Oestrogen receptor-alpha gene polymorphism is associated with endometriosis, adenomyosis and leiomyomata

Jo Kitawaki1,4, Hiroshi Obayashi3, Hiroaki Ishihara1, Hisato Koshiba1, Izumi Kusuki1, Noriko Kado1, Katsumi Tsukamoto1, Goji Hasegawa2, Naoto Nakamura2 and Hideo Honjo1

1 Department of Obstetrics and Gynecology and 2 First Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 and 3 Department of Clinical Research, Kyoto Microbiological Institute, Kyoto 607-8482, Japan


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Endometriosis, adenomyosis and leiomyomata develop in women of reproductive age and regress after menopause or ovariectomy, suggesting that they grow in an oestrogen-dependent fashion. We investigated whether polymorphism in the oestrogen receptor-alpha (ER{alpha}) gene is related to oestrogen-dependent benign uterine disease. A total of 203 women with regular menstrual cycles underwent laparotomy or laparoscopy and were diagnosed histologically with endometriosis, adenomyosis and/or leiomyomata. Patients with cervical carcinoma in situ, tubal occlusion or adhesion but no other gynaecological disease were considered to be disease-free. A total of 179 women undergoing annual health examination were grouped as reference population. The distribution of PvuII genotypes (PP, Pp, and pp) of the ER{alpha} gene was different between each pair of the four groups of endometriosis, adenomyosis/leiomyomata, disease-free, and reference population (P = 0.022–0.0005), except between the former two groups. The PP genotype was less frequent in the groups of endometriosis (P = 0.0002) and adenomyosis/leiomyomata (P = 0.002) as compared to that in the disease-free group. In the endometriosis group, there was no difference in the distribution of PvuII genotypes due to complicating diseases (adenomyosis and/or leiomyomata) or severity of the clinical stages. These results suggest that the PvuII polymorphism of the ER{alpha} gene is associated with the risk for endometriosis, adenomyosis, and leiomyomata.

Key words: adenomyosis/endometriosis/leiomyomata/oestrogen receptor-alpha gene/restriction fragment length polymorphism


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Endometriosis develops mostly in women of reproductive age and regresses after menopause or ovariectomy. Suppression of the oestrogen level by danazol or gonadotrophin-releasing hormone (GnRH) agonists provides the regression of lesions; however, recovery of the oestrogen level after discontinuation of the therapies results in relapse of lesions. This evidence suggests that endometriosis grows and regresses in an oestrogen-dependent fashion. Indeed, ectopic endometriotic implants contain oestrogen receptor (ER), progesterone receptor and androgen receptor as shown by hormone-ligand binding assays (Bergqvist and Ferno, 1993Go), immunohistochemistry (Bergqvist et al., 1993Go; Howell et al., 1994Go; Jones et al., 1995Go), reverse transcription-polymerase chain reaction (Rey et al., 1998Go; Brandenberger et al., 1999Go; Fujimoto et al., 1999Go) and in-situ hybridization (Fujishita et al., 1997Go). The implants also express aromatase cytochrome P450, an enzyme that catalyses the conversion of androgens to oestrogens, suggesting that local oestrogen production may increase the oestrogen concentration, which together with circulating oestrogen stimulates the growth of tissue mediated by the ER (Kitawaki et al., 1997Go, 1999Go). Adenomyosis and leiomyomata are separate entities from endometriosis, but they share a common pathophysiology in that they develop primarily in women of reproductive age, their growth is oestrogen dependent, and they have a complicated pattern of occurrence. Adenomyosis and leiomyomata tissue also express ER (Kitawaki et al., 1997Go) and aromatase (Kitawaki et al., 1997Go).

Recent studies have postulated that ER gene polymorphisms may influence its action as a modulator of the ligand oestrogens. Oestrogens are known to be important for the preservation of bone mass in females during menopause. Several studies have shown that among ER{alpha} genotypes assessed by PvuII restriction fragment length polymorphism (RFLP), the PP genotype has higher bone mineral density than the Pp and pp genotypes (Kobayashi et al., 1996Go; Ongphiphadhanakul et al., 1998Go; Willing et al., 1998Go; Kurabayashi et al., 1999Go), and that in adolescent boys PP genotype has a greater body height than the others (Lorentzon et al., 1999Go). These findings may suggest that the local oestrogenic action is more potent in those with PP genotype than in those with the Pp or pp genotypes. This is also supported by the presence of an association between ER{alpha} gene polymorphisms and oestrogen-dependent disease, including endometriosis (Georgiou et al., 1999Go), and the risk of pre-menopausal hysterectomy and onset of natural menopause (Weel et al., 1999Go).

The purpose of the present study was to investigate whether the polymorphism in the ER{alpha} gene was related to oestrogen-dependent benign uterine disease such as endometriosis, adenomyosis and leiomyomata.


    Materials and methods
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 Materials and methods
 Results
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Subjects
The patients who had undergone laparotomy or laparoscopy at the Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine were diagnosed by both macroscopic and histological examinations. All patients were of reproductive age (24–48 years) with cyclic menstruation. Prior to the operation the patients had not received any endocrine therapy such as GnRH analogue, danazol, or oestrogen-progestin that might mask the presence of disease. Cases with malignant uterine neoplasms other than cervical carcinoma in situ, ovarian neoplasms, pelvic inflammation, or pregnancy were excluded from the study. A total of 203 patients met the criteria for enrolment. The patients were classified into three groups defined as endometriosis (n = 109), adenomyosis/leiomyomata (n = 67), and disease-free (n = 27) (Kitawaki et al., 1997Go, 1999Go). The endometriosis group consisted of patients with endometriosis subclassified into three different categories and each category subdivided into two groups: pure or complicated, with or without chocolate cysts, and light or severe; the pure subgroup consisted of patients who had only endometriosis but no other gynaecological disease, and the complication subgroup consisted of patients who had endometriosis associated with adenomyosis and/or leiomyomata. The stage of endometriosis was assigned according to the revised American Fertility Society scoring system (American Fertility Society, 1985Go): stages I and II were grouped as light while stages III and IV were grouped as severe. The adenomyosis/leiomyomata group consisted of patients with adenomyosis and/or leiomyomata but without endometriosis. The disease-free group consisted of patients with cervical cancer in situ but showing no other gynaecological disease, or patients with tubal occlusion or adhesion but without endometriosis, adenomyosis or leiomyomata. These were diagnosed at laparoscopy performed during examinations for infertility. Since the number of women in the disease-free group was small (n = 27), in addition, 179 women living in Japan, Kyoto prefecture, who underwent annual health examination, were enrolled as reference population for this study. The study protocol was approved by the Kyoto Prefectural University of Medicine institutional review board, and informed consent was obtained from each patient.

Genomic DNA analysis
Peripheral blood was drawn from each patient and collected in a tube with EDTA added. Genomic DNA was extracted from peripheral blood with DNA extractor WB kit (Wako pure chemical, Osaka, Japan) according to the manufacturer's instructions (Obayashi et al., 1999Go). Genotyping of the PvuII polymorphism in intron 1, 0.4 kb upstream of exon 2 of the ER{alpha} gene was determined by polymerase chain reaction (PCR)-RFLP analysis, essentially as previously described (Yaich et al., 1992Go). Briefly, an aliquot of 100 ng DNA was mixed with 0.5 µmol/l each of the primers (forward, 5'-CTGCCACCCTATCTGTATCTTTTCCTATTCTCC-3'; and reverse, 5'-TCTTTCTCTGCCACCCTGGCGTCGATTATCTGA-3'), 0.2 mmol/l dNTPs, and 1.25 units Taq polymerase (Takara Premix Ex Taq; Takara Biochemicals, Tokyo, Japan), in a total volume of 50 µl of PCR buffer provided by the manufacturer. The PCR procedure was as follows: an initial denaturation step at 95°C for 5 min, and then amplified for 30 cycles at 94°C for 1 min, at 62°C for 1 min, and at 72°C for 1 min, followed by a final extension step at 72°C for 6 min. The PCR products were digested with restriction enzyme PvuII (Takara Biochemicals), separated by 4% agarose gel electrophoresis, and identified by ethidium bromide staining. Genotypes were defined as PP, Pp, or pp. Upper-case letters represent the absence of, and lower-case letters represent the presence of, restriction sites.

Statistics
Differences in the items of baseline characteristics of patients were analysed with one-factor analysis of variance and multiple comparisons were performed using Scheffé's procedure. The distributions of the ER{alpha} genotypes and allele frequencies were evaluated by {chi}2-test with 2x3 table (for genotypes) or 2x2 table (for alleles). The odds ratio (OR) and 95% confidence interval (CI) were calculated using logistic regression models. P < 0.05 was considered significant.


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 Results
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Table IGo summarizes the baseline characteristics of patients enrolled in this study. The mean age (P < 0.001), height (P < 0.001), body weight (P < 0.001) and body mass index (P < 0.05–0.01) of the reference population differed from those of endometriosis, adenomyosis/leiomyomata and disease-free since the reference population consisted of elderly women, whereas the other three groups of women were of reproductive age. The mean age of the adenomyosis/leiomyomata group was higher than the groups of endometriosis (P < 0.001) and disease-free (P < 0.001). However, the mean height, body weight and body mass index were comparable among the three groups of reproductive age.


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Table I. Baseline characteristics
 
The distribution of PvuII genotypes of the ER{alpha} gene in the endometriosis group was different from those in the groups of disease-free ({chi}2 = 15.32, P = 0.0005) and reference population ({chi}2 = 7.63, P = 0.022), the adenomyosis/leiomyomata group was different from the disease-free group ({chi}2 = 10.81, P = 0.005), and the disease-free group was different from the reference population ({chi}2 = 8.31, P = 0.016) (Table IIGo). However, the distribution was not different between endometriosis and adenomyosis/leiomyomata groups, or between adenomyosis/leiomyomata and the reference population (Table IIGo). In the endometriosis group, the PP genotype was less frequent than the Pp + pp genotypes as compared to that in the reference population [OR = 0.47 (95% CI, 0.24–0.90), P = 0.021] and that in the disease-free group [OR = 0.18 (95% CI, 0.07–0.47), P = 0.0002]. Similarly, in the adenomyosis/leiomyomata group, the PP genotype was less frequent than the Pp + pp genotypes as compared to that in the disease-free group [OR = 0.22 (95% CI, 0.08–0.60), P = 0.002]. On the other hand, in the disease-free group, the pp genotype was less frequent than the PP + Pp genotypes as compared to that in the reference population [OR = 0.22 (95% CI, 0.064–0.76), P = 0.009], the endometriosis group [OR = 0.25 (95% CI, 0.072–0.90), P = 0.024], and the adenomyosis/leiomyomata group [OR = 0.26 (95% CI, 0.069–0.94), P = 0.031]. The distribution of the genotypes in the reference population was in-between those of the disease and disease-free groups. The frequency of the heterozygous Pp genotype did not differ between the groups (Table IIGo). The p allele was less frequent than the P allele in the disease-free group as compared to that in the groups of endometriosis (P = 0.0004), adenomyosis/leiomyomata (P = 0.001) and reference population (P = 0.002) (Table IIGo).


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Table II. Distribution of the PvuII genotypes of the ER{alpha} gene
 
The patients with endometriosis were subclassified into three categories (Table IIIGo). However, there was no difference in the distribution of the PvuII genotypes between the groups with and without complication of other oestrogen-dependent disease including adenomyosis and/or leiomyomata, between the groups with and without chocolate cysts, or between the groups of light and severe stages (Table IIIGo).


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Table III. Lack of differences in the distribution of the PvuII genotypes of the ER{alpha} gene between various forms of endometriosis
 

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 Materials and methods
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In the present study we showed that the allelic variants of the ER{alpha} gene are associated with endometriosis, adenomyosis and leiomyomata. The frequency of PP genotype was low in the patients with endometriosis, adenomyosis and leiomyomata, whereas it was high in the disease-free patients, suggesting that the P allele is protective against endometriosis, adenomyosis and leiomyomata. In the endometriosis group, the distribution of ER{alpha} gene genotypes was similar, regardless of whether or not the patient had complications of adenomyosis and/or leiomyomata, chocolate cysts, or whether the clinical stage was light or severe. The distribution of ER{alpha} gene genotypes was similar between the groups of endometriosis and adenomyosis/leiomyomata. These findings suggest that polymorphism in the ER{alpha} gene is associated, in part, with the onset or growth of oestrogen-dependent benign uterine disease.

Similar results were reported (Georgiou et al., 1999Go) in 57 Greek patients with endometriosis, who had a significantly lower frequency of PP genotype in the ER{alpha} gene compared with that in the control group. In contrast, a number of studies (Hill et al., 1989Go; Parl et al., 1989Go; Yaich et al., 1992Go; Andersen et al., 1994Go; Southey et al., 1998Go) failed to show an association between polymorphisms in the ER{alpha} gene and breast cancer.

It is unclear how the anonymous intronic polymorphism of the ER{alpha} gene influences its protein function. However, recent studies have postulated that ER{alpha} gene polymorphisms may influence its action as a modulator of the ligand oestrogens. Oestrogens are known to be important for preservation of bone mass in females during menopause. Several studies have shown that the PP genotype has higher bone mineral density than the Pp and pp genotypes (Kobayashi et al., 1996Go; Ongphiphadhanakul et al., 1998Go; Willing et al., 1998Go; Kurabayashi et al., 1999Go), while studies of Korean (Han et al., 1999Go) and Caucasian (Vandevyver et al., 1999Go) women have observed no association between ER{alpha} gene polymorphisms and bone mineral density. The PP genotype has a greater body height in adolescent boys (Lorentzon et al., 1999Go), whereas bone density was not reduced in patients with endometriosis (Lane et al., 1991Go; Ulrich et al., 1998Go). These findings suggest that the local oestrogenic action is more potent in women carrying the P allele than those carrying the p allele. This is also supported by the finding that the PP genotype has a higher risk of premenopausal hysterectomy and earlier onset of natural menopause due to menorrhagia and fibroids than the Pp and pp genotypes (Weel et al., 1999Go). These findings, however, contradict those of the present and the Greek studies (Georgiou et al., 1999Go). The present study shows that women carrying the PP genotype have a lower risk for oestrogen-dependent uterine disease. Indeed, endometriotic implants and adenomyotic tissues express a reduced amount of ER protein (Prentice et al., 1992Go; Bergqvist and Ferno, 1993Go; Bergqvist et al., 1993Go; Nisolle et al., 1994Go) and lose the cyclic change of ER expression (Prentice et al., 1992Go). This suggests that aberrant expression of ER may be partly involved in the onset or growth of oestrogen-dependent benign uterine disease. Similarly, women carrying the PP genotype show poorer response to ovarian stimulation (Georgiou et al., 1997Go; Sundarrajan et al., 1999Go).

Previously the distinction between normal or disease-free cases and those with endometriosis, adenomyosis and leiomyomata had not been clearly defined. In our laboratory (Kitawaki et al., 1997Go, 1999Go), we demonstrated that the mRNA and protein of aromatase cytochrome P450, responsible for oestrogen biosynthesis, are expressed in the eutopic endometria of patients with endometriosis, adenomyosis, and/or leiomyomata, whereas neither are expressed in endometrial specimens obtained from normally menstruating women with cervical carcinoma in situ but no other gynaecological disease. Similarly, we demonstrated that the enzyme activity of 17ß-hydroxysteroid dehydrogenase type 2, which inactivates oestradiol to oestrone, is induced during the secretory phase in the endometrium of patients with endometriosis, adenomyosis, and/or leiomyomata, whereas the induction does not occur in disease-free endometrium (Kitawaki et al., 2000Go). These findings indicate that, although the endometria of patients with endometriosis, adenomyosis, or leiomyomata resemble those of women without gynaecological disease, the oestrogen metabolism of these tissues is remarkably different. We carried out the present study using these criteria, strictly distinguishing disease-free cases from disease cases by abdominal examination and histopathology. This grouping is important for analysing studies relating to oestrogen and the disease-free group is an ideal control. However, we could only collect a small number of disease-free patients, because establishing a disease-free state was difficult in women undergoing surgery. This would have resulted in less statistically significant data. Alternatively, we enrolled the reference population, which consisted of a common female population in Japan. This group might include some with oestrogen-dependent uterine disease. Although the group consisted mostly of postmenopausal women with different profiles in physical constitution, such difference might not be related to genetic background. Indeed, the distribution of the ER{alpha} gene genotypes was compatible to that in Caucasians (Willing et al., 1998Go; Weel et al., 1999Go) and Japanese (Kobayashi et al., 1996Go; Kurabayashi et al., 1999Go). Furthermore, the distribution of the genotypes in the reference population was in-between those of the disease groups comprising the less frequent PP genotype and the disease-free group comprising the less frequent pp genotype. This supports the finding that the distribution of the ER{alpha} gene genotypes is different between the disease and disease-free groups.

The present study included cases who had undergone surgery but not those at early stages without symptoms or those who were not infertile. The higher mean age in the adenomyosis/leiomyomata group compared to that in the disease-free and endometriosis groups also adds to the problem. However, in the endometriosis group, the distribution of the ER{alpha} gene genotypes was not related to complications of other diseases or to the clinical stage. We combined adenomyosis and leiomyomata into one group, because of the comparative numbers of cases that complicated both diseases, and the difficulty of being able to discriminate between the two diseases. Similarly, the disease-free group was not necessarily strictly `normal', but was associated with cervical carcinoma in situ or infertility. We have used this criterion previously and showed the lack of aromatase expression (Kitawaki et al., 1997Go, 1999Go) and the lack of 17ß-hydroxysteroid dehydrogenase type 2 induction (Kitawaki et al., 2000Go) in the disease-free endometrium. We therefore conclude that the present study does not contain serious selection biases.

In conclusion, the PvuII polymorphism of the ER{alpha} gene is associated with the risk for endometriosis, adenomyosis, and leiomyomata. The mechanism by which the anonymous intronic polymorphism affects protein function needs to be clarified. This will provide better understanding of the pathogenesis and pathophysiology of oestrogen-dependent uterine diseases.


    Notes
 
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan. E-mail: kitawaki{at}koto.kpu-m.ac.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on June 23, 2000; accepted on September 29, 2000.