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Effects of dydrogesterone and norethisterone, in combination with oestradiol, on lipoproteins and inflammatory markers in postmenopausal women
Maturitas 53 (2006) 439–446
Effects of dydrogesterone and norethisterone, in combination with oestradiol, on lipoproteins and inflammatory markers in postmenopausal women See Kwok a,b,∗ , Valentine Charlton-Menys b , Philip Pemberton c , Patrick McElduff d , Paul N. Durrington b a Barlow Medical Centre, 8 Barlow Moor Road, Manchester M20 6TR, UK University Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK c Clinical Research Laboratory, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK Department of Epidemiology, Stopford Building, University of Manchester, Oxford Road, Manchester M13 9P, UK b
d
Received 13 January 2005; received in revised form 8 July 2005; accepted 20 July 2005
Abstract Objective: The lack of cardiovascular benefit from postmenopausal hormone replacement therapy (HRT) in randomised controlled trials is not readily explained. The androgenic properties of progestogens could be crucial in understanding the results of these studies, all of which employed medroxyprogesterone. We have previously reported that medroxyprogesterone has some androgenic effects intermediate between those of the more androgenic norethisterone and the less androgenic desogestrel. To examine the androgenicity of progestogens further, we compared the effects of dydrogesterone (DGT) that is even less androgenic than desogestrel, and norethisterone (NET), on lipoproteins and inflammatory markers while maintaining the same oestrogen dose. Method: In a crossover trial, 25 non-hysterectomised postmenopausal women were randomised to two preparations of HRT each for three 28-day treatment cycles. Both HRT regimens comprised oestradiol (1 mg). One also included DGT (10 mg) and the other NET (1 mg). Oestradiol was taken continuously and the progestogens sequentially. Measurements were made at baseline and on the last day of the oestradiol phase and the last day of the progestogen phase in the third treatment cycle of each regimen. Results: NET was more effective than DGT in significantly reducing lipoprotein (a) (p < 0.05). NET was, however, associated with significantly lower levels of high-density lipoprotein (HDL) cholesterol (p = 0.001) and triglycerides (p < 0.05). NET was less effective in opposing the oestrogen-related increase in C-reactive protein (CRP). Interleukin-6 levels did not change with either progestogen.
∗
Corresponding author. Tel.: +44 161 445 4649; fax: +44 161 445 9560. E-mail address: [email protected] (S. Kwok).
0378-5122/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.maturitas.2005.07.006
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Conclusion: The effect of androgenic progestogens on cardiovascular risk factors may not be as deleterious as previously assumed, especially if the lower HDL levels result from more efficient reverse cholesterol transport. The hormone related rise in C-reactive protein, without a corresponding increase in interleukin-6, may not represent a systemic inflammatory response. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Androgenicity; Progestogens; HDL cholesterol; Inflammatory markers
1. Introduction The view that postmenopausal hormone replacement therapy (HRT) has a cardioprotective role has had to be revised following the publication of randomised controlled trials [1–3], which did not confirm the cardiovascular advantage anticipated from observational studies [4]. HRT is known to cause changes in lipoprotein levels such as raising high-density lipoprotein (HDL) cholesterol and lowering low-density lipoprotein (LDL) cholesterol [5], which could be potentially beneficial in the prevention of coronary heart disease (CHD). It is intriguing that these changes failed to be translated into reduced CHD risk in randomised trials. In the general population serum HDL cholesterol levels relate inversely to cardiovascular risk, and LDL cholesterol, triglycerides and lipoprotein (a) (Lp (a)) are positively related to risk [6–8]. The postmenopausal lipoprotein profile is thus proatherogenic, with lower levels of HDL cholesterol and higher levels of LDL cholesterol, triglycerides and Lp (a) [9–11]. LDL is heterogeneous and raised levels of the class of LDL with a density of 1.044–1.060 g/ml, known as smalldense LDL (SD-LDL), are strongly associated with an increased cardiovascular risk, regardless of the total level of LDL present [12]. Postmenopausal women not only have increased levels of LDL, they also have proportionately more small dense LDL particles [13], which possibly further increase their CHD risk. The inflammatory marker C-reactive protein (CRP) has been shown to be an independent predictor of CHD risk in healthy women [14]. The menopause has been associated with increasing levels of interleukin-6 (IL-6) [15], the cytokine principally regulating the hepatic synthesis of CRP. The adverse changes in cardiovascular risk factors after the menopause have been attributed to oestrogen deficiency, and the apparent reversal of some of these changes by HRT contributed to the anticipation that it would decrease cardiovascular risk.
Progestogens are combined with oestrogen in HRT preparations for non-hysterectomised postmenopausal women in order to reduce the risk of endometrial carcinoma due to unopposed oestrogen. The effects of oestrogens on lipoproteins are well documented [5,16]. The major outcome studies have all used the combination of conjugated equine oestrogens and medroxyprogesterone. It is questionable whether the results from these studies can be generalized to include all other hormone combinations since progestogens are a heterogeneous group. The biological effects of progestogens are determined to a large extent by their androgenicity. Progestogens with varying androgenicity oppose or enhance the effects of oestrogen differently, and may yet be critical in the understanding of the complex relationship of sex hormones and cardiovascular risk factors. In our previous study of three progestogens [17], we demonstrated that desogestrel was the least and norethisterone the most androgenic, while the androgenic properties of medroxyprogesterone were intermediate. In the present study we compared two progestogens of markedly contrasting androgenicity: dydrogesterone (DGT) that is even less androgenic than desogestrel; and norethisterone (NET) that is strongly androgenic. We measured lipoproteins including SD-LDL and the inflammatory markers CRP and IL-6. 2. Subjects Twenty-five healthy postmenopausal women were recruited from the Barlow Medical Centre. None of the participants had previously used HRT. Natural menopause was defined as at least 12 months of amenorrhoea with raised levels of follicle stimulating hormone. Women were excluded if they were obese (defined as body mass index (BMI) >30 kg m−2 ), if they had undergone hysterectomy, if they had any contraindications to hormone
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therapy, if they were diabetic or had any other coexisting illnesses, which might affect the study outcome measures. They were also excluded if they were on concomitant medications likely to affect outcome variables. All participants gave their informed consent and the study was approved by the Central Manchester Research Ethics Committee. Women were asked to fast from 10 p.m. the evening before they attended for each visit. At each visit, women had their height and weight measured. After resting in a sitting position for 5 min, their blood pressure was measured using an Omron 711 sphygmomanometer (Omron Healthcare Europe B.V., Hoofddorp, Netherlands). Fasting blood samples were taken between 9:00 and 11:00 a.m. and analysed in the Lipid Laboratory and the Clinical Research Laboratory at the Manchester Royal Infirmary (MRI).
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the day of their first visit. Femoston is administered as 14 days of E followed by 14 days of E combined with DGT. Elleste duet 1 mg consists of 16 days of E followed by 12 days of E combined with NET. Measurements were repeated in the third treatment cycle at the end of the oestrogen-only course and at the end of the combined oestrogen and progestogen course. This corresponded to day 70 and day 84 for women randomised to femoston and day 72 and day 84 for women randomised to elleste duet 1 mg. Women were then given their second HRT regime of 84 days. They started the second HRT regime on the day following their third visit and measurements were repeated in the third treatment cycle as before. There was no washout because this would provoke unacceptable menstrual irregularity.
4. Methods 3. Study design The study was a randomised, crossover study using two formulations of HRT commonly prescribed in general practice in the UK. The formulations both contain oestradiol (E) 1 mg daily; the two progestogens in the HRT are DGT 10 mg (femoston) and NET 1 mg (elleste duet 1 mg) daily. All the women in the study received 84 days (three 28-day treatment cycles) of each of the two preparations in random order. Fig. 1 shows the study design. Baseline measurements were taken at the first visit (day 1). Women were randomised to 84 days (3 × 28 days) to either femoston or elleste duet 1 mg, which they started on
Total cholesterol and triglycerides were measured by the CHOD/PAP and GPO/PAP methods respectively on a Cobas Mira S analyzer (ABX Diagnostics, Shefford, UK). Reagents for the assays were obtained from the same source. Serum HDL cholesterol was measured by the heparin-manganese precipitation method and LDL cholesterol was calculated using the Friedewald formula. Lp (a) was measured by an ELISA method (Mercodia, Uppsala, Sweden). Plasma was adjusted to density of 1.044 g/ml with K Br solution and ultracentrifuged at 144,000 × g for 18 h at 4 ◦ C in a Beckman L8-55M ultracentrifuge (Beckman Coulter United Kingdom, High Wycombe, UK). The tube was sliced at mid-point (Beckman Coulter tube slicer) and apo B
Fig. 1. Study design. (*) Repeated measurements were made on day 70 or 72 and 154 or 156 according to whether receiving DGT or NET.
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was measured in the infranatant using the Cobas Mira S analyzer. SD-LDL apo B results at density >1.044 g/ml were corrected for 11% Lp (a). This was shown in our earlier study to be a reliable measurement of SD-LDL [18]. C-reactive protein was measured by an in-house method using anti-human CRP antibodies; calibrators and controls were obtained from Dakocytomation (Glostrop, Denmark). Interleukin-6 was measured by an ELISA method (R&D Systems Europe Ltd., Abington, UK).
5. Statistical methods The period effect was tested by two-sample t-test comparing the differences between the two treatment periods in the two groups. The average response to the two treatments in the groups was tested by twosample t-test to check for treatment-period interaction. Statistical analysis was performed per protocol. For the comparison of baseline measurements with each of the treatment phases, paired t-tests were used when the data were normally distributed and Wilcoxon ranked sign tests were used for data with a non-Gaussian distribution. Results from the 15 women who completed all phases of the trial were analysed. A power calculation indicated that there was an 80% likelihood of finding a statistically significant (p < 0.05) difference of 0.35 mmol/l in HDL cholesterol between DGT and NET with 15 subjects.
6. Results Twenty-five women were recruited for the study, and 15 women completed it. Four women left the study due to hormonal side effects: depression, mood swings, migraine and constipation on DGT, and heavy periods on NET. Two women were confused by the medications and took the tablets in the wrong order. One woman changed her mind about HRT without giving any reasons. Two women left the study after reading newspaper reports about the Women’s Health Initiative study. One woman left due to inconvenience of visits. The average age of the women was 51 ± 5 (mean ± S.D.) years. The number of years since menopause was 2 ± 2 (mean ± S.D.). The results of the treatment phases of both HRT were compared with baseline, and the results of treatment with the two progestogens were compared (Table 1). No order effects were observed. At the end of the treatment phase of E of both HRT there was a significant reduction in LDL cholesterol and SD-LDL (both p < 0.05), and a significant increase in CRP (p < 0.05). Whilst on DGT there were significant reductions in total cholesterol, LDL cholesterol and SD-LDL (all p < 0.05); serum triglycerides were significantly increased (p < 0.05) and CRP level reverted closer to the baseline level. NET significantly lowered total cholesterol (p < 0.01), LDL cholesterol, Lp (a), SD-LDL
Table 1 Comparison of treatment phases with baseline and comparison of two progestogens BMI (kg/m2 ) Systolic BP (mmHg) Diastolic BP (mmHg) Total cholesterol (mmol/l) HDL cholesterol (mmol/l) LDL cholesterol (mmol/l) Triglycerides (mmol/l) Total Lp (a) (mg/dl) SD-LDL apo B (mg/dl) C-reactive protein (mg/l) Interleukin-6 (pg/ml)
Baseline
EDGT
E + DGT
ENET
E + NET
25 ± 5 130 ± 16 79 ± 11 5.8 ± 0.8 2.1 ± 0.8 3.1 ± 0.9 1.29 (0.98, 1.60) 11.1 (4.5, 60.7) 13.9 ± 5.3 1.14 (0.05, 4.98) 1.0 (0.9, 1.9)
26 ± 5 132 ± 25 81 ± 12 5.6 ± 0.8 2.3 ± 1.0 2.7 ± 0.9* 1.29 (0.84, 1.63) 7.8 (3.9, 51.3) 11.2 ± 5.5* 2.69 (0.69, 7.10)* 1.2 (0.7, 3.7)
26 ± 5 128 ± 25 77 ± 13 5.3 ± 0.8* 2.1 ± 0.8 2.5 ± 0.9* 1.41 (0.99, 1.99)* 7.5 (3.0, 51.5) 11.4 ± 5.7* 1.99 (0.43, 4.02) 1.6 (1.0, 3.5)
26 ± 5 126 ± 22 78 ± 12 5.3 ± 0.8* 2.0 ± 0.6 2.8 ± 0.8* 1.11 (0.83, 1.77) 6.5 (2.7, 51.6) 11.4 ± 4.4* 3.49 (0.70, 7.12)* 1.4 (0.9, 3.0)
26 ± 5 130 ± 21 79 ± 11 5.1 ± 0.7* 1.8 ± 0.6***‡ 2.8 ± 0.7* 1.31 (0.68, 1.65) † 6.6 (3.0, 42.9)* † 11.8 ± 5.4* 3.31 (0.47, 7.73)** 1.4 (1.0, 2.8)
Baseline values were obtained before commencing HRT. EDGT results were obtained on the 14th day of the third cycle of treatment before commencement of DGT and E + DGT after 14 days of combined treatment. ENET results were obtained on the 16th day of the third treatment cycle before addition of NET and E + NET after 12 days of combined regimen. Data presented as mean ± S.D. except triglycerides, Lp (a), Creactive protein and interleukin-6, which are presented as medians (interquartile range). p-value vs. baseline: * p < 0.05, ** p < 0.01, *** p < 0.001; p-value comparing two progestogens, † p < 0.05, ‡ p < 0.01.
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Fig. 2. Comparison of the effects of two hormone treatments on lipoproteins and inflammatory markers. Data presented as median (interquartile range) () E + DGT; () E + NET; (*) statistically significant.
(all p < 0.05) and HDL cholesterol (p < 0.001). CRP remained significantly raised (p < 0.01). Comparison of the two progestogens (Fig. 2) showed that treatment with NET resulted in significantly lower levels of HDL cholesterol (p = 0.001), triglycerides (p < 0.05) and Lp (a) (p < 0.05) than DGT.
7. Discussion The crossover design of the present study allowed the comparison of the two progestogens using the women as their own controls. No carryover effect was demonstrated. This was consistent with other crossover studies [17,19]. The two treatments were given without a washout period as this would cause irregular bleeding which would be distressing to the women, and had been shown to make no difference to the results in studies with similar designs [17,20]. A comparison of the two progestogens in the present study showed that the more androgenic NET resulted in significantly lower HDL cholesterol levels. Androgenic progestogens up-regulate hepatic scavenger receptor B1 (SRB1) and increase hepatic lipase activity [21], the resulting reduced HDL levels are perhaps indicative of more effective reverse cholesterol transport. On the other hand, a progestogen with
no androgenic properties such as DGT has minimal effect on hepatic lipase activity and HDL level is thus unaffected. A low HDL level is frequently regarded as cardiovascular risk [9], but the import of low HDL level depends on the mechanism by which it is caused; and sex hormone induced changes in HDL levels are for the reasons discussed above difficult to interpret. DGT was associated with an increase in triglyceride levels whereas triglyceride levels with NET were similar to pre-treatment values. This is consistent with other studies [17,22,23]. Oestrogen is known to cause a rise in triglycerides [17,24], and androgenic progestogens are more successful in reversing this potentially unfavourable oestrogenic effect [17]. Triglycerides have been found to be a cardiovascular risk factor in women and diabetic subjects [7]. Progestogens that are more androgenic have been reported to increase hepatic lipase activity [21] thus reducing the pool size of triglyceride-rich lipoproteins. This may be the explanation for the effects observed in the present study. Some studies of exogenous oestrogens administered to postmenopausal women have shown an increase in small LDL particles [25,26], although others have not reported similar changes [27,28]. Progestogen effects on SD-LDL have been little investigated. LDL cholesterol and SD-LDL levels in the present study decreased with oestradiol and remained significantly lower with
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the addition of both progestogens. Oestrogen causes reduced plasma residence time of both VLDL and LDL particles [29]. Oral oestrogen also up-regulates LDL receptors [30]. The clearance rates of both VLDL and LDL thus increase and the formation of SD-LDL particles may be discouraged. Progestogens did not oppose the oestrogen effect on SD-LDL in the present study, and the results with either progestogen were similar. HRT is one of the few means of decreasing Lp (a) concentrations [31]. Lp (a) has been shown to be associated with cardiovascular risk [8]. In the Heart and Estrogen/progestin Replacement Study, women with elevated baseline levels of Lp (a) who were randomised to HRT had greater reductions of Lp (a) levels and lower risk of CHD events compared to the placebo group [32]. In this present study the more androgenic NET was associated with significant Lp (a) reduction. Oral oestradiol in the present study caused an increase in CRP that was consistent with other studies [33]. Any such effect in our study was opposed by DGT, but NET was less effective in this respect. Similar results were obtained in other studies [34,35]. The increase in CRP resulting from oral oestrogen is considered to be a hepatic first pass effect, which is not seen when transdermal oestrogen is administered [36]. Some of the metabolites of NET have been found to have oestrogenic properties [37] that could explain the high CRP level when this agent is combined with oestrogen. In our previous study comparing progestogens of varying androgenicity [17], we showed that although all the androgenic progestogens attenuated the oestrogen-induced rise in CRP, CRP levels remained significantly increased. In the present study, DGT, which binds almost exclusively to progesterone receptor, is able to successfully oppose the oestrogen effect on CRP. Hormone related CRP increase is undoubtedly an oestrogenic effect. The progestogenic influence on oestrogen-induced CRP rise is likely to be a progesterone/anti-oestrogenic effect, and appears not to be affected by the androgenic properties of progestogens. Neither oestrogen nor progestogens in the present study affected the levels of interleukin-6. This is consistent with the findings of the Women’s Health Initiative study [33]. It suggests that the raised CRP levels in hormone users may not represent a systemic inflammatory response but is rather a direct hepatic effect of oral oestrogen. Its significance is thus uncertain.
Women and their physicians have been troubled by the negative results of randomised studies on HRT, and HRT is no longer recommended to women with established CHD [1]. However, HRT remains an extremely effective treatment for menopausal symptoms and there are women who will continue to benefit symptomatically from taking it. Thus it is essential to expand our knowledge on HRT and cardiovascular risk factors. Although oestrogen has favourable effects on lowering LDL cholesterol and SD-LDL levels, it also causes potentially unfavourable increases in triglycerides and CRP. Whilst raised triglyceride levels have been associated with increased CHD risk [7], CRP has not been shown to modify the effects of HRT on cardiovascular risk [38]. The present study showed that the more androgenic NET maintained the reductions in LDL and SD-LDL, and moreover was more successful in lowering Lp (a) and opposing triglyceride increase. NET, however, did result in a significantly lower HDL cholesterol level. Nevertheless, there is no evidence that the reduced HDL levels caused by progestogens are directly associated with increased cardiovascular risk. If the lower HDL levels are indicative of a speedier reverse cholesterol transport, it might not ultimately be a deleterious effect. The CRP increase appears to be an oestrogenic effect and androgenicity has little impact on it. The significance of CRP in HRT users requires further elucidation. The use of the more androgenic progestogens may not be as harmful as previously assumed. The choice of progestogens in HRT regimens should take into account their varying androgenic properties. Acknowledgements We thank Ms. C. Price for helping to prepare this manuscript. We are grateful to the patients at the Barlow Medical Centre who participated in this study. S.K. received a research fellowship from the United Kingdom Department of Health. References [1] Hulley S, Grady D, Bush T, et al. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. JAMA 1998;280:605–13.
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[19] Koh KK, Blum A, Hathaway L, et al. Vascular effects of estrogen and Vitamin E therapies in postmenopausal women. Circulation 1999;100:1851–7. [20] Koh KK, Ahn JY, Jin DK, et al. Effects of continuous combined hormone replacement therapy on inflammation in hypertensive and/or overweight postmenopausal women. Arterioscler Thromb Vasc Biol 2001;22:1459–64. [21] Jansen H, Verhoeven AJM, Sijbrands JG. Hepatic lipase: a pro- or anti-atherogenic protein? J Lipid Res 2002;43:1352– 62. [22] Manassiev NA, Godsland IF, Crook D, Proudler AJ, Whitehead MI, Stevenson JC. Effect of postmenopausal oestradiol and dydrogesterone therapy on lipoproteins and insulin sensitivity, secretion and elimination in hysterectomised women. Maturitas 2002;42:233–42. [23] Pornel B, Chevallier O, Netelenbos JC. Oral 17ß-estradiol (1 mg) continuously combined with dydrogesterone improves the serum lipid profile of postmenopausal women. Menopause 2002;9:171–8. [24] Barrett-Connor E, Wingard DL, Criqui MH. Postmenopausal estrogen use and heart disease risk factors in the. JAMA 1989;261:2095–100. [25] Wakatsuki A, Ikenoue N, Sagara Y. Effect of estrogen on the size of low-density lipoprotein particles in postmenopausal women. Obstet Gynecol 1997;90:22–5. [26] Wakatsuki A, Ikenoue N, Sagara Y. Estrogen-induced small low-density lipoprotein particles in postmenopausal women. Obstet Gynecol 1998;91:234–40. [27] Campos H, Sacks FM, Walsh BW, Schiff I, O’Hanesian MA, Krauss RM. Differential effects of estrogen on low-density lipoprotein subclasses in healthy postmenopausal women. Metabolism 1993;42(9):1153–8. [28] Farish E, Spowart K, Barnes JF, et al. Effects of postmenopausal hormone replacement therapy on lipoproteins including lipoprotein (a) and LDL subfractions. Atherosclerosis 1996;126:77–84. [29] Campos H, Walsh BW, Judge H, Sacks FM. Effect of estrogen on very low density lipoprotein and low density lipoprotein subclass metabolism in postmenopausal women. J Clin Endocrinol Metab 1997;82:3955–63. [30] Parini P, Angelin B, Rudling M. Importance of estrogen receptors in hepatic LDL receptor regulation. Arterioscler Thromb Vasc Biol 1997;17:1800–5. [31] Farish E, Rolton HA, Barnes JF, Hart DM. Lipoprotein (a) concentrations in postmenopausal women taking norethisterone. Br Med J 1991;303:694. [32] Shlipak MG, Simon JA, Vittinghoff E, et al. Estrogen and progestin, Lipoprotein (a), and the risk of recurrent coronary heart disease events after menopause. JAMA 2000;283:1845– 52. [33] Pradhan AD, Manson JE, Roussouw JE, et al. Inflammatory biomarkers, hormone replacement therapy, and incident coronary heart disease. JAMA 2002;288:980–7. [34] Marchien van Baal W, Kenemans P, Emeis JJ, et al. Long-term effects of combined hormone replacement therapy on markers of endothelial functionand inflammatory activity in healthy postmenopausal women. Fert Steril 1999;71:663–70.
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Effects of dydrogesterone and norethisterone, in combination with oestradiol, on lipoproteins and inflammatory markers in postmenopausal women See Kwok a,b,∗ , Valentine Charlton-Menys b , Philip Pemberton c , Patrick McElduff d , Paul N. Durrington b a Barlow Medical Centre, 8 Barlow Moor Road, Manchester M20 6TR, UK University Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK c Clinical Research Laboratory, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK Department of Epidemiology, Stopford Building, University of Manchester, Oxford Road, Manchester M13 9P, UK b
d
Received 13 January 2005; received in revised form 8 July 2005; accepted 20 July 2005
Abstract Objective: The lack of cardiovascular benefit from postmenopausal hormone replacement therapy (HRT) in randomised controlled trials is not readily explained. The androgenic properties of progestogens could be crucial in understanding the results of these studies, all of which employed medroxyprogesterone. We have previously reported that medroxyprogesterone has some androgenic effects intermediate between those of the more androgenic norethisterone and the less androgenic desogestrel. To examine the androgenicity of progestogens further, we compared the effects of dydrogesterone (DGT) that is even less androgenic than desogestrel, and norethisterone (NET), on lipoproteins and inflammatory markers while maintaining the same oestrogen dose. Method: In a crossover trial, 25 non-hysterectomised postmenopausal women were randomised to two preparations of HRT each for three 28-day treatment cycles. Both HRT regimens comprised oestradiol (1 mg). One also included DGT (10 mg) and the other NET (1 mg). Oestradiol was taken continuously and the progestogens sequentially. Measurements were made at baseline and on the last day of the oestradiol phase and the last day of the progestogen phase in the third treatment cycle of each regimen. Results: NET was more effective than DGT in significantly reducing lipoprotein (a) (p < 0.05). NET was, however, associated with significantly lower levels of high-density lipoprotein (HDL) cholesterol (p = 0.001) and triglycerides (p < 0.05). NET was less effective in opposing the oestrogen-related increase in C-reactive protein (CRP). Interleukin-6 levels did not change with either progestogen.
∗
Corresponding author. Tel.: +44 161 445 4649; fax: +44 161 445 9560. E-mail address: [email protected] (S. Kwok).
0378-5122/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.maturitas.2005.07.006
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Conclusion: The effect of androgenic progestogens on cardiovascular risk factors may not be as deleterious as previously assumed, especially if the lower HDL levels result from more efficient reverse cholesterol transport. The hormone related rise in C-reactive protein, without a corresponding increase in interleukin-6, may not represent a systemic inflammatory response. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Androgenicity; Progestogens; HDL cholesterol; Inflammatory markers
1. Introduction The view that postmenopausal hormone replacement therapy (HRT) has a cardioprotective role has had to be revised following the publication of randomised controlled trials [1–3], which did not confirm the cardiovascular advantage anticipated from observational studies [4]. HRT is known to cause changes in lipoprotein levels such as raising high-density lipoprotein (HDL) cholesterol and lowering low-density lipoprotein (LDL) cholesterol [5], which could be potentially beneficial in the prevention of coronary heart disease (CHD). It is intriguing that these changes failed to be translated into reduced CHD risk in randomised trials. In the general population serum HDL cholesterol levels relate inversely to cardiovascular risk, and LDL cholesterol, triglycerides and lipoprotein (a) (Lp (a)) are positively related to risk [6–8]. The postmenopausal lipoprotein profile is thus proatherogenic, with lower levels of HDL cholesterol and higher levels of LDL cholesterol, triglycerides and Lp (a) [9–11]. LDL is heterogeneous and raised levels of the class of LDL with a density of 1.044–1.060 g/ml, known as smalldense LDL (SD-LDL), are strongly associated with an increased cardiovascular risk, regardless of the total level of LDL present [12]. Postmenopausal women not only have increased levels of LDL, they also have proportionately more small dense LDL particles [13], which possibly further increase their CHD risk. The inflammatory marker C-reactive protein (CRP) has been shown to be an independent predictor of CHD risk in healthy women [14]. The menopause has been associated with increasing levels of interleukin-6 (IL-6) [15], the cytokine principally regulating the hepatic synthesis of CRP. The adverse changes in cardiovascular risk factors after the menopause have been attributed to oestrogen deficiency, and the apparent reversal of some of these changes by HRT contributed to the anticipation that it would decrease cardiovascular risk.
Progestogens are combined with oestrogen in HRT preparations for non-hysterectomised postmenopausal women in order to reduce the risk of endometrial carcinoma due to unopposed oestrogen. The effects of oestrogens on lipoproteins are well documented [5,16]. The major outcome studies have all used the combination of conjugated equine oestrogens and medroxyprogesterone. It is questionable whether the results from these studies can be generalized to include all other hormone combinations since progestogens are a heterogeneous group. The biological effects of progestogens are determined to a large extent by their androgenicity. Progestogens with varying androgenicity oppose or enhance the effects of oestrogen differently, and may yet be critical in the understanding of the complex relationship of sex hormones and cardiovascular risk factors. In our previous study of three progestogens [17], we demonstrated that desogestrel was the least and norethisterone the most androgenic, while the androgenic properties of medroxyprogesterone were intermediate. In the present study we compared two progestogens of markedly contrasting androgenicity: dydrogesterone (DGT) that is even less androgenic than desogestrel; and norethisterone (NET) that is strongly androgenic. We measured lipoproteins including SD-LDL and the inflammatory markers CRP and IL-6. 2. Subjects Twenty-five healthy postmenopausal women were recruited from the Barlow Medical Centre. None of the participants had previously used HRT. Natural menopause was defined as at least 12 months of amenorrhoea with raised levels of follicle stimulating hormone. Women were excluded if they were obese (defined as body mass index (BMI) >30 kg m−2 ), if they had undergone hysterectomy, if they had any contraindications to hormone
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therapy, if they were diabetic or had any other coexisting illnesses, which might affect the study outcome measures. They were also excluded if they were on concomitant medications likely to affect outcome variables. All participants gave their informed consent and the study was approved by the Central Manchester Research Ethics Committee. Women were asked to fast from 10 p.m. the evening before they attended for each visit. At each visit, women had their height and weight measured. After resting in a sitting position for 5 min, their blood pressure was measured using an Omron 711 sphygmomanometer (Omron Healthcare Europe B.V., Hoofddorp, Netherlands). Fasting blood samples were taken between 9:00 and 11:00 a.m. and analysed in the Lipid Laboratory and the Clinical Research Laboratory at the Manchester Royal Infirmary (MRI).
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the day of their first visit. Femoston is administered as 14 days of E followed by 14 days of E combined with DGT. Elleste duet 1 mg consists of 16 days of E followed by 12 days of E combined with NET. Measurements were repeated in the third treatment cycle at the end of the oestrogen-only course and at the end of the combined oestrogen and progestogen course. This corresponded to day 70 and day 84 for women randomised to femoston and day 72 and day 84 for women randomised to elleste duet 1 mg. Women were then given their second HRT regime of 84 days. They started the second HRT regime on the day following their third visit and measurements were repeated in the third treatment cycle as before. There was no washout because this would provoke unacceptable menstrual irregularity.
4. Methods 3. Study design The study was a randomised, crossover study using two formulations of HRT commonly prescribed in general practice in the UK. The formulations both contain oestradiol (E) 1 mg daily; the two progestogens in the HRT are DGT 10 mg (femoston) and NET 1 mg (elleste duet 1 mg) daily. All the women in the study received 84 days (three 28-day treatment cycles) of each of the two preparations in random order. Fig. 1 shows the study design. Baseline measurements were taken at the first visit (day 1). Women were randomised to 84 days (3 × 28 days) to either femoston or elleste duet 1 mg, which they started on
Total cholesterol and triglycerides were measured by the CHOD/PAP and GPO/PAP methods respectively on a Cobas Mira S analyzer (ABX Diagnostics, Shefford, UK). Reagents for the assays were obtained from the same source. Serum HDL cholesterol was measured by the heparin-manganese precipitation method and LDL cholesterol was calculated using the Friedewald formula. Lp (a) was measured by an ELISA method (Mercodia, Uppsala, Sweden). Plasma was adjusted to density of 1.044 g/ml with K Br solution and ultracentrifuged at 144,000 × g for 18 h at 4 ◦ C in a Beckman L8-55M ultracentrifuge (Beckman Coulter United Kingdom, High Wycombe, UK). The tube was sliced at mid-point (Beckman Coulter tube slicer) and apo B
Fig. 1. Study design. (*) Repeated measurements were made on day 70 or 72 and 154 or 156 according to whether receiving DGT or NET.
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was measured in the infranatant using the Cobas Mira S analyzer. SD-LDL apo B results at density >1.044 g/ml were corrected for 11% Lp (a). This was shown in our earlier study to be a reliable measurement of SD-LDL [18]. C-reactive protein was measured by an in-house method using anti-human CRP antibodies; calibrators and controls were obtained from Dakocytomation (Glostrop, Denmark). Interleukin-6 was measured by an ELISA method (R&D Systems Europe Ltd., Abington, UK).
5. Statistical methods The period effect was tested by two-sample t-test comparing the differences between the two treatment periods in the two groups. The average response to the two treatments in the groups was tested by twosample t-test to check for treatment-period interaction. Statistical analysis was performed per protocol. For the comparison of baseline measurements with each of the treatment phases, paired t-tests were used when the data were normally distributed and Wilcoxon ranked sign tests were used for data with a non-Gaussian distribution. Results from the 15 women who completed all phases of the trial were analysed. A power calculation indicated that there was an 80% likelihood of finding a statistically significant (p < 0.05) difference of 0.35 mmol/l in HDL cholesterol between DGT and NET with 15 subjects.
6. Results Twenty-five women were recruited for the study, and 15 women completed it. Four women left the study due to hormonal side effects: depression, mood swings, migraine and constipation on DGT, and heavy periods on NET. Two women were confused by the medications and took the tablets in the wrong order. One woman changed her mind about HRT without giving any reasons. Two women left the study after reading newspaper reports about the Women’s Health Initiative study. One woman left due to inconvenience of visits. The average age of the women was 51 ± 5 (mean ± S.D.) years. The number of years since menopause was 2 ± 2 (mean ± S.D.). The results of the treatment phases of both HRT were compared with baseline, and the results of treatment with the two progestogens were compared (Table 1). No order effects were observed. At the end of the treatment phase of E of both HRT there was a significant reduction in LDL cholesterol and SD-LDL (both p < 0.05), and a significant increase in CRP (p < 0.05). Whilst on DGT there were significant reductions in total cholesterol, LDL cholesterol and SD-LDL (all p < 0.05); serum triglycerides were significantly increased (p < 0.05) and CRP level reverted closer to the baseline level. NET significantly lowered total cholesterol (p < 0.01), LDL cholesterol, Lp (a), SD-LDL
Table 1 Comparison of treatment phases with baseline and comparison of two progestogens BMI (kg/m2 ) Systolic BP (mmHg) Diastolic BP (mmHg) Total cholesterol (mmol/l) HDL cholesterol (mmol/l) LDL cholesterol (mmol/l) Triglycerides (mmol/l) Total Lp (a) (mg/dl) SD-LDL apo B (mg/dl) C-reactive protein (mg/l) Interleukin-6 (pg/ml)
Baseline
EDGT
E + DGT
ENET
E + NET
25 ± 5 130 ± 16 79 ± 11 5.8 ± 0.8 2.1 ± 0.8 3.1 ± 0.9 1.29 (0.98, 1.60) 11.1 (4.5, 60.7) 13.9 ± 5.3 1.14 (0.05, 4.98) 1.0 (0.9, 1.9)
26 ± 5 132 ± 25 81 ± 12 5.6 ± 0.8 2.3 ± 1.0 2.7 ± 0.9* 1.29 (0.84, 1.63) 7.8 (3.9, 51.3) 11.2 ± 5.5* 2.69 (0.69, 7.10)* 1.2 (0.7, 3.7)
26 ± 5 128 ± 25 77 ± 13 5.3 ± 0.8* 2.1 ± 0.8 2.5 ± 0.9* 1.41 (0.99, 1.99)* 7.5 (3.0, 51.5) 11.4 ± 5.7* 1.99 (0.43, 4.02) 1.6 (1.0, 3.5)
26 ± 5 126 ± 22 78 ± 12 5.3 ± 0.8* 2.0 ± 0.6 2.8 ± 0.8* 1.11 (0.83, 1.77) 6.5 (2.7, 51.6) 11.4 ± 4.4* 3.49 (0.70, 7.12)* 1.4 (0.9, 3.0)
26 ± 5 130 ± 21 79 ± 11 5.1 ± 0.7* 1.8 ± 0.6***‡ 2.8 ± 0.7* 1.31 (0.68, 1.65) † 6.6 (3.0, 42.9)* † 11.8 ± 5.4* 3.31 (0.47, 7.73)** 1.4 (1.0, 2.8)
Baseline values were obtained before commencing HRT. EDGT results were obtained on the 14th day of the third cycle of treatment before commencement of DGT and E + DGT after 14 days of combined treatment. ENET results were obtained on the 16th day of the third treatment cycle before addition of NET and E + NET after 12 days of combined regimen. Data presented as mean ± S.D. except triglycerides, Lp (a), Creactive protein and interleukin-6, which are presented as medians (interquartile range). p-value vs. baseline: * p < 0.05, ** p < 0.01, *** p < 0.001; p-value comparing two progestogens, † p < 0.05, ‡ p < 0.01.
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Fig. 2. Comparison of the effects of two hormone treatments on lipoproteins and inflammatory markers. Data presented as median (interquartile range) () E + DGT; () E + NET; (*) statistically significant.
(all p < 0.05) and HDL cholesterol (p < 0.001). CRP remained significantly raised (p < 0.01). Comparison of the two progestogens (Fig. 2) showed that treatment with NET resulted in significantly lower levels of HDL cholesterol (p = 0.001), triglycerides (p < 0.05) and Lp (a) (p < 0.05) than DGT.
7. Discussion The crossover design of the present study allowed the comparison of the two progestogens using the women as their own controls. No carryover effect was demonstrated. This was consistent with other crossover studies [17,19]. The two treatments were given without a washout period as this would cause irregular bleeding which would be distressing to the women, and had been shown to make no difference to the results in studies with similar designs [17,20]. A comparison of the two progestogens in the present study showed that the more androgenic NET resulted in significantly lower HDL cholesterol levels. Androgenic progestogens up-regulate hepatic scavenger receptor B1 (SRB1) and increase hepatic lipase activity [21], the resulting reduced HDL levels are perhaps indicative of more effective reverse cholesterol transport. On the other hand, a progestogen with
no androgenic properties such as DGT has minimal effect on hepatic lipase activity and HDL level is thus unaffected. A low HDL level is frequently regarded as cardiovascular risk [9], but the import of low HDL level depends on the mechanism by which it is caused; and sex hormone induced changes in HDL levels are for the reasons discussed above difficult to interpret. DGT was associated with an increase in triglyceride levels whereas triglyceride levels with NET were similar to pre-treatment values. This is consistent with other studies [17,22,23]. Oestrogen is known to cause a rise in triglycerides [17,24], and androgenic progestogens are more successful in reversing this potentially unfavourable oestrogenic effect [17]. Triglycerides have been found to be a cardiovascular risk factor in women and diabetic subjects [7]. Progestogens that are more androgenic have been reported to increase hepatic lipase activity [21] thus reducing the pool size of triglyceride-rich lipoproteins. This may be the explanation for the effects observed in the present study. Some studies of exogenous oestrogens administered to postmenopausal women have shown an increase in small LDL particles [25,26], although others have not reported similar changes [27,28]. Progestogen effects on SD-LDL have been little investigated. LDL cholesterol and SD-LDL levels in the present study decreased with oestradiol and remained significantly lower with
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the addition of both progestogens. Oestrogen causes reduced plasma residence time of both VLDL and LDL particles [29]. Oral oestrogen also up-regulates LDL receptors [30]. The clearance rates of both VLDL and LDL thus increase and the formation of SD-LDL particles may be discouraged. Progestogens did not oppose the oestrogen effect on SD-LDL in the present study, and the results with either progestogen were similar. HRT is one of the few means of decreasing Lp (a) concentrations [31]. Lp (a) has been shown to be associated with cardiovascular risk [8]. In the Heart and Estrogen/progestin Replacement Study, women with elevated baseline levels of Lp (a) who were randomised to HRT had greater reductions of Lp (a) levels and lower risk of CHD events compared to the placebo group [32]. In this present study the more androgenic NET was associated with significant Lp (a) reduction. Oral oestradiol in the present study caused an increase in CRP that was consistent with other studies [33]. Any such effect in our study was opposed by DGT, but NET was less effective in this respect. Similar results were obtained in other studies [34,35]. The increase in CRP resulting from oral oestrogen is considered to be a hepatic first pass effect, which is not seen when transdermal oestrogen is administered [36]. Some of the metabolites of NET have been found to have oestrogenic properties [37] that could explain the high CRP level when this agent is combined with oestrogen. In our previous study comparing progestogens of varying androgenicity [17], we showed that although all the androgenic progestogens attenuated the oestrogen-induced rise in CRP, CRP levels remained significantly increased. In the present study, DGT, which binds almost exclusively to progesterone receptor, is able to successfully oppose the oestrogen effect on CRP. Hormone related CRP increase is undoubtedly an oestrogenic effect. The progestogenic influence on oestrogen-induced CRP rise is likely to be a progesterone/anti-oestrogenic effect, and appears not to be affected by the androgenic properties of progestogens. Neither oestrogen nor progestogens in the present study affected the levels of interleukin-6. This is consistent with the findings of the Women’s Health Initiative study [33]. It suggests that the raised CRP levels in hormone users may not represent a systemic inflammatory response but is rather a direct hepatic effect of oral oestrogen. Its significance is thus uncertain.
Women and their physicians have been troubled by the negative results of randomised studies on HRT, and HRT is no longer recommended to women with established CHD [1]. However, HRT remains an extremely effective treatment for menopausal symptoms and there are women who will continue to benefit symptomatically from taking it. Thus it is essential to expand our knowledge on HRT and cardiovascular risk factors. Although oestrogen has favourable effects on lowering LDL cholesterol and SD-LDL levels, it also causes potentially unfavourable increases in triglycerides and CRP. Whilst raised triglyceride levels have been associated with increased CHD risk [7], CRP has not been shown to modify the effects of HRT on cardiovascular risk [38]. The present study showed that the more androgenic NET maintained the reductions in LDL and SD-LDL, and moreover was more successful in lowering Lp (a) and opposing triglyceride increase. NET, however, did result in a significantly lower HDL cholesterol level. Nevertheless, there is no evidence that the reduced HDL levels caused by progestogens are directly associated with increased cardiovascular risk. If the lower HDL levels are indicative of a speedier reverse cholesterol transport, it might not ultimately be a deleterious effect. The CRP increase appears to be an oestrogenic effect and androgenicity has little impact on it. The significance of CRP in HRT users requires further elucidation. The use of the more androgenic progestogens may not be as harmful as previously assumed. The choice of progestogens in HRT regimens should take into account their varying androgenic properties. Acknowledgements We thank Ms. C. Price for helping to prepare this manuscript. We are grateful to the patients at the Barlow Medical Centre who participated in this study. S.K. received a research fellowship from the United Kingdom Department of Health. References [1] Hulley S, Grady D, Bush T, et al. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. JAMA 1998;280:605–13.
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