Significant differential effects of lower doses of hormone therapy or tibolone on markers of cardiovascular disease in post-menopausal women: a randomized, double-blind, crossover study

Kwang Kon Koh1,*, Seung Hwan Han1, Mi-Seung Shin1, Jeong Yeal Ahn2, Yonghee Lee3 and Eak Kyun Shin1

1Division of Cardiology, Gil Heart Center, Gachon Medical School, 1198 Kuwol-dong, Namdong-gu, Incheon 405-760, Korea
2Department of Laboratory Medicine, Gil Heart Center, Gachon Medical School, Incheon, Korea
3Department of Statistics, Ewha Womans University, Seoul, Korea

Received 27 January 2005; revised 26 March 2005; accepted 7 April 2005; online publish-ahead-of-print 4 May 2005.

* Corresponding author. Tel: +82 32 460 3683; fax: +82 32 460 3117. E-mail address: kwangk{at}ghil.com

See page 1345 for the editorial comment on this article (doi:10.1093/eurheartj/ehi354)


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 References
 
Aims We have previously reported that lower doses of hormone therapy (L-HT) and tibolone have different effects on markers of cardiovascular disease when compared with conventional doses of HT. The objective was to compare the effects of L-HT and tibolone on lipid profile, vasodilation, and factors associated with inflammation and haemostasis.

Methods and results Forty-one women received a combination of micronized progesterone 100 mg with conjugated equine estrogen 0.3 mg vs. tibolone 2.5 mg alone daily in random order during 2 months with 2 months washout period. When compared with L-HT, tibolone significantly reduced total cholesterol (P<0.001), triglyceride (P<0.001), HDL cholesterol (P<0.001) levels, and triglyceride/HDL cholesterol ratios (P=0.004) except total cholesterol/HDL cholesterol ratios. Tibolone improved flow-mediated response to hyperaemia from baseline values (P<0.001) by a similar magnitude to L-HT. L-HT and tibolone did not increase high-sensitivity C-reactive protein relative to baseline values. L-HT reduced antithrombin III from baseline values (P=0.037), compared with tibolone showing no changes. However, there was no difference between either. In contrast, tibolone increased pro-thrombin fragment 1+2 (F1+2) from baseline values (P=0.002), compared with L-HT showing no changes. Tibolone significantly reduced plasma plasminogen activator inhibitor type 1 (PAI-1) antigen levels from baseline values (P=0.004), compared with L-HT showing no changes. The effects of L-HT and tibolone on F1+2 and PAI-1 were significantly different (P=0.045 and P=0.008, respectively).

Conclusion Both tibolone and L-HT improved flow-mediated response by a similar magnitude and did not significantly increase high-sensitivity C-reactive protein. However, tibolone significantly reduced PAI-1, but increased F1+2 more than L-HT.

Key Words: Hormone therapy • Lower doses • Tibolone • Endothelial function • Inflammation • Haemostasis


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 References
 
Prospective cohort surveys suggest that hormone therapy (HT) decreases the risk of coronary artery disease (CAD) in relatively young and healthy post-menopausal women.1,2 In contrast, two recent randomized studies, the Heart and Estrogen/progestin Replacement Study (HERS)3 and the Women's Health Initiative (WHI)4 reported that HT did not reduce the risk of cardiovascular events and further demonstrated some trends towards an increased risk of cardiovascular events. The increased risk of coronary heart disease was surprising given that LDL cholesterol levels decreased and that HDL cholesterol levels increased. The reasons may result from the effects of HT on increase in triglyceride and C-reactive protein levels and thromboembolism risk through activating coagulation pathways evidenced by decreased antithrombin III and increased prothrombin fragment 1+2 (F1+2).5,6

It has been reported that ~59% of women discontinue HT within 2 years and the use of lower doses of HT has been proposed to improve long-term compliance with HT.7 Recently, lower doses of HT demonstrated comparable effects to conventional doses of HT on menopausal symptoms,8 plasma lipoproteins, carbohydrate metabolism,9 bone mineral density,10 and endothelial function.11,12 Indeed, we reported that the effects of lower doses [conjugated equine estrogen (CEE) 0.3 mg] of HT (L-HT) on lipoproteins and flow-mediated dilation (FMD) are comparable to those of conventional doses (CEE 0.625 mg) of HT.13

Tibolone, a synthetic steroid with estrogenic, androgenic, and progestogenic properties relieves climacteric symptoms and prevents post-menopausal bone loss. Furthermore, tibolone has no effect on breast cancer incidence and does not promote endometrial hyperplasia, and rare vaginal bleeding when compared with HT.14 Tibolone has complex effects on lipids, some of which might be expected to improve vasomotor function and others that might worsen vasomotor function.14 Recently, we demonstrated that tibolone significantly improved flow-mediated brachial artery dilator response by a similar magnitude to conventional doses of HT; however, tibolone did not significantly change high-sensitivity C-reactive protein and antithrombin III.15

However, no studies have compared the effects of L-HT and tibolone. Therefore, the objective of this study was to compare the effects of L-HT and tibolone on lipid profile, vasodilation, and factors associated with inflammation and haemostasis.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 References
 
Study population and design
Forty-one post-menopausal women participated in the present study; all had plasma 17ß-estradiol levels <50 pg/mL and reported cessation of menses for at least 1 year. Eleven and 12 women had participated previously in studies assessing the effect of L-HT13 and tibolone,15 respectively. No subject had taken any cholesterol-lowering agent, estrogen therapy, antioxidant vitamin supplements, or angiotensin-converting enzyme-inhibitors during the preceding 2 months. Baseline 17ß-estradiol and lipoprotein levels are shown in Table 1. No subject had diabetes, was a smoker, or had previous angina. This study utilized a randomized, double-blind, crossover design. Forty-one women received a combination of micronized progesterone (MP) 100 mg with CEE 0.3 mg vs. tibolone 2.5 mg alone daily during 2 months with a 2-month washout period. No one was withdrawn because of side effects. We used the National Heart, Lung, and Blood Institute's definitions16 for overweight as the cutoff points, body mass index ≥25.0 and <30.0 kg/m2. We used WHO/ISH definitions17 for hypertension: systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg. Severe and moderate hypertension was excluded to avoid drug effects. Twenty and 14 women were hypertensive and overweight, respectively, and 16 women were normotensive and normal weight. The average time past menopause was 7.5 years. The study was approved by the Gil Hospital Institute Review Board, and all participants gave written, informed consent.


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Table 1 Effects of L-HT or tibolone in 41 post-menopausal women
 
Laboratory assays
Blood samples for laboratory assays and vascular studies were obtained at approximately 8:00 a.m. following an overnight fast, at each baseline and at the end of each treatment period, and immediately coded so that investigators performing laboratory assays were blinded to subject identity or study sequence. Assays for lipids, fibrinogen, antithrombin III, F1+2, and plasminogen activator inhibitor type 1 (PAI-1) antigens were measured as previously described.1821 In all patients, plasma was collected for the measurement of high-sensitivity C-reactive protein levels by commercially available kits (Immundiagnostik CRP ELISA test, Bensheim, Germany). The lower limit of detection was 0.001 mg/L. All samples from the same patient (batch samples) were measured in blinded pairs on the same ELISA kit to minimize run-to-run variability. The interassay and intra-assay coefficients of variation were <6%.

Vascular studies
Imaging studies of the right brachial artery were performed using a ATL HDI 3000 ultrasound machine equipped with a 10 MHz linear-array transducer, based on a previously published technique.19,20,22 Measurements were performed by two independent investigators blinded to the subject's identity and medication status. Measurements of maximum diameter and per cent FMD were made in 10 studies selected at random. The interobserver and intraobserver variability for repeated measurement of maximum diameter were 0.01±0.06 and 0.008±0.05 mm, respectively. The interobserver and intraobserver variability for repeated measurement of per cent FMD were 0.12±1.31 and 0.10±1.29%, respectively.

Statistical analysis
Data are expressed as mean±SEM or median (range: 25–75%). After testing data for normality, we used Student's paired t-test or Wilcoxon signed-rank test to compare values at each baseline and after each therapy and the relative changes in values in response to treatment, as reported in Table 1. We set a 2-month washout period based on our previous studies.13,15 However, to assess the possibility of a common carryover effect from the initial treatment period to the next treatment period, we compared the baseline values before the first treatment phase with those before the second treatment phase by using Student's unpaired t or Mann–Whitney rank sum test. No significant differences were found between the earlier two comparisons (Table 2). In other words, there were no detectable carryover effects. Pearson or Spearman correlation coefficient analysis was used to assess associations between measured parameters. We calculated that 40 subjects would provide 80% power for detecting difference of absolute increase, 1.5% or greater FMD of the brachial artery between baseline and tibolone, with {alpha}=0.05 and standard deviation of 3.39 based on our previous studies.15,22 The comparison of endothelium-dependent dilation among L-HT and tibolone treatment schemes was prospectively designated as the primary endpoint. All other comparisons were considered secondary endpoints. Values of P<0.05 were deemed as statistically significant.


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Table 2 Carryover effects of L-HT and tibolone
 

    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 References
 
Baseline values before L-HT or tibolone treatment period were compared: no significant differences were noted (Table 1). After 2 months of L-HT, plasma levels of 17ß-estradiol significantly increased from baseline values in contrast to no changes following tibolone therapy (Table 1).

Effects of therapies on lipids
The effects of therapies on lipids are shown in Table 1. L-HT and tibolone reduced total cholesterol levels by 2±2 and 15±2%, respectively, from baseline values; L-HT increased triglyceride levels by 46±12% and tibolone reduced triglyceride levels by 37±4% from baseline values; L-HT increased HDL cholesterol levels by 8±3% and tibolone reduced HDL cholesterol levels by 26±2% from baseline values; L-HT and tibolone reduced non-HDL cholesterol levels by 5±2 and 10±2%, respectively, from baseline values; L-HT reduced total cholesterol/HDL cholesterol ratios by 7±2% and tibolone increased total cholesterol/HDL cholesterol ratios by 20±3% from baseline values; L-HT increased triglyceride/HDL cholesterol ratios by 46±14% and tibolone reduced triglyceride/HDL cholesterol ratios by 6±8% from baseline values. As a result, when compared with L-HT, tibolone significantly reduced total cholesterol levels (P<0.001), triglyceride levels (P<0.001), HDL cholesterol levels (P<0.001), and triglyceride/HDL cholesterol ratios (P=0.004) (Figure 1) and increased total cholesterol/HDL cholesterol ratios (P<0.001). L-HT and tibolone reduced LDL cholesterol levels by 8±4 and 2±3%, respectively, from baseline values.



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Figure 1 L-HT (micronized progesterone 100 mg combined with conjugated equine estrogen 0.3 mg, CEE0.3+MP) and tibolone increased triglyceride levels by 46±12 and –37±4%, respectively, from baseline values (P=0.006 and P<0.001, respectively) and triglyceride/HDL cholesterol ratios by 46±14 and –6±8%, respectively, from baseline values (P=0.116 and P=0.004, respectively). When compared with L-HT, tibolone significantly reduced triglyceride (P<0.001) and triglyceride/HDL cholesterol ratios (P=0.004). Standard error of the mean is identified by the bars.

 
Effects of therapies on vasomotor function
Basal brachial artery diameter and forearm blood flows were similar during the two treatment periods, as were the peak brachial artery diameters and forearm blood flows during reactive hyperaemia and the per cent increase in flow during hyperaemia. L-HT and tibolone improved the per cent flow-mediated dilator response to hyperaemia by 50±8 and 51±7%, respectively, from baseline values (both P<0.001) (Figure 2) by a similar degree (P=0.892). The brachial artery dilator response to nitroglycerin between each therapy was not significantly changed from baseline measurements.



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Figure 2 L-HT (CEE0.3+MP) and tibolone improved the per cent flow-mediated dilator response to hyperaemia by 50±8 and 51±7%, respectively, from baseline values (both P<0.001) by a similar degree (P=0.892). The brachial artery dilator response to nitroglycerin between each therapy was not significantly changed from baseline measurements (P=0.118 and P=0.495, respectively). Standard error of the mean is identified by the bars.

 
Effects of therapies on high-sensitivity C-reactive protein and haemostasis
Both L-HT and tibolone did not significantly increase high-sensitivity C-reactive protein from baseline values. There were significant inverse correlations between the pre-treatment C-reactive protein levels and the degree of change in C-reactive protein after L-HT (r=–0.510, P<0.001) and after tibolone (r=–0.647, P<0.001). L-HT decreased antithrombin III by 4±2% from baseline values (P=0.037), compared with tibolone showing no changes (P=0.599). However, there was no difference between both (P=0.273). There were significant inverse correlations between the pre-treatment antithrombin III levels and the degree of changes in antithrombin III after L-HT (r=–0.559, P<0.001) and after tibolone (r=–0.372, P=0.017). In contrast, tibolone increased F1+2 from baseline values (P=0.002), compared with L-HT showing no changes (P=0.238). Tibolone reduced plasma PAI-1 antigen levels by 15±8% from baseline values (P=0.004), compared with L-HT showing no changes (P=0.228). The effects of L-HT and tibolone on F1+2 and PAI-1 were significantly different (P=0.045 and P=0.008, respectively). There were significant inverse correlations between the pre-treatment PAI-1 levels and the degree of changes in PAI-1 after L-HT (r=–0.590, P<0.001) and after tibolone (r=–0.473, P=0.002). Neither therapies significantly changed fibrinogen levels from baseline values.

There were significant correlations between body mass index and high-sensitivity C-reactive protein levels before L-HT and tibolone (r=0.328, P=0.037 and r=0.317, P=0.043, respectively). However, there were no significant correlations between the degree of change in FMD and the degree of change in lipoprotein or high-sensitivity C-reactive protein levels after L-HT (–0.124≤r≤0.195) and after tibolone (–0.080≤r≤–0.024). There were no significant correlations between pre-treatment triglyceride levels and pre-treatment PAI-1 levels. There were no significant correlations between the degree of change in antithrombin III or F1+2 and the degree of change in PAI-1 antigen levels after L-HT (–0.245≤r≤–0.200) and after tibolone (–0.190≤r≤0.056).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 References
 
In the present study, we observed that when compared with L-HT, tibolone significantly reduced total cholesterol levels, triglyceride levels, HDL cholesterol levels, and triglyceride/HDL cholesterol ratios except total cholesterol/HDL cholesterol ratios. Both L-HT and tibolone did not significantly change high-sensitivity C-reactive protein. L-HT reduced antithrombin III, compared with tibolone showing no changes. In contrast, tibolone significantly increased F1+2 from baseline values, compared with L-HT showing no changes. Tibolone significantly reduced plasma PAI-1 antigen levels from baseline values, compared with L-HT showing no changes. The effects of L-HT and tibolone on F1+2 and PAI-1 were significantly different. Tibolone improved flow-mediated brachial artery dilator response to a similar magnitude to L-HT. It may be argued that further protection by HDL may not be required and that the reduction in HDL level seen with tibolone may therefore have no adverse consequences. In support of this speculation, we observed that tibolone significantly reduced triglyceride/HDL cholesterol ratio, which is a powerful predictor of both insulin resistance and coronary heart disease risk.2325

Elevated blood triglyceride levels are an important risk factor for coronary heart disease, especially among women. The elevated triglycerides are associated with higher levels of dense LDL cholesterol.26 In this regard, oral CEE significantly increased plasma triglyceride and decreased LDL particle size, which counteracted the antioxidant effect of estrogen, in contrast to tibolone having antioxidant effect.27,28

Tibolone significantly improved flow-mediated brachial artery dilator response. Clarkson et al.29 demonstrated that tibolone neither increased nor reduced coronary artery atherosclerosis despite reductions in HDL cholesterol levels in cynomolgus monkeys. In this study, tibolone's potential for inhibiting coronary artery atherosclerosis might be underestimated because tibolone increased total cholesterol, LDL cholesterol, and triglyceride levels and decreased HDL cholesterol levels to greater extent, which cannot be observed in women. Indeed, we observed no correlations between the degree of change in FMD and the degree of change in lipoprotein levels after L-HT or tibolone. Several recent studies have demonstrated that tibolone lowers HDL cholesterol by increasing hepatic lipase activity but does not impair cholesterol efflux capacity or paraoxonase activity in post-menopausal women.30,31 In other words, anti-atherogenic activities of HDL remained unchanged despite decreased HDL cholesterol. Simoncini et al.32 demonstrated that tibolone and its estrogenic metabolites activate endothelial nitric oxide synthase through genomic and non-genomic, estrogen receptor-dependent mechanism and that tibolone inhibits leucocyte adhesion molecule expression in human endothelial cells.33 Indeed, tibolone prevented cholesterol accumulation and fatty streak formation in the aorta and the impairment of endothelium-dependent smooth muscle relaxation of the aorta, and these beneficial effects were plasma lipid-independent.34 Three months of tibolone treatment decrease the intima-media thickness of the carotid artery by 28% in 28 healthy post-menopausal women.35 In fact, there is no clinical evidence that tibolone increases coronary heart disease rates.14 Tibolone has similar effects to probucol in the aspect of antioxidant effect and HDL cholesterol lowering effect. Despite the reduction of HDL cholesterol, probucol reduced the rate of restenosis and complications after percutaneous intervention in patients with CAD.36,37

However, one study compared the effect of CEE combined with medroxyprogesterone acetate and tibolone for 3 months on FMD in healthy post-menopausal women.38 This study observed that HT improved FMD; however, tibolone did not. We discussed this issue in our previous paper15 and speculate that some differences of study design and patients' characteristics may result in different observations.

We observed that high-sensitivity C-reactive protein levels were significantly correlated with body mass index, which is consistent with another study.39 We observed that both L-HT and tibolone did not significantly increase high-sensitivity C-reactive protein levels. These effects of L-HT and tibolone may be clinically very relevant. Epidemiologic studies have consistently shown that elevated C-reactive protein is a risk factor for coronary heart disease among women.40 C-reactive protein increases the expression of tissue factor41 and decreases endothelial nitric oxide synthase expression and activity.42 Tissue factor activates the extrinsic coagulation cascade, providing a link between inflammation and thrombosis. In this regard, we recently reported that a conventional dose of HT significantly increased C-reactive protein and tissue factor activity and increased thrombosis in post-menopausal women.18 These observations have led many investigators to suggest that the disappointing results of the recent clinical trials may be because of conventional dose of HT-induced increases in high-sensitivity C-reactive protein.3,43,44 In addition, we observed that the highest pre-treatment C-reactive protein levels increased to the least extent following L-HT and tibolone. We do not know exactly how tibolone does not change high-sensitivity C-reactive protein, but it may be because of its androgenic property. In support of this speculation, Wakatsuki et al.45 observed that medroxyprogesterone acetate having androgenic effect attenuated the increase of C-reactive protein with oral CEE in women; as a consequence, its effect on C-reactive protein was not different from controls. Liver is a major source of coagulants and C-reactive protein46 and we demonstrate that oral administration of L-HT activates coagulation pathway and C-reactive protein less than conventional dose of HT.13

In this study, we observed significant different effects of L-HT and tibolone on haemoastasis. Tibolone did not reduce antithrombin III levels and significantly reduced PAI-1 antigen levels, but increased F1+2 more than L-HT. Meanwhile, L-HT did not increase F1+2 but did not reduce PAI-1 antigen levels and significantly reduced antithrombin III from baseline values, albeit with no difference when compared with tibolone. Tibolone lowered PAI activity and antigen levels and increased plasminogen and increased fibrinolytic activity.14,47 We previously observed that L-HT significantly reduced PAI-1 antigen levels.13 Taken together, both L-HT and tibolone may increase fibrinolysis potential with little changes on coagulation unlike conventional dose of HT. In support of this fact, there have been no clinical studies to report the risk of thrombosis with tibolone.14


    Conclusions
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 References
 
Our data suggest that L-HT and tibolone have significantly different effects on markers of cardiovascular disease; however, both may be considered as an alternative to conventional dose of HT in post-menopausal women. Clinical recommendations regarding the effects of L-HT and tibolone on cardiovascular outcomes, however, must await the performance of additional studies with clinical endpoints.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 References
 

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