Tohoku University School of Medicine, Sendai, Japan

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1 FERTILITY AND STERILITY VOL. 74, NO. 4, OCTOBER 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. REPRODUCTIVE BIOLOGY Quantitative analysis of estrogen receptor alpha and beta messenger ribonucleic acid levels in normal endometrium and ovarian endometriotic cysts using a real-time reverse transcription-polymerase chain reaction assay Sachiko Matsuzaki, M.D., a Shigeki Uehara, M.D., a Takashi Murakami, M.D., a Junko Fujiwara, B.S., b Tadao Funato, M.D., b and Kunihiro Okamura, M.D. a Tohoku University School of Medicine, Sendai, Japan Received January 14, 2000; revised and accepted April 4, Reprint requests: Sachiko Matsuzaki, M.D., Department of Obstetrics and Gynecology, Tohoku University School of Medicine, 1-1, Seiryomachi, Aoba-ku, Sendai, , Japan (FAX: ; matsuzaki@ob-gy.med.tohoku.ac.jp). a Department of Obstetrics and Gynecology. b Department of Laboratory Medicine /00/$20.00 PII S (00) Objective: To quantify messenger RNA (mrna) levels of the two estrogen receptor isoforms, estrogen receptor- (ER- ) and estrogen receptor- (ER- ) in the eutopic endometrium and ovarian endometriotic cysts. Design: Prospective study. Setting: University hospital. Patient(s): Patients with endometriosis and patients with uterine leiomyoma or carcinoma in situ. Intervention(s): Gonadotropin-releasing hormone agonist (GnRH-a)-treated (n 12) or untreated (n 24) endometriotic cysts were obtained from 36 patients during laparoscopic cystectomy. Eutopic endometrium tissues were obtained from 24 patients during or immediately after surgery. Main Outcome Measure(s): ER- and ER- mrna levels, using a real-time reverse transcription (RT)- polymerase chain reaction (PCR) assay, TaqMan RT-PCR. Result(s): Eutopic endometrium and ovarian endometriotic cysts showed predominantly higher levels of ER- mrna than ER- mrna. Although ER- and ER- mrna levels in the eutopic endometrium were affected by a cyclic change in ovarian hormones, ovarian endometriotic cysts were less affected. Moreover, a long-term hypoestrogenic state induced by GnRH-a especially decreased ER- mrna levels in endometriotic cysts. Consequently, the relative ratios of ER- to ER- mrna levels in both GnRH-a-treated and untreated endometriotic cysts were significantly lower than those in the eutopic endometrium. Conclusion(s): The results suggest that the principal and regulatory effects of estrogens may be mediated mainly via ER- rather than ER- in both the eutopic endometrium and endometriotic cysts. (Fertil Steril 2000;74: by American Society for Reproductive Medicine.) Key Words: Estrogen receptor-, estrogen receptor-, endometriosis, gonadotropin-releasing hormone agonist (GnRH-a), TaqMan reverse transcription-polymerase chain reaction Endometriosis occurs almost exclusively in menstruating women of reproductive age. It is highly likely, therefore, that estrogen stimulates the growth of endometriotic tissue. Until recently it was considered that estrogen acts via a single receptor, the classical estrogen receptor (ER- ). However, the recent identification of ER- (1, 2) has indicated that pathophysiological actions of estrogen are mediated within target cells by the two estrogen receptors, ER- and ER-. Therefore, it is important to study the possible significance of these two estrogen receptors in the development and growth of endometriotic lesions. We previously evaluated the expression of ER- and ER- messenger RNA (mrna) in both the eutopic endometrium and ovarian endometriotic cysts with use of the reverse transcription-polymerase chain reaction (RT-PCR) method and in situ hybridization technique (3, 4). Our results showed that although ER- 753

2 mrna was detected in all analyzed samples of ovarian endometriotic cysts, ER- mrna was not because it was apparently more restricted. These findings suggested that the predominant expression of ER- in glandular epithelial and stromal cells may be essential in the development and growth of ovarian endometriosis (4). However, because of the qualitative nature of the method used, further research using more quantitative methods was needed to clarify differential expression of ER- and ER- isoforms. Therefore, we have developed a rapid, accurate, and highly sensitive method to determine levels of ER- and ER- mrna using a real-time RT-PCR assay with the TaqMan detection system (5, 6). This technique can generate yes-or-no quantitative results much faster than Northern blotting, ribonuclease protection assay, and several other quantitative RT-PCR techniques (7, 8). To characterize the biological significance of ER- and ER- isoforms, we quantified and compared their mrna levels in the eutopic endometrium and ovarian endometriotic cysts at different phases of the menstrual cycle with use of the TaqMan RT-PCR method. In addition, to further characterize the two isoforms, we quantified and compared mrna levels of ER- and ER- in gonadotropin-releasing hormone agonist (GnRH-a)-treated and untreated ovarian endometriotic cysts. Endometriosis has been widely treated with GnRH-a (9), which induces hypogonadism by causing a reversible block of ovarian cyclicity, yet its hypoestrogenic effects on ER- and ER- expression are unknown. MATERIALS AND METHODS Experimental Subjects During laparoscopic cystectomy, a sample of ovarian endometriotic cyst tissue was obtained from each of 36 patients (20 39 years of age). The endometriotic stage for each sample was determined with use of the revised American Fertility Society classification scheme (10). Twentythree of the samples were identified as class III, whereas the remaining were identified as class IV. All of the endometriotic cysts were 3 cm in diameter and contained a granular epithelium surrounded by stromal tissue. The presence of these features was sufficient to meet the criteria for histopathological diagnosis of endometriosis. We microscopically confirmed that cyst samples were not contaminated with normal ovarian tissues. Patients who had undergone ovarian endometriotic cystectomy were divided into two basic categories. The first group (n 12) received GnRH-a (depot leuprolide acetate; Takeda, Tokyo, Japan) treatment 5 months before the surgery, whereas the second group (n 24) received no hormonal treatment during the 6 months period preceding the surgery. Patients in the first group, in which serum 17 -estradiol and progesterone levels were markedly low, were in amenorrhea. Patients in the second group had regular menstrual cycles, confirmed by menstrual history and measurement of serum 17 -estradiol and progesterone levels. Within the second group, 12 patients were in follicular phase and 12 in luteal phase. Control tissues were obtained from normal eutopic endometrium of 24 patients (12 in proliferative phase, 12 in secretory phase, as determined with use of the criteria of Noyes et al. (11). These control samples were collected from patients who had never received hormonal therapy but who had undergone hysterectomy due to uterine leiomyoma or carcinoma in situ of the cervix. Samples of endometriotic cysts and the normal eutopic endometrium were divided into two portions. The first tissue portion was fixed in 4% paraformaldehyde (ph 7.4) and embedded in paraffin to permit the histopathological examinations. The second portion was immediately frozen in liquid nitrogen and stored at 80 C for further analysis. All tissue samples were obtained with the full and informed consent of the patients, and the research protocol was approved by the human research board of the ethical committee of Tohoku University School of Medicine. Theoretical Basis of TaqMan RT-PCR The TaqMan RT-PCR method is based on the use of fluorogenic probes that hybridize to the targeted gene sequence of the two PCR primers (5, 6). It exploits the 5 nuclease activity of the recombinant Tth (rtth) DNA polymerase to cleave a TaqMan probe during PCR. Each intact probe contains a fluorescent dye reporter at the 5 end and a quencher dye at the 3 end, which inhibits the reporter emission by quenching the energy emission. During the amplification reaction, the 5 nuclease activity of the rtth DNA polymerase cleaves the probe between the reporter and the quencher, separating them. The separation of dyes results in increased fluorescence emission of the reporter dye, which can be detected in real-time by monitoring fluorescent energy with the ABI PRISM 7700 Sequence Detector (Perkin-Elmer Applied Biosystems, Chiba, Japan). This increase in fluorescence is proportional to the concentration of the target sequence in the initial samples. To measure sample RNA levels, it is necessary to determine the threshold cycle (CT value), which represents the PCR cycle at which a statistically significant increase (above a baseline signal) in reporter fluorescence energy is first detected. A standard curve of CT initially obtained from known RNA concentrations (copy numbers) is generated, and concentrations of unknown samples can be determined by interpolation. Oligonucleotide Primers and TaqMan Probes for ER- and ER- Oligonucleotide primers and TaqMan probes for ER- and ER- were designed with use of Primer Express (version 1.0, Perkin-Elmer Applied Biosystems) from the GenBank database. Table 1 in Primer Express lists the primer and TaqMan probe sequences and the locations of the comple- 754 Matsuzaki et al. ER- and ER- in ovarian endometriosis Vol. 74, No. 4, October 2000

3 mentary DNA (cdna) corresponding to the human ER- (12) and ER- (2). Synthesis of Recombinant RNA of ER- and ER- for Generation of a Standard Curve Plasmids, in which the full-length wild-type cdna of each ER- or ER- gene was ligated, were used as DNA templates for PCR amplification. The PCR was performed in a 50- L volume. The PCR mixture contained 2 L of the RT reaction product, 5 L of10 PCR buffer, 2.5 mm MgCl 2, 0.5 mm dntps, 0.4 M of each of the sense and antisense primers for ER- or ER-, and 2.5 U Taq DNA polymerase (Takara, Otsu, Japan). Amplification was performed in a Takara PCR Thermocycler (Takara) with use of a hot start. The same cycle profiles were used for both ER- and ER- : an initial denaturation period of 5 minutes at 94 C, followed by 35 cycles of 1 minute at 94 C, 1 minute at 60 C, and 1.5 minutes at 72 C, with a final extension period of 10 minutes at 72 C. The PCR products (ER- : 101 bp; ER- : 202 bp) were eluted from a 2% agarose gel with use of the QIAEX II Gel Extraction kit (QIAgen, Tokyo, Japan). The products were then subcloned in the pgem T-Easy vectors (Promega, Tokyo, Japan) and linearized with Apa I (for ER- ) or Nco I (for ER- ). Recombinant RNA for ER- or ER- was generated by in vitro transcription reaction with T6 RNA polymerase with use of the Dig labeling transcription kit (Boehringer Mannheim, Tokyo, Japan), following the manufacturer s instructions. Recombinant ER- and ER- RNA were quantified by spectrophotometery, aliquoted, and stored at 80 C for future use in the TaqMan RT- PCR assay. RNA Extraction and TaqMan RT-PCR Each of the frozen tissue samples subjected to TaqMan RT-PCR analysis was first homogenized, and the total RNA was extracted with ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer s instructions. With use of a single-tube, single-enzyme system, reverse transcription and DNA polymerization take place without the addition of subsequent enzymes or buffers. The RT-PCR reaction was performed in a 50- L volume of the reaction solution containing 5 TaqMan EZ buffer, 3 mm Mn(OAc) 2, 300 M da/dc/dg/dutp, 5.0 U rtth DNA polymerase, 400 nm primers (forward and reverse; the same primer sets described above), 100 nm TaqMan probe, 0.5 U AmpErase UNG, and 1 g total RNA. The TaqMan EZ RT-PCR Core Reagents kit for RT-PCR reaction was purchased from Perkin-Elmer Applied Biosystems. TaqMan RT reaction conditions were 50 C for 2 minutes, 60 C for 30 minutes, and 95 C for 10 minutes for 1 cycle; TaqMan PCR conditions were 95 C for 15 seconds, 60 C for 1 minute for 40 cycles on an ABI PRISM 7700 Sequence Detector (Perkin-Elmer Applied Biosystems). Each RT-PCR experiments included a standard curve assay with five RNA concentrations in duplicate (10-fold serially diluted recombinant RNA; ER- : g, ER- : g) and a blank assay without RNA template. A strong linear relationship between the threshold cycle (Ct) and the log of the starting RNA-copy number was always shown (r ). (Fig. 1A and B). Statistical Analysis Statistical analysis was performed with the Stat View 4.5 program (Abacus Concepts, Inc., Berkeley, CA). Mann- Whitney U test was applied to compare results from different groups. Statistical significance was defined as P.05. RESULTS The final ER- and ER- mrna levels were shown as the copy number per 1 g of total RNA extracted from each tissue sample. Copy numbers for standard curves of ER- and ER- were calculated with use of the mean molecular weight of recombinant RNA of ER- and ER-. ER- mrna Levels The ER- mrna was detected in all samples of the eutopic endometrium and ovarian endometriotic cysts analyzed. Distribution of ER- mrna levels in each condition (the proliferative phase, the secretory phase, and the GnRHa treatment) is shown in Figure 2A, and the mean mrna levels are summarized in Table 1. The mean ER- mrna level in the eutopic endometrium was significantly higher in the proliferative phase than in the secretory phase. On the other hand, in the endometriotic cysts, the distribution of ER- mrna levels was significantly restricted despite the conditions. The mean ER- mrna levels in endometriotic cysts were significantly less than those in the proliferative eutopic endometrium. Comparison of mean ER- mrna levels of endometriotic cysts showed no statistically significant differences between the proliferative and the secretory phases, but in the GnRH-atreated cysts, the mean level was significantly higher than in the untreated cysts. ER- mrna Levels The ER- mrna was detected in all eutopic endometrial tissue samples but was limited to 9 of 12 of the endometriotic cysts in the proliferative phase, 11 of 12 in the secretory phase, and 11 of 12 of the GnRH-a-treated cysts. Distributions of ER- mrna levels in each condition of the samples are shown in Figure 2B, and the mean mrna levels are summarized in Table 2. In all the samples in which ER- mrna was detected, ER- mrna levels were lower than ER- mrna levels. The eutopic endometrium showed a statistically significant difference in the mean ER- mrna levels between the proliferative and the secretory phases. On the other hand, the endometriotic cysts showed no statistically significant differences between the proliferative phase and the secretory FERTILITY & STERILITY 755

4 FIGURE 1 An ER- mrna standard curve by TaqMan RT-PCR. (A), Amplification plots for reactions with the five points (A, B, C, D, and E) of the ER- standard curve (10-fold serially diluted recombinant RNA for ER- ) and for a blank assay without template RNA (F). Rn represents the normalized reporter signal (Rn) minus the baseline signal. Rn increases during PCR as ER- PCR product copy number increases until the reaction reaches a plateau. (B), A standard curve plotting log starting copy number vs. threshold cycle (Ct) to determine the initial template concentration. Ct represents the PCR cycle at which a significant increase in Rn above a baseline signal can first be detected. Two replicates for each standard curve point sample were performed, but the data for only one are shown here. Matsuzaki. ER- and ER- in ovarian endometriosis. Fertil Steril phase. Compared with untreated endometriotic cysts, the mean ER- mrna level was lower in the GnRH-a-treated endometriotic cysts, but it was not statistically significant. Relative Ratios of ER- : ER- mrna Levels A total of five ER- mrna negative samples of ovarian endometriotic cyst were excluded from this analysis. Therefore, 24 samples of the eutopic endometrium and the remaining 31 samples of ovarian endometriotic cysts, which were positive for both ER- and ER- mrnas, were included for further analysis. Distributions of relative ratios are shown in Figure 2C, and mean relative ratios are summarized in Table 1. Relative ratios of the proliferative eutopic endometrium were widely distributed, whereas those of the secretory eutopic endometrium showed a restricted distribution. The mean relative ratio of the proliferative eutopic endometrium was significantly higher than that of secretory eutopic endometrium. Although there were some exceptional cases (n 4), relative ratios of ovarian endometriotic cysts were limited to 100 in both the proliferative and secretory phases. A statistically significant difference was observed between the mean relative ratios of the proliferative eutopic endometrium and endometriotic cysts. Relative ratios of GnRH-a-treated endometriotic cysts were markedly restricted within low 756 Matsuzaki et al. ER- and ER- in ovarian endometriosis Vol. 74, No. 4, October 2000

5 FIGURE 2 (A), ER- mrna levels in the eutopic endometrium and ovarian endometriotic cysts. Results are expressed as log 10 of copy numbers. (B), ER- mrna levels in the eutopic endometrium and ovarian endometriotic cysts. Results are expressed as log 10 of copy numbers. (C), ER- to ER- mrna ratios in the eutopic endometrium and ovarian endometriotic cysts. Values represent type of samples and its conditions: 1, the eutopic endometrium in proliferative phase (n 12); 2, the eutopic endometrium in secretory phase (n 12); 3, endometriotic cysts in proliferative phase (n 12 in A, n 10 in B and C); 4, endometriotic cysts in secretory phase (n 12 in A, n 10 in B and C); 5, GnRH-a-treated endometriotic cysts (n 12 in A, n 11 in B and C). The horizontal lines represent median value. Matsuzaki. ER- and ER- in ovarian endometriosis. Fertil Steril values, and the mean relative ratio of GnRH-a-treated cysts was significantly lower than that of untreated cysts. DISCUSSION TaqMan RT-PCR analyses successfully revealed quantitative mrna levels of the two ER isoforms (ER- and ER- ) in the eutopic endometrium and ovarian endometriotic cysts and indicated that ER- mrna was predominantly expressed. In addition, we showed that the mean ER- mrna levels in endometriotic cysts were significantly less than those in the proliferative eutopic endometrium. These findings support the previous results of the immunohistochemical studies (13, 14). Although ER- and ER- mrna expression in the proliferative eutopic endometrium showed high levels, these levels in the secretory eutopic endometrium were significantly lower than those of the proliferative endometrium. On the other hand, ovarian endometriotic cysts showed restricted ER- and ER- mrna levels in both the proliferative and secretory phases. These suggest an apparent differential effect of cyclical changes in ovarian hormones on ER- and ER- mrna expression between the eutopic endometrium and ovarian endometriotic cysts. Moreover, these findings support our previous results with in situ hybridization techniques (3, 4). Because of the different response to ovarian hormones, the relative ratio of ER- to ER- mrna in the proliferative phase was significantly higher in the eutopic endometrium than in ovarian endometriotic cysts. These results are totally in agreement with the report by Brandenberger et al. (15) and in partial agreement with the report by Fujimoto et al. (16). Therefore, our results provide further evidence that regulation of ER- and ER- levels in the eutopic endometrium is diminished in ovarian endometriotic cysts. Comparison of ER- and ER- mrna expression in GnRH-a-treated and untreated endometriotic cysts showed a significant decrease in ER- mrna levels in the GnRH-atreated cysts and a slight (nonsignificant) decrease in ER- mrna levels. These results suggest that in ovarian endo- FERTILITY & STERILITY 757

6 metriotic cysts, a long-term hypoestrogenic state results in down-regulation of ER- rather than ER- mrna expression. Furthermore, it is also likely that GnRH-a has a therapeutic effect mainly through a significant decrease in ER- mrna expression. Thus, we consider that in endometriotic cysts, the principal regulatory effects of estrogens are mainly via ER-. Pathogenesis of ovarian endometriotic cysts is a source of controversy. Previous reports have suggested that ovarian endometriosis may develop from a single precursor cell (17, 18). The endometrial cell and the celomic cell (including an ovarian surface mesothelial cell) have been suggested as candidates for the precursor cell; a precursor cell progresses to form a cyst, becoming transplanted, undergoing metaplasia, or combining these two processes (9, 19, 20). If ovarian endometriotic cysts are the consequence of metaplasia, it could be expected that the levels of estrogen receptors would be lower in endometriotic cysts than in precursor cells. The present study clearly showed that mrna levels of the two ER isoforms in ovarian endometriotic cysts were lower than those in the eutopic endometrium. Therefore, the present results and our previous results with in situ hybridization techniques (3, 4) support the theory of metaplasia. It is possible that an endometrial cell or an ovarian surface mesothelial cell, both of which originally expressed ER- and ER- mrna (4), diminished their expression through metaplasia in formation of ovarian endometriotic cysts. The possible role of ER- in the eutopic endometrium and ovarian endometriotic cysts remains elusive. Previous studies have shown putative differences of function between ER- and ER-, and the two isoforms seem to display both cooperative and contradictory effects through the formation of homodimers and/or heterodimers in various organs (21 23). Because ER- mrna levels were contained much more than ER- mrna in the eutopic endometrium and endometriotic cysts, we speculate that ER- may predominantly form homodimers in these tissues and that homodimers of ER- or heterodimers of ER- and ER- are much less than homodimers of ER-. However, ER- might play a role in the eutopic endometrium and ovarian endometriotic cysts. If the effects of estrogens via ER- and ER- are cooperative, ER- may play a minor role in regulation mechanisms of pathophysiology in the eutopic endometrium and ovarian endometriotic cysts. On the other hand, if the effects of estrogens via ER- and ER- are contradictory, it is possible that ER- could have an important role in the mechanism that controls the ER- -mediated effects. In the present study, ER- mrna expression was not detected by TaqMan PCR of 40 cycles in five samples of endometriotic cysts. Because it can be speculated that in these cysts estrogen bioaction via ER- is extremely low or null, detailed characterization of the ER- negative endometriotic cysts may provide further information about the proliferating mechanism and therapeutic methods for treatment of endometriotic cysts. Acknowledgments: The authors thank G. L. Greene, M.D. (Ben May Laboratory for Cancer Research, University of Chicago, Chicago, IL) and S. Mosselman, M.D. (N.V. Organon, Oss, The Netherlands) for providing the ER- and ER- cdnas, respectively. REFERENCES 1. Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. 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