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Effects of Estrone Sulfate Alone or With Medroxyprogesterone Acetate on Serum Lipoprotein Levels in Postmenopausal Women

From the Center for Fertility and Reproductive Endocrinology at New Britain General Hospital, New Britain, Connecticut, and Pharmacia Upjohn Inc., Kalamazoo, Michigan.

Address reprint requests to: Anthony A. Luciano, MD New Britain General Hospital Center for Fertility and Reproductive Endocrinology 100 Grand Street New Britain, CT 06050 E-mail: aluciano{at}nbgh.org

	Abstract

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Objective: To determine the effects of estrone sulfate alone or with different doses of medroxyprogesterone acetate on serum lipid and lipoprotein levels.

Methods: A multicenter, double-masked, randomized trial for 1 year involved 682 postmenopausal women, aged 53.8 ± 0.2 years (mean ± standard deviation) with intact uteri. Subjects received fixed daily doses of 0.625 mg of estrone sulfate and one of the following regimens: placebo; 2.5 mg daily of medroxyprogesterone acetate; 5 mg daily of medroxyprogesterone acetate; or 10 mg of medroxyprogesterone acetate for the first 12 days of each 28-day cycle. Fasting lipid and lipoprotein levels were measured at baseline and weeks 12, 16, 24, 30, 36, and 52 of treatment. Absolute mean changes from baseline were determined by paired t test, and treatment effects were determined by analysis of variance.

Results: Total cholesterol levels decreased significantly (P < .05) from baseline in all study groups; however, reduction was significantly greater (P < .001) in the 2.5-, 5-, and 10-mg groups (-13.3%, -15.2%, and -14.1%) than in the placebo group (-4.9%). Low-density lipoprotein cholesterol levels decreased significantly and equally in all groups (-10.1% to -12.3%). High-density lipoprotein cholesterol levels increased by 3.2% with unopposed estrogen (P < .05) and did not change from baseline with combined therapy. Triglyceride and very low-density lipoprotein cholesterol levels increased by 13.4% and 2.7%, respectively, in the placebo group, did not change in the 2.5-mg group, decreased by 10.2% and 2.0% and by 11.4% and 2.2% in the 5- and 10-mg groups, respectively (P < .05).

Conclusion: Estrone sulfate at the daily dose of 0.625 mg alone or with medroxyprogesterone acetate significantly improved lipoprotein levels. Combined therapy with medroxyprogesterone acetate and estrone sulfate was associated with statistically significantly greater reduction in total cholesterol and statistically significantly less increase in triglyceride levels than unopposed estrone sulfate therapy.

Incidence of cardiovascular disease is lower in women than in men because of differences in risk factors and hormonal milieu.¹ Postmenopausal women have a higher incidence of atherosclerotic heart disease compared with premenopausal or postmenopausal women on hormone replacement therapy (HRT).^2,3 The atheroprotective effects of estrogen include increased myocardial contractility,⁴ vasodilation and decreased response to injury,^5,6 and favorable changes in serum lipoprotein levels.^6,7 The effect of estrogen therapy on serum lipid concentrations results largely from estrogen receptor–mediated effects on hepatic expression of apoprotein genes.⁶ It is estimated that at least one-third of the cardioprotective effects of estrogen is mediated by favorable lipid changes.⁷ In a longitudinal, populationbased study of healthy middle-aged women, natural menopause led to a rise in low-density lipoprotein (LDL) cholesterol and a decline in high-density lipoprotein (HDL) cholesterol, which was prevented or reversed in women who received HRT.⁸ Several studies reported that estrogen replacement therapy in postmenopausal women decreases serum levels of total cholesterol and LDL cholesterol and increases serum HDL cholesterol and triglyceride levels.^7–11 However, the route of estrogen administration, the type and dose of estrogen, and the coadministration of a progestin might influence dramatically the effects of HRT on serum lipids.^9–15

Most studies that evaluated effects of oral estrogen in postmenopausal women used conjugated equine estrogen,^7–10 which is composed of several estrogenic compounds that might have different effects on serum lipoprotein levels and other target organs of estrogen action, such as reproductive tissues, breast, bone, liver, and brain. Although conjugated equine estrogen is the most widely prescribed preparation in the United States, other estrogens are used widely also and their effects, presumed similar to conjugated equine estrogen, have not been adequately studied.

Ogen (Pharmacia-Upjohn Inc., Kalamazoo, MI) is prepared from purified crystalline estrone and is available as an oral tablet containing sodium estrone sulfate at doses of 0.625 mg, 1.25 mg, and 2.5 mg. It is used at doses similar to those of conjugated equine estrogen, but no comparative studies have been done to confirm comparable effects. In this study we evaluated the effects of a fixed oral dose of Ogen (0.625 mg of estrone sulfate) alone or with various doses of medroxyprogesterone acetate on menopausal symptoms, reproductive tissues, skeleton, and serum lipoprotein levels. The dosage was selected because it is considered the minimal clinically effective dosage to prevent bone loss. We report the effects of estrone sulfate with and without medroxyprogesterone acetate on lipoprotein levels in healthy postmenopausal women.

	Methods

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This randomized, double-masked study was conducted in 28 research centers throughout the United States from September 20, 1994 through February 9, 1998. The protocol was approved by the institutional review board at each site. Eligible women had to be postmenopausal, between 45 and 65 years old, have intact uteri, be willing to have at least two and perhaps three endometrial biopsies, and keep diaries of uterine bleeding and vasomotor symptoms. Their serum FSH and estradiol (E2) concentrations also had to be consistent with postmenopausal status. Women were excluded if they had any contraindications to estrogen or progestin use, if they had used oral estrogen or progestin during the previous month, or had injectable sex hormones within the previous 3 months. Other exclusion criteria included use of systemic steroids, lipid-lowering agents, or other investigational drugs, and major illnesses including liver, kidney, or thyroid disease, or significant abnormalities of cervical cytology, endometrial biopsy, mammograms, or renal or liver function studies. Subjects were screened by general medical history, dietary calcium intake, physical and pelvic examinations with Papanicolaou smears, and baseline endometrial biopsies. If biopsy showed inadequate tissue for diagnosis, transvaginal ultrasound was used to measure endometrial thickness. If thickness was less than 5 mm, the woman was included if other criteria were met. If thickness was at least 5 mm, a repeat biopsy had to show nonhyperplastic endometrium. Mammograms had to be done and show normal results within the previous 6 months. Other baseline tests included serum levels of fasting lipoprotein, serum chemistry, total calcium, thyroid function studies, complete blood count, and urinalysis.

After giving informed consent, 866 women were randomly assigned for 1 year to one of four treatment groups: 0.625 mg of oral estrone sulfate plus placebo (days 1–28), 0.625 mg of estrone sulfate plus 2.5 mg of medroxyprogesterone acetate (days 1–28), 0.625 mg of estrone sulfate plus 5.0 mg of medroxyprogesterone acetate (days 1–28), 0.625 mg of estrone sulfate (days 1–28) plus placebo (days 1–16), and 10 mg of medroxyprogesterone acetate (days 17–28). The investigators were not masked to the study drugs.

Study drugs were masked, packaged in 28-day blister cards, and randomized with sequential patient identification numbers by Upjohn. A commercial software package, Drug Labeling System (Almedica Services Inc., Allendale, NJ), was used to randomize study medication in forced blocks of eight. All subjects received the same number of tablets daily. Estrone sulfate tablets were open labeled (not masked), and medroxyprogesterone acetate and placebo tablets were double masked. Blister packs of drugs for one cycle (28 days) looked the same for each treatment group, and participants were instructed to take medication daily, in the morning with food, and return unused drug at the next scheduled visit.

Blood samples were collected in the morning, after overnight fast, from the antecubital vein while the women were at rest. Serum lipoprotein levels were determined before (baseline levels) and during treatment on days 23–28 of cycles 12, 16, 24, 30, 36, and 52. Frozen serum samples were sent on dry ice to the research laboratories of Pharmacia and Upjohn and assayed in batches to avoid interassay variability. High-density lipoprotein and its fractions, HDL-2 and HDL-3, were measured by the dextran sulfate–magnesium chloride precipitation technique of Talameh et al.¹⁶ Total cholesterol was measured as postoxidative chromogenic quinoneimine dye detected at absorbance of 520 nm. Triglycerides were measured as postlipase, oxidized glycerol-3-phosphate product that was condensed by 4-aminoantipyrine with 3,5-dichloro-2-hydroxybenzene sulfonic acid and peroxidase to form a quinoneimine chromophore measured at 520 nm. Friedwald calculations were used for LDL and very LDL determinations. The coefficients of variation within and between assays for lipoproteins were less than 3% and 7%, respectively.

The sample was based on the primary efficacy variable of endometrial hyperplasia. To establish adequacy of the sample, the binomial test of proportions was used. With two-sided tests, = 05 was used and the power of detection was 80%. Assuming 2% rate of hyperplasia in each of the three medroxyprogesterone groups and 10% rates of hyperplasia in the estrogenonly group, 161 subjects per arm were needed. Eight hundred seventy subjects were recruited to attain the required 644-subject population. Lipids were secondary variables in the study. Actual lipid data and changes from baseline were analyzed at each time point. Paired t tests were used to evaluate changes from baseline within each group. Analysis of variance model with an effect for treatment was used to compare treatment groups at baseline and each time point and changes from baseline at each time point. Pairwise assessment of treatment comparability between each of the four treatment groups was done using least square mean analysis. Treatment comparisons, including overall and pairwise comparisons, were made using pooled estimates of the variances. All statistical tests were based on two-sided alternative hypotheses, at , = .05, because no a priori assumptions were made. Statistical significance was set at P .05.

	Results

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Among 870 postmenopausal women randomized to treatment, 682 completed the study. The groups were similar in demographic characteristics of age, weight, height, race, medical and family histories, use of nicotine, and dietary and nutritional habits (Table 1). Dropout rates were 18%, 21%, 26%, and 19% for groups 1, 2, 3, and 4, respectively, and demographic characteristics of the dropout population were similar to those of subjects who completed the study.

View larger version (15K): [in this window] [in a new window]   Figure 1. Mean changes (% ± standard deviation) from baseline in serum levels of total cholesterol at the indicated interval for each group. P values indicate differences from the overall tests. MPA = medroxyprogesterone acetate.

View larger version (14K): [in this window] [in a new window]   Figure 4. Mean changes (% ± standard deviation) from baseline in serum levels of triglycerides at the indicated interval for each group. P values indicate differences from the overall tests. MPA = medroxyprogesterone acetate.

The mean changes in LDL cholesterol levels from baseline are shown in Figure 2. Mean LDL cholesterol levels decreased significantly from baseline in all groups. There were no statistically significant differences among groups at baseline or during treatment. The percentage changes in mean HDL cholesterol levels from baseline are shown in Figure 3. Combined therapy tended to reduce serum HDL cholesterol levels, whereas estrone sulfate alone therapy increased mean HDL cholesterol levels (P < .05). Comparison analyses by pairwise testing showed statistically significant differences between the unopposed estrone sulfate therapy group and the 2.5-mg (P = .012), 5-mg (P < .001), and 10-mg (P = .005) medroxyprogesterone acetate groups. The results were similar for HDL-2 cholesterol subfraction (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 2. Mean changes (% ± standard deviation) from baseline in serum levels of low-density lipoprotein cholesterol at the indicated intervals for each treatment group. P values indicate differences from the overall tests. MPA = medroxyprogesterone acetate.

View larger version (14K): [in this window] [in a new window]   Figure 3. Mean changes (% ± standard deviation) from baseline in serum levels of high-density lipoprotein cholesterol at the indicated intervals for each group. P values indicate differences from the overall tests. MPA = medroxyprogesterone acetate.

Mean levels of very LDL cholesterol followed the same pattern as triglycerides, as shown in Table 2, which illustrates the mean percentage changes from baseline to week 52. There were no statistically significant differences among treatment groups at baseline, but statistically significant differences were noted among groups during treatment. Pairwise comparisons showed that the differences primarily were between women in all the groups who received combined therapy and those who took estrone sulfate only (P < .001). As with triglycerides, the mean percentage changes were significantly positive for the unopposed estrone sulfate group, indifferent for the low-dose medroxyprogesterone acetate group, and significantly negative for the combined higher medroxyprogesterone acetate groups.

	Discussion

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The relationship between hypercholesterolemia and coronary heart disease has been established through several clinical trials that involved lipid-lowering interventions, including diet, drugs, and hormonal manipulations.^13–15 The Lipid Research Clinics Trial showed that an 11% decrease in LDL cholesterol levels reduced incidence of coronary heart disease by 19%.¹⁷ In the present study, women in all groups had significant reductions in LDL cholesterol levels, indicating a substantial decrease in the risk of coronary heart disease associated with 0.625 mg of estrone sulfate with or without medroxyprogesterone acetate. Similar changes in LDL cholesterol were reported in clinical studies in which 0.625 mg of conjugated equine estrogen was used alone (unopposed) or with medroxyprogesterone acetate continuously or sequentially at doses similar to those in the present study.^9,10

Hypertriglyceridemia also might be a risk factor for coronary heart disease.^17–19 Oral estrogen alone significantly increases serum triglyceride levels, as reported in this and other studies.^20–24 The estrogen-induced increase in plasma triglycerides was reported to cause smaller LDL cholesterol particles and greater prevalence of LDL subclass pattern B,^20,21 which might be associated with higher incidence of coronary heart disease.²² In the present study, unopposed administration of estrone sulfate resulted in a significant increase in mean triglyceride level. The addition of medroxyprogesterone acetate at a low dose, however, abolished the increase in triglycerides and very LDL cholesterol levels that occurred with estrone sulfate alone. Higher doses of medroxyprogesterone acetate resulted in significant reduction in mean levels of triglycerides and very LDL cholesterol, which might decrease adverse effects of estrogen on LDL particle size, as reported with unopposed estrogen.^20–24 Combination therapy of estrone sulfate and medroxyprogesterone acetate resulted in significant decrease in LDL and very LDL cholesterol and prevented the estrogen-induced increase in serum triglyceride levels that might be atherogenic.

As with other estrogens, unopposed estrone sulfate caused progressively increasing serum levels of HDL cholesterol up to 5.5% by the end of the study. However, the addition of medroxyprogesterone acetate attenuated those positive changes, causing insignificant decreases from baseline in all three combined treatment groups. In several prospective studies, HDL cholesterol was the best predictor of cardiovascular disease in women.^25–27 Using data from four prospective studies, Gordon et al²⁷ estimated that for each milligram-perdeciliter increase in HDL cholesterol, there was a 3% decrease in coronary heart disease in women. Based on those data and data published for conjugated estrogen,^9,10 unopposed estrogen therapy should confer 5–15% additional reduction in cardiovascular disease risk above the combined therapy of estrogen and progestin. There are no clinical trial data showing that isolated improvements in HDL cholesterol significantly reduce the risk of coronary heart disease. Moreover, there is no evidence that the regimen most favorable for HDL cholesterol is also most favorable for other biologically plausible mechanisms proposed for hormonemediated cardioprotection, such as effects on oxidized LDL,^28,29 blood flow, arterial stiffness, and nitric oxide.^4–6 However, there are clinical data suggesting that the combination of estrogen and progestin is associated with equal¹⁴ or greater¹³ reduction in cardiovascular disease risk than estrogen therapy alone. Therefore, the clinical significance of the less favorable HDL cholesterol pattern associated with combined therapy is uncertain, especially when the baseline HDL cholesterol levels exceed 55 mg/dL, as was the case for all groups in this study. The changes in the serum levels of HDL-2 and -3 subfractions were positive for the estrone sulfate only group, but diverged for the medroxyprogesterone acetate groups by decreasing for HDL-2 and increasing for HDL-3 subfractions (Table 2). However, the changes from baseline for either subfraction were not significant.

Total cholesterol levels decreased significantly from baseline in all treatment groups, but the largest decrease was in the three groups on combined therapy in which the mean percentage decrease was more than twice that in the unopposed estrone sulfate group. That difference might be attributed to changes in triglyceride and HDL cholesterol levels. In the unopposed estrone sulfate group, the decrease in total cholesterol levels was attenuated by the significant increase in triglyceride and the modest increase in HDL cholesterol levels. The significant decrease in triglyceride levels associated with addition of medroxyprogesterone acetate contributed to the greater decline in total cholesterol levels in the combined-therapy groups.

	Footnotes

 
Financial Disclosure

Supported by Pharmacia-Upjohn Inc., Kalamazoo, Michigan, which markets estrone sulfate as Ogen and medroxyprogester-one acetate as Provera, both used in this study. Michael J. Schoenenfeld and Robert J. Schaser are employees of Pharmacia-Upjohn Inc., and own stock in that company. Grants for the study were made individually to participating institutions.

PII S0029-7844(00)01081-4

Received December 8, 1999. Received in revised form July 21, 2000. Accepted September 13, 2000.

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