© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Aortic Plaque Size and Endometrial Response in Cholesterol-Fed Rabbits Treated With Estrogen Plus Continuous or Sequential Progestin
From the Department of Occupational and Social Medicine (U.B., B.B., N.G., F.W.S.), and the Department of Pathology (M.W.), University of Tübingen; and the Department of Internal Medicine, Division of Cardiology, University of Ulm (S.H., G.F., H.H.), Germany.
Correspondence to Ute Brehme, PhD, Department of Occupational and Social Medicine, Wilhelmstr. 27, 72074 Tübingen, Germany. E-mail ute.brehme{at}uni-tuebingen.de
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Abstract |
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Abstract—ERT is associated with a reduced incidence of coronary risk and cardiac events in postmenopausal women, but increases the risk of endometrial hyperplasia and carcinoma. Combined estrogen and progestin therapy protects the endometrium; however, its effects on heart disease risk factors are not completely known. In our study, 56 ovariectomized New Zealand White rabbits in 7 groups received a 0.5% cholesterol diet for 12 weeks. Controls were not treated with hormones. All other animals received (per kilogram body weight per week) intramuscular injections of either 0.3 mg estrogen (estradiol valerate) alone, 8.3 mg progestin (hydroxyprogesterone caproate) alone, estrogen and progestin continuously in 3 different dosages (0.3 and 8.3 mg; 1 and 8.3 mg; or 1 and 2.8 mg; estrogen and progestin, respectively), or 1 mg estrogen with 25 mg progestin sequentially in 2-week cycles. Eight non-ovariectomized animals served as further controls for endometrial analysis. Morphometric analysis of plaque size in the aortic arch showed that estrogen monotherapy, and the 3 combined therapies with 1 mg estrogen, significantly reduced intimal thickening (P<0.05). The application of progestin alone had no effect on plaque size. The endometrium was enlarged by 3-fold after estrogen treatment, and was decreased by half after progestin treatment, compared with control uteri (P<0.05). In all groups with combined hormone regimens, endometrial size was not significantly different from control uteri. However, these uteri showed more inflammatory reactions, especially when higher doses of hormones were given. In this animal model, doses of progestin that are able to successfully reduce the proliferative effect of estrogen on endometrium do not diminish the desirable antiatherosclerotic properties of estrogen.
Key Words: atherosclerosis • estrogen • progestin • endometrium • rabbits
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Introduction |
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Natural menopause is associated with an increased incidence of coronary risk and cardiac events.1 Numerous studies have evaluated the relationship between estrogen replacement therapy (ERT) and the risk of coronary heart disease (CHD) in postmenopausal women. ERT is thought to reduce morbidity and mortality from CHD
50% in women using unopposed oral
estrogen.2 3 Proposed mechanisms involve favorable changes
in lipid and lipoprotein levels,4 5 6 as well as less
well-understood nonlipid effects, including direct actions on the
vascular endothelium and vascular smooth muscle
cells.7 8 Because unopposed long-term estrogen therapy
considerably increases the risk of developing endometrial hyperplasia
and endometrial cancer, the concomitant use of progestogens has been
recommended for women with an intact uterus.9 10 11 Combined
estrogen-progestin regimens effectively relieve menopausal
symptoms,12 13 and protect the endometrium from the
carcinogenic effects of estrogen.10 However, possible
adverse effects of progestins on the atheroprotective properties of
estrogen are a matter of debate.14 Compared with unopposed
estrogen treatment, both favorable15 and
unfavorable16 17 18 effects of combined hormone replacement
therapy (HRT) on HDL cholesterol have been reported.
Recently published data from the Nurses' Health Study, however,
suggest that the beneficial effects of estrogen therapy for the
prevention of CHD are not attenuated by addition of
progestin.19 Animal studies involving combined estrogen-progestin therapy have focussed predominantly on hormone effects on coronary vessels or the aorta. Depending on the type and dose of the progestational agents added, in different experimental studies using mostly monkeys and rabbits, the beneficial lipid or cardiovascular effects of estrogen were found to be maintained as well as reduced. In studies using natural progesterone, no attenuation of the estrogenic effect was found,20 21 except for a study by McKinney et al,22 where progesterone impeded the positive influence of estrogen on LDL oxidation. When medroxyprogesterone acetate or other progestins were used, the cardioprotective effects of estrogen were reduced in most,23 24 25 26 27 but not all,28 studies. The uterus was rarely analyzed in these studies.
The present study was designed to investigate the effects of estradiol valerate plus hydroxyprogesterone caproate on intimal plaque development in the aortic arch, and on the endometrium, of rabbits. Three regimens with continuous-combined, and one regimen with sequential-combined, progestin application were tested. Progestational agents are added to ERT to counteract the risk of increased endometrial hyperplasia and carcinoma. Because the endometrium is the main target organ of steroid hormones, it is important to analyze in what way hormone doses used in animal models for evaluating atherogenesis affect the endometrium.
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Methods |
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Adult female New Zealand White rabbits (n=56) with body weights between 2.5 and 3.0 kg were supplied by Charles River (Kißlegg, Germany). They were allowed to acclimatize in the laboratory environment for 3 weeks. Animals were housed individually with 12-hour light periods and had free access to food and water throughout the study period. At the beginning of the study, they were ovariectomized bilaterally under anesthesia (ketamine hydrochloride, xylazine hydrochloride, and atropine sulfate). They were then randomized into 7 groups of 8 rabbits each and were fed a standard diet supplemented with 0.5% cholesterol (Altromin) for the study period of 12 weeks. Once a week, estrogen and/or progestin were administered intramuscularly. Estrogen was given as estradiol valerate (Progynon-Depot-10), and progestin as hydroxyprogesterone caproate (Proluton Depot), both from Schering AG. Group 1, the controls, received no hormones; group 2 received estrogen; group 3 received progestin; and groups 4 to 7 received both hormones. In groups 4, 5, and 6, hormones were given continuously, once a week. In group 7, a sequential regimen was followed, with estrogen given continuously, and progestin given sequentially, in 2-week cycles during weeks 3 and 4, weeks 7 and 8, and weeks 11 and 12 (Table 1
).
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Blood was collected at the beginning of the study, and on days 28, 56, and 84 (1 week after the last hormone injection). After an overnight fast of 18 hours, animals were anesthetized and blood was collected from the central ear vein into EDTA-containing tubes, and was centrifuged at 4°C for 10 minutes at 3000 rpm. All experimental procedures were approved by the Animal Research Committee of the regional governmental authorities and conformed to the American Heart Association Guidelines for Use of Animals in Research.
Plasma cholesterol concentrations were determined enzymatically (CHOD-PAP-Method, Boehringer Mannheim). The area under the curve (AUC) was calculated using the 4 different measured concentrations of total plasma cholesterol. AUC represents the total plasma cholesterol to which the arteries were exposed over the duration of the study period. For determination of plasma triglyceride concentrations, an enzymatic method (Peridochrom Triglyceride GPO-PAP-Method, Boehringer Mannheim) was also used.
Plasma levels of 17ß-estradiol and 17
-hydroxyprogesterone were
determined by radioimmunoassay (DPC Biermann GmbH). The 4 measured
values of 17ß-estradiol and 17-hydroxy-progesterone were also
calculated as AUC.
On day 84, animals were euthanized with an intravenous injection of 2 mL of the barbiturate T61 (Hoechst Roussel Vet GmbH). The aortic arch and uterus were excised and immersion-fixed in 0.1 M cacodylate-buffered 2% paraformaldehyde solution for at least 24 hours.
The aortic arch was divided into 3 sections: the first section was
0.5 cm proximal to the ostium of the innominate artery; the second
section was located between the left carotid artery and the left
subclavian artery; and the third section was a descending segment of
the aortic arch 0.7 cm beneath the subclavian artery.
Paraffin-embedded tissue samples were sliced 4 µm thick and
stained with Elastica-van-Gieson (EvG). The extent of the
neointimal plaque was measured in the 3 sections using a
digital image analyzer (software from Bilany Consultants GmbH).
The arithmetic mean was calculated and used for further statistical
analyses of the plaque size in the aortic arch.
The bicornus uterus was cut transversally at its broadest part. After paraffin embedding, 4-µm-thick slices of tissue were cut and hematoxylin-eosin (HE) stained. Both endometrial areas were measured in as many slices as necessary to determine the maximum area. A blinded histopathologic analysis of the HE-stained uterus sections was performed by one of the authors (M.W.), evaluating inflammation, necrosis, branching of mucosal folds, edema, and vascularization (0, not present; +, <25%; ++, 25% to 50%; +++, >50%). Uteri with major pathological changes were stained with a mouse monoclonal antibody against rabbit macrophages (RAM 11, DAKO). Immunohistological detection of macrophages was performed with the avidin-biotin method combined with hemalaune staining. The uteri of the control animals were not included in the analysis, but the 8 uteri from non-ovariectomized rabbits of similar age fed a normal standard diet without cholesterol supplementation were included, representing normal organs from untreated animals.
Statistical Analysis
One-way analysis of variance (ANOVA) was performed on
the data. AUCs of 17-hydroxyprogesterone and final
triglyceride concentrations were analyzed by Welch
ANOVA, allowing unequal variances. Data representing
intimal plaque size, endometrium area, and AUC of 17ß-estradiol were
used after log10 transformation because of the
nonhomogenous variance between groups. Equality of group variances was
analyzed by Bartlett's test. Although statistical
analyses were performed with transformed data, original data
are shown in the figures and cited in the text. For multiple
comparisons between the different treatment groups, the Tukey-Kramer
test was used when ANOVA showed an F-value <0.05. Intimal plaque size
was also analyzed in an analysis of covariance
(ANCOVA) by adding AUC of total plasma cholesterol as a
covariate of the model.
Log10 transformed data of intimal plaque size were correlated with the parameter AUC of total plasma cholesterol using Pearson's correlation coefficient.
Statistical analyses were carried out with JMP (Version 3.1.6.2, SAS). Data are presented as mean±SD, unless stated otherwise. P<0.05 was considered to indicate statistical significance for all tests.
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Results |
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Body Weight
At the beginning of the study, animals had a mean body weight of 2.75±0.16 kg with no significant differences between the single groups. At the end of the study, animals with estrogen monotherapy (group 2) had the highest body weight, whereas animals with progestin monotherapy (group 3) had the lowest body weight. Compared with controls, body weights in groups 2 and 4 were significantly higher (Table 2
).
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Plasma Hormone Concentrations
Initial values of 17ß-estradiol and 17-hydroxyprogesterone
were below detectable limits in all animals.
In animals of group 1 and group 3, which were not treated with
estrogen, levels of plasma 17ß-estradiol remained below the detection
limit throughout the study period. After 12 weeks, final
17ß-estradiol levels in the groups receiving the high estrogen dosage
and continuous progestin treatment reached values of 1420±723 pmol/L
(group 5) and 1397±803 pmol/L (group 6). The concentration of
17ß-estradiol was lower in animals with sequential progestin
treatment, but not significantly different from groups 5 and 6. Animals
in groups 2 and 4, receiving the lower estrogen dosage, showed final
17ß-estradiol concentrations of 414±184 pmol/L and 185±81 pmol/L,
respectively, which were significantly lower than those of groups 5 and
6 (Table 2
). AUC of 17ß-estradiol was significantly lower in
groups 2 and 4, compared with groups 5, 6, and 7 (Figure 1).
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In group 1, group 2, and 3 of the 5 progestin-treated groups (3, 4, and
6), 17-hydroxyprogesterone levels were not detectable one week after
the last hormone injection. An increase in 17-hydroxyprogesterone
concentrations was observed in groups 5 and 7, with final values of
1.5±0.6 nmol/L and 0.4±0.1 nmol/L, respectively (P=0.001,
Table 2). AUC of 17-hydroxyprogesterone was also
significantly higher in group 5 compared with group 7 (16.2±7.2 versus
7.1±2.3 nmol/L · 12 weeks; P=0.009).
Total Plasma Cholesterol
Before cholesterol feeding, animals had an average
total plasma cholesterol concentration of 1.6±0.5
mmol/L. No significant differences (ANOVA, P=0.15) were
found between the groups (Table 2).
Final total cholesterol concentrations, which ranged from
61.4±10.2 mmol/L (group 2) to 35.2±12.3 mmol/L (group 7),
were significantly different by ANOVA (P=0.0003). With the
exception of the control group, all other groups had significantly
lower final cholesterol values compared with group 2, which
received estrogen monotherapy (P<0.05). When the whole
period of cholesterol exposure was considered, AUC of total
plasma cholesterol also showed significant differences
(ANOVA, P<0.0001). It was lower in animals administered 1
mg/kg body weight per week of estrogen (groups 5, 6, and 7) compared
with controls and animals administered 0.3 mg/kg body weight per week
of estrogen (P<0.05); progestin-treated animals and group 4
animals (administered progestin combined with the lower estrogen
dosage) were not significantly different from the other groups. There
were no significant differences in AUC of total plasma
cholesterol between the 4 groups administered the combined
treatment (Figure 2).
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Plasma Triglycerides
Initial plasma triglyceride levels, after an overnight
fast, were between 0.5±0.1 mmol/L (group 6) and 0.8±0.2
mmol/L (group 1), and were not significantly different (ANOVA,
P=0.13). Final values were in the same range as initial
values, and did not differ significantly between the groups (Welch
ANOVA, P=0.06; Table 2).
Intimal Plaques in the Aortic Arch
Plaque development in the aortic arch after 12 weeks of
cholesterol feeding was significantly different between the
groups (ANOVA, P<0.0001). Animals treated with estrogen
alone (group 2) showed a significantly smaller plaque development
(0.9±0.7 mm2) compared with controls
(3.5±1.7 mm2). The largest plaques
(3.9±1.6 mm2) were observed in the group
treated with progestin alone, but they were not significantly different
from controls. Compared with controls, plaque development was not
significantly reduced in group 4, administered estrogen at the lower
dosage of 0.3 mg/kg per week combined with 8.3 mg/kg per week of
progestin (1.7±1.2 mm2). However, the
extent of atherosclerotic lesions after administration of continuous
(group 5, 0.8±0.7 mm2; and group 6,
0.7±0.4 mm2) or sequential (group 7,
0.5±0.4 mm2) progestin application in
combination with the higher dosage of estrogen (1 mg/kg per week), was
significantly smaller than in controls and progestin-alone treated
animals. No differences in plaque development were seen between
continuous and sequential hormone regimens with identical estrogen
dosages (Figure 3).
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As mentioned above, AUC of total plasma cholesterol differed widely between the groups. Because there was a correlation between AUC of total cholesterol and intimal plaque size of r=0.51 (P=0.0001, n=56), we analyzed the intimal plaque size, taking into consideration the differences in AUC of plasma cholesterol as a covariate in an ANCOVA model. All statistically significant differences in plaque size from ANOVA remained significant in the ANCOVA model.
Endometrium
The endometrial area of untreated animals had an extension
of 8.7±2.7 mm2. Compared with these normal
controls, a significant increase was observed after single estrogen
treatment (23.7±3.7 mm2) and a significant
decrease was observed after single progestin treatment (3.9±0.7
mm2). In the 4 groups with combined hormone
treatment, the extent of endometrium was not significantly different
from those of control animals (P>0.05). When progestin was
administered in 2-week cycles, significantly larger endometria were
seen versus groups 5 and 6 (continuous progestin treatment; Figure 4).
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Inflammatory, necrotic, and other changes of endometria were not
observed in control animals (group 1) and rarely observed in animals
treated with one hormone alone (groups 2 and 3). Moderate changes were
seen in group 4, which was administered both hormones in the same
dosages as in groups 2 and 3; and in group 6, where the lowest
progestin dose was used. In the 2 other groups with combined treatment
(groups 5 and 7), inflammatory and necrotic alterations were more
frequent. A comparison of groups 5 and 6, which had received the same
estrogen dose but different progestin dosages, showed that uteri were
in better histopathologic condition after exposure to the lower
progestin dosage. Comparing groups 4 and 5, which had received the same
progestin treatment but different estrogen dosages, uteri of animals
administered the lower estrogen dosage exhibited fewer pathological
transformations (Table 3).
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The typical structure of rabbit endometrium, showing 6 major
extensively branched mucosal folds, persisted under hormone
monotherapy. With combined treatment, this morphology vanished
completely in groups 5 and 7, and to a lesser extent in groups 4 and 6.
In groups 5 and 7, large necrotic and inflammatory areas with
calcification and expression of macrophages were detected
(Figure 5).
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); (I) group 7, necrosis of the endometrium with inflammatory
reaction of macrophages ( |
Discussion |
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In this animal study, estrogen monotherapy, progestin monotherapy, and 4 different combined estrogen-progestin treatment regimens (3 with continuous and one with sequential progestin application), were investigated. All 4 combined regimens were effective at reducing the action of estrogen on endometrium proliferation; three of them also resulted in a significantly reduced neointimal plaque size in the aortic arch compared with controls.
In agreement with previous animal studies,29 30 31 estrogen monotherapy reduced plaque development, whereas progesterone alone had no significant effect. Haarbo et al28 found no inhibiting effect of norethisterone acetate and levonorgestrel on estrogen action when orally applied to rabbits in a 3- and 6-fold lower dosage than 17ß-estradiol, respectively. In group 4 animals, which were treated with the lower estrogen (0.3 mg/kg per week) and the higher progestin dose (8.3 mg/kg per week), progestin attenuated the beneficial estrogen effect on plaque growth. The same progestin dose, however, showed no attenuating effect in group 5 when combined with 1 mg/kg per week of estrogen. In a similar study, which used higher hormone dosages of 1 mg/kg per week of estrogen and 25 mg/kg per week of progestin, the protective estrogen effect was diminished.25 In the present study, a 25-mg/kg dose of progestin was also used, but applied in 2-week cycles. This 50% lower dosage did not cause inhibition of the atheroprotective estrogen action. These results show that the absolute estrogen dose, as well as the relation between estrogen and progestin dosages, is important for the maintenance of the atheroprotective estrogen action.
The hormone doses used in the present study resulted in hormone
concentrations quite higher than physiological
levels usually observed in rabbits. Batra and
Källstrand32 showed that 17ß-estradiol levels of
adult rabbits are between 40 and 228 pmol/L. In
postmenopausal women, 17ß-estradiol concentrations are 300 pmol/L
with ERT and 50 pmol/L without ERT.33 After
intramuscular injection of 5 mg estradiol valerate, a high estradiol
peak level of 2200 pmol/L was observed within 2 days. After 6 days,
estradiol levels were down to 350 pmol/L.34 In rabbits
treated with 0.3 mg estrogen valerate, we observed similar
17ß-estradiol levels of 200 to 400 pmol/L seven days after
injection. The estrogen dosage of 1 mg/kg per week, however, resulted
in concentrations 2- to 8-fold higher.
In addition to hormone dose and timing of administration, different types of estrogen and progestational agents can have different effects on the cardiovascular system. Oral estrogens are usually given as conjugated equine estrogen. Parenteral preparations include conjugated estrogens and estradiol esters, such as estradiol valerate.35 Oral estrogen monotherapy, in the form of conjugated estrogens or estradiol, is known to reduce LDL cholesterol and increase HDL cholesterol.5 Progestational agents used in HRT are derivatives of either progesterone, eg, medroxyprogesterone acetate, or testosterone, eg, 19-nortestosterone derivatives such as norethisterone acetate or levonorgestrel.34 Most 19-nortestosterone derivatives have androgenic activity, and, in combination with estrogen, LDL cholesterol levels are reduced to a lesser extent than with unopposed estrogen. Derivatives of progesterone do not seem to have adverse effects on lipid metabolism. The combination of natural estrogens, such as estradiol valerate, and cyclical progesterone, seem to have the most favorable impact on lipids and lipoproteins in women.35 In rabbits, orally administered estrogen or progestin did not produce significant differences in total serum cholesterol.31 In our study, intramuscular monotherapy with these hormones also showed no effect on the AUC of total cholesterol compared with controls. However, animals administered high dose estrogen in combined treatment showed a decrease in AUC of total cholesterol. Because animals had free access to food, and thus cholesterol intake was not controlled, it is difficult to exactly determine to what extent hormone treatment was responsible for the decrease in plasma cholesterol concentrations. Because there was a significant reduction in AUC of total cholesterol in group 5 (which had received the same progestin dose but the 3-fold higher estrogen dose compared with animals in group 4), but not in group 4, it can be hypothesized that estrogen affected plasma cholesterol concentrations.
Three studies29 36 37 provided information on the lipid effects of intramuscular injection of estrogens to cholesterol-fed rabbits. In the studies by Hough and Zilversmit,29 and Fischer and Swain,36 estrogen given as 0.5 mg/kg per week of 17ß-estradiol cypionate and 50 µg per week of estradiol, respectively, did not affect lipid metabolism. In contrast, Kushwaha and Hazzard37 found that a dosage of estradiol cypionate between 0.2 and 0.5 mg/kg per week considerably decreased the diet-induced rise in total plasma cholesterol, compared with cholesterol-fed rabbits not treated with hormones. In women, nonoral estrogen therapies produce less-pronounced favorable effects on LDL and HDL cholesterol than their oral counterparts.38 However, triglyceride concentrations, known to be increased by oral HRT therapy,11 are not altered by transdermal hormone application.39 40 In rabbits, no change in triglycerides was observed with intramuscular hormone treatment. Although our results of total plasma cholesterol and triglycerides were comparable to the situation in humans, one disadvantage of the rabbit model has to be considered. The lipid and lipoprotein profile of rabbits is different from that of humans. VLDL cholesterol predominates in rabbits, LDL cholesterol in humans.41 Therefore, results of hormone treatment on changes in lipid metabolism obtained in rabbits should be cautiously extrapolated to humans.
A number of nonlipid effects of estrogen for protection against atherosclerosis have been discussed, ie, endothelium dependent and independent effects, hemostasis and direct antiatherosclerotic effects.42 In the rabbit model used here, the majority of atheroprotective estrogen effects must be lipid-independent because the neointima was significantly smaller in groups with combined treatment compared with controls, even when AUC of total cholesterol was added as a covariate to the statistical analyses.
Prolonged use of unopposed estrogen was found to be associated with a
10-fold increase in the risk of endometrial cancer.43
Therefore, the concomitant use of sequential or continuous progestins
is considered mandatory for women who have an intact
uterus.44 A number of studies showed that endometrial
hyperplasia, caused by treatment with unopposed estrogen, can be
effectively prevented when estrogen is combined with a
progestin.45 46 47 In the endometrium of the rabbits
studied, the proliferative activity of estrogen was seen in group 3
animals, who underwent estrogen monotherapy. Compared with untreated
intact controls, endometrium size was increased 2.7-fold. This result
is in agreement with a study examining the uteri of ovariectomized or
sham-operated cholesterol-fed rabbits after a 45-week study
period. Uteri were found to be 2.5-fold bigger in rabbits fed a diet
supplemented with estrogen, and >50% smaller in ovariectomized
animals without hormone treatment, compared with
controls.48 In the group treated with progestin alone, we
observed a 55% reduction in endometrial size. Thus, progestin did not
seem to have a stimulating effect on uterine tissue proliferation.
Batra and Källstrand,32 who observed a tendency for
a cyclic pattern in 17ß-estradiol levels of rabbits, supposed that a
minimal estrogen concentration is necessary for optimal response of
endometrium to progesterone.
When applied in combination with estrogen, progestin doses were clearly
able to confer protection against the undesired proliferative action of
estrogen. In all 4 groups with combined treatment, uteri showed neither
an increase, nor a reduction, in endometrial size, compared with
controls. This was also true for animals without measurable plasma
progesterone concentrations in groups 4 and 6. Considered over the
whole study period, animals treated with 1 mg/kg per week of estrogen
were found to have 4- to 5-fold higher plasma levels of
17ß-estradiol than animals treated with 0.3 mg/kg of estrogen.
Nevertheless, progestin was able to counteract estrogenic actions on
the uterus at both plasma concentrations. The lowest dosage of
continuously administered progestin, 2.8 mg/kg per week in group 6, was
as effective as the 3-fold higher continuous dosage in group 5. Because
the lowest dose of progestin used in this study was fully capable of
attenuating proliferative estrogen action on the uterus, even lower
doses might be adequate. Although no statistically significant
difference in endometrium size was observed between controls and
sequentially treated rabbits, the continuous regimens were advantageous
over sequential therapy with respect to endometrial response. Despite
the fact that the progestin dose was highest in the sequential therapy,
the endometrium was significantly enlarged compared with the 2
analogous continuous-application groups.
Furthermore, histopathologic evaluation showed that uteri of rabbits treated with combined therapy exhibited inflammatory or necrotic changes less frequently when the lowest progestin dose was administered. However, uteri of animals treated with one hormone alone showed no signs of inflammation or necrosis. Thus, in the rabbit uterus, combined hormone treatment has more side effects than the identical doses given as monotherapy.
The dosage of 25 mg/kg of progestin applied in 2-week cycles (group 7)
caused scores of pathological transformations higher than those of the
other 3 groups with combined treatment. Although the dosage of 8.3
mg/kg per week of progestin (group 5) added up to a 33% lower dose
over the entire study period, AUC of 17-hydroxyprogesterone was
significantly lower in group 5 than in group 7. It could be speculated
that progesterone concentrations after the injection of 25 mg/kg of
hydroxyprogesterone caproate reached higher peaks, which might have
caused the damage. From our results, it could not be determined with
certainty whether the sequential application form, or the high dose of
progestin, was responsible for the undesired changes seen in group 7.
Generally, continuous treatment seems to be superior to sequential
treatment with respect to endometrial reactions in rabbit uterus.
Considering results of all groups, however, hormone dose seems to be
more important than the timing of administration.
The parallel examination of hormone effects on plaque development in aortic vessels and on the uterus of rabbits revealed that there could be a different hormone threshold for cardiovascular and endometrial effects. Results in group 4 showed that the estrogen dose required for protective effects in the aortic arch was substantially higher than the dose able to produce effects on the endometrium. On the other hand, progestin doses able to attenuate negative estrogen effects on the endometrium did not impede beneficial estrogen actions at the vessel wall (groups 5, 6, and 7). A study in apoE-deficient mice also showed that the atheroprotective effects are achieved at higher 17ß-estradiol levels than those required by other estradiol target tissues, such as uterus.49
The rabbit model provided information about atheroprotective estrogen properties in relation to different progestin doses, and about continuous versus sequential progestin application regimens. Doses applied for the investigation of estrogen and progesterone action in cholesterol-fed animals are reasonable because they are in ranges not only appropriate for cardiovascular factors, but also for the uterus.
In this animal model, doses of progestin were able to successfully reduce the proliferative effect of estrogen on endometrium without diminishing the desirable antiatherosclerotic properties of estrogen. The rabbit model could therefore also be valuable for testing new antiestrogenic substances as well as HRT regimens involving different types of estrogens and progestogens with respect to cardiovascular and endometrial effects.
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Acknowledgments |
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The authors wish to thank H. Bacher, A. Glückman, M. Heilig, M. Holz, G. Kaletta, C. Lenz, and J. Schatz for technical assistance; Mag. A. Rosenberger, Institute of Medical Information Processing, University of Tübingen, for his help with the statistical analysis; and Dr P.F. Kahle for his help with the English manuscript.
Received September 1, 1998; accepted December 29, 1998.
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