Ali Serdar Fak, M.D.,* Mithat Erenus, M.D., Hakan Tezcan, M.D.,* Oguz Caymaz, M.D.,* Sule Oktay, M.D., Ph.D., and Ahmet Oktay, M.D.

FERTILITY AND STERILITY VOL. 73, NO. 1, JANUARY 2000 Copyright 1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Effects of a single dose of oral estrogen on left ventricular diastolic function in hypertensive postmenopausal women with diastolic dysfunction Ali Serdar Fak, M.D.,* Mithat Erenus, M.D., Hakan Tezcan, M.D.,* Oguz Caymaz, M.D.,* Sule Oktay, M.D., Ph.D., and Ahmet Oktay, M.D.,* Marmara University Medical School, Istanbul, Turkey Received April 19, 1999; revised and accepted July 30, 1999. Reprint requests and present address: Ali Serdar Fak, M.D., Centre Hospitalier Universitaire Vaudois, Electrophysiologie 219, Exploration Coeur, BH Niv 07, Bugnon 46, 1011 Lausanne, Switzerland (FAX: 41-21-341-00-13). * Department of Cardiology. Department of Obstetrics and Gynecology. Department of Clinical Pharmacology. 0015-0282/99/$20.00 PII S0015-0282(99)00451-3 Objective: To evaluate the acute effects of a single dose of oral estrogen on left ventricular diastolic function in hypertensive postmenopausal women with diastolic dysfunction. Design: Prospective, double-blind, placebo-controlled, clinical study. Setting: Cardiology and postmenopausal outpatient clinics of a university hospital. Patient(s): Thirty postmenopausal women with hypertension (diastolic blood pressure of 90 mm Hg) and left ventricular diastolic dysfunction (mitral E/A ratio [the ratio of peak velocity of early mitral diastolic filling to late diastolic filling] of 1 and isovolumic relaxation time of 100 ms) were included in the study. Thirty normotensive postmenopausal women with normal left ventricular diastolic function served as the control group. Intervention(s): Conjugated equine estrogen (0.625 mg) was given orally. Left ventricular diastolic function was assessed by Doppler echocardiography at baseline and 3 hours after the administration of estrogen. Main Outcome Measure(s): Left ventricular diastolic filling as assessed by Doppler echocardiography. Result(s): Estrogen had no effect on heart rate or blood pressure in either study group. The baseline E/A ratios were 0.72 0.26 and 1.22 0.30, and the isovolumic relaxation times were 122 18 ms and 89 14 ms in the hypertensive and normotensive groups, respectively. Estrogen had no significant effect on any of the Doppler parameters in the normotensive group. In the hypertensive group, there was a trend toward normalization of the E/A ratio (from 0.73 0.11 to 0.84 20) and a significant improvement in the isovolumic relaxation time (from 124 20 ms to 105 13 ms) in response to the administration of estrogen compared with placebo. Conclusion(s): A single dose of oral estrogen caused a significant improvement in left ventricular diastolic filling in hypertensive postmenopausal women with diastolic dysfunction. (Fertil Steril 2000;73:66 71. 1999 by American Society for Reproductive Medicine.) Key Words: Estrogen, diastolic dysfunction, postmenopausal women The use of hormone replacement therapy (HRT) to reduce cardiovascular risks in postmenopausal women has gained popularity (1). The mechanisms by which estrogen exerts its cardioprotective effects are multifactorial (2). Short-term administration of estrogen in postmenopausal women has been reported to improve endothelium-dependent vasodilation in both the coronary and peripheral vasculatures (3, 4). However, there are conflicting reports on the effects of estrogen on left ventricular diastolic function in postmenopausal women (5, 6). It has become increasingly clear that abnormalities of left ventricular diastolic dysfunction have a major role in producing signs and symptoms in patients with heart failure (7, 8). Left ventricular diastolic function is abnormal in patients with coronary artery disease, systemic hypertension, hypertrophic and dilated cardiomyopathy, and valvular heart disease, and it precedes systolic dysfunction in most cases (9). The incidence of diastolic dysfunction is age-related, and the incidence of heart failure caused by diastolic dysfunction rises dramatically with age (10, 11). Thus, either as a consequence of aging or as a result of the underlying pathology, left ventricular diastolic function may be a sign of increased risk of cardiovascular disease. 66

Estrogen may improve left ventricular diastolic function as one of the mechanisms of its cardioprotective effects. Estrogen replacement was shown to have some beneficial effects on left ventricular diastolic filling properties in a retrospective study (5). However, the acute effect of estrogen on diastolic filling, especially in patients with diastolic dysfunction, is not well documented. Diastolic function of the left ventricle can be assessed with transmitral Doppler flow measurements. However, the time course of transmitral velocity is related closely to physical and physiologic factors such as heart rate, left atrial pressure, the inertia of the blood across the mitral valve, the area of the mitral valve, and the systolic function of both the left ventricle and the left atrium (12, 13). Although Doppler echocardiographic measurements of left ventricular filling are affected by many variables, the technique is simple, inexpensive, and reproducible. Therefore, the aim of the present study was to examine the acute effects of a single dose of oral estrogen on left ventricular diastolic function in hypertensive postmenopausal women with diastolic dysfunction documented by Doppler echocardiography. MATERIALS AND METHODS Subjects Postmenopausal women with elevated systemic blood pressure (diastolic blood pressure of 90 mm Hg), impaired diastolic left ventricular filling, and either a normal or an increased left ventricular mass index as assessed by twodimensional echocardiography were included in the study (hypertensive group). Age-matched postmenopausal women with normal blood pressure (diastolic blood pressure of 90 mm Hg) and normal left ventricular diastolic filling served as the control group (normotensive group). None of the study subjects had ever used postmenopausal HRT. The postmenopausal state was defined as the presence of amenorrhea for 1 year and a serum FSH level of 45 IU/L. All subjects were required to be free of any clinical, electrocardiographic, or echocardiographic evidence of coronary artery disease, valvular heart disease, or any cardiomyopathy that could affect the systolic and/or diastolic functions of the left ventricle. All subjects underwent routine physical examination. Systemic blood pressure was measured by the same physician, with a mercury sphygmomanometer and with the patient in the supine position, and the average of three measurements taken at three consecutive outpatient clinic visits was calculated. A 12-lead surface electrocardiogram was obtained, and complete two-dimensional and color flow Doppler echocardiography was performed in all study subjects. The study protocol was in accordance with the ethical standards of the Helsinki Declaration of 1975, as revised in 1983, and was approved by the institutional review board at our institution. Informed written consent was obtained from all subjects. Administration of the Study Drug This was a double-blind, placebo-controlled, crossover study in which all study subjects were randomized to receive a single oral dose (0.625 mg) of conjugated estrogen (Premarin; Wyeth Ayerst, St. Davids, PA) or placebo as the first agent, with a washout period of at least 48 hours ( 5t 1/2 [more than five serum half-lives of conjugated oral estrogen] of conjugated estrogen) between the two treatments. Antihypertensive therapy was continued in the hypertensive patients during the study. Echocardiographic examination was performed 3 hours after the administration of the estrogen or placebo, which was the expected approximate time of the peak plasma concentration of estrogen. The heart rate was monitored and the blood pressure was measured every 5 minutes during the echocardiographic examination. Echocardiographic Examination All echocardiographic examinations were performed 2 3 hours after the last meal and after 20 30 minutes of rest with the patient in quiet respiration in the partial left decubitus position using an Ultramark-9 echocardiograph (Advanced Technology Laboratories, Bothell, WA) with a 2.25-MHz transducer. The echocardiograms were recorded on videotape and analyzed at the completion of the study by two cardiologists who were blind to the type of drug administered to the patient. M-mode and Two-Dimensional Measurements M-mode measurements were performed according to the principles of the American Society of Echocardiography (14). Left ventricular mass was calculated according to the Penn convention, and normal left ventricular mass in women was defined as 110 g/m 2 (15). Doppler Echocardiographic Examination Doppler echocardiographic measurements were performed from the apical four-chamber view that gave the maximal excursion of the mitral valve leaflets. The angle between the presumed direction of the diastolic blood flow observed during color-flow examination of the left ventricular inflow and the orientation of the ultrasonic beam (cursor) was estimated to be zero or 20 degrees in each subject. The sample volume was placed in the inflow area of the left ventricle, and the position of the sample along the cursor line was adjusted at the tips of the mitral valves until the maximal early diastolic flow velocities were recorded and the graphic quality of the Doppler waveform was clearly defined. Five cardiac cycles were analyzed and averaged for each measurement. The Doppler echocardiographic measurements that were of particular interest were as follows: peak velocity of early mitral diastolic filling (E), measured as the height of the early diastolic flow velocity; peak velocity of late mitral diastolic filling (A), measured as the height of the late FERTILITY & STERILITY 67

TABLE 1 The clinical characteristics of the study groups and their left ventricular mass indexes at study entry. Study group Variable Hypertensive subjects Normotensive subjects P value Mean ( SD) age (y) 54 5 52 6 NS Mean ( SD) heart rate (bpm) 78 10 72 16 NS Mean ( SD) systolic blood pressure (mm Hg) 158 19 124 11.005 Mean ( SD) diastolic blood pressure (mm Hg) 102 10 81 9.005 Mean ( SD) LVMI (g/m 2 ) 108 13 99 14 NS Note: bpm beats per minute; LVMI left ventricular mass index; NS not significant. diastolic flow velocity; mitral E/A (the ratio between maximal early and late diastolic flow velocities); and isovolumic relaxation time (IVRT; the time from aortic valve closure to mitral valve opening on the continuous-wave Doppler signal that intersects both the left ventricular outflow velocity and the mitral inflow). The pattern of diastolic dysfunction was defined as the combination of an IVRT of 100 msec and an E/A of 1 (16, 17). Statistical Analysis The echocardiographic findings with placebo and estrogen treatment were compared by the Student s t-test to determine whether estrogen had a significant effect on left ventricular diastolic function in the normotensive and hypertensive postmenopausal women. P.05 was considered statistically significant. The data are expressed as means SD. RESULTS Seventy hypertensive postmenopausal women were screened for the study and 30 of these who also had diastolic dysfunction were included in the hypertensive group (mean [ SD] age, 54 5 years). Thirty normotensive postmenopausal women (mean [ SD] age, 52 6 years) served as the control group. The clinical characteristics of the study groups are shown in Table 1. Both systolic and diastolic blood pressure were significantly higher in the hypertensive group at the baseline examination, but there was no significant difference in heart rate. Left ventricular mass index was similar in the two groups. Effects of Estrogen on Blood Pressure and Heart Rate There was no significant effect of estrogen on heart rate and blood pressure compared with placebo (Table 2). Effects of Estrogen on Doppler Parameters Peak E velocity and peak A velocity were similar at baseline in both groups. However, the E/A ratio was significantly lower and the IVRT was higher in the hypertensive group, which is indicative of left ventricular diastolic dys- TABLE 2 Effects of a single dose of oral estrogen on clinical and Doppler echocardiographic parameters in the study groups. Study group Hypertensive subjects Normotensive subjects Variable Baseline Placebo Estrogen Baseline Placebo Estrogen Heart rate (bpm) 74 14 76 18 78 12 77 15 72 14 71 20 Systolic BP (mm Hg) 160 20 162 18 160 18 126 13 128 10 118 28 Diastolic BP (mm Hg) 102 10 104 12 106 14 78 9 78 10 80 8 E (m/s) 0.65 0.21 0.66 0.22 0.78 0.20 0.85 0.23 0.84.026 0.84 0.24 A (m/s) 0.91 0.18 0.90 0.16 0.86 0.18 0.67 0.23 0.66 0.24 0.68 0.20 E/A ratio 0.73 0.11 0.71 0.10 0.84 0.20 1.23 0.28 1.24 0.28 1.22 0.24 IVRT (ms) 124 20 123 20 105 13* 93 12 90 12 89 14 Note: All values are means SD. A peak velocity of mitral late filling; BP blood pressure; bpm beats per minute; E peak velocity of mitral early filling. * P.005 vs. placebo. 68 Fak et al. Estrogen and diastolic dysfunction Vol. 73, No. 1, January 2000

FIGURE 1 The effect of a single dose of oral estrogen on the mitral E/A ratio in postmenopausal women. FIGURE 2 The effect of a single dose of oral estrogen on the IVRT in postmenopausal women. *P.005 vs. placebo. function. The effects of estrogen and placebo on the Doppler flow parameters are given in Table 2. Estrogen had no significant effect on any of the Doppler parameters or ratios in the normotensive group. Likewise, peak E and peak A velocities did not change significantly with estrogen treatment, whereas a slight, albeit not significant, improvement in the E/A ratio was observed in the hypertensive group (Fig. 1). Further, estrogen treatment significantly reduced the IVRT in the hypertensive group from 124 20 ms to 105 13 ms (P.005 compared with placebo) (Fig. 2). DISCUSSION Epidemiologic observations suggest that postmenopausal women who take HRT have less coronary heart disease (18, 19) and even lower rates of cardiac-related mortality (20). The mechanism by which HRT may prevent cardiovascular disease after menopause is most likely multifactorial and includes the effects of estrogen on lipid metabolism and hemostatic factors as well as direct hemodynamic effects (2). The presence of estrogen receptors has been demonstrated in the cardiovascular system of baboons (21), rats (22, 23), and humans (24, 25). Therefore, it has been postulated that the cardiovascular system is a target tissue for estrogen, and that estrogen may play a significant role in the regulation of cardiovascular function (22). Left ventricular diastolic function indices seem to be affected in the postmenopausal state. Premenopausal women have been shown to have lower left ventricular wall thickness (26) and higher peak E velocities and E/A ratios on Doppler mitral inflow (27) compared with postmenopausal women. Diastolic dysfunction that can be described as an abnormality in left ventricular relaxation is common in patients with hypertensive and ischemic heart disease, and in those with hypertrophic cardiomyopathy (28). Both noninvasive and invasive studies suggest that left ventricular diastolic function also deteriorates with age (29). Long-term administration of numerous antihypertensive drugs has been shown to improve diastolic function in hypertensive patients (28). This effect has been ascribed mainly to the reversal of coexisting left ventricular hypertrophy. However, regression of left ventricular hypertrophy has not always been associated with improvement in diastolic function in other studies (30, 31). Further, improvement in diastolic function also has been observed in hypertensive patients after the acute administration of nifedipine (32, 33) and verapamil (34), which suggests an acute systemic and/or direct myocardial effect. Similarly, long-term estrogen use also has been found to improve diastolic filling in postmenopausal women. Voutilainen et al. (5) reported an association of hormonal therapy with a higher early-to-atrial peak transmitral velocity ratio. In this retrospective study, postmenopausal women who took HRT had younger-looking echocardiographic diastolic flow parameters compared with those who did not take HRT. In addition, hormonal replacement had significant effects on these parameters when the confounding factors were considered. Another retrospective study reported normalization of menopause-induced diastolic dysfunction indices (35). However, Pines et al. (6) reported that 8 weeks of estrogen therapy had no influence on Doppler velocities in healthy postmenopausal women. FERTILITY & STERILITY 69

In the present study, the acute effect of a single dose of estrogen was investigated and an improvement in IVRT was found in patients with systemic hypertension and impaired left ventricular diastolic filling. There was no significant effect of estrogen on diastolic filling in normotensive postmenopausal women who had normal diastolic filling parameters; however, a trend toward normalization of peak E and peak A velocities and a significant reduction in IVRT in hypertensive postmenopausal women was observed. The finding that estrogen had no effect on Doppler velocities in healthy postmenopausal women is compatible with the findings of Pines et al. (6). However, the improvement in IVRT in the hypertensive group who had impaired left ventricular filling is remarkable. The improvement in diastolic filling could be related to the effects of estrogen on systemic hemodynamics or the heart rate, or to its direct action on the myocardium. A systemic effect of estrogen on peripheral vascular resistance was suggested in a study with long-term estrogen replacement therapy using conventional doses (36). In this study, no effect on systemic blood pressure was noticed, but a significant decrease in carotid artery impedance was demonstrated after 9 weeks of estrogen therapy in postmenopausal women. The acute administration of 17 -E 2 also was shown to increase forearm blood flow and to decrease resistance (37). Likewise, 10 weeks of HRT has been reported to improve aortic peak flow velocity, the velocity time integral, and mean acceleration (38). All these parameters are known to decrease with menopause (39). The finding that estrogen replacement reduces arterial impedance (36) could result, at least in theory, in more rapid left ventricular relaxation as a result of reduced late systolic loading of the ventricle, as reported by Ochi et al. (40). However, this was not the case in our study, as there was no effect of estrogen on blood pressure. On the other hand, HRT has been reported to exert some positive inotropic effects (41, 42). Sites et al. (43) recently reported that short-term HRT increased cardiac output and the ejection fraction, possibly through a direct myocardial effect. The inotropic state induced by estrogen could contribute to the improvement in the IVRT in hypertensive patients; however, cardiac output and hemodynamic parameters were not examined in our study. Estrogen also has been regarded as having a calcium antagonistic effect (44, 45), and this, in addition to its effect on arterial impedance, could be one of the mechanisms underlying the improvement in the IVRT in hypertensive postmenopausal women. However, because of the absence of invasive measurements, the data do not conclusively prove any of these explanations. Additional short- and longterm studies involving hemodynamic measurements are needed to prove that this reflects direct myocardial effects of estrogen as a sex steroid rather than, or in addition to, indirect effects through changes in arterial impedance. Although women who take long-term estrogen therapy have been reported to have slower heart rates (46), we could not detect a significant change in the heart rate with a single dose of estrogen in the present study and therefore excluded it from consideration as another mechanism of action in the estrogen-induced improvement in the IVRT. Our study has some limitations. First, the number of patients studied was relatively small, so the results of the study should be interpreted cautiously. Second, a low dose of estrogen was given and the drug was administered only once. Thus, the possibility that higher doses of estrogen over a longer period could have different effects needs to be investigated in further studies. 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