1 Department of Obstetrics and Gynecology, University of Freiburg School of Medicine, Freiburg, Germany and 2 Department of Obstetrics and Gynecology, University of Vienna School of Medicine, Vienna, Austria
3 To whom correspondence should be addressed at: University of Freiburg School of Medicine, Freiburg, Hugstetter Strasse 55, D-79106 Freiburg, Germany. Email: pietrowski{at}frk.ukl.uni-freiburg.de
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Abstract |
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Key words: apoptosis/genotyping/idiopathic recurrent miscarriage/p53/pregnancy
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Introduction |
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Pregnancy is dependent on adequate placental circulation. The development of a normal functioning placental vascular network requires a remarkable degree of coordination between different vascular endothelial cell-specific growth factors and cell types and is exquisitely dependent upon signals exchanged between these cells. Abnormalities of placental vasculature may result in a number of gestational pathologies including pregnancy loss, intrauterine fetal death (IUFD), intrauterine growth restriction (IUGR), placental abruption, and pre-eclampsia (Salafia et al., 1995).
The p53 tumour suppressor gene encodes a multifunctional transcription factor that is activated by stressful stimuli, including DNA damage and hypoxia. It is a well-known factor regulating apoptosis in a wide variety of cells and alterations in p53 are among the most common genetic changes in human cancers. However, beside its role as a tumour suppressor gene, p53 plays a critical role in regulating angiogenesis (Ravi et al., 2000; Yuan et al., 2002
). Alterations in the p53 gene product have been shown to be a potent inducer of angiogenesis via the Hypoxia inducible factor 1
(Hif1
) and vascular endothelial growth factor (VEGF) pathway (Dameron et al., 1994
). Recently, it was shown that p53 is a potential mediator of pregnancy by estrogen and progesterone activation (Sivaraman et al., 2001
). Dysfunction of p53 may lead to accumulation of cytoplasmic p53 which in turn may lead to immunity to abnormally expressed p53 as revealed by autoantibodies in the blood. Alterations in the p53 gene product may be caused by polymorphic sites within the gene. A common sequence polymorphism is located within the proline-rich domain of p53 encoding either proline or arginine at position 72. These variants have been reported to differ in functional activity. The Arg72 variant is believed to be a better suppressor of cellular transformation and was found to be more susceptible to degradation by the human papilloma virus (HPV) 18 E6 protein (Storey et al., 1998
; Thomas et al., 1999
). These studies imply that there is a functional difference between the codon 72 polymorphic variants and suggests that a closer analysis of the Pro72 and Arg72 variants is warranted. We hypothesized that the polymorphism in the p53 gene is associated with IRM by influencing p53-mediated function during pregnancy. We thus investigated the frequency of the p53 codon 72 variants in a Middle-European white population of women with a history of IRM and in control population of women with no history of recurrent miscarriage.
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Materials and methods |
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To avoid confounding by ethnicity, only white Caucasian women were included in the study and control groups. To avoid confounding by genetic admixture, only women whose parents were of the same ethnicity were included in the study and control groups. A total of 175 women was included in the study group.
Primary RM was defined as no history of a pregnancy carried beyond 20 weeks of gestation and at least three spontaneous miscarriages. Secondary RM was defined as a history of at least one pregnancy carried beyond 20 weeks of gestation and at least three spontaneous, consecutive miscarriages before 20 weeks of gestation with the same partner.
The control group consisted of 143 women with at least one live birth and no history of RM. Controls were recruited from the Outpatients Clinic for Post-menopausal Disorders at the University of Vienna School of Medicine. The reason for referral was evaluation of, counselling on, and therapy of peri- and post-menopausal signs and symptoms. All women in the control group were post-menopausal to rule out possible future miscarriages after inclusion in the study. Written informed consent was obtained from participating women. Local ethics committees approved the study.
Genetic studies
Blood was drawn from the antecubital vein and DNA was extracted using the Puregene System (Gentra Systems, USA). DNA were stored at 4°C until analysed. Analysis of the p53 genotype at codon 72 was performed as described by Nagpal et al. (2002) with some modifications. We used the primers GCCAGAGGCTGCTCCCCC and CGTGCAAGTCACAGACTT for amplification of the Pro codon and primers TCCCCCTTGCCGTCCCAA and CTGGTGCAGGGGCCACGC for amplification of the Arg codon.
PCR conditions comprised an initial denaturing step at 94°C for 5 min, followed by 45 cycles of 94°C for 30 s 55°C for 30 s and 72°C for 45 s and a final extension at 72°C for 5 min. Reaction products were fractionated on a 2% agarose gel and visualized after ethidium bromide staining with a raytest digital camera system (Raytest, Germany).
ELISA analysis
Enzyme-linked immunosorbent assay (ELISA) for p53 autoantibody detection was performed according to the manufacturer's instructions (Biodiagnostics, Germany).
Statistical analysis
Differences in the frequencies of the p53 alleles in the study and control groups were analysed by use of 2-test. The odds ratio (OR) was used as a measure of the strength of the association between allele frequencies and IRM. We calculated the power to detect a difference between IRM and control women for the p53 Arg
Pro polymorphism. For the calculation, we used a total of 318 women and we achieved a power of 30% to detect a 30% difference in genotype frequencies at an
of 0.05 using the Yates correction factor. All P-values are two-tailed and 95% confidence intervals (CI) were calculated. P<0.05 was considered statistically significant.
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Results |
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In a subset analysis comparing the genotype frequencies between women with primary and secondary IRM, no statistically significant difference was found. The genotype frequencies of the homozygous Pro/Pro genotypes and the heterozygous Arg/Pro genotypes were not significantly different between women with primary IRM (8.1 and 32.0% respectively) and secondary IRM (11.5 and 41% respectively) (P=0.1).
Serum levels of p53 autoantibodies were determined in a subset of 30 women with IRM and 30 controls. We did not detect p53 autoantibodies in any of these serum samples.
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Discussion |
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Our findings of an increased risk of IRM for women carrying the Pro allele is in line with the hypothesis that changes of the proline-rich region of p53 may alter the potential of the protein to regulate processes in the cell involved in apoptosis or cell cycle arrest. Fetal growth and development depend on intact placental function. Maintenance of placental structure and differentiation is essential for the provision of adequate gas, nutrient and waste exchange between the fetus and its mother. Placental trophoblast and placental reorganization is a ongoing process during pregnancy. Therefore apoptosis and cell proliferation is frequently observed during pregnancy in blood vessel cells, cytotrophoblasts and trophoblasts of the placenta. Imbalances in these highly regulated processes of tissue or cell differentiation caused by an increased number of cells arrested at the G1 checkpoint, as may occur in the p53 Pro variants compared to the p53 Arg variants, might to some extent cause inadequate supply of nutrients, gases or waste exchange between mother and fetus, leading to preterm abortion.
A second possible explanation for the observed increase of IRM for p53 Pro carriers might be their higher potential to resist apoptosis. Because placental developmental is a dynamic process of cell proliferation and cell degradation, it might be a disadvantage if cells respond with a later onset or to a lower extent in terms of necessary physiological apoptosis in the p53 Pro allele carriers compared to the Arg carriers. The lower level of apoptosis in these carriers might lead to misguided growth of cells or tissues determined to be degraded by intrinsic apoptotic stimuli. However, these possible explanations are largely speculative and a more extensive study on how the p53 Pro variant contributes to preterm abortion is required.
It has to be acknowledged that the design of our study has limitations. The selection of the control group, namely post-menopausal women with proven fertility, may cause selection bias in that these women are not representative of the general population. In addition, a number of association studies investigating polymorphisms as possible risk factors for IRM have been published by our group and others (Tempfer et al., 2001; Pietrowski et al., 2003
). Looking for associations between numerous different polymorphisms implies also detecting a significant association accidentally. However, the important impact the Arg/Pro polymorphism seems to exert on the placental and embryonic development warrants closer attention to this polymorphism in further studies and meta-analysis of comparable studies. This has to be taken into account when assessing the results of this study.
In summary, this is the first report which demonstrates an association of IRM with the p53 codon Pro72 variant in 318 Caucasian women. This study adds to the growing list of genetic factors that may increase the susceptibility to miscarriage in otherwise healthy women.
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References |
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Submitted on June 8, 2004; resubmitted on November 3, 2004; accepted on November 30, 2004.
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