health over the lifetime of the exposed female mice at both high and environmentally relevant doses. Specifically, I investigated the effects of short-term exposure to DEHP and DiNP during adulthood on estrous cyclicity, fertility, sex steroid hormones, peptide hormones, and ovarian follicle populations at time points including immediately post-dosing and 3, 6, 9, 12, 15, and 18 months post-dosing First, I tested the hypothesis that short-term exposure (10 days) to DEHP and DiNP during adulthood in the mouse was sufficient to disrupt estrous cyclicity, fertility, and weights of reproductive organs at time points during the prime reproductive age of the mouse, including immediately post-dosing and 3 and 9 months post-dosing. Immediately post-dosing and 3 and 9 months post-dosing, fertility was assessed via breeding trials and weights of reproductive organs were measured. In addition, estrous cyclicity was monitored at 3 and 9 months post-dosing. Assessing estrous cyclicity disruptions and changes in weights of organs can give insight to possible avenues through which DEHP and DiNP may disrupt fertility. I found that 10 days of DEHP exposure was enough to significantly reduce the uterine weight immediately post-dosing. Additionally, at 3 months post-dosing, females treated with the lowest dose of DEHP and DiNP had significant difficulty achieving pregnancy from a mating, resulting in significantly fewer females producing offspring in these groups when compared to control. I also observed that several treatment groups had increased male to female ratios of pups at 9 months post-dosing. Further, 10 days of phthalate exposure was enough to significantly affect the estrous cyclicity at both 3 and 9 months post-dosing, indicating that short-term exposure can have long lasting impacts. Next, I tested the hypothesis that short-term exposure (10 days) to DEHP and DiNP during adulthood in the mouse was sufficient to alter levels of sex steroid hormones, levels of the peptide hormones follicle-stimulating hormone (FSH) and inhibin B, and ovarian follicle populations at time points during the prime reproductive life of the mouse, including immediately post-dosing and 3, 6, and 9 months post-dosing. Proper maintenance of ovarian follicle maturation is necessary for proper fertility and maximization of the reproductive lifespan. Further, changes in circulating hormones can alter cyclicity and behavior and can directly impact fertility itself. I found that transient exposure to DEHP and DiNP during adulthood was sufficient to cause disruption of ovarian folliculogenesis by altering numbers of follicle types, percentages of follicle types, and percent of unhealthy follicles immediately post-dosing and up to 9 months post-dosing. Further, I found that DEHP and DiNP disrupted several hormones and that disruptions were present at all time points tested in this study. Further, I tested the hypothesis that short-term exposure (10 days) to DEHP and DiNP during adulthood in the mouse was sufficient to disrupt fertility and estrous cyclicity and alter levels of sex steroid hormones, levels of the peptide hormones follicle-stimulating hormone (FSH) and inhibin B, and ovarian follicle populations at time points during late-life in the mouse, including 12, 15, and 18 months post-dosing. Maintenance of the finite pool of ovarian follicles is required for maximization of the reproductive lifespan in females. Dysregulation of the ovarian follicular pool or hormones could lead to abnormalities in reproductive behavior, fertility complications, and shortening of the reproductive lifespan. I found that DEHP and DiNP increased pregnancy loss, reduced fertility, and altered follicular populations and circulating hormone levels in late-life in female mice. Collectively, data from my dissertation research indicate that short-term exposure to DEHP and DiNP during adulthood has consequences on a variety of aspects of female fertility at time points ranging from immediately post-dosing to 18 months following completion of the dosing period