Serum unconjugated bisphenol A
concentrations in women may adversely
influence oocyte quality during in vitro
Bisphenol A (BPA) is an endocrine disruptor with estrogenic properties that can adversely affect meiotic spindle
assemblies. Our data indicate that BPA exposure in female patients may interfere with oocyte quality during
IVF, as suggested by the inverse association between serum unconjugated BPA concentration and normal
fertilization. (Fertil Steril?2011;95:1816–9. ?2011 by American Society for Reproductive Medicine.)
Key Words: Bisphenol A (BPA), in vitro fertilization (IVF), oocyte maturation, fertilization, infertility
Bisphenol A(BPA) isgainingglobal attention asan environmental
contaminant of human health relevance owing to its widespread
exposure and endocrine-disrupting properties (1, 2). Although
knowledge gaps exist regarding the inadvertent BPA exposure of
human oocytes, Hunt et al. (3, 4) suggested a relationship
between BPA and meiotic disruption of both adult and fetal
murine oocytes. BPA has been detected in human follicular fluid,
providing evidence that human oocytes are exposed during
folliculogenesis (5, 6). Moreover, an inverse association was
reported between urinary BPA levels and number of oocytes
retrieved and peak estradiol (E2) levels during in vitro
fertilization (IVF), but without comment on oocyte quality (7).
To address the dearth of evidence implicating BPA as a disruptor
of human oocyte development, we measured serum BPA levels
in women undergoing IVF and correlated these levels with oocyte
maturation and fertilization outcomes.
This studycohort hasbeen previouslydetailed(8) and consists of
58 infertile female patients and 37 male partners undergoing a first
IVF cycle at the University of California at San Francisco (UCSF)
Center for Reproductive Health between September 1, 2007, and
August 31, 2008. Women and men received infertility evaluations,
including medical and reproductive histories, and their informed
consents were obtained before participation. The study protocol
was approved by the UCSF Committee for Human Research.
Female participants underwent gonadotropin-induced ovarian
stimulation per clinic protocols. When at least two follicles
measured 17 mm in diameter, human chorionic gonadotropin
(hCG) (5,000–10,000 IU) was administered subcutaneously and
oocytes retrieved 36 hours later. A fasting blood specimen was ob-
tained from women at the time of oocyte retrieval, whereas men,
when available, provided a nonfasting blood specimen. Specimens
were collected into serum separator Vacutainer tubes (Becton
Dickinson and Co., Franklin Lakes, NJ) and allowed to stand for
conventional insemination or by intracytoplasmic sperm injection
(ICSI) using fresh sperm from male partners or frozen sperm from
a male partner or donor. Approximately 16–18 hours after insem-
ination, zygotes were identified by the appearance of two pronu-
clei. For each ICSI patient, the proportion of mature oocytes
collected was defined as the total number of oocytes in metaphase
II (MII) arrest divided by the total number of oocytes collected
from a patient. The proportion of oocytes fertilized comprised:
(1) the total number ofzygotes formed divided by the total number
of mature oocytes injected from ICSI patients; and (2) the total
number of zygotes formed divided by the total number of oocytes
with a visible polar body from conventional insemination patients.
To maximize statistical power the denominators were collapsed
and a single value generated.
Serum was shipped on dry ice to the Endocrine Disruptors
Laboratory at the University of Missouri (Columbia, MO) in
September 2008. Serum specimens of R1–2 mL (44 women and
Victor Y. Fujimoto, M.D.a
Dongsul Kim, B.S.b
Frederick S. vom Saal, Ph.D.d
Julie D. Lamb, M.D.a
Julia A. Taylor, Ph.D.d
Michael S. Bloom, Ph.D.b,c
aDepartment of Obstetrics, Gynecology, and Reproductive
Sciences, University of California, San Francisco, California
bDepartment of Environmental Health Sciences, University at
Albany, State University of New York, Rensselaer, New York
cDepartment of Epidemiology and Biostatistics, University at
Albany, State University of New York, Rensselaer, New York
dDivision of Biological Sciences, University of Missouri,
Received August 22, 2010; revised October 5, 2010; accepted
November 3, 2010; published online December 3, 2010.
V.Y.F. has nothing to disclose. D.K. has nothing to disclose. F.S.v.S. has
nothing to disclose. J.D.L. has nothing to disclose. J.A.T. has nothing
to disclose. M.S.B. has nothing to disclose.
Funding for BPA measurements provided by the National Institute of
Environmental Health Sciences (grant no. ES018764).
Reprint requests: Victor Y. Fujimoto, M.D., Department of Obstetrics,
Gynecology, and Reproductive Sciences, 2356 Sutter Street, J707,
San Francisco, CA 94115-0916 (E-mail: firstname.lastname@example.org).
Fertility and Sterility?Vol. 95, No. 5, April 2011
Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
31 men) were assayed for concentrations of unconjugated BPA by
high-performance liquid chromatography, using known standards,
with an ESA Coularray 5600 detector (ESA, Chelmsford, MA), as
described in detail elsewhere (9). Recoveries averaged 89%, and
was blinded to all clinical and end point data, and machine-read
values were reported for samples below the LOD based on extrapo-
lation from the standard curve; these are different from samples for
which there was no evidence for the presence of BPA, which were
assigned zero values by the machine (10).
Log binomial regression models (11) were used to estimate
adjusted associations between log-transformed serum BPA con-
centrations and oocyte maturity for 31 women undergoing ICSI
and fertilization for 26 IVF couples; only participants with
complete covariate data were included. Removal of the cumulus
mass from retrieved oocytes during ICSI facilitated direct visuali-
zation of oocytes before insemination, thereby minimizing
misclassification of MII arrest in the analysis of this end point.
Age, race/ethnicity, and cigarette smoking were included as cova-
riates contingent on associations with BPA exposure and IVF end
the unit of analysis, and generalized estimating equations were
used to accommodate correlations among measures within
couples (14). A forward stepwise selection procedure was used
to retain statistically significant interactions between serum BPA
concentrations and covariates in each model where P<.05,
terion. Regression coefficients and 95% confidence intervals (CIs)
ciated with a doubling of BPA concentrations. Statistical analysis
was conducted using SAS v.9.2 (SAS Institute, Cary, NC).
The demographics of the study population have been previously
described (8). In brief, an average of 13.1 ? 7.8 oocytes were col-
lected during each of 59 initiated study cycles (i.e., only the male
partner participated for one cycle), 0.77 ? 0.22 of which were in
MII arrest and 0.64 ? 0.27 of which were fertilized. The median
BPA concentrations were 2.53 ng/mL for women (range
0.0–67.4, 86.4% >LOD) and 0.34 ng/mL for men (range
0.0–22.7, 51.6% >LOD), respectively. The results of the multivar-
iable statistical analysis are presented in Table 1. There was no
association between BPA and oocyte maturation when all cases
were considered (aRR 1.01, 95% CI 0.98–1.05; data not shown).
However, in ICSI-only cases, we found a 9% decrease in the prob-
ability for a mature oocyte for a doubling of female serum BPA
concentration, but only among the nine Asian women (aRR
0.91, 95% CI 0.83–1.00). Moreover, a 55% decrease in the proba-
bility for fertilization among the 26 cases of ICSI or conventional
insemination was associated with a doubling in female serum BPA
concentration (aRR 0.45, 95% CI 0.21–0.66), an effect which was
reduced by 2% for each year increase in female age (aRR 1.02,
95% 1.01–1.03) and further reduced by 6% for each doubling of
male BPA (aRR 1.06, 95% CI 1.02–1.10). A doubling in male se-
rum BPA concentration was itself associated with a 12% reduction
in the probability for fertilization, but only for the five Asian men.
tent BPA exposure may negatively affect oocyte development
potential. Although the findings are preliminary, inverse associa-
tions detected between BPA in Asian women and oocyte maturity
and between BPA in all women and normal fertilization implicate
BPA as having a disruptive influence on oocyte developmental
competence and oocyte quality.
We can only speculate on the mechanisms responsible for these
suggested effects of BPA on human oocytes. It is well established
that follicle-stimulating hormone (FSH) interacts synergistically
with E2to increase luteinizing hormone (LH) receptor and ste-
roidogenic enzyme expression in granulosa cells (15–17). BPA
has been demonstrated to bind and activate estrogen receptor
(ER) isoforms a and b (18–20). The first indication of an
adverse effect of BPA on oocyte development described
Log-binomial regression models describing the adjusted relative risk (aRR) for a doubling in serum unconjugated bisphenol A
(BPA) concentrations on oocyte maturity and fertilization during IVF.
Model for oocyte maturitya
Model for oocyte fertilizationb
aRR Low 95% CLHigh 95% CLP valueaRR Low 95% CL High 95% CLP value
BPA-female (ng/mL serum)
BPA-male (ng/mL serum)
Race-female (not Asian/Asian)
Race-male (not Asian/Asian)
BPA-female ? race-female
BPA-female ? age-female
BPA-female ? BPA-male
BPA-male ? race-male
Note: CL ¼ confidence limit; — ¼ not included in the relevant log-binomial regression model.
an ¼ 31 women undergoing intracytoplasmic sperm injection (ICSI).
bn ¼ 26 couples undergoing ICSI or conventional IVF.
Fujimoto. Correspondence. Fertil Steril 2011.
Fertility and Sterility?
a dramatic increase in meiotic abnormalities as a direct result of
coincidental BPA exposure (3). It has been demonstrated that
BPA altered murine oocyte development by inducing greater
meiotic arrest at either the germinal vesicle (GV) stage or the
metaphase I stage, but only at the highest exposure concentration
of 30 mmol/L (21). Interestingly, the highest rate of MII abnormal-
ities in that study were found at the lowest BPA dose tested
(3 nmol/L), suggesting nonlinear effects (21). Our preliminary
dataset focused on human oocyte developmental competence but
did not include nondisjunction end points.
Estrogen receptor b is the predominant estrogen receptor found
in granulosa cells at all stages of follicular development (22).
A functional ERb is necessary for adequate LH-induced cyclic
tion of the LH receptor (23–25). That we detected a significant
negative association between normal fertilization and female
serum BPA concentrations suggests that oocyte quality is
impaired to some degree by the presence of BPA (26). However,
our limited dataset prevented confirmation of the negative associa-
tion between normal fertilization and female BPA concentrations
owing to statistical instability when only ICSI cases were consid-
ered. Although it is not known if meiotic markers are adversely
influenced by BPA in humans, our data provide preliminary evi-
dence that cytoplasmic competence and meiotic progression may
be affected by BPA. Based on the current literature, it is interesting
to speculate that our observations may have occurred due to BPA
disruption of ERb-mediated LH receptor induction.
sive evaluation of the associations detected between BPA and
oocyte quality, and furthermore, it restricted the number of cova-
riates for which these associations could be adjusted and for which
sample size for the fertilization end point, we treated cases of ICSI
and conventional insemination as a homogeneous group. This ap-
proach may have biased the results if misclassification occurred in
the identification of MII arrest in cases of conventional insemina-
tion. Another limitation is that BPA was the only environmental
exposure considered in the statistical analyses. It is possible that
exposures including other phenols may have interactions with
BPA effects on oocyte development. We consider one significant
advantage of these data to be the measurements of unconjugated
serum BPA, rather than total BPA, which includes both conjugated
and unconjugated BPA. Thus, we were measuring only the circu-
lating bioactive BPA component. It is not clear if other exposure
assessment approaches of BPAwould have similar findings, given
the variability in BPA metabolism in humans (2).
This preliminary study suggests an overall negative association
tential of human oocytes. The complexity of statistical interactions
in our regression models suggests that the effects of female BPA
are modified by other factors, such as female age, race/ethnicity,
and male BPA. Further studies are needed to confirm our findings
and elucidate the biologic mechanisms mediating the effects of
BPA on human oocyte development during folliculogenesis.
Acknowledgments: The authors thank Giulia Conti and Natasha Narayan
for their assistance in conducting the study. They acknowledge the support
of the IVF laboratory staff at the UCSF Center for Reproductive Health
during the collection of samples.
1. Crain DA, Janssen SJ, Edwards TM, Heindel J,
Ho S-m, Hunt P, et al. Female reproductive
disorders: the roles of endocrine-disrupting com-
pounds and developmental timing. Fertil Steril
2. VandenbergLN, Chauhoud
Padmanabhan V, Paumgartten FJR, Schoenfelder G.
studies indicate widespread exposure to bisphenol
A. Environ Health Perspect 2010;118:1055–70.
3. Hunt PA, Koehler KE, Susiarjo M, Hodges CA,
Ilagan A, Voigt RC, et al. Bisphenol A exposure
causes meiotic aneuploidy in the female mouse.
Curr Biol 2003;13:546–53.
4. Susiarjo M, Hassold TJ, Freeman E, Hunt PA.
Bisphenol A exposure in utero disrupts early
oogenesis in the mouse. PLoS Genetics 2007;3:e5.
Determination of bisphenol A concentrations in
human biological fluids reveals significant early
prenatal exposure. Hum Reprod 2002;17:2839–41.
6. Kaddar N, Bendridi N, Harth? e C, de Ravel MR,
Bienvenu A-L, Cuilleron C-Y, et al. Development
of a radioimmunoassay for the measurement of
bisphenol A in biological samples. Anal Chim
7. Mok-Lin E, Ehrlich S, Williams PL, Petrozza J,
Wright DL, Calafat AM, et al. Urinary bisphenol A
concentrations and ovarian response among women
undergoing IVF. Int JAndrol 2010;33:385–93.
8. Bloom MS, Parsons
Schisterman EF, Browne RW, Kim K, et al. Toxic
trace metals and human oocytes during in vitro
9. Taylor JA, vom Saal FS, Welshons WV, Drury B,
of bisphenol A pharacokinetics in rhesus monkeys
and mice: relevance for human exposure. Environ
Health Perspect 2010. doi: 10.1289/ehp.1002514.
10. Schisterman EF, Vexler A, Whitcomb BW, Liu A.
The limitations due to exposure detection limits
for regression models. Am J Epidemiol 2006;163:
relative risk in cohort studies and clinical trials of
12. Calafat AM,Ye X,
Needham LL. Exposure of the U.S. population to
bisphenol A and 4-tertiary-octylphenol: 2003–
2004. Environ Health Perspect 2008;116:39–44.
et al. Bisphenol A levels in blood and urine in
a Chinese population and the personal factors
affecting the levels. Environ Res 2009;109:629–33.
14. Zeger SL, Liang KY. Longitudinal data analysis for
discrete and continuous outcomes. Biometrics
of estrogens in the augmentation of luteinizing
granulosa cells. Biol Reprod 1985;32:1038–50.
16. Knecht M, Brodie AMH, Catt KJ. Aromatase
inhibitors prevent granulosa cell differentiation: an
17. Segaloff DL, Wang H, Richards JS. Hormonal
gonadotropin receptor mRNA in rat ovarian cells
during follicular development and luteinization.
Mol Endocrinol 1990;4:1856–65.
Newbold RR, Rubin BS, Talsness CE, et al. In
vivo effects of bisphenol A in laboratory rodent
studies. Reprod Toxicol 2007;24:199–224.
19. Routledge EJ, White R, Parker MG, Sumpter JP.
coactivator recruitment by estrogen receptor (ER)
a and ERb. J Biol Chem 2000;275:35986–93.
20. Wetherill YB,Akingbemi
McLachlan JA, Nadal A, Sonnenschein C, et al. In
vitro molecular mechanisms of bisphenol A
action. Reprod Toxicol 2007;24:178–98.
Continuous exposure to bisphenol A during in vitro
abnormalities. Mutat Res 2008;651:71–81.
22. Pelletier G, El-Alfy M. Immunocytochemical
localization of estrogen receptors a and b in the
human reproductive organs. J Clin Endocrinol
23. Couse JF, Yates MM, Deroo BJ, Korach KS.
Estrogen receptor b is critical to granulosa cell
differentiation and the ovulatory response to
gonadotropins. Endocrinology 2005;146:3247–62.
Fujimoto et al.
Vol. 95, No. 5, April 2011
24. Emmen JMA, Couse JF, Elmore SA, Yates MM, Download full-text
Kissling GE, Korach KS. In Vitro growth and
ovulation of follicles from ovaries of estrogen
receptor (ER)a and ERb null mice indicate a role
for ERb in follicular maturation. Endocrinology
25. Rodriguez KF, Couse JF, Jayes FL, Hamilton KJ,
Burns KA, Taniguchi F, et al. Insufficient luteinizing
tor-b. Endocrinology 2010;151:2826–34.
26. Rosen MP, Shen S, Dobson AT, Fujimoto VY,
McCulloch CE, Cedars MI. Triploidy formation
after intracytoplasmic sperm injection may be
a surrogate marker for implantation. Fertil Steril
Fertility and Sterility?