1 Department of Gynaecology and Reproductive Medicine, 2 Department of Clinical Chemistry, 3 Department of Hematology and Clinical Epidemiology, Leiden University Medical Center, P.O.Box 9600, 2300 RC Leiden and 4 Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, P.O.Box 616, 6200 MD Maastricht, The Netherlands
5 To whom correspondence should be addressed. Email: f.m.helmerhorst{at}lumc.nl
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Abstract |
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Key words: APC resistance/oral contraceptives/SHBG/venous thrombosis
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Introduction |
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Use of oral contraceptives causes changes in procoagulant, anticoagulant and fibrinolytic parameters, resulting in a net prothrombotic effect (Vandenbroucke et al., 2001). This prothrombotic effect can be measured globally by a thrombin generation-based APC resistance test (Rosing et al., 1999
). The test outcome predicts the risk of venous thrombosis, in users of oral contraceptives as well as in non-users and men (Tans et al., 2003
). Supporting the epidemiological observations, users of oral contraceptives containing desogestrel, gestodene or cyproterone acetate were found more resistant to the anticoagulant action of activated protein C (APC) by this test than users of oral contraceptives containing levonorgestrel (Rosing et al., 1999
; van Vliet et al., 2004
). We found the same for users of a new oral contraceptive containing drospirenone, for which no post-marketing data are currently available, but which safety with regard to thrombosis has been questioned (Sheldon, 2002
; van Vliet et al., 2004
).
Recently a literature study and a randomized controlled trial postulated that the effect of an oral contraceptive on sex hormone-binding globulin (SHBG) levels could be an indicator for the thrombotic risk of that oral contraceptive (Odlind et al., 2002; van Rooijen et al., 2004
). SHBG produced in the liver is a carrier protein for estrogen and testosterone. Estrogens cause a dose-related increase in SHBG, whereas progestogens induce a decrease of SHBG, the extent of which depends on both dose and type of progestogen (Anderson, 1974
; El Makhzangy et al., 1979
; van der Vange et al., 1990
; Knopp et al., 2001
). The type-related differences in progestogen-induced decrease of SHBG might be interpreted as differences in anti-estrogenic properties of progestogens. Thus, the effect of an oral contraceptive on SHBG levels can be seen as the sum of the estrogenic effect of ethinylestradiol and the anti-estrogenic effect of the progestogen resulting in the total estrogenicity of the pill (van Kammen et al., 1975
; Odlind et al., 2002
).
The literature study demonstrated a relationship between the known thrombotic risk of second-generation, third-generation and cyproterone acetate-containing oral contraceptives and the effect of the various types of oral contraceptives on SHBG (Odlind et al., 2002). In agreement with the increased risk of thrombosis, gestodene- and desogestrel-containing contraceptive pills were more estrogenic, i.e. increased SHBG more, than levonorgestrel-containing pills (Odlind et al., 2002
). Oral contraceptives containing cyproterone acetate were associated with the highest SHBG levels (Odlind et al., 2002
). A randomized controlled trial comparing SHBG levels in women using desogestrel-containing oral contraceptives and women using levonorgestrel-containing oral contraceptives confirmed the higher levels of SHBG in desogestrel-containing pill users (van Rooijen et al., 2004
). In addition, an association between SHBG levels and the resistance to APC measured with the classical activated partial thromboplastin time-based APC resistance test was reported (van Rooijen et al., 2004
).
To test the usefulness of SHBG as a marker for the thrombotic risk of an oral contraceptive, we compared the plasma levels of SHBG and the resistance to APC determined with a thrombin generation-based APC resistance test in users of oral contraceptives containing either second- or third-generation progestogens or drospirenone or cyproterone acetate. Our a priori hypotheses were: (i) the plasma levels of SHBG in women using oral contraceptives known to confer an increased risk of venous thrombosis are higher than in women using contraceptive pills with levonorgestrel; and (ii) the resistance to APC in women using oral contraceptives follows the same pattern and is associated with SHBG levels.
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Materials and methods |
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The Medical Ethics Committee of the Leiden University Medical Center, Leiden, The Netherlands approved the study. All volunteers gave written informed consent.
Risk ranking per oral contraceptive
Based on the risk of thrombosis reported in the literature, we can rank oral contraceptives by estrogen dose and progestogen type. High-dose oral contraceptives containing 50 µg ethinylestradiol have been associated with a higher risk of thrombosis than low-dose oral contraceptives containing 2030 µg ethinylestradiol (Gerstman et al., 1991
). Data from the ongoing Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis (MEGA) study indicate a 1.9-fold higher risk of thrombosis with a 10 µg increase in estrogen dose (van Hylckama Vlieg, 2003
). Concerning the progestogen component, it was shown that combined oral contraceptives containing the third-generation progestogens gestodene and desogestrel or the progestogen cyproterone acetate are more thrombogenic than oral contraceptives containing the second-generation progestogen levonorgestrel. A recent meta-analysis concluded that gestodene- and desogestrel-containing oral contraceptives increases the risk of thrombosis by a factor 1.51.7 compared to levonorgestrel-containing oral contraceptives (Kemmeren et al., 2001
). Oral contraceptives containing cyproterone acetate confer the highest thrombotic risk, 3.9-fold higher than oral contraceptives containing levonorgestrel (Vasilakis-Scaramozza and Jick, 2001
).
Laboratory methods
Blood samples were taken from the antecubital vein in the morning after an overnight abstinence from intake of food, caffeine and alcohol. Nine parts of blood were collected in one part 0.106 mol/l sodium citrate (pH 5.8). Cell-free, citrated plasma was prepared by centrifuging plasma at 2100g for 10 min at 18°C, coded and centrally stored at 80°C.
Normalized APC sensitivity ratios (nAPCsr) were determined by quantifying the effect of APC on thrombin generation (thrombin generation-based or ETP-based APC resistance test) as described before (Rosing et al., 1997). SHBG was measured with an immunometric assay (Immulite; DPC, USA). The sensitivity is 0.2 nmol/l; the variation coefficient according to the manufacturer ranges from 5.8 to 13% for the inter-assay variation from high to very low levels.
The samples were analysed in one series in random order. SHBG levels and APC resistance were measured without knowledge of the oral contraceptive used or any other of the participant's characteristics. The APC resistance test was performed in duplicate.
Statistical analysis
Mean SHBG plasma levels and mean nAPCsr in users of oral contraceptives containing norethindrone, levonorgestrel, norgestimate, gestodene, desogestrel, drospirenone or cyproterone acetate were calculated. A scatter diagram and a regression line were constructed with SHBG levels as the independent variable and nAPCsr as the dependent variable. The degree of association between SHBG levels and nAPCsr was measured by Pearson's correlation coefficient.
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Results |
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For the analyses of the first blood donation 156 women were included, of whom 39 were included in the analysis of the second blood donation. The various groups of oral contraceptive users did not differ with respect to age and body mass index (Table I).
Users of oral contraceptives with a moderately increased risk, i.e. gestodene- and desogestrel-containing pills, had SHBG plasma levels that were higher than for users of low-risk oral contraceptives containing levonorgestrel (Table II). Likewise, for higher doses of estrogen in oral contraceptives we found higher SHBG levels, i.e. women using oral contraceptives containing 30 µg ethinylestradiol rendered higher SHBG levels than women using oral contraceptives containing 20 µg ethinylestradiol. The difference in SHBG levels between third-generation pills containing gestodene and desogestrel and second-generation pills containing levonorgestrel was observed for both 20 µg and 30 µg ethinylestradiol oral contraceptives. In the group of women taking pills containing 20 µg ethinylestradiol, mean SHBG plasma levels were 111 nmol/l (95% CI 90131) for users of gestodene pills and 143 nmol/l (95% CI 110175) for users of desogestrel pills, while for users of levonorgestrel pills mean SHBG levels were 63 nmol/l (95% CI 3691). Similarly, in the group of women taking oral contraceptives containing 30 µg ethinylestradiol, mean SHBG levels were 136 nmol/l (95% CI 66205) for users of gestodene pills and 164 nmol/l (95% CI 144185) for users of desogestrel pills compared to 63 nmol/l (95% CI 5967) for users of levonorgestrel pills. For oral contraceptives with the highest risk of thrombosis, i.e. cyproterone acetate-containing pills, we found the highest SHBG levels (mean 210 nmol/l; 95% CI 182238).
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Discussion |
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In addition, SHBG plasma levels were positively associated with the resistance to the anticoagulant action of APC determined with a thrombin generation-based APC resistance test, i.e. high SHBG levels were related to a high resistance to APC. The thrombin generation-based APC resistance test that we used in this study predicts the risk of thrombosis in users of oral contraceptives as well as in non-users and men, so the relationship between SHBG plasma levels and APC resistance supports the hypothesis that SHBG is a marker for the thrombotic risk of an oral contraceptive (Tans et al., 2003).
The differences in SHBG levels and resistance to APC were not the result of differences between women rather than between type of oral contraceptive, as indicated by the results in women who switched from oral contraceptive type. Switching from a highly thrombogenic pill containing cyproterone acetate to a less thrombogenic pill containing levonorgestrel resulted in a decrease of SHBG levels as well as APC resistance.
Recently, Kemmeren et al., (2004) postulated that the different effects of third- and second-generation oral contraceptives on the anticoagulant pathway might be explained by the observation that the effect of ethinylestradiol on anticoagulant parameters is less well counteracted by desogestrel than by levonorgestrel. In their study, progestogen-only pills did not induce changes of anticoagulant parameters or induce changes opposite to those of combined oral contraceptives containing the same dose of progestogen (Kemmeren et al., 2004
). The divergent effects of estrogen and progestogens have also been observed with regard to SHBG, i.e. progestogen-only pills decrease SHBG in contrast with combined oral contraceptives or estrogen alone which increase SHBG levels (van Kammen et al., 1975
; El Makhzangy et al., 1979
; Crona et al., 1984
). In agreement with the effect on anticoagulant parameters, the lowering effect on SHBG is more pronounced in pills containing only levonorgestrel compared to pills containing only desogestrel (Crona et al., 1984
; Barkfeldt et al., 2001
; Kemmeren et al., 2004
).
In conclusion, our findings support the hypothesis that the increase of SHBG induced by a combined oral contraceptive could be interpreted as a measure of estrogenicity of a combined oral contraceptive and that estrogenicity is a factor influencing the thrombotic risk of an oral contraceptive.
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Acknowledgements |
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References |
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Submitted on August 12, 2004; accepted on October 21, 2004.
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