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Original Studies |
Department of Clinical Science, Section of Obstetrics and Gynecology (K.E., A.B., I.G., P.S.), Karolinska Institute, Huddinge University Hospital, SE-14186 Huddinge, Sweden; and Department of Clinical Physiology (A.N.) and Department of Women’s and Children’s Health (U.L., B.L.), Section of Obstetrics and Gynecology, Uppsala University, SE-751 85 Uppsala, Sweden
Address all correspondence and requests for reprints to: Bo Lindblom, M.D., Department of Women’s and Children’s Health, Section of Obstetrics and Gynecology, Akademiska Hospital, S-751 85 Uppsala, Sweden.
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Abstract |
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Myometrial and fibroid tissues were recovered from women undergoing gynecological operations during different phases of the menstrual cycle and during treatment with an analogue of GnRH (GnRHa). Contents of ER and PR in the tissue cytosol were determined by enzyme immunoassay. The ER levels were significantly higher in fibroid tissue than in homologous myometrium in all the endocrine conditions. During the secretory phase, when luteal progesterone production is prominent, the ER levels in the myometrium and fibroids were lower than during the proliferative phase. During GnRHa treatment, the ER levels in both tissues were similar to those in the proliferative phase but significantly higher than in the secretory phase. The PR levels were also significantly higher in fibroids than in myometrium in all the different endocrine conditions. In both tissues, the PR levels were lower in the secretory phase and during GnRHa treatment, compared with the proliferative phase. Our data suggest that, in these categories of women, both ER and PR are overexpressed in fibroid tissue. Apparently, high progesterone levels down-regulate the ER in both fibroids and myometrium, whereas estrogen mediates the up-regulation of the PR during the proliferative phase. Increased knowledge about the mechanisms by which sex steroids regulate their own receptors in uterine tissues might provide a basis for development of new treatment strategies for women with fibroid disease.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Most fibroids grow slowly, and the mechanisms responsible for their neoplastic potential are not completely known. Recently, increasing interest has been directed towards pharmacological treatment of fibroids. Long-term treatment with analogues of GnRH (GnRHa) is seldom used because of the side effects, mainly the reduction of the bone mineral content.
Conversely, short-term treatment with GnRHa is usually well tolerated and is effective in reducing the size and symptoms of fibroids, including correction of anemia secondary to menorrhagia. Considerable experience has shown, however, that rapid regrowth takes place after discontinuation of such treatment (2). At present, the most widely accepted use of GnRHa in this context is for short-term preoperative therapy. The rationale for such treatment is the proposed advantage of increasing hemoglobin levels and reducing tumor size, which may facilitate removal of the uterus by the vaginal route.
The present investigation was undertaken: 1) to compare the contents of estrogen and progesterone receptors (ER, PR) in human myometrium and fibroids obtained during different phases of the menstrual cycle and also after pretreatment with a GnRHa; and 2) to elucidate the influence of these endocrine conditions on the receptor levels in myometrial and fibroid tissue.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The study comprised 27 women who had a diagnosis of uterine fibroids and were admitted for myomectomy or hysterectomy. The clinical diagnosis was based on pelvic examination and an ultrasound scan. The indication for surgery was one or more of the following: menorrhagia, dysmenorrhea, pelvic discomfort, rapid tumor growth, and uncertainty about the diagnosis. Some of the patients were offered and received preoperative treatment with a GnRHa (goserelin) to reduce the symptoms and uterine size before surgery. Six patients with a desire for pregnancy received goserelin before myomectomy, and another 6 received goserelin before hysterectomy. This selection of patients was done randomly in the majority of cases as a part of a controlled clinical trial. In addition, a few cases received GnRHa treatment on clinical grounds only. As a rule, this treatment was initiated 2–3 months before admission and during the time the patient was on the waiting list. The dose of goserelin was 3.6 mg sc every 4 weeks. From 3 of the GnRHa-treated women, only fibroid tissue was obtained.
The patients not treated with a GnRHa were subclassified into
proliferative and secretory groups, according to the phase of the
menstrual cycle. This classification was based on the menstrual cycle
pattern, the LMP, and serum levels of estradiol and
progesterone (Table 1
). None
of these patients were given any hormonal treatment before surgery.
Several untreated women could not be classified into these categories
because of menstrual irregularity, hormone levels indicating a
postmenopausal state, or both. These cases, as well as those with
adenomyosis, were not included in the present analysis. All
GnRHa-treated women showed markedly decreased serum estrogen
levels.
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All operations were performed under general anesthesia. Tissue was collected from fibroids and myometrium immediately after removal of the uterus or fibroid. In case of multiple fibroids, biopsies were taken from the largest tumors. However, tumors smaller than 2 cm in diameter were not included in the present study. Material was collected from 52 fibroids (18 and 14 from patients in the proliferative and secretory phases, respectively, and 20 from GnRHa-treated women). The specimens were immediately frozen in liquid nitrogen and transported to the laboratory. They were stored at -70 to -80 C until the experiments were conducted.
Hormone analyses
Blood samples from an antecubital vein were taken in the morning on the day of surgery. For determination of the endocrine status, serum levels of estradiol 17ß and progesterone were measured by the Amerlite assay kit (Kodak Clinical Diagnostics Ltd., Amersham, UK).
Steroid receptor determinations
Specimens (50–150 mg) were cut from the frozen biopsies with a prechilled scalpel. Each specimen was weighed and cut into smaller pieces, avoiding thawing. The minced tissue was transferred to a prechilled (dry ice) capsule containing a tungsten ball. The capsule was frozen in liquid nitrogen and, thereafter, shaken at 1,600 rpm in a dismembranation apparatus for 30 sec. This procedure was repeated once with intermediate freezing in nitrogen. The pulverized tissue was then transferred to a prechilled tube and suspended in 10 vol Tris buffer. The suspension was centrifuged at 24,000 x g for 30 min at +4 C.
The ER and PR contents in the tissues were determined by a commercial monoclonal receptor enzyme immunoassay kit (Abbott - ER-EIA and PR-EIA, Abbott Diagnostica, Wiesbaden, Delkenheim, Germany), which is based on the sandwich principle. Beads coated with one monoclonal antireceptor antibody are incubated with cytosol or standards containing receptors. During incubation, receptors bind to the beads. Unbound material is then removed by washing. A second monoclonal antireceptor antibody is incubated with the beads to measure the amount of bound receptor. The intensity of the color resulting from the enzyme reaction is proportional to the amount of receptor and is determined spectrophotometrically.
Each assay included control samples for estimation of between-assay variability, and the coefficient of variation was found to be below 9% in all ranges. The results were expressed as fmol receptor/mg protein. The cytosol fraction was used for protein, ER, and PR assays. The cytosolic protein was determined according to the method of Bradford (3) using the Bio-Rad Protein Assay (Bio-Rad, Munich, Germany).
Statistics
The mean value of 1–5 fibroids from each patient was used for all further calculations. To compare differences between tissues, the Wilcoxon signed-rank test was used; and for comparison between groups, the Wilcoxon rank-sum test was applied. A P value of less than 0.05 was considered significant.
Ethics
The design of the study was approved by the Ethics Committee of the Huddinge University Hospital and the corresponding committee of the Uppsala University Hospital. Informed consent was obtained from all participating women.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In women having menstrual cycles, the steroid concentrations in
serum were as expected for the corresponding cycle phase. Conversely,
in women treated with GnRHa, the steroid levels were markedly depressed
(Table 1
).
Sex steroid receptor analyses
The mean ER content in fibroid tissue was significantly higher than that in the myometrium in all three groups of women.
The ER content in both myometrium and fibroids was significantly lower
in the secretory phase, as compared with both the proliferative phase
and GnRHa treatment (Fig. 1a). The levels
in the latter groups were similar.
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). A marked variation in the levels of both receptors between fibroids from the same patient was observed in several subjects. The maximal difference was 70-fold for ER and 26-fold for PR.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The present immunoassay procedure, which is based on the so-called sandwich principle and on direct antigenic recognition, is a specific and sensitive method. Good concordance with a steroid binding assay on uterine tissues has been demonstrated (4). An advantage of the method is that the results are not influenced by the tissue levels of the steroids.
The marked variation in ER and PR levels in tumors from the same subject illustrates the heterogenity between fibroids, which may account for some of the discrepancies between previous reports on ER and PR content in fibroids, compared with homologous myometrium.
Our data suggest that progesterone, and not withdrawal of sex steroid influence, down-regulates the ER in both fibroids and myometrium. The concept that progesterone suppresses the ER in fibroid tissue is further supported by the findings of Sadan et al. (5), who found an inverse correlation between serum progesterone levels and fibroid ER concentration, using a ligand binding assay. A report by Kawaguchi et al. (6), using immunohistochemistry, is also compatible with this concept.
In contrast to our data, Rein et al. (7) reported that the fibroid ER content was higher in GnRHa-treated women than in women given placebo. Conversely, Vollenhoven et al. (8) found no difference in ER binding between GnRHa-treated and untreated subjects. In these two studies, no subclassification of the untreated patients was made according to phase of the menstrual cycle. The present study indicates that such a classification is of the utmost importance for an appropriate interpretation of the steroid receptor status in both fibroid and myometrial tissue.
It is worth noting that half of the patients in the GnRH group underwent myomectomy because of a desire for preserved fertility. This subgroup had a lower mean age (35.0 yr vs. 45.0 yr) and a lower mean parity (0.17 vs. 1.50) than the subgroup receiving GnRHa before hysterectomy. These subgroups did not differ in ER or PR content in any of the tissues, indicating that the contribution of younger women with lower parity in the GnRHa group did not induce a selection bias.
Earlier studies of sex steroid receptor levels in fibroids vs. myometrium have been contradictory, showing increased, decreased, or similar receptor levels (for review, see Ref. 9). Differences in methodology, characterization of patients, and number of fibroids collected from each patient might contribute to these discrepancies. Two recent reports by Brandon and co-workers (10, 11), demonstrating that both messenger RNA and protein levels of ER and PR are higher in fibroids than in the corresponding myometrium, are in concordance with our data.
The higher PR content observed in fibroids and myometrium obtained during the proliferatory phase, as compared with tissue obtained from the secretory phase and from GnRHa-treatment, suggest that estrogen up-regulates the PR. This view gains support from the positive correlation between serum estradiol levels and myometrial PR levels in the proliferative phase reported by Schmidt-Gollwitzer et al. (12) and from a study in ovariectomized rhesus monkeys demonstrating induction of PR in myometrial smooth muscle cells after estradiol treatment (13). However, because the estrogen levels during the secretory phase in general are higher than during GnRHa-treatment, where all sex steroids are low, the data may also indicate that progesterone down-regulates its own receptor. The previously reported inverse relationship between serum progesterone and myometrial PR levels favors this view (12). It is worth noting that fibroids, in general, seem to respond in a way similar to that of myometrium to the alterations in the endocrine conditions studied here.
It has been suggested that the higher levels of ER and PR in fibroids than in myometrium could contribute to the relative growth advantage of fibroids, as compared with myometrium. Several studies indicate that fibroid volume decreases more than mean uterine volume during GnRHa-treatment (14, 15), whereas other investigators claim that this is not the case (16). It is feasible that an enhanced responsiveness of fibroids to steroid withdrawal represents a reflection of an increased responsiveness of fibroids to steroid stimulation, as compared with myometrium, in women of fertile age.
The relative importance of the individual sex steroids is a matter of controversy. Whereas early studies focussed on the growth-promoting effect of estrogens, there is now increasing evidence for a central role of progesterone in the regulation of fibroid growth (17). It is possible that progesterone and progestins have dual actions on fibroids: first, they may limit the tissue response to estrogen by blocking the replenishment of the ER; and second, they may exert an intrinsic, growth-stimulating action (11, 17, 18). The net effect of progesterone would be dependent on which of these actions was predominant.
Against this background, it is of interest that antiprogestin therapy has been found to be as effective as GnRHa in reducing fibroid size and uterine volume (19). As an explanation, the authors postulated that antiprogestins might influence the ER in fibroid tissue. The effect may also be exerted through a decrease in uterine blood flow, which in turn, could be mediated by steroid receptors in vascular smooth muscle and/or endothelium (20). Certainly, the further exploration of the influence of antiprogestins on uterine fibroid growth is of great interest, both from a biological viewpoint and from a clinical perspective.
The present findings suggest that progestins down-regulate the ER in uterine fibroids. It is reasonable to assume that fibroid growth may be governed by the interplay between both steroids. Unopposed estrogen influence may be one mechanism for promotion of growth, both by a direct stimulating action and by increasing the expression of the PR. High-dose progestin therapy may down-regulate the ER, but without occurrence of tumor shrinkage, because of the intrinsic growth-promoting action of progesterone (6). According to this theory, antiprogestin therapy could inhibit fibroid growth, both by blocking the progesterone effect and by interfering with the ER in fibroid tissue and, possibly, in the uterine vasculature (20). Further investigations are needed to clarify the possible significance of a critical temporal relationship between these actions and to identify the target genes responsible for sex steroid stimulation of fibroid growth.
It is clear that the concentration of both estrogen and progesterone and their receptors influence growth mechanisms in human myometrium and fibroids. Consequently, the endocrine status of the patient, as well as the phenotypic characteristics of the fibroid, will determine whether each individual lesion will grow, remain at equilibrium, or shrink.
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Acknowledgments |
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Footnotes |
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Received December 31, 1997.
Revised July 7, 1998.
Accepted August 11, 1998.
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References |
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