Bisphenol A and Reproductive Health: Update of Experimental and Human Evidence, 2007–2013

1Bisphenol A (BPA) is a high production volume chemical used in a variety of common consumer products. Most notably, BPA is present in polycarbonate plastics, the epoxy resin liners of aluminum cans, and thermal receipts. BPA is an endocrine-disrupting chemical that can act through a variety of physiological receptors, such as genomic estrogen receptors 1 and 2, membrane-bound estrogen receptors, androgen receptor, peroxisome proliferator–activated receptor γ, and thyroid hormone receptor (reviewed by ).

2To address research gaps in the published BPA studies, in 2006, an organized committee sponsored by the National Institute of Environmental Health Sciences (NIEHS), National Institute of Dental Craniofacial Research (NIDCR), U.S. Environmental Protection Agency (EPA), and Commonweal reviewed the associations between BPA exposure and reproductive health outcomes and published their analyses in 2007. This committee was organized into five topic-driven panels that evaluated the available data and developed guidelines for the conduct of future in vivo and in vitro studies that would facilitate comparisons across studies and extrapolations to human health outcomes. Overall, the panels recommended the use of oral and dietary exposure routes, doses of BPA similar to human exposure levels, and further evaluations of BPA concentrations in animal and human tissues and fluids with the aim of using doses that would result in human-relevant serum concentrations of unconjugated BPA in studies with experimental animals.

3With respect to reproductive effects, the subpanel evaluating in vivo animal studies found contradictory results among studies but concluded that, based on the available evidence, a) they were confident that BPA impacts the male reproductive system, and b) they thought it likely—although it required confirmation—that both developmental and adult exposure affect the female reproductive system. At the time, the prostate was the most extensively studied reproductive tissue, with both developmental and adult exposures reported to increase prostate size [reviewed by and ]. However, not all studies reported effects, and controversy remained regarding the doses necessary to elicit effects. In females, effects on the oocyte, developing reproductive tract, and timing of sexual maturation were available, but many remained largely unconfirmed [reviewed by and ]. Little epidemiological information was available, and the few published studies were limited in their design, restricting the strength of the conclusions that could be drawn about the reproductive hazards of BPA exposure [reviewed by and ].

4Between 2007 and 2013, new studies on the impact of BPA on the reproductive system have been conducted and are summarized in this review. Specifically, we focus on summarizing a) reproductive findings from human studies and how they compare with animal studies; b) detailed information about the experimental effects of BPA on a variety of reproductive end points, taking into account species, dose, route of exposure, and timing of exposure; and c) mechanisms of BPA action in the reproductive system, whenever possible. Thus, this update is a compilation of the more recent literature detailing the effects of BPA exposure on the female and male reproductive systems. We hope that the insights and conclusions of this review will be used to direct future studies and be used in developing consensus statements about the effects of BPA on the reproductive system based on the state of the evidence.

5Search strategy. We searched Pubmed (http://www.ncbi.nlm.nih.gov/pubmed) to identify journal articles published between 2007 and 2013 that were related to BPA and reproduction. Search terms included the following:

6Ovary, follicle, oocyte, oocyte competence, meiosis, granulosa cell, theca cell, ovulation, oviduct, uterus, endometrial, stroma, implantation, pregnancy, miscarriage, spontaneous abortion, embryo, blastocyst, placenta, trophoblast, cytotrophoblast, syncytiotrophoblast, birth, birth weight, gestation, transgeneration, testes, sperm, Leydig cell, Sertoli cell, germ cell, epididymis, prostate, ventral lobe, dorsal lobe, ejaculation, fertilization, infertility, sexual dysfunction, estradiol, testosterone, androstenedione, estrone, dihydrotestosterone, dehydroepiandrosterone, or pregnenolone.

7After the aforementioned search, we searched the terms “bisphenol A” and “BPA” to identify any papers that did not include the other selected search terms or term variants.

8Article inclusion criteria. Journal articles found using the search terms and in the selected time frame were considered for inclusion. All human and experimental animal in vivo studies were included. In vitro studies were included if there was a clear mechanism for an effect or if the studies supported in vivo or human findings. Journal articles were not omitted due to a paucity of research in any category.

9Dose designation. Similar to the 2007 expert panel and to, we defined “low dose” as a BPA dose of ≤ 50 mg/kg/day. This is the currently accepted lowest adverse effect level (LOAEL) used by the U.S. EPA. Thus, “high-dose” BPA studies are those using concentrations > 50 mg/kg/day. Throughout the text, we use the terms “low dose” and “high dose” to provide a simple description of the doses used in each study. However, specific doses used in each study are listed in the Supplemental Material.

10Exposure timing. Here, we define BPA exposure as gestational when it occurred in utero, neonatal when it took place after birth but before weaning, and postnatal when it occurred any time after weaning.

11Article strength determination. Similar to the 2007 expert panel, we considered the evidence to be strong when multiple studies in multiple species indicated a similar effect of BPA on a reproductive tissue or end point, even if concordance was not 100% across all studies, given that species and strain differences can lead to differences in dose response and magnitude of effect. We considered the evidence to be limited when some studies—but not the majority—indicated a similar effect of BPA on a reproductive tissue or end point, and/or when data from in vitro, in vivo animal, and human studies were discordant. Finally, we considered the evidence to be inconclusive when a limited number of studies had examined the effect of BPA on a selected reproductive tissue or end point, and/or when the studies were conducted in only one species or by in vitro studies alone. We recognize that in vitro studies have played critical roles in identifying how BPA affects specific tissues; however, because it is difficult to correlate human and animal health outcomes with in vitro outcomes alone, studies performed in vitro only were classified as inconclusive.

12All included studies on BPA exposure and early oogenesis and follicle formation were conducted using animal models or in vitro systems. These studies indicate that developmental BPA exposure has the potential to affect two stages of oogenesis: a) the onset of meiosis in the fetal ovary, and b) germ cell nest breakdown and follicle formation (see Supplemental Material, Table S1). Several reports have confirmed the original findings from studies in mice (reviewed by ) that gestational exposure affects the onset of meiosis and induces nondisjunction in meiosis in the fetal ovary, but does not induce aneuploidy. In a macaque study designed to mimic serum levels of unconjugated BPA reported in human biomonitoring studies, observed that daily low-dose BPA exposure (measured < 1 ng/mL in maternal serum) significantly disrupted synapsis and recombination between homologous chromosomes at the onset of meiosis, which was consistent with previous findings. Three studies reported that gestational low-dose BPA exposure of mice induced changes in gene expression in germ cells and early meiocytes. BPA exposure increased expression of Stra8 (stimulated by retinoic acid 8 homolog) and a variety of meiotic genes in C57BL/6 mice. However, longer gestational BPA exposure down-regulated the expression of Stra8, Dazl (deleted in azoospermia-like), and Nobox (newborn ovary homeobox) in CD-1 mice. Last, BPA exposure commencing after the onset of meiosis induced a meiotic delay at gestational day (GD) 17.5 in CD-1 females. Collectively, these studies provide strong evidence that BPA exposure disrupts meiosis in mice and macaques, and alters gene expression in germ cells and early meiocytes in two different strains of mice.

13In vitro studies provide further evidence that BPA affects the onset of meiosis., ) reported that BPA (1–30 μM) increased oocyte degeneration by impairing meiotic progression in cultured human fetal oocytes and that, similar to mouse studies, human fetal oocytes that progressed to prophase exhibited increased levels of recombination (MLH1 foci) and gene expression changes. BPA also increased methylation errors in differentially methylated regions of maternally imprinted genes of oocytes in cultured preantral follicles of C57/BL6xCBA/Ca mice. Thus, the data from different mouse strains, macaques, and in vitro studies consistently provide strong evidence to conclude that BPA exposure has detrimental effects on the meiotic process, both at the gene expression and phenotypic levels.

14Studies have also suggested that BPA interferes with germ cell nest breakdown in animal models. In neonatally exposed lambs, low-dose BPA was reported to increase the incidence of multioocyte follicles. Similarly, in gestationally exposed macaques, dietary low-dose BPA exposure increased the number of oocytes present in secondary and antral follicles at birth, and continuous BPA exposure (measured < 1 ng/mL in maternal serum) increased the incidence of unenclosed oocytes. Further, in gestationally exposed CD-1 mice, low-dose BPA increased the number of unenclosed oocytes, whereas it decreased the number of primordial follicles in a dose-dependent manner. In another study, reported that prenatal BPA exposure altered the fetal ovarian steroidogenic gene and microRNA expression that mediate gonadal differentiation and folliculogenesis in sheep. Collectively, these studies provide strong evidence that gestational BPA exposure—across multiple exposure routes, doses, and species—impairs proper germ cell nest breakdown, leading to the formation of multioocyte follicles. The presence of multioocyte follicles is of concern because they are considered a pathologic condition that may lead to ovulatory problems.

15BPA exposure also appears to accelerate follicle transition and growth in several species. reported that neonatal BPA exposure accelerated follicle transition in lambs, decreasing numbers of primordial follicles and increasing primary follicles without affecting total follicle numbers. A similar enhanced activation of follicular recruitment was observed by in neonatally exposed Wistar rats. BPA exposure also increased cell proliferation, indicative of follicular growth, in small antral follicles in neonatally exposed lambs and Wistar rats. Taken together, the data suggest that BPA enhances the recruitment and growth of primordial and primary follicles across species. Combined with the effects on germ cell nest breakdown, there is strong evidence that BPA induces ovotoxicity by acting on developing and immature follicle stages in animals models. However, the consequences of these effects on reproductive potential and longevity are unclear. In one study, low-dose neonatal BPA exposure decreased numbers of all follicle types and increased atretic follicles in rats during adulthood. These effects of BPA on follicles could lead to premature reproductive senescence, but this needs to be confirmed in future studies.

16In vitro studies on the effects of BPA have focused on mature ovarian follicles. In murine preantral follicles, BPA (0.003 μM) accelerated development to antral follicles. In murine antral follicles, exposure to BPA (440 μM) aberrantly up-regulated expression of cell cycle regulators and the proatretic and antiatretic factors Bax (BCL2-associated X protein), Trp53 (tumor protein 53), and Bcl2 (B-cell lymphoma 2), inhibiting follicle growth and inducing apoptosis. Further, BPA (110–440 μM) inhibited antral follicle growth in mice. These studies suggest that low-dose BPA exposure may alter follicle formation, but high-dose BPA may directly inhibit growth, cause atresia, and induce changes in gene expression in rodent antral follicles. In future studies, it will be important to validate findings from these in vitro studies at the in vivo level and determine the consequences of BPA-induced follicle toxicity on reproduction function.

17Multiple studies have investigated the association between BPA exposure and ovarian steroid hormone production in women (see Supplemental Material, Table S2). In three publications based on women undergoing in vitro fertilization (IVF), BPA exposure was associated with a decrease in peak serum estradiol levels prior to oocyte retrieval. A case–control study by showed that BPA was associated with increased testosterone and androstenedione levels in women with polycystic ovary syndrome (PCOS). In a study of 60 women undergoing IVF, found that urinary BPA concentrations were not associated with a negative linear dose–response in the expression of the steroidogenic enzyme Cyp19 in granulosa cells collected at the time of oocyte retrieval, but instead were associated with a suggested nonmonotonic dose–response. Conversely, BPA was not associated with estradiol or testosterone levels in women in the INChianti study, a prospective, population-based study of adults living in Chianti, Italy. Given the limited information on BPA exposure and ovarian steroidogenesis in women and the discrepant study results, additional studies utilizing sensitive and reliable steroid hormone and BPA assays are required to delineate whether BPA levels negatively impact reproductive hormonal patterns in women. Further, given that most existing studies on BPA exposure and steroid levels were conducted in IVF populations, it is critical to examine the association between BPA exposure and steroidogenesis in women from the general population.

18Several experimental studies have examined the effect of BPA exposure on ovarian steroidogenesis in laboratory animals (see Supplemental Material, Table S1). In three rodent studies, perinatal and postnatal low-dose BPA exposure was reported to increase serum estradiol levels. Further, in one study of Sprague-Dawley (SD) rats and one of pregnant ICR mice, low-dose BPA increased testosterone and progesterone levels. Interestingly, reported that postnatal BPA exposure alone did not affect serum hormone levels in the mice. Similarly, in other studies using rats, mice, and lambs, gestational or gestational plus neonatal BPA exposure had no effect on steroidogenesis. In these four studies, the doses used were lower than those in studies reporting BPA effects, indicating that BPA doses < 20 mg/kg may not increase hormone production in animal models. In contrast, another study found that although BPA did not alter estradiol levels, low-dose BPA decreased progesterone levels in adult mice during early pregnancy. In addition, in adult rats, low-dose BPA (< 0.1 mg/kg/day) decreased estradiol, testosterone, Cyp19 (aromatase), and Star (steroidogenic acute regulatory protein). Further, in adult mice, low-dose BPA decreased expression of estrogen and progesterone receptors but did not alter hormone levels. The differences in study results may be a function of exposure times, internal doses of BPA, or species.

19The results of in vitro studies on the effects of BPA on steroidogenesis are also equivocal. BPA exposure (44 and 440 μM) inhibited estradiol, testosterone, androstenedione, estrone, dehydroepiandrosterone, and progesterone production and decreased StAR and Cyp11a1 (cytochrome P450 side-chain cleavage) expression in cultured intact murine antral follicles. However, in isolated rat theca-interstitial cells, BPA (0.1–100 μM) had opposite effects, increasing testosterone synthesis and Cyp17a (cytochrome P450 17α-hydroxylase/lyase), Cyp11a1, and StAR expression. In isolated rat granulosa cells, BPA (100 μM) decreased progesterone synthesis and increased StAR expression. In a separate study using porcine granulosa cells, 0.1 μM BPA increased estradiol levels, whereas higher doses (1 and 10 μM) decreased estradiol levels. All three concentrations of BPA resulted in decreased progesterone levels. Collectively, these studies indicate that BPA adversely affects steroidogenesis in vitro (with the effects dependent on BPA concentration) and that BPA’s effects on steroidogenesis vary by whether intact follicles or isolated cells are used in the cultures.

20A few human studies have analyzed the association between urinary BPA levels and oocyte yield, maturation, and fertilization (see Supplemental Material, Table S2). In a small prospective study of 58 infertile women and 37 male partners undergoing intracytoplasmic sperm injection or conventional IVF, found an association between serum BPA concentrations and oocyte maturation only among Asian women, but an overall correlation between increasing serum concentrations and the developmental potential of human oocytes. In two publications from the same prospective cohort of 84 women and 174 women undergoing IVF, increasing urinary BPA concentration was associated with decreased numbers of retrieved oocytes, mature oocytes (MII), and normally fertilized oocytes (2PN). These results suggest that BPA is associated with impaired oocyte yield, maturation, and fertilization, adversely affecting the success of IVF treatment.

21Several recent experimental results support the findings from earlier studies that suggested BPA exposure affects the resumption of meiosis in the periovulatory oocyte (reviewed by ) (see Supplemental Material, Table S1). reported that neonatal exposure to low-dose BPA inhibited germinal vesicle breakdown (GVBD) in CD-1 F1 hybrid female mice, confirming the previous work of. Conversely, low-dose BPA administered to MF-1 (C57BL/6 × CBA/Ca F1 hybrid) mice on postnatal days 22–28 did not reduce GVBD or polar body extrusion nor did it increase spindle aberrations. Similarly, low-dose BPA given orally to 4- or 9-week-old superovulated female C57Bl/6 mice did not affect oocyte retrieval, meiotic maturation, or induce aneuploidy. Although the exact reasons for the subtle discrepancies among studies are unknown, there are numerous possible reasons for the variations in the window of neonatal exposure (reviewed by ) and potential coexposure to phytoestrogens that may modulate the effects of BPA on the periovulatory oocyte.

22In in vitro studies, BPA (43.8 μM) has been reported to significantly alter spindle formation, distribution of pericentriolar material at spindle poles, and induce congression failure in MF-1 mouse oocytes isolated from antral follicles. In a study in follicle-enclosed oocytes from adult mice, reported that BPA (30 μM) impaired spindle alignment and caused meiotic arrest after GVBD but before polar body extrusion. Importantly, a recent study of human oocytes exposed during in vitro maturation, with doses of BPA within the range measured in human follicular fluid (0.02, 0.2, and 20 μg/mL), found a dose-dependent increase in the incidence of meiotic arrest, disturbances in spindle formation and chromosome alignment, and spontaneous oocyte activation. Because the doses used were in the range measured in human follicular fluid, these data, together with the results of experimental studies, provide compelling evidence that BPA adversely affects the maturing oocyte.

23Studies on the effects of BPA on PCOS are limited (see Supplemental Material, Table S2). Women with PCOS are characterized by oligo-anovulation, functional hyperandrogenism, and multifollicular ovaries (accumulation of several small-sized antral follicles), with the majority of women showing insulin resistance and excess luteinizing hormone. One case–control study of 71 women with PCOS and 100 women without PCOS showed an association between serum BPA levels and increased testosterone, androstenedione, and insulin resistance in PCOS (see Supplemental Material, Table S2). Given the limited number of studies assessing the association between BPA exposure and PCOS symptoms, more studies are needed in order to make firm conclusions about the impact of BPA exposure on PCOS or PCOS symptoms.

24The ovarian phenotype in BPA-treated rodents (cystic-appearing follicles) differs from the ovarian phenotype of women with PCOS (accumulation of small antral follicles). In rodents, prenatal and neonatal exposure to low-dose BPA lead to disruption of estrous cyclicity, increased testosterone production, and ovarian cysts (see Supplemental Material, Table S1). High-dose BPA exposure in rodents also leads to ovarian cysts and an accumulation of large antral follicles. In addition, reported that BPA exposure decreased gonadotropin-releasing hormone (GnRH) levels measured from hypothalamic explants in vitro. Additional studies in other animal models are needed to fully understand whether BPA exposure causes PCOS or PCOS-like conditions, and to determine why outcomes differ in animal models and women.

25Only one study we reviewed investigated the effects of BPA on the oviduct (see Supplemental Material, Table S1). observed progressive proliferative lesions in the oviducts of adult CD-1 mice prenatally exposed to low-dose BPA. Because alterations in oviduct morphology would be expected to adversely affect both fertilization and embryo transport, future studies are clearly warranted to examine the impact of BPA not only on the oviduct but also on fertilization and embryo transport.

26No studies have reported the impact of gestational BPA exposure on uterine morphology in women because of the extreme difficulty of monitoring adult outcomes of prenatal exposure to BPA; however, animal studies suggest that gestational BPA exposure perturbs gross morphology of the uterus in the adult (see Supplemental Material, Table S3). Specifically, low-dose BPA exposure has been reported to induce benign and malignant lesions and endometrial polyps in the uterus and to perturb Wölffian duct regression in prenatally exposed adult mice. Further, adult hens that were exposed in ovo on day 4 of incubation to BPA (134 ng/kg) had decreased thickness of their tunica mucosa and density of uterine glandular structures compared with unexposed hens. Together, these studies indicate that gestational BPA exposure may be potentially deleterious to uterine morphology in adult females. Because abnormalities have been identified in middle age that were not observed in young adult mice, future studies should assess effects in exposed animals at different life stages.

27Limited data exist on BPA exposure and uterine endometrium in women. One case–control study of 69 women suggests that serum BPA concentrations may be associated with the occurrence of endometriosis (see Supplemental Material, Table S2). In another study of 495 individuals in an operative cohort and 131 women in population cohort, found no association between BPA exposure and endometriosis. However, that study was not originally intended to investigate BPA exposure, nor was it was appropriately powered to assess endometriosis; that study also included a lapse between sample collection and endometriosis evaluation, further confounding these data. Given the lack of studies on BPA and endometrial disorders in humans, more studies are required before making conclusions about whether BPA adversely impacts the uterine endometrium in women.

28Experimental studies support the findings of (see Supplemental Material, Table S3). reported that adult female Balb-c mice exposed prenatally and neonatally to low-dose BPA developed endometrial-like structures with both glands and stroma in adipose tissue surrounding the genital tract. These structures expressed Hoxa10 (homeobox A10), a transcription factor that mediates proliferation of stromal tissue prior to implantation. Two other studies reported that BPA, at both low- and high doses, increased expression of Hoxa10 in prenatally exposed adult CD-1 and ICR mice.

29Other in vivo studies have provided evidence that BPA impairs proliferation in the uterus. In rodents, BPA exposure over a wide range of doses, times, and routes decreased expression of uterine Esr1 (estrogen receptor α), which may lead to inhibited endometrial proliferation in the uterine epithelium and stroma. In two separate studies of adult rats, prenatal and neonatal exposure to low-dose BPA decreased uterine epithelium proliferation in response to hormone treatments. In a study in non-human primates, reported that although low-dose BPA did not affect progesterone receptor expression, it dampened glandular and stromal progesterone receptor expression in response to estradiol stimulus. Low-dose BPA was also reported to impair apoptosis of the uterine epithelium during estrus in neonatally exposed adult rats.

30In vitro studies also support the hypothesis that BPA exposure adversely affects the uterus. BPA (50 or 100 μM) has been reported to significantly decrease proliferation of human endometrial stromal fibroblasts cultured for 48 hr. BPA (50 μM) was also found to decrease the proliferation of cultured human endometrial endothelial cells. Further, in cultured primary heterogeneous populations of uterine cells, BPA (10 μM) significantly inhibited uterine cell contractions, increased oxytocin-related pathways, and decreased prostaglandin-related signaling after 48 hr. Taken together, the existing animal and in vitro studies provide strong support that BPA impairs uterine cell proliferation.

31Only one study has reported on the association between BPA exposure and uterine receptivity/implantation in women (see Supplemental Material, Table S2). In a study of 137 women undergoing IVF, found that higher quartiles of urinary BPA concentrations were associated with increased odds of implantation failure. Given the limited information on this topic, future studies need to be conducted to determine whether BPA exposure is associated with adverse uterine receptivity/implantation outcomes in women.

32Several experimental studies indicate that BPA exposure impairs uterine receptivity and implantation (see Supplemental Material, Table S3). Repeated exposure of pregnant mice to low-dose BPA during early gestation was reported to completely ablate embryo implantation. A single exposure to high-dose BPA on GD0 or GD1, but not on GD2, was found to decrease implantation in mice. Neonatal exposure to low-dose BPA also decreased implantation sites in pregnant rats. Interestingly, low-dose BPA has been reported to increase preimplantation loss in unexposed female rats mated to neonatally exposed males and decrease implantation sites in females mated to adult exposed male rats. Further, when untreated and healthy embryos were transplanted into the uteri of low-dose-BPA–exposed mice, implantation failed. Together, these studies provide strong evidence that BPA exposure, in both males and females, affects uterine receptivity in females. However, a need still exists to explore whether BPA-exposed embryos attach to the uterine epithelium and initiate implantation.

33Neonatal exposure to low-dose BPA was also reported to decrease pregnancy maintenance in experimental studies. Low-dose BPA increased resorption rates in uteri of unexposed female rats mated to males that were neonatally exposed, gestationally plus neonatally exposed, or exposed as adults, suggesting that male exposure can significantly contribute to pregnancy loss. These effects were also found in F1 and F2 offspring, suggesting transgenerational effects of BPA. The potential ability of BPA to cause transgenerational effects in animal models is further supported by, who observed reduced uterine weight and expanded uterine lumen in mice developmentally exposed to high-dose BPA, as well as demethylation of the Hoxa10 gene in the F2 generation. Collectively, these experiments indicate that BPA exposure adversely affects implantation and pregnancy maintenance in animal models, which may be transgenerational in nature.

34Few studies have examined the effects of parental BPA levels on subsequent in vitro embryo development in humans (see Supplemental Material, Table S2). In one prospective preconception cohort study of 174 women, total urinary BPA concentration was associated with a decreased rate of blastocyst formation. Further, a prospective cohort study involving 27 couples undergoing IVF treatment found that increasing urinary BPA concentrations in the male—but not the female partner—was associated with decreased odds of a high embryo fragmentation score, suggesting that the embryos were low quality for IVF treatments. Collectively, these studies indicate adverse associations of parental BPA levels on the development of early embryos in vitro, but further studies are required to determine whether the observed effects occur in vivo.

35Although only two experimental studies have investigated the effects of BPA exposure on early embryo development in vivo, the results are intriguing. reported delayed embryo development in pregnant C57BL/6 mice administered high-dose BPA from GD0.5 to 3.5 (see Supplemental Material, Table S3). A more recent study of F1 female C57BL/6 Cast7 mice showed that low-dose BPA exposure initiated prior to breeding and continuing through pregnancy disrupted the expression of imprinted genes in midgestation embryos and placentas. Given the limited number of studies on the effects of BPA on embryo development in vivo, additional studies are needed in order to make firm conclusions about the effects of BPA on early embryos.

36Although epidemiological studies on BPA and the placenta have not been published, one experimental study suggested that both low and high doses of BPA increase levels of plasma estradiol, testosterone, and corticotropin-releasing hormone due to an increase in mRNA expression of corticotrophin-releasing hormone and activation of protein kinase C ζ/λ and δ in the placenta (see Supplemental Material, Table S3). A few in vitro studies have indicated that BPA affects placental cell proliferation. After 24 hr or 48 hr of culture, BPA (1–10 μM) increased apoptosis and decreased proliferation of cultured human trophoblast cells from first trimester placentas, whereas 10-μM BPA decreased cell viability. Similarly, after 24 hr, BPA (0.0877–8.77 μM) increased apoptosis in cultured human cytotrophoblasts. In addition, recent data from mouse studies suggest that BPA alters gene expression in the placenta. Taken together, these in vitro data suggest that BPA may affect placental function, but additional animal and human studies are required to substantiate these data.

37Only one study has examined the association between BPA exposure and pregnancy outcome in humans. Specifically, in a nested case–control study of 60 pregnant women, found a positive association between urinary BPA concentration and preterm birth (see Supplemental Material, Table S4). Given the limited information available, more studies are needed before making conclusions about whether BPA exposure is associated with adverse pregnancy outcomes in humans.

38In contrast, several experimental studies have investigated the effects of BPA exposure on pregnancy outcomes in animal models, such as mice and rats, which are altrical species in which pups are born at a stage of development equivalent to midgestation in humans. As shown in Supplemental Material, Table S5, several studies have reported that low-dose BPA did not alter gestation length in gestationally plus neonatally exposed mice, adult-exposed mice, or gestationally plus neonatally exposed SD rats. reported that low-dose BPA decreased the number of pregnancies and successful deliveries in gestationally exposed mice. BPA also decreased the percent of hatchings of chickens exposed in ovo to BPA (134 ng/kg) on day 4 on incubation. Conversely, neither low- nor high-dose BPA affected the number of litters born to unexposed female SD rats mated to gestationally exposed males, suggesting that maternal, but not paternal, BPA exposure may influence successful delivery of offspring.

39Many experimental studies have reported that low-dose BPA does not affect the number of live pups or total number of delivered pups in mice and rats. In a few studies, however, low-dose BPA exposure was reported to decrease the number of live pups born to prenatally plus neonatally exposed CD-1 mice and Holtzman rats. Low-dose BPA also decreased the total number of pups born to gestationally and neonatally exposed CD-1 mice. Interestingly, BPA acted differently from the positive control (diethylstilbestrol; DES), suggesting that the effects of BPA may differ from those of DES in CD-1 mice. Low-dose BPA also decreased the total number of pups born to unexposed female Holtzman rats mated to neonatally and gestationally exposed males of the same strain. In these two studies, BPA exposure decreased pup numbers after multiple litters, similar to DES. Collectively, these studies suggest that BPA exposure affects pregnancy outcomes in many, but not all, studies depending on experimental protocol. Clearly, more studies are required to determine why results differ and to determine whether BPA exposure does indeed affect pregnancy outcomes in animal models.

40Human studies of BPA and birth weight are equivocal (see Supplemental Material, Table S4). In a cross-sectional study of 97 pregnant women, observed that women with serum BPA concentrations > 2.51 ng/mL had a higher risk for having male babies that were small for gestational age than did women with lower serum BPA concentrations. However, these authors found no associations with female babies (see Supplemental Material, Table S4). These results are consistent with a retrospective cohort study that included 587 births in which maternal and paternal occupational exposure to BPA were both associated with low birth weight and small gestational size; the association was stronger for maternal exposure than paternal exposure. Further, in a Dutch population-based prospective cohort study of 219 pregnant women, urinary BPA was positively associated with lower growth rates and smaller head circumference. Similarly, in a birth cohort study of 757 Korean pregnant women participating in the Mothers and Children’s Environmental Health (MOCEH) study, BPA was associated with increased birth weight in males and neonatal length in females. However, in a case–control study of 191 pregnant women, BPA had an inverse U-shaped association with birth weight. In a cross-sectional study of 40 pregnant women, urinary BPA levels at delivery were not associated with birth weights of the offspring. Further, in a prospective cohort study of 404 mother–infant pairs, BPA exposure was not associated with increased birth weight. Given some of these discrepant results, future studies should assess whether BPA exposure is associated with birth weight.

41In previous work, gestational exposure to low doses of BPA did not alter the birth weight of mice or rats. However, recent animal studies suggest that the effect of BPA on offspring birth weight is dose dependent (see Supplemental Material, Table S5). reported that high-dose BPA increased the weight of offspring born to gestationally exposed mice. Conversely, low-dose BPA decreased the weight of offspring born to neonatally exposed mice. However, at doses below the U.S. EPA reference dose (50 μg/kg), BPA did not affect the weight of offspring born to gestationally plus neonatally exposed mice or rats, suggesting that BPA has a specific dose effect on birth weight of offspring.

42Human studies of the effect of adult exposure to BPA on sperm quality are very limited (see Supplemental Material, Table S6). Studies of occupationally exposed men and men recruited from an infertility clinic reported that higher urinary BPA levels were associated with a decrease in sperm count and motility. However, in a study of fertile men, urinary BPA concentration was not associated with changes in any semen parameters, but urinary BPA levels were significantly associated with markers of free testosterone. Although the results of these few epidemiological studies are consistent, there is insufficient evidence to draw conclusions about the association between BPA and semen quality in humans.

43The earliest experimental studies of the effects of BPA on sperm suggested adverse effects on spermatogenesis in the adult following either prenatal or early postnatal exposure (reviewed by ). Subsequent experimental studies have provided further evidence that prenatal and early postnatal exposure to BPA adversely affect spermatogenesis and have provided new evidence that exposure in adult rodents affects sperm quality (see Supplemental Material, Table S7). In rodents, exposure during the time of testis development—either during gestation or in the early postnatal period—or to young adult males has been associated with a range of adverse effects in the adult testis. However, variability in exposure timing, species and strains, end points, and life stage makes a simple analysis of the data difficult. Despite the variation in study design, several findings have emerged repeatedly, including decreased sperm counts, increased apoptotic cells within the seminiferous tubules, and decreased sperm motility; alterations in hormone levels and/or steroidogenic enzymes; and evidence of sperm DNA damage.

44The evidence that BPA exposure adversely affects sperm production and quality in the adult rodent is supported by data from recent studies (see Supplemental Material, Table S7). Gestational exposure to low-dose BPA resulted in a decreased number of elongated spermatids present in seminiferous tubules in pubertal ICR mice and decreased sperm counts in Holtzman rats. Similarly, both low- and high-dose BPA exposure during early postnatal development or around the time of puberty increased apoptosis and/or decreased spermatogenesis in male mice and rats. Further, adult rats exposed to low-dose BPA for either 6 or 14 days showed increased apoptosis and decreased sperm counts.

45In addition, a number of studies using various routes of exposure (oral and subcutaneous) and different exposure times (embryonic, fetal, perinatal, and adult) have reported that low-dose BPA impairs sperm motility in rats and mice (see Supplemental Material, Table S7). Interestingly, reported that the BPA-induced decrease in rat sperm motility was prevented by coadministration of the antioxidant n-acetyl cysteine, suggesting that impaired sperm motility may be related to increases in reactive oxygen species (ROS). As reviewed by, antioxidant use has been suggested to increase sperm quality in men and increase pregnancy rates of infertile couples.

46Spermatogenesis is dependent upon androgens, and changes that impact the endocrine environment of the testes have the potential to adversely affect sperm production and quality. Endocrine-related changes have been reported in several experimental studies: Rodent studies of gestational exposure alone and continuous gestational–neonatal exposure to BPA have reported evidence of impaired steroidogenesis, a decreased number of steroid receptors, or a decrease in Star expression (see Supplemental Material, Table S7). However, the most compelling data have come from studies of male rats exposed in the postnatal/adult period: Most of these studies reported a decrease in testosterone levels and/or steroidogenic enzymes in BPA-exposed males (low or high dose) compared with controls. Only one study in ICR mice reported no change in testosterone levels in low-dose-BPA–exposed males compared with controls, despite the fact that the exposed males had impaired spermatogenesis.

47In addition to endocrine changes, sperm DNA damage has been reported in BPA-exposed rodents (see Supplemental Material, Table S7). Consistent with studies before 2007, recent studies have shown that continuous exposure to low-dose BPA exposure induces DNA breaks and the production of ROS in mice and rats (see Supplemental Material, Table S7). reported that oral exposure to low-dose BPA for 6 days also induced sperm DNA damage. Similarly, oral low-dose BPA exposure has been reported to decrease testicular glucose levels and the expression and translation of glucose transporter-8 in spermatocytes and spermatids and to increase testicular hydrogen peroxide levels in Wistar rats and oxidative stress in SD rats.

48Unfortunately, the relevance of the doses used in the experimental studies to human exposure is not certain because internal or serum BPA levels were not determined in the studies. Nevertheless, these data add to the evidence that BPA exposure in the adult rodent adversely impacts the testis and sperm quality at levels below the LOAEL of 50 mg/kg. Although similar reproductive effects have been reported in multiple studies, a few studies failed to find any adverse reproductive effects in rodent models after gestational plus neonatal BPA exposure (see Supplemental Material, Table S7). These discordant findings may be related to a variety of factors, including dose, exposure route, internal dose, timing, and selected end points. Nevertheless, commonalities among studies finding no adverse effects raise several concerns. First, two of the four studies used the same inbred mouse strain, C57BL/6, suggesting that this strain may be insensitive or less sensitive to the effects of BPA on sperm than other strains. Second, dietary exposure has been used in several no-effect studies, but it was not used in any studies reporting adverse effects. These results add to the concern about the role that route of exposure plays in BPA studies and substantiates calls to replace exposure route with measurements of internal dose. Indeed, recent studies have shown that the route of BPA administration results in markedly different ratios of unconjugated (bioactive) to conjugated BPA and provided evidence that much of human exposure must be from non-oral routes. Last, two of the studies focused on end points such as body/organ weight, which alone may not accurately measure effects on male fertility. Indeed, some studies reporting detrimental effects (e.g., increased germ cell apoptosis, reduction in sperm counts) did not find significant changes in testis or epididymal weight (see Supplemental Material, Table S7). Nevertheless, these negative findings underscore the need for comparative experimental studies involving different strains of animals and routes of exposure. In addition, assessing the importance of effects during different life stages is important, particularly because identified abnormalities in middle age that were not seen in young adult female rodents.

49Three epidemiological studies investigated the association between BPA exposure and serum hormone levels in men (see Supplemental Material, Table S6). In a study of 307 Italian men participating in the INChianti study (a prospective, population-based study of adults living in Chianti, Italy), observed an association between increased urinary BPA concentrations and increased serum testosterone levels, but not estradiol levels. This observation was not supported by two cross-sectional studies of 167 and 302 fertile men, which reported no correlation between BPA exposure and testosterone levels. However, found associations between urinary BPA concentrations and decreased free androgen index (FAI), the ratio of FAI to luteinizing hormone, and the ratio of free testosterone to luteinizing hormone in male partners of pregnant women. found an association between high BPA concentrations and increased serum levels of follicle-stimulating hormone, and between high BPA concentrations and decreased levels of inhibin B, the ratio of follicle-stimulating hormone to inhibin B, and the ratio of estradiol to testosterone. These data suggest that BPA can alter steroid hormone pathways in men; however, because of limited association studies and likely multifactorial responses produced by BPA, more studies are needed to determine how BPA affects steroidogenesis. Further, because most studies conducted before 2007 focused on infertile men, it is important to conduct studies on fertile men to confidently determine if BPA affects male steroidogenesis and whether findings in infertile men can be extrapolated to the general population.

50Experimental studies indicate that BPA exposure decreases hormone levels in male animals, but the data are discordant (see Supplemental Material, Table S7). Low-dose BPA was reported to decrease testosterone levels in gestationally through postnatally exposed CD-1 mice but not in adult C57BL/6 mice exposed in utero. Low-dose BPA decreased testosterone levels in gestationally or neonatally exposed Holtzman rats, adult-exposed albino rats, and adult-exposed Wistar rats. Conversely, gestational plus neonatal exposure to low-dose BPA did not affect testosterone levels in Long Evans (LE) rats or SD rats. However, although reported that low dose BPA did not decrease testosterone in Wistar rats, low-dose BPA decreased dihydrotestosterone levels. Mechanistic underpinnings for the ability of BPA to inhibit testosterone levels may be found in the fact that BPA decreases expression of steroidogenic enzymes and levels of follicle-stimulating hormone, which are required to directly and indirectly stimulate steroidogenesis. Collectively, these data indicate that BPA exposure decreases sex steroid hormone levels in male rodents, but strain and species, as well as other confounders (e.g., time, route of exposure, age at analysis), may modulate the sensitivity to BPA effects.

51Because Leydig cells are responsible for most steroid hormone production in males, experimental studies have been designed to examine whether BPA directly affects Leydig cells. In pubertal Wistar/ST rats, continuous exposure to high-dose BPA was reported to decrease cell numbers and expression of steroidogenic enzymes in the Leydig cells.. Conversely, low-dose BPA increased Leydig cell numbers in gestationally plus neonatally exposed LE rats in adulthood by up-regulating mitogenic factors. Although BPA exposure increased Leydig cell proliferation, it did not alter circulating testosterone levels. In fact, in in vitro Leydig cell isolates from adult-exposed males, low-dose BPA decreased the expression of steroidogenic enzymes and testosterone production. Similarly, BPA decreased testosterone levels in human, mouse, and rat fetal testes in vitro. In another study, reported that gestational exposure to low- and high-dose BPA increased Leydig cell numbers without affecting serum testosterone levels in SD rats in adulthood. Collectively, these data strongly suggest that BPA negatively affects gonadal function through changes in steroid-synthesizing cells/enzymes, which then affect steroid synthesis and circulating steroid levels.

52Disruptions in testicular testosterone production during development may lead to phenotypic abnormalities after birth, such as shortened anogenital distance (AGD) and undescended testes. In a retrospective occupational study of 587 Chinese children, found a dose-dependent association between parental occupational exposure to BPA and shortened AGD in male offspring (see Supplemental Material, Table S4). This inverse correlation was strengthened with increasing maternal BPA concentrations. Interestingly, observed that BPA concentrations in cord blood samples were not correlated with cryptorchidism in a matched case–control study of 152 newborn males. Although these studies suggest that BPA exposure may be associated with shortened AGD and undescended testes, it is important to confirm these findings in other epidemiological studies.

53In several animal studies, BPA exposure did not affect AGD, but it did influence testes descent (see Supplemental Material, Table S7). In four recent studies, gestational or gestational plus neonatal exposure to low-dose BPA did not alter AGD in male rodents. In another study, reported that gestational exposure to high-dose BPA decreased absolute AGD in F1 CD-1 males at birth, but not in the F2 generation and not when normalized to relative body size. In the same study, high-dose BPA delayed preputial separation and increased the incidence of treatment-related undescended testes in F1 and F2 offspring. Notably, the doses used by were very high and not physiologically relevant to human exposure. Collectively, these data suggest that BPA exposure may not affect AGD in animal models, but it may affect other developmental aspects such as undescended testes.

54To date, no epidemiological studies have reported associations between BPA exposure and alterations in the male urinary tract, and only a few experimental studies have evaluated the effects of BPA on the male urinary tract (see Supplemental Material, Table S7). In the adult rat prostate, low- and high-dose BPA exposure has been reported to adversely increase clusterin levels and expression of aromatase, while decreasing levels of 5α-reductase type 1 and 2. Interestingly, and reported that BPA exposure increased the plasma estradiol to testosterone ratio, which has been implicated in the development of benign prostate hyperplasia. Further, gestational exposure to low-dose BPA increased gene expression of androgen receptor, Esr1, aromatase, and estrogen-related receptor γ in the mouse prostate.

55Other studies have indicated that BPA exposure plays a role in prostate pathogenesis in animal models. reported that neonatal exposure of rats to low-dose BPA increased the incidence of the precursor for prostate cancer, prostate intraepithelial neoplasia, in adulthood. Further, reported that in rats with induced prostatic hyperplasia, high-dose BPA increased prostate gland mass and increased relative weight of the dorsolateral prostate lobe. High-dose BPA exposure also increased epithelial cell heights in the ventral prostate and dorsolateral prostate lobes. Last, neonatal exposure to low-dose BPA led to transient and permanent hypomethylations in the rat epigenome, implicated in the manifestation of prostatic carcinogenesis. Collectively, these studies support the concept that both low and high doses of BPA promote changes in steroidogenic pathways and morphology of the prostate, which may in turn affect homeostasis and pathogenesis.

56Currently, no information is available about whether BPA exposure causes benign urologic disease. However, studies have shown that estrogens and estrogen receptor pathways negatively affect the lower urinary tract, supporting a potential role for estrogen-like molecules, including BPA, in the manifestation of urological diseases. Future experimental studies evaluating the effects of BPA on the lower urinary tract, as well as epidemiological studies in humans, need to gain insight into the role BPA plays in disease processes in the lower male urinary tract.

57Two epidemiological studies investigated the effect of BPA on puberty and measured BPA levels in girls at similar ages (see Supplemental Material, Table S2). In these studies, one of 1,151 girls 6–8 years of age and another of 192 girls 9 years of age, BPA exposure was not associated with accelerated breast or pubic hair development. In a study of 82 patients with precocious puberty and 32 patients without precocious puberty, BPA exposure was not associated with precocious puberty. However, in a study of 110 girls with precocious puberty and 100 girls without precocious puberty, BPA was associated with increased uterine and ovarian volume. These studies suggest that BPA exposure may not be associated with onset of puberty in girls, but given the limited number of studies, these results should be confirmed in future studies.

58In animal models, the effects of BPA on factors such as puberty onset and sexual receptivity are equivocal (see Supplemental Material, Table S8). In one study, low-dose BPA did not affect the timing of puberty onset as measured by vaginal opening in LE rats orally exposed to BPA gestationally plus neonatally. Conversely, low- and high-dose BPA exposure accelerated vaginal opening in neonatally exposed ICR mice, SD rats, and LE rats. Low-dose BPA also decreased the time neonatally exposed ICR mice and SD rats spent in estrus. However, low-dose BPA did not affect estrous cyclicity in gestationally plus neonatally exposed CD-1 mice and LE rats. Further, low-dose BPA had no effect on lordosis behavior in gestationally plus neonatally exposed LE rats or neonatally exposed female LE rats. Collectively, these studies indicate that the effects of BPA on the onset of puberty and sexual receptivity in animal models are unclear and likely differ depending on strain and species. Further studies should examine differences in puberty onset and sexual receptivity in various strains and species of animal models.

59Few epidemiological or experimental studies have examined the association between BPA exposure and sexual dysfunction. One cross-sectional study of 425 men occupationally exposed to BPA reported an increased risk of self-reported impaired sexual abilities compared to 284 unexposed men (see Supplemental Material, Table S6). Some animal studies have indicated that BPA may impair sexual ability (see Supplemental Material, Table S8). Low-dose BPA increased the time taken for copulation in neonatally or gestationally exposed Holtzman rats and increased latency to insemination in perinatally exposed CF-1 mice. Low-dose BPA also decreased intromission and ejaculations of perinatally exposed male CF-1 mice mated to unexposed females. However, in another study, low- or high-dose BPA did not affect the time taken for copulation in gestationally plus neonatally exposed adult CD-1 mice or their offspring. Given the limited number of studies conducted on BPA and sexual function and the equivocal nature of the results, it is not possible to make firm conclusions on the effects of BPA on sexual function. However, it is important to note that all but one study conducted to date suggest that BPA negatively impacts sexual function.

60Here we summarized the strength of the evidence for associations between BPA and adverse reproductive outcomes based on literature published from 2007 through 2013. The data presented in this review build on the overall conclusions of the expert panel report that the widespread effects of BPA in experimental animal studies are a concern for overall human health and may be involved in human reproductive disease. Similar to the 2007 expert panel report, we considered the evidence to be strong when multiple studies in multiple species indicated a similar effect of BPA on a reproductive tissue or end point, even if concordance was not 100% across all studies, given that species and strain differences can lead to differences in dose response and magnitude of effect. These conclusions, however, are not to be considered definitive without further investigation, especially with the gaps in clear results detailed throughout the review. In the experimental studies, strong, definitive conclusions often were difficult because study designs were so different. Experimental studies rarely used the same doses, timing, positive controls, and exposure routes; thus, the effects of BPA on exposure among animal strains and species could not effectively be compared. However, one unifying strength of these studies related to human health is that most of the studies we evaluated used BPA doses below the LOAEL. In the epidemiological studies, strong conclusions were difficult to determine because of study design and exposure parameters. For example, exposure assessments in most human studies relied on a single urine sample, which may introduce exposure misclassification and attenuate associations if they are present. Given the continuous and variable exposure to BPA, a single urine sample may not represent longer-term exposure or exposure in the relevant etiological window. Finally, most of the human studies were cross-sectional, making it difficult to discern the temporal relationship of exposure with response. These limitations affect the interpretation of human studies. Future human studies need to consider improvements in exposure assessment to represent longer-term BPA exposure assessed at etiologically relevant windows. Given recent data, it will be critical in future studies to assess effects of different routes of exposure and to analyze effects at different stages in the life of the exposed individual. In addition, for experimental studies, the inclusion of positive controls should be considered essential.

61However, given the data included in this review, we have drawn some insights and conclusions that add to the conclusions drawn based on reviews of the BPA literature prior to 2007, categorized by the strength of evidence presented.

62Strong evidence exists that

63BPA is an ovarian toxicant in animal models and women. It adversely affects the onset of meiosis in ovaries from both animal models and humans, interferes with germ cell nest breakdown in animal models, accelerates follicle transition in several animal species, alters steroidogenesis in multiple animal models and in women, and reduces oocyte quality in animal models and in women undergoing IVF.

64BPA is a uterine toxicant in animal models because it impairs uterine endometrial cellular proliferation, decreases uterine receptivity, alters gene expression, and increases implantation failure in several strains and species. However, human studies have not adequately addressed these end points.

65BPA is a prostate toxicant in animal models, impairing the steroidogenic capacity and altering dorsal and ventral lobe morphology, potentially leading to prostate pathogenesis. Human studies, however, are lacking.

66The effects of BPA on the reproductive system are variable and evident at doses below the LOAEL of 50 mg/kg and the proposed safe level of 50 μg/kg/day.

67Limited evidence exists that

68Relative to the impact of BPA on birth rate, birth weight, and length of gestation, human data on birth weight are inconsistent; although animal studies suggest such an association, there are limited human studies on gestational length and preterm birth.

69BPA exposure is associated with hyperandrogenism, such as in PCOS in women. However, data in rodent models do not support the development of symptoms of human PCOS.

70BPA is a testicular toxicant in animal models because it decreases sperm quality and motility, causes oxidative stress, and alters steroidogenesis. Human data, however, are inconsistent.

71BPA is associated with impaired implantation in women undergoing IVF. The associations between BPA and implantation failure in women may be due to the effects of BPA on the embryo or the uterus, or both.

72BPA exposure in male rats is associated with implantation failure in nonexposed female rats. Human data, however, are lacking.

73BPA is associated with sexual dysfunction among men exposed to high occupational levels and in experimental studies on males.

74There is insufficient evidence to draw conclusions regarding effects of BPA on the oviduct, placenta, and pubertal development.

75Future studies need to

76Consider the critical period of differentiation of the organ system in question and the reproductive life span of the animal model or human.

77Use continuous exposure to BPA in view of the ubiquitous and continuous exposure to BPA in humans.

78Target internal dose levels of BPA that are achieved by human exposures.

79Recognize the potential interaction of BPA with other hormone-altering chemicals and lifestyle factors such as diet and stress.

80Distinguish organizational (permanent) versus activational (transient) effects of BPA.

81Determine whether preterm and maternal morbidities (e.g., preeclampsia, gestational diabetes), as well as paternal factors, modify or mediate the effects of BPA on the reproductive system.