Evidence for bisphenol A-induced female infertility - Review (2007–2016)

Abstract

We summarized the scientific literature published from 2007 to 2016 on the potential effects of bisphenol A (BPA) on female fertility. We focused on overall fertility outcomes (e.g., ability to become pregnant, number of offspring), organs that are important for female reproduction (i.e., oviduct, uterus, ovary, hypothalamus, and pituitary), and reproductive related processes (i.e., estrous cyclicity, implantation, and hormonal secretion). The reviewed literature indicates that BPA may be associated with infertility in women. Potential explanations for this association can be generated from experimental studies. Specifically, BPA may alter overall female reproductive capacity by affecting the morphology and function of the oviduct, uterus, ovary, and hypothalamus-pituitary-ovarian axis in animal models. Additionally, BPA may disrupt estrous cyclicity and implantation. Nevertheless, further studies are needed to better understand the exact mechanisms of action and to detect potential reproductive toxicity at earlier stages.

Introduction

Female infertility is generally defined as the inability to get pregnant naturally and to deliver a live healthy newborn. According to the Center for Disease Control and Prevention (CDC; http://www.cdc.gov/nchs/nsfg/key_statistics/i.htm#infertility), 6.1% of married women were considered to be infertile between 2011 and 2013 in the USA alone. The percentage of infertile women can reach 30% world-wide (1). Infertility in women can be the result of various factors, including physical problems, endocrine problems, lifestyle habits, and environmental factors. Environmental factors such as exposure to chemicals with endocrine disrupting properties can mimic or block the endocrine activity of endogenous hormones and thus, adversely affect reproduction.

One of the most extensively studied endocrine disrupting chemicals is bisphenol A (BPA). BPA is incorporated in many daily used products as it is used by the manufacturers of polycarbonate plastics and epoxy resins. Despite the relatively short half-life of BPA (6–24 hours) (2), it was measured in various reproductive tissues (3), including ovarian follicular fluid, placenta, breast milk, and colostrum. Findings from previous publications suggest that BPA is a reproductive toxicant (4–6).

The current review focuses on the scientific evidence for BPA-induced fertility problems in females. We summarized the main findings of epidemiological and experimental studies that examined the potential effects of BPA on female fertility and that were published between 2007 and 2016. We included the morphological and mechanistic findings reported in the reviewed manuscripts. We focused on the reported outcomes of BPA exposure on overall: 1) fertility, 2) reproductive related processes including the ovarian cycle, and 3) reproductive tissues.

Methods

Pubmed (http://www.ncbi.nlm.nih.gov/pubmed) searches for the years 2007–2016 were conducted using the following key words: BPA, bisphenol A, fertility, female, reproduction, ovary, pregnancy, oviduct, ovulation, fertilization, uterus, implantation, hypothalamus, and pituitary. We focused on manuscripts published in 2007–2016 to expand upon previous review papers on the same topic (4, 5, 7–11). Additionally, references included in other review papers were examined for relevant information. We included manuscripts that dealt with fertility/infertility outcomes related to overall fertility, implantation, uterine morphology and function, estrous cyclicity, hypothalamus-pituitary, hormone levels (luteinizing hormone; LH, follicular stimulating hormone; FSH, and prolactin; PRL), oviduct, and ovary. We excluded manuscripts about topics that were out of the scope of this review paper or ones that will be reviewed by other authors in this special issue (e.g., sexual maturation/behavior, oocyte quality and maturation, ovarian steroidogenesis, pregnancy, miscarriage, endometriosis, polycystic ovarian syndrome, and uterine fibroids/leiomyoma).

BPA studies have used various study designs and included a wide range of doses. Based on the definitions in other studies, we considered a “low dose” of BPA as follows: a dose below the lowest observable adverse effect level (LOAEL) of 50 mg/kg/day in animal models (4, 5, 12, 13), 17.2 mg/l for aquatic animals (5, 14), 1 × 10⁻⁷ M for cell culture experiments (5, 15), and a dose in the range of typical (not occupational) human exposures for epidemiological studies (5, 16). The majority of the studies described in this review used doses that are within the category of “low dose”. Throughout the text of this review, we indicated if the doses were considered low or high based on the categories above. In the tables, the specific doses that were used in each study are described in detail. Lastly, similar to Peretz et al. (4), we defined exposure time during pregnancy as “in utero”; exposure after birth that ended before weaning as “neonatal”; and exposure any time after weaning as “postnatal” or adult exposure.

Results

Overall fertility

In recent years, several research groups have examined the effects of BPA on overall fertility. Epidemiological studies examined if BPA levels are higher in infertile women than in fertile women (Table 1). Findings from these studies indicate that infertile women have higher serum BPA levels compared to fertile women (17, 18). Further, studies conducted in women undergoing in vitro fertilization (IVF) treatments show that BPA levels (total or unconjugated BPA) were inversely associated with peak estradiol levels, number of oocytes retrieved, oocyte maturation, fertilization rates, and embryo quality (19–23). Thus, increased levels of BPA may decrease the success rate of IVF treatments. Nevertheless, these studies did not take into account potential modifying factors such as co-exposure to other chemicals and the location of sample collection as pointed out by Teeguarden et al (24, 25). Thus, additional studies are needed to fully understand the associations between BPA exposure and fertility outcomes in women.

Table 1.

BPA and fertility (epidemiological studies)

Study design	Study population	Sample size	Time of BPA measurement	BPA concentration	Outcome	Ref
Cross-sectional	Women undergoing in vitro fertilization	44	Day of oocyte retrieval	Median unconjugated serum BPA 2.53 ng/ml (range 0.3–67.36 ng/ml)	BPA inversely associated with peak estradiol; No association with number of oocytes retrieved	(19)
Matched cohort	Women discontinuing contraception	210	On the day of expected menstruation	Urine (total BPA), mean (95% confidence interval): Pregnant: 0.63 ng/ml (0.54–0.73); Non pregnant: 0.68 ng/ml (0.53–0.87)	No association with impaired fecundity or time to pregnancy	(30)
Cross-sectional	Fertile and infertile Italian women	12 fertile 35 infertile	Enrollment	Not indicated; limit of detection 0.5 ng/ml (serum samples)	Higher percentage of infertile women with detectable serum BPA levels	(17)
Prospective	Women undergoing in vitro fertilization	239 (63 no soy food intake); 347 cycles	Between day 3–9 of gonadotropin phase and on day of oocyte retrieval	Urine (total BPA). Range <0.4 μg/l to 16.6 μg/l; median 1.3 μg/l (interquartile range 0.9 – 1.9 μg/l)	Soy food consumption modifies the correlation between BPA and infertility treatment outcomes	(29)
Prospective	Women undergoing in vitro fertilization	174 (237 cycles)	Day of oocyte retrieval	Urinary (total BPA) geometric mean 1.50 (standard deviation 2.22) μg/l	Higher BPA levels associated with lower serum peak E2, oocyte yield, MII oocyte count, and number of normally fertilizing oocytes	(20)
Prospective	Women undergoing intracytoplasmic sperm injection and in vitro fertilization	58	Day of oocyte retrieval	Median unconjugated serum BPA 2.53 ng/ml (range 0.0–67.4 ng/ml)	Inverse associations between BPA and oocyte maturation (Asian women) and normal fertilization (all women)	(22)
Pregnancy-based retrospective	Women in 1st trimester	1,742	Spot urine during the 1st trimester visit	Urinary (total BPA) geometric mean 0.78 (0.73 – 0.82) ng/ml	No association with diminished fecundity	(31)
Prospective	Women undergoing in vitro fertilization	35 of total of 58	Day of oocyte retrieval	Median unconjugated serum BPA 2.4 μg/l serum (range 0.0 – 67.4)	Up-regulation of TSP50 with increased BPA levels due to a loss of methylation	(26)
Prospective	Fertile women that discontinued contraception	221	Pooled urine throughout menstrual cycles	Urinary (total BPA) median 2.7 ng/ml (interquartile range 1.8, 4.3), not adjusted	BPA associated with shorter luteal phase; Null associations with follicular-phase length, time to pregnancy, and early pregnancy loss	(32)
Cross-sectional	Women who volunteered	110 infertile, 43 fertile	Whole blood sample prior to any treatment	Mean (total BPA, serum) (ng/ml): fertile 4.8 infertile 10.6	BPA levels higher among infertile women (odds ratio 8.3) in metropolitan area	(18)
Prospective	Women undergoing in vitro fertilization	256 (375 cycles)	Between day 3–9 of gonadotropin phase; and on day of oocyte retrieval	Urinary (total BPA) geometric mean 1.87 μg/l	No associations with oocyte yield, endometrial thickness, embryo quality, fertilization rates, implantation, clinical pregnancy and live birth rates	(33)
Prospective	Women undergoing in vitro fertilization	84 (112 cycles)	Day of oocyte retrieval	Urinary (total BPA). Range <0.4 to 25.5 μg/l; urinary geometric mean 2.52 μg/l (standard deviation 3.2)	BPA levels inversely associated with the number of oocytes retrieved and peak serum estradiol levels	(21)
Prospective	Women undergoing in vitro fertilization	36	Day of oocyte retrieval	Median unconjugated serum BPA 3.3 ng/ml (range 0.0–67.4 ng/ml)	No association with embryo quality	(23)

Limited information is available on the potential molecular targets of BPA in infertile women, but Hanna et al. reported an association between higher serum levels of unconjugated BPA and decreased methylation within the TSP50 gene promoter in whole blood samples of women undergoing IVF treatments (26). However, the researchers did not provide any mechanistic explanations of these findings other than to indicate that TSP50 may be an oncogene based on previous research by other groups (27, 28). Interesting findings reported by Chavarro et al. suggest a potential modifying effect of soy food consumption on the inverse correlations between urinary total BPA concentrations and fertility treatment outcomes (29). Overall, these studies are suggestive for potential associations between BPA and infertility. However, additional studies are needed to determine possible cause and effect relationship and the mechanism of action of BPA-mediated effects on fertility in healthy women.

Not all epidemiological studies found an association between BPA exposure and fertility outcomes. Null associations were reported between urinary total BPA concentrations and impaired fecundity or time to pregnancy in generally healthy women (30–32). In another study, null associations between urinary total BPA concentrations and number of oocytes retrieved, embryo quality, and fertilization rates were reported in women undergoing IVF treatments (33). The differences in the results may be explained by differences in sample characteristics (i.e., generally healthy women without any reported infertility issues versus women undergoing IVF treatments) and by differences in sample size.

Studies utilizing animal models provide further insights on the effects of BPA exposure on female fertility (Table 2). In mice, Berger et al. reported that low dose BPA exposure of pregnant dams during the pre-implantation period significantly reduced the number of litters and litter size compared to controls (34). Further, in utero post-implantation low dose BPA exposure affected the fertility of the females in the subsequent generations (35, 36). Cabaton et al. performed a forced breeding study and found that low dose BPA-exposed females had fewer pregnancies and overall reduced cumulative number of pups compared to controls (37). Moore-Ambriz et al. examined the effects of BPA exposure in young adult mice on fertilization capacity later in adult life (38). The fertilization rate of BPA exposed females was reduced compared to controls (38). Further, impaired fertility was also reported in a study that examined the effects of in utero low dose BPA exposure in three subsequent generations of mice (35, 36). Specifically, F1 females that were gestationally exposed to BPA had reduced fertility, reduced litter size (36), and reduced ability to maintain pregnancy to term (i.e., reduced gestational index) compared to controls (35). Further, F2 females had a reduced gestational index compared to controls (35). In addition, F3 females exhibited reduced fertility and decreased ability to become pregnant compared to controls, indicating a potential transgenerational effect of BPA on female reproduction (35). In chickens, in ovo high dose BPA exposure reduced hatchability (39), whereas in fish, low dose BPA exposure increased the observed hatching rate (40).

Table 2.

BPA and fertility (experimental studies)

Source	Strain	Exposure route	Time of exposure	Doses	Time of observation	Outcome	Ref
Mouse	CF-1	Subcutaneous injection	GD1-4	0–10.125 mg/day	After birth	BPA10.125 reduced percent of females giving birth; BPA3.4 and 10.125 reduced number of pups born	(34)
Mouse	CD-1	Alzet osmotic pump	GD8 – PND16	25 ng, 250 ng, 25 μg/kg/day	F1: from 8 weeks until 32 weeks (forced breeding)	BPA 25ng females had fewer pregnancies and lower cumulative number of pups per dam that got worse with age	(37)
Mouse	C57BL/6NHsd, Hsd:ICR (CD-1 Swiss)	Extruded pellet diets	Prior to breeding-PND21	0.03, 0.3, 30 ppm	After birth	No effect on fertility of C57BL6; Decreased fertility of CD-1 (BPA0.03 and 0.3)	(44)
Mouse	C57BL/6J	Dietary	GD6 – PND21	0.33, 3.3, 33 ppm	After birth	No effect on number of births or litter size	(43)
Rat	Sprague-Dawley	Dietary	GD6 – PND21	0.33, 3.3, 33 ppm	After birth	No effect on number of births or litter size	(42)
Fish	Pimephales promelas	Aquarium water	164 days exposure	1, 16, 64, 160, 640 μg/l	On exposure days 85–105, 135–155	No effect on fecundity	(48)
Mouse	C57BL/6J (39 days)	Oral	12–15 days (first 3 reproductive cycles)	50 μg/kg/day	Age 51–54 days	Reduced fertilization (in vitro fertilization or mating); Did not alter zygotic stages	(38)
Fish	Transgenic Zebrafish	Aquarium	embryonically	0.1, 1, 10, 100, 1000 μg/l	48, 55 hours post fertilization	BPA1 and 10 increased hatching rate	(40)
Rat	Long Evans	Oral Gavage + lactation	GD7-PND18	2, 20, 200 μg/kg/day	After birth and over 4 months	No effect on litter size	(47)
Rat	Wistar	Oral, drinking water	GD9-birth	0.5, 50 μg/kg/day	After birth	No effect on litter size or number of births	(41)
Rat	Wistar	Drinking water + lactation	GD9 – PND21	0.5, 50 μg/kg/day	After birth	No effect on litter size	(60)
Mouse	FVB	Oral	GD11-birth	0.5, 20, 50 μg/kg/day	F1: 3, 6, 9 months	BPA0.5 reduced fertility with age, increased % dead pups with time (3–9 months); BPA50 reduced litter size (6 months)	(36)
Mouse	CD-1	Oral gavage	F0: GD1 – PND20	12, 25, 50 mg/kg/day	After birth	No effect on litter size	(46)
Chicken	White Leghorn	In ovo injection	Incubation day 4	67, 134 μg/g egg	Hatching	Decreased hatchability (BPA 67, 134)	(39)
Mouse	CD-1	Oral	GD12.5 – PND18.5	0.2, 0.04, 0.08 mg/kg	PND1	No effect on litter size	(45)
Mouse	FVB	Oral of pregnant dams (F0)	GD11-birth	0.5, 20, 50 μg/kg/day	F1, F2, F3: 3, 6, 9 months	BPA50 reduced gestational index (F1, F2); BPA0.5 reduced fertility index (F3); Reduced % of dead pups (F3, BPA20, 50); Decreased ability to maintain pregnancy with age	(35)

In contrast, some experimental studies reported that BPA exposure does not affect fertility outcomes. Specifically, a few studies indicate that gestational low dose BPA exposure did not alter number of litters (41–44) or litter size (41–48) in mice, rats, and fish. Xi et al., also indicate that gestational BPA exposure at a dose of 50 mg/kg/day (i.e. LOAEL) did not alter litter size in mice (46). Similarly, Moore-Ambriz et al. reported that low dose BPA exposure did not affect the size of preovulatory follicles, the number of shed oocytes, and zygotes in adult mice that were exposed to BPA at a younger age (38). One of the reasons for differences between the reported results may be the age of the animals. Studies that examined reproductive capacity in older animals were more likely to observe a difference between the BPA treated females and controls.

In summary, several studies indicate that BPA levels may be higher among infertile women than fertile women and that BPA exposure may reduce fertility in animal models. However, further studies are needed to link findings from epidemiological studies and experimental studies.

To provide information about the potential mechanisms by which BPA impairs fertility, below we focus on reproductive organs that are targeted by BPA in a manner that could reduce fertility. Specifically, the sections below focus on BPA-induced abnormalities in reproductive organs that stem from changes in morphology, function, gene expression, and levels of proteins or hormones related to reproduction. We review the recent studies on the effects of BPA on the oviduct, uterus and implantation, estrous cyclicity, ovary, and hypothalamic-pituitary axis.

Oviduct

Following ovulation, the oocyte travels from the ovary through the oviduct to allow fertilization. Upon successful fertilization, the conceptus will continue to travel through the oviduct until it is settled in the uterus. Thus, a normal functioning oviduct is required for fertility. The available recent evidence on the effects of BPA exposure on the oviduct is extremely limited and is based only on experimental studies in mice (Table 3). Specifically, in utero low dose BPA exposure resulted in the appearance of progressive proliferative lesions in the oviduct and remnants of the Wolffian duct during adult life (49, 50). Further, studies indicate that in utero high dose BPA exposure delayed development and transport of the conceptus compared to controls (51, 52). Taken together, the current data indicate that gestational BPA exposure may affect both oviduct morphology and function; however, further studies are needed to confirm whether this is the case in women and to examine potential molecular mechanisms of BPA action on the oviduct.

Table 3.

BPA and oviduct (experimental studies)

Source	Strain	Exposure route	Time of exposure	Doses	Time of observation	Outcome	Ref
Mouse	CD-1	Subcutaneous injection	GD 1-5	10, 100, 1,000 μg/kg/day	18 months	Increased progressive proliferative lesions; remnants of Wolffian ducts	(50)
Mouse	CD-1	Subcutaneous injection	GD 9-16	0.1, 1, 10, 100, 1,000 μg/kg/day	18 months	Increased progressive proliferative lesions	(49)
Mouse	Not indicated	Oral gavage	GD0.5 – 3.5	200, 400, 600, 800 mg/kg/day	GD4	BPA 400 – 800 delayed transfer of embryos from fallopian tubes to uterus (GD4)	(51)
Mouse	C57BL6	Subcutaneous injection	GD 0.5-3.5	0.025, 0.5, 10, 40, 100 mg/kg/day	GD3.5	BPA100 retention of embryos and delayed embryo development	(52)

Uterus

Implantation

Implantation is required for the establishment of pregnancy. During this stage, the blastocyst attaches to the uterine wall. Thus, exposures that interfere with implantation have the potential to impact fertility. The scientific evidence for a possible link between BPA exposure and impairments in implantation is based on one epidemiological study (Table 4) and several experimental studies (Table 5). Specifically, higher urinary total BPA levels were associated with increased implantation failure defined by a serum β-human chorionic gonadotropin test (β-hCG < 6 IU/L) conducted 17 days after egg retrieval in women undergoing IVF treatments (53).

Table 4.

BPA and implantation (epidemiological study)

Study design	Study population	Sample size	Time of BPA measurement	BPA concentration	Outcome	Ref
Prospective	Women undergoing in vitro fertilization	137 (180 in vitro fertilization cycles)	Day of oocyte retrieval	Urinary (total BPA) geometric mean 1.53 (standard deviation 2.22) μg/l	Higher BPA levels associated with increased implantation failure	(53)

Table 5.

BPA and implantation (experimental studies)

Source	Strain	Exposure route	Time of exposure	Doses	Time of observation	Outcome	Ref
Mouse	CF-1	Subcutaneous injection	GD1 - 4	0–10.125 mg/day	GD6	BPA10.125 decreased number of implantation sites	(34)
Mouse	CF-1	Subcutaneous injection	GD0, 1, 2	0–10.125 mg/day	GD6	BPA10.125 and 6.75 decreased implantation sites	(56)
Mouse	CF-1	Subcutaneous injection	GD1 - 4	0–10.125 mg/day	GD6	BPA6.75 and 10.125 decreased number of implantation sites	(56)
Mouse	CF-1	Subcutaneous injection	GD1 - 4	3.375, 6.75, 10.125 mg/day	GD6	BPA6.75 and 10.125 decreased number of implantation sites	(55)
Mouse	CF-1	Subcutaneous injection	GD1 - 4	3, 4, 5 mg/day	GD6	No difference in number of implantation sites	(58)
Mouse	CF-1	Subcutaneous injection	GD1 - 3	2, 4 mg/day (61, 122 mg/kg/day)	GD6	No difference in number of implantation sites	(59)
Mouse	CD-1	3 times a day feeding	PND22-GD9	60, 600 μg/kg/day	Through GD9	Impaired implantation and impaired PGR-HAND2 pathway	(54)
Mouse	Not indicated	Oral gavage	GD0.5 – 3.5	200, 400, 600, 800 mg/kg/day	GD4.5	BPA 400 – 800 delayed transfer of embryos from fallopian tubes to uterus and decreased number of implantation sites	(51)
Rat	Wistar	Subcutaneous injection	PND1, 3, 5, 7	0.05, 20 mg/kg/day	GD5, GD18	Reduced implantation sites	(57)
Mouse	C57BL6	Subcutaneous injection	GD0.5-3.5	0.025, 0.5, 10, 40, 100 mg/kg/day	GD4.5	No implantation sites (BPA100); delayed implantation (BPA40)	(52)
Mouse	C57BL6	Subcutaneous injection	GD0.5-3.5	100 mg/kg/day	GD4.5	No implantation sites when untreated embryos transferred to pseudo-pregnant females pre-treated with BPA100	(52)

Experimental studies examining the effects of BPA exposure at early gestational stages report a reduced number (34, 51, 54–57) or complete ablation of implantation sites (52) in mice and rats when compared to controls. These studies include both low (34, 54–57) and high (51) BPA doses. Further, a recent study by Li et al. (54) demonstrated that low dose BPA exposure reduced uterine levels of leukemia inhibitory factor (Lif), progesterone receptor (Pgr), heart and neural crest derivatives expressed transcript 2 (Hand2), and homeobox A10 (Hoxa10) compared to controls. These observed BPA mediated effects can impair fertility because these factors are part of the progesterone-mediated signaling pathway and are important in uterine receptivity and implantation.

Although the majority of studies indicate that BPA alters implantation, two experimental studies report no effect of low dose (58) or a relatively high dose (122 mg/kg/day) (59) of BPA on the number of implantation sites. The reasons for these discordant results are unclear, but it may be that the effects of BPA on implantation are ablated at high doses.

Uterine morphology and function

For proper blastocyst invasion, implantation, and successful pregnancy, the uterine endometrium transforms and reorganizes under the influence of estrogen and progesterone. Thus, exposures that interfere with uterine function have the potential to adversely impact fertility. Based on our search criteria, we did not locate epidemiological studies that focus on BPA exposures and associations with uterine outcomes. However, several in vivo and in vitro experimental studies have examined the effects of BPA exposure on the uterus or uterine cells (Tables 6 and 7).

Table 6.

BPA and uterus (morphology and function) experimental studies (in vivo)

Source	Strain	Exposure route	Time of exposure	Doses	Time of observation	Outcome	Ref
Non-human primate	African green monkey	Alzet minipump	Adult	50 μg/kg/day	End of 28 days of treatment	BPA increased levels of glandular and stromal PR; BPA + estradiol benzoate decreased PR expression; BPA may antagonize estradiol effects on PR expression	(66)
Rat	Sprague-Dawley	Subcutaneous injection	PND 17-19	10, 100, 500 mg/kg/day	PND20	BPA500 changed levels of contraction-associated proteins and decreased uterine contractility	(75)
Rat	Sprague-Dawley	Cultured uterine tissue	PND 20	10−5 M	24, 48 hours	Decreased uterine contractility	(75)
Mouse	CF-1	Subcutaneous injection	GD1-4	3.375, 6.75, 10.125 mg/day	GD6	BPA6.75 and 10.125 increased uterine luminal area and cell height	(55)
Rat	Wistar	Subcutaneous injection	PND1, 3, 5, 7	0.05, 20 mg/kg/day	(Ovx PND80) PND94	BPA0.05 decreased endometrial proliferation, decreased levels of VEGF and ERa in subepithelial cells, and ERa in endothelial cells; BPA0.05, 20 increased expression of NCOR1 in subepithelial cells	(63)
Mouse	CD-1	Intraperitoneal injection	GD 9 – 16	5 mg/kg	2, 6 weeks old	Increased Hoxa10 mRNA and HOXA10 protein levels; Hypomethylation of promoter and intron of Hoxa10	(70)
Non-human primates	Macaca mulatta	Oral	GD50-100 or GD100 - 165	400 μg/kg/day	GD100 or GD 165 (fetuses)	Differences in levels of HOX and Wnt/Fzd family genes on GD 165	(67)
Rat	Sprague-Dawley	Oral gavage	GD6 –birth	2.5, 8, 25, 80, 260, 840, 2,700, 100,000, 300,000 μg/kg/day	F1 PND90	No effect on Vegfa, Pgr, S100g, or C3 expression	(71)
Mouse	ICR	Subcutaneous injection	GD12-16	100, 200, 500, 1,000 mg/kg/day	Not directly exposed F2 (8 weeks)	BPA100 decreased uterine weight; Possible effects on Hoxa10 methylation	(61)
Chicken	White Leghorn	In ovo injection	Incubation day 4	67, 134 μg/g egg	21 weeks old	Decreased uterine tubular glandular density and tunica mucosa thickness (BPA134)	(39)
Mouse	CD-1 Swiss	Extruded pellet diets		0.03, 0.3, 30 ppm		BPA0.3 increased sensitivity to develop pyometra (C57BL6)	(44)
Mouse	ICR	Injection	Adult (OVX)	10–500 mg/kg	2 hours post injection	BPA regulated Egr1 expression through ER-ERK1/2 pathways; BPA induced phosphorylation of AKT andERK1/2 via non-genomic actions of ERs	(68)
Rat	Wistar	Drinking water and lactation	GD6 – PND 21	10 mg/l (1.2 mg/kg/day)	F1 4 months	Increased thickness of uterine epithelia and stroma	(64)
Mouse	ICR	Subcutaneous injection	PND 8	0.1, 1, 10, 100 mg/kg	PND25, 30, 70	Reduced uterine weight (BPA100)	(62)
Mouse	CD-1	Subcutaneous injection	GD1-5	10, 100, 1000 μg/kg/day	18 months	Cystic endometrial hyperplasia (BPA100)	(50)
Mouse	CD-1	Subcutaneous injection	GD9-16	0.1, 1, 10, 100, 1000 μg/kg/day	18 months	Adenomatous hyperplasia (BPA1 and 100), stromal polyps (BPA100), endometrial polyps (BPA0.1, 1 and 10), squamous metaplasia (BPA1 and 10), and remnants of Wolffian duct (all except BPA100)	(49)
Mouse	CD-1	Intraperitoneal injection	GD9-16	0.5, 1.0, 5.0, 50, 200 mg/kg	F1 2–6 weeks after birth	Increased in HOXA10 in dose response fashion (BPA0.5-5)	(69)
Rat	Wistar	Subcutaneous injection	Neonatal PND1, 3, 5, and 7	0.05, 20 mg/kg/day	PND8, (OVX at PND80) PND94	PND8: downregulation of Hoxa10, Hoxa11; PND94: downregulation of Era, Hoxa11 (BPA0.05), and Hoxa10 (BPA 0.05, 20); Impaired proliferative response to P+E (BPA0.05 and 20)	(65)
Rat	Wistar	Subcutaneous injection	Neonatal PND1, 3, 5, and 7	0.05, 20 mg/kg/day	F1 GD5 or GD18	Reduced implantation sites, decreased Hoxa10, ITGB3, Pr and Esr1, increased EMX-2 (BPA20); Decreased ESR1 (BPA0.05 and 20) and PRa and PRb (BPA20)	(57)
Rat	Wistar	Drinking water + lactation	GD9 – PND21	0.5, 50 μg/kg/day	F1 PND90, PND360	PND 90: decreased glandular epithelium proliferation (BPA0.5 and 50) and decreased percentage of α-SMA-positive stromal cells (BPA50); PND 360: increased anomalies in uterine luminal epithelium (BPA0.5, 50) and glands (BPA50)	(60)

Table 7.

BPA and uterus (morphology and function) experimental studies (in vitro)

Source	Strain	Exposure route	Doses	Time of observation	Outcome	Ref
Human	Endometrial stromal fibroblasts	Culture	5–100 μmol/l	48 hours	Decreased proliferation, induced IGFB1 (BPA50), decreased CYP11A, HSD17B1, HSD17B2; No effect on PRL levels	(9)
Human	Ishikawa cells	Culture	1 μM	24 hours	BPA or BPA + estradiol increased levels of PR; BPA + estradiol decreased PR expression compared to estradiol only; BPA may antagonize estradiol effects on PR expression	(66)
Human	Endometrial endothelial cells	Culture	0.1, 50, 100 nM	24 hours	BPA0.1-100 decreased proliferation and viability; BPA100 increased necrosis	(74)
Human	Endometrial endothelial cells	Culture	50 μM	24 hours	Decreased cell proliferation	(72)
Human	Primary stromal endometrial cells (proliferative phase)	Culture	0.01 mM, 0.01 μM, 0.01 nM; decidualization induced by P4	24, 48, 72 hours	No effect on proliferation; BPA (0.01 μM and 0.01 nM) increased G2/M and decreased G0/G1 fractions; BPA increased PRL (0.01 μM and 0.01 nM), LEFTY (0.01 μM), and IGFBP1 (0.01 mM, 0.01 μM and 0.01 nM)	(76)
Human	5 cycling women undergoing hysterectomy	Culture	10 μM, 0.1 μM, 1 nM, 0.01 nM	24 hours	No effect on cell viability or proliferation; Increased angiogenic activity (10 μM); No difference in VEGF, ESR2, and GPR30 levels	(78)
Human	Decidualized stromal cells	Culture	1–100 pM, 1–100 nM, 1–100 μM	24, 48 hours	No effect on cell viability; Increased proliferation (48 hours; 100 nM)	(73)
Human	Decidualized stromal cells	Culture	1 pM, 1 nM, 1 μM	24, 48 hours	BPA1 μM: decreased PRL; no effect on IGFBP1, increased ESR1 and ESR2 (1nM) BPA1 μM increased PR protein and PRA PRB mRNA; BPA1pM decreased hCG/LH-R protein and increased MIF protein secretion	(73)
Human	Ishikawa cells	Culture	1 nM, 100 nM, 10 μM, 100 μM	8, 24, 48 hours	No effect on cell viability; Affected multiple molecular pathways associated with cell organization and biogenesis, translation, proliferation, and intracellular transport	(77)
Human	Ishikawa cells	Culture	0.1 nM–25 μM, 1 μM	24 hours	Increase in HOXA10 in a dose response manner	(69)

In mice, in utero low dose BPA exposure increased uterine anomalies in the luminal epithelium and glands (60) and caused uterine hyperplasia, stromal polyps, and retention of remnants of the Wolffian duct in the adult offspring compared to controls (49, 50). Further, in utero high dose BPA exposure reduced uterine weight in the second generation of pups compared to controls (61). Neonatal high dose BPA exposure (single dose, 100 mg/kg) reduced uterine weight in young adult mice compared to controls (62), and neonatal low dose BPA exposure decreased endometrial proliferation in adult ovariectomized rats compared to controls (63). In young adult rats, low dose BPA exposure from gestation day 6 until weaning increased the thickness of the uterine epithelia and stroma compared to controls (64). In adult mice, dietary supplementation with low dose BPA resulted in clinical signs that are typical of pyometra (44). Lastly, in hens, in ovo high dose BPA exposure resulted in abnormal uterine morphology compared to controls (39). Overall, the results from in vivo studies are suggestive for impaired morphology of the uterus following early life stage BPA exposures at both low and high doses.

Some of the molecular factors in the uterus that were altered following in vivo BPA exposure include members of the Hoxa family, vascular endothelial growth factor (Vegf), estrogen receptor alpha and beta (Esr1 and Esr2), and Pgr (57, 63, 65–70). These factors are important for endometrial proliferation and receptivity. In contrast, in utero high dose BPA exposure did not affect expression levels of coding complement component 3 (C3), Pgr, calbindin D9K (S100g), and Vegfa in the adult offspring of rats (71); hence, it is unclear whether they are primary targets for BPA-induced uterine toxicity.

Findings from in vitro studies utilizing various human cell lines indicate that low dose BPA exposure decreased endothelial cell proliferation (9, 72) and increased decidualized stromal cell proliferation (73) compared to controls. Similarly, high dose BPA exposure decreased endothelial cell proliferation compared to controls (74). Some of the potential mechanisms through which BPA may affect cell proliferation in the uterus include alterations in insulin-like growth factor binding protein 1 (IGFBP1), macrophage migration inhibitory factor (MIF), HOXA10, and left right determination factor 1 (LEFTY), steroidogenic receptors (e.g., ESR1, ESR2, PGR), and enzymes (e.g., cytochrome P450, family 11, subfamily a, polypeptide 1; CYP11A1, hydroxysteroid (17-beta) dehydrogenase 1; HSD17B1, hydroxysteroid (17-beta) dehydrogenase 2; HSD17B2), or other hormones (e.g., PRL, LH) (9, 61, 62, 64, 66, 69, 73, 75–77). Further, Nacif et al. (77) performed a microarray on Ishikawa cells that were cultured with a range of low and high BPA doses (1nM–100μM) and found that multiple molecular pathways (e.g., cell organization and biogenesis, proliferation, and intracellular transport) were altered in response to BPA compared to controls.

In contrast, two studies on human cell lines cultured with low and high BPA doses found no effect on proliferation of primary stromal endometrial cells (76, 78). Differences in cell viability or proliferation may be due to the study design and experimental model. For example, differences in the source of the cells (carcinogenic tissue versus normal) and differences in the experimental cell lines (primary versus established/immortal cell line lines such as Ishikawa). Nevertheless, the majority of the in vitro studies support the observations reported in the above-mentioned in vivo studies.

Lastly, at the end of pregnancy, the uterus needs to contract to induce labor. Uterine contractions are under the control of endogenous hormones such as estradiol, progesterone, oxytocin, and prostaglandins (79). The effects of BPA on uterine contractility were investigated in one study in rats (75). Findings from this study suggest that BPA exposure decreased uterine contractility and altered transcript and protein levels of contraction-associated factors (75). Specifically, high dose BPA exposure increased oxytocin and oxytocin receptor, and decreased prostaglandin (PG)-F2α receptor compared to controls (75). Overall, the current literature suggests that BPA exposure selectively affects uterine cell proliferation and function, depending on the study model. These effects of BPA on uterine function could lead to adverse effects on fertility.

Estrous cyclicity

Estrous cyclicity is crucial for ovulation and the preparation of the uterus for potential implantation. Hence, chemical exposures that disrupt estrous cyclicity can impair fertility. Multiple experimental studies examined the effects of BPA exposure on estrous cyclicity (Table 8). Early neonatal low (80–82) and high (62) dose BPA exposure caused increased or decreased days in estrus and overall altered cycles in adult mice or rats (70–90 days) compared to controls. In contrast, low dose BPA exposure at earlier time points (51–54 days old) had no effect on estrous cyclicity (38). In rats, in utero low dose (64) and high dose (83, 84) BPA exposure resulted in irregular estrous cycles in the offspring compared to controls. Moreover, studies published by Wang et al. (36) and Ziv-Gal et al. (35) examined the effects of in utero exposure of low dose BPA on estrous cyclicity in subsequent generations of mice. Interestingly, BPA-induced altered cyclicity was observed in both the F1 and F3 generations, but not in the F2 generation compared to controls. In contrast, other studies reported no effect of in utero low dose BPA exposure or 50 mg/kg/day (i.e. LOAEL) on estrous cyclicity of rats and mice offspring (41, 46, 60). Differences in study design and timing of evaluation of estrous cyclicity may explain the disagreement between the results. Overall, neonatal BPA exposure may affect estrous cyclicity in older animals. However, the evidence regarding the effects of in utero BPA exposure on estrous cyclicity is inconclusive. Further studies that examine the effects of in utero BPA exposure on estrous cyclicity and that encompass multiple generations are needed to fully understand the effects of BPA on estrous cyclicity.

Table 8.

BPA and estrous cyclicity

Source	Strain	Exposure route	Time of exposure	Doses	Time of observation	Outcome	Ref
Rat	Long Evans	Injections	PND0 - 3	50μg/kg/day, 50 mg/kg/day	Post weaning and vaginal opening	By week, 15 only 33% of females (BPA50 mg/kg) cycled regularly	(84)
Rat	Sprague Dawley	Oral gavage	GD6-birth+ PND1-15 or 21	2.5, 8, 25, 80, 260, 840 μg/kg/day, 2.7, 100, 300 mg/kg/day	F1 PND15, 21, 90, PND69-90, 150-170 (estrous)	BPA300: abnormal cyclicity (PND90 and 150)	(83)
Rat	Sprague Dawley	Subcutaneous injection	PND1 - 10	50 μg/50 μl, 500 μg/50 μl	PND21 PND60-120	BPA500 showed irregular cycles with more days at estrus, after PND90	(80)
Rat	Sprague Dawley	Oral gavage	90 days	0.001, 0.1 mg/kg/day	30 days after the age of 21 weeks	Extended estrous phase (2–7 days)	(81)
Rat	Wistar	Drinking water and lactation	GD6 – PND21	10 mg/l (1.2 mg/kg/day)	F1 3 months of age for 4 consecutive weeks	Irregular estrous cycles	(64)
Rat	Wistar	Subcutaneous injection	PND1, 3, 5, and 7	0.05, 20 mg/kg/day	PND85-100	BPA0.05: more time at proestrus-estrus	(82)
Mouse	C57BL6J	Oral	12–15 days (first 3 reproductive cycles)	50 μg/kg/day	51–54 days	No effect on estrous cyclicity	(38)
Mouse	ICR	Subcutaneous injection	PND8	0.1, 1, 10, 100 mg/kg	Observed: PND20-29; scarified: PND25, 30, 70	BPA100: decreased number of days in estrus	(62)
Rat	Wistar	Oral, drinking water	GD9-birth	0.5, 50 μg/kg/day	F1: PND45, 90	No effect on estrous cyclicity	(41)
Rat	Wistar	Drinking water + lactation	GD9 – PND21	0.5 or 50 μg/kg/day	F1: PND90, PND360	No effect on estrous cyclicity	(60)
Mouse	FVB	Oral	GD11-birth	0.5, 20, 50 μg/kg/day	F1 on PND21	PND21: shorter time span between vaginal opening and first estrus (BPA50); BPA0.5 less time in proestrus and estrus and more in diestrus and metestrus; BPA20 shortened estrus	(36)
Mouse	CD-1	Oral gavage	GD1 – PND20	12, 25, 50 mg/kg/day	F1 on PND50	No effect on estrous cyclicity	(46)
Mouse	CD-1	Oral gavage	PND21 - 49	25, 50 mg/kg/day	PND50	No effect on estrous cyclicity	(46)
Mouse	FVB	Oral of pregnant dams (F0)	GD11-birth	0.5, 20, 50 μg/kg/day	F1, F2, F3: 3, 6, 9 months	F3: delayed age at first estrus (BPA50)	(35)

Ovary

The ovary is required for normal production of ova for fertilization and for production of sex steroid hormones that regulate estrous cyclicity and fertility. Thus, BPA exposures that target the ovary can interfere with fertility. One epidemiological study examined the associations between BPA levels and ovarian volume and mature follicle counts (Table 9) (85). Results from this study indicate that urinary BPA exposure was negatively correlated with antral follicle counts in women undergoing IVF treatments (85).

Table 9.

BPA and ovary, epidemiological study

Study design	Study population	Sample size	Time of BPA measurement	BPA concentration	Outcome	Ref
Prospective	Women undergoing in vitro fertilization	Overall 209; Antral follicle count=154; FSH=120; Ovarian volume= 114)	Urine: upon entry into the study and at subsequent treatment cycle visits; Ultrasound: 3rd day of an unstimulated menstrual cycle	Urinary geometric mean (geometric standard deviation). Antral follicle count= 1.6 (2.0); FSH= 1.7 (2.1); Ovarian volume= 1.5 (1.8)	BPA not associated with day 3-FSH or ovarian volume; Higher urinary BPA concentrations associated with lower antral follicle counts	(85)

Findings from experimental studies indicate that in utero or neonatal low and high dose BPA exposures resulted in abnormal ovarian morphology and histology compared to controls (Table 10). Specifically, BPA exposure increased the number of multi-oocyte follicles (86), inhibited germ cell nest breakdown (36, 45, 87), decreased the number of primordial follicles (45, 87), increased apoptotic oocytes (87), and increased primordial follicular recruitment (36, 88). It also affected follicle type distribution by reducing the number of antral follicles and increasing the numbers of primary and secondary follicles (89).

Table 10.

BPA and ovary

Source	Strain	Exposure route	Time of exposure	Doses	Time of observation	Outcome	Ref
Rat	Long Evans	Subcutaneous injection	PND0 - 3	50 μg/kg/day, 50 mg/kg/day	Post weaning and vaginal opening	Abnormal ovarian morphology – multinucleated and hemorrhagic tissue (BPA50 mg/kg)	(84)
Mouse	FVB	Oral	GD11-birth	0.5, 20, 50 μg/kg/day	F1, F2, F3: PND4, 21	PND4: no effect on germ cell nest breakdown or % of primordial follicles; Reduced Bcl2 (BPA0.5, F2), oxidative stress, autophagy, and altered gene expression; PND21: some effect on follicle numbers and oxidative stress. Altered expression of apoptotic, steroidogenic, Esr1, Ar, and Igf family genes	(98)
Mouse	CD-1	Subcutaneous injection	PND7 – 14	20, 40 μg/kg/day	PND15	Accelerated primordial to primary follicle transition	(91)
Mouse	CD-1	Subcutaneous injection	PND5 – 20 (every 5 days)	20, 40 μg/kg/day	PND21	Accelerated primordial to primary follicle transition	(91)
Rat	Sprague Dawley	Oral gavage	GD6-birth+ PND1-15 or 21	2.5, 8, 25, 80, 260, 840 μg/kg/day, 2.7, 100, 300 mg/kg/day	PND15, 21, 90, PND69-90, PND150-170	BPA300: Small ovaries with depletion of corpora lutea and antral follicles	(83)
Human	In vitro fertilization patients	Granulosa-lutein cells culture	72 hours	1–10,000 ng/ml	End of culture	BPA 1,000 and 10,000: decreased cell viability; BPA 100 and 1,000: increased MMP-9; BPA 10,000: decreased MMP-9	(114)
Rat	Wistar	Drinking water + lactation	GD0 – PND21	3 μg/kg/day	PND30	No difference in ovarian weight, higher total follicle number, higher primary, secondary, and lower antral follicle numbers; Increased atresia	(89)
Swine		Granulosa cells culture	48 hours	0.1, 1, 10 μM	End of culture	No effect on cell proliferation; VEGF secretion stimulated (BPA1, 10); No effect on oxidative stress	(113)
Non-human primate	Rhesus macaque	Silastic pump	GD100 - term	2.2–3.3 ng/ml serum levels	PND0	Increased number of multi-oocyte follicles, impaired oocyte development (unenclosed oocytes)	(86)
Human	In vitro fertilization patients	Granulosa-lutein cells culture	48 hours	40, 60, 80, 100 μM	End of culture	BPA 40-100: inhibited proliferation, decreased FSH induced genes (IGF-1, aromatase) and altered aromatase regulators (GATA4, SF-1, PPARγ)	(112)
Rat	Sprague Dawley	Oral gavage	90 days	0.001, 0.1 mg/kg/day	21 weeks old	Increased apoptosis and CASP3, decreased aromatase expression (granulosa cells)	(81)
Rat	Wistar	Intraperitoneal injection	PND28 - 35	10, 40, 160 mg/kg	PND35	Decreased follicle numbers, increased atretic follicles; Decreased H1FOO and FIGLA(BPA160), increased AMH	(92)
Zebrafish	Danio rerio	Aquarium water	14 days	1, 10, 100, 1000 μg/l	End of exposure	Abnormal ovarian follicles; Dose dependent increased atresia and decreased primordial follicles	(93)
Mouse	C57BL6J	Oral	12–15 days (during first 3 cycles)	50 μg/kg/day	51–54 days	No differences in follicle type distribution	(38)
Mouse	ICR	Subcutaneous injection	PND8	0.1, 1, 10, 100 mg/kg	Observed: PND20-29; scarified: PND25, 30, 70	All BPA groups: reduced ovarian weight (PND25, 30)	(62)
Mouse	CD-1	Subcutaneous injection	GD9-16	0.1, 1, 10, 100, 1000 μg/kg/day	18 months	Ovarian cysts (BPA 1)	(49)
Mouse	FVB	Cultured antral follicles	24-120 hours	4.4 – 440 μM (1–100 μg/ml)	End of culture	BPA 440: Inhibited follicle growth	(107)
Mouse	FVB	Cultured antral follicles	24–96 hours	4.4 – 440 μM (1– 100 μg/ml)	End of culture	BPA 440: Inhibited follicle growth, increased atresia rating, Bcl2, Bax, Cdk4, Ccne1, and Trp53, and decreased Ccnd2	(105)
Mouse	C57BL/6, FVB, CD-1	Cultured antral follicles	24–120 hours	4.4 – 440 μM (1– 100 μg/ml)	End of culture	All strains: BPA440 inhibited follicle growth, increased expression of Cdk4, Ccne1, Trp53, Bax, and Bcl2	(106)
Human	HOSEpiC	Culture	3, 24, 48 hours	0.1, 1, 40 nM	End of culture	Increased expression of VEGF-R2	(111)
Lamb	Hampshire Down	Subcutaneous injection	PND1– 14	50 μg/kg/day	PND30	PND30: increased primordial-to-primary follicle transition but no difference in total follicle numbers; Increased multi-oocyte follicles; Increased granulosa and theca cell proliferation, induced atresia in small antral follicles	(90)
Lamb	Corriedale X Hampshire	Subcutaneous injection	PND1-14	0.5, 50 μg/kg/day	PND30 or PND34 post FSH stimulation	Decreased number of follicles >2mm, impaired response to FSH as evident by decreased percent of atretic follicles	(94)
Rat	Wistar	Subcutaneous injection	PND1 - 7	0.05, 20 mg/kg	PND8	BPA20: Increased primordial follicular recruitment, increased granulosa cell proliferation	(88)
Rat	Wistar	Oral, drinking water	GD9-birth	0.5, 50 μg/kg/day	PND21, 45, or 90	Lower ovarian weight; Reduced number of growing follicles and inhibited transition of primordial to primary follicles; Higher numbers of corpora lutea; Increased Fshr (BPA0.5); No difference in Lhcgr	(41)
Mouse	C57/Bl6J x CBA/Ca	Cultured pre-antral follicles	12 days	3, 300 nM	End of culture	BPA 3: accelerated follicle growth	(109)
Sheep	Suffolk	Subcutaneous injection	GD30-90	0.5 mg/kg/day	Fetal ovaries on GD65, 90	Age-dependent increase in mRNA of 3β1HSD, 3β2HSD, AR, ESR1, GDF9, IR, mTOR, PPARα, and IGF1R; Altered miRNA	(101)
Sheep	Suffolk	Subcutaneous injection	GD30-90	0.05, 0.5, 5 mg/kg/day	19 months	Similar number or size of corpora lutea; Differences in follicular count trajectories	(95)
Mouse	FVB	Oral	GD11-birth	0.5, 20, 50 μg/kg/day	PND4, 21	PND4: inhibited germ cell nest breakdown, decreased primordial follicles (BPA0.5 and 50); altered levels of apoptotic genes	(36)
Mouse	CD-1	Oral gavage	GD1 – PND20	12, 25, 50 mg/kg/day	PND50	Similar number of growing follicles	(46)
Mouse	CD-1	Oral gavage	PND21 - 49	25, 50 mg/kg/day	PND50	Similar number of growing follicles	(46)
Chinese Hamster	V79	Cell culture	12 or 24 hours	40, 80, 100, 120 μM	End of culture	Increased cell viability (BPA40), cytotoxicity (BPA 80, 100, 120). Induced DNA damage; Micronucleus (BPA100, 120)	(110)
Mouse	CD-1	Oral	GD12.5 – PND18.5	0.2, 0.04, 0.08 mg/kg	GD15.5, 17.5, 19.5 PND3, 5, 7	BPA0.08: Higher percentage of oocytes in cysts, higher oocyte number, and fewer primordial follicle numbers on PND3; Decreased Stra8 associated with altered DNA methylation	(45)
Mouse	CD-1	Cultured neonatal ovaries	Not specified	10, 100 μM	3 days of culture	Inhibition of germ cell nest breakdown and reduced primordial follicles (BPA10, 100); Increased apoptotic oocytes and increased Bax (BPA100); Reduced Nobox (BPA100), NOBOX, Lhx8 and protein, Sohlh2, Figla (BPA10, 100); Increased Lhx8 methylation	(87)
Fish	Gobiocypris rarus	Aquarium water	8 months old	15, 50 μg/l	After 14, or 35 days	Increased ovarian weight (BPA15); No histological effects at 14 days; Increased atretic follicles and perinuclear oocytes (BPA50); Increased Gdf9, Bmp15 (BPA15)	(97)
Fish	Gobiocypris rarus	Aquarium water	6 months old	1, 15, 225 μg/l	After 7 days	Increased ovarian weight (BPA15). Increased H2O2 ovarian levels (BPA1 and 225): Decreased glutathione	(96)
Mouse	C57BL/6	Cultured neonatal ovaries	PND4-14	0.1, 1, 10 μM	End of culture	<5 days culture: increased primary follicle number and decreased primordial follicle number (BPA10); Day 10: decreased primordial follicle number and increased primary follicle number (BPA1 and 10);Reduced proliferation (Ki67), apoptosis (Casp3), and activation of the PI3K/Akt pathway	(100)
Mouse	CD-1	Cultured neonatal ovaries	PND0-8	0.1, 1, 5, 10 μg/ml	2, 4, or 8 day of culture	PND 4: Inhibited germ cell nest breakdown, decreased primordial follicles; Random fluctuations in levels of anti-oxidant genes (Gpx, Cat, Gsr); Limited effects on apoptotic related genes. PND8: Increased ROS production (BPA5) yet post germ cell nest breakdown	(99)
Mouse	C57BL/6	Cultured antral follicles	96 hours	0.004 – 438 μM	End of culture	BPA 110-438: Inhibited follicular growth BPA 43.8-110: increased Bcl2	(108)
Mouse	Ah/rtm1Bra; C57BL/6 background	Cultured antral follicles	96 hours	0.004 – 438 μM	End of culture	BPA 219-438: Inhibited follicular growth	(108)
Mouse	CD-1	Subcutaneous injection	PND7 – 14	20, 40 μg/kg/day	PND15	Accelerated primordial to primary follicle transition	(91)
Mouse	CD-1	Subcutaneous injection	PND5 – 20 (every 5 days)	20, 40 μg/kg/day	PND21	Accelerated primordial to primary follicle transition	(91)

Molecular analysis revealed that low dose BPA exposure affected levels of genes related to apoptosis. Specifically, it BPA increased levels of B cell leukemia/lymphoma 2 (Bcl2), BCL2-like 1 (Bcl2l1) (36, 87). BPA exposure also decreased levels of BCL2-antagonist/killer 1 (Bak1), tumor necrosis factor receptor superfamily, member 11b (Tnfrsf11b), tumor necrosis factor receptor superfamily, member 1a (Tnfrsf1a), tumor necrosis factor (ligand) superfamily, member 12 (Tnfsf12), and lymphotoxin B receptor (Ltbr) (36, 87). Additionally, BPA exposure altered BCL2-associated X protein (Bax) levels by either increasing (87) or decreasing (36) its levels. Differences in the effects of BPA on Bax can result from differences in study designs (e.g., ovarian transplant versus excised neonatal ovaries).

Further, BPA exposure decreased expression of factors that control folliculogenesis such as NOBOX oogenesis homeobox (Nobox) (mRNA and protein), LIM homeobox protein 8 (Lhx8) (mRNA and protein), spermatogenesis and oogenesis specific basic helix-loop-helix 2 (Sohlh2), stimulated by retinoic acid gene 8 (Stra8), DNA meiotic recombinase 1 (Dmc1), REC8 meiotic recombination protein (Rec8), synaptonemal complex protein 3 (Scp3), and folliculogenesis specific basic helix-loop-helix (Figlα) (45, 87). In addition, BPA exposure prevented DNA methylation in CpG sites of Lhx8, indicating that BPA may impair normal processes of folliculogenesis (87) and ovarian dynamics.

Similar effects of exposure to both low and high doses of BPA during neonatal life on the ovary were observed at older ages/post weaning. Specifically, researchers reported findings such as BPA-induced multinucleated and hemorrhagic tissue (84), multi-oocyte follicles (90), altered follicle type distribution or numbers (41, 83, 89–95), reduced ovarian weight (41, 62), and ovarian cysts (49) compared to controls. Molecular analysis revealed that high dose BPA exposure decreased the expression of Figla and oocyte-specific histone H1 variant (H1f00), and increased the levels of anti-Müllerian hormone (Amh) genes (92). Additionally, Lee et al. reported that low dose BPA increased apoptosis in ovarian follicles that was coupled with increased levels of the apoptotic protein caspase-3 (81). Hence, in rodents, BPA affects ovarian development and dynamics via molecular pathways that involve apoptosis, folliculogenesis, and oocyte specific factors.

Similarly, in fish, low dose BPA exposure increased ovarian weight, increased levels of hydrogen peroxide, and decreased glutathione levels compared to controls, indicating that BPA may alter the oxidative stress mechanism in the ovary (96). Another study in fish found that low dose BPA exposure increased ovarian weight, atretic follicles, perinuclear oocytes, and expression of factors involved in folliculogenesis (Gdf9 and Bmp15) compared to controls (97).

Other studies in mice found no effect on follicle type distribution in the adult ovary following low dose BPA exposure (38, 46); however, it is possible that BPA did not affect ovarian follicle distribution because of the timing of BPA exposure and differences in study design. Further, some of the effects of BPA exposure on the ovary do not persist in subsequent generations. Wang et al. (36) reported that in utero low dose BPA exposure inhibited germ cell nest breakdown in the F1 generation of mice compared to controls; however, these changes were not observed in the subsequent generations examined by Berger et al. (98). Further, BPA exposure caused several generation-specific differences in gene expression, but not all were transgenerational (i.e., genes related to oxidative stress, autophagy, and apoptosis) (98). Interestingly, BPA-induced changes in steroidogenic genes and Esr1, androgen receptor (Ar), and insulin-like growth factor (Igf) family genes were suggested to be carried transgenerationally (98). It is possible that the changes in the genes related to oxidative stress and apoptosis are activated in an acute manner and thus, effects on these factors were not carried over to the subsequent generations. In contrast, steroidogenic factors are crucial to the function of the ovary (as an endocrine organ) and thus, some of BPA effects on these factors were carried over to the subsequent generations. Overall, these experiments provide strong evidence that BPA acts via mechanisms related to apoptosis, folliculogenesis, and oxidative status.

In mice, in vitro studies of isolated neonatal ovaries indicate that high dose BPA exposure inhibited germ cell nest breakdown and accelerated primordial follicle recruitment compared to controls (99, 100), similar to some of the observations in in vivo studies. Specifically, BPA exposure decreased levels antigen KI-67 (Ki67), tumor necrosis factor receptor superfamily member 6 (Fas), and caspases (Casp3 and 8) (99, 100). Further, BPA increased levels of Bcl2 and factors related to the phosphatidylinositol 3-kinase/thymoma viral proto-oncogene (PI3K/Akt) signaling pathway (99, 100). In sheep fetal ovaries, low dose BPA exposure resulted in an age-dependent increase in expression of steroidogenic genes, mammalian target of rapamycin (mTor), peroxisome proliferator-activated receptor (Pparα), and Igf1r compared to controls (101). The overall findings suggest that BPA may act via mechanisms that are related to follicle dynamics and apoptosis. Further, studies suggest that BPA exposure may act via mechanisms involving altered miRNA levels (101). BPA downregulated miR-137 that may decrease sex steroid hormone synthesis (102) and miR-765 that may be associated with premature ovarian failure (103, 104). Additional findings included variable levels of miRNAs related to insulin signaling, without changing levels of miRNA processing enzymes (101).

In vitro studies of isolated mouse ovarian follicles indicate that high dose BPA exposure selectively inhibited antral follicular growth (105–108), but increased preantral follicle growth (109) compared to controls. Molecular analysis revealed that BPA exposure affected the expression of genes related to the cell cycle, apoptosis, and steroidogenesis (105–108). Specifically, BPA exposure increased Bcl2, cyclin-dependent kinase 4 (Cdk4), cyclin E1 (Ccne1), transformation related protein 53 (Trp53), Bax, and downregulated cyclin D2 (Ccnd2). In short-term cultures (up to 24 hours) of Chinese hamster ovarian cells, high doses of BPA selectively increased cell viability, increased cytotoxicity, and induced DNA damage and the appearance of micronuclei (110). In short-term cultures (3–48 hours) of human ovarian cells, low dose BPA increased expression of VEGF-R2 (111). In short-term cultures (48 hours) of human granulosa-lutein cells, high dose BPA inhibited cell proliferation and decreased levels of IGF-1, aromatase, GATA binding protein 4 (GATA4), steroidogenic factor-1 (SF-1), and PPARγ (112). In contrast, in short-term porcine granulosa cell cultures (48 hours), high dose BPA did not affect cell proliferation or expression of oxidative stress genes (113). Similar concentrations of BPA over longer culture times (72 hours) decreased viability and disrupted matrix metallopeptidase 9 (MMP-9) secretion in human granulosa cells (114). It is plausible that some of BPA-mediated effects can be detected only at the end of longer culture times, or in a species specific manner.

Overall, current studies indicate that BPA affects the ovary, mainly during the ovarian developmental window as well as in early neonatal life via multiple pathways that include cell cycle, apoptosis, oxidative stress, and proliferation. More epidemiological studies are warranted to better understand the specific associations of BPA exposure and ovarian outcomes in women. Further, more experimental studies are warranted to better understand the specific mechanisms of action and the specific effects of low and high doses of BPA on the ovary.

Hypothalamic-pituitary-ovarian axis

Overall, reproductive function is dependent on the hypothalamic-pituitary-ovarian axis. Following sexual maturation, coordinated feedback loops along the hypothalamic-pituitary-ovarian axis control the ability of the mammalian female to ovulate and to prepare the reproductive organs to support potential pregnancy. In the hypothalamus, sex steroid hormones (estradiol and progesterone) activate the kisspeptin neurons that in turn, relay the secretion of gonadotrophic releasing hormone (GnRH). GnRH stimulates the anterior pituitary to secrete gonadotrophic hormones (FSH and LH). FSH and LH act on the ovary to support folliculogenesis. Increased levels of ovarian sex steroid hormones feed-back to the hypothalamic kisspeptin neurons to induce the LH surge that is needed for ovulation. Therefore, any alteration in proper levels/function of the hypothalamic-pituitary axis including the kisspeptin neurons can alter female fertility. The sections below describe the current data on the effects of BPA on the hypothalamus (Table 11), pituitary (Table 11), and gonadotrophic hormones (Tables 12 and 13).

Table 11.

BPA and hypothalamic-pituitary ovarian axis

Source	Strain	Exposure route	Time of exposure	Doses	Time of observation	Outcome	Ref
Sheep	Suffolk	Subcutaneous injection	GD30-90	5 mg/kg/day	F1: OVX at 21 months, testing at 23–26 months	No effect on steroid feedback and/or increased pituitary responsiveness to GnRH	(123)
Mouse	Mixed FVB X C57BL/6	Oral	GD10.5-18.5	0.5, 50 μg/kg/day	Birth	Increased number of pituitary mKi67-immunoreactive cells, increased gonadotroph cell number (LHβ, FSHβ positive); Increased Lhβ and Fshβ (BPA0.5); Decreased Lhβ and Fshβ, Nr5a1 (BPA50); Decreased Gnrhr (BPA0.5, 50); No effect on hormone synthesis by pituitary cells	(125)
Rat	Long Evans	Subcutaneous injection	PND0-2	50 μg/kg/day, 50 mg/kg/day	PND4 or 10	PND4: increased Esr1, no effect on Esr2, diminished Kiss1; PND10: Esr1 decreased to male typical levels, decreased/eliminated Esr2, diminished Kiss1	(117)
Rat	Long Evans	Subcutaneous injection	PND0-2	50 μg/kg/day, 50 mg/kg/day	PND4 or 10	Altered Esr2 expression and reversed sex differences in expression	(120)
Rat	Sprague-Dawley	Oral gavage	GD6-PND21	2.5, 25.0 μg/kg/day	PND21	No effect on density of anteroventral periventricular nucleus tyrosine hydroxylase immunoreactivity	(122)
Mouse	BALB/c	Oral	GD0-19	2, 20, 200 μg/kg/day	PND28	BPA20 altered methylation patterns in the hypothalamus	(121)
Sheep	Suffolk	Subcutaneous injection	GD30-90	5 mg/kg/day	Adult prior to onset of pre-ovulatory LH surge	Decreased hypothalamic levels of GnRH; Increased ESR1; Decreased ESR2 (medial preoptic area)	(119)
Rat	Wistar	Subcutaneous injection	PND1, 3, 5, and 7	0.05, 20 mg/kg/day	PND100	Anteroventral periventricular nucleus expression of ESR1 increased (BPA0.05, 20), PR decreased (BPA0.05); Arcuate nucleus expression of ESR1 decreased (BPA0.05, 20), no effect on PR	(82)
Rat	Wistar	Subcutaneous injection	PND1-5	100, 500 μg/kg/day	PND30	Decreased hypothalamic Kiss1	(115)
Rat	Long Evans	Subcutaneous injection	PND0 - 3	50 μg/kg/day, 50 mg/kg/day	Post weaning and vaginal opening	BPA 50 mg/kg reduced density of hypothalamic kisspeptin immunoreactive fibers; more profound in arcuate nucleus	(116)
Fish	Transgenic Zebrafish	Aquarium		0.1, 1, 10, 100, 1000 μg/l	25, 120 hours post fertilization	Selective increase of Kiss1, Kiss1r, Gnrh3, Lhβ, Fshβ, synaptic vesicle protein-2 (Sv2)	(40)
Mouse	ICR	Oral	Proestrus of 4th/5th estrous	20 μg/kg/day	6 hours post administration	Elevated plasma Gnrh; Increased Kiss1 in anteroventral periventricular nucleus	(118)
Mouse	ICR	Injection into right lateral ventricle	Proestrus of 4th/5th estrous	0.02, 0.2, 2, 20, 200 nM/3μl	6 hours post administration	Anteroventral periventricular nucleus - Kiss1 altered (BPA EC50 2.754 nM) and arcuate nucleus (BPA>20nM); BPA increased GnRH mRNA, but effect blocked by pretreatment with GPR54 blocker	(118)
Mouse	CD-1	Oral gavage	GD1 – PND20	12, 25, 50 mg/kg/day	PND50	Dose-dependent increase in the expression levels of KiSS-1 and GnRH in hypothalamus, no difference in Gpr54	(46)
Mouse	CD-1	Oral gavage	PND21 - 49	25, 50 mg/kg/day	PND50	No effect on expression levels of KiSS-1 and GnRH in the hypothalamus	(46)

Table 12.

BPA and gonadotrophic hormones (epidemiological studies)

Study design	Study population	Sample size	Time of BPA measurement	BPA concentration	Outcome	Ref
Retrospective	Women exposed to BPA in workplace	106 exposed, 250 unexposed	Air sampling+ spot urine sample	Geometric (total BPA) mean (95% confidence interval) Exposed: 22.3 (12.4, 39.8 μg/g cr); unexposed: 0.9 (0.7, 1,1)	Positive association between prolactin and urine BPA levels (all women); negative association between FSH and BPA (unexposed group)	(124)
Prospective	Women undergoing in vitro fertilization	Overall 209 (antral follicle count=154; FSH=120; ovarian volume= 114)	Urine: upon entry into the study and subsequent treatment visits; Ultrasound: 3rd day of an unstimulated menstrual cycle	Urinary geometric mean (geometric standard deviation); Antral follicle count= 1.6 (2.0); FSH= 1.7 (2.1); ovarian volume= 1.5 (1.8)	No association with day 3-FSH or ovarian volume; Higher urinary BPA concentrations associated with lower antral follicle counts	(85)

Table 13.

BPA and gonadotrophic hormones (experimental studies)

Source	Strain	Exposure route	Time of exposure	Doses	Time of observation	Outcome	Ref
Rat	Sprague Dawley	Subcutaneous injection	PND1 - 10	50 μg/50 μl, 500 μg/50 μl	PND13	In vivo: BPA500 lower basal LH levels and lower GnRH induced LH release after 15 min; No difference in FSH or PRL levels; In vitro: higher GnRH pulse frequency and effects on ERK1/2 and IP3 signaling pathway	(80)
Rat	Wistar	Drinking water + lactation	GD0 – PND21	3 μg/kg/day	PND30	LH higher; no effect on FSH levels	(89)
Rat	Sprague Dawley	Oral gavage	90 days	0.001, 0.1 mg/kg/day	21 weeks old	Increased LH (protein levels in pituitary cells and serum); no effect on FSH	(81)
Zebrafish	Japanese medaka	Aquarium water	21 days	2, 20, 200 μg/l	5 months old	Reduced FSH and LH (BPA200)	(127)
Rat	Wistar	Subcutaneous injection	PND1, 3, 5, 7	0.05, 20 mg/kg/day	PND100	BPA20: dampened LH surge; Mature LHRH mRNA increased (BPA0.5) and decreased (BPA20); Unprocessed intron A containing LHRH decreased (BPA0.05 and 20)	(82)
Mouse	C57BL6J	Oral	12–15 days (first 3 reproductive cycles)	50 μg/kg/day	51–54 days	No differences in FSH, LH levels	(38)
Sheep	Suffolk	Subcutaneous injection	GD30-90	0.05, 0.5, 5 mg/kg/day	19 months	No difference in LH surge levels	(95)
Mouse	ICR	Injection into right lateral ventricle	Proestrus of 4th/5th estrous	0.02, 0.2, 2, 20, 200 nM/3μl	6 hours post administration	BPA increased plasma LH but blocked by pretreatment with GPR54 blocker; No change in timing or peak concentration of LH surge	(118)
Mouse	CD-1	Oral gavage	GD1 – PND20	12, 25, 50 mg/kg/day	PND50	BPA25: increased Fsh; BPA50: increase Fsh mRNA; No difference in levels of LH, thyroid stimulating hormone, growth hormone, PRL	(46)
Mouse	CD-1	Oral gavage	PND21 - 49	25, 50 mg/kg/day	PND50	No difference in levels of LH, thyroid stimulating hormone, growth hormone, PRL	(46)
Rat	Sprague Dawley	Oral	5 weeks old	50 mg/kg/day	After 6 weeks	Decreased serum FSH, no effect on serum LH	(126)

Hypothalamus

Few experimental studies have examined the effects of BPA on the hypothalamus, and findings vary between the studies. For example, neonatal BPA exposure increased (40, 46) or decreased levels of Kiss1 (115–117) in fish, mice, and rats. Differences in species and age of the animals in which observations were made can partially explain these opposite results rather than the doses that were used (i.e. low or high). Neonatal BPA exposure also decreased levels of gonadotropin releasing hormone (Gnrh) (40, 118), Esr1, and Esr2 (82, 117, 119, 120) in sheep and rats compared to controls. Interestingly, Wang et al. (118) reported that the effects of low dose BPA on Gnrh can be ablated by pretreatment with a specific blocker of the receptor of KISS1. These findings imply that the effects of BPA exposure are mediated via the kisspeptin signaling pathway. Future studies that utilize molecular techniques including kisspeptin knock-out mice can aid in further elucidating BPA-induced effects in the hypothalamus.

Similar to the prominent effects of BPA on neonatal animals, in utero BPA exposure (low doses and LOAEL) that continued until the age of weaning increased levels of Kiss1 and GnRH on postnatal day 50 compared to controls in mice (46). Interestingly, the same exposure levels at a later age (postnatal days 21–49) did not affect hypothalamic gene expression (46), indicating that the timing of exposure has a large influence on the outcome. Other researchers reported that in utero low dose exposure to BPA disrupted methylation patterns in the hypothalamus that can affect the expression of Esr1 (121). In contrast, low dose BPA exposure did not affect the density of tyrosine hydroxylase cells that are involved in sexual dimorphism of the brain (122). Similarly, Abi Salloum et al. reported that sheep that were exposed to low dose BPA in utero still maintained a well-functioning neuroendocrine system in response to steroid feedback at adulthood (123). Overall, the effects of BPA exposure on the hypothalamus are likely to be dependent on the timing of exposure and type of animal model that is used. Potential factors that may be mediating BPA effects are kisspeptin and GnRH.

Pituitary

Limited data on the association between BPA exposure and pituitary outcomes are available from human studies (Table 12). Miao et al. reported a positive association between creatinine adjusted urine BPA levels and PRL, and a negative association with FSH levels in women exposed to BPA in their work place (124). In contrast, Souter et al. found no association between specific-gravity adjusted BPA levels and day-3 FSH levels in women undergoing IVF treatments (85). The differences between the two cohorts may explain the inconsistent results.

Few experimental studies have examined the effects of BPA exposure on the pituitary (Table 13). The majority of the studies examined the effects of low dose BPA, whereas only two studies included high doses as well (46, 125). In utero low dose BPA exposure increased proliferation of pituitary cells, and increased gonadotroph cell number (LHβ, FSHβ positive) compared to controls in the pituitaries of mice (125). In the same study, both BPA doses (0.5 and 50 μg/kg/day) decreased gonadotropin releasing hormone receptor (Gnrhr) levels, whereas 0.5 μg/kg/day of BPA increased the levels of Lhβ and Fshβ, and 50 μg/kg/day of BPA decreased the levels of Lhβ, Fshβ, and Nr5a1 compared to controls (125). Further, longer BPA exposure (25 and 50 mg/kg/day) from gestation through weaning increased Fsh levels in mice compared to controls (46). These findings indicate that BPA exposure can affect the pituitary, but it is likely to be dependent on the timing of exposure and dose.

Several of the BPA-induced changes in pituitary gene expression are accompanied by altered serum levels of the pituitary hormones. Specifically, low and high dose BPA exposure decreased serum FSH and LH levels in adult rats and fish compared to controls (80, 126, 127). Low dose BPA exposure also increased serum LH levels compared to controls in mice and rats (81, 89, 118). Lastly, early neonatal low dose BPA exposure resulted in a dampened LH surge compared to controls in adult rats (82). Differences in study design including BPA doses may contribute to the variability in the results. Nevertheless, additional studies are needed to examine if the effects on FSH, LH, and LH surge can explain some of the effects on ovarian function and estrus cyclicity.

In contrast to the effects of BPA described above, a few studies reported that BPA exposure does not affect serum levels of FSH (38, 46, 80, 81, 89) or LH (38, 118, 126) in mice and rats. Similarly, one study found that low dose BPA exposure did not affect the LH surge in adult sheep (95). Overall, the majority of the current studies suggest that BPA exposure affects the function of the anterior pituitary. However, the scientific evidence needs to be supported with additional studies. The secretion of LH and FSH may need to be measured over several time points to delineate the mechanisms through which BPA selectively targets impairs its function.

Conclusions

The current literature fairly consistently shows the following:

• Infertile women have higher measurable BPA levels than fertile women, and these higher BPA levels are correlated with fertility problems in women undergoing IVF treatment.

• Based on animal studies, it is likely that:

  • BPA alters oviduct morphology and gene expression. These changes may impair development and transport of the conceptus from the oviduct to the uterus; however further studies are needed to elucidate the mechanism of action.

  • BPA can reduce and/or impair implantation. These effects may be mediated via the Pgr-Hand2 signaling pathway.

  • BPA affects uterine morphology and function and may cause these changes over several generations via mechanisms that involve cell proliferation and receptivity.

  • BPA may cause abnormal estrous cyclicity.

  • BPA affects cell proliferation in the pituitary and the expression of factors related to the pituitary gonadotrophs.

  • BPA affects the expression of major determinants in the hypothalamic-pituitary axis, including kisspeptin and Gnrh.

  • BPA is an ovarian toxicant that is likely to act via multiple pathways including apoptosis, oxidative stress, and folliculogenesis.

Despite the number of studies that have examined potential BPA effects on female fertility in the past years, it is still difficult to layout the exact mechanism of BPA action. BPA is a model endocrine disrupting chemical with a complicated mechanism of action that is yet to be fully elucidated. BPA effects are highly dependent on various factors in the study design such as timing of exposure, species, dose, route of exposure, and mode of quantification/assessment. Moreover, other modifying factors including co-exposures, study location/setting (e.g. hospital), and study sample (e.g. potentially unhealthy/previously exposed participants) should also be taken into consideration when possible as proposed by Teeguarden et al., (24, 25).

In summary, further studies are needed to better understand the mechanisms of action of BPA on female fertility. These studies will need to include some integrated endpoints to assure reproducibility of previous findings while taking into account relevance of the doses to human exposure, the ability of BPA to bind certain receptors, and study design/setting. Because female fertility relies on several organs and feedback loops, studies that utilize in vivo methods should aim for a multi-organ/disciplinary approach.