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Bisphenol A Exposure, Ovarian Follicle Numbers, and Female Sex Steroid Hormone Levels: Results From a CLARITY-BPA Study

Shreya Patel, Emily Brehm, Liying Gao, Saniya Rattan, Ayelet Ziv-Gal, and Jodi A. Flaws.
Endocrinology (2017) DOI: https://doi.org/10.1210/en.2016-1887 PMID: 28324068

Publication

Abstract

Bisphenol A (BPA) is an industrial chemical found in thermal receipts and food and beverage containers. Previous studies have shown that BPA can affect the numbers and health of ovarian follicles and the production of sex steroid hormones, but they often did not include a wide range of doses of BPA, used a small sample size, focused on relatively short-term exposures to BPA, and/or did not examine the consequences of chronic BPA exposure on the ovaries or steroid levels. Thus, this study was designed to examine the effects of a wide range of doses of BPA on ovarian morphology and sex steroid hormone production. Specifically, this study tested the hypothesis that prenatal and continuous BPA exposure reduces ovarian follicle numbers and sex steroid hormone levels. To test this hypothesis, rats were dosed with vehicle, ethinyl estradiol (0.05 and 0.5 μg/kg body weight/d), or BPA (2.5, 25, 250, 2500, and 25,000 μg/kg body weight/d) from gestation day 6 until 1 year as part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA). Ovaries and sera were collected on postnatal days 1, 21, and 90, and at 6 months and 1 year. The ovaries were subjected to histological evaluation of follicle numbers and the sera were subjected to measurements of estradiol and progesterone. Collectively, these data indicate that BPA exposure at some doses and time points affects ovarian follicle numbers and sex steroid levels, but these effects are different than those observed with ethinyl estradiol exposure and some previous studies on BPA.

Figures

Figure 1. Effects of EE2 and BPA on ovarian morphology on PND 1.

On PND 1, pups from each group were euthanized and one ovary from each animal was fixed for histological evaluation of germ cell and ovarian follicle numbers. The graph represents the means ± SEM of the number of healthy germ cells, apoptotic germ cells, and primordial follicles present in 9 to 10 ovaries per treatment group.

Figure 2. Effects of EE2 and BPA on ovarian morphology and sex steroid hormones on PND 21.

On PND 21, rats from each group were euthanized and one ovary from each animal was fixed for histological evaluation of different ovarian follicle types. Additionally, serum was collected from the blood of each of the animals and used in the measurement of sex steroid hormones.
(a) Graph represents the means ± SEM of the number of the different follicle types and total healthy follicles present in 8 to 10 ovaries per treatment group.
(b) Graph represents the means ± SEM of the amount of estradiol present in the serum of 8 to 10 animals per group.
(c) Graph represents the means ± SEM of the amount of progesterone present in the serum of 8 to 10 animals per group.
*P ≤ 0.05, difference between the control group and doses of BPA or EE2 (n = 8 to 10).

Figure 3. Effects of prenatal and preweaning exposure to EE2 and BPA on ovarian morphology and sex steroid hormones on PND 90.

On PND 90, rats from each group were euthanized and one ovary from each animal was fixed for histological evaluation of different ovarian follicle types. Additionally, serum was collected from the blood of each of the animals and used in the measurement of sex steroid hormones.
(a) Graph represents the means ± SEM of the number of the different follicle types and total healthy follicles present in 8 to 10 ovaries per treatment group.
(b) Graph represents the means ± SEM of the amount of estradiol present in the serum of four to nine animals per group.
(c) Graph represents the means ± SEM of the amount of progesterone present in the serum of four to nine animals per group.
^P > 0.05.

Figure 4. Effects of continuous exposure to EE2 and BPA on ovarian morphology and sex steroid hormones on PND 90.

On PND 90, rats from each group were euthanized and one ovary from each animal was fixed for histological evaluation of different ovarian follicle types. Additionally, serum was collected from the blood of each of the animals and used in the measurement of sex steroid hormones.
(a) Graph represents the means ± SEM of the number of the different follicle types and total healthy follicles present in 8 to 10 ovaries per treatment group.
(b) Graph represents the means ± SEM of the amount of estradiol present in the serum of six to nine animals per group.
(c) Graph represents the means ± SEM of the amount of progesterone present in the serum of six to nine animals per group.
*Significant difference between the control group and doses of BPA or EE2 (n = 8 to 10; P ≤ 0.05).

Figure 5. Effects of prenatal and preweaning exposure to EE2 and BPA on ovarian morphology and sex steroid hormones at 6 months.

At 6 months, rats from each group were euthanized and one ovary from each animal was fixed for histological evaluation of different ovarian follicle types. Additionally, serum was collected from the blood of each of the animals and used in the measurement of sex steroid hormones.
(a) Graph represents the means ± SEM of the number of the different follicle types and total healthy follicles present in 9 to 10 ovaries per treatment group.
(b) Graph represents the means ± SEM of the amount of estradiol present in the serum of five to nine animals per group.
(c) Graph represents the means ± SEM of the amount of progesterone present in the serum of five to nine animals per group.
*P ≤ 0.05, difference between the control group and doses of BPA or EE2 (n = 9 to 10).

Figure 6. Effects of continuous exposure to EE2 and BPA on ovarian morphology and sex steroid hormones at 6 months.

Effects of continuous exposure to EE2 and BPA on ovarian morphology and sex steroid hormones at 6 months. At 6 months, rats from each group were euthanized and one ovary from each animal was fixed for histological evaluation of different ovarian follicle types. Additionally, serum was collected from the blood of each of the animals and used in the measurement of sex steroid hormones.
(a) Graph represents the means ± SEM of the number of the different follicle types and total healthy follicles present in 10 ovaries per treatment group.
(b) Graph represents the means ± SEM of the amount of estradiol present in the serum of three to eight animals per group.
(c) Graph represents the means ± SEM of the amount of progesterone present in the serum of three to eight animals per group.
*Significant difference between the control group and EE2 (n = 9 to 10; P < 0.05).

Figure 7. Effects of prenatal and preweaning exposure to EE2 and BPA on ovarian morphology and sex steroid hormones at 1 year.

At 1 year, rats from each group were euthanized and one ovary from each animal was fixed for histological evaluation of different ovarian follicle types. Additionally, serum was collected from the blood of each of the animals and used in the measurement of sex steroid hormones.
(a) Graph represents the means ± SEM of the number of the different follicle types and total healthy follicles present in 9 to 10 ovaries per treatment group.
(b) Graph represents the means ± SEM of the amount of estradiol present in the serum of 5 to 10 animals per group.
(c) Graph represents the means ± SEM of the amount of progesterone present in the serum of 5 to 10 animals per group.
*Significant difference between the control group and EE2 (n = 9 to 10; P ≤ 0.05).

Figure 8. Effects of continuous exposure to EE2 and BPA on ovarian morphology and sex steroid hormones at 1 year.

At 1 year, rats from each group were euthanized and one ovary from each animal was fixed for histological evaluation of different ovarian follicle types. Additionally, serum was collected from the blood of each of the animals and used in the measurement of sex steroid hormones.
(a) Graph represents the means ± SEM of the number of the different follicle types and total healthy follicles present in 8 to 10 ovaries per treatment group.
(b) Graph represents the means ± SEM of the amount of estradiol present in the serum of three to nine animals per group.
(c) Graph represents the means ± SEM of the amount of progesterone present in the serum of three to nine animals per group.
*P ≤ 0.05, difference between the control group and doses of BPA or EE2 (n = 3 to 9); ^P > 0.05.

Figure 9. Heat map depicting the effects of EE2 and BPA exposure on ovarian morphology and sex steroid hormones.

Data from Figs. 1–8 were used to generate a heat map. The light shading shows nonsignificant findings between controls and treatment groups. Darker shading corresponds to significant differences between control and treatment group (with the lowest P values being the darkest colors).