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Department of Women’s and Children’s Health, Section for Obstetrics and Gynecology (T.N., K.R.-M.), and Department of Geriatrics (H.L.), University Hospital, SE-751 85 Uppsala, Sweden
Address all correspondence and requests for reprints to: Tord Naessen, M.D., Ph.D., Section for Obstetrics and Gynecology, University Hospital, SE-751 85 Uppsala, Sweden. E-mail: Tord.Naessen{at}kbh.uu.se
Abstract
To determine whether ultra-low doses of estradiol (E2) affect the serum lipid profile in elderly women, we analyzed changes in serum lipids and lipoproteins in 70 healthy women, 60 yr and older, randomly assigned to parenteral E2 (7.5 µg per 24 h) delivered by a vaginal ring (Estring; Pharmacia-Upjohn, Malmö, Sweden) or no treatment for 12 months. Baseline serum estrone sulfate (E1S), but not E2 or serum FSH, was negatively associated with serum total cholesterol (P = 0.026), low-density lipoprotein (LDL) cholesterol (P = 0.053), and apolipoprotein B levels (P = 0.023). Compared with no treatment, Estring treatment yielded nonsignificant increases within the normal postmenopausal range in serum E1S (+16%) and E2 (+13%), but significantly reduced serum LDL cholesterol by 7.6% (-0.32 mmol/L; 95% confidence interval, -0.58, -0.07; P = 0.014) and LDL to high-density lipoprotein (HDL) ratio by 7.3% (-0.19 mmol/L; 95% confidence interval, -0.44, -0.06; P = 0.030). In Estring users values were significantly reduced in total cholesterol (by 4%), LDL cholesterol (by 7%), LDL to HDL ratio (by 7%), and apolipoprotein B (by 4%), and significantly increased in serum HDL triglyceride (by 25%) but not triglycerides. No significant changes were found in the untreated group. There was a significant interaction between age and both baseline serum E2/sex hormone-binding globulin (P = 0.006) and sex hormone-binding globulin (P = 0.009) and a marginal interaction between age and E1S (P = 0.083) with regard to effects on changes in LDL cholesterol levels during Estring treatment. We conclude that ultra-low doses of E2, which previously were considered to have only local effects, may improve serum lipid profile in elderly women with a pattern and magnitude similar to that reported after conventional estrogen doses or first-generation lipid-lowering agents. The reduction in LDL cholesterol tended to be greater with a combination of high age and low baseline levels of biologically active estrogens.
THE LOW RISK of cardiovascular diseases in women before menopause and the increased risk thereafter, approaching that in men (1), has been attributed to effects of endogenous estrogens (2). Menopause is associated with changes toward a more atherogenic serum lipid profile (2), but menopausal estrogen or estrogen/progestin therapy restores the profile to one similar to that in premenopausal women (3, 4). In epidemiological studies estrogen therapy has been associated with a 40–70% reduction in the incidence of and mortality from coronary heart disease (CHD) events (5, 6, 7, 8). Estrogen therapy lowers the low-density lipoprotein (LDL) cholesterol level by 10–15% (4, 9). Each percent reduction of serum LDL cholesterol is associated with a 2% reduction of the risk of CHD (6, 10). Thus, about 30% of the decrease produced by estrogen therapy in the risk of CHD has been attributed to the improved serum lipid profile (5). The mechanism of the remaining cardioprotective effect is probably multifactorial, including direct effects on the vessel wall structures (11) and effects on atherosclerotic accumulation independently of changes in serum lipids (12), and on endothelium-dependent vasodilation (13).
National guidelines for both primary and secondary prevention of CHD events focus mainly on the reduction of LDL cholesterol (14). Modern generations of lipid-lowering agents reduce LDL cholesterol by about 20–30% (15, 16, 17), but their beneficial effect on the risk of cardiovascular disease perhaps levels off at about 20% reduction or at a LDL cholesterol level of 3.2 mmol/L (16, 17). A lipid-lowering diet may reduce LDL cholesterol by up to 15% under optimal conditions (18), an effect similar to or greater than that after intervention with oral estradiol (E2) therapy (19, 20) or with the first generation of lipid-lowering agents (21).
Estrogen therapy with standard doses at higher ages has poor long-term adherence. The lowest dose of estrogens needed to substantially improve the serum lipid profile in elderly women is not known. One aim of the present study was to determine whether ultra-low doses of parenteral E2 improve the serum lipid and lipoprotein profiles, especially the LDL cholesterol level, in elderly women.
Subjects and Methods
Subjects
Seventy women, 60 yr of age and above, recruited by advertisement in the local media, were randomly assigned either to treatment with a vaginal ring (Estring) releasing 7.5 µg per 24 h of E2, or to no treatment, for 12 months, with 35 women in each group. The randomization procedure was carried out by computer-based assignment, within blocks, of numbered sealed envelopes that were opened consecutively after inclusion of each subject. Women with diseases or medications known to affect bone or lipid metabolism were excluded, as were women who had used E2 or estriol vaginal pessaries in the past 6 months. In the control group, a placebo vaginal ring was not used, because of the risk of vaginal ulceration in women without estrogen therapy.
During the 12-month study period, 7 women dropped out; 6 of 35 in the Estring group and 1 of 35 in the control group, 5 during the first 6 months and another 2 during the last 6 months. The reasons given were: sweatings (4 women), lack of time (one), had entered another clinical study (one), and desire for active treatment (one). The baseline characteristics of the dropout group were similar to and did not differ significantly from those in the remaining participants. The study followed the Good Clinical Practice recommendations, and each subject gave written informed consent. The study was approved by the local ethics committee of the Faculty of Medicine, Uppsala University (Uppsala, Sweden).
An ultra-low dose of E2, registered for local treatment of urogenital estrogen deficiency symptoms, was administered in the form of a vaginal ring (Estring), which was replaced every third month. After an initial surge in the delivery of E2 to the circulation during the first 24 h, the mean rate of delivery is about 7.5 µg per 24 h, with minor or no increase in the serum level of E2 but an increase in serum estrone sulfate (E1S) (22, 23, 24).
Methods
Duplicate serum samples were drawn some days apart between 0700 and 1000 h after an overnight fast, at baseline, and after 6 and 12 months before the E2 vaginal ring was changed. Serum samples were assayed consecutively for their cholesterol and triglyceride (TG) concentrations by enzymatic techniques (Instrumentation Laboratory SpA, Milano, Italy) in a Monarch 2000 centrifugal analyzer (Instrumentation Laboratories, Lexington, MA), with an interassay coefficient of variation (CV) of 4%. Serum high-density lipoprotein (HDL) was separated after precipitation of lipoproteins with magnesium chloride/phosphotungstate. LDL cholesterol was calculated using Friedewald’s formula (25). Serum apolipoproteins (Apo) A1 and B were assayed with use of monospecific polyclonal antibodies (Orion Diagnostica, Espoo, Finland), with an interassay CV of less than 3%. Serum Apo (a) was measured by an Elisa-kit (Mercodia, Uppsala, Sweden).
Additional biochemical parameters were assessed in single samples of serum frozen at -70 C until analyzed in batch. Serum E2 was measured by RIA, using a commercial kit (Estradiol Double Antibody; Diagnostic Products, Los Angeles, CA) with one crucial modification; instead of direct analysis of untreated serum, the serum samples were extracted with diethyl ether, the ether phase was evaporated and the dry extract was dissolved in zero calibrator. The dissolved extracts were then analyzed according to the manufacturer’s specifications. This modification was undertaken to minimize effects of differences in nonspecifically bound radioactivity between calibrators and serum samples (26). The detection limit was 6 pmol/L and the intraassay CV was 6% (26). Serum E1S was measured by RIA after enzymatic hydrolysis with Helix pomatia sulfatase-glucuronidase preparation and extraction with diethyl ether as described by Carlström and Skoldefors (27), with minor modifications. The detection limit was 0.3 nmol/L, and the intraassay CV was 7%. Serum FSH was assessed by Auto-Delfia human FSH, and sex hormone-binding globulin (SHBG) by Auto-Delfia (Pharmacia-Wallac, Inc., Turku, Finland), with intraassay CV of 2 and 4%, respectively.
Statistical methods
Baseline descriptions of the two randomized groups are presented
as mean ± SD and main differences in changes in
percentage (%). The intraindividual variation in serum LDL cholesterol
levels was estimated as CV% from the duplicate measurements according
to the following formula: CV% = 100 (SD/x) and
SD = 
d2/2n, where
x = mean of all measurements, d = difference
between duplicate measurement values, and n = number of duplicate
determinations. Means of duplicate samples were used in the analysis to
further reduce the variability. Fisher’s exact test was used for
comparing distributions of categorical parameters. Within-group changes
were analyzed by paired Student’s t test for normally
distributed variables and by Wilcoxon signed-ranks test for those not
normally distributed; between-group differences in changes were
analyzed by Student’s t test for independent samples or the
Mann-Whitney U test, respectively. Between-groups
differences in changes (%) were calculated as the difference between
the within-group percentage changes in the two groups. We also tested
the interaction between age and baseline sex hormone levels, with
regard to effects on changes in serum lipid levels during Estring
treatment. Associations between variables were assessed by Spearman’s
rank correlation test. All statistical analyses were performed with the
statistical program package JMP or Statistical Analysis System
(SAS Institute, Inc., Cary, NC).
Results
The two study groups were similar regarding a number of
descriptive variables at baseline (Table 1
), but baseline serum levels of
cholesterol, LDL cholestrol and TG were higher in the untreated group
(P < 0.02) (Table 2). With the sensitive methods
used, estrogen therapy (Estring) for 12 months, yielded minor increases
in serum E1S (+0.27 nmol/L; P = 0.004) and
E2 (+1.93 pmol/L; P = 0.027),
both within the normal postmenopausal range (Fig. 1), and not significantly different from
changes in the untreated women (+16%; P = 0.09 and
+13%; P = 0.15, respectively) (Table 2). The
intraindividual variation (CV%) in serum LDL cholesterol between
duplicate samples was 6.5%. Means of the duplicate values were used in
the analysis to further reduce the variability.
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). There was a significant interaction between age and baseline serum E2/SHBG (P = 0.006) or SHBG (P = 0.009) and a marginal interaction between age and E1S level (P = 0.083), indicating a change toward a greater reduction in LDL cholesterol levels in association with high age combined with low baseline levels of unbound estrogens.
The baseline serum E1S level was negatively associated with the serum level of total cholesterol (r = -0.27; P = 0.026), LDL cholesterol (r = -0.24; P = 0.053), and Apo B (r = -0.27; P = 0.023). Serum SHBG was positively associated with the serum HDL cholesterol (r = 0.31; P = 0.009) and Apo A1 (r = 0.28; P = 0.018) and negatively associated with TG (r = -0.25; P = 0.040). In contrast, baseline serum E2 and FSH were not significantly associated with baseline serum lipid and lipoprotein levels or their changes over time.
Body mass index (BMI) did not differ significantly between groups at baseline, and changes in BMI within and between study groups over the study period were not significant. No significant associations were found between changes in BMI and changes in serum lipid fractions.
Discussion
To our knowledge, this is the first report to indicate that ultra-low doses of parenteral estrogens, intended for local treatment of vaginal atrophy and yielding serum estrogen levels within the normal postmenopausal ranges, have systemic effects and improve the serum lipid profile in elderly women. In the E2 group, the serum total, the LDL cholesterol levels, the LDL to HDL ratio, and the Apo B level were significantly reduced and serum HDL-TG (but not serum TG) significantly increased, compared with baseline. When compared with the values in the untreated women, the LDL cholesterol level and LDL to HDL ratio still showed significant reductions. Moreover, data indicated a greater reduction in LDL cholesterol in association with high age combined with low baseline levels of unbound estrogens. Changes in BMI did not seem to explain the changes in serum lipid levels.
The magnitudes and pattern of the changes in serum lipid levels
were similar to those observed after conventional doses of parenteral
estrogens (patches) in early menopausal women (4, 9) or of
the first-generation lipid-lowering agents (21). Compared
with the postmenopausal estrogen/progestin interventions (PEPI) data
(oral conjugated equine estrogens 0.625 mg/day for 3 yr)
(3) and adjusted for changes in the placebo groups, the
change in total cholesterol was similar in the present study (-3.2%
vs. -3.5%), the change in LDL cholesterol also similar
(-7.6% vs. -10%), the increase in HDL cholesterol was
smaller (+2.4% vs. 8.8%), and the TG level showed a
smaller absolute increase (0.16 vs. 0.19 mmol/L) but a
larger percentage change (+13% vs. 5.9%). The participants
in the PEPI trial were younger than those in the present study and
had lower baseline mean levels of serum lipids except for a higher mean
level of TG. Compared with changes after 9 weeks of oral
E2 (1 mg daily), in women on average 8 yr after
menopause (20), the decrease in the total cholesterol
level in our study was smaller (-3.2% vs. -5%), the
decrease in LDL cholesterol was similar (-7.6% vs.
-10%), the increase in HDL cholesterol was smaller (+2.4%
vs. +8%), and the change in total TG was similar (+13%
vs. +14%). The increase in HDL TG was similar to that
induced by E2 patches (+12% vs. 10%)
but lower than that resulting from oral 0.625 mg conjugated equine
estrogens daily (+12% vs. 26%) (Table 3).
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Baseline serum E1S, but not E2 or FSH, was negatively associated with the serum total and LDL cholesterol and Apo B levels. This observation support the view that E1S is quantitatively an important estrogen in elderly menopausal women and that it may be a good indicator of the total estrogen status in menopausal women (26, 30). Our findings of minor increases in the serum E1S and E2 levels, totally within the normal postmenopausal range, are consistent with previous reports on this low-dose regimen (22, 23).
Subtle differences in endogenous serum estrogen levels, within the
normal postmenopausal range, may be of clinical relevance. Lower serum
E2 levels, compared with higher ones within this
range, are associated with lower bone densities (31) and
higher fracture rates in elderly women (31, 32). In a
previous pilot study serum markers for bone metabolism and bone density
were positively affected by ultra-low doses of vaginally administered
E2 (24). The longer the period of
estrogen deprivation (i.e. time after the menopause), the
greater is the capacity of estrogen-sensitive tissues to respond to
exogenous estrogens (33). The number of estrogen
-receptors increases with advancing age (34).
Furthermore, estrogen receptor sensitivity is higher in the post- than
in the premenopausal period and increases when hypoestrogenism is
induced by treatment with GnRH agonists (35). Hence,
systemic effects of ultra-low doses of E2 may
tentatively be explained by an increase in the number of estrogen
-receptors (34) and/or by an up-regulation of the
estrogen receptor sensitivity after long-standing hypoestrogenism
(35).
This low-dose regimen is registered in many countries, including the United States, for the treatment of urogenital atrophy and administered without addition of a progestin. No proliferation of the endometrium, as measured by ultrasonography, was found with this low-dose regimen during 6 months of exposure in two previous studies in 30 women with mean ages of 57 and 60 yr, respectively (23, 36). However, caution with regard to endometrial effects is warranted until the long-term effects on the endometrium are documented in a large number of women.
The doses of estrogen recommended to elderly women have long been similar to those prescribed early after the menopause. Such estrogen doses and regimens are usually associated with breast tenderness and vaginal bleeding, resulting in poor adherence to long-term therapy and to therapy started at a high age (37). Ultra-low doses of estrogen, yielding serum E2 levels within the normal postmenopausal range, may tentatively reduce the otherwise reported initial increase in thromboembolism (38) observed with ordinary doses of both conjugate equine estrogens and E2 (19, 39), have better long-term compliance (23, 40), and still maintain beneficial effects on the bone metabolism (24) and the serum lipid profile.
Acknowledgments
We are indebted to Kiki Synnöve Moen, R.N., for excellent technical assistance, to Associate Professor Kjell Carlström for expert help with the assays of serum E2 and E1S, and to Lars Berglund, B.S.c., for expert statistical advice.
Footnotes
1 This study was supported by the Swedish Society of Medicine (Grant
99-02-0248) and Pharmacia & Upjohn, Inc., Sweden. 
Received December 5, 2000.
Revised January 30, 2001.
Accepted February 5, 2001.
References
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