1 Department of Obstetrics and Gynecology, Nagoya City University Medical School, Nagoya, 2 Department of Genetics, Institute for Developmental Research, Aichi Human Service Center and 3 Department of Obstetrics and Gynecology, Tokai University School of Medicine, Kanagawa, Japan
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Nagoya City University Medical School, Mizuho-ku, Nagoya, 4678601 Japan. Email: og.mym{at}med.nagoya-cu.ac.jp
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: aneuploidy/antinuclear antibody/bisphenol A/embryonic karyotype/recurrent miscarriage
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bisphenol A [BPA; 2, 2-bis(4-hydroxyphenyl) propane] is well known as an endocrine disruptor having estrogen activity. About 350 000 t/year is produced for use in polycarbonate 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 to alter post-natal development and sexual maturity (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 4070% 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 (Hertz-Picciotto and Samuels, 1998). An abnormal embryonal karyotype of the conceptus has been found in 1050% 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
). We hypothesed that elevated exposure to BPA might induce meiotic aneuploidy in females and cause recurrent miscarriages. 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.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 neighbourhood. None was from specific geographical areas or taking oral contraceptives. 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.
|
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 embryonal karyotype.
Antinuclear antibodies, antiphospholipid antibodies, natural killer cell activity, TSH, free T4, progesterone and prolactin measurement
Blood samples were taken 59 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 again.
ANAs were assessed by indirect immunofluorescence on Hep-2 cell slides. aPLs were measured by lupus anticoagulant using five times diluted aPTT regents and 2-glycoprotein I-dependent anticardiolipin 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 reagents supplied by the Diagnostic Products Corporation (Los Angeles, CA). A midluteal phase single serum progesterone level <10 ng/ml was used as the criterion for a luteal phase defect. Prolactin was measured by immunoradiometric assay with a kit supplied by Daiichi Radioisotope Laboratories, Ltd. (Tokyo, Japan).
Bisphenol A ELISA measurement
Serial samples were collected from individuals at Nagoya City University Hospital and kept frozen at 20°C until analysis. Briefly, goat anti-rabbit IgG was added to 96-well microtitre plates and, after standing for 2 h at room temperature, the plates were then blocked with 25% Block Ace and stored at 4°C until use. Aliquots of 50 µl of standard antigen (0.55000 ng/ml) or samples, BPA-mono-glutanylhorseradish peroxidase (HRP) and rabbit anti-BPA serum were added in duplicate and the wells were incubated for 2 h at room temperature. Phenylenediamine solution was added and the absorbance was measured at 492 nm (Kodaira et al., 2000).
Statistical analysis
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 MannWhitney 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.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mean±SD values for BPA in patients were 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, 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.
|
A total of 35 patients became pregnant subsequently and 17 (48.6%) of these miscarried again. One suffered an ectopic pregnancy and thus there were 17 live births. The BPA level of patients who miscarried subsequently (mean±SD, 4.39±8.08; range, 0.2829.43; 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, 0.223.85; median, 0.91; quartiles, 1.28).
Thirteen karyotypes of the miscarried conceptus could be analysed. Four were abnormal: 47, XX,+7, 47, XY,+9, 47, XY,+10 and 47, XY, +13. 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 versus a normal embryonal karyotype) are shown in Figure 1.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has been a matter of controversy whether chemical compounds 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 0.00010.0005 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 meiotic disturbance, including aneuploidy, 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 recapitulate the meiotic abnormalities by intentionally 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 concentration of 2.0±0.8 ng/ml and in human follicular fluid at a concentration of 2.4±0.8 ng/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 chromatography (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 4070% 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 1050% 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 number of previous miscarriage (Hertz-Picciotto et al., 1998; Ogasawara et al., 2000
). An abnormal karyotype of the conceptus 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 provided 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 provide any support for an action in this way (Steinmetz et al., 1997
). Exposure to BPA does not influence prolactin production, 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 lupus erythematosus, including estrogen-containing oral contraceptives, 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 contribute to autoimmune disease.
This was a preliminary study and the sample size was relatively 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 warranted.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ashby J and Tinwell (1998) Uterotrophic activity of bisphenol A in the immature rat. Environ Health Perspect 106, 719720.[ISI][Medline]
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, 46974700.[CrossRef][ISI]
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. 293304.
Christiansen OB, Pedersen B, Rosgaard A and Husth M (2002) A randomized, double-blind, placebo-controlled trial of intravenous immunoglobulin in the prevention of recurrent miscarriage: evidence for a therapeutic effect in women with secondary recurrent miscarriage. Hum Reprod 17, 809816.
Hertz-Picciotto I and Samuels SJ (1998) Incidence of early loss of pregnancy. N Engl J Med 319, 14831484.[CrossRef]
Hirahara F, Ando N, Sawai K, Hirabuki T, Uemura T and Minaguchi H (1998) Hyperprolactinemic recurrent miscarriage and results of randomized bromocriptine treatment trials. Fertil Steril 70, 246252.[CrossRef][ISI][Medline]
Hiroi H, Tsutsumi O, Momoeda M, Takai Y, Osuga Y and Taketani Y (1999) Differential interactions of bisphenol A and 17-estradiol with estrogen receptor
and ER
. Endocr J 46, 773778.[ISI][Medline]
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 diethylstilbestrol on female mouse reproduction. Reprod Toxicol 16, 117122.[CrossRef][ISI][Medline]
Howdeshell KL, Hotchkiss AK, Thayer KA, Vandenbergh JG and vom Saal FS (1999) Exposure to bisphenol A advances puberty. Nature 401, 763764.[CrossRef][ISI][Medline]
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, 546553.[CrossRef][ISI][Medline]
Ikezuki Y, Tsutsumi O, Takai Y, Kamei Y and Taketani Y (2002) Determination of bisphenol A concentrations in human biological fluids reveals significant early prenatal exposure. Hum Reprod 17, 28392841.
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, 117121.[ISI]
Kogevinas M (2001) Human health effects of dioxin: cancer, reproductive endocrine system effects. Hum Reprod Update 7, 331339.
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 organochlorine compounds in blood of women with or without miscarriage: a hypothesis of correlation. Ecotox Environ Safe 17, 111.[CrossRef][ISI]
Longnecker MP, Klebanoff MA, Zhou H and Brock JW (2001) Association between maternal serum concentration of the DDT metabolite DDE and preterm and small-for-gestational-age babies at birth. Lancet 358, 110114.[CrossRef][ISI][Medline]
Milligan SR, Balasubramanian AV and Kalita JC (1998) Relative potency of xenobiotic estrogen in an acute in vivo mammalian assay. Environ Health Perspct 106, 2326.
Ogasawara M, Aoki K, Kajiura S and Yagami Y (1996a) Are antinuclear antibodies predictive of recurrent miscarriage? Lancet 347, 11831184.[CrossRef][Medline]
Ogasawara M, Aoki K, Matsuura E, Sasa H and Yagami Y (1996b) Anti2glycoprotein I antibodies and lupus anticoagulant in patients with recurrent pregnancy loss: prevalence and clinical significance. Lupus 5, 587592.[ISI][Medline]
Ogasawara M, Aoki K, Katano K, Aoyama T, Kajiura S and Suzumori K (1999) Prevalence of autoantibodies in patients with recurrent miscarriages. Am J Reprod Immunol 41, 8690.[ISI][Medline]
Ogasawara MS, Aoki K, Okada S and Suzumori K (2000) Embryonic karyotype of abortuses in relation to the number of previous miscarriages. Fertil Steril 73, 300304.[CrossRef][ISI][Medline]
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 Perspect 104, 298305.[ISI][Medline]
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, 17801786.
Stephenson MD, Awartani KA and Robinson WP (2002) Cytogenetic analysis of miscarriages from couples with recurrent miscarriage: a case-control study. Hum Reprod 17, 446451.
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 65, 250253.[ISI][Medline]
Sugiura-Ogasawara M, Ozaki Y, Sonta S-I, Makino T and Suzumori K (2003) PCBs, hexachlorobenzene and DDE are not associated with recurrent miscarriage. Am J Reprod Immunol 50, 485489.[CrossRef][ISI][Medline]
Takai Y, Tsutsumi O, Ikezuki Y, Hiroi H, Osuga Y, Momoeda M, Yano T and Taketani Y (2000) Estrogen receptor-mediated effects of a xenoestrogen, bisphenol A, on preimplantation mouse embryos. Biochem Biophys Res Commun 270, 918921.[CrossRef][ISI][Medline]
Tian X, Takamoto M and Sugane K (2003) Bisphenol A promotes IL-4 production by Th2 cells. Int Arch Allergy Immunol 132, 240247.[CrossRef][ISI][Medline]
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 81, 13021307.[CrossRef][ISI][Medline]
Submitted on September 30, 2004; resubmitted on February 22, 2005; accepted on March 3, 2005.