GnRH II as a possible cytostatic regulator in the development of endometriosis

Chieko Morimoto, Yutaka Osuga*, Tetsu Yano, Yuri Takemura, Miyuki Harada, Tetsuya Hirata, Yasushi Hirota, Osamu Yoshino, Kaori Koga, Koji Kugu and Yuji Taketani

Department of Obstetrics and Gynecology, Faculty of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

* To whom correspondence should be addressed. E-mail: yutakaos-tky{at}umin.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: GnRH II is the second form of GnRH and is widely distributed in peripheral tissues of the female reproductive tract as well as in the central nervous system. In the present study, we studied the possible implication of GnRH II in endometriosis. METHODS: Effects of GnRH II on 5-bromo-2'-deoxyuridine (BrdU) uptake by cultured endometriotic stromal cells were examined. Effects of GnRH II on interleukin (IL)-1{beta}-induced expression of cyclooxygenase (COX)-2 and IL-8 were also studied. mRNA levels of GnRH I, GnRH II, type I GnRH receptor and type II GnRH receptor were determined by real-time quantitative RT-PCR in endometrial tissues of women with or without endometriosis and in endometriotic tissues. RESULTS: GnRH II dose-dependently suppressed BrdU uptake by endometrial stromal cells. Treatment with IL-1{beta} markedly increased mRNA levels of COX-2 and IL-8 in endometrial stromal cells and IL-8 protein secretion by these cells, while these increments were significantly suppressed by supplementation with GnRH II. The mRNA levels of GnRH II were lower in endometrial and endometriotic tissues of women with endometriosis than in endometrial tissues of women without endometriosis, both in the proliferative phase and the secretory phase. In addition, as for GnRH I, type I GnRH receptor and type II GnRH receptor, the mRNA levels were lower in endometrial tissues of women with endometriosis than in those without endometriosis in the secretory phase. CONCLUSIONS: In the light of the demonstrated antiproliferative and anti-inflammatory effects of GnRH II on endometrial stromal cells, the lower expression of GnRH II in eutopic and ectopic endometrium of women with endometriosis suggests that endogenous GnRH II-mediated cytostatic regulation may be impaired in the development of endometriosis.

Key words: cytokines/endometriosis/GnRH/immuno-inflammatory response/proliferation/receptor


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Endometriosis, an enigmatic disease, affects women of reproductive age, causing pelvic pain and infertility. Implantation and growth of endometrial tissues in retrograde menstrual flux is a widely-believed aetiology of the disease. As the development of endometriosis is oestrogen-dependent, a GnRH analogue has been used for treatment, to suppress the pituitary–ovarian axis. In addition, the direct effect of the classical form of GnRH (GnRH I) on endometriotic cells has been proposed, based on the findings that the GnRH agonists exerts antiproliferative and apoptotic effects on cultured endometriotic cells (Borroni et al., 2000Go; Imai et al., 2000Go). The antiproliferative effects of GnRH I and GnRH analogues have also been demonstrated in some cancer cells from reproductive organs (Tang et al., 2002Go; Limonta et al., 2003Go).

The second form of GnRH (GnRH II), which was originally cloned from the chicken, has a structure that has been conserved during evolution from fish to mammals (White et al., 1998Go; Millar 2003Go). GnRH II is widely distributed in the central nervous system as well as in peripheral tissues of the female reproductive tract, such as the placenta, endometrium and ovarian granulosa cells (Cheon et al., 2001Go; Choi et al., 2001Go; Kang et al., 2001Go; Siler-Khodr and Grayson, 2001Go, Khosravi and Leung, 2003Go). Remarkably, GnRH II has a more potent antiproliferative effect than GnRH I in human endometrial and ovarian cancer cells (Grundker et al., 2002Go).

These findings prompted us to study whether GnRH II has any effect on the proliferation of endometriotic stromal cells (ESC). In addition, to address a possible implication of GnRH II in the pathogenesis of endometriosis, the expression of GnRH II and its possible human receptors, type I GnRH receptor (GnRHR) and type II GnRHR, was examined in eutopic endometrial tissues of women with or without endometriosis, and in endometriotic tissues. The effects of GnRH II on the expression of interleukin (IL)-8 and cyclooxygenase 2 (COX-2) were also examined in ESC, as these molecules are suggested to be involved in the immunoinflammatory responses that are pivotal in the pathophysiology of endometriosis (Gazvani et al., 1998Go; Iwabe et al., 1998Go; Akoum et al., 2001Go; Ota et al., 2001Go; Chishima et al., 2002Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Sample collection
In total, 59 women undergoing gynaecological surgery were recruited to the study. Forty women (aged 33.7 ± 5.2 years; mean ± SD) had endometriosis and 19 women (aged 34.8 ± 8.3 years) did not have endometriosis but had uterine fibroid (n = 10) and mature cystic teratoma of the ovary (n = 9). Endometrial tissues were collected by curettage at the time of surgery. In women with ovarian endometrioma, endometriotic tissues were collected from the walls of endometriomas. All women with or without endometriosis had had regular menstrual cycles without hormonal treatment for at least 6 months before surgery. The endometrial tissues obtained were dated with a standard histological dating method. The samples were snap-frozen and stored at –80°C for RT-PCR analysis, or were used for cell culture experiments.

Written informed consent was obtained from each woman, and the study protocol was approved by the institutional review board of the University of Tokyo.

Isolation and culture of human ESC
Primary ESC culture was conducted as described previously (Yoshino et al., 2004Go). Endometriotic tissue was dissected free of underlying parenchyma, minced into small pieces, incubated in DMEM/F12 (Gibco, Grand Island, NY, USA) with type I collagenase (Sigma, St Louis, MO, USA) (2.5 mg/ml) and deoxyribonuclease I (Takara, Tokyo, Japan) (15 U/ml) for 1–2 h at 37°C, and separated using serial filtration. Debris was removed with a 100-µm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ, USA), and dispersed epithelial glands were eliminated with a 70-µm nylon cell strainer (Becton Dickinson). Stromal cells remaining in the filtrate were collected by centrifugation, resuspended in DMEM/F12, and plated onto 100-mm dishes (Iwaki, Chiba, Japan) and allowed to adhere at 37°C for 30 min. Non-adhering epithelial cells and blood cells were then removed by rinsing with phosphate-buffered saline. The cells were cultured in DMEM/F12 medium reconstituted with 10% charcoal-stripped fetal bovine serum (Hyclone, Logan, UT, USA), penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (250 ng/ml) (Sigma). At the first passage, the cells were seeded into six-, 24- or 96-well culture plates (Becton Dickinson) at a density of 2 x 105 cells/ml in medium supplemented with 10% fetal bovine serum. After 24 h, the medium was replaced with serum-free medium for 24 h, and then the cells were used for experiments in medium with 2.5% fetal bovine serum.

Purification of the stromal cell population was confirmed by immunocytochemical staining for antibodies against vimentin (stromal cells), cytokeratin (epithelial cells), CD45 and CD68 (monocytes and other leucocytes), and von Willebrand factor (endothelial cells). The purity of the stromal cells was greater than 98%, as judged by positive cellular staining for vimentin and negative cellular staining for cytokeratin, CD45, CD68 and von Willebrand factor.

5-Bromo-2'-deoxyuridine (BrdU) incorporation
The effect of GnRH II (Peptide Institute, Osaka, Japan) on the proliferation of ESC was examined by measuring incorporation of BrdU into DNA. The assay was performed using the Biotrak cell proliferation enzyme-linked immunosorbent assay (ELISA) system (Amersham Biosciences, Little Chalfont, UK), as previously described (Tang et al., 2002Go). Briefly, endometriotic cells were seeded into 96-multiwell plates at a density of 104 cells per well in 100 µl of culture medium. After 24 h, the medium was replaced with fresh medium containing GnRH II, which was dissolved in dimethylsulphoxide and diluted with the medium to yield the desired concentrations. The final concentration of dimethylsulphoxide in the medium never exceeded 0.1%. In the experiment to see the effects of the GnRH I antagonist Antide (Sigma), Antide was added 1 h before the treatment with GnRH II. After 24 h of treatment with GnRH II, 10 µl BrdU solution was added. Incubation was at 37°C for an additional 2 h. The culture medium was then removed and the cells were fixed and the DNA was denatured by the addition of fixative at 200 µl/well. The peroxidase-labelled anti-BrdU bound to the BrdU incorporated in newly synthesized, cellular DNA. The immune complexes were detected by the subsequent substrate reaction, and the resultant colour was read at 450 nm in a Digiscan Microscope Reader (ASYS Hitech, Eugendorf, Austria).

Treatment of ESC to study COX-2 and IL-8 mRNA expression and IL-8 secretion
IL-1{beta} was used as a prototype proinflammatory cytokine to stimulate ESC in view of the finding that it is suggested to play essential roles in the pathophysiology of endometriosis (Taketani et al., 1992Go; Lebovic et al., 2001Go; Yoshino et al., 2004Go). To examine the effects of GnRH II on IL-1{beta}-induced expression of COX-2 and IL-8 mRNA, ESC were preincubated in the culture medium with IL-1{beta} (5 ng/ml; Genzyme, Cambridge, MA, USA) in 24-well plates for 1 h, and then incubated with various doses of GnRH II. As a preliminary study had indicated that the maximum increase in IL-1{beta}-induced mRNA expression of COX-2 and IL-8 was observed at 2 and 4 h, respectively, the effects of GnRH II were examined at 2 h for COX-2 and at 4 h for IL-8. To examine the effects of GnRH II on IL-1{beta}-induced IL-8 secretion by ESC, the cells were incubated with GnRH II for 24 h.

RNA extraction and RT-PCR
Total RNA was extracted from the cultured cells in six-well plates using the RNAeasy Mini Kit (Qiagen, Hilden, Germany). Total RNA was extracted from 22 samples of endometrial tissue and 16 ovarian endometriomas by the acid guanidinium–phenol–chloroform method using Isogen (Nippongene, Toyama, Japan). One microgram of total RNA was reverse-transcribed in a 20-µl volume using Rever Tra Dash (Toyobo, Tokyo, Japan) according to the manufacturer’s instructions. Human glyceraldehyde dehydrogenase (GAPDH) primers (Toyobo) were used to ensure RNA quality and amounts.

The primer pairs of GnRH II, GnRH I, type I GnRHR, type II GnRHR, COX-2, IL-8 and GAPDH used in PCR are shown in Table I. In standard PCR, the conditions for amplification for GnRH I, GnRH II, type I GnRHR and type II GnRHR were all as follows: denaturing, 98°C for 10 s, annealing 60 C for 2 s, extension 74 C for 20 s x30 cycles. PCR products were analysed by agarose gel electrophoresis with ethidium bromide.


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Table I. Primer pairs used for PCR analysis

 

Real-time quantitative PCR was performed as previously reported (Hirota et al., 2003Go; Koga et al., 2000Go; Yoshino et al., 2001Go). Real-time quantitative PCR and data analysis were performed using the Light Cycler (Roche Applied Science, Mannheim, Germany), according to the manufacturer’s instructions. Expression of each mRNA was normalized to RNA loading for each sample using GAPDH mRNA as an internal standard. In real-time quantitative PCR, conditions were as follows: for GnRH II, 40 cycles at 95°C for 15 s, 64°C for 8 s, 72°C for 8 s; for GnRH I, 40 cycles at 95°C for 15 s, 64°C for 8 s, 72°C for 8 s; for type I GnRHR, 45 cycles, at 95°C for 15 s, 64°C for 10 s, 72°C for 12 s; for type II GnRHR, 30 cycles at 95°C for 15 s, 64°C for 8 s, 72°C for 12 s; for IL-8, 30 cycles at 95°C for 15 s, 66°C for 8 s, 72°C for 10 s; for COX-2, 45 cycles at 95°C for 15 s, 64°C for 10 s, 72°C for 12 s; for GAPDH, 30 cycles, at 95°C for 15 s, 64°C for 8 s, 72°C for 10 s. All these PCR conditions were followed by melting curve analysis.

Each PCR product was purified with a Qiaex II gel extraction kit (Qiagen, Tokyo, Japan), and their identities were confirmed by using a DNA sequencer (ABI Prism 310 Genetic Analyzer; Perkin-Elmer Applied Biosystems, Foster City, CA, USA).

IL-8 measurement
Concentrations of IL-8 were measured using a specific ELISA kit (Quantikine; Genzyme/Techne, Minneapolis, MN, USA). The sensitivity of the assay was 3.9 pg/ml. The intra-assay and inter-assay coefficients of variation were less than 5%.

Statistical analysis
Data from the cell culture experiments were analysed using ANOVA with Scheffé’s post hoc analysis for multiple comparisons. For comparison of mRNA levels of the tissue samples, the Mann–Whitney U-test was used. P values less than 0.05 were considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Expression of mRNA for GnRH I, GnRH II, type I GnRHR and type II GnRHR in endometrial tissues, endometriotic tissues and cultured ESC
As shown in Figure 1, standard RT-PCR analysis demonstrated that amplified products with the predicted size of 119 bp for GnRH II, 240 bp for GnRH I, 319 bp for type I GnRHR and 314 bp for type II GnRHR were all detected in endometrial tissues of women with or without endometriosis, endometriotic tissues and cultured ESC. Each PCR product was sequenced and confirmed to be identical to the sequence of GnRH II (White et al., 1998Go), GnRH I (Hayflick et al., 1989Go), type I GnRHR (Chi et al., 1993Go) or type II GnRHR (GenBank NM 057163).



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Figure 1. Standard RT-PCR analysis of the expression of GnRH II, GnRH I, type I GnRH receptor and type II GnRH receptor mRNA in endometrial tissues of women without endometriosis (lane 1) and with endometriosis (lane 2), endometriotic tissues (lanes 3 and 4) and cultured ESC (lanes 5 and 6). The PCR products show the predicted sizes on the basis of cDNA sequences. M, molecular marker. Lane 7 shows a negative control. The result is representative of three independent experiments.

 

Effects of GnRH II on DNA synthesis in ESC
As depicted in Figure 2, GnRH II, at concentrations between 10–12 and 10–6 M, produced a dose-dependent inhibition of BrdU incorporation into DNA in ESC at 24 h of treatment, the maximal effect (28% decrease below the control) being observed at 10–6 M.



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Figure 2. Effects of GnRH II on BrdU incorporation in ESC. ESC were treated with GnRH II at the indicated concentrations for 24 h. Results are shown as the mean percentage of the untreated control ± SEM (bars) of eight independent experiments using samples from different women. *P < 0.01; **P < 0.05 (both versus control).

 

Treatment with Antide (10–8 M) abrogated the inhibitory effect of GnRH II (10–8 M) on BrdU incorporation of ESC, while Antide by itself showed no effect on BrdU incorporation (Figure 3).



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Figure 3. Effects of Antide on GnRH II-induced reduction in BrdU incorporation of ESC. ESC were treated with GnRH II (10–8 M) and/or Antide (10–8 M) for 24 h. Results are shown as the mean percentage of the untreated control ± SEM (bars) of four independent experiments using samples from different women. *P < 0.01; **P < 0.05.

 

Effect of GnRH II on IL-1{beta}-induced mRNA expression of COX-2 and IL-8 in ESC, and IL-1{beta}-induced secretion of IL-8 by ESC
Effects of GnRH II on IL-1{beta}-induced mRNA expression of COX-2 and IL-8 are shown in Table II. IL-1{beta} (5 ng/ml) significantly increased mRNA levels of COX-2 and IL-8 in ESC, and IL-8 protein secretion by ESC. GnRH II, at concentrations between 10–10 and 10–6 M, caused a marked decrease in IL-1{beta}-induced COX-2 mRNA levels at 2 h of treatment, the maximal effect (33.2 % of the control) being observed at 10–6 M. Likewise, GnRH II, at concentrations between 10–10 and 10–6 M, suppressed IL-1{beta}-induced IL-8 mRNA levels at 4 h of treatment, to 68.2% of the control level at 10–6 M. The IL-1{beta}-induced increase in IL-8 secretion by ESC was significantly diminished by treatment with GnRH II (10–10 to 10–6 M) at 24 h.


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Table II. Effects of GnRH II on IL-1{beta}-induced gene expression of COX-2 and IL-8 in ESC and IL-8 protein secretion by ESC

 

Expression of GnRH I, GnRH II, type I GnRHR and type II GnRHR mRNA in endometrial and endometriotic tissues
Expression of GnRH I, GnRH II, type I GnRHR and type II GnRHR mRNA in endometrial and endometriotic tissues was analysed by real-time quantitative RT-PCR (Figure 4A–D). In the proliferative phase, the levels of GnRH I mRNA were not significantly different among endometrial tissues of women with or without endometriosis and endometriotic tissues (Figure 4A). In the secretory phase, mRNA levels in endometrial tissues of women with endometriosis were significantly lower than those without the disease. The variation of mRNA levels of type I GnRHR and type II GnRHR in each tissue showed substantially the same pattern as that of GnRH I (Figure 4A, C and D). On the other hand, the pattern of GnRH II expression in the tissues was unique (Figure 4B). GnRH II mRNA levels in endometrial tissues of women with endometriosis were significantly lower than in tissues of women without endometriosis, both in the proliferative phase and in the secretory phase. The levels in endometriotic tissues were significantly reduced in the proliferative phase and tended to be low in the secretory phase, compared with those in endometrial tissues of women without endometriosis.



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Figure 4. Expression levels of mRNA of GnRH I (A), GnRH II (B), type I GnRH receptor (C) and type II GnRH receptor (D) in endometrial tissues and endometriotic tissues. Total RNA isolated from endometrial tissues with or without endometriosis and endometriotic tissues was reverse-transcribed and amplified by real-time quantitative PCR, and data analysis was performed using the Light Cycler. Expression of GnRH I, GnRH II, type I GnRH receptor and type II GnRH receptor mRNA was normalized to RNA loading for each sample using GAPDH mRNA as an internal standard. The data were calculated by subtracting the signal threshold cycles (CT) of the internal standard (GAPDH) from the CT of GnRH I, GnRH II, type I GnRH receptor and type II GnRH receptor. Symbols represent samples from women in the proliferative phase (open circles) and those in the secretory phase (closed circles). The values represent relative ratios of the expression levels, standardized by the median of those in endometrium without endometriosis. The horizontal bar illustrates the median for each study group. *P < 0.05 versus endometrium of non-endometriosis in the secretory phase; **P < 0.01, ***P < 0.05 vs. endometrium of non-endometriosis in the proliferative phase.

 


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the present study, we demonstrated that GnRH II inhibited the proliferation of ESC. GnRH II also inhibited IL-1{beta}-induced expression of IL-8 and COX-2 in ESC. The expression levels of GnRH II were lower in endometrial tissues and in endometriotic tissues of women with endometriosis compared with those in endometrial tissues of women without the disease, irrespective of the menstrual phases.

GnRH I has been implicated in the pathogenesis of endometriosis on the basis of the findings that a GnRH agonist inhibited the proliferation and stimulated the apoptosis of endometriotic cells (Borroni et al., 2000Go; Imai et al., 2000Go) and endometrial cells (Meresman et al., 2003aGo, bGo). The present study provides the novel notion that GnRH II, as well as GnRH I, may play roles in the pathophysiology of endometriosis.

It is known that both GnRH I and GnRH II can react with type I GnRHR and marmoset’s type II GnRHR with different binding affinities (Millar, 2003Go). In functional studies that measured inositol phosphate production, primate type I GnRHR demonstrated approximately 48-fold selectivity for GnRH I versus GnRH-II, whereas type II GnRHR demonstrated a 421-fold preference for GnRH II versus GnRH I (Harrison et al., 2004Go). Though some investigators suggested that a functional full-length type II GnRHR is absent in the human genome (Morgan et al., 2003Go), a recent study demonstrated that both human type I and type II GnRHRs are involved in GnRH II-induced effects on cell proliferation (Enomoto et al., 2004Go). In the present study, we detected the expression of mRNAs of type I and type II GnRHRs in the endometrial and endometriotic tissues, suggesting the possible involvement of both GnRHRs in the effects of GnRH II in these tissues.

It has been shown that the antiproliferative and apoptotic effects of GnRH I on the endometrial cells were blocked by Antide (GnRH I antagonist), suggesting that type I GnRHR is responsible for the effects (Meresman et al., 2003aGo, bGo). Similarly, as shown in this study, the antiproliferative effect of GnRH II on endometriotic cells was inhibited by Antide. Thus, type I GnRHR is suggested to mediate these antiproliferative effects of both GnRH I and GnRH II, while type II GnRHR may be co-functioning. Notably, Antide has been reported to inhibit GnRH I- and GnRH II-dependent suppression of progesterone secretion by human granulosa cells (Kang et al., 2001Go) and of the proliferation of immortalized ovarian surface epithelium (Choi et al., 2001Go).

On the other hand, type I GnRHR-independent functions of GnRH II have been indicated. Although both GnRH I and GnRH II up- or down-regulate urokinase-type plasminogen activator, plasminogen activator inhibitor type 1, matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitor of metalloproteinase-1 in human cytotrophoblasts, the GnRH I antagonist Cetrorelix is reported to abolish the effect of GnRH I, but not that of GnRH II, on the expression of these molecules (Chou et al., 2002Go, 2003Go). In addition, antiproliferative effects of GnRH II on human endometrial and ovarian cancer cells have been shown to be independent of type I GnRHR (Grundker et al., 2004Go).

Accumulating evidence suggests that eutopic endometrium from women with endometriosis has aberrant properties compared with that from women without the disease, and that these aberrant properties may play central roles in the pathogenesis and pathophysiology associated with the disease (Sharpe-Timms, 2001Go). For example, cell proliferation was shown to be increased in endometrial cells of women with endometriosis, suggesting that the elevated proliferative property may increase the likelihood that refluxed endometrial cells will implant and survive in the peritoneal cavity (Wingfield et al., 1995Go). The present study showed that the expression of GnRH II was significantly decreased in eutopic endometrium of women with endometriosis, both in the proliferative phase and in the secretory phase. Likewise, endometriotic cells expressed lower levels of GnRH II than endometrial cells of women without the disease. In the light of the antiproliferative effect of GnRH II on endometriotic cells, these findings may be consistent with the notion that endometrial cells in women with endometriosis have enhanced proliferative activities.

In addition to the proliferation of endometriotic cells, inflammation associated with endometriotic lesions is proposed to play essential roles in the pathophysiology of endometriosis (Lebovic et al., 2001Go; Wu and Ho, 2003Go). IL-1{beta}, the levels of which are increased in the peritoneal fluid of women with endometriosis (Taketani et al., 1992Go; Ho et al., 1996Go), is suggested to be a key proinflammatory molecule in endometriosis. The IL-1{beta}-induced increase of COX-2 and IL-8 in endometriotic cells has been suggested to be involved in the development of the disease (Yoshino et al., 2004Go). The present findings that GnRH II suppresses the IL-1{beta}-induced expression of IL-8 and COX-2 in ESC imply another mechanism by which GnRH II inhibits the development of endometriosis. In this context, again it may make sense that the expression of GnRH II in eutopic and ectopic endometrium of women with endometriosis is reduced.

Given that both type I and type II GnRHRs mediate the effects of GnRH II, the low expression levels of the two receptors in endometrial tissues of women with endometriosis in the secretory phase imply that the anti-endometriosis effects of GnRH II may be defective in the secretory endometrium of these women. The diminished expression of the receptors, in conjunction with decreased GnRH II expression, may further predispose these women to endometriosis.

In summary, the present study demonstrates that GnRH II exerts the antiproliferative and anti-inflammatory effects in ESC. The lower expression levels of GnRH II in eutopic and ectopic endometrium of women with endometriosis suggest that endogenous GnRH II-mediated cytostatic mechanisms may be impaired in the process of the development of endometriosis.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank Emi Nose for her technical assistance.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on January 4, 2005; resubmitted on May 1, 2005; accepted on June 9, 2005.





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