Exposure to bisphenol A is associated with recurrent
Mayumi Sugiura-Ogasawara1,4, Yasuhiko Ozaki1, Shin-ichi Sonta2, Tsunehisa Makino3
and Kaoru Suzumori1
1Department of Obstetrics and Gynecology, Nagoya City University Medical School, Nagoya,2Department of Genetics, Institute for
Developmental Research, Aichi Human Service Center and3Department of Obstetrics and Gynecology, Tokai University School of
Medicine, Kanagawa, Japan
4To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Nagoya City University Medical
School, Mizuho-ku, Nagoya, 4678601 Japan. E-mail: firstname.lastname@example.org
BACKGROUND: Little is known about the influence of high exposure to bisphenol A on recurrent miscarriage
and immunoendocrine abnormalities. METHODS: Serum bisphenol A, antiphospholipid antibodies (aPLs), anti-
nuclear antibodies (ANAs), natural killer cell (NK) activity, prolactin, progesterone, thyroid-stimulating hormone
(TSH) and free T4 were examined in 45 patients with a history of three or more (3–11) consecutive first-trimester
miscarriages and 32 healthy women with no history of live birth and infertility. Subsequent pregnancy outcome
and embryonic karyotype of abortuses were examined prospectively. RESULTS: The mean 6 SD values for
bisphenol A in patients were 2.59 6 5.23ng/ml, significantly higher than the 0.77 6 0.38ng/ml found for control
women (P 5 0.024). High exposure to bisphenol A was associated with the presence of ANAs but not hypothyroid-
ism, hyperprolactinaemia, luteal phase defects, NK cell activity or aPLs. A high level of bisphenol A in itself did
not predict subsequent miscarriage. CONCLUSION: Exposure to bisphenol A is associated with recurrent
Key words: aneuploidy/antinuclear antibody/bisphenol A/embryonic karyotype/recurrent miscarriage
Many chemical compounds introduced into the environment
by human activity are known to influence the endocrine sys-
tem of various animals and humans. Kogevinas (2001) has
reported dioxin to cause cancer and endometriosis, and
Longnecker et al. (2001) recently suggested that DDT [1,1,1,-
trichloro-2,2-bis (p-chlorophenyl) ethane] use increases pre-
term birth and small for gestational age babies in humans.
Endocrine disruptors such as dioxin are well established as
reproductive toxicants. However, it is a matter of controversy
whether they might be linked with first-trimester spontaneous
abortion because there is limited information concerning this
issue, especially recurrent miscarriages (Leoni et al., 1989).
We previously studied the serum concentration of polychloro-
biphenyls (PCBs), hexachlorobenzene (HCB) and the DDT
metabolite DDE [1,1-dichloro-2,2-bis (p-chlorophenyl) ethyl-
ene] and found no association with recurrent miscarriage
(Sugiura-Ogasawara et al., 2003).
Bisphenol A [BPA; 2, 2-bis(4-hydroxyphenyl) propane] is
well known as an endocrine disruptor having estrogen
activity. About 350000 t/year is produced for use in poly-
carbonate plastics, epoxy resins, dental sealants and food
package lacquers to coat food cans in Japan (Biles et al.,
1997; Olea et al., 1996; Biles et al., 1999). We are
surrounded by materials made from BPA and exposed to this
environmental contaminant worldwide. BPA administered
to pregnant mice is known to be transferred to fetuses and
(Howdeshell et al., 1999). Recently, Hunt et al. (2003)
demonstrated that daily oral dosing with the compound
causes meiotic aneuploidy in the female mouse.
About 40–70% of sporadic spontaneous abortions are
linked to chromosomal abnormalities of the conceptus,
especially aneuploidy (Creasy, 1988; Ogasawara et al.,
2000). No unequivocal cause is currently available for
more than half of the cases suffering recurrent miscarriages
embryonal karyotype of the conceptus has been found in
10–50% of recurrent miscarriage cases (Stern et al., 1996;
Ogasawara et al., 2000;
Stephenson et al., 2002) and meiotic aneuploidy in 29.9%
in one series of such cases (Ogasawara et al., 2000). We
hypothesed that elevated exposure to BPA might induce
meiotic aneuploidy in females and cause recurrent miscar-
riages. Thus, we conducted a prospective study. Also, we
studied the association between serum levels of BPA and
endocrine and immunological abnormalities in a series of
recurrent miscarriage patients.
Christiansenet al., 2002;
Human Reproduction Vol.20, No.8 pp.2325–2329, 2005
Advance Access publication April 7, 2005
q The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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We studied 45 patients with a history of three or more (3–11)
chromosome analysis for both partners, immunological tests for
parameters such as antinuclear antibodies (ANAs), antiphospholipid
antibodies (aPLs) and natural killer (NK) cell activity, and blood
tests for hypothyroidism, diabetes mellitus and hyperprolactinaemia
were performed for all cases before subsequent pregnancy.
Cases with any uterine anomaly or chromosome abnormality in
either partner were excluded from the study. None had a history of
live birth. All were seen at Nagoya City University Hospital
between August 2001 and December 2002. Fasting blood samples
were taken at the same time.
Thirty- two healthy non-pregnant women with no history of live
birth, infertility and miscarriage were examined as controls. Patients
and controls lived in Nagoya City and the surrounding neighbour-
hood. None was from specific geographical areas or taking oral con-
traceptives. Half of the patients were housewives and the others had
many different occupations. Healthy volunteers were staff such as
doctors of Obstetrics and Gynaecology and other departments,
nurses and secretaries in Nagoya City University Medical School.
Data regarding place of residence and occupation for patients and
controls is shown in Table I. Informed consent approved by the
Institutional Review Board was obtained from all subjects.
Mean ^ SD values for serum concentrations of BPA in patients
were compared statistically with data for controls. Correlations with
the presence of ANAs, aPLs, hypothyroidism, luteal phase defects
and hyperprolactinaemia were also assessed. Correlations among
serum progesterone, prolactin and NK cell activity were examined
by Pearson’s correlation coefficient.
Subsequent pregnancy outcome was examined prospectively.
Ultrasonography was performed twice a week during pregnancy,
and dilation and curettage were undertaken when a miscarriage was
diagnosed. The karyotype of each aborted conceptus was ascertained
using a standard G-banding technique.
Serum BPA levels were compared between cases with successful
pregnancy and miscarriages with a normal and an abnormal embryo-
Antinuclear antibodies, antiphospholipid antibodies, natural killer
cell activity, TSH, free T4, progesterone and prolactin
Blood samples were taken 5–9 days after ovulation in at least two
cycles. For progesterone and prolactin analysis, they were collected
at least 3 months after their last abortion and before they conceived
ANAs were assessed by indirect immunofluorescence on Hep-2
cell slides. aPLs were measured by lupus anticoagulant using five
times diluted aPTT regents and b2-glycoprotein I-dependent anticar-
diolipin antibodies by enzyme-linked immunosorbent assay (ELISA)
methods (Ogasawara et al., 1996b). NK cell activity was measured
by a chromium-51 release cytotoxicity assay, with K562 human
myeloid leukaemia cells as the targets.
Progesterone levels were determined by radioimmunoassay, using
reagentssupplied bythe Diagnostic
(Los Angeles, CA). A midluteal phase single serum progesterone
level ,10ng/ml was used as the criterion for a luteal phase defect.
Prolactin was measured by immunoradiometric assay with a kit sup-
plied by Daiichi Radioisotope Laboratories, Ltd. (Tokyo, Japan).
Bisphenol A ELISA measurement
Serial samples were collected from individuals at Nagoya City Uni-
versity Hospital and kept frozen at 2208C until analysis. Briefly,
goat anti-rabbit IgG was added to 96-well microtitre plates and,
after standing for 2h at room temperature, the plates were then
blocked with 25% Block Ace and stored at 48C until use. Aliquots
of 50ml of standard antigen (0.5–5000ng/ml) or samples, BPA-
mono-glutanyl–horseradish peroxidase (HRP) and rabbit anti-BPA
serum were added in duplicate and the wells were incubated for 2h
at room temperature. Phenylenediamine solution was added and the
absorbance was measured at 492nm (Kodaira et al., 2000).
Differences in group values were analysed using Stat view and DA
stats with an Apple Macintosh computer. A significance level of
P , 0.05 was applied for all tests. We used Welch’s test to compare
BPA levels between patients and controls because the distribution of
the two groups might have differed. The Mann–Whitney test was
employed for comparisons of BPA levels between patients who had
successful pregnancies and patients who miscarried again with and
without an abnormal embryonal karyotype and between those with
and without hypothyroidism, aPLs, ANAs, luteal phase defect or
hyperprolactinaemia. Correlations were analysed with reference to
Pearson’s correlation coefficients.
The 45 study patients had a mean (^SD) age of 31.6 ^ 4.4
years and 4.0 ^ 1.7 previous miscarriages (range, 3–11 mis-
carriages). The 32 controls were 32.0 ^ 4.8 years of age
The mean ^ SD values for BPA in patients
2.59 ^ 5.23, significantly higher than the 0.77 ^ 0.38 found
for control women (P = 0.024). No linkage was evident with
the body mass index (kg/m2).
Of the 45 patients, eight (15.6%) had hypothyroidism, and
six (13.3%) and 10 (22.2%) patients had aPLs and ANAa,
Table I. Demographic characteristics and bisphenol A values in patients and
(n = 45)
(n = 32)
No. of previous
Place of residence
31.6 ^ 4.4
4.0 ^ 1.7 0
32.0 ^ 4.8NS
20.9 ^ 2.7
20.81 ^ 2.25
Office workers 17
Medical co-workers 3
Worker in industry 1
2.59 ^ 5.23
0.77 ^ 0.38
Values are means ^ SD.
BMI = body mass index (kg/m2).
M.Sugiura-Ogasawara et al.
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respectively (Table II). Five (11.1%) and nine (20.5%)
demonstrated hyperprolactinaemia and a luteal phase defect.
No linkage with NK cell activity, progesterone and prolactin
level was evident.
There were no differences in mean values for BPA
between patients with and without hypothyroidism, hyperpro-
lactinaemia, a luteal phase defect and the presence of
aPLs. However, ANA-positive patients had significantly
higher BPA levels than their ANA-negative counterparts
(P = 0.025).
A total of 35 patients became pregnant subsequently and
17 (48.6%) of these miscarried again. One suffered an ecto-
pic pregnancy and thus there were 17 live births. The BPA
level of patients who miscarried subsequently (mean ^ SD,
4.39 ^ 8.08; range, 0.28–29.43; median, 0.71; quartiles,
3.055) tended to be higher, albeit without significance, than
that of patients whose subsequent pregnancy was successful
(mean ^ SD, 1.22 ^ 1.07; range, 0.22–3.85; median, 0.91;
Thirteen karyotypes of the miscarried conceptus could be
analysed. Four were abnormal: 47, XX, þ 7, 47, XY, þ 9,
47, XY, þ 10 and 47, XY, þ13. The BPA levels of cases
with abnormal embryonal karyotypes were 0.66, 19.09, 0.69
and 29.43, respectively (mean ^ SD, 12.47 ^ 14.26). The
individual BPA levels in each group (patients versus controls,
patients who had successful pregnancies versus patients who
miscarried again, with an abnormal embryonal karyotype ver-
sus a normal embryonal karyotype) are shown in Figure 1.
The present study provided the first concrete evidence that
high exposure to BPA may be associated with recurrent mis-
carriage, especially with ANA-positive patients.
It has been a matter of controversy whether chemical com-
pounds are linked with first-trimester spontaneous abortion,
and our previous study of serum concentrations of PCBs,
HCB and the DDT metabolite DDE found no association
between exposure and recurrent miscarriage (Leoni et al.,
1989; Sugiura-Ogasawara et al., 2003).
BPA is well known as a chemical compound having
0.00025 times the estrogen activity of estradiol, binding to
the estrogen receptor. Steinmetz et al. (1997) reported that
BPA has 0.0001–0.0005 times the estrogen activity in vitro,
but with an increase of 0.05 times in terms of stimulating
prolactin in rats. BPA may be more potent in vivo than
in vitro. Honma et al. (2002) reported that low dose exposure
to BPA caused earlier vaginal opening in female mice. Many
reports have been published concerning its influence on the
female mammalian reproductive system (Ashby and Tinwell,
1998; Milligan et al., 1998; Hiroi et al., 1999; Howdeshell
et al., 1999; Takai et al., 2000).
Hunt et al. (2003) demonstrated that daily oral dosing
exposure causes meiotic aneuploidy in the female mouse
(Hunt et al., 2003). They reported a sudden, spontaneous
increase in meioticdisturbance,
in studies of oocytes from control female mice in their
laboratory, coinciding with accidental exposure of the
animals to an environmental source of BPA. They identified
damaged caging material as the source, as they were able to
Figure 1. The mean ^ SD values for BPA in 45 patients were
significantly higher than that in 32 control women. The BPA level
of 17 patients who miscarried subsequently tended to be higher,
albeit without significance, than that of 17 patients whose sub-
sequent pregnancy was successful.
Table II. Bisphenol A values in patients with and without hypothyroidism,
antiphospholipid antibodies, antinuclear antibodies, luteal phase defect and
aPL = antiphospholipid antibody; ANA = antinuclear antibody; LPD = luteal
Bisphenol A and recurrent miscarriage
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damaging cages and water bottles. In further studies, they
administered daily oral doses of BPA to test directly the
hypothesis that low levels of BPA disrupt female meiosis.
They concluded that the meiotic effects were dose dependent
and inducible by environmentally relevant doses. BPA has
been found in blood of non-pregnant women at a concen-
tration of 2.0 ^ 0.8ng/ml and in human follicular fluid at a
concentration of 2.4 ^ 0.8ng/ml by ELISA methods (Ikezuki
et al., 2002). The results further indicated that the oocyte and
its meiotic spindle can provide a sensitive assay system for
the study of reproductive toxins. Ikezuki et al. also showed a
significant linear correlation between BPA values obtained
by conventional reverse-phase high-performance liquid chro-
matography (HPLC) and ELISA data (Kodaira et al., 2000;
Ikezuki et al., 2002). The BPA values of non-pregnant
women in the previous study tended to be slightly higher
than in the present study.
About 40–70% of sporadic spontaneous abortions appear
to be caused by chromosomal abnormalities of the conceptus,
and aneuploidy is especially important (Creasy, 1988;
Ogasawara et al., 2000). An abnormal embryonal karyotype
of the conceptus has been found in 10–50% of recurrent
miscarriage cases (Stern et al., 1996; Ogasawara et al., 2000;
Christiansen et al., 2002; Stephenson et al., 2002) and meiotic
aneuploidy in 29.9% in one series of such cases (Ogasawara
et al., 2000). Both miscarriage and the aneuploidy rate depend
on women’s age. No unequivocal cause is currently available
for more than half of the cases with recurrent miscarriages
and, in recurrent aborters, the miscarriage rate increases and
abnormal embryonal karyotype rate decreases with the num-
ber of previous miscarriage (Hertz-Picciotto et al., 1998;
Ogasawara et al., 2000). An abnormal karyotype of the con-
ceptus was also detected in .50% of cases with #4 recurrent
miscarriages, but less in cases with $5 miscarriages
(Ogasawara et al., 2000; Christiansen et al., 2002). There are
very few cases with an abnormal embryonal karyotype in
patients with $10 miscarriages (Ogasawara et al., 2000).
Thus, the rate decreases with the number of abortions. We
speculate that in ,18% of recurrent aborters the cause is
embryonal chromosome abnormalities. The present study pro-
vided the possibility that elevated exposure to BPA might
play a role in these cases.
Hyperprolactinaemia, hypothyroidism, elevated NK cell
activity and the presence of autoantibodies are well known to
be associated with recurrent miscarriages (Aoki et al., 1995;
Hirahara et al., 1998; Ogasawara et al., 1999). While BPA is
reported to stimulate prolactin release, our results do not pro-
vide any support for an action in this way (Steinmetz et al.,
1997). Exposure to BPA does not influence prolactin pro-
duction, thyroid function, NK cell activity and aPLs in the
human body, in contrast to the case with animals, and the
only link here was shown with ANAs. The frequency of
ANAs is significantly elevated in recurrent miscarriage cases,
but ANA itself does not predict subsequent miscarriage
(Ogasawara et al., 1996a). This is the first evidence of a link
between BPA and ANA production, to our knowledge. Some
drugs have been shown to exacerbate idiopathic systemic
the meioticabnormalities byintentionally
lupus erythematosus, including estrogen-containing oral con-
traceptives, and BPA, which has estrogen activity, could
induce autoimmunity. In fact, Tian et al. (2003) demonstrated
that interleukin-4 production was increased both in vitro and
in vivo by treatment with BPA, suggesting that it might con-
tribute to autoimmune disease.
This was a preliminary study and the sample size was rela-
tively small. Preimplantation diagnosis might be introduced
to reduce the miscarriages caused by abnormal embryonal
karyotype, although in many countries, including Japan, this
is not presently permitted for ethical reasons (Wilding et al.,
2004). However, the results suggest that further analysis of
the effects of BPA on human reproduction is definitely
Supported by the Ministry of Health, Labour and Welfare, Japan.
Aoki K, Kajiura S, Matsumoto Y, Ogasawara M, Okada S, Yagami Y and
Gleicher N (1995) Elevated natural killer cell activity at preconception as
a predictor of a subsequent miscarriage: a prospective cohort study. Lancet
Ashby J and Tinwell (1998) Uterotrophic activity of bisphenol A in the
immature rat. Environ Health Perspect 106,719–720.
Biles JE, McNeal TP and Begley TH (1997) Determination of bisphenol A
migrating from epoxy can coating to infant formula liquid concentrates.
J Agric Food Chem 45,4697–4700.
Creasy R (1988) The cytogenetics of spontaneous abortion in humans. In
Beard RW and Sharp F (eds) Early Pregnancy Loss: Mechanisms and
Treatment. Springer-Verlag, London, pp. 293–304.
Christiansen OB, Pedersen B, Rosgaard A and Husth M (2002) A random-
ized, double-blind, placebo-controlled trial of intravenous immunoglobulin
in the prevention of recurrent miscarriage: evidence for a therapeutic effect
in womenwith secondary recurrent
Hertz-Picciotto I and Samuels SJ (1998) Incidence of early loss of preg-
nancy. N Engl J Med 319,1483–1484.
Hirahara F, Ando N, Sawai K, Hirabuki T, Uemura T and Minaguchi H
(1998) Hyperprolactinemic recurrent miscarriage and results of random-
ized bromocriptine treatment trials. Fertil Steril 70,246–252.
Hiroi H, Tsutsumi O, Momoeda M, Takai Y, Osuga Y and Taketani Y
(1999) Differential interactions of bisphenol A and 17b-estradiol with
estrogen receptor a and ERb. Endocr J 46,773–778.
Honma S, Suzuki S, Buchanan DL, Katsu Y, Watanabe H and Iguchi T
(2002) Low dose effect of in utero exposure to bisphenol A and diethylstil-
bestrol on female mouse reproduction. Reprod Toxicol 16,117–122.
Howdeshell KL, Hotchkiss AK, Thayer KA, Vandenbergh JG and vom Saal
FS (1999) Exposure to bisphenol A advances puberty. Nature 401,
Hunt PA, Koehler KE, Susiarjo M, Hodges CA, Ilagan A, Voigt RC,
Thomas S, Thomas BF and Hassold TJ (2003) Bisphenol A exposure
causes meiotic aneuploidy in the female mouse. Curr Biol 13,546–553.
Ikezuki Y, Tsutsumi O, Takai Y, Kamei Y and Taketani Y (2002) Determi-
nation of bisphenol A concentrations in human biological fluids reveals
significant early prenatal exposure. Hum Reprod 17,2839–2841.
Kodaira T, Kato I, Li J, Mochizuki T, Hoshino M, Usuki Y, Oguri H and
Yanaihara N (2000) Novel ELISA for the measurement of immunoreactive
bisphenol A. Biomed Res 21,117–121.
Kogevinas M (2001) Human health effects of dioxin: cancer, reproductive
endocrine system effects. Hum Reprod Update 7,331–339.
Leoni V, Fabiani L, Marinelli G, Puccetti G, Tarsitani GF, De Carolis A,
Vescia N, Morini A, Aleandri V and Pozzi V (1989) PCB and other orga-
nochlorine compounds in blood of women with or without miscarriage:
a hypothesis of correlation. Ecotox Environ Safe 17,1–11.
Longnecker MP, Klebanoff MA, Zhou H and Brock JW (2001) Association
between maternal serum concentration of the DDT metabolite DDE
miscarriage. Hum Reprod
M.Sugiura-Ogasawara et al.
by guest on June 2, 2013
and preterm and small-for-gestational-age babies at birth. Lancet 358, Download full-text
Milligan SR, Balasubramanian AV and Kalita JC (1998) Relative potency of
xenobiotic estrogen in an acute in vivo mammalian assay. Environ Health
Ogasawara M, Aoki K, Kajiura S and Yagami Y (1996a) Are antinuclear
antibodies predictive of recurrent miscarriage? Lancet 347,1183–1184.
Ogasawara M, Aoki K, Matsuura E, Sasa H and Yagami Y (1996b) Anti-
b2glycoprotein I antibodies and lupus anticoagulant in patients with recur-
Ogasawara M, Aoki K, Katano K, Aoyama T, Kajiura S and Suzumori K
(1999) Prevalence of autoantibodies in patients with recurrent miscar-
riages. Am J Reprod Immunol 41,86–90.
Ogasawara MS, Aoki K, Okada S and Suzumori K (2000) Embryonic karyo-
type of abortuses in relation to the number of previous miscarriages. Fertil
Olea N, Pulgar R, Perez P, Olea-Serrano F, Rivas A, Novillo-Fertrell A,
Pedraza V, Soto AM and Sonnenschein C (1996) Estrogenicity of
resin-based composites and sealants used in dentistry. Environ Health
Steinmetz R, Brown NG, Allen DL, Bigsby RM and Ben-Jonathan N (1997)
The environmental estrogen bisphenol A stimulates prolactin release
in vitro and in vivo. Endocrinology 138,1780–1786.
Stephenson MD, Awartani KA and Robinson WP (2002) Cytogenetic analy-
sis of miscarriages from couples with recurrent miscarriage: a case-control
study. Hum Reprod 17,446–451.
Stern JJ, Dorfmann AD, Gutierrez-Najar AJ, Cerrillo M and Coulam CB
(1996) Frequency of abnormal karyotypes among abortuses from women
with and without a history of recurrent spontaneous abortion. Fertil Steril
Sugiura-Ogasawara M, Ozaki Y, Sonta S-I, Makino T and Suzumori K
(2003) PCBs, hexachlorobenzene and DDE are not associated with recur-
rent miscarriage. Am J Reprod Immunol 50,485–489.
Takai Y, Tsutsumi O, Ikezuki Y, Hiroi H, Osuga Y, Momoeda M, Yano T
and Taketani Y (2000) Estrogen receptor-mediated effects of a xenoestro-
gen, bisphenol A, on preimplantation mouse embryos. Biochem Biophys
Res Commun 270,918–921.
Tian X, Takamoto M and Sugane K (2003) Bisphenol A promotes IL-4 pro-
duction by Th2 cells. Int Arch Allergy Immunol 132,240–247.
Wilding M, Forman R, Hogewind G, Di Matteo L, Zullo F, Cappiello F and
Dale B (2004) Preimplantation genetic diagnosis for the treatment of failed
in vitro fertilization-embryo transfer and habitual abortion. Fertil Steril
Submitted on September 30, 2004; resubmitted on February 22, 2005;
accepted on March 3, 2005
Bisphenol A and recurrent miscarriage
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