Aromatase Expression in the Eutopic Endometrium of Myomatous Uteri

By Maia, Hugo Jr Pimentel, Kleber; Casoy, Julio; Correia, Tania; Et al

Abstract Aims. To detect aromatase expression in the endometrium of myomatous uteri and to correlate it with the location of the myoma, phase of the menstrual cycle, the presence of menorrhagia and oral contraceptive use.

Method. Aromatase p450 expression was measured using immunohistochemical methods in the endometrium of 116 patients. Sixty-one patients had menorrhagia associated with intramural/ submucous myomas and nine had subserous myomas and no excessive bleeding. Forty-six patients had no uterine pathology and served as controls. Nineteen out of 61 patients with menorrhagia were oral contraceptive users at the time of the examination. Endometrial samples were obtained by hysteroscopy in all cases.

Results. Aromatase p450 expression was detected more frequently in the eutopic endometrium of patients with submucous or intramural myomas than in those in the subserous group, and was significantly greater during the proliferative phase than during the luteal phase or following the use of oral contraceptives. In normal uteri, aromatase expression was detected in the endometrium in less than 10% of users.

Conclusions. Aromatase expression in the endometrium was affected by the location of the myoma, the presence of symptoms, and the phase of the menstrual cycle. Oral contraceptives, on the other hand, inhibited aromatase expression in the eutopic endometrium of patients with submucous/intramural myomas.

Keywords: Aromatase, endometrium, oral contraceptives, menorrhagia, myomas

Introduction

Estrogens and possibly progesterone are involved in the regulation of myoma growth, although many as yet unresolved issues remain in relation to the role played by each steroid hormone in the development of these neoplasms [1,2]. Differences in the etiology and physiopathology of myomas showing different rates of growth further complicate this issue. Subserous myomas may grow to a considerable size and abnormalities in karyotype are more common in myomas that have attained large sizes, therefore suggesting that cytogenetic abnormalities affect the growth potential of tumors [3]. Submucous myomas, on the other hand, are more susceptible to the influence of hormones and they have fewer clonal cytogenetic abnormalities than subserous myomas, although they are associated with more menstrual disturbances [4,5]. Vascular changes observed in the endometrium during hysteroscopy were also more marked when the myoma was located closer to the uterine cavity [6]. The causal relationship between locally produced estrogens and the excessive menstrual bleeding associated with myoma development remains to be fully clarified. Myomas not only contain more estrogen than progesterone receptors but the aromatase gene and enzyme are also expressed in these neoplasms at significant levels, thereby indicating a greater dependency on estrogenic stimuli for their growth [7,8]. Aromatase activity has also been detected in the eutopic endometrium of patients with endometrial polyps, endometriosis and adenomyosis, but not in the endometrium of disease- free uteri, thus indicating that a local hyperestrogenic milieu may play a pivotal role in the development of corpus uterine pathology [9-11]. However, it is not known whether endometrial intracrine estrogen production antedates the increase in menorrhagia associated with the presence of myomas, although estrogens are known to increase angiogenesis and vascular endothelium growth factor (VEGF) expression in the endometrium [12,13].

The purpose of the present study was to investigate the association between myoma-related menstrual bleeding disorders and the presence of aromatase expression in the eutopic endometrium during both the proliferative and luteal phases of the menstrual cycle, using immunohistochemical methods. The effect of oral contraceptives on aromatase expression in the eutopic endometrium of the myomatous uteri of patients with menorrhagia was also investigated. Oral contraceptives are known to reduce not only the excessive menstrual bleeding associated with the presence of myomas but also the relative risk of developing this pathology [14,15]. In patients with adenomyosis, endometriosis and myomas, danazol decreased aromatase expression in the eutopic endometrium and this inhibitory effect on local estrogen production may play a pivotal role in the control of the menstrual disorders associated with these pathological conditions [16]. Likewise, oral contraceptives are effective in decreasing aromatase expression in the eutopic endometrium of uteri with adenomyosis, but it remains to be seen whether or not a similar inhibition is likewise observed in myomatous uteri [17].

Methods

This was a retrospective study carried out on paraffin-embedded tissue obtained from 116 patients submitted to endometrial resection or biopsy in our institute between January 2005 and September 2006. In the myoma group, 61 patients had a history of menorrhagia and a previous diagnosis of intramural or submucous myomas, as detected by transvaginal sonography, while the remaining nine patients had only subserous myomas, also diagnosed by transvaginal sonography, and no history of abnormal uterine bleeding. Patients with normal uteri at sonography but who were referred to our unit for hysteroscopic evaluation of the uterine cavity, including endometrial biopsy, as part of the diagnostic workup prior to in vitro fertilization procedures because of male factor infertility, served as controls (n = 46). Endometrial resection (w = 40), myomectomy (w=15) or a combined procedure (n = 6) was carried out in patients with intramural/submucous myomas using the bipolar resectoscope (Versapoint). Using this technique, it is possible to resect not only submucous myomas but also, when indicated, the surrounding endometrium and approximately 5 mm of the myometrium. Two patients with submucous myomas with an intramural component were submitted to a two-step myomectomy and used oral contraceptives containing gestodene for 2 months during the interval between the two procedures. In the case of subserous myomas, diagnostic hysteroscopy with endometrial biopsy was carried out to confirm the normality of the uterine cavity. In patients with normal uteri, as defined by transvaginal sonography, only ambulatory hysteroscopy with endometrial biopsy was performed. All patients were retrospectively included in the present study and had progesterone levels compatible with ovulatory cycles and normal thyroid function as inferred by their admission medical records. At the time of endometrial resection or myomectomy, 19 patients in the intramural/submucous myoma group were using an oral contraceptive containing 30 [mu]g of ethinyl estradiol and 75 [mu]g of gestodene (Gestinol (R) ; Libbs Farmaceutica, Brazil) in a continuous regimen for at least 56 days, as prescribed by their attending physician to reduce bleeding and thin the endometrium. All patients were premenopausal, and were in the 34- to 52-year age bracket. In the case of submucous myomas, diagnosis was confirmed histologically and hysteroscopically. In the intramural/submucous myoma group, 23 patients were in the proliferative phase and 19 were in the luteal phase of the menstrual cycle, while 19 patients were using oral contraceptives at the time of the hysteroscopic procedure. In the subserous group, five patients were in the proliferative phase and four were in the luteal phase.

All tissue samples were fixed in 10% formalin before being sent to pathology. Immunohistochemistry was carried out following antigen retrieval to detect the presence of aromatase p450. Aromatase expression was investigated using a commercially available monoclonal antibody (MCA2077, clone H4; Serotech, Raleigh, NC, USA). Antigen retrieval was carried out using the Tris- ethylenediaminetetraacetic acid buffer at pH 8.0. The reaction was revealed using the streptavidin-biotin method. The presence of aromatase expression was rated either as positive if there was any detectable staining reaction or negative when no reaction was observed. Placental tissue and an atrophic endometrial sample were used as positive and negative controls, respectively, in all immunostaining reactions for aromatase p450.

Statistical analysis was carried out using the StatsDirect software program, version 2.3.8 (StatsDirect Ltd, Cheshire, UK, 2004). The ?2 and Fisher’s exact tests were used to compare proportions of aromatase expression among the various groups. Significance was established as p < 0.05. Patients included in the present study gave their informed consent for the immunohistochemical studies to be performed on the biopsy specimens.

Results

Aromatase expression was detected by immunohistochemistry in the endometrium of patients with submucous and intramural myomas during both the proliferative and luteal phases of the menstrual cycle. In the aromatase-positive endometria, the reaction was detected in the stroma and epithelium, albeit more frequently in the former (Figure 1 and Table I). The percentage of endometrium (stroma + epithelium) displaying a positive staining reaction for aromatase varied according to the phase of the menstrual cycle, being present in a significantly higher number of endometria during the proliferative phase (90.9%) compared with the luteal phase of the cycle (30.4%) (p < 0.001). In patients with subserous myomas but without symptoms of menorrhagia, on the other hand, the endometrium was negative for aromatase p450 expression in all nine cases irrespective of the phase of the menstrual cycle. The difference between the submucous/ intramural and subserous groups was statistically significant when the total numbers of positive endometria (epithelium + stroma) in both phases of the menstrual cycle were pooled together and the differences between the two groups were compared (58.7% vs. 0%) (p = 0.0018). In control uteri, the percentage of aromatase-positive endometria was 8.0% (2/25) during the proliferative phase and 9.5% (2/21) during the luteal phase, a difference that was not statistically significant (p=l). When the percentages of aromatase- positive endometria in the control group were compared with the percentages in the group with myomatous uteri, the values were not significantly different from those observed in cases of subserous myomas. They were significantly lower, however, compared with those observed in the intramural/submucous group during the proliferative phase (p < 0.01) but not during the luteal phase (p = 0.136). These results are summarized in Table II. In patients with intramural/ submucous myomas who were using oral contraceptives containing gestodene prior to hysteroscopy, aromatase expression in the eutopic endometrium was detected in the endometrium in 10.5% of cases (2/ 19). This percentage was significantly lower than in the intramural/ subserous group during the proliferative (p < 0.001) but not during the luteal phase of the cycle (p = 0. 15). Two patients in the oral contraceptive group were submitted to a two-step hysteroscopic myomectomy because of the intramural component of the submucous myoma. These patients used a continuous regimen of oral contraceptives for 2 months between the two procedures in order to induce endometrial atrophy and tumor shrinkage. In both cases, aromatase expression was detected in the pretreatment endometrial biopsies, but expression was negative in the endometrium at the time of the second hysteroscopic procedure carried out when the patients were still using the oral contraceptive (Figure 2). Figure 1 . Aromatase p450 expression in the stroma of the eutopic endometrium of a patient with intramural myoma during the proliferative phase of the menstrual cycle.

Discussion

The present study shows that, in the endometrium of patients with submucous/intramural myomas, aromatase expression is detected more frequently during the proliferative phase than in the luteal phase of the menstrual cycle. In patients with subserous myomas but without menorrhagia, aromatase expression was not detected in the eutopic endometrium. Although the present data are not conclusive with respect to whether the presence of aromatase expression in the endometrium may account for the presence of abnormal menstrual bleeding in patients with myomatous uteri, aberrant aromatase expression has been detected in the presence of other uterine pathologies associated with excessive uterine bleeding, such as adenomyosis and endometrial polyps [9,1 1]. There is evidence that local estrogen production in myomas or in the eutopic endometrium is more important than systemic estrogens with respect to growth stimulation, since myoma growth cannot be fully restored by exogenous estrogens in patients using gonadotropin-releasing hormone (GnRH) analogs because of the suppressive effects of GnRH analogs on aromatase expression in myomatous uteri [18]. Our results are in agreement with these findings and suggest that estrogens acting in an intracrine fashion either in the endometrium or in the myoma may play a pivotal role not only in stimulating their growth but also in the pathogenesis of abnormal menstrual bleeding. It is also noteworthy that aromatase expression was negative in the eutopic endometrium of patients bearing subserous myomas but without symptoms of menorrhagia. This is in agreement with previous findings that the growth of subserous myomas, which can attain very large sizes, is more dependent on cytogenetic rearrangements than hormonal factors [5]. However, it still remains to be established whether aromatase expression in the endometrium antedates the development of myomas or other uterine pathologies associated with abnormal menstrual bleeding, or not.

Table I. Changes in aromatase expression in the stroma and epithelium in the endometrium of patients with submucous/ intramural myomas during the menstrual cycle.

Figure 2. Suppression of aromatase p450 expression in the stroma of the eutopic endometrium in a patient with submucous myoma. Note that aromatase expression becomes negative after 2 months of oral contraceptive use.

Estrogens are known to upregulate cyclooxygenase2 (Cox-2) expression in the endometrium, thereby increasing prostaglandin production and creating a vicious circle of augmented Cox-2 and aromatase expression [19-21]. Prostaglandin E^sub 2^ is a potent inducer of the promoter gene for aromatase in both endometriotic lesions and the corresponding eutopic endometrium, although it is devoid of any stimulatory effect in the normal endometrium [19]. The hyperestrogenic milieu in the endometrium caused by the aberrant expression of aromatase may theoretically stimulate the synthesis of angiogenic factors such as VEGF, ultimately leading to increased bleeding. However, the time course of events that will ultimately lead to myoma development still remains to be elucidated. It would be an oversimplification to consider local estrogen production to be the only stimulus of myoma development, since other factors may act synergistically in association with aromatase expression, including cytogenetic abnormalities that might affect growth rates [3,5]. Experimental stuthes carried out in primates have shown that chronic exposure of the uterus to estrogens leads to the development of myomas, endometrial polyps, hyperplasia and adenomyosis [22]. Although these findings suggest that excessive estrogenic stimulation may be an important factor in the genesis of various uterine pathologies, thus explaining their common association, genetic and other factors probably play important roles in determining which pathology will develop in response to excessive estrogenic stimuli. The presence of aberrant aromatase expression in the eutopic endometrium of patients with adenomyosis, myomas, endometrial polyps and endometriosis corroborates these experimental findings in primates and further suggests a common pathway for the menstrual bleeding abnormalities associated with the presence of these pathologies [8-11]. The growth of myomas is known to be stimulated by the presence of estrogens, since the use of GnRH analogs or danazol is associated not only with regression of these neoplasms but also with the inhibition of aromatase expression in the eutopic endometrium of these uteri [16].

The use of oral contraceptives is effective in reducing the menstrual bleeding associated with the presence of myomas without stimulating their growth [15]. A reduction in the relative risk of developing myomas has also been reported in women using oral contraceptives [23,24]. The mechanisms by which oral contraceptives exert a protective effect on the development of myomas are not fully understood, but one likely explanation may be their suppressive effects on aromatase p450 expression in the endometrium, thus reducing local estrogen production and hampering the development of myomas. This effect may be a consequence of the progestational component of the oral contraceptive, since the percentages of endometria positive for aromatase expression while under progesterone dominance, such as during the luteal phase, were significantly lower compared with the proliferative phase, but not statistically different from those in the gestodene group, as reported here. It is noteworthy that aromatase expression in the eutopic endometrium of these patients was associated with the presence of abnormal menstrual bleeding, although a causal relationship cannot be inferred from these data. However aberrant aromatase expression has been previously detected in the eutopic endometrium of patients with endometriosis, adenomyosis and endometrial polyps [8,9,11]. This may suggest a common etiology in these conditions, involving chronic local exposure to estrogens that will lead to the development of these pathologies, thus corroborating previous stuthes carried out in rhesus monkeys [22].

We have recently reported that aromatase expression could not be detected in the eutopic endometria of patients with adenomyosis following the use of oral contraceptives containing gestodene, thereby suggesting that a 1 9-nortestosterone derivative may also suppress aromatase expression in adenomyotic uteri in a similar way to that of danazol [17]. However, it still remains to be seen whether or not other progestins share this same effect, and if inhibition of aromatase gene expression occurs at gene transcriptional level as it does with danazol [16]. The suppression of aromatase expression in the eutopic endometrium of myomatous uteri may explain some of the health benefits of oral contraception such as the treatment of abnormal menstrual bleeding caused by myomas and possibly other uterine pathology [15]. These findings are also important in explaining the preventive effects of oral contraceptives on the development of endometrial pathologies and other gynecological conditions [15].

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HUGO MAIA JR1, KLEBER PIMENTEL2, JULIO CASOY1, TANIA CORREIAZIA2,3, LUIS ANTONIO R. FREITAS3, BELA ZAUSNER4, CELIA ATHAYDE1, & ELSIMAR COUTINHO1

1 Centro de Pesquisa e Assistencia em Reproducao Humana (CEPARH), Salvador, Bahia, Brazil, 2 Escola Bahiana de

Medicina, Salvador, Bahia, Brazil, 3 Fiocruz, Salvador, Bahia, Brazil and School of Medicine, Federal University of Bahia,

Bahia, Brazil, and 4 Genese, Salvador, Bahia, Brazil

(Received 5 December 2006; revised 26 February 2007; accepted 2 March 2007)

Correspondence: H. Maia Jr, CEPARH, Rua Caetano Moura 35, 40210- 341 Salvador, Bahia, Brazil. Tel: 55 71 3247 8216. Fax: 55 71 3235 3442. E-mail: ceparhfwuol.com.br

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