The cardiovascular safety of triphasic contraceptive steroids

ELSEVIER

The Cardiovascular Safety of Triphasic Contraceptive Steroids Mohamed Mamdouh

Shaarawy,*t Sheibat -

Sami Nafea,t

Omar Abdel-Aziz,t

It was hypothesized that estrogen-induced cardioprotection is mediated by up-regulation and down-regulation of expression of nitric oxide (NO) and P-selectin, respectively. Published data on circulating levels of the vasodilator NO, atherogenic glycoprotein P-selectin, and lipoprotein-a [Lp(a)] in users of triphasic contraceptive steroids are lacking. A total of 30 healthy women (nonusers, controls) and 82 women using oral triphasic contraceptive steroids (ethinyl estradiol and levonorgestrel: Triovlar, Schering AG) for 18 to 24 cycles participated in this study. Fasting blood samples were obtained from users and nonusers for the determination of P-selectin and Lp(a) by enzyme immunoassay and NO by a calorimetric method. The serum Lp(a) levels in OC users were significantly higher than those of nonusers. On the other hand, the serum NO levels in OC users were significantly elevated when compared to nonusers. Plasma P-selectin was significantly lowered in OC users p <0.005. These results demonstrate the beneficial effects of ethinyl estradiol in the triphasic contraceptive regimen. Ethinyl estradiol may afford a degree of anti-atherogenic-cardioprotective effect by up-regulation of the expression of the vasodilator NO and down-regulation of the expression of the atherogenic P-selectin. This may outweigh the cardiovascular risk of the increased atherogenic Lp(a). This study may explain the verylowrate of mortality from venous thromboembolism in OC users, which compares favorably with the risks that many people accept in daily life. CONTRACEPTION 1997$6:157-163 0 1997 Elsevier Science Inc. All rights reserved. WORDS: triphasic contraceptive steroids, nitric oxide, P-selectin, lipoprotein [a), cardiovascular safety KEY

*Endocrinology and Maternal Biochemistry Unrt, TDepartment of Obstetrics and Gynecology, Faculty of Medicrne. Cairo University, Cairo, Egypt Name and address for correspondence Prof. Mohamed Shaarawy, 21 El-Khalifa El-Maamoun Street, Apt. 701. Roxy Tower, Roxy. Heliopolrs, Cairo, Egypt Tel /Fax: +20-Z-245-3906 Submitted for publicatron December 16, 1996 Revised April 28, 1997 Accepted for publicatron May 30, 1997

0 1997 Elsevier Science Inc. All rights reserved 655 Avenue of the Americas, New York, NY 10010

Khalid Rahseed,?

and

Introduction

I

nitial concerns about the relationship between contraceptive steroids and heart problems or stroke stemmed from early studies involving women who took the high-dose formulations. The risk of cardiovascular disease among pill users is largely limited to women over age 35 who smoke.’ In 1995, preliminary research was released from a World Health Organization (WHO) study and other research that indicated that venous thromboembolism might be slightly higher for women using pills with the new progestins? Fortunately, the additional risk of mortality from venous thromboembolism is very low, estimated to be no higher than 2 to 3 deaths per million users. This compares favorably with all causes of mortality in pregnancy and with the risks that many people accept in daily life.3 Combined oral contraceptives exist in biphasic and triphasic forms, where the ratio of estrogen and progestin varies among the active pills, twice during the cycle for the biphasic and three times for the triphasic. This variation allows the pill to mimic a woman’s natural hormonal cycle more closely in the hope of reducing side effects, although research has not generally shown this to be true.4 Currently used formulations appear to be associated with fewer adverse clinical events. However, in view of increasing knowledge of the metabolic precursors of cardiovascular disease (CVD), risk factor assessment has continued to command attention in studies of steroid contraceptives. The growing recognition that CVD is a major cause of morbidity and mortality in women underscores the need for continuing evaluation of cardiovascular risk in oral contraceptive users.’ Elevated serum lipoprotein-a [Lp(a)] was positively associated with premature coronary heart disease.6 This association was independent of the known risk factors of smoking, hypertension, and diabetes mellitus as well as being independent of serum concentrations of triglycerides, LDL-cholesterol, and total cholesterol.7 Brown and Goldstein* reported that when the level of serum Lp(a) is above 30 mg/dL, the risk of ISSN 0010.7824/97/$17 PII SOOlO-7824(97)00114-5

00

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Shaarawy

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et al

coronary atherosclerosis rises about two-fold. Recently, serum Lpa has been used as a predictor for the gravity of coronary disease in myocardial infarction9 and a predictor of restenosis after percutaneous transluminal coronary angioplasty. lo Steroidal contraceptives can reduce HDL-cholester01,l1 raise triglycerides,” and elevate blood pressurel’ and these changes might contribute to the increased risk of arterial disease reported in users of these formulations. In two studies of triphasic formulations, LDL-cholesterol and apolipoprotein B concentrations were found to be increased.13f14 Low-estrogen dose, monophasic levonorgestrel-containing combinations have been reported to increase blood pressure, depending on the levonorgestrel dose,15 but triphasic formulations appear to have relatively few such effects. On the other hand, Jilma et a1.l” hypothesized that 17B-estradiol might down-regulate P-selectin expression in endothelial cells and platelets and subsequently decrease circulating P-selectin levels. Given the role of I?-selectin in atherosclerosis, such an effect may contribute to the well documented anti-atherogenic-cardioprotective effect of estradi01.‘~ Does this hold true for the synthetic estrogen ethinyl estradiol, a component of oral contraceptive steroids? Moreover, it was hypothesized that estrogen-induced cardioprotection is mediated by estrogen-induced increases in the release of nitric oxide [NO) from the vascular endothelium. The rationale for this hypothesis is that NO release is reduced in cardiovascular diseases, such as hypertension and atherosclerosis,ls and NO has several actions that are cardioprotective, such as vasodilation” and inhibition of platelet adhesion and aggregation?’ It was reported that estrogen regulates coronary blood flow, in part by up-regulating nitric oxide synthase in the coronary vasculature. ” Estrogen an d L-arginine, the precursor of NO, can partly correct impaired endotheliumdependent vasodilation, a pathophysiologic hallmark of hypertensive states. 22Genes regulating the production of two important vasodilators synthesized by the vessel wall (prostaglandin and NO) can be up-regulated by estrogens.23 Although many authors attributed cardiovascular risk in oral contraceptive users to the estrogen component of the pills, others focused on the cardioprotective effect of estrogens through up-regulation of the expression of the vasodilator NO and downregulation of the expression of the atherogenic glycoprotein I?-selectin. This promoted our interest in measuring circulating levels of NO and P-selectin in women using triphasic oral contraceptive steroids, and in measuring Lp(a) in their sera in an attempt to

investigate the discrepancy between the disturbed lipid metabolism in pill users, which is the most frequent etiologic factor for cardiovascular diseases, and the very low mortality from venous thromboembolism in pill users.

Material

and Methods

Subjects A total of 30 healthy women [controls, aged 1935 years (mean ? 1 SD, 25 + 2 years) with a body mass index of 22.6 + 2.1 kg/m21 and 82 women using triphasic oral contraceptive pills [aged 1938 years (mean & 1 SD, 26 2 5 years) with a body mass index of 21.9 + 3.0 kg/m21 were included in the study. All subjects reported regular menstrual cycles and none of them had taken drugs or suffered from conditions likely to affect lipid or carbohydrate metabolism. All women participating in this study were nonsmokers, nondiabetics, and without cardiovascular or hepatorenal disorders. Nonuser controls had taken no steroid contraceptives for at least 3 months and oral contraceptive users had used their current formulation for 18-24 cycles. The formulation used was a triphasic ethinyl estradiol/levonorgestrel formulation (Triovlar, Schering AG, Berlin, Germany). The progestogen is given in increments: 50 kg for the first 6 days, 75 Fg for the next 5 days, and 125 kg for the last 10 days. The ethinyl estradiol dose is 30 pg daily except during the middle 5 days when it is 40 pg. Nonusers [controls) were studied on days 21-27 of their menstrual cycles and oral contraceptive users were studied on days 15-21 of their pill-taking cycles. Studies were carried out in the Endocrinology and Maternal Biochemistry Unit, Department of Obstetrics and Gynecology, Cairo University Hospitals. Procedures Fasting blood samples were obtained by venipuncture through a 2 1-gauge needle into vacutainer tubes (Becton Dickinson Vacutainer System Europe, Meylan, France), one without any additives (plain) and the other containing 0.5 mL 0.129 mol trisodium citrate. All plasma samples were placed on ice for 10 min and then centrifuged. Both serum and plasma samples were centrifuged at 2000 g for 15 min at 4”C, aspirated and stored at -80°C until analysis. All samples were assayed in duplicate. Plasma concentration of P-selectin was determined with a commercially available enzyme immunoassay (EIA) kit for P-selectin (GMP140, R & D Systems, Oxford, England) following the instruction manual supplied. The detection limit of the assay was 40 ng/mL; inter- and intra-assay coefficients of variation (CV) were <9%.

Contraception 1997;56: 157-l 63

Cardiovascular

Serum total nitrite concentration, which is the index of total NO production, was determined by a calorimetric assay using a reagent kit purchased from Cayman Chemical Company (Ann Arbor, MI). The method is based on a two-step process. The first step is the conversion of nitrate to nitrite using nitrate reductase. The second step is the addition of Greiss reagent, which converts nitrite into a deep purple azo compound, where photometric measurement using a plate reader of the absorbance at 540 nm due to this azo chromophore accurately determines NO-2 concentration. The detection limit of the Cayman Chemical Nitrate/Nitrite Assay Kit was 1 kmol/L. The inter- and intra-assay coefficients of variation were 5.5% and 4%, respectively. It is acknowledged that serum nitrite levels may be altered by dietary factors. However, questioning women who participated in the study about their dietary habits did not reveal any obvious differences between the groups. Serum Lp(a) was determined with commercially available solid phase EIA (Coaliza Lpa, Chromogenix AB, Molndal, Sweden). Microplate wells coated with anti-Lp(a) monoclonal antibody were incubated with samples or Lp(a) standard solution. After incubation, unbound substances were then removed by washing wells. An enzyme-labelled anti-apo B polyclonal antibody (conjugate) bound to the antibody-antigen complex formed in the previous step was added to the wells. Unbound conjugate was then removed by washing the wells and finally the enzyme substrate was added. The absorbance of color produced in wells was read in a microplate reader at 450 nm. The assay allows detection of 2 mg/dL. The inter- and intraassay coefficients of variation were 2.9% and 4%, respectively.

Statistical

Analysis

Results are presented as mean 2 SE, with the 95% confidence intervals. Values of controls and pill users were compared using unpaired, two-tailed Student’s t

Table

1. Serum Lp(a) levels (mg/dL) in oral contraceptive

pill users

Controls No. of cases Mean ? SE 95 % confidence interval t/df

p value Percent abnormal values (>31)

56/82

2. Serum nitrate/nitrite traceptive pill users Table

No. of cases Mean ? SE 95 % confidence interval

(68.3%)

OC

159

level (Fmol/L) in oral con-

Controls

Oral Contraceptive Pills Users

30 6.97 2 0.706

82 12.57 ? 0.628

up to 14.7

t/df

1.19-23.95

5.9266/l

p value Percent abnormal values

10

<0.0005

(>14.7)

38/82

(46.3%)

test (with unequal variances). The level of significance was defined as ~0.05.

Results Serum Lp(a) levels of oral contraceptive pill users was significantly higher than that of nonusers (controls) at p <0.0005. The mean value of serum Lp(a) in pill users was 3-fold that of controls. Serum Lp(a) values ~31 mg/dL (the upper limit of the 95% confidence interval for controls) were encountered in 56 of 82 pill users representing an incidence of 68% (Table 1). Serum nitrate/nitrite level in oral contraceptive pill users was significantly increased when compared to the corresponding level of controls (p <0.0005). The mean value of serum nitrate/nitrite concentrations of pill users was 1.8 times that of controls. Abnormal values of serum nitrate/nitrite, values more than 14.7 p,mol/L, the upper limit of 95% confidence intervals of control group, were encountered in 38 of 82 pill users representing 46% (Table 2). In oral contraceptive pill users, the circulating level of P-selectin was significantly lower than that of healthy nonusers (controls) at p <0.0005. The mean percent decrease amounted to about 18%. Abnormally low values (~98 ng/mL) was encountered in 47 of 82 pill users (Table 3).

3. Plasma P-selectin level (ng/mL) in oral contraceptive pill users

Table

Oral Contraceptive Pill Users

30 82 15.1 + 1.45 45.52 -+ 1.567 up to31 17.14-73.9 14.2486/l 10 to.0005

Safety of Triphasic

Controls No. of cases Mean + SE 95% confidence interval tldf

p value Percent abnormal values (>98.06)

0

Oral Contraceptive Pills Users

118 t 1.82

82 96.8 2 1.55

98.06-137.94

68.8-124.8

8.8681/l 10 <0.0005 47/82

(57.3%)

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et al.

Discussion There is corroborating evidence that biochemical imbalances are a major factor in the emergence of cardiovascular disease (CVD) and that these abnormalities are interlinked. Contributing factors that are implicated in CVD include reduced HDL cholesterol, specifically the HDL, subfraction, elevated triglyceride-rich lipoprotein concentrations, elevated blood pressure, and high levels of the antithrombolytic factor plasminogen activator inhibitor-l .24 Lp(a) is also implicated in CVD. 25 The structural homology between human apoprotein a of Lp(a) [ape(a)] and human plasminogen has given rise to the hypothesis that the increased risk of premature atherosclerosis and thrombotic disease with elevated Lp(a) level arises from the molecular mimicry plasminogen by ape(a). By interfering with the fibrinolytic functions of plasminogen, Lp( a) could promote thrombotic events.26 Thus, Lp(a) could be the long-sought for link between thrombosis and atherosclerosis. Lp(a) represent an independent risk factor for myocardial infarction with a possible threshold value of approximately 30 mg/dL in normolipidemic subjects.” The atherogenie effect of Lp(a) is related to targeting LDL away from the regulated LDL receptor pathway and its ready permeation through endothelial cells with accumulation in arterial tissue.28 The thrombogenic effect of Lp(aJ is attributed to both competition for plasminogen binding sites on peripheral blood cells and endothelial cells and competition with plasminogen binding to fibrin.29 The growing recognition that CVD is a major cause of morbidity and mortality in women underscores the need for continuing evaluation of cardiovascular risk in oral contraceptives users.5 Reduction in estrogen dose and modification of progestin content have resulted in formulations with no adverse effect on HDL and blood pressure, but insulin resistance and hypertriglyceridemia remain. 3o In the present study, the circulating level of Lp(a) in women using triphasic oral contraceptive steroids was significantly elevated when compared with that of healthy controls. In two studies of triphasic formulations, LDL cholesterol and apolipoprotein B (the protein component of LDL) were found to be significantly increased.13,14 Oral contraceptive induced increases in blood pressure have been recognized since the 1960s. Lowestrogen, monophasic, levonorgestrel-containing combinations have been reported to increase blood pressure depending on the levonorgestrel dose, but triphasic formulations and the formulations containing the more selective progestins appear to have relatively little effect. l5 The introduction of triphasic regimens in the early 1980s provided formulations

Contraception 1997:56:157-163

with reduced progestin doses and no apparent effect on HDL.13 However, this lack of effect masked reciprocal changes in the HDL, and HDL, subfractions, so that these formulations still reduced the important HDL, subfraction.31 Over a period of 12 triphasic oral contraceptive cycles, there was an increase of LDL cholesterol of 15 % .32 The present study demonstrates an increase of atherogenic and thrombogenic Lp(a) in women using oral triphasic contraceptive steroids. There is a large, coherent, and consistent body of information showing that women who use estrogen replacement therapy have a substantially reduced risk of coronary artery disease. There has been some concern that combined estrogen-progestin therapy may not convey the same degree of cardioprotection as unopposed estrogens. 33 Several studies have shown that estrogen administration decreases serum QbJ. 34-36 Although these studies used different estrogen preparations and doses, one can not overlook the favorable changes in Lp(a) induced by oral estrogen use. On the other hand, a single previous study showed that the androgenic progestogen norethindrone decreases serum Lp(a) in menopausal women.37 However, there are no published data on the effect of levonorgestrel on serum Lp(a) levels. Before condemning the progestational component of the oral contraceptive as a possible cause of increased serum Lp(a), further studies are needed to elucidate this assumption. In the present study, the increased serum Lp(a) may indicate that patients on oral triphasic contraceptive steroids are at a higher risk of developing CVD. This is not the case because the number of mortalities from venous thromboembolism among pill users is very low, being 2 or 3 deaths per million users. This may raise the question of other possible mechanisms counteracting this metabolic high risk lipid abnormality. This spurred our interest in looking for the cardioprotective effect of estrogen through up-regulation of expression of the vasodilator NO and down-regulation of expression of the atherogenic glycoprotein P-selectin. In the present study the circulating level of NO in pill users was significantly increased when compared to the corresponding level in controls. Several studies, using acetylcholine (an endotheliumdependent dilator), support the NO hypothesis for estrogen-induced cardioprotection.38-40 Further support for the NO hypothesis is found in the fact that estradiol-induced constitutive NO synthase activity in cultured endothelial cells41 and administration of N6-nitro-L-arginine methyl ester, a competitive inhibitor of NO synthase, to oophorectomized ewes attenuated the uterine artery flow augmentation induced by estradiol. 42 Moreover Henry and Cabelloz2 reported that estrogens and L-arginine, the precursor of

Contraception 1997;56: 157-163

NO, can partly correct impaired endothelium-dependent vasodilatation, a pathophysiologic hallmark of hypertensive states. Genes regulating the production of two important vasodilators synthesized by the vessel wall (prostaglandin and NO) can be up-regulated by estrogens. 23 However, the precise mechanism by which estrogen may induce/stimulate NO release still remains unclear. Anatomical evidence for an association of estrogen with nitric oxide synthase in the rat ventromedial hypothalamic nucleus was reported,43 suggesting that nitric oxide synthase activity can be modulated by estradiol in estrogensensitive neurons. It was reported that estradiol could stimulate NO synthase activity and induce the expression of constitutive NO synthase and NO, + NO, production in cultured human aortic endothelial cells.41 Additionally, estradiol might decrease the level of NO scavengers such as superoxide anions, factors that are known to accelerate the inactivation/ breakdown of intracellular NO. Thus, the increase of the circulating level of NO in oral contraceptive pill users in our study may represent one of the mechanisms explaining the very low incidence of CVD amongst pill users in spite of the significantly elevated serum Lp(a). This concept seems reasonable because estrogen augments the contribution of NO to blood pressure regulation in transgenic hypertensive rats expressing the mouse Rem-2 gene.44 Moreover, using technetium scanning, it was found that women at an early phase of menopause have a stronger myocardial contractility than women of a similar age whose menopause is of longer duration and these effects of estrogens on hemodynamic characteristics might be controlled by vasoregulatory factors such as endothelin and N0.4” In the present study, serum P-selectin level in oral contraceptive pill users was significantly lower than that of controls at p <0.0005. Several lines of evidence suggest that the glycoprotein I?-selectin tethers leukocytes to endothelial cells and activated platelets and may play a role in atherosclerosis.46 Increased IJ-selectin expression has been shown on endothelial cells of atherosclerotic plaques.47 Although I?-selectin is almost specifically expressed on activated cell surfaces, a soluble form is found in the circulation.48 This may be explained by the release of circulating P-selectin from endothelium or platelets. Lower plasma levels of P-selectin have been observed in women during the reproductive period than in men,48 but no significant sex difference was observed when the average age was 55 years.49 These observations would be compatible with a down-regulation of circulating P-selectin by female sex hormones in premenopausal women. Jilma et a1.16hypothesized that estradiol might down-regulate P-selectin expression

Cardiovascular

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OC

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on endothelial cell or platelets and subsequently decrease circulating P-selectin levels. Given the role of P-selectin in atherosclerosis, such an effect may contribute to the well documented antiatherogeniccardioprotective effect of estradiol.” Higher than normal plasma levels of P-selectin have been found in various diseases, including peripheral vascular disease,50unstable angina pectoris,51 myocardial infarction,52 and thrombotic diseases,49 These disease states are associated with damage of endothelial cells and thrombus formation, processes that could be mediated by P-selectin. Further release of soluble P-selectin into the coronary circulation has been acetylcholine-induced observed after coronary spasm.53 In addition, soluble P-selectin seems to retain biological activity and has been shown to enhance superoxide generation by activated polymorphic neutrophils at physiologic concentrations.54 Yet, the contribution of circulating P-selectin to the pathogenesis of the above disease states remains to be determined. However, it is reasonable to assume that an estradiol-induced decrease in circulating P-selectin reflects either decreased activation or damage to platelets or endothelial cells in vivo and thus is a beneficial effect of estradiol on either endothelium or hemostasis.55 There is increasing evidence that estrogens have direct beneficial effects on the artery wa1150,5”,s7and these effects may outweigh any adverse effects of metabolic disturbance. The principle evidence for this comes from studies in grossly hypercholesterolemic, fat-fed, cynomolgus monkeys in which reduction in HDL-cholesterol by a combination of estrogen and progestin are accompanied by a reduction in arterial lesions.58 The present study adds further evidence for the beneficial effects of estrogens that counteracts or outweighs any adverse effects of disturbed lipid metabolism. Data from this study demonstrate the possible cardioprotective mechanisms by which the estrogen component of contraceptive pills oppose the cardiovascular risk of increased circulatory levels of Lp(a) through up-regulation of the vasodilator NO and down-regulation of the expression of the atherogenic glycoprotein P-selectin. Thus, the formulation of the triphasic ethinyl estradiollevonorgestrel combination may result in metabolically safe oral contraceptive steroids that could conceivably afford a degree of cardiovascular protection. Moreover, our data may explain the contradiction between the higher cardiovascular risk attributed to disturbed lipid metabolism versus the very low mortality rate from cardiovascular diseases in oral contraceptive users, which compares favorably with all causes of mortality in pregnancy and with the risks that many people accept in daily life.

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