Estrogen in the Prevention of Atherosclerosis

Coronary heart disease is the leading cause of death in women, and mortality rates from this disease substantially and steadily increase after menopause (13). Population studies indicate that estrogen reduces the incidence of coronary heart disease in women. Bilateral oophorectomy before natural menopause increases the risk for coronary heart disease (4). This pattern of risk for coronary heart disease suggests that endogenous estrogens, including 17-estradiol, play a cardioprotective role before menopause. More than 40 observational studies have suggested that hormone replacement therapy (HRT) reduces cardiovascular morbidity and mortality in postmenopausal women (5, 6). Most of these studies were conducted in healthy postmenopausal women who used unopposed estrogen replacement therapy (ERT). Although observational studies are important, selection bias is a potential problem, especially when studying HRT, since healthier women tend to use hormones (7). Only randomized, controlled trials can ensure that patients are assigned to treatment in an unbiased manner and can establish the efficacy of HRT for reducing the progression of atherosclerosis and its clinical sequelae. The effect of unopposed ERT on progression of atherosclerosis in healthy postmenopausal women without preexisting cardiovascular disease remains untested in randomized, controlled trials. We report the results of the Estrogen in the Prevention of Atherosclerosis Trial (EPAT), a randomized, double-blind, placebo-controlled trial designed to test whether unopposed micronized 17-estradiol reduces progression of subclinical atherosclerosis in healthy postmenopausal women without preexisting cardiovascular disease. Our primary hypothesis was that unopposed ERT significantly reduces the progression of subclinical atherosclerosis. Methods Study Design Potential participants were prescreened by telephone and seen at three screening visits 2 to 4 weeks apart to collect baseline data and to determine final study eligibility. Women were eligible if they were postmenopausal (serum estradiol level < 73.4 pmol/L [<20 pg/mL]), 45 years of age or older, and had a low-density lipoprotein (LDL) cholesterol level of 3.37 mmol/L or greater ( 130 mg/dL). Women were excluded if breast or gynecologic cancer had been diagnosed in the past 5 years or if these cancers were identified during screening; if they had previously used HRT for more than 10 years or had used HRT within 1 month of the first screening visit; if they had five or more hot flushes daily that interfered with daily activity and precluded randomization, diastolic blood pressure greater than 110 mm Hg, untreated thyroid disease, life-threatening disease with a survival prognosis of less than 5 years, total triglyceride level of 4.52 mmol/L or greater ( 400 mg/dL), high-density lipoprotein (HDL) cholesterol level less than 0.78 mmol/L (<30 mg/dL), or serum creatinine concentration greater than 221 mol/L (>2.5 mg/dL); or if they were current smokers. All women, including those with diabetes mellitus, were included provided that their fasting blood glucose level was less than 11.1 mmol/L (<200 mg/dL). All participants gave written informed consent, and the study protocol was approved by the University of Southern California Institutional Review Board. Packets of study medications were prepared in a blinded manner (to both the clinical staff and participants) before the start of the study. Computer-generated random numbers were used to assign participants to unopposed estradiol or placebo in one of eight strata, defined by LDL cholesterol level (<4.15 mmol/L [<160 mg/dL] or 4.15 mmol/L), previous duration of HRT use (<5 years or 5 years), and diabetes mellitus (yes or no). As a new participant was determined to be eligible for randomization, the next packet in sequence in the appropriate stratum was obtained and recorded. The Data Coordinating Center monitored adherence to sequential assignment of medication packets. The participants, gynecologists, clinical staff, and image analysts were blinded to treatment assignment. The data monitor and data analyst were blinded to treatment assignment until analyses were completed. Participants were followed every month for the first 6 months and every other month thereafter for a total of 2 years. All participants received dietary counseling according to step II American Heart Association dietary recommendations: 200 mg of cholesterol or less per day, 25% of energy as total-fat calories, and 7% of energy as saturated-fat calories. Dietary intake was monitored at each clinic visit by using 3-day dietary booklets (Nutrition Scientific, South Pasadena, California). Participants received lipid-lowering medication (primarily hydroxymethylglutaryl coenzyme A reductase inhibitors) if their LDL cholesterol level exceeded 4.15 mmol/L (160 mg/dL). Vital signs; clinical events; adherence; and use of nonstudy medications, dietary supplements, and nutriceuticals were ascertained at each visit. Carotid artery ultrasonography was performed at baseline (two visits 1 to 3 weeks apart) and every 6 months thereafter. The baseline intimamedia thickness was the average of the two measurements. Pelvic examination (and uterine ultrasonography in participants with a uterus), Papanicolou smear, and mammography were done yearly in all participants. Uterine biopsy was performed if endometrial thickness was 5 mm or more. Adverse clinical symptoms and bleeding were assessed by the study gynecologist, who was blinded to treatment assignment. The primary trial end point was the rate of change in intimamedia thickness of the right distal common carotid artery far wall in computer image processed B-mode ultrasonograms (815). Power calculations indicated that a sample size of 200 (100 participants per treatment group) was needed to detect a treatment effect size (the standardized difference in progression rates between the two treatment groups) of 0.40 or greater with 80% power. Two hundred twenty-two participants (111 per treatment group) were recruited to accommodate the anticipated dropout rate. Assessment of the Progression of Subclinical Atherosclerosis High-resolution B-mode ultrasonograms of the right common carotid artery were obtained by using a 7.5-MHz linear-array transducer attached to a Toshiba SSH 140A ultrasonography system (Toshiba Corp., Tokyo, Japan). The ultrasonographers were blinded to treatment assignment. Participants were placed in a supine position with the head rotated to the left by using a 45-degree head block. The jugular vein and carotid artery were located in the transverse view, with the jugular vein stacked above the carotid artery according to modification of a procedure described by Beach and colleagues (16). The transducer was then rotated 90 degrees around the central line of the transverse image of the stacked jugular vein and carotid artery to obtain a longitudinal image while the stacked position of the vessels was maintained. All images contained anatomic landmarks for reproducing probe angulation, and a hard copy of each participant's baseline image was used as a guide for follow-up examinations. For each participant, the depth of field, gain, monitor intensity setting, and other instrumentation settings used at baseline examination were used at all follow-up examinations. These techniques significantly reduce measurement variability (14, 15). All images were recorded with the electrocardiogram tracing on super-VHS video tape. An image analyst who was blinded to treatment assignment measured the intimamedia thickness of the distal common carotid artery far wall with automated computerized edge detection using an in-house software package (Prosound, University of Southern California, Los Angeles, California), as described elsewhere (14, 15). Carotid intimamedia thickness was the average of approximately 70 to 100 individual measurements between the intimalumen and mediaadventitia interfaces along a 1-cm length just distal to the carotid artery bulb. This method standardized the location and the distance over which intimamedia thickness was measured and ensured that the same portion of the arterial wall was measured in each image and compared within and across all participants. Laboratory Measurements Participants fasted for 8 hours before sample collection. Total plasma cholesterol and triglyceride levels were measured by using an enzymatic method of the Standardization Program of the National Centers for Disease Control and Prevention. High-density lipoprotein cholesterol levels were measured after lipoproteins containing apolipoprotein B were precipitated in whole plasma by using heparin manganese chloride. Low-density lipoprotein cholesterol levels were estimated by using the Friedewald equation (17). Serum estradiol and fasting insulin levels were measured by using radioimmunoassay. Fasting serum glucose levels were measured by using the glucose oxidase technique on a Beckman Glucose II analyzer (Beckman Instruments, Brea, California). Hemoglobin A1c levels were measured by using high-performance liquid chromatography (Bio-Rad Diamat, Bio-Rad Corp., Hercules, California). Statistical Analysis We compared demographic and baseline clinical and laboratory values between the estradiol and placebo groups by using a t-test for independent samples for means or a chi-square test for proportions. At each study visit, adherence to study treatment was determined by calculating the percentage pill adherence (number of pills consumed divided by the number that should have been consumed) and by measuring estradiol levels. The average percentage pill adherence (over the entire study and by 6-month study period) and average serum estradiol levels (over the entire study) were compared between treatment groups by using a t-test for independent samples. The preplanned intention-to-treat analysis of the primary study end point, progression of subclinical atherosclerosis (def