Hum. Reprod. Advance Access originally published online on February 14, 2006
Human Reproduction 2006 21(6):1473-1476; doi:10.1093/humrep/del015
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Estrogen receptor 1 haplotype does not regulate oral contraceptive-induced changes in haemostasis and inflammation risk factors for venous and arterial thrombosis
1 Department of Thrombosis Research, University of Southern Denmark and Department of Clinical Biochemistry, Ribe County Hospital, Esbjerg, Denmark, 2 Department of Hematology, Erasmus University Medical Center, Rotterdam, 3 Department of Biomedical Research, TNO Prevention and Health, Leiden, The Netherlands, 4 Klinikum Wetzlar-Braunfels, Wetzlar, Germany, 5 Organon B.V., Oss, The Netherlands and 6 Department of Obstetrics and Gynecology, Frederiksberg Hospital, University of Copenhagen, Copenhagen, Denmark
7 To whom correspondence should be addressed at: Department of Hematology, Room Ee13.93, Erasmus MC, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: m.demaat{at}erasmusmc.nl
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Abstract |
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BACKGROUND: Administration of oral contraceptives (OCs) has profound effects on the plasma levels of haemostasis and inflammation variables, resulting in an increased thrombosis risk. Individuals show large differences in the response of these variables to OCs. Polymorphism in the estrogen receptor-1 (ER1) gene may explain part of this inter-individual response. METHODS: We investigated the relationship between variants (c.454-397T>C and c.454-351A>G polymorphisms and the combined haplotype) in the ER1 gene in relation to changes in haemostasis and inflammation variables that are known risk factors for thrombosis in 507 healthy, nonsmoking, nulliparous women receiving six cycles of monophasic OCs with 20, 30 or 50 µg/day estrogen. RESULTS: A significant relationship was observed between the ER1 haplotype and changes in tissue-type plasminogen activator activity (P = 0.006), but no clear interaction pattern between the genotypes or between the estrogen doses was seen. No relationships were observed for the other variables, neither in the haplotype nor in the single polymorphism analysis. CONCLUSION: The ER1 haplotype does not have a strong effect on the estrogen-induced changes in haemostasis and inflammation risk markers for arterial and venous thrombosis.
Key words: estrogen receptor 1/haemostasis/inflammation/oral contraceptives/polymorphism
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Introduction |
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The levels of haemostasis proteins in blood are sensitive to estrogens, and their levels are modulated by oral contraceptives (OCs) and HRT (Tans et al., 2003
; Thijs and Stehouwer, 2003). The effect of estrogens on the haemostasis proteins shows a considerable variation between individuals, resulting from genetic and environmental factors. Estrogen signalling is mediated by estrogen receptors (ERs), which are ligand-activated transcription factors composed of several domains important for hormone binding, DNA binding and activation of transcription. After binding of an estrogen molecule to the receptor in the cytosol they migrate to the nucleus where they affect transcription of genes that contain estrogen responsive motifs. These motifs are present in numerous genes, including haemostasis and inflammatory genes.
A number of genetic variants in the ER1 gene have been identified, and it has been described recently that effects of HRT on high-density lipoprotein (HDL), HDL3, sex hormone-binding globulin and E-selectin (human CD62E) depend on the different genotypes of the ER1 intervening sequence 1 (IVS1) c.454-397T>C (previously called intron A -401T/C) polymorphism (Herrington et al., 2002a; Herrington, 2003). It may be expected that a similar dependence of the ER1 polymorphism is seen for the effects of estrogen treatment on haemostasis and inflammation variables. The effect of the polymorphism may even be stronger for OC than for HRT, because the estrogen dose is higher.
In this study, we analysed the relationship between genetic variations in the ER1 gene and the effect of OC on the plasma levels of haemostasis and inflammation variables associated with venous and arterial thrombosis risk. These variables are affected by OC, and some of the OC-induced changes are dependent on the type and dose of estrogen (The Oral Contraceptive and Hemostasis Study Group, 2003). We hypothesize that ER1 gene variants also affect the response.
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Subjects and methods |
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Subjects and study design
The study was designed as a randomized, open-label, parallel group, comparative study in seven study centres in five countries (The Netherlands, Germany, Belgium, Ireland and USA) and has been described in detail previously (The Oral Contraceptive and Hemostasis Study Group, 2003). Briefly, the study was performed between October 1997 and June 1999 and was conducted in compliance with the current revision of the Declaration of Helsinki (Hong Kong, 1989), International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Harmonized Tripartite Guideline: Guideline for Good Clinical Practice, and within the current national regulations in the countries where the study was conducted.
In the original study, 707 women were treated for a period of six cycles with monophasic OCs with 20, 30 or 50 µg/day estrogen. Subjects included were healthy, nonsmoking, nulliparous women who had not used an OC for at least two complete cycles preceding the start of intake of the study drug, regular menstrual cycles (mean cycle length 
24 and 
35 days) for the two cycles preceding the start of study medication, no malignancy in the last 5 years or history of such, no undiagnosed abnormal genital bleeding in the last 6 months, no history or presence of active thrombophlebitis or (venous and/or arterial) thromboembolic disorders, no family history of thrombovascular disease in first degree relatives <55 years and no coagulation disorder, hyperlipidemia, pregnancy or other contraindications to OC use (The Oral Contraceptive and Hemostasis Study Group, 2003). In the present study, only Caucasian women in the per-protocol dataset who did not have the factor V Leiden mutation or the factor II 20210 gene variant were included, resulting in a study population of 507 women. Characteristics of the population are presented in Table I.
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Blood sampling and plasma analyses
Blood samples were collected at baseline and after six cycles of OC-treatment. Baseline samples were collected on or between cycle days 18 and 21, counted from the onset of the last menstrual bleeding. Blood samples during treatment cycle 6 were collected on or between days 18 and 21 of tablet intake. Blood sampling and plasma analyses have been described in detail elsewhere (The Oral Contraceptive and Hemostasis Study Group, 1999). Venous thrombosis risk factors that were measured include activated protein C (APC)[activated partial thromboplastin time (aPTT) based], APC [endogenous thrombin potential (ETP) based], protein C antigen, total protein S, antithrombin antigen, factor VIII (FVIII) antigen. Arterial thrombosis risk factors include von Willebrand factor ristocetin cofactor (vWF:Rco) activity, D-dimer, plasminogen activator inhibitor-type 1 (PAI-1) antigen, tissue-type plasminogen activator (tPA) activity, fibrinogen, FVII coagulant activity (FVII:C), C-reactive protein (CRP).
Genotyping
DNA was isolated from white blood cells with the use of a standard salting-out procedure, and ER1 c.454-397T>C (intron A -401T/C, PvuI; dbSNP rs#2234693) and c.454-351A>G (intron A -354A/G, XbaI; dbSNP rs#9340799) genetic variants were determined using PCR and restriction fragment length polymorphism analysis as reported by Kobayashi et al. (1996).
As quality control, all genotypes were determined from the gel by two independent technicians; results were entered in the database in duplicate, samples of known genotype were analysed in each run, and 10% of the samples (at random) was re-analysed. Our quality control program has been described in detail elsewhere (Bladbjerg et al., 2002).
Statistical analysis
Deviations of the genotype distributions in the samples from that expected for a population in Hardy–Weinberg equilibrium was analysed using a chi-square test. Allele frequencies for the individual polymorphisms and for the haplotypes were determined by gene counting; 95% confidence intervals of the allele frequencies were calculated from sample allele frequencies.
Descriptive statistics were calculated using the per-protocol dataset as defined previously (The Oral Contraceptive and Hemostasis Study Group, 2003). The relation between haplotype and genotype groups and the baseline levels of haemostasis and inflammation risk factors for venous and arterial thrombosis was analysed using analysis of variance (ANOVA). The relation of the changes in these levels during OC use was calculated using repeated measures ANOVA. When the distribution of the variables was skewed (CRP, D-dimer, FVIII activity, PAI-antigen and tPA activity), the baseline levels were logarithmically transformed before the analysis, and geometric means and coefficients of variation are given. The relative changes were always analysed using untransformed data.
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Results |
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Relationship between ER1 gene variants and baseline levels of haemostasis and inflammation variables
The distribution of the c.454-397T>C and the c.454-351A>G polymorphisms was in Hardy–Weinberg equilibrium. The frequency of the c.454-351G-allele was 0.33 (0.31–0.36) and of the c.454-397C-allele 0.46 (0.42–0.49) (Table I).
For vWF:Rco, there appeared to be a haplotype effect (P = 0.05) on the baseline plasma levels (Table II). For vWF:Rco, the highest levels were observed in carriers of the c.454-397C-allele who were homozygous for the c.454-351A allele. For the other variables there was no association between baseline levels and the ER1 haplotype (Tables II and III).
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When the relationships were studied for the polymorphisms separately, similar results were obtained (results not shown).
Relationship between ER1 gene variants and OC-induced changes in levels of haemostasis and inflammation variables
No significant differences were seen between the haplotype groups for the changes in venous thrombosis risk factors (Table III). A significant relationship between the change in tPA activity (0.006) and ER1 haplotype was seen, but no clear pattern in effects of the genotypes was observed (Table II). For vWF:Rco and FVIII there was a borderline significant difference between the haplotype groups, but since no clear genotype pattern was seen, and since a significant haplotype effect was also observed for vWF:Rco at baseline, this observation has to be handled with care (Table II). For the other arterial thrombosis risk factors there was no association between relative changes and the ER1 haplotype (Table II).
When the relationships were studied for the polymorphisms separately, similar results were obtained (results not shown). It was also studied whether the observed effects depended on the estrogen dose in the OCs, but no significant dose–effect interactions were seen (results not shown).
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Discussion |
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The results of this study show no strong effect of the ER1 IVS1 c.454-397T>C and c.454-351A/G haplotypes (or polymorphisms) on the OC-induced changes of the haemostasis and inflammation risk markers for arterial and venous thrombosis. For tPA activity there was a difference between haplotypes for the changes in levels during OC treatment. Furthermore, at baseline the ER1 haplotype was significantly associated with the levels of vWF:Rco, but no allele dose effect was observed for neither baseline levels nor for OC-induced changes.
Recently, Herrington et al., 2002a reported an association between the ER1 c.454-397T>C (-401C/T) polymorphism and the effect of HRT on levels of HDL cholesterol. Since haemostasis variables are also modulated by estrogen treatment, we expected that the ER1 c.454-397T>C polymorphism would also be a determinant of the change in levels in haemostasis variables. However, no clear association was observed, not even for APC-resistance, a variable that has been shown to be very sensitive to estrogen (Rosing et al., 1997). As described, we did find differences between haplotypes in the OC-induced changes of tPA activity, but the effect was unique for the heterozygous groups which makes it difficult to understand the underlying mechanism. There is no explanation for such a relationship, and we therefore look at this observation as a chance finding.
It has recently been reported in population-based studies that the ER1 haplotype is associated with an increased risk of myocardial infarction (MI) and ischaemic heart disease in postmenopausal women, independent of known cardiovascular risk factors (Schuit et al., 2004). In another study (Shearman et al., 2003), a substantial increase in risk of MI was seen in individuals with the common ER1 c.454-397CC genotype (-401CC). These studies support the importance of ERs in cardiovascular disease susceptibility. However, other studies did not observe this association (Koch et al., 2005; Straczek et al., 2005). Since thrombosis is one of the precipitating events of cardiovascular events, and since estrogen affects the levels of haemostasis variables, our hypothesis was that the ER1 haplotype may influence the levels of haemostatic cardiovascular risk factors, such as fibrinogen, FVII:C, PAI-1, vWF:Rco and D-dimer. We did not observe such relationships, which indicates that it is unlikely that the up-regulation of haemostasis variables plays a major role in the mechanism underlying the relationship between the ER1 haplotype and cardiovascular disease.
Our results do not exclude the possibility that other genetic variations in the ER1 gene are associated with the response of haemostasis variables to estrogen administration. The ER1 gene is a very large (291 kb) gene and many other polymorphisms have been described in the ER1 gene and its promoter region, and they give a complex haplotype structure (Herrington, 2003). Thus, a more comprehensive haplotype analysis will give a better estimation of the true effect of genetic variation in the ER1 gene on estrogen-dependent haemostasis changes. Another possibility is that other genes in the same regulatory pathway act together with the ER1 gene and that the effect is only present when a set of genetic variants in different genes is present (gene–gene interaction). We think here about the myb family of transcription factors.
To understand the relationship between a genetic variant and a possible effect, it is important to think about the functionality of the genetic variation. For the ER1 gene one may expect that the haplotype affects the function or the level of the ERs. Unfortunately, in our study we were not able to determine such an intermediate phenotype. Recently, Herrington et al. (2002b) reported that the IVS1-401C allele produces a functional binding site for the transcription factor B-myb, but the impact of this observation has not yet been established.
A limitation of our study is that we were only able to evaluate the effects of different estrogen dosage and not of the different OC regimens, because the groups are too small. This would be an interesting analysis, because different OC regimens have been reported to differently affect haemostasis variables (Schindler, 2003; Kemmeren et al., 2004).
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Conclusion |
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TopAbstractIntroductionSubjects and methodsResultsDiscussionConclusionReferences |
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The results of this study do not indicate that there is a strong effect of genetic variation in IVS1 of the ER1 gene on the estrogen-induced changes in haemostasis and inflammation risk factors for arterial and venous thrombosis.
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References |
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Submitted on October 12, 2005; resubmitted on December 28, 2005; accepted on January 8, 2006.
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