Routine Tests of Haemostasis and Low-Oestrogen Oral Contraceptives: What to Expect?

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1 Clin. Lab. 2014;60: Copyright ORIGINAL ARTICLE Routine Tests of Haemostasis and Low-Oestrogen Oral Contraceptives: What to Expect? RENATA ZADRO 1 AND SANDRA OSLAKOVIC 2 1 Department of Laboratory Diagnosis, Zagreb University Hospital Centre and Faculty of Pharmacy and Biochemistry, Zagreb, Croatia 2 Department of Transfusion Medicine, Cakovec County Hospital, Cakovec, Croatia SUMMARY Background: Frequent requests from gynaecologists for testing of haemostatic parameters in women using hormonal contraception in order to predict development of thromboembolic disease, along with advancement of testing quality and accuracy, created the need to assess and compare the overall effect of oral contraceptives on today s routine haemostatic tests. Methods: This 6-month prospective study included 195 women, first time users of oral contraceptives or with at least a 3-month pause before their entry in the study. Haemostatic changes were assessed by the following tests performed at three study time-points: at baseline, after three and after six months of taking oral contraceptives: prothrombin time, activated partial thromboplastin time, fibrinogen, resistance to activated protein C ratio, protein C activity, protein S activity, FVIII activity, antithrombin activity, plasminogen activity, α2-antiplasmin activity, inhibitor of plasminogen activator type 1 (PAI-1), and D-dimers. In addition, study subjects underwent genetic testing for FV Leiden mutation, FII G20210A mutation, and PAI-1 4G/5G polymorphism. Results: Significant changes were found in all haemostatic variables except for FVIII activity. With in-depth analysis, we showed that PAI-1 concentration decreased only in subjects with PAI-1 4G/5G genotype. None of the subjects developed venous thromboembolic disease during the study. Conclusions: As most changes were within reference ranges, routine laboratory testing still does not appear to be recommendable in women without a known risk of thromboembolic disease. (Clin. Lab. 2014;60: DOI: /Clin.Lab ) KEY WORDS oral contraceptives, blood coagulation, fibrinolysis INTRODUCTION Oral contraceptives (OCs) are widely used in Croatia, not only as a birth control method but also as therapy for irregular menstrual cycles and/or skin problems. A number of OCs are currently available on the Croatian market, all with a low dose (35 g or less) of ethinyl estradiol as oestrogen component. These OCs are associated with a low risk of venous thromboembolism (VTE) due to less pronounced haemostatic changes [1]; however, the risk still exceeds that in OC non users (relative risk ) [2]. A rise in public interest in the side effects caused by oral contraceptives has imposed increased demands for laboratory testing of various haemostatic parameters. Changes in selected haemostatic variables influenced by OCs have recently been intensively investigated, showing variations associated with several variables and confirming the impact of ethnic and environmental differences [3]. The aim of this study was to assess and compare the overall effect of oral contraceptives on today s routine haemostatic tests. MATERIALS AND METHODS Study subjects The study included 227 women referred by their gynaecologists for blood testing on voluntary basis to Cakovec County Hospital in the period from June 2009 to Manuscript accepted July 19, 2013 Clin. Lab. 5/

2 RENATA ZADRO AND SANDRA OSLAKOVIC February The inclusion criterion was first time OC user or at least 3 months free from OC use without contraindications; a parental consent was required for underage subjects. Of these 227 women, 195 were included in per protocol analysis (21 women prematurely discontinued OCs, 4 women did not take any dose of oral contraceptives, 6 women did not follow the protocol correctly, and 1 woman became pregnant before starting OCs). The age range of study subjects was years (mean 23.51). 29.7% were smokers and 58.5% were first time OC users. Detailed subject characteristics are shown in Table 1. Study design This prospective study was performed at three timepoints. At baseline, before starting OCs, the subjects were asked to fill out a standardized questionnaire concerning their medical history. In addition, their body height and weight were measured, and blood samples were obtained for laboratory testing. After three and six cycles of OCs, the subjects filled out an additional questionnaire in which they specified the OC used, described the potential adverse effects of OC, and underwent body weight measurement and blood sampling. Among several types suggested by gynaecologist, study subjects selected a type of contraceptive. The haemostatic assays were performed in both baseline and repeat blood samples. Genetic testing for FV Leiden mutation, FII G20210A mutation and PAI-1 4G/5G polymorphism was only performed in baseline samples. The study protocol was approved by the Ethics Committee of Faculty of Pharmacy and Biochemistry, University of Zagreb and by the local hospital ethics committee. An informed consent in writing was obtained from all study subjects. Samples Blood samples were collected between 8:00 and 10:00 a.m. after overnight fast. Blood samples were collected three times (baseline, after three and six OC cycles) from an antecubital vein into two plastic tubes containing 1/10 volume of 3.2% sodium citrate and thoroughly mixed several times before double centrifugation (10 minutes at 2500 x g). One sample was intended for genetic testing and another one for haemostatic variables. Within one hour, 1 ml aliquots of platelet depleted plasma for further testing in series were frozen and stored in plastic tubes (Eppendorf micro tubes) at -25 C for a maximum of 4 weeks. Before testing, they were thawed at 37 C within 10 minutes. Samples for genetic testing were transported on ice to the Division of Haematology and Coagulation, Department of Laboratory Diagnostics, University Hospital Centre Zagreb, Zagreb, Croatia. Assays for haemostatic variables All tests were performed on a BCS (Behring coagulation system) automated analyzer using reagents manufactured by Siemens Healthcare Diagnostics, Germany, as follows: PT (Thromborel S, reference range s), aptt (Actin FS, reference range s), and fibrinogen (Multifibren U, reference range g/l) were determined in fresh citrated plasma; Factor VIII activity (FVIII chromogenic assay, reference range 50% - 150%), apcr (ProC AcR, reference value ratio > 1.8 ), protein C activity (Berichrom Protein C, reference range 70% - 140%), protein S activity (Protein S Ac, reference range 59% - 118%), and antithrombin activity (Berichrom ATIII reference range 75% - 125%) were determined in thawed plasma; D-dimers (D-dimer Innovance, reference value < 550 µg/l FEU) were determined routinely in fresh citrated plasma; plasminogen (Plasminogen, reference range 75% - 150%), α2-antiplasmin (Berichrom α2-antiplasmin, reference range 80% - 120%) and PAI-1 (Berichrom PAI, reference range U/mL) were determined in thawed plasma. Genetic testing Genomic DNA was extracted according to standard procedures using the salting out method [4]. FV Leiden and FII G20210A were identified after amplification by polymerase chain reaction using primers according to Zöller et al. [5] and Poort et al. [6], respectively, followed by digestion of amplified 287 bp fragments with MnlI and 345 bp fragments with Hind III, as previously reported. The PAI-1 4G/5G polymorphism was determined by real-time PCR using melting curve analysis according to Nauck et al. [7] on a LightCycler (Roche Diagnostics, Mannheim, Germany). Statistical analysis Results were statistically processed using MedCalc ver statistical software. All haemostatic variables showed normal distribution determined by Kolmogorov-Smirnov test, so repeated measures ANOVA analysis of variance test was employed in further analysis. RESULTS Comparison of baseline results and results recorded after three and six cycles of OCs showed statistically significant differences in all haemostatic variables except for FVIII activity (Table 2). In three general tests, PT and aptt showed decreased levels, while fibrinogen was increased (p < 0.001). In thrombosis tests, apc resistance and PS activity showed decreased levels, while PC activity increased (p < 0.001). The tests assessing the fibrinolytic pathway, i.e. plasminogen, α2-antiplasmin, and D-dimers, also showed increased levels (p < 0.001), but PAI-1 concentration was decreased. Comparison of results obtained after cycles 3 and 6 showed an absence of significant differences for most variables except aptt, protein S, PAI-1, and D-dimers. Only eight study subjects were diagnosed with inherited thrombophilia, four with mutation - FV Leiden, three with FII G20210A, and one double heterozygote (Table 3). None of the subjects showed congenital deficiency of 828 Clin. 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3 ROUTINE TESTS OF HEMOSTASIS AND ORAL CONTRACEPTION Table 1. Clinical and biochemical characteristics of study groups. Age (years, mean ± standard deviation, range) ± 6.16 (15-43) < (10.3%) (61.5%) (13.8%) > (14.4%) BMI (kg/m 2, mean ± standard deviation, range) ± 3.03 ( ) < (7.2%) (81.5%) (8.7%) 30 5 (2.6%) Smokers 58 (29.7%) Non-smokers 137 (70.3%) First time OC users 114 (58.5%) Previous OC users 81 (41.5%) BMI - body mass index, OC - oral contraceptive. Table 2. Values of haemostatic variables at baseline and after three and six cycles of oral contraceptives (OC) (mean ± standard deviation). p-value Variable Baseline Cycle 3 Cycle 6 Baseline vs. Cycle 3 Baseline vs. Cycle 6 Cycle 3 vs. Cycle 6 Prothrombin time - PT (s) ± ± ± 0.06 < < Activated partial thromboplastin ± ± ± 0.19 < < < time - aptt (s) Fibrinogen (g/l) 2.80 ± ± ± 0.05 < < Resistance to activated protein C - apcr (ratio) 4.24 ± ± ± 0.07 < < Protein C activity (%) ± ± ± 1.42 < < Protein S activity (%) ± ± ± 1.31 < < FVIII activity (%) ± ± ± Antithrombin activity (%) ± ± ± < Plasminogen activity (%) ± ± ± 1.28 < < Alpha2-antiplasmin activity (%) ± ± ± 0.64 < < Inhibitor of plasminogen activator type 1 - PAI ± ± ± < (U/mL) D-dimer (μg/l FEU) ± ± ± < < antithrombin, protein C and/or protein S. PAI-1 4G/5G genotype was the most frequent polymorphism, found in 51.8% of study subjects. As overall PAI-1 concentration showed significant difference according to OC use, we performed additional comparison of PAI-1 gene polymorphism and PAI-1 concentrations (Table 4), which yielded a significant difference in PAI-1 concentrations associated exclusively with PAI-1 4G/5G genotype in both measuring points (p < 0.05 and p < 0.001, respectively). There was no significant difference in PAI-1 concentrations among study subjects according to the presence of PAI-1 4G/5G polymorphism at baseline or after OC use. None of the participants developed venous thromboembolic disease during the study. Clin. Lab. 5/

4 RENATA ZADRO AND SANDRA OSLAKOVIC Table 3. Frequency of FV Leiden, FII G20210A mutation, and PAI-1 4G/5G polymorphism in study population. FV Leiden FII G20210A PAI-1 4G/5G Genetic test N (%) GG 190 (97.4%) GA 5 (2.6%) GG 191 (97.9%) GA 4 (2.1%) 5G/5G 43 (22.1%) 4G/5G 101 (51.8%) 4G/4G 51 (26.2%) N - sample size. Table 4. Comparison of PAI-1 4G/5G polymorphism and PAI-1 concentration at baseline and after three and six cycles of OCs (mean ± standard deviation). Genotype N PAI-1 (U/mL) Baseline Cycle 3 Cycle 6 Baseline vs. Cycle 3 p-value Baseline vs. Cycle 6 4G/4G ± ± ± G/5G ± ± ± < G/5G ± ± ± N - sample size, PAI-1 - inhibitor of plasminogen activator type 1. DISCUSSION Many studies have investigated the effect of oral contraceptives in different segments of haemostasis. The effect of two contraceptives and/or their oestrogen and progesterone components has been most extensively investigated. This prospective study was conducted to assess the overall effects of the most widely administered oral contraceptives with low-dose ethinyl estradiol (35 μg or less) on haemostatic variables in Croatian women. The results recorded after three and after six months of taking combined oral contraceptives indicated that all haemostasis segments (procoagulant, anticoagulant, profibrinolytic, and antifibrinolytic) were altered. The rate of changes was moderate and, although investigated parameters mostly remained within the established reference ranges, observed changes can be interpreted as a shift towards a new equilibrium of haemostasis [8-10]. When compared with other similar studies [9,11-13], results varied for PT, aptt, PAI-1, FVIII, apc resistance, and antithrombin activity, but were mostly consistent for protein S activity, protein C activity, plasminogen, α2-antiplasmin, fibrinogen, and D-dimer levels. Decreased resistance to activated protein C corresponded to previous data for women who are not carriers of FV Leiden mutation [14-16]. Although we used an aptt based test, which is considered to be less sensitive, our results showed significant difference [17]. One of the factors that influence this test is FVIII activity [15,18], which was not statistically changed in our study, suggesting that other factors of the intrinsic pathway may have greater influence [13]. The result for aptt confirmed these findings as well, showing considerably lower values. Increased FVIII activity (over 150%) is considered one of the factors that contribute to the development of thromboembolic disease [19]. However, contradictory results are reported from studies that measured FVIII activity in relation to oral contraceptives. Some showed increased FVIII activity [20,21]; however, our results are consistent with Luxembourg et al. [13] and Winkler et al. [22], reporting a non-significant change in FVIII activity. One of the reasons may be high FVIII heritability, with the gene explaining 40% of this change [23]. In addition, it should be taken into consideration that we investigated the overall effect of oral contraceptives. Alpha-2 antiplasmin or plasmin inhibitor, along with alpha-2 macroglobulin, is the main regulator of plasmin degradation. Increased alpha-2 antiplasmin activities in our study could be explained by increased plasminogen activity, but also by increased protein C activity as one 830 Clin. Lab. 5/2014

5 ROUTINE TESTS OF HEMOSTASIS AND ORAL CONTRACEPTION of its inhibitors [24]. PAI-1 level is related to PAI-1 4G/5G polymorphism and its increased concentrations are considered to be a risk factor for myocardial infarction [25]. In our study, comparison of PAI-1 levels revealed significant differences in baseline levels and the levels determined after two consequent measurements of oral contraceptives among different genotypes. Although some studies have reported more significant decreases of PAI-1 level [26, 27], we cannot confirm these findings, because further intragroup analysis showed that the only significant difference was a decrease in PAI-1 level, exclusively in subjects with PAI-1 4G/5G genotype. These results sustain the inconsistencies found in earlier studies, suggesting greater environmental impact or an additional unidentified polymorphism at the PAI-1 locus or other quantitative trait loci [28,29]. Decreased PAI-1 levels are also affected by hepatic synthesis and clearance influenced by the oestrogen component of oral contraceptives, so a reduced oestrogen impact could be expected with lowering its concentration in oral contraceptives [30,31]. Previous studies have also shown that coagulation and fibrinolytic response to the use of oral contraceptives differ among ethnic groups [3,32,33]. Lippi et al. [34] investigated 137 Italian women using third-generation oral contraceptives. Their results are consistent with ours concerning activated protein C resistance, antithrombin, protein C and protein S. In a German study including 91 women, Wiegratz et al. [8] analysed the effect of four low-dose oral contraceptives during six months and reported a significant decrease in PAI-1 levels and no change in resistance to activated protein C (except for 20 μg ethinyl estradiol + 2 mg dienogest), which is in contrast to our data. Nevertheless, data for fibrinogen, plasminogen, D-dimers, protein S, and protein C activity are consistent with our results. Some variables such as prothrombin fragment 1+2, FVII activity, and antithrombin showed diversity in results among oral contraceptives. Uchinkova et al. [35] investigated the effect of gestodene on haemostasis over 12 months in 70 Bulgarian women. They found no significant differences in PT and aptt, while α2-antiplasmin and antithrombin levels were increased in women using 75 g gestodene with 30 g of ethinyl estradiol but not with 20 g of ethinyl estradiol. A tendency to minimal haemostatic changes with the ethinyl estradiol component lower than 35 μg is well described but statistical and clinical significance has not been established [9,12]. Results for fibrinogen, plasminogen, protein C, protein S, and D-dimers correspond to our data. 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