Evidence for Bisphenol A-Induced Female Infertility: a Review (2007–2016)

Evidence for bisphenol A-induced female infertility: a review (2007–2016)

Ayelet Ziv-Gal, Ph.D.a and Jodi A. Flaws, Ph.D.b a School of Food and Nutrition, Massey University, Palmerston North, New Zealand; and b Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, Illinois

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. In addition, 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. (Fertil SterilÒ 2016;106:827–56. Ó2016 by American Society for Reproductive Medicine.) Key Words: Infertility, female, bisphenol A, ovary, uterus, implantation, hypothalamus, pituitary

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emale infertility is generally One of the most extensively studied and 2016. We included the morphological F defined as the inability to get preg- endocrine disrupting chemicals is bisphe- and mechanistic findings reported in the nant naturally and to deliver a live nol A (BPA). Bisphenol A is incorporated reviewed articles. We focused on the re- healthy newborn. According to the Cen- in many daily used products; it is used ported outcomes of BPA exposure on ters for Disease Control and Prevention by the manufacturers of polycarbonate overall: [1] fertility, [2] reproductive- (CDC; http://www.cdc.gov/nchs/nsfg/ plastics and epoxy resins. Despite the related processes including the ovarian key_statistics/i.htm#infertility), between relatively short half-life of BPA cycle, and [3] reproductive tissues. 2011 and 2013, 6.1% of married women (6–24 hours) (2),itwasmeasuredin were considered to be infertile in the various reproductive tissues (3),including MATERIALS AND METHODS United States alone. The percentage of ovarian follicular fluid, placenta, breast infertile women can reach 30% world- milk, and colostrum. Findings from previ- PubMed (http://www.ncbi.nlm.nih.gov/ wide (1). Infertility in women can be the ous publications suggest that BPA is a pubmed) searches for the years 2007– result of various factors, including phys- reproductive toxicant (4–6). 2016 were conducted using the ical problems, endocrine problems, life- The current review focuses on the sci- following key words: BPA, bisphenol style habits, and environmental factors. entific evidence for BPA-induced fertility A, fertility, female, reproduction, ovary, Environmental factors, such as exposure problems in females. We summarized the pregnancy, oviduct, ovulation, fertiliza- to chemicals with endocrine disrupting main findings of epidemiological and tion, uterus, implantation, hypothala- properties, can mimic or block the endo- experimental studies that examined the mus, and pituitary. We focused on crine activity of endogenous hormones potential effects of BPA on female fertility articles published in 2007–2016 to and thus adversely affect reproduction. and that were published between 2007 expand on previous review reports on thesametopic(4, 5 ,7–11). In addition, references included in other review

Received April 26, 2016; revised May 25, 2016; accepted June 15, 2016; published online July 12, 2016. articles were examined for relevant A.Z.-G. has nothing to disclose. J.A.F. has nothing to disclose. information. We included articles that Supported by National Institutes of Health P01 ES022848 and EPA RD-83459301. dealt with fertility/infertility outcomes Reprint requests: Jodi A. Flaws, Ph.D., Department of Comparative Biosciences, University of Illinois, 2001 South Lincoln Avenue, Urbana, Illinois 61802 (E-mail: jﬂ[email protected]). related to overall fertility, implantation, uterine morphology and function, Fertility and Sterility® Vol. 106, No. 4, September 15, 2016 0015-0282/$36.00 Copyright ©2016 American Society for Reproductive Medicine, Published by Elsevier Inc. estrous cyclicity, hypothalamus- http://dx.doi.org/10.1016/j.fertnstert.2016.06.027 pituitary, hormone levels (luteinizing

VOL. 106 NO. 4 / SEPTEMBER 15, 2016 827 ENDOCRINE DISRUPTING CHEMICALS: FEMALE AND MALE REPRODUCTION hormone [LH], follicular stimulating hormone [FSH], and pro- Overall, these studies are suggestive for potential associations lactin [PRL]), oviduct, and ovary. We excluded articles about between BPA and infertility. However, additional studies are topics that were out of the scope of this review or ones that needed to determine a possible cause and effect relationship will be reviewed by other investigators in this special issue and the mechanism of action of BPA-mediated effects on (e.g., sexual maturation/behavior, oocyte quality and matura- fertility in healthy women. tion, ovarian steroidogenesis, pregnancy, miscarriage, endome- Not all epidemiological studies found an association be- triosis, polycystic ovarian syndrome [PCOS], and uterine tween BPA exposure and fertility outcomes. Null associations fibroids/leiomyoma). were reported between urinary total BPA concentrations and The BPA studies have used various study designs and impaired fecundity or time to pregnancy in generally healthy included a wide range of doses. Based on the definitions in women (18, 23, 25). In another study (27), null associations other studies, we considered a ‘‘low dose’’ of BPA as follows: between urinary total BPA concentrations and number of a dose below the lowest observable adverse effect level of oocytes retrieved, embryo quality, and fertilization rates 50 mg/kg/d in animal models (4, 5, 12, 13), 17.2 mg/L for were reported in women undergoing IVF treatments. The aquatic animals (5, 14),1 107 M for cell culture differences in the results may be explained by differences in experiments (5, 15), and a dose in the range of typical (not sample characteristics (i.e., generally healthy women occupational) human exposures for epidemiological studies without any reported infertility issues vs. women (5, 16). Most studies described in this review used doses that undergoing IVF treatments) and by differences in sample size. are within the category of low dose. Throughout the text of Studies using animal models provide further insights on this review, we indicated if the doses were considered low the effects of BPA exposure on female fertility (Table 2). In or high based on these categories. In the Tables, the specific mice, Berger et al. (34) reported that low dose BPA exposure doses that were used in each study are described in detail. of pregnant dams during the preimplantation period signifi- Last, similar to Peretz et al. (4),wedefined exposure time cantly reduced the number of litters and litter size compared during pregnancy as in utero; exposure after birth that with controls. Furthermore, in utero low dose BPA exposure ended before weaning as neonatal; and exposure any time after implantation affected the fertility of the females in the after weaning as postnatal or adult exposure. subsequent generations (45, 49). Cabaton et al. (35) performed a forced breeding study and found that low dose RESULTS AND DISCUSSION BPA-exposed females had fewer pregnancies and overall reduced cumulative number of pups compared with controls. Overall Fertility Moore-Ambriz et al. (40) examined the effects of BPA expo- In recent years, several research groups have examined the ef- sure in young adult mice on fertilization capacity later in fects of BPA on overall fertility. Epidemiological studies adult life. The fertilization rate of BPA exposed females was examined whether BPA levels are higher in infertile women reduced compared with controls. Furthermore, impaired than in fertile women (Table 1). Findings from these studies fertility was also reported in a study that examined the effects (19, 26) indicate that infertile women have higher serum of in utero low dose BPA exposure in three subsequent gener- BPA levels compared with fertile women. Furthermore, ations of mice (45, 49). Specifically, F1 females that were studies (17, 21, 22, 28, 29) conducted in women undergoing gestationally exposed to BPA had reduced fertility, reduced IVF treatments show that BPA levels (total or unconjugated litter size (45), and reduced ability to maintain pregnancy to BPA) were inversely associated with peak E2 levels, number term (i.e., reduced gestational index) compared with of oocytes retrieved, oocyte maturation, fertilization rates, controls (49). Furthermore, F2 females had a reduced and embryo quality. Thus, increased levels of BPA may gestational index compared with controls (49). In addition, decrease the success rate of IVF treatments. Nevertheless, F3 females exhibited reduced fertility and decreased ability these studies did not take into account potential modifying to become pregnant compared with controls, indicating a factors such as co-exposure to other chemicals and the loca- potential transgenerational effect of BPA on female tion of sample collection as pointed out by Teeguarden et al. reproduction (49). In chickens, in ovo high dose BPA (30, 31). Thus, additional studies are needed to fully exposure reduced hatchability (47), whereas in fish, low understand the associations between BPA exposure and dose BPA exposure increased the observed hatching rate (41). fertility outcomes in women. In contrast, some experimental studies (36–39, 42, 43 ,46, Limited information is available on the potential molecular 48) reported that BPA exposure does not affect fertility targets of BPA in infertile women. Hanna et al. (24) reported an outcomes. Specifically, a few studies (36–38,43) indicate that association between higher serum levels of unconjugated BPA gestational low dose BPA exposure did not alter number of and decreased methylation within the TSP50 gene promoter in litters or litter size (36–39,42,43,46,48) in mice, rats, and fish. whole blood samples of women undergoing IVF treatments. Xi et al. (46) also indicate that gestational BPA exposure at a However, the researchers did not provide any mechanistic ex- dose of 50 mg/kg/d (i.e., lowest observable adverse effect planations of these findings other than to indicate that TSP50 level) did not alter litter size in mice. Similarly, Moore- may be an oncogene based on previous research by other Ambriz et al. (40) reported that low dose BPA exposure did groups (32, 33). Interesting findings reported by Chavarro not affect the size of preovulatory follicles, the number of et al. (20) suggest a potential modifying effect of soy food shed oocytes, and zygotes in adult mice that were exposed to consumption on the inverse correlations between urinary BPA at a younger age. One of the reasons for differences be- total BPA concentrations and fertility treatment outcomes. tween the reported results may be the age of the animals.

828 VOL. 106 NO. 4 / SEPTEMBER 15, 2016 O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. TABLE 1

BPA and fertility (epidemiological studies). Time of BPA Reference Study design Study population Sample size measurement BPA concentration Outcome no. Cross-sectional Women undergoing IVF 44 Day of oocyte retrieval Median unconjugated BPA inversely associated (17) serum BPA with peak estradiol; 2.53 ng/mL (range, no association with 0.3–67.36 ng/mL) number of oocytes retrieved Matched cohort Women discontinuing 210 On the day of expected Urine (total BPA), mean No association with (18) contraception menstruation (95% conﬁdence impaired fecundity or interval): pregnant: time to pregnancy 0.63 ng/mL (0.54–0.73); nonpregnant: 0.68 ng/mL (0.53–0.87) Cross-sectional Fertile and infertile Italian 12 fertile Enrollment Not indicated; limit of Higher percentage of (19) women 35 infertile detection 0.5 ng/mL infertile women with (serum samples) detectable serum BPA levels Prospective Women undergoing IVF 239 (63 no soy food Between days 3 and 9 of Urine (total BPA); Range, Soy food consumption (20) intake); 347 cycles gonadotropin phase <0.4–16.6 mg/L; modiﬁes the and on day of oocyte median, 1.3 mg/L correlation between retrieval (interquartile range, BPA and infertility 0.9–1.9 mg/L) treatment outcomes Prospective Women undergoing IVF 174 (237 cycles) Day of oocyte retrieval Urinary (total BPA) Higher BPA levels (21) geometric mean 1.50 associated with lower (SD 2.22) mg/L serum peak estradiol, oocyte yield, MII oocyte count, and number of normally fertilizing oocytes Prospective Women undergoing ICSI 58 Day of oocyte retrieval Median unconjugated Inverse associations (22) and IVF serum BPA between BPA and 2.53 ng/mL (range, oocyte maturation 0.0–67.4 ng/mL) (Asian women) and normal fertilization (all women) Pregnancy-based Women in 1st trimester 1,742 Spot urine during the 1st Urinary (total BPA) No association with (23) retrospective trimester visit geometric mean 0.78 diminished fecundity

(0.73–0.82) ng/mL Sterility® and Fertility Prospective Women undergoing IVF 35 of 58 Day of oocyte retrieval Median unconjugated Up-regulation of TSP50 (24) serum BPA 2.4 mg/L with increased BPA serum (range, levels due to a loss of 0.0–67.4) methylation Ziv-Gal. BPA and female infertility. Fertil Steril 2016. 829 NORN IRPIGCEIAS EAEADML REPRODUCTION MALE AND FEMALE CHEMICALS: DISRUPTING ENDOCRINE 830 TABLE 1

Continued. Time of BPA Reference Study design Study population Sample size measurement BPA concentration Outcome no. Prospective Fertile women that 221 Pooled urine throughout Urinary (total BPA) BPA associated with (25) discontinued menstrual cycles median 2.7 ng/mL shorter luteal phase; contraception (interquartile range null associations with 1.8, 4.3), not follicular-phase adjusted length, time to pregnancy, and early pregnancy loss Cross-sectional Women who 110 infertile, 43 fertile Whole blood sample Mean (total BPA, serum) BPA levels higher among (26) volunteered prior to any (ng/mL): fertile 4.8 infertile women treatment infertile 10.6 (odds ratio 8.3) in metropolitan area Prospective Women undergoing IVF 256 (375 cycles) Between days 3 and 9 of Urinary (total BPA) No associations with (27) gonadotropin phase; geometric mean oocyte yield, and on day of oocyte 1.87 mg/L endometrial retrieval thickness, embryo quality, fertilization rates, implantation, clinical pregnancy and live birth rates Prospective Women undergoing IVF 84 (112 cycles) Day of oocyte retrieval Urinary (total BPA); BPA levels inversely (28) Range, <0.4–25.5 associated with the mg/L; urinary number of oocytes geometric mean retrieved and peak 2.52 mg/L (SD 3.2) serum estradiol levels Prospective Women undergoing IVF 36 Day of oocyte retrieval Median unconjugated No association with (29) serum BPA 3.3 ng/mL embryo quality (range, 0.0–67.4 ng/mL) Note: BPA [ bisphenol A; ICSI ¼ intracytoplasmic sperm injection. Ziv-Gal. BPA and female infertility. Fertil Steril 2016. O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. TABLE 2

BPA and fertility (experimental studies). Reference Source Strain Exposure route Time of exposure Doses Time of observation Outcome no. Mouse CF-1 Subcutaneous injection GD1-4 0–10.125 mg/d After birth BPA10.125 reduced (34) percent of females giving birth; BPA3.4 and 10.125 reduced number of pups born Mouse CD-1 Alzet osmotic pump GD8– PND16 25 ng, 250 ng, F1: from 8 wk until 32 wk BPA 25 ng females had (35) 25 mg/kg/d (forced breeding) fewer pregnancies and lower cumulative number of pups per dam that got worse with age Mouse C57BL/6NHsd, Hsd:ICR Extruded pellet diets Prior to breeding-PND21 0.03, 0.3, 30 ppm After birth No effect on fertility of (36) (CD-1 Swiss) C57BL6; decreased fertility of CD-1 (BPA0.03 and 0.3) Mouse C57BL/6J Dietary GD6–PND21 0.33, 3.3, 33 ppm After birth No effect on number of (37) births or litter size Rat Sprague-Dawley Dietary GD6–PND21 0.33, 3.3, 33 ppm After birth No effect on number of (38) births or litter size Fish Pimephales promelas Aquarium water 164 d exposure 1, 16, 64, 160, On exposure days No effect on fecundity (39) 640 mg/L 85–105, 135–155 Mouse C57BL/6J (39 d) Oral 12–15 d (ﬁrst 3 50 mg/kg/d Age 51–54 d Reduced fertilization (40) reproductive cycles) (in vitro fertilization or mating); did not alter zygotic stages Fish Transgenic zebraﬁsh Aquarium Embryonically 0.1, 1, 10, 100, 48, 55 h after fertilization BPA1 and 10 increased (41) 1,000 mg/L hatching rate Rat Long Evans Oral gavage þ lactation GD7–PND18 2, 20, 200 mg/kg/d After birth and over 4 mo No effect on litter size (42) Rat Wistar Oral, drinking water GD9–birth 0.5, 50 mg/kg/d After birth No effect on litter size or (43) number of births Rat Wistar Drinking water þ GD9–PND21 0.5, 50 mg/kg/d After birth No effect on litter size (44) lactation Mouse FVB Oral GD11–birth 0.5, 20, 50 mg/kg/d F1: 3, 6, 9 mo BPA0.5 reduced fertility (45) with age, increased % dead pups with time (3–9 mo); BPA50 reduced litter size (6 mo) Sterility® and Fertility Mouse CD-1 Oral gavage F0: GD1–PND20 12, 25, 50 mg/kg/d After birth No effect on litter size (46) Chicken White Leghorn In ovo injection Incubation d 4 67, 134 mg/g egg Hatching Decreased hatchability (47) (BPA 67, 134) Mouse CD-1 Oral GD12.5–PND18.5 0.2, 0.04, 0.08 mg/kg PND1 No effect on litter size (48) Ziv-Gal. BPA and female infertility. Fertil Steril 2016. 831 ENDOCRINE DISRUPTING CHEMICALS: FEMALE AND MALE REPRODUCTION

Studies that examined reproductive capacity in older animals were more likely to observe a difference between the BPA- no. (49) treated females and controls.

Reference 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. Howev- er, 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, we focus on reproductive organs that are targeted by BPA in a manner that could reduce gestational index (F1, F2); BPA0.5 reduced fertility index (F3); Reduced % of dead pups (F3, BPA20, 50); decreased ability to maintain pregnancy with age fertility. Speciﬁcally, the later sections 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-ovarian axis.

Oviduct After 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 settles in the uterus. Thus, a normal functioning oviduct is g/kg/d F1, F2, F3: 3, 6, 9 mo BPA50 reduced

m 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). Spe- ciﬁcally, 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 (50, 51). Furthermore, studies (52, 53) indicate that in utero high dose BPA exposure delayed development and transport of the conceptus compared with controls. Taken together, the current data indicate that gestational BPA exposure may

birth 0.5, 20, 50 affect both oviduct morphology and function; however, – further studies are needed to conﬁrm whether this is the

GD11 case in women and to examine potential molecular mechanisms of BPA action on the oviduct.

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 (F0) 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). Specif- ically, higher urinary total BPA levels were associated with increased implantation failure deﬁned by a serum b-hCG (<6 IU/L) conducted 17 days after egg retrieval in women undergoing IVF treatments (54). Experimental studies (34, 52, 55, 56, 59, 60) examining the effects of BPA exposure at early gestational stages bisphenol A. report a reduced number or complete ablation of [ implantation sites (53) in mice and rats when compared BPA with controls. These studies include both low (34, 55, 56, Ziv-Gal. BPA and female infertility. Fertil Steril 2016. Continued. Source StrainNote: Exposure route Time of exposure Doses Time of observation Outcome Mouse FVB Oral of pregnant dams TABLE 2 59, 60) and high (52) BPA doses. Furthermore, a recent

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TABLE 3

BPA and oviduct (experimental studies). Exposure Time of Time of Reference Source Strain route exposure Doses observation Outcome no. Mouse CD-1 Subcutaneous GD 1–5 10, 100, 1,000 18 mo Increased progressive (50) injection mg/kg/d proliferative lesions; remnants of Wolffian ducts Mouse CD-1 Subcutaneous GD 9–16 0.1, 1, 10, 100, 18 mo Increased progressive (51) injection 1,000 mg/kg/d proliferative lesions Mouse Not indicated Oral gavage GD 0.5–3.5 200, 400, 600, GD4 BPA 400–800 delayed (52) 800 mg/kg/d transfer of embryos from fallopian tubes to uterus (GD4) Mouse C57BL6 Subcutaneous GD 0.5–3.5 0.025, 0.5, 10, 40, GD3.5 BPA100 retention of (53) injection 100 mg/kg/d embryos and delayed embryo development Note: BPA [ bisphenol A. Ziv-Gal. BPA and female infertility. Fertil Steril 2016. study by Li et al. (59) demonstrated that low dose BPA In mice, in utero low dose BPA exposure increased uterine exposure reduced uterine levels of leukemia inhibitory anomalies in the luminal epithelium and glands (44) and factor (Lif), P receptor (PR; Pgr), heart and neural crest caused uterine hyperplasia, stromal polyps, and retention of derivatives expressed transcript 2 (Hand2), and homeobox remnants of the Wolffian duct in the adult offspring compared A10 (Hoxa10) compared with controls. These observed with controls (50, 51). Furthermore, in utero high dose BPA BPA-mediated effects can impair fertility because these fac- exposure reduced uterine weight in the second generation of tors are part of the P-mediated signaling pathway and are pups compared with controls (67). Neonatal high dose BPA important in uterine receptivity and implantation. exposure (single dose, 100 mg/kg) reduced uterine weight in Although most studies indicate that BPA alters implanta- young adult mice compared to controls (70), and neonatal tion, two experimental studies report no effect of low dose low dose BPA exposure decreased endometrial proliferation (57) or a relatively high dose (122 mg/kg/d) (58) of BPA on in adult ovariectomized rats compared with controls (63).In the number of implantation sites. The reasons for these young adult rats, low dose BPA exposure from gestation discordant results are unclear, but it may be that the effects day 6 until weaning increased the thickness of the uterine of BPA on implantation are ablated at high doses. epithelia and stroma compared with controls (69). In adult mice, dietary supplementation with low dose BPA resulted Uterine morphology and function. For proper blastocyst in- in clinical signs that are typical of pyometra (36). Last, in vasion, implantation, and successful pregnancy, the uterine hens, in ovo high dose BPA exposure resulted in abnormal endometrium transforms and reorganizes under the influ- uterine morphology compared with controls (47). Overall, ence of estrogen (E) and P. Thus, exposures that interfere the results from in vivo studies are suggestive for impaired with uterine function have the potential to adversely impact morphology of the uterus after early life stage BPA fertility. Based on our search criteria, we did not locate exposures at both low and high doses. epidemiological studies that focus on BPA exposures and Some of the molecular factors in the uterus that were associations with uterine outcomes. However, several altered after in vivo BPA exposure include members of in vivo and in vitro experimental studies have examined the Hoxa family, vascular endothelial growth factor (Vegf), the effects of BPA exposure on the uterus or uterine cells E receptor (ER) alpha and beta (Esr1 and Esr2), and Pgr (Tables 6 and 7). (60, 61, 63–65, 68, 71, 72). These factors are important for

TABLE 4

BPA and implantation (epidemiological study). Study Sample Time of BPA Reference Study design population size measurement BPA concentration Outcome no. Prospective Women 137 (180 IVF Day of oocyte Urinary (total BPA) geometric Higher BPA levels (54) undergoing IVF cycles) retrieval mean 1.53 (SD 2.22) mg/L associated with increased implantation failure Note: BPA [ bisphenol A. Ziv-Gal. BPA and female infertility. Fertil Steril 2016.

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TABLE 5

BPA and implantation (experimental studies). Time of Time of Reference Source Strain Exposure route exposure Doses observation Outcome no. Mouse CF-1 Subcutaneous injection GD 1–40–10.125 mg/d GD 6 BPA10.125 decreased (34) number of implantation sites Mouse CF-1 Subcutaneous injection GD 0, 1, 2 0–10.125 mg/d GD 6 BPA10.125 and 6.75 (55) decreased implantation sites Mouse CF-1 Subcutaneous injection GD 1–40–10.125 mg/d GD 6 BPA6.75 and 10.125 (55) decreased number of implantation sites Mouse CF-1 Subcutaneous injection GD 1–4 3.375, 6.75, 10.125 GD 6 BPA6.75 and 10.125 (56) mg/d decreased number of implantation sites Mouse CF-1 Subcutaneous injection GD 1–4 3, 4, 5 mg/d GD 6 No difference in number of (57) implantation sites Mouse CF-1 Subcutaneous injection GD 1–3 2, 4 mg/d (61, 122 GD 6 No difference in number of (58) mg/kg/d) implantation sites Mouse CD-1 3 times a day feeding PND 22–GD 9 60, 600 mg/kg/d Through GD 9 Impaired implantation and (59) impaired PGR-HAND2 pathway Mouse Not indicated Oral gavage GD 0.5–3.5 200, 400, 600, 800 GD 4.5 BPA 400–800 delayed (52) mg/kg/d transfer of embryos from fallopian tubes to uterus and decreased number of implantation sites Rat Wistar Subcutaneous injection PND 1, 3, 5, 7 0.05, 20 mg/kg/d GD 5, GD 18 Reduced implantation sites (60) Mouse C57BL6 Subcutaneous injection GD 0.5–3.5 0.025, 0.5, 10, 40, 100 GD 4.5 No implantation sites (53) mg/kg/d (BPA100); delayed

O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. implantation (BPA40) Mouse C57BL6 Subcutaneous injection GD 0.5–3.5 100 mg/kg/d GD 4.5 No implantation sites when (53) untreated embryos transferred to pseudopregnant females pretreated with BPA100 Note: BPA [ bisphenol A. Ziv-Gal. BPA and female infertility. Fertil Steril 2016. O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. TABLE 6

BPA and uterus (morphology and function) experimental studies (in vivo). Time of Reference Source Strain Exposure route exposure Doses Time of observation Outcome no. Non-human primate African green Alzet minipump Adult 50 mg/kg/d End of 28 d of BPA increased levels of (61) monkey treatment glandular and stromal PR; BPA þ E2 benzoate decreased PR expression; BPA may antagonize estradiol effects on PR expression Rat Sprague-Dawley Subcutaneous PND 17–19 10, 100, 500 mg/kg/d PND20 BPA500 changed levels (62) injection of contraction- associated proteins and decreased uterine contractility Rat Sprague-Dawley Cultured uterine tissue PND 20 10 5 M 24, 48 h Decreased uterine (62) contractility Mouse CF-1 Subcutaneous GD 1–4 3.375, 6.75, GD6 BPA6.75 and 10.125 (56) injection 10.125 mg/d increased uterine luminal area and cell height Rat Wistar Subcutaneous PND 1, 3, 5, 7 0.05, 20 mg/kg/d (Ovx PND80) PND94 BPA0.05 decreased (63) injection endometrial proliferation, decreased levels of Vegf and Esr1 in subepithelial cells, and Esr1 in endothelial cells; BPA0.05, 20 increased expression of Ncor1 in subepithelial cells Mouse CD-1 Intraperitoneal GD 9–16 5 mg/kg 2, 6 wk old Increased Hoxa10 (64) injection mRNA and HOXA10 protein levels;

hypomethylation Sterility® and Fertility of promoter and intron of Hoxa10 Non-human primates Macaca mulatta Oral GD 50–100 or GD 400 mg/kg/d GD100 or GD 165 Differences in levels of (65) 100–165 (fetuses) HOX and Wnt/Fzd family genes on GD 165 Ziv-Gal. BPA and female infertility. Fertil Steril 2016. 835 NORN IRPIGCEIAS EAEADML REPRODUCTION MALE AND FEMALE CHEMICALS: DISRUPTING ENDOCRINE 836 TABLE 6

Continued. Time of Reference Source Strain Exposure route exposure Doses Time of observation Outcome no. Rat Sprague-Dawley Oral gavage GD 6–birth 2.5, 8, 25, 80, 260, F1 PND90 No effect on Vegfa, (66) 840, 2,700, Pgr, S100g,orC3 100,000, expression 300,000 mg/kg/d Mouse ICR Subcutaneous GD 12–16 100, 200, 500, Not directly exposed F2 BPA100 decreased (67) injection 1,000 mg/kg/d (8 wk) uterine weight; possible effects on Hoxa10 methylation Chicken White Leghorn In ovo injection Incubation d 4 67, 134 mg/g egg 21 wk old Decreased uterine (47) tubular glandular density and tunica mucosa thickness (BPA134) Mouse CD-1 Swiss Extruded pellet diets 0.03, 0.3, 30 ppm BPA0.3 increased (36) sensitivity to develop pyometra (C57BL6) Mouse ICR Injection Adult (OVX) 10–500 mg/kg 2 h after injection BPA regulated Egr1 (68) expression through ER-ERK1/2 pathways; BPA induced phosphorylation of AKT and ERK1/2 via non-genomic actions of ERs Rat Wistar Drinking water and GD 6–PND 21 10 mg/L (1.2 mg/kg/d) F1 4 mo Increased thickness of (69) lactation uterine epithelia and stroma Mouse ICR Subcutaneous PND 8 0.1, 1, 10, 100 mg/kg PND 25, 30, 70 Reduced uterine (70) injection weight (BPA100) Mouse CD-1 Subcutaneous GD 1–5 10, 100, 1000 mg/kg/d 18 mo Cystic endometrial (50) injection hyperplasia

O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. (BPA100) Mouse CD-1 Subcutaneous GD 9–16 0.1, 1, 10, 100, 18 mo Adenomatous (51) injection 1,000 mg/kg/d 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) Ziv-Gal. BPA and female infertility. Fertil Steril 2016. O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. TABLE 6

Continued. Time of Reference Source Strain Exposure route exposure Doses Time of observation Outcome no. Mouse CD-1 Intraperitoneal GD 9–16 0.5, 1.0, 5.0, F1 2–6 wk after birth Increased HOXA10 in (71) injection 50, 200 mg/kg dose response fashion (BPA0.5-5) Rat Wistar Subcutaneous Neonatal PND 1, 3, 5, 0.05, 20 mg/kg/d PND 8, (OVX at PND PND8: downregulation (72) injection and 7 80) PND 94 of Hoxa10, Hoxa11; PND94: downregulation of Esr1, Hoxa11 (BPA0.05), and Hoxa10 (BPA 0.05, 20); impaired proliferative response to PþE (BPA0.05 and 20) Rat Wistar Subcutaneous Neonatal PND 1, 3, 5, 0.05, 20 mg/kg/d F1 GD 5 or GD 18 Reduced implantation (60) injection and 7 sites, decreased Hoxa10, ITGB3, Pr and Esr1, increased Emx-2 (BPA20); Decreased Esr1 (BPA0.05 and 20) and PRa and PRb (BPA20) Rat Wistar Drinking water þ GD 9–PND 21 0.5, 50 mg/kg/d F1 PND 90, PND 360 PND 90: decreased (44) lactation glandular epithelium proliferation (BPA0.5 and 50) and decreased percentage of a-SMA-positive stromal cells (BPA50); PND 360: increased anomalies in uterine luminal epithelium (BPA0.5, 50) and Sterility® and Fertility glands (BPA50) Note: BPA [ bisphenol A. Ziv-Gal. BPA and female infertility. Fertil Steril 2016. 837 ENDOCRINE DISRUPTING CHEMICALS: FEMALE AND MALE REPRODUCTION

TABLE 7

BPA and uterus (morphology and function) experimental studies (in vitro). Exposure Time of Reference Source Strain route Doses observation Outcome no. Human Endometrial stromal Culture 5–100 mmol/L 48 h Decreased proliferation, (9) ﬁbroblasts induced IGFB1 (BPA50), decreased CYP11A, HSD17B1, HSD17B2; no effect on PRL levels Human Ishikawa cells Culture 1 mM 24 h BPA or BPA þ estradiol (61) increased levels of PR; BPA þ estradiol decreased PR expression compared to estradiol only; BPA may antagonize estradiol effects on PR expression Human Endometrial endothelial Culture 0.1, 50, 100 nM 24 h BPA0.1-100 decreased (73) cells proliferation and viability; BPA100 increased necrosis Human Endometrial endothelial Culture 50 mM 24 h Decreased cell proliferation (74) cells Human Primary stromal Culture 0.01 mM, 0.01 mM, 24, 48, 72 h No effect on proliferation; (75) endometrial cells 0.01 nM; decidualization BPA (0.01 mM and (proliferative phase) induced by P4 0.01 nM) increased G2/M and decreased G0/G1 fractions; BPA increased PRL (0.01 mM and 0.01 nM), LEFTY (0.01 mM), and IGFBP1 (0.01 mM, 0.01 mM and 0.01 nM) Human 5 cycling women Culture 10 mM, 0.1 mM, 1 nM, 24 h No effect on cell viability or (76) undergoing 0.01 nM proliferation; Increased hysterectomy angiogenic activity (10 mM); no difference in VEGF, ESR2, and GPR30 levels Human Decidualized stromal Culture 1–100 pM, 1–100 nM, 24, 48 h No effect on cell viability; (77) cells 1–100 mM Increased proliferation (48 h; 100 nM) Human Decidualized stromal Culture 1 pM, 1 nM, 1 mM 24, 48 h BPA1 mM: decreased PRL; no (77) cells effect on IGFBP1, increased ESR1 and ESR2 (1nM) BPA1 mM increased PR protein and PRA PRB mRNA; BPA1pM decreased hCG/LH-R protein and increased MIF protein secretion Human Ishikawa cells Culture 1 nM, 100 nM, 10 mM, 8, 24, 48 h No effect on cell viability; (78) 100 mM Affected multiple molecular pathways associated with cell organization and biogenesis, translation, proliferation, and intracellular transport Human Ishikawa cells Culture 0.1 nM–25 mM, 1 mM 24 h Increase in HOXA10 in a (71) dose response manner Note: BPA [ bisphenol A. Ziv-Gal. BPA and female infertility. Fertil Steril 2016. endometrial proliferation and receptivity. In contrast, in utero it is unclear whether they are primary targets for BPA- high dose BPA exposure did not affect expression levels of induced uterine toxicity. coding complement component 3 (C3), Pgr, calbindin D9K Findings from in vitro studies using various human cell (S100g), and Vegfa in the adult offspring of rats (66); hence, lines indicate that low dose BPA exposure decreased

838 VOL. 106 NO. 4 / SEPTEMBER 15, 2016 Fertility and Sterility® endothelial cell proliferation (9, 74) and increased decidualized published by Wang et al. (45) and Ziv-Gal et al. (49) examined stromal cell proliferation (77) compared with controls. the effects of in utero exposure of low dose BPA on estrous Similarly, high dose BPA exposure decreased endothelial cell cyclicity in subsequent generations of mice. Interestingly, proliferation compared with controls (73). Some of the BPA-induced altered cyclicity was observed in both the F1 potential mechanisms through which BPA may affect cell and F3 generations, but not in the F2 generation compared proliferation in the uterus include alterations in insulin-like with controls. In contrast, other studies reported no effect of growth factor binding protein 1 (IGFBP1), macrophage migra- in utero low dose BPA exposure or 50 mg/kg/d (i.e., lowest tion inhibitory factor (MIF), HOXA10, and left right determi- observable adverse effect level) on estrous cyclicity of rats nation factor 1 (LEFTY), steroidogenic receptors (e.g., ESR1, and mice offspring (43, 44, 46). Differences in study design ESR2, PGR), and enzymes (e.g., cytochrome P450, family 11, and timing of evaluation of estrous cyclicity may explain subfamily a, polypeptide 1; CYP11A1, hydroxysteroid (17- the disagreement between the results. Overall, neonatal BPA beta) dehydrogenase 1; HSD17B1, hydroxysteroid (17-beta) exposure may affect estrous cyclicity in older animals. dehydrogenase 2; HSD17B2), or other hormones (e.g., PRL, However, the evidence regarding the effects of in utero BPA LH) (9, 61, 62, 67, 69–71, 75, 77, 78). Furthermore, Naciff exposure on estrous cyclicity is inconclusive. Further studies et al. (78) performed a microarray on Ishikawa cells that that examine the effects of in utero BPA exposure on estrous were cultured with a range of low and high BPA doses (1 cyclicity and that encompass multiple generations are nM–100 mM) and found that multiple molecular pathways needed to fully understand the effects of BPA on estrous (e.g., cell organization and biogenesis, proliferation, and cyclicity. intracellular transport) were altered in response to BPA compared with controls. Ovary In contrast, two studies (75, 76) on human cell lines cultured with low and high BPA doses found no effect on The ovary is required for normal production of ova for fertil- proliferation of primary stromal endometrial cells. ization and for production of sex steroid hormones that regu- Differences in cell viability or proliferation may be due to late estrous cyclicity and fertility. Thus, BPA exposures that the study design and experimental model. For example, target the ovary can interfere with fertility. One epidemiolog- differences in the source of the cells (carcinogenic tissue vs. ical study (85) examined the associations between BPA levels normal) and differences in the experimental cell lines and ovarian volume and mature follicle counts (Table 9). Re- (primary vs. established/immortal cell line lines such as sults from this study indicate that urinary BPA exposure was Ishikawa). Nevertheless, most in vitro studies support the negatively correlated with antral follicle counts in women un- observations reported in these in vivo studies. dergoing IVF treatments. Last, at the end of pregnancy, the uterus needs to contract Findings from experimental studies indicate that in utero or to induce labor. Uterine contractions are under the control of neonatal low and high dose BPA exposures resulted in endogenous hormones such as E2, P, oxytocin, and prosta- abnormal ovarian morphology and histology compared with glandins (PG) (79). The effects of BPA on uterine contractility controls (Table 10). Specifically, BPA exposure increased the were investigated in one study (62) in rats. Findings from this number of multi-oocyte follicles (91), inhibited germ cell nest study suggest that BPA exposure decreased uterine contrac- breakdown (45, 48, 106), decreased the number of primordial tility and altered transcript and protein levels of follicles (48, 106), increased apoptotic oocytes (106),and contraction-associated factors. Specifically, high dose BPA increased primordial follicular recruitment (45, 101).Italso exposure increased oxytocin and oxytocin receptor, and affected follicle type distribution by reducing the number of decreased PG F2a receptor compared with controls (62). Over- antral follicles and increasing the numbers of primary and all, the current literature suggests that BPA exposure selec- secondary follicles (89). tively affects uterine cell proliferation and function, Molecular analysis revealed that low dose BPA exposure depending on the study model. These effects of BPA on uter- affected levels of genes related to apoptosis. Specifically, BPA ine function could lead to adverse effects on fertility. increased levels of B-cell leukemia/lymphoma 2 (Bcl2), BCL2- like 1 (Bcl2l1) (45, 106). BPA exposure also decreased levels of BCL2-antagonist/killer 1 (Bak1), tumor necrosis factor (TNF) Estrous Cyclicity receptor superfamily, member 11b (Tnfrsf11b), TNF receptor Estrous cyclicity is crucial for ovulation and the preparation of superfamily, member 1a (Tnfrsf1a), TNF (ligand) superfamily, the uterus for potential implantation. Hence, chemical expo- member 12 (Tnfsf12), and lymphotoxin B receptor (Ltbr) (45, sures that disrupt estrous cyclicity can impair fertility. Multiple 106). In addition, BPA exposure altered BCL2-associated X experimental studies examined the effects of BPA exposure on protein (Bax) levels by either increasing (106) or decreasing estrous cyclicity (Table 8). Early neonatal low (82–84) and high (45) its levels. Differences in the effects of BPA on Bax can (70) dose BPA exposure increased or decreased days in estrus result from differences in study designs (e.g., ovarian trans- and altered cycles in adult mice or rats (70–90 days) compared plant vs. excised neonatal ovaries). with controls. In contrast, low dose BPA exposure at earlier Furthermore, BPA exposure decreased expression of fac- time points (51–54 days old) had no effect on estrous tors that control folliculogenesis, such as NOBOX oogenesis cyclicity (40). In rats, in utero low dose (69) and high dose homeobox (Nobox) (messenger RNA [mRNA] and protein), (80, 81) BPA exposure resulted in irregular estrous cycles in LIM homeobox protein 8 (Lhx8) (mRNA and protein), sper- the offspring compared with controls. In addition, studies matogenesis and oogenesis-specific basic helix-loop-helix 2

VOL. 106 NO. 4 / SEPTEMBER 15, 2016 839 NORN IRPIGCEIAS EAEADML REPRODUCTION MALE AND FEMALE CHEMICALS: DISRUPTING ENDOCRINE 840

TABLE 8

BPA and estrous cyclicity. Exposure Time of Reference Source Strain route exposure Doses Time of observation Outcome no. Rat Long Evans Injection PND 0–350mg/kg/d, 50 mg/kg/d Post weaning and vaginal By wk, 15 only 33% of (80) opening females (BPA50 mg/kg) cycled regularly Rat Sprague Dawley Oral gavage GD 6–birthþ 2.5, 8, 25, 80, 260, F1 PND 15, 21, 90, PND 69– BPA300: abnormal cyclicity (81) PND 1–15 or 21 840 mg/kg/d, 2.7, 90, 150–170 (estrous) (PND90 and 150) 100, 300 mg/kg/d Rat Sprague Dawley Subcutaneous PND 1–10 50 mg/50 ml, 500 PND 21 BPA500 caused irregular (82) injection mg/50 ml PND 60–120 cycles with more days in estrus, after PND90 Rat Sprague Dawley Oral gavage 90 d 0.001, 0.1 mg/kg/d 30 d after the age of 21 wk Extended estrous phase (83) (2–7d) Rat Wistar Drinking water GD 6–PND 21 10 mg/l (1.2 mg/kg/d) F1 3 mo of age for 4 Irregular estrous cycles (69) and lactation consecutive weeks Rat Wistar Subcutaneous PND 1, 3, 5, and 7 0.05, 20 mg/kg/d PND 85–100 BPA0.05: more time in (84) injection proestrus-estrus Mouse C57BL6J Oral 12–15 d (ﬁrst 3 50 mg/kg/d 51–54 d No effect on estrous cyclicity (40) reproductive cycles) Mouse ICR Subcutaneous PND 8 0.1, 1, 10, 100 mg/kg Observed: PND 20–29; BPA100: decreased number (70) injection euthanized: PND 25, 30, of days in estrus 70 Rat Wistar Oral, drinking GD 9–birth 0.5, 50 mg/kg/d F1: PND 45, 90 No effect on estrous cyclicity (43) water Rat Wistar Drinking water þ GD 9–PND 21 0.5 or 50 mg/kg/d F1: PND 90, PND 360 No effect on estrous cyclicity (44) lactation Mouse FVB Oral GD 11–birth 0.5, 20, 50 mg/kg/d F1 on PND 21 PND21: shorter time span (45) between vaginal opening and ﬁrst estrus (BPA50); BPA0.5 less time in proestrus and estrus and

O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. more in diestrus and metestrus; BPA20 shortened estrus Mouse CD-1 Oral gavage GD 1–PND 20 12, 25, 50 mg/kg/d F1 on PND 50 No effect on estrous cyclicity (46) Mouse CD-1 Oral gavage PND 21–49 25, 50 mg/kg/d PND 50 No effect on estrous cyclicity (46) Mouse FVB Oral of pregnant GD 11–birth 0.5, 20, 50 mg/kg/d F1, F2, F3: 3, 6, 9 mo F3: delayed age at ﬁrst estrus (49) dams (F0) (BPA50) Note: BPA [ bisphenol A. Ziv-Gal. BPA and female infertility. Fertil Steril 2016. Fertility and Sterility®

TABLE 9

BPA and ovary (epidemiological study). Study Time of BPA Reference Study design population Sample size measurement BPA concentration Outcome no. Prospective Women Overall 209; antral Urine: upon entry Urinary geometric BPA not associated (85) undergoing follicle count¼154; into the study mean (geometric with d 3-FSH or IVF FSH¼120; ovarian and at subsequent SD). Antral follicle ovarian volume; volume¼ 114) treatment cycle count ¼ 1.6 (2.0); higher urinary BPA visits; Ultrasound: FSH ¼ 1.7 (2.1); concentrations 3rd d of an ovarian volume ¼ associated with unstimulated 1.5 (1.8) lower antral menstrual cycle follicle counts Ziv-Gal. BPA and female infertility. Fertil Steril 2016.

(Sohlh2), stimulated by retinoic acid gene 8 (Stra8), DNA specific differences in gene expression, but not all were meiotic recombinase 1 (Dmc1), REC8 meiotic recombination transgenerational (i.e., genes related to oxidative stress, auto- protein (Rec8), synaptonemal complex protein 3 (Scp3), and phagy, and apoptosis) (86). Interestingly, BPA-induced folliculogenesis-specific basic helix-loop-helix (Figla) (48, changes in steroidogenic genes and Esr1, androgen receptor 106). In addition, BPA exposure prevented DNA methylation (Ar), and insulin-like growth factor (Igf) family genes were in CpG sites of Lhx8, indicating that BPA may impair normal suggested to be carried transgenerationally (86). It is possible processes of folliculogenesis (106) and ovarian dynamics. that the changes in the genes related to oxidative stress and Similar effects of exposure to both low and high doses of apoptosis are activated in an acute manner and thus, effects BPA during neonatal life on the ovary were observed at older on these factors were not carried into the subsequent genera- ages/after weaning. Specifically, researchers reported find- tions. In contrast, steroidogenic factors are crucial to the ings such as BPA-induced multinucleated and hemorrhagic function of the ovary (as an endocrine organ) and thus tissue (80), multi-oocyte follicles (99), altered follicle-type some of BPA effects on these factors were carried into the sub- distribution or numbers (43, 81, 87, 89, 93, 94, 99, 100, sequent generations. Overall, these experiments provide 104), reduced ovarian weight (43, 70), and ovarian cysts strong evidence that BPA acts through mechanisms related (51) compared with controls. Molecular analysis revealed to apoptosis, folliculogenesis, and oxidative status. that high dose BPA exposure decreased the expression of In mice, in vitro studies (109, 110) of isolated neonatal Figla and oocyte-specific histone H1 variant (H1f00), and ovaries indicate that high dose BPA exposure inhibited increased the levels of antimullerian€ hormone (Amh) genes germ cell nest breakdown and accelerated primordial follicle (93). In addition, Lee et al. (83) reported that low dose BPA recruitment compared with controls, similar to some of the increased apoptosis in ovarian follicles that was coupled observations in in vivo studies. Specifically, BPA exposure with increased levels of the apoptotic protein caspase-3. decreased levels antigen KI-67 (Ki67), TNF receptor super- Hence, in rodents, BPA affects ovarian development and dy- family member 6 (Fas), and caspases (Casp3 and 8). Further- namics by molecular pathways that involve apoptosis, folli- more, BPA increased levels of Bcl2 and factors related to the culogenesis, and oocyte-specific factors. phosphatidylinositol 3-kinase/thymoma viral proto- Similarly, in fish, low dose BPA exposure increased oncogene (PI3K/Akt) signaling pathway (109, 110). In sheep ovarian weight, increased levels of hydrogen peroxide, and fetal ovaries, low dose BPA exposure resulted in an age- decreased glutathione levels compared with controls, indi- dependent increase in expression of steroidogenic genes, cating that BPA may alter the oxidative stress mechanism in mammalian target of rapamycin (mTor), peroxisome the ovary (108).Anotherstudyinfish found that low dose proliferator-activated receptor (Ppara), and Igf1r compared BPA exposure increased ovarian weight, atretic follicles, peri- with controls (103). The overall findings suggest that BPA nuclear oocytes, and expression of factors involved in follicu- may act through mechanisms that are related to follicle dy- logenesis (Gdf9 and Bmp15) compared with controls (107). namics and apoptosis. Furthermore, studies suggest that Other studies in mice found no effect on follicle type dis- BPA exposure may act through mechanisms involving altered tribution in the adult ovary after low dose BPA exposure (40, microRNA levels (103). BPA down-regulated miR-137 may 46). However, it is possible that BPA did not affect ovarian decrease sex steroid hormone synthesis (112) and miR-765 follicle distribution because of the timing of BPA exposure may be associated with premature ovarian failure (113, and differences in study design. Furthermore, some of the 114). Additional findings included variable levels of effects of BPA exposure on the ovary do not persist in microRNAs related to insulin signaling, without changing subsequent generations. Wang et al. (45) reported that in levels of microRNA processing enzymes (103). utero low dose BPA exposure inhibited germ cell nest In vitro studies of isolated mouse ovarian follicles indicate breakdown in the F1 generation of mice compared with that high dose BPA exposure selectively inhibited antral follic- controls. However, these changes were not observed in the ular growth (95–97,111), but increased preantral follicle growth subsequent generations examined by Berger et al. (86). (102) compared with controls. Molecular analysis revealed that Furthermore, BPA exposure caused several generation- BPA exposure affected the expression of genes related to the cell

VOL. 106 NO. 4 / SEPTEMBER 15, 2016 841 NORN IRPIGCEIAS EAEADML REPRODUCTION MALE AND FEMALE CHEMICALS: DISRUPTING ENDOCRINE 842 TABLE 10

BPA and ovary. Reference Source Strain Exposure route Time of exposure Doses Time of observation Outcome no. Rat Long Evans Subcutaneous PND 0–350mg/kg/d, After weaning and Abnormal ovarian (80) injection 50 mg/kg/d vaginal opening morphology: multinucleated and hemorrhagic tissue (BPA50 mg/kg) Mouse FVB Oral GD 11–birth 0.5, 20, 50 mg/kg/d F1, F2, F3: PND 4, 21 PND4: no effect on (86) 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 Mouse CD-1 Subcutaneous PND 7–14 20, 40 mg/kg/d PND 15 Accelerated primordial (87) injection to primary follicle transition Mouse CD-1 Subcutaneous PND 5–20 (every 5 d) 20, 40 mg/kg/d PND 21 Accelerated primordial (87) injection to primary follicle transition Rat Sprague Dawley Oral gavage GD 6–birthþ 2.5, 8, 25, 80, 260, PND 15, 21, 90, BPA300: small ovaries (81) PND 1–15 or 21 840 mg/kg/d, PND 69–90, with depletion of 2.7, 100, PND 150–170 corpora lutea and 300 mg/kg/d antral follicles

O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. Human In vitro fertilization Granulosa-lutein cell 72 h 1–10,000 ng/mL End of culture BPA 1,000 and 10,000: (88) patients culture decreased cell viability; BPA 100 and 1,000: increased MMP-9; BPA 10,000: decreased MMP-9 Ziv-Gal. BPA and female infertility. Fertil Steril 2016. O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. TABLE 10

Continued. Reference Source Strain Exposure route Time of exposure Doses Time of observation Outcome no. Rat Wistar Drinking water þ GD 0–PND 21 3 mg/kg/d PND 30 No difference in (89) lactation ovarian weight, higher total follicle number, higher primary, secondary, and lower antral follicle numbers; Increased atresia Swine Granulosa cells culture 48 h 0.1, 1, 10 mM End of culture No effect on cell (90) proliferation; VEGF secretion stimulated (BPA1, 10); no effect on oxidative stress Non-human primate Rhesus macaque Silastic pump GD 100–term 2.2–3.3 ng/mL serum PND 0 Increased number of (91) levels multi-oocyte follicles, impaired oocyte development (unenclosed oocytes) Human IVF patients Granulosa-lutein cell 48 h 40, 60, 80, 100 mM End of culture BPA 40–100: inhibited (92) culture proliferation, decreased FSH induced genes (IGF-1, aromatase) and altered aromatase regulators (GATA4, SF-1, PPARg) Rat Sprague Dawley Oral gavage 90 d 0.001, 0.1 mg/kg/d 21 wk old Increased apoptosis (83) and CASP3, decreased aromatase expression (granulosa cells) Rat Wistar Intraperitoneal PND 28–35 10, 40, 160 mg/kg PND 35 Decreased follicle (93) injection numbers, increased Sterility® and Fertility atretic follicles; decreased H1FOO and FIGLA(BPA160), increased AMH Ziv-Gal. BPA and female infertility. Fertil Steril 2016. 843 NORN IRPIGCEIAS EAEADML REPRODUCTION MALE AND FEMALE CHEMICALS: DISRUPTING ENDOCRINE 844 TABLE 10

Continued. Reference Source Strain Exposure route Time of exposure Doses Time of observation Outcome no. Zebrafish Danio rerio Aquarium water 14 d 1, 10, 100, 1,000 mg/L End of exposure Abnormal ovarian (94) follicles; dose dependent increased atresia and decreased primordial follicles Mouse C57BL6J Oral 12–15 d (during first 3 50 mg/kg/d 51–54 d No differences in (40) cycles) follicle type distribution Mouse ICR Subcutaneous PND 8 0.1, 1, 10, 100 mg/kg Observed: PND 20–29; All BPA groups: (70) injection scarified: PND 25, reduced ovarian 30, 70 weight (PND 25, 30) Mouse CD-1 Subcutaneous GD 9–16 0.1, 1, 10, 100, 18 mo Ovarian cysts (BPA 1) (51) injection 1,000 mg/kg/d Mouse FVB Cultured antral follicles 24–120 h 4.4–440 mM End of culture BPA 440: inhibited (95) (1–100 mg/mL) follicle growth Mouse FVB Cultured antral follicles 24–96 h 4.4–440 mM(1–100 End of culture BPA 440: inhibited (96) mg/mL) follicle growth, increased atresia rating, Bcl2, Bax, Cdk4, Ccne1, and Trp53, and decreased Ccnd2 Mouse C57BL/6, FVB, CD-1 Cultured antral follicles 24–120 h 4.4–440 mM(1–100 End of culture All strains: BPA 440 (97) mg/mL) inhibited follicle growth, increased expression of Cdk4, Ccne1, Trp53, Bax, and Bcl2 Human HOSEpiC Culture 3, 24, 48 h 0.1, 1, 40 nM End of culture Increased expression of (98) VEGF-R2 Lamb Hampshire Down Subcutaneous PND 1–14 50 mg/kg/d PND 30 PND30: increased (99) injection primordial-to- O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. 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 Ziv-Gal. BPA and female infertility. Fertil Steril 2016. O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. TABLE 10

Continued. Reference Source Strain Exposure route Time of exposure Doses Time of observation Outcome no. Lamb Corriedale X Subcutaneous PND 1–14 0.5, 50 mg/kg/d PND 30 or PND34 after Decreased number of (100) Hampshire injection FSH stimulation follicles >2 mm, impaired response to FSH as evident by decreased percent of atretic follicles Rat Wistar Subcutaneous PND 1–7 0.05, 20 mg/kg PND 8 BPA20: Increased (101) injection primordial follicular recruitment, increased granulosa cell proliferation Rat Wistar Oral, drinking water GD 9–birth 0.5, 50 mg/kg/d PND 21, 45, or 90 Lower ovarian weight; (43) 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 Mouse C57/Bl6J x CBA/Ca Cultured pre-antral 12 d 3, 300 nM End of culture BPA 3: accelerated (102) follicles follicle growth Sheep Suffolk Subcutaneous GD 30–90 0.5 mg/kg/d Fetal ovaries on GD 65, Age-dependent (103) injection 90 increase in mRNA of 3b1hsd, 3b2hsd, AR, Esr1, Gdf9, IR, mTOR, Ppara, and Igf1r; altered miRNA Sheep Suffolk Subcutaneous GD 30-90 0.05, 0.5, 5 mg/kg/d 19 mo Similar number or size (104) injection of corpora lutea; differences in

follicular count Sterility® and Fertility trajectories Mouse FVB Oral GD 11–birth 0.5, 20, 50 mg/kg/d PND 4, 21 PND4: inhibited germ (45) cell nest breakdown, decreased primordial follicles (BPA0.5 and 50); altered levels of 845 apoptotic genes Ziv-Gal. BPA and female infertility. Fertil Steril 2016. NORN IRPIGCEIAS EAEADML REPRODUCTION MALE AND FEMALE CHEMICALS: DISRUPTING ENDOCRINE 846 TABLE 10

Continued. Reference Source Strain Exposure route Time of exposure Doses Time of observation Outcome no. Mouse CD-1 Oral gavage GD 1–PND 20 12, 25, 50 mg/kg/d PND 50 Similar number of (46) growing follicles Mouse CD-1 Oral gavage PND 21–49 25, 50 mg/kg/d PND 50 Similar number of (46) growing follicles Chinese hamster V79 Cell culture 12 or 24 h 40, 80, 100, 120 mM End of culture Increased cell viability (105) (BPA40), cytotoxicity (BPA 80, 100, 120). induced DNA damage; micronucleus (BPA100, 120) Mouse CD-1 Oral GD 12.5–PND 18.5 0.2, 0.04, 0.08 mg/kg GD 15.5, 17.5, 19.5 BPA0.08: higher (48) PND 3, 5, 7 percentage of oocytes in cysts, higher oocyte number, and fewer primordial follicle numbers on PND3; decreased Stra8 associated with altered DNA methylation Mouse CD-1 Cultured neonatal Not speciﬁed 10, 100 mM 3 d of culture Inhibition of germ cell (106) ovaries nest breakdown and reduced primordial follicles (BPA10, 100); increased apoptotic oocytes and increased Bax (BPA100); reduced Nobox (BPA100), Nobox, Lhx8 and O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. protein, Sohlh2, Figla (BPA10, 100); increased Lhx8 methylation Fish Gobiocypris rarus Aquarium water 8 mo old 15, 50 mg/L After 14 or 35 d Increased ovarian (107) weight (BPA15); no histological effects at 14 d; increased atretic follicles and perinuclear oocytes (BPA50); increased Gdf9, Bmp15 (BPA15) Ziv-Gal. BPA and female infertility. Fertil Steril 2016. O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. TABLE 10

Continued. Reference Source Strain Exposure route Time of exposure Doses Time of observation Outcome no. Fish Gobiocypris rarus Aquarium water 6 mo old 1, 15, 225 mg/L After 7 d Increased ovarian (108) weight (BPA15). increased H2O2 ovarian levels (BPA1 and 225): decreased glutathione Mouse C57BL/6 Cultured neonatal PND 4–14 0.1, 1, 10 mM End of culture <5 d culture: increased (109) ovaries primary follicle number and decreased primordial follicle number (BPA10); D 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 Mouse CD-1 Cultured neonatal PND 0–8 0.1, 1, 5, 10 mg/mL 2, 4, or 8 d of culture PND 4: inhibited germ (110) ovaries cell nest breakdown, decreased primordial follicles; random ﬂuctuations in levels of anti- oxidant genes (Gpx, Cat, Gsr); limited effects on apoptotic related genes. PND8: Sterility® and Fertility increased ROS production (BPA5) yet post germ cell nest breakdown Ziv-Gal. BPA and female infertility. Fertil Steril 2016. 847 ENDOCRINE DISRUPTING CHEMICALS: FEMALE AND MALE REPRODUCTION

cycle, apoptosis, and steroidogenesis (95–97,111).Speciﬁcally, BPA exposure increased Bcl2, cyclin-dependent kinase 4 no. (87) (87) (111) (111) (Cdk4), cyclin E1 (Ccne1), transformation-related protein 53

Reference (Trp53), Bax, and down-regulated 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

Bcl2 appearance of micronuclei (105). In short-term cultures (3– 48 hours) of human ovarian cells, low dose BPA increased expression of VEGF-R2 (98). In short-term cultures (48 hours) inhibited follicular growth increased to primary follicle transition inhibited follicular growth to primary follicle transition of human granulosa (GC) cells and lutein cells, high dose BPA

BPA 43.8-110: inhibited cell proliferation and decreased levels of IGF-1, aromatase, GATA-binding protein 4 (GATA4), steroidogenic factor-1 (SF-1), and PPARg (92). In contrast, in short-term porcine GC cultures (48 hours), high dose BPA did not affect cell proliferation or expression of oxidative stress genes (90). Similar concentrations of BPA during longer culture times (72 hours) decreased viability and disrupted matrix metallopep- tidase 9 (MMP-9) secretion in human GCs (88). 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 M End of culture BPA 110-438: M End of culture BPA 219-438: well as in early neonatal life through multiple pathways m m g/kg/d PND 15 Accelerated primordial g/kg/d PND 21that include Accelerated primordial cell cycle, apoptosis, oxidative stress, and prolif- m m 438 438 – – eration. More epidemiological studies are warranted to better understand the specific associations of BPA exposure and ovarian outcomes in women. Furthermore, additional 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. 14 20, 40 20 (every 5 d) 20, 40 Hypothalamic-pituitary-ovarian Axis – – Overall, reproductive function is dependent on the PND 7 PND 5 hypothalamic-pituitary-ovarian axis. After 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 (E2 and P) activate the kisspeptin neurons that in turn relay the secretion of GnRH. The GnRH stimulates the anterior pituitary injection injection to secrete gonadotrophic hormones (FSH and LH). The FSH and

Cultured antral follicles 96 h 0.004 LH act on the ovary to support folliculogenesis. Increased levels of ovarian sex steroid hormones feedback to the hypothalamic kisspeptin neurons to induce the LH surge, which 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 following sections describe the current data on the effects of BPA on the hypothal-

tm1Bra amus (Table 11), pituitary (Table 11), and gonadotrophic hor- C57BL/6 background Ahr mones (Tables 12 and 13). 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 (41, bisphenol A. 46) or decreased levels of Kiss1 (117, 122, 123) in ﬁsh, mice, and [ rats. Differences in species and age of the animals in which BPA observations were made can partially explain these opposite Mouse Mouse C57BL/6 Cultured antral follicles 96 h 0.004 Ziv-Gal. BPA and female infertility. Fertil Steril 2016. Continued. Source Strain ExposureNote: route Time of exposure Doses Time of observation Outcome Mouse CD-1 Subcutaneous Mouse CD-1 Subcutaneous TABLE 10 results rather than the doses that were used (i.e., low or high).

848 VOL. 106 NO. 4 / SEPTEMBER 15, 2016 O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. TABLE 11

BPA and hypothalamic-pituitary-ovarian axis. Time of Reference Source Strain Exposure route exposure Doses Time of observation Outcome no. Sheep Suffolk Subcutaneous injection GD 30–90 5 mg/kg/d F1: OVX at 21 mo, No effect on steroid (115) testing at 23–26 mo feedback and/or increased pituitary responsiveness to GnRH Mouse Mixed FVB X C57BL/6 Oral GD 10.5–18.5 0.5, 50 mg/kg/d Birth Increased number of (116) pituitary mKi67- immunoreactive cells, increased gonadotroph cell number (LHb, FSHb positive); increased Lhb and Fshb (BPA0.5); Decreased Lhb and Fshb, Nr5a1 (BPA50); decreased Gnrhr (BPA0.5, 50); no effect on hormone synthesis by pituitary cells Rat Long Evans Subcutaneous injection PND 0–250mg/kg/d, 50 mg/kg/d PND 4 or 10 PND4: increased Esr1,no (117) effect on Esr2, diminished Kiss1; PND10: Esr1 decreased to male typical levels, decreased/eliminated Esr2, diminished Kiss1 Rat Long Evans Subcutaneous injection PND 0–250mg/kg/d, 50 mg/kg/d PND 4 or 10 Altered Esr2 expression and (118) reversed sex differences in expression Rat Sprague-Dawley Oral gavage GD 6–PND 21 2.5, 25.0 mg/kg/d PND 21 No effect on density of (119) anteroventral periventricular nucleus tyrosine hydroxylase immunoreactivity Mouse BALB/c Oral GD 0–19 2, 20, 200 mg/kg/d PND 28 BPA20 altered methylation (120) patterns in the hypothalamus Sheep Suffolk Subcutaneous injection GD 30–90 5 mg/kg/d Adult prior to onset Decreased hypothalamic (121) of pre-ovulatory levels of GnRH; increased

LH surge ESR1; decreased ESR2 Sterility® and Fertility (medial preoptic area) Ziv-Gal. BPA and female infertility. Fertil Steril 2016. 849 NORN IRPIGCEIAS EAEADML REPRODUCTION MALE AND FEMALE CHEMICALS: DISRUPTING ENDOCRINE 850 TABLE 11

Continued. Time of Reference Source Strain Exposure route exposure Doses Time of observation Outcome no. Rat Wistar Subcutaneous injection PND 1, 3, 5, and 7 0.05, 20 mg/kg/d PND 100 Anteroventral periventricular (84) 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 Rat Wistar Subcutaneous injection PND 1–5 100, 500 mg/kg/d PND 30 Decreased hypothalamic (122) Kiss1 Rat Long Evans Subcutaneous injection PND 0–350mg/kg/d, 50 mg/kg/d After weaning and BPA 50 mg/kg reduced (123) vaginal opening density of hypothalamic kisspeptin immunoreactive ﬁbers; more profound in arcuate nucleus Fish Transgenic zebraﬁsh Aquarium 0.1, 1, 10, 100, 1,000 mg/L 25, 120 h after Selective increase of Kiss1, (41) fertilization Kiss1r, Gnrh3, Lhb, Fshb, synaptic vesicle protein-2 (Sv2) Mouse ICR Oral Proestrus of 20 mg/kg/d 6 h after Elevated plasma Gnrh; (124) 4th/5th estrous administration Increased Kiss1 in anteroventral periventricular nucleus Mouse ICR Injection into right lateral Proestrus of 0.02, 0.2, 2, 20, 200 6 h after Anteroventral periventricular (124) ventricle 4th/5th estrous nM/3 mL administration 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 O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. Mouse CD-1 Oral gavage GD 1–PND 20 12, 25, 50 mg/kg/d PND 50 Dose-dependent increase in (46) the expression levels of Kiss-1 and Gnrh in hypothalamus, no difference in Gpr54 Mouse CD-1 Oral gavage PND 21–49 25, 50 mg/kg/d PND 50 No effect on expression (46) levels of Kiss-1 and Gnrh in the hypothalamus Note: BPA [ bisphenol A. Ziv-Gal. BPA and female infertility. Fertil Steril 2016. Fertility and Sterility®

Neonatal BPA exposure also decreased levels of GnRH (Gnrh) (41, 124), Esr1,andEsr2 (84, 117, 118, 121) in sheep and rats no. (85)

(125) compared with controls. Interestingly, Wang et al. (124)

Reference 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 by the kisspeptin signaling pathway. Future studies that use 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 lowest observ- PRL and urine BPA levels (all women); negative association between FSH and BPA (unexposed group) or ovarian volume; higher urinary BPA concentrations associated with lower antral follicle counts able adverse effect level) that continued until the age Positive association between No association with d 3-FSH of weaning increased levels of Kiss1 and Gnrh on postnatal day 50 compared with 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

1.6 (2.0); on the outcome. Other researchers reported that in utero ¼

dence low dose exposure to BPA disrupted methylation patterns 1.5 (1.8) ﬁ

¼ in the hypothalamus that can affect the expression of Esr1 1.7 (2.1); ovarian g/g cr); m

¼ (120). In contrast, low dose BPA exposure did not affect the density of tyrosine hydroxylase cells, which are involved Unexposed: 0.9 (0.7, 1,1) (geometric SD); antral follicle count FSH volume interval) 39.8 (95% con in sexual dimorphism of the brain (119). Similarly, Abi Sal- Urinary geometric mean Exposed: 22.3 (12.4, Geometric (total BPA) mean loum et al. (115) 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. 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 spot urine

þ factors that may be mediating BPA effects are kisspeptin and GnRH. study and subsequent treatment visits; Ultrasound: 3rd d of an unstimulated menstrual cycle sample Pituitary. Limited data on the association between BPA expo-

Air sampling Urine: upon entry into the sure and pituitary outcomes are available from human studies (Table 12). Miao et al. (125) reported a positive association between creatinine-adjusted urine BPA levels and PRL, and

120; a negative association with FSH levels in women exposed to 114) ¼ BPA in their work place. In contrast, Souter et al. (85) found ¼ no association between speciﬁc gravity adjusted BPA levels and day 3 FSH levels in women undergoing IVF treatments. 154; FSH

¼ The differences between the two cohorts may explain the inconsistent results. unexposed count ovarian volume Few experimental studies have examined the effects of BPA

106 exposed, 250 Overall 209 (antral follicle exposure on the pituitary (Table 13). Most studies examined the effects of low dose BPA, whereas only two studies (46, 116) included high doses as well. In utero low dose BPA exposure increased proliferation of pituitary cells, and increased gonadotroph cell number (LHb,FSHb positive) compared with controls in the pituitaries of mice (116). In the same

Study study, both BPA doses (0.5 and 50 mg/kg/d) decreased GnRH population Sample size Time of BPA measurement BPA concentration Outcome to BPA in workplace IVF receptor (Gnrhr)levels, whereas 0.5 mg/kg/d of BPA increased the levels of Lhb and Fshb,and50mg/kg/d of BPA decreased the levels of Lhb, Fshb,andNr5a1 compared with controls (116). Furthermore, longer BPA exposure (25 and 50 mg/kg/d) bisphenol A. from gestation through weaning increased Fsh levels in mice [ compared with controls (46).Theseﬁndings indicate that BPA BPA exposure can affect the pituitary, but it is likely to be Retrospective Women exposed Prospective Women undergoing Ziv-Gal. BPA and female infertility. Fertil Steril 2016. BPA and gonadotrophic hormones (epidemiological studies). Study design Note: TABLE 12 dependent on the timing of exposure and dose.

VOL. 106 NO. 4 / SEPTEMBER 15, 2016 851 NORN IRPIGCEIAS EAEADML REPRODUCTION MALE AND FEMALE CHEMICALS: DISRUPTING ENDOCRINE 852 TABLE 13

BPA and gonadotrophic hormones (experimental studies). Time of Time of Reference Source Strain Exposure route exposure Doses observation Outcome no. Rat Sprague Dawley Subcutaneous injection PND 1–10 50 mg/50 mL, PND 13 In vivo: BPA500 lower basal (82) 500 mg/50 mL 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 Rat Wistar Drinking water þ lactation GD 0–PND 21 3 mg/kg/d PND 30 LH higher; no effect on FSH (89) levels Rat Sprague Dawley Oral gavage 90 d 0.001, 0.1 mg/kg/d 21 wk old Increased LH (protein levels (83) in pituitary cells and serum); no effect on FSH Zebraﬁsh Japanese medaka Aquarium water 21 d 2, 20, 200 mg/L 5 mo old Reduced FSH and LH (126) (BPA200) Rat Wistar Subcutaneous injection PND 1, 3, 5, 7 0.05, 20 mg/kg/d PND100 BPA20: dampened LH surge; (84) mature LHRH mRNA increased (BPA0.5) and decreased (BPA20); unprocessed intron A containing LHRH decreased (BPA0.05 and 20) Mouse C57BL6J Oral 12–15 d (ﬁrst 3 50 mg/kg/d 51–54 d No differences in FSH, LH (40) reproductive levels cycles) Sheep Suffolk Subcutaneous injection GD 30–90 0.05, 0.5, 5 mg/kg/d 19 mo No difference in LH surge (104) levels Mouse ICR Injection into right lateral Proestrus of 0.02, 0.2, 2, 20, 6 h after BPA increased plasma LH but (124) ventricle 4th/5th 200 nM/3mL administration blocked by pretreatment estrous with GPR54 blocker; no change in timing or peak concentration of LH surge O.16N.4/SPEBR1,2016 15, SEPTEMBER / 4 NO. 106 VOL. Mouse CD-1 Oral gavage GD 1–PND 20 12, 25, 50 mg/kg/d PND 50 BPA25: increased Fsh; (46) BPA50: increased Fsh mRNA; No difference in levels of LH, thyroid stimulating hormone, growth hormone, PRL Mouse CD-1 Oral gavage PND 21–49 25, 50 mg/kg/d PND 50 No difference in levels of LH, (46) thyroid stimulating hormone, growth hormone, PRL Rat Sprague Dawley Oral 5 wk old 50 mg/kg/d After 6 wk Decreased serum FSH, no (127) effect on serum LH Note: BPA [ bisphenol A. Ziv-Gal. BPA and female infertility. Fertil Steril 2016. Fertility and Sterility®

Several of the BPA-induced changes in pituitary gene action. Bisphenol A is a model endocrine-disrupting chem- expression are accompanied by altered serum levels of the pi- ical with a complicated mechanism of action that is yet to tuitary hormones. Specifically, low and high dose BPA expo- be fully elucidated. Its effects are highly dependent on sure decreased serum FSH and LH levels in adult rats and fish various factors in the study design such as timing of expo- compared with controls (82, 126, 127). Low dose BPA sure, species, dose, route of exposure, and mode of quantifi- exposure also increased serum LH levels compared with cation/assessment. In addition, other modifying factors controls in mice and rats (83, 89, 124). Last, early neonatal including co-exposures, study location/setting (e.g., hospi- low dose BPA exposure resulted in a dampened LH surge tal), and study sample (e.g., potentially unhealthy/previ- compared with controls in adult rats (84). Differences in ously exposed participants) should also be taken into study design including BPA doses may contribute to the consideration when possible, as proposed by Teeguarden variability in the results. Nevertheless, additional studies are et al. (30, 31). needed to examine whether the effects on FSH, LH, and LH In summary, further studies are needed to better under- surge can explain some of the effects on ovarian function stand the mechanism of action of BPA on female fertility. and estrus cyclicity. These studies will need to include some integrated end points In contrast to these effects of BPA described, a few studies to assure reproducibility of previous findings, yet taking into reported that BPA exposure does not affect serum levels of account relevance of the doses to human exposure, the ability FSH (40, 46, 82, 83, 89) or LH (40, 124, 127) in mice and of BPA to bind certain receptors, and study design/setting. rats. Similarly, one study (104) found that low dose BPA Because female fertility relies on several organs and feedback exposure did not affect the LH surge in adult sheep. Overall, loops, studies that use in vivo methods should aim for a multi- most current studies suggest that BPA exposure affects the organ/disciplinary approach. function of the anterior pituitary. However, the scientific evidence needs to be supported with additional studies. The REFERENCES secretion of LH and FSH may need to be measured for 1. Inhorn MC, Patrizio P. Infertility around the globe: new thinking on gender, several time points to delineate the mechanisms through reproductive technologies and global movements in the 21st century. Hum which BPA selectively targets its function. Reprod Update 2015;21:411–26. In conclusion, the current literature fairly consistently 2. Volkel W, Colnot T, Csanady GA, Filser JG, Dekant W. 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