Department of Obstetrics and Gynecology, Niigata University School of Medicine, 1-757 Asahimachi-dori, Niigata 951-8510, Japan
1 To whom correspondence should be addressed. e-mail: takumi{at}med.niigata-u.ac.jp
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Abstract |
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Key words: bone mineral density/estrogen receptor/genetic polymorphism/pregnancy/vitamin D receptor
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Introduction |
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In this study, we investigated women who underwent two successive pregnancies. We repeatedly measured their lumbar BMD after each delivery and compared its longitudinal change with that of non-pregnant control women. Our focus is whether all women with multiple pregnancies are safe from the loss of peak bone mass, and the aim of this study was to examine the associations of the VDR and ER gene polymorphisms on the bone changes of women with multiple pregnancies.
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Materials and methods |
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Height was measured at the first scan, and weights were measured at each scan of BMD. Their backgrounds between the two deliveries, including lactational information, were collected by questionnaire immediately after the next delivery. Between the deliveries, three women had formula-fed, and the others breast-fed their infants with or without formula feeding. Fourteen women became pregnant while they were still lactating.
Before enrolment in the study, written informed consent was obtained from the women, and the study was approved by the Niigata University Human Investigation Committee.
Measurement
Bone mineral content (BMC) and bone area (BA) of the lumbar spine (L2L4) were repeatedly measured by dual-energy X-ray absorptiometry (DXA) (QDR-2000; Hologic, Inc., USA) within 7 days of each delivery (range: 1.15.3 years; mean: 2.5 ± 1.2 years), and BMD was calculated as the BMC divided by the BA (BMD1 and BMD2 respectively). The average coefficients of variation (CV) of phantom measurements for BMC, BA and BMD during the study period were 1.1, 0.7 and 0.6% respectively. In addition, the CV for BMD in-vivo precision between two measurements (mean interval: 2.6 ± 1.2 years) of the control women was 0.9%. There was no scanner drift during the study period.
Genomic DNA analysis
Peripheral blood samples were obtained from each woman, and genomic DNA was extracted from peripheral blood leukocytes. DNA analyses were carried out to identify restriction fragment length polymorphisms (RFLP) using restriction endonucleases as previously reported by Morrison et al. (1992, 1994) (TaqI and ApaI) and Gross et al. (1996) (FokI) for VDR gene, and Yaich et al. (1992
) (PvuII) and Zuppan et al. (1989
) (XbaI) for ER gene followed by PCR amplification. Genotypes detected by using the enzymes TaqI, ApaI, FokI, PvuII and XbaI were defined as T, A, F, P and X (indicating the absence of the respective restriction site) or t, a, f, p and x (indicating the presence of the restriction site).
Statistical analysis
All data were expressed as the mean ± SD, and all data management and statistical analyses were performed with StatView 5.0 (Abacus Concepts, USA).
The percentage change in BMD (BMD%) was calculated by the formula: (BMD2 BMD1)/BMD1*100.
BMD% was considered significant if >1.96
2*CV (i.e. 2.5%) (Garnero et al., 1999
; Holmberg-Marttila et al., 1999
), so we interpreted
BMD% between 3% and +3% as no significant change.
Group characteristics were compared with analysis of variance (ANOVA), and BMD% were with analysis of covariance (ANCOVA), with Fishers protected least significant difference (PLSD) test. Since the frequency of the genotype tt for VDR TaqI is rare in Japanese, the Tt and tt were analysed together versus TT (Tokita et al., 1996
). In addition, TT is highly associated with aa for ApaI polymorphisms, so AA and Aa were compared together versus aa. The XX for ER XbaI was also analysed together with Xx versus xx due to its rarity in our population. The following covariates were considered for ANCOVA analysis: age at each delivery, interval between the scans, and BMD1, because our previous study showed that age was a significant determinant of the
BMD% (Matsushita et al., 2002
). In addition, the interval between the scans was included because of its wide scan range, and BMD1 to avoid regression to the mean.
If a significant difference was found in BMD%, multiple regression analysis was carried out to evaluate the contribution of the genotype. Genotypes were coded numerically as 0, 1 or 2 representing the absence of the restriction site in neither allele, one allele or both alleles respectively. Differences of P < 0.05 were considered significant.
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Results |
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All of the genotype distributions were in accordance with HardyWeinberg equilibrium, and the PvuII and XbaI ER gene polymorphisms were in strong linkage disequilibrium in the cases as well as in the control group. The genotype frequencies and characteristics for the VDR (TaqI, ApaI and FokI) and the ER (PvuII and XbaI) gene polymorphisms of the women with multiple pregnancies are shown in Tables I and II respectively. It was not significant but there was a trend that BMD% of the women with the AA/Aa genotype was higher than that of the women with the aa genotype, when adjusted for Age 2 and BMD1 (2.0 ± 3.5 versus 0.6 ± 4.5%; P = 0.056, ANCOVA) (Table I).
BMD% of the women with the XX/Xx genotype was lower than that of the women with the xx genotype after adjusting for Age 1, Age 2, BMD1 and interval between the scans (0.2 ± 3.3 versus 2.0 ± 4.2%; P = 0.030, ANCOVA) (Table II). In the control group,
BMD% of the women with the Xx genotype (n = 32; no XX genotype) was not significantly different from that of the women with the xx genotype (n = 41) (0.4 ± 2.3 versus 0.4 ± 4.3%; P = 0.33, ANOVA). There were no differences in
BMD% for TaqI, FokI and PvuII, and
BMD% in all genotype groups were considered non-significant (within 3% to 3%) (Tables I and II).
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Discussion |
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Previously, we had reported that the percentage decrease in lumbar BMD of women with multiple pregnancies had been greater for women whose age at the latter delivery were higher, and that the length of lactation, body weight change, or the interval between the scans did not affect the percentage change in lumbar BMD (Matsushita et al., 2002). In the present study, we investigated the possible relationships between VDR and ER gene polymorphisms and changes in lumbar BMD, and found that lumbar
BMD% was significantly influenced by the XbaI polymorphism of the ER gene, and furthermore, the percentage decrease in lumbar BMD was greater for women whose ER gene alleles lacked the XbaI restriction site.
The functional roles and the effects of these genetic polymorphisms on bone metabolism remain controversial. Thus the mechanisms by which the bone changes may have an association with the ER gene polymorphism (XbaI), but not with the VDR gene polymorphisms, are unclear. During PPA and lactation, postpartum women are assumed to be in an equally hypoestrogenic status (Battin et al., 1985) similar to the peri-menopausal period, when a higher rate of bone loss is observed. Mizunuma et al. (1997
) reported that Japanese late pre-menopausal women with the Xx and XX genotype had been likely to have a faster bone loss rate over 1 year than women with the xx genotype. However, the cross-sectional study of Han et al. (1999
) failed to find an association between the XbaI polymorphism of the ER gene and bone turnover in peri-menopausal Korean women. On the other hand, a recent meta-analysis of the ER gene polymorphisms and BMD showed that XX homozygotes had significantly higher BMD compared with carriers of the x haplotypes (Ioannidis et al., 2002
). In the present study, a similar trend of XX homozygotes (0.041 g/cm2 higher compared with Xx, and 0.057 g/cm2 compared with xx) was observed, although the difference was not significant. Though the mechanism remains unclear, speculation might be that our result was due to the differences in susceptibility to their estrogen status. As for VDR gene polymorphisms, there was a trend between ApaI polymorphisms and lumbar
BMD%, which did not reach statistical significance. Although the result might have been different according to their calcium and vitamin D status (Ferrari et al., 1998
), we could not include this information in this study.
A limitation of this study is that it is impossible to know when the ER XbaI gene polymorphism may interfere to make a difference in the lumbar BMD%. In the present study,
BMD% should ideally have been determined from the bone loss during lactation and PPA, the recovery after the cessation of breast-feeding and resumption of menstruation, and the substantial change during the successive pregnancy. The
BMD% for a comparison group should have been similarly measured at equivalent time-points.
Association studies between the bone change with pregnancy or its related events and genetic variations are extremely limited, and to the best of our knowledge, there are only two studies in the literature. Laskey et al. (1998) reported that the changes in BMC after 3 months of lactation were not related to the BsmI genotype of the VDR gene. Holmberg-Marttila et al. (2000
) reported that they failed to find any association between bone mineral changes during PPA or 1 year after resumption of menstruation, and the BsmI polymorphisms of the VDR gene and PvuII polymorphisms of the ER gene. Though the incompleteness of our study might be partly compensated by these studies, genetic involvement in the bone changes with pregnancy and its related events should be resolved further. On the other hand, since it could be that future larger population studies or meta-analyses may fail to replicate genetic associations with the susceptibility to human diseases (Ioannidis et al., 2001
; Lohmueller et al., 2003
), our findings should also be interpreted with much caution.
In summary, our current study demonstrated that lumbar BMD of the women was not decreased with multiple pregnancies, suggesting that multiple pregnancies are not a risk for reducing peak bone mass of women. On the other hand, the BMD% was significantly influenced by the XbaI polymorphism of the ER gene, and the percentage decrease was greater for the women with ER alleles lacking the XbaI restriction site. Further work is needed to clarify the factors which affect the changes in BMD with multiple pregnancies.
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Submitted on February 14, 2003; resubmitted on July 18, 2003; accepted on September 19, 2003.