Thus, what has emerged from results of studies conducted in experimental animals and what can be identified as a critical difference between previous observational studies in humans and the WHI hormone trials was the time for initiation of treatment relative to menopause [43]. In particular, the mean age of menopause in the USA is 51 years, which corresponds to the age of women in the Nurses Health Study (NHS) who initiated MHT as in usual clinical practice for menopausal symptoms in the perimenopause [31]. This age and timing of MHT initiation contrasts with that of participants in the WHI who were an average of 63 years of age at MHT initiation, approximately 12 years past menopause [46, 59]. Because atherosclerosis progresses gradually before declaring itself with a clinical event [60, 64], a significant number of women in the WHI likely had subclinical atherosclerosis at randomization.

The presence of subclinical disease might have accounted for the tendency for CVD events to be increased early in the WHI, just as in the major secondary CVD prevention study of MHT, the Heart and Estrogen/Progestin Replacement Study (HERS) which began concurrently with the WHI. The HERS showed that in women with preexisting disease, there was an increase in CVD events in the first year or two of the trial. Although continued use of MHT in HERS was associated with a reversal of this trend, the major conclusion from that trial, contrary to conclusions drawn from meta-analyses of the literature [29] was that women with preexisting CHD were not candidates for preventive therapy with MHT [47]. This conclusion changed clinical practice.

Analysis of the NHS and post hoc analysis of data from the WHI based on timing of initiation of treatment relative to menopause supports the concept that initiation of treatment (up to 10 years after the menopause) reduces risk of adverse cardiac events [33, 46, 59]. Furthermore, as in the animal studies, MHT administered to individuals with established atherosclerosis did not induce regression of established lesions [11, 38, 42, 47]. Collectively, these studies suggest that MHT may provide primary prevention for CVD in women by preventing or reducing (slowing) early stages of atherogenic disease processes in the immediate time of menopause when endogenous estrogen shows the steepest decline [43]. Studies in humans, of course, were disadvantaged by the inability to obtain accurate histological assessment of disease progression, and noninvasive techniques to monitor progression of the atherosclerosis, such as B-mode ultrasound imaging of carotid artery wall thickness and plaque or computer-based tomography of coronary artery calcification, were not widely available nor were they sufficiently validated in 1991 to be used as endpoints when the WHI was designed. Advances in these technologies [30, 40] have prompted their use in a small subgroup of the CEE only group of the WHI at the end of the trial [58] and as the major endpoints in ongoing trials of MHT (see below).

An important difference between studies conducted in laboratory animals and clinical trials in humans is that most studies in animals, especially rodents, did not administer CEE but rather 17β-estradiol by a nonoral route. Therefore, possible differences between efficacy and safety of complex conjugated estrogen preparations and transdermal preparations of MHT on cardiovascular parameters remain to be clarified [22, 66, 74, 75, 77].

From these historical considerations, two hypotheses emerge which require rigorous testing in humans: (1) MHT initiated early after menopause (i.e., prior to the appearance of advanced atherosclerotic lesions) will prevent progression of atherosclerosis and (2) oral CEE and transdermal 17β-estradiol will be similarly efficacious in their arterial effects. These hypotheses are the basis of the Kronos Early Estrogen Prevention Study (KEEPS). Findings of the KEEPS have the potential to affect the health of many millions of women who may endure cardiac events and bone fractures that could be prevented. If there proves to be a window of time in the early postmenopause when initiation of long-term MHT has a net beneficial effect, then women can feel reassured that treatment of their menopausal symptoms when they are likely to be the most severe, can concurrently protect their vascular system, and can reduce bone resorption.

KEEPS was designed as a randomized, placebo-controlled, double-blinded, prospective trial (KEEPS; NCT000154180) to evaluate effects of MHT on progression of atherosclerosis as defined by carotid intima–media thickness (CIMT) [44] and coronary arterial calcification (CAC) [8, 73] in women who more closely match the age of initiation of MHT reported by prior observational studies. Women meeting inclusion criteria subsequently were randomized to daily placebo, oral CEE, or transdermal 17β-estradiol with placebo or pulsed progesterone for 12 days/month. The detailed inclusion and exclusion criteria for KEEPS have been published elsewhere [36]. In brief, women between the ages of 42 and 58 years of age who were at least 6 months and no more than 36 months from their last menses with plasma follicle-stimulating hormone (FSH) level ≥35 ng/mL and/or E₂ levels <40 pg/mL were eligible. A history of clinical CVD including myocardial infarction, angina, congestive heart failure, or thromboembolic disease excluded women from KEEPS. Other major cardiovascular risk factors excluding participation were current heavy smoking (more than ten cigarettes/day by self-report), morbid obesity [body mass index (BMI) >35 mm²/kg], dyslipidemia (LDL cholesterol >190 mg/dL), hypertriglyceridemia (triglycerides >400 mg/dL), and uncontrolled hypertension (systolic blood pressure >150 mm Hg and/or diastolic blood pressure >95 mm Hg) and glucose >126 mg/dL. Complete blood count and chemistry panel, estradiol, and FSH were performed at the clinical laboratories at each recruiting center. Lipid profiles and thyroid-stimulating hormone (TSH) were performed at the Kronos Science Laboratories (Phoenix, AZ, USA). At screening, women were asked to rank their menopausal symptoms (hotflashes, night sweats, vaginal dryness, dyspareunia, palpitations, insomnia, depression, mood swings, and irritability) as either none, mild, moderate, or severe. Finally, all subjects were screened for CAC and women with Agatston score ≥50 U, indicating significant subclinical coronary artery disease, were excluded. All women meeting inclusion criteria underwent baseline measurements of CIMT by B-mode ultrasound [44]. All imaging results are read centrally by individuals blinded to participant demographics (CAC at the Los Angeles Biomedical Research Institute at Harbor-UCLA, Torrance, CA, USA under the direction of Dr. M. Budoff and CIMT at the Atherosclerosis Research Unit Core Imaging and Reading Center, University of Southern California, Los Angeles, CA, USA under the direction of Dr. H. Hodis).

Subjects were recruited through nine clinical study centers (Brigham and Women’s Hospital, Boston, MA, USA; Columbia University College of Physicians and Surgeons, New York, NY, USA; Mayo Clinic, Rochester, MN, USA; Montefiore Medical Center, Bronx, NY, USA; Kronos Longevity Research Institute, Phoenix, AZ, USA; University of California at San Francisco, San Francisco, CA, USA; University of Utah School of Medicine, Salt Lake City, UT, USA; University of Washington School of Medicine; Tacoma, WA, USA; Yale University School of Medicine, New Haven, CT, USA) beginning in July, 2005 with complete enrollment of 728 participants qualified for randomization in June, 2008. Subjects were recruited using a variety of strategies including mass mailings, recruiting posters in hospitals and clinics, print and electronic media advertisements, and an internet web page (www.​keepstudy.​org).

Contacts were asked a series of standard questions from a Phone Screen Interview Form. Of 4,533 phone enquiries, 2,528 callers were disqualified and 927 others, who were qualified, either failed to make or show up for screening visit appointments or did not complete screening for personal reasons. Women who qualified by phone screen gave written informed consent at their screening visit. Of these 1,078 women, 290 candidates were excluded based on history, physical examination, laboratory tests, or imaging procedures (Table 1). Some subjects had more than one disqualification factor (45 with two, seven with three, and three with four exclusions). The major cardiovascular risk factor disqualifying women was a CAC score >50 AU, which represented 3.7% of women qualified by phone screen and 14.5% of disqualified women (Table 1).

The average level of education and income were greater in women meeting eligibility criteria compared to those women who were disqualified. Women who were disqualified also were more likely to have never married or declined to report marital status than women who were qualified (Table 2). Menopausal symptoms scores were similar between women excluded and those meeting eligibility criteria (9.0 ± 5.4, n = 257 and 9.4 ± 5.4, n = 728, respectively). Women meeting eligibility criteria were younger, had lower LDL cholesterol, and had lower systolic blood pressure than women excluded (Table 3).

CIMT in women subsequently disqualified from KEEPS did not differ significantly from that of women meeting eligibility requirements [0.725 ± 0.084 mm (n = 113) and 0.726 ± 0.90 mm (n = 718), respectively]. Repeat CIMT readings on the same individual performed within 90 days had a coefficient of variation between scan 1 and scan 2 of less than 1% (0.84 ± 0.99%; minimum of 0% and a maximum of 11%).

Women qualified for KEEPS represent a similar ethnic mix to women enrolled in the CEE+MPA arm of the WHI (Table 4). However, women who qualified for KEEPS had higher levels of education and more declined to report their income level than women in the WHI (Table 2). The percentage of women who qualified for KEEPS who report partnered/separated and divorced status is similar to that reported in the WHI with the exception that fewer women who qualified for KEEPS reported a widowed status while more reported never having been married. This finding may reflect the difference of nearly a decade in mean ages of participants between the two studies (Tables 2 and 3). Prior hormone use did not differ between participants in KEEPS and WHI but the incidence and severity of menopausal symptoms were strikingly different, with 67% of KEEPS-qualified women reporting severe symptoms compared to 61% of WHI women reporting no symptoms. These differences were also characteristic of women in the 50–59-year grouping of the WHI with the exception of the number of participants that were current hormone users and were higher in the WHI than in women qualified for KEEPS (Table 2).

A major modifiable CVD risk factor, smoking, was also about 50% less in qualified KEEPS participants than women of the WHI. All other conventional cardiovascular risk factors comprising the Framingham Risk Score including systolic blood pressure, total cholesterol, and HDL-C were significantly different between women meeting eligibility for KEEPS and those in the WHI (Table 3). The same was true of CVD risk factors not included in the Framingham score such as BMI and fasting triglycerides and glucose, reflecting a reduced 10-year CVD risk for women in KEEPS compared to those in the WHI.

KEEPS is an example of a study that leveraged characteristics of populations in observational and epidemiological studies and incorporated design elements from basic science studies into its trial design. Thus, women who qualified for KEEPS are younger, healthier, reported more menopausal symptoms, and have fewer cardiovascular risk factors than women in the WHI. Therefore, women in KEEPS more closely match the demographics of women in observational studies. Although observational studies have been criticized for reflecting a “healthy user bias” [72], KEEPS is randomized and blinded so outcomes will not be biased by participant group assignment. Also, KEEPS is more representative of the typical clinical experience, i.e., women who are relatively healthy but who are experiencing menopausal symptoms and who seek MHT around the time of their menopausal transition.

One potential limitation of KEEPS is that the majority of participants are Caucasian and results may not be applicable to diverse ethnic populations. However, as medicine moves toward a pharmacogenomic approach to treatment choice, personal definition of ethnicity may not be as relevant as a genotypic definition. Alternatively, KEEPS may help to define the subset of women for whom MHT may provide the most favorable benefit/risk ratio. Furthermore, because a large percentage of women in KEEPS are never smokers and are otherwise at lower risk of CVD, as was the case in the Estrogen for Prevention of Atherosclerosis Trial [44], KEEPS results should more accurately reflect actions of MHT on arteries less confounded by preexisting atherosclerosis as was the case in the WHI. It might also be expected that as KEEPS proceeds, various biochemical markers and soluble cytokines known to be affected by estrogenic treatments will show less variability among participants because fewer participants are current smokers than in other studies. Smoking is well recognized as a risk factor for CVD and many of the components of tobacco smoke (particulate matter, nicotine, carbon dioxide, oxygen-derived free radicals, and other chemicals) negatively impact the same biochemical pathways affected by estrogen [5, 63, 83, 96].

In the post-WHI era, recruiting for KEEPS was challenging and the resultant socioeconomic and ethnicity demographics of qualified participants reflect difficulties in recruiting diverse groups of women. Negative reports in the lay media that accompanied the scientific reporting of results from the WHI scared many women away from use of MHT, even those who were highly symptomatic. As in observational studies where women most likely initiated MHT for menopausal symptoms, the majority of women in KEEPS reported moderate to severe menopausal symptoms, unlike the WHI in which symptomatic women were discouraged from participation. It is in women seeking treatment for symptoms that cardiovascular benefit has been reported in observational studies. However, even in the relatively asymptomatic women in the WHI, CAC scores, an outcome of KEEPS, were significantly lower in women between the ages of 50 and 59 years after 7 years of blinded randomized to CEE compared to those receiving placebo [58]. In the future, it will be important to better understand biochemical and physiological differences between women with and without menopausal symptoms and to clarify if even asymptomatic women would receive benefit from MHT for chronic diseases of aging.

An unexpected finding from the KEEPS screening processes was the number (nearly 4%) of women in this age group, usually considered at low risk for CVD that had to be excluded because of significant CAC, even in the absence of other clinically significant cardiovascular risk factors (i.e., elevated BMI and cholesterol, hypertension, and diabetes). These observations are in agreement with other studies and support the emerging concept that CAC may be a common endpoint of multiple etiologies and may be a sensitive indicator of atherosclerosis in middle-aged humans [20, 21, 26, 54, 94]. As KEEPS continues, new information will be gained regarding how MHT affects progression of CAC and if the progression is related to conventional risk factors or new biomarkers.

KEEPS will provide the first direct comparison of effects of long-term use of oral versus transdermal MHT on atherosclerosis in women. Such information is essential in order for women to select treatments which best suit their needs and lifestyle.

A major difference between KEEPS and studies in experimental animals is that KEEPS participants underwent natural menopause and not a surgical one. However, there are now established animal models of ovarian failure which should provide useful information about the similarities in vascular actions of estrogen between types of menopause [4, 45]. Furthermore, data are emerging from observational studies of women postovariectomy suggesting that MHT reduces overall mortality and maintains cardiovascular health and neurological function [3, 25, 29, 48, 51, 57, 67‐70]. Prospective randomized trials are needed in this group of women to validate the effectiveness of MHT for women independent of the type of menopause [57]. In addition, more information is needed about the type, dose, and duration of progestogen therapies to reduce risk of uterine cancer but still provide cardiovascular benefit [13]. Basic science studies of the combined and pulsed use of progestogens and vascular function are scarce [2, 55, 61, 93].

KEEPS has the advantage of being able to incorporate mechanistic data from basic science studies which were not available at the time of the design of the WHI. Therefore, ancillary studies to the parent KEEPS protocol are ongoing to examine new biomarkers of disease progression and thrombotic risk [49, 50], genetic characterization of estrogen receptors relative to response to MHT and outcomes of KEEPS, measures of endothelial function, and bone health which should provide prospective data to complement data obtained from the WHI and Study of Women Across the Nation [18, 56, 78‐81, 90].

Finally, there are lessons to be learned from these experiences of basic scientists and clinical investigators involved in the study of cardiovascular actions of estrogen. First, observational and epidemiological studies are hypothesis generating and do not establish cause and effect. Second, design of prospective clinical studies to test these hypotheses should mimic, as closely as possible, the study population and treatment circumstances of the observational studies. The design should also take into account data from experimental animals in which the basic information on drug action was obtained without confounding variables. Separate studies may be needed to account for such confounding variables (i.e., environmental pollutants, obesity, preexisting conditions like diabetes). Third, the design should not compromise integrity of primary outcomes for unrelated secondary outcomes. Fourth, advancements in understanding the basic mechanisms of action of sex steroids was possible in the environment of sustained funding by National Institutes of Health which attracted the best scientists to tackle difficult questions. And finally, collaboration among interdisciplinary teams will facilitate translation of information from basic science to design of clinical trials and ultimately improve the evidence upon which to base clinical practice. These lessons may be considered obvious and common sense, but are often overlooked.

The KEEPS is an example of a study that has incorporated these lessons. KEEPS has matched its sample population to that of observational studies, controlled for confounding variables such as smoking and pre-clinical vascular disease, and will evaluate a mode of treatment delivery similar to what has been used in the majority of basic science studies. Noninvasive measures of vascular health as outcomes also will allow for analysis of disease progression and help to support a paradigm shift in evaluating primary prevention [7]. Taking advantage of the latest technological advances in imaging, diagnostics, and testing, KEEPS will provide valuable information for women making decisions about MHT and their health as they age [41]. The KEEPS cohort, stored serum/plasma, and other data are valuable resources. However, additional support from funding agencies will be required in order to leverage these resources to address new questions generated by KEEPS and to translate new discoveries from basic science to further investigations of this defined cohort.