Estrogen Therapy and Coronary-Artery Calcification

WHI Trial of Estrogen Alone

Detailed descriptions of the design of the WHI trial of conjugated equine estrogens and the baseline characteristics of the participants have been published previously.^4,20,21 Briefly, the participants were postmenopausal women who were 50 to 79 years of age at randomization and had undergone hysterectomy. We randomly assigned the participants to receive oral conjugated equine estrogens (0.625 mg per day) (Premarin, Wyeth Pharmaceuticals) or placebo. Methods regarding data collection, data management, and assurance of the quality of the data have been published previously.²²

The WHI trial of conjugated equine estrogens was originally scheduled to continue until close-out visits between October 2004 and March 2005. However, the National Institutes of Health stopped the trial approximately 1 year early, owing to an increased risk of stroke in the absence of apparent benefit for the risk of CHD.⁴ Study participants were informed of this decision on March 1, 2004, were instructed to discontinue the study medication, and were informed of their group assignment. Although the evidence suggested a reduced risk of CHD among the women aged 50 to 59 years who were receiving conjugated equine estrogens,^4,5 the statistical power of the study was inadequate to provide conclusive results. Therefore, an explanatory ancillary substudy (WHI-CACS) was proposed to provide mechanistic information that might elucidate this finding. WHI-CACS was restricted to women aged 50 to 59 years, both to clarify the results concerning conjugated equine estrogens in this age group and because this age group is the most clinically relevant with regard to initiation of hormone therapy for menopausal symptoms.

Design of the Ancillary Study (WHI-CACS)

A total of 28 of the 40 WHI clinical centers were in close proximity to the requisite imaging facilities and had the ability to mobilize quickly to participate in WHI-CACS. A central reading center at Wake Forest University School of Medicine was selected by means of a competitive bidding process. After the study was approved by central and local institutional review boards, we mailed invitational letters to the 1742 eligible women among the 2271 women who had participated in the trial of conjugated equine estrogens at these 28 centers and who were 50 to 59 years old at the time of randomization in the WHI trial. Exclusion criteria were a request by the participant for no further clinic visits, a weight of 300 lb (136 kg) or higher (owing to technical restrictions), or a loss to follow-up or death since randomization. A total of 529 of the original participants (23.3%) were excluded for one or more of these reasons. A total of 1079 women (61.9% of the 1742 eligible participants at the 28 clinical centers) provided written informed consent and underwent CT examinations of the heart between May 2005 and September 2005. Because the study period was a mean of 7.4 years and coronary-artery calcium was measured on average 1.3 years after the trial, the women had a mean age of 64.8 years at the time of the coronary-artery calcium measurements.

Measurement of Calcified Plaques

Noninvasive imaging of the coronary arteries was performed with the use of electron-beam or multidetector-row CT at the 28 participating centers, all of which used CT systems with the capability to acquire cardiac images in approximately 250 msec or less. A standardized protocol was developed on the basis of previous multicenter experience with CT of the heart.^13,14 Phantom and test images were obtained with the use of each CT system to verify technical settings and system performance. The measurements were analyzed at a central reading center at Wake Forest University without knowledge of randomization status.¹³ The Agatston scores²³ were calculated at a computer workstation (TeraRecon) by experienced image analysts using established criteria.^8,13,23

Twelve women were excluded owing to a history of coronary revascularization procedures before randomization or missing data on this variable, and three women were excluded because of incomplete scans. In addition, the reading protocol specified the exclusion of data from patients with coronary stents, pacemakers, metallic clips, and other surgical remnants. The final data set represented 1064 participants.

Statistical Analysis

Baseline cardiovascular risk factors and other characteristics of participants receiving conjugated equine estrogens and those receiving placebo were tested for differences with the use of t-tests for continuous variables and chi-square tests for categorical variables. Coronary-artery calcium scores in the group receiving estrogen and the placebo group were compared with the use of the Kruskal–Wallis rank test. Because the distribution of coronary-artery calcium scores was skewed, with 53% of participants having scores of 0, we also performed tobit regression analyses for left-censored data, using a cube root transformation (to the coronary-artery calcium score + 1).²⁴ Coronary-artery calcium scores were also grouped as follows: 0 (no calcification), 1 to less than 10 (minimal calcification), 10 to 100 (mild calcification), 101 to 300 (moderate calcification), and more than 300 (extensive calcification).^8,25,26 Associations between study group and coronary-artery calcium score were assessed with the use of dichotomous logistic-regression analysis for coronary-artery calcium scores of more than 0 (vs. 0), 10 or more (vs. <10), and 100 or more (vs. <100), as well as ordinal (polychotomous) logistic-regression analysis for higher scores.

Primary analyses were conducted according to the intention-to-treat design, with and without further adjustment for coronary risk factors. Additional models also involved adjustment for educational level, presence or absence of a history of oophorectomy, reproductive status, and presence or absence of randomization in the WHI diet-modification trial, the WHI calcium and vitamin D trial, or both trials. Inverse-probability-of-censoring weighted analyses²⁷ were also conducted to estimate the results for all eligible women in the WHI trial of conjugated equine estrogens. Secondary analyses, restricted to women with at least 80% adherence to the study medication for at least 5 years, were performed with and without multivariate adjustment. All P values are two-sided, and P values of less than 0.05 were considered to indicate statistical significance. Statistical analyses were performed with the use of SAS statistical software, version 9 (SAS Institute).

Table 1. Table 1. Baseline Characteristics and Cardiovascular-Risk-Factor Status, According to Randomized-Group Assignment.

Cardiovascular risk factors and other characteristics at baseline were similar among WHI-CACS participants receiving conjugated equine estrogens and those receiving placebo (Table 1). There were no significant differences between the two groups on the basis of age, race or ethnic group, traditional coronary risk factors, or key lifestyle or reproductive variables. In addition, the coronary-risk-factor status among the participants was similar to that among all women of eligible age in the WHI trial of conjugated equine estrogens.

Table 2. Table 2. Distribution of Coronary-Artery Calcium Scores after Trial Completion, According to Randomized-Group Assignment.

Among the 1064 participants for whom coronary-artery calcium scores were available, the mean (±SD) score was 102.9±303.5, with a range of 0.0 to 4506.6. The mean score was 83.1 among women receiving conjugated equine estrogens and 123.1 among women receiving placebo (P=0.02 by the Kruskal–Wallis rank test) (Table 2). The 50th, 60th, 75th, and 95th percentile values of the coronary-artery calcium scores were 0, 3, 43, and 452 for conjugated equine estrogens and 0, 17, 84, and 689 for placebo, respectively.

The tobit regression analyses, which were performed to assess the overall distribution of coronary-artery calcium scores,²⁴ showed that the overall distribution of scores was significantly lower in the group receiving estrogen than in the placebo group (Table 2). After adjustment for age, race or ethnic group, and coronary-risk-factor status, the multivariate P value for this difference was 0.03 in intention-to-treat analyses. The multivariate P value was 0.002 when analyses were restricted to participants who had at least 80% adherence to the study treatment (estrogen or placebo) for at least 5 years (Table 2).

Table 3. Table 3. Coronary-Artery Calcium Scores after Trial Completion, According to Score Category.

In analyses of the prevalence of coronary-artery calcium, multivariate odds ratios for coronary-artery calcium scores of more than 0 (vs. 0), 10 or more (vs. <10), and 100 or more (vs. <100) were 0.78, 0.74, and 0.69, respectively (Table 3). In secondary analyses restricted to data for women with at least 80% adherence for at least 5 years, the corresponding multivariate odds ratios were substantially reduced: 0.64 (P=0.01), 0.55 (P<0.001), and 0.46 (P=0.001), respectively (Table 3).

Table 4. Table 4. Odds Ratios for Various Categories of Elevation in the Coronary-Artery Calcium Score.

To examine higher levels of coronary-artery calcium, we conducted prespecified ordinal logistic-regression analyses, using a coronary calcium score of less than 10 as the reference category (Table 4).^10,25 In intention-to-treat analyses, women receiving estrogen had a multivariate odds ratio of 0.58 (P=0.03) for extensive coronary-artery calcification (score >300); in secondary analyses restricted to women with at least 80% adherence, the multivariate odds ratio was 0.39 (P=0.004) (Table 4).

Further adjustment for additional variables — including educational level, presence or absence of a history of oophorectomy, reproductive status, and presence or absence of randomization in the WHI diet-modification trial, the WHI calcium and vitamin D trial, or both trials — did not materially alter the results. In ordinal logistic-regression analyses, with a coronary-artery calcium score of less than 10 as the reference category, the odds ratios for coronary-artery calcium scores of more than 100 and more than 300 in the estrogen group were 0.66 and 0.57, respectively (P=0.04 for both comparisons), and were 0.44 (P=0.002) and 0.41 (P=0.009), respectively, among women with at least 80% adherence. Inverse-probability-of-censoring weighted analyses,²⁷ conducted to estimate the results for all eligible women in the WHI trial of conjugated equine estrogens, provided similar findings.

Figure 1. Figure 1. Multivariate Odds Ratios for a Coronary-Artery Calcium Score of More Than 100, According to Randomized-Group Assignment and Coronary-Risk-Factor Status.

Multivariate odds ratios were adjusted simultaneously for coronary risk factors. The reference category for the calcium score was a score of less than 10. Data for some risk factors were missing for some participants. For a family history of myocardial infarction, premature age was defined as younger than 55 years for a male first-degree relative and younger than 65 years for a female first-degree relative. The body-mass index is the weight in kilograms divided by the square of the height in meters.

The odds ratios for a coronary-artery calcium score of more than 100, among participants receiving conjugated equine estrogens as compared with those receiving placebo, are shown in Figure 1, as are the associations between traditional risk factors for CHD and coronary-artery calcium scores. Past or current smoking and the presence of hypertension, a high cholesterol level, and diabetes were all strongly predictive of elevated coronary-artery calcium scores. However, these risk factors did not significantly alter the relationship between treatment with conjugated equine estrogens and coronary-artery calcium scores (all P values for interaction >0.30).

The WHI-CACS assessed the post-trial burden of calcified atheroma in the coronary arteries in women 50 to 59 years old at the time of randomization in the WHI trial of conjugated equine estrogens. An average of 8.7 years after randomization, women receiving estrogen had a lower prevalence and quantity of coronary-artery calcium than those receiving placebo, with odds ratios for high levels of coronary-artery calcium generally 30 to 40% lower in intention-to-treat analyses and 60% lower in analyses among women with at least 80% adherence to the study medication for at least 5 years. The results remained robust and significant in analyses that involved diverse analytic approaches. These findings, in conjunction with the suggestion of a reduced risk of clinical coronary events among women treated with conjugated equine estrogens in this age group,⁵ are consistent with previous evidence from laboratory, animal, and observational studies.^2,3,6

Previous studies of postmenopausal hormone therapy and coronary-artery calcium have been observational only. Like observational studies of hormone therapy and clinical coronary events that have suggested cardiac benefits,^1,28 most previous coronary imaging studies indicate that users of hormone therapy have less coronary-artery calcium than nonusers.^17-19,29 However, observational studies may be susceptible to bias — in particular, confounding by health-promoting behaviors associated with the choice to use hormone therapy — underscoring the need to examine these relationships in the context of randomized clinical trials.^6,30

Coronary-artery calcification serves as a marker of calcified atheroma and total plaque burden.^7,8,13,16 The presence of calcium in atherosclerotic lesions reflects the progression from simple fatty streaks to complex plaques, and coronary calcium measurements have been shown to be directly related to histologic measures of atheromatous plaques.^7,31 In a large cross-sectional study, the risk of CHD increased by a factor of 30 from the lowest to the highest quartile of coronary-artery calcium scores.²⁶ In our study, traditional coronary risk factors were strongly associated with increased quantities of coronary-artery calcium, providing support for the role of this measure as a marker of atherosclerosis. Moreover, coronary-artery calcium measurements have been shown to be highly predictive of future cardiovascular events in several studies, independently of traditional risk factors.^8-11

The new findings from WHI-CACS indicate that estrogen therapy initiated in women at 50 to 59 years of age is related to a reduced plaque burden in the coronary arteries and a reduced prevalence of subclinical coronary artery disease, providing support for the hypothesis that estrogen therapy may have cardioprotective effects in younger women. Although the WHI trial of conjugated equine estrogens suggested that younger women, but not older women, may have a reduced risk of myocardial infarction and coronary revascularization when using estrogen, statistical tests for an interaction according to age were nonsignificant (range of P values, 0.07 to 0.09).⁵ Estrogen has complex biologic effects that may vary according to the underlying state of the vasculature and other tissues.^30,32-34 Conclusive answers, however, can be derived only from large-scale trials involving sufficient numbers of clinical events among women in early menopause.

The strengths of our study include the randomized design of the main WHI trial of conjugated equine estrogens, the relatively long duration of treatment with estrogen, the standardized assessment of coronary-artery calcium at a central reading center, and the large number of women studied, providing good statistical power to detect moderate associations. However, limitations of the study also warrant consideration. In the WHI trial of conjugated equine estrogens, a large percentage of women had stopped taking the study medication before the trial was terminated, and an average of 1.3 years had elapsed between completion of the trial and coronary-artery calcium measurement. Both of these limitations, however, would lead to an attenuation of the association between treatment with conjugated equine estrogens and coronary-artery calcium scores and would not explain our findings.

Although WHI-CACS did not include all participants who had undergone randomization in the estrogen trial and coronary-artery calcium measurements were not available before randomization, the distributions of coronary risk factors and behavioral characteristics at baseline were similar among the women receiving estrogen and those receiving placebo. In addition, adjustment for a large number of variables potentially related to participation or adherence did not result in a weakening of the associations. It would have been of interest to have coronary-artery calcium measurements for women in the older age groups, to allow a comparison of findings for younger and older women. Logistic and operational constraints precluded imaging of the full cohort without a substantial extension of the interval between the discontinuation of study medication and the measurement of coronary-artery calcium.

Moreover, coronary-artery calcium measurements in the older women would not have necessarily informed or elucidated our findings with respect to CHD among participants 50 to 59 years of age. It is possible that estrogen could reduce coronary-artery calcium scores but still increase the risk of clinical CHD events, owing to adverse effects on thrombosis and plaque rupture, which are more likely in older women with advanced stages of atherosclerosis. Such a duality of effects would not necessarily apply to younger women with lower burdens of atherosclerosis. Data from studies of nonhuman primates suggest that estrogen inhibits the progression of atherosclerosis during the early period after bilateral oophorectomy but not later,³ and previous angiographic trials involving women with CHD have suggested that hormone therapy does not prevent atherosclerosis progression in high-risk women.^35,36 The WHI-CACS focused on women 50 to 59 years old because women in this age group are most likely to be engaged in decision making about whether to use hormone therapy for menopausal symptoms.

In conclusion, the results of WHI-CACS indicate that women 50 to 59 years old when they were randomly assigned to receive conjugated equine estrogens had a lower coronary-plaque burden and a lower prevalence of subclinical coronary artery disease after completion of the WHI trial of estrogen than did women receiving placebo. These findings, in conjunction with the data on clinical CHD events among younger women in the WHI trial, provide some reassurance that estrogen is unlikely to have an adverse effect on the risk of coronary events among women who have recently undergone menopause and are considering hormone therapy for the treatment of menopausal symptoms. However, the possibility of a favorable effect of estrogen on atherosclerosis in younger women requires confirmation in future studies,^37,38 and other risks and benefits of treatment^4,39 must be considered. We could not address the question of whether any vascular benefits of treatment with estrogen initiated at a younger age will, with prolonged use, persist at older ages. Additional research on various formulations and regimens of hormone therapy will also be important. In the meantime, hormone therapy should not be initiated (or continued) for the express purpose of preventing cardiovascular disease in either younger or older postmenopausal women. The current recommendations from many organizations that hormone therapy be limited to the treatment of moderate-to-severe menopausal symptoms, with the lowest effective dose used for the shortest duration necessary, remain appropriate.

The Women's Health Initiative Coronary-Artery Calcium Study Investigators are as follows: Program Office, National Heart, Lung, and Blood Institute, Bethesda, MD: J.E. Rossouw, S. Ludlam; Clinical Coordinating Center, Fred Hutchinson Cancer Research Center, Seattle: B.B. Cochrane, J.R. Hunt, B. Lund, R. Prentice; Computed Tomography Reading Center, Wake Forest University, Winston-Salem, NC: J.J. Carr, C. O'Rourke, L. Du, S. Pillsbury, C. Hightower, R. Ellison, J. Tan; Clinical Centers: Albert Einstein College of Medicine, Bronx, NY — S. Wassertheil-Smoller, M. Magnani, D.H. Noble, T. Dellicarpini; Brigham and Women's Hospital, Harvard Medical School, Boston — J.E. Manson, M. Bueche, A.D. McGinnis, F.J. Rybicki; Brown University, Providence, RI — A.R. Assaf, G. Sloane; Emory University, Atlanta — L.S. Phillips, V. Butler, M. Huber, J. Vitali; George Washington University Medical Center, Washington, DC — J. Hsia, C. LeBrun, R. Palm, D. Embersit; Kaiser Permanente Center for Health Research, Portland, OR — E. Whitlock, K. Arnold; Kaiser Permanente Division of Research, Oakland, CA — S. Sidney, V. Cantrell; Medical College of Wisconsin, Milwaukee — J.M. Kotchen, C. Feltz; MedStar Research Institute–Howard University, Washington, DC — B.V. Howard, A. Thomas-Geevarghese, G. Boggs, J.S. Jelinick; Northwestern University, Chicago and Evanston, IL — P. Greenland, A. Neuman, G. Carlson-Lund, S.M. Giovanazzi; Stanford Prevention Research Center, Stanford, CA — M.L. Stefanick, S. Swope; Ohio State University, Columbus — R. Jackson, K. Toussant; University of Alabama at Birmingham, Birmingham — C.E. Lewis, P. Pierce, C. Stallings; University at Buffalo, Buffalo, NY — J. Wactawski-Wende, S. Goel, R. Laughlin; University of California at Davis, Sacramento — J. Robbins, S. Zaragoza, D. Macias, D. Belisle; University of California at Los Angeles, Los Angeles — L. Nathan, B. Voigt, J. Goldin, M. Woo; University of California at San Diego, LaJolla and Chula Vista — R.D. Langer, M.A. Allison, X. Lien, C.M. Wright; University of Cincinnati, Cincinnati — M. Gass, S. Sheridan; University of Iowa, Iowa City and Davenport — J.G. Robinson, D. Feddersen, K. Kelly-Brake, J. Carroll; University of Massachusetts–Fallon Clinic, Worcester — J. Ockene, L. Churchill; University of Medicine and Dentistry of New Jersey, Newark — N.L. Lasser, B. Miller, P.D. Maldjian, J. Pierre-Louis; University of Miami, Miami — J. Fishman, M.J. O'Sullivan, D. Fernandez; University of Minnesota, Minneapolis — K.L. Margolis, C.L. Bjerk, C. Truwit, J.A. Hearity; University of North Carolina, Chapel Hill — W.B. Hyslop, K. Darroch, C. Murphy, G. Heiss; University of Pittsburgh, Pittsburgh — L.H. Kuller, D. Edmundowicz, D. Ives; University of Tennessee, Memphis — K.C. Johnson, S. Satterfield, S.A. Connelly, E.L. Jones; University of Texas Health Science Center, San Antonio — R. Brzyski, M.A. Nashawati, S. Torchia, A. Rodriguez, R. Garza, P. Nentwich; University of Wisconsin, Madison — G.E. Sarto, L. Broderick, N.K. Sweitzer.

The Women's Health Initiative Study Investigators are as follows: Program Office, National Heart, Lung, and Blood Institute, Bethesda, MD: E. Nabel, J.E. Rossouw, S.E. Ludlam, L. Pottern, J. McGowan, L. Ford, N. Geller; Clinical Coordinating Center: Fred Hutchinson Cancer Research Center, Seattle — RL. Prentice, G. Anderson, A. LaCroix, C.L. Kooperberg, R.E. Patterson, A. McTiernan; Wake Forest University School of Medicine, Winston-Salem, NC — S. Shumaker; Medical Research Labs, Highland Heights, KY — E. Stein; University of California at San Francisco, San Francisco — S. Cummings; Clinical Centers: Albert Einstein College of Medicine, Bronx, NY — S. Wassertheil-Smoller; Baylor College of Medicine, Houston — J. Hays; Brigham and Women's Hospital, Harvard Medical School, Boston — J.E. Manson; Brown University, Providence, RI — A.R. Assaf; Emory University, Atlanta — L. Phillips; Fred Hutchinson Cancer Research Center, Seattle — S. Beresford; George Washington University Medical Center, Washington, DC — J. Hsia; Los Angeles Biomedical Research Institute at Harbor–University of California at Los Angeles Medical Center, Torrance — R. Chlebowski; Kaiser Permanente Center for Health Research, Portland, OR — E. Whitlock; Kaiser Permanente Division of Research, Oakland, CA — B. Caan; Medical College of Wisconsin, Milwaukee — J.M. Kotchen; MedStar Research Institute–Howard University, Washington, DC — B.V. Howard; Northwestern University, Chicago and Evanston, IL — L. Van Horn; Rush Medical Center, Chicago — H. Black; Stanford Prevention Research Center, Stanford, CA — M.L. Stefanick; State University of New York at Stony Brook, Stony Brook — D. Lane; Ohio State University, Columbus — R. Jackson; University of Alabama at Birmingham, Birmingham — C.E. Lewis; University of Arizona, Tucson and Phoenix — T. Bassford; University of Buffalo, Buffalo, NY — J. Wactawski-Wende; University of California at Davis, Sacramento — J. Robbins; University of California at Irvine, Irvine — F.A. Hubbell; University of California at Los Angeles, Los Angeles — H. Judd; University of California at San Diego, La Jolla/Chula Vista — R.D. Langer; University of Cincinnati, Cincinnati — M. Gass; University of Florida, Gainesville and Jacksonville — M. Limacher; University of Hawaii, Honolulu — D. Curb; University of Iowa, Iowa City and Davenport — R. Wallace; University of Massachusetts–Fallon Clinic, Worcester — J. Ockene; University of Medicine and Dentistry of New Jersey, Newark — N. Lasser; University of Miami, Miami — M.J. O'Sullivan; University of Minnesota, Minneapolis — K.L. Margolis; University of Nevada, Reno — R. Brunner; University of North Carolina, Chapel Hill — G. Heiss; University of Pittsburgh, Pittsburgh — L.H. Kuller; University of Tennessee, Memphis — K.C. Johnson; University of Texas Health Science Center, San Antonio — R. Brzyski; University of Wisconsin, Madison — G.E. Sarto; Wake Forest University School of Medicine, Winston-Salem, NC — D. Bonds; Wayne State University School of Medicine–Hutzel Hospital, Detroit — S. Hendrix.