GnRH agonist-suppressed expression of nitric oxide synthases and generation of peroxynitrite in adenomyosis

Yasuhiko Kamada, Mikiya Nakatsuka1, Kazuo Asagiri, Soichi Noguchi, Toshihiro Habara, Masayo Takata and Takafumi Kudo

Department of Obstetrics and Gynecology, Okayama University Medical School, Okayama-city, Okayama, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Because overproduction of nitric oxide (NO) and peroxynitrite is known to cause tissue injury, the expression of NO synthases (NOS) and generation of peroxynitrite were investigated in adenomyosis. Immunoreactivities to endothelial and inducible NOS demonstrated phase-dependent changes in normal endometrium, and in eutopic endometrium of adenomyosis. However, NOS were expressed throughout the menstrual cycle in ectopic endometrium from the majority of patients with adenomyosis. Nitrotyrosine, a footprint of peroxynitrite, was detected concomitantly with NOS protein. This suggested that high doses of NO and superoxide are produced in the ectopic endometrium, presumably by stimulation with bioactive molecules such as cytokines and growth factors. The expression of NOS and generation of peroxynitrite were markedly reduced by administration of gonadotrophin-releasing hormone agonists (GnRHa). The suppression of serum concentrations of nitrite/nitrate, stable metabolites of NO, by long-term administration of GnRHa was also demonstrated. The suppression of synthesis of NO and/or peroxynitrite may be part of both the therapeutic and adverse effects of GnRHa therapy.

Key words: adenomyosis/GnRH agonist/nitric oxide/peroxynitrite


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Adenomyosis is a common disease among women of reproductive age, and often causes severe dysmenorrhoea, hypermenorrhoea and infertility. However, the pathogenesis and pathophysiology of adenomyosis remain unexplained. Although adenomyosis has some unique properties from endometriosis, the existence of ectopic endometrial gland and stroma is a common characteristic of both diseases, and they have aspects of both inflammatory disease and oestrogen-induced and/or oestrogen-dependent tumour. Endometriotic implants are known to activate local immune responses and cause the release of bioactive mediators including free radicals, cytokines and growth factors, followed by inflammation (Ishikawa et al., 1993Go; McLaren et al., 1996Go; Overton et al., 1996Go; Ota et al., 1998bGo). It has been suggested that even minimal endometriotic change by these molecules in the peritoneal cavity might damage spermatozoa and ovum and cause infertility.

In inflammatory diseases, nitric oxide (NO) is overproduced and forms a potent and relatively long-lived oxidant, peroxynitrite, by reaction with superoxide (Beckman, 1996Go). Because NO has been known to play important roles in reproductive processes such as fertilization (Heck et al., 1994Go), implantation (Chwalisz et al., 1999Go; Purcell et al., 1999Go) and ovulation (Ellman et al., 1993Go), any disorder of NO synthesis has the potential to cause reproductive failure. Peroxynitrite is generated in various pathological conditions, including ischaemia/reperfusion, atherosclerosis and septic shock, and induces lipid peroxidation, protein modification and DNA damage (Beckman, 1996Go). Oestrogen, which is involved in the growth of adenomyosis and endometriosis, is also known to induce nitric oxide synthases (NOS) in various organs (Okamura et al., 1994Go; Weiner et al., 1994Go; Huang et al., 1995Go). In contrast, NO is known to suppress aromatase, which is a key enzyme in the synthesis of sex steroid hormones (Snyder et al., 1996Go). Progesterone is known to regulate expression of NOS in the uterus during pregnancy (Ali et al., 1997Go). In the proliferative phase, progesterone may also affect NO synthesis in the uterus.

In the present study, we determined the phase-dependent changes of expression of NOS in human uterus during a menstrual cycle and the differential expression of this enzyme between eutopic and ectopic endometrium of adenomyotic uterus. We also demonstrated the generation of peroxynitrite in adenomyosis. In addition, we investigated the effect of gonadotrophin-releasing hormone agonists (GnRHa), which are widely used clinical agents for adenomyosis and endometriosis, on the expression of NOS and formation of peroxynitrite in the uterus from patients with adenomyosis. Finally, serum concentrations of nitrite/nitrate, which are stable metabolites of NO, in healthy volunteers with normal menstrual cycles were compared with those in patients treated with GnRHa.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Collection of uterine tissue
Fifty patients of reproductive age, who were undergoing total hysterectomy due to adenomyosis or carcinoma in situ (CIS) of the uterine cervix were recruited into the study. Informed consent was obtained from each patient involved in this study. No patients had other gynaecological complications. Menstrual phase was determined by basal body temperature and histological analysis of eutopic endometrium.

Adenomyosis was diagnosed by the presence of endometrial glands and stroma within the myometrium. Diagnosis was made only when the distance between the lower border of the endometrium and the adenomyosis exceeded one-half of a lower-power field (about 2.5 mm). A total of 38 patients was confirmed histologically as having adenomyosis; 10 of these were in the proliferative phase, and 21 in the secretory phase (Table IGo). The remaining patients (n = 7) were treated with a GnRHa, buserelin (Hoechst Pharmaceuticals, Tokyo, Japan) or nafarelin (Yamanouchi, Tokyo, Japan) for 4–6 months before surgical operation. Hysterectomy was performed without discontinuation of medication. Twelve patients with CIS were designated as controls. These control patients did not have any symptoms related to menstruation, and they did not have endometrial abnormality, leiomyoma, endometriosis or any other diseases of the reproductive organs except CIS in the uterine cervix. Six control patients were in the proliferative phase, and six were in the secretory phase.


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Table I. Ages and indications for 50 subjects undergoing total hysterectomy of the uterine cervix
 
Uterine tissue was fixed in 10% buffered formalin for ~24 h. Specimens were embedded in paraffin, cut into 4 µm thick sections, and mounted on silanized slides. For histological confirmation, the sections were stained with haematoxylin and eosin (Merck, Darmstadt, Germany).

Immunohistochemistry
For immunohistochemistry, slides were processed with the peroxidase-conjugated streptavidin–biotin system (LSAB kit; Dako Corp., Carpinteria, CA, USA) with diaminobenzidine (DAB) chromogen (Biomeda Corp., Foster, CA, USA) following the manufacturer's instructions as described previously (Nakatsuka et al., 1999Go).

Polyclonal antibodies to endothelial NOS (eNOS) and inducible NOS (iNOS) (Affinity BioReagents, Neshanic Station, NJ, USA) were used as primary antibodies. These antibodies were raised against a synthetic peptide of a sequence derived from bovine eNOS or mouse macrophage iNOS by immunization of rabbits. The concentrations used were 1:250 and 1:200 respectively. Large vascular vessels were prepared from normal rats, and liver from lipopolysaccharide (LPS)-treated rats as positive control slides for eNOS and iNOS respectively (Figure 1A and BGo).



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Figure 1. Positive and negative controls in immunohistochemistry. (A) Endothelial nitric oxide synthase (eNOS) protein was detected in the endothelia of rats. (B) Inducible nitric oxide synthase (iNOS) protein was detected in liver from lipopolysaccharide (LPS)-treated rats. (C) Nitrotyrosine was detected in liver from LPS-treated rats. (D) Eutopic endometrium in secretory phase as negative control. Preimmune rabbit sera were used as primary antibodies. After staining with diaminobenzidine (DAB), the sections were counterstained with Mayer's haematoxylin solution. Scale bars: (A) 20 µm; (B–D) 50 µm.

 
To detect formation of peroxynitrite, polyclonal antibodies to nitrotyrosine were used, as peroxynitrite specifically causes nitration of tyrosine residue in protein (Beckman, 1996Go). A rabbit polyclonal antibody raised against nitrated keyhole limpet haemocyanin (KLH) (Upstate Biotechnology Inc., Lake Placid, NY, USA) was used as a primary antibody at 1:500 dilution. As a positive control slide, we used liver from LPS-treated rats (Figure 1CGo). As a negative control, preimmune rabbit sera were used instead of the primary antibodies to NOS (Figure 1DGo). After staining with DAB, the sections were counterstained with Mayer's haematoxylin solution.

In the present study we termed eutopic endometrium of patients with CIS as control endometrium. Eutopic endometrium of adenomyosis was termed eutopic endometrium, while ectopic endometrium located inside the myometrium was termed ectopic endometrium.

Immunostaining was evaluated by scoring the frequency of positive cells and the intensity of staining in the endometrium. Frequency was scored as: 0 = no positive cells; 1 = 0–10% positive; 2 = 10–50% positive; and 3 = 50–100% positive. Intensity was scored as 0 = no staining; 1 = weak staining (distinct from negative controls); 2 = moderate staining (similar to positive control); and 3 = strong staining (stronger than positive control). Sections were assigned a score in more than three high-power (x400) fields by a first observer, then confirmed by a second observer. Positive immunostaining in glandular epithelium or stromal cells was determined when total score was higher than 4 or 2 respectively.

Collection of peripheral blood
Peripheral blood was drawn from patients with adenomyosis or endometriosis during treatment with the GnRHa, buserelin (n = 8). These patients were diagnosed by laparoscopy and/or magnetic resonance imaging (MRI). We also collected blood samples from healthy volunteers as controls (n = 15). Transvaginal ultrasonography revealed normal-sized uterus and ovaries without any characteristic findings of adenomyosis or endometriosis in all control women. Dysmenorrhoea, hypermenorrhoea, dyspareunia, dyschezia, chronic pelvic pain and other characteristic symptoms of adenomyosis or endometriosis were not observed in any of the control women. Informed consent was obtained from each patient or healthy volunteer involved in the study. The phase of the menstrual cycle was determined by basal body temperature and transvaginal ultrasonography. To minimize the influence of nitrate in food, samples were collected in the morning after an overnight fasting. The serum fraction was obtained by centrifugation and stored at –70°C until analysis.

Measurement of nitrite/nitrate in serum
Nitrite/nitrate concentrations in serum were determined as described previously (Nakatsuka et al., 1999Go). The serum was deproteinized by a filter (Ultrafree®-MC 10 000 NMWL Filter Unit; Millipore, Bedford, MA, USA), which was thoroughly washed in advance by ultrapure water (Milli-Q water purification system; Millipore). Nitrate in the sample was reduced to nitrite by nitrate reductase prepared from Escherichia coli (Boehringer Mannheim GmbH, Mannheim, Germany) in 20 mmol/l Tris–HCl buffer containing 40 µmol/l NADPH (Sigma Chemicals, St Louis, MO, USA). Griess reagents were used to determine nitrite concentration. Sodium nitrate was also used as the standard to ensure the efficacy of the nitrate reductase. These reactions were performed in a 96-well microplate and optical density at 540 nm was measured using a microplate reader (Bio-Rad Model 3550, Osaka, Japan).

Measurement of oestradiol in serum
Oestradiol concentrations in sera from the patients with adenomyosis or endometriosis during treatment with GnRHa were determined by enzyme immunoassay (EIA) (Serono SR1TM system, Serono Japan, Tokyo, Japan).

Statistical analysis
Statistical significance in Tables II, III and IVGoGoGo were determined by Fisher's exact probability test. Statistical significance in Figures 4 and 5A and BGoGo were determined by one-way analysis of variance (ANOVA) and Scheffé's multiple comparison post hoc test. Data were presented as mean ± SD, and a P value < 0.05 was considered statistically significant.


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Table II. Immunoreactivities to endothelial nitric oxide synthase (eNOS) in endometrium. Values are presented as percentage of patients with positive staining
 

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Table III. Immunoreactivities to inducible nitric oxide synthase (iNOS) in endometrium. Values are presented as percentage of patients with positive staining
 

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Table IV. Immunoreactivities to nitrotyrosine in endometrium. Values are presented as percentage of patients with positive staining
 


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Figure 4. Localization of nitrotyrosine in eutopic and ectopic endometrium of adenomyosis. Immunoreactivities to nitrotyrosine were detected by brown DAB staining. Mayer's haematoxylin was used for counterstaining. (AC) Eutopic endometrium; (DF) ectopic endometrium. Left column (A, D), proliferative phase; middle column (B, E), secretory phase; right column (C, F), GnRHa-treated. The endometrium shown is representative of the majority in each group. The detailed rate of detection of nitrotyrosine by immunohistochemistry is shown in Table IVGo. Scale bar 50 µm.

 


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Figure 5. Nitrite/nitrate concentrations in serum from normal women over the menstrual cycle. Concentrations were elevated in the mid-follicular and mid-luteal phases. A transient decrease was observed after ovulation. Values are mean ± SD. Statistical significance: *P < 0.01; **P < 0.05.

 

    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
Localization of nitric oxide synthases
Immunoreactivities to eNOS were detected in luminal and glandular epithelium of endometrium and in endothelium of vascular vessels. Intense immunoreactivities to eNOS were detected in control endometrium during the secretory phase (100% of subjects), but not during the proliferative phase (Table IIGo). A similar observation was made in eutopic endometrium of adenomyosis (Figure 2Go). In ectopic endometrium from a proportion (60%) of patients with adenomyosis however, eNOS protein was detected even in the proliferative phase. Staining of some stromal cells was observed in ectopic endometrium in the proliferative phase and also in eutopic endometrium in the secretory phase, although their immunohistochemical score did not fulfil the criteria for positive staining. Treatment with GnRHa significantly decreased the proportion of patients with adenomyosis who expressed eNOS in eutopic endometrium (P < 0.004). GnRHa had less effect in ectopic endometrium.



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Figure 2. Localization of endothelial nitric oxide synthase (eNOS) protein in eutopic and ectopic endometrium of adenomyosis. Immunoreactivities to eNOS were detected by brown DAB staining. Mayer's haematoxylin was used for counterstaining. (AC) Eutopic endometrium; (DF) ectopic endometrium. Left column (A, D), proliferative phase; middle column (B, E), secretory phase; right column (C, F), GnRHa-treated. The endometrium shown is representative of the majority in each group. The detailed rate of eNOS expression detected by immunohistochemistry is shown in Table IIGo. Scale bar 50 µm.

 
Cells expressing iNOS were detected in stromal lesions of endometrium. In control endometrium and eutopic endometrium of adenomyosis, iNOS proteins were detected in more subjects in the secretory phase than in the proliferative phase (Table IIIGo). Ectopic endometrium of adenomyosis contained cells that expressed iNOS protein in the proliferative phase at a higher rate than control or eutopic endometrium of adenomyosis (Figure 3Go). A faint staining of glandular epithelial cells was also observed in eutopic and ectopic endometrium in the secretory phase, although their immunohistochemical score did not fulfil the criteria for positive staining. Expression of iNOS was suppressed by treatment with GnRHa in eutopic endometrium of adenomyosis (P < 0.05). There was also a tendency for GnRHa treatment to suppress expression of iNOS in ectopic endometrium.



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Figure 3. Localization of inducible nitric oxide synthase (iNOS) protein in eutopic and ectopic endometrium of adenomyosis. Immunoreactivities to iNOS were detected by brown DAB staining. Mayer's haematoxylin was used for counterstaining. (AC) Eutopic endometrium; (DF) ectopic endometrium. Left column (A, D), proliferative phase; middle column (B, E), secretory phase; right column (C, F), GnRHa-treated. The endometrium shown is representative of the majority in each group. The detailed rate of iNOS expression detected by immunohistochemistry is shown in Table IIIGo. Scale bar 50 µm.

 
Generation of peroxynitrite
Immunoreactivities to nitrotyrosine (a footprint of peroxynitrite) were detected in cells that expressed eNOS or iNOS. Both in control endometrium and in eutopic endometrium of adenomyosis, immunoreactivities to nitrotyrosine were intense in the secretory phase as compared with the proliferative phase (Figure 4Go). Nitrotyrosine was detected in ectopic endometrium of the majority of the patients with adenomyosis throughout the menstrual cycle (Table IVGo). Treatment with GnRHa reduced the immunoreactivities in both eutopic and ectopic endometrium of adenomyosis (P < 0.002 and P < 0.002 respectively).

Nitrite/nitrate concentrations in serum
Concentrations of nitrite/nitrate in serum varied depending on the menstrual phase (Figure 5Go). Serum nitrite/nitrate concentrations in mid-follicular phase and late follicular phase were 36.7 ± 11.6 µmol/l and 33.2 ± 9.5 µmol/l respectively, which were significantly higher than that in early follicular phase (19.7 ± 5.4 µmol/l, P < 0.01) or early luteal phase (22.9 ± 6.0 µmol/l, P < 0.01). The nitrite/nitrate concentration in mid-luteal phase was 30.8 ± 10.0 µmol/l, and tended to be higher than that in early luteal phase (not significant).

Treatment with GnRHa abolished cyclic change of serum nitrite/nitrate concentrations (Figure 6AGo). Nitrite/nitrate was elevated significantly to 48.6 ± 29.0 µmol/l at 2 weeks after initiation of GnRHa administration (P < 0.05), but decreased to the value at early follicular phase by 4 weeks. Similar changes in oestradiol concentration were observed during GnRHa treatment (Figure 6BGo).



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Figure 6. Oestradiol and nitrite/nitrate concentrations in serum from women treated with GnRHa. Concentrations of both oestradiol (A) and nitrite/nitrate (B) were increased significantly after 2-week treatment with GnRHa. Values are mean ± SD. Statistical significance: *P < 0.05.

 

    Discussion
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 Materials and methods
 Results
 Discussion
 References
 
A large number of women of reproductive age suffer from adenomyosis and have severe dysmenorrhoea or hypermenorrhoea, in addition to troublesome reproductive failure such as sterility or miscarriage. Although the pathophysiology of adenomyosis has not been fully elucidated, bioactive molecules including inflammatory cytokines, growth factors and oestrogen are considered to be key regulators of its growth (Ishikawa et al., 1993Go; McLaren et al., 1996Go; Overton et al., 1996Go). These molecules have also been known to regulate expression of NOS and release of NO (Okamura et al., 1994Go; Weiner et al., 1994Go; Förstermann et al., 1995Go; Huang et al., 1995Go; Hood et al., 1998Go). We therefore investigated the expression and localization of NOS in uterus with adenomyosis, and the effects of GnRHa administration on them.

The present study demonstrated that both eNOS and iNOS may be found in endometrium, and that the expression of eNOS and iNOS proteins is the phase-dependent factor in control endometrium and in eutopic endometrium of adenomyosis. This suggests that these proteins may be under regulation by the hypothalamo-pituitary-ovarian axis. It has been reported that ovariectomy reduces immunoreactive iNOS in mast cells and luminal epithelial cells in mouse uterus, and that it is reversed by treatment with oestradiol (Huang et al., 1995Go). Furthermore, L-NAME, an inhibitor of NOS, is known to suppress uterine enlargement in the oestradiol-treated immature rat (Chaves et al., 1993Go). These reports indicate that oestradiol causes uterine changes at least in part by up-regulating NOS. However, our observation of enhanced expression of NOS in the secretory phase suggests that both progesterone and oestrogen may affect NO synthesis.

It has also been reported that expression of eNOS in control endometrium is evaluated in the secretory phase (Ota et al., 1998aGo), which supports our observation. Because of the short-lived fate of NO, this molecule is likely to be generated in the uterus and act as a paracrine or autocrine regulator in flow and permeability of blood vessels, stromal oedema, glandular secretion and acceptance of the conceptus. In the present study, immunoreactivities to eNOS were detected in glandular epithelial cells and vascular endothelium, whereas those to iNOS were detected in cells located in stromal lesions of the endometrium, including cells with morphological features of leukocytes. Therefore, eNOS and iNOS may function in a different manner.

Although expression of NOS was menstrual cycle-dependent in control endometrium and eutopic endometrium of adenomyosis, it was enhanced throughout the menstrual cycle in ectopic endometrium from the majority of the patients with adenomyosis. It is known that the ectopic endometrium rarely shows functional changes consonant with the ovarian hormonal cycle and with the appearance of the eutopic endometrium. It has also been reported that oestrogen receptors are always present in foci of adenomyosis, while progesterone receptor numbers are low or absent (Tamaya et al., 1979Go) and that aromatase concentrations in ectopic endometrium are lower than those in eutopic endometrium (Yamamoto et al., 1993Go). The disrupted expression of steroid receptors and aromatase may thus be involved in altered hormonal regulation of NOS expression in the ectopic endometrium. Furthermore, tumour necrosis factor and interleukin-1 have been reported to be produced at high concentrations in the peritoneal fluid of patients with adenomyosis (Halme, 1989Go). These cytokines may also affect expression of iNOS in ectopic endometrium.

Under pathological conditions, overproduced NO reacts with superoxide anion at near-diffusion-limited rates to form peroxynitrite anion, a relatively long-lived oxidant (Beckman, 1996Go). We demonstrated that nitrotyrosine, a footprint of peroxynitrite, is present throughout the menstrual cycle in ectopic endometrium. Peroxynitrite is known to cause lipid peroxidation, protein oxidation and DNA damage (Beckman, 1996Go). Therefore, generation of peroxynitrite throughout the menstrual cycle in ectopic endometrium may also be involved in the pathophysiology of adenomyosis.

Reactions of NO or peroxynitrite with transition metals may result in inactivation of iron-sulphur centres in aconitase of mitochondria (Castro et al., 1994Go), lipoxygenase of platelets (Nakatsuka and Osawa, 1994Go), and in inactivation of the haem centre in cytochrome P-450s, including aromatase (Osawa et al., 1995Go). NO also reacts with thiols in enzymes (Snyder et al., 1996Go), and its inactivation may cause cell dysfunction. Locally produced NO or peroxynitrite may also modulate the environment in eutopic endometrium (Ota et al., 1998aGo,bGo), and affect both spermatozoa and ovum, causing infertility in patients with adenomyosis or minimal endometriotic change.

It has also been reported (Telfer et al., 1997Go) that eNOS protein is expressed throughout the menstrual cycle in glandular epithelial cells of eutopic endometrium from patients undergoing hysterectomy for benign diseases. Although these authors did not specify the diseases of the patients in their study, their observation suggests that some benign diseases may also enhance expression of NOS in eutopic endometrium. Such a situation may affect implantation and cause infertility. Furthermore, inhibitory effects of NO on platelet function and myometrial contractility may control menstrual bleeding, although this point remains controversial (Telfer et al., 1997Go; Cameron and Campbell, 1998Go).

Our study indicated that administration of GnRHa suppressed the expression of both eNOS and iNOS and formation of peroxynitrite in adenomyosis. This suppressive effect may be involved in part of the therapeutic effect of GnRHa on adenomyosis and endometriosis. We therefore investigated the effect of GnRHa on concentrations of oestrogen and NO in women of reproductive age.

Our data on healthy volunteers with normal menstrual cycle support oestradiol-mediated NO release. It has also been reported that circulating nitrite/nitrate concentrations are increased in accordance with follicular development (Rosselli et al., 1994Go). These authors also observed a decrease in serum nitrite/nitrate concentrations 12 h after induction of ovulation with human chorionic gonadotrophin. From this, they postulated that post-ovulatory increases in serum progesterone and LH reduce serum nitrite/nitrate concentrations, even in the presence of high oestradiol concentrations. However, we observed a tendency for an increase in mid-luteal phase. Since it has been reported that nitrite/nitrate concentrations are increased during pregnancy (Sladek et al., 1997Go), progesterone may not always be suppressive to the formation of NO.

It is known that GnRHa transiently stimulates and then antagonizes oestrogen secretion. We detected a transient elevation in serum oestrogen concentration, followed by suppression. During treatment with GnRHa, serum concentrations of nitrite/nitrate changed in parallel with those of serum oestradiol, which suggests that GnRHa may affect NO synthesis by regulating oestradiol concentrations. The long-term administration of GnRHa is likely to inhibit NO synthesis, presumably by suppression of oestradiol secretion.

Lastly, protective effects of oestradiol on the cardiovascular system (Farhat et al., 1996Go) and bone (Barbieri, 1992Go) have been reported, and it is believed these effects against atherosclerosis and bone loss are both mediated in part by NO synthesis (Farhat et al., 1996Go; Wimalawansa et al., 1996Go; Holm et al., 1997Go). Therefore, although suppression of NO and peroxynitrite overproduced under pathological conditions may be involved in the therapeutic effect of GnRHa, suppression of constitutive NO synthesis in the cardiovascular system and bone may be involved in its adverse effects.

Further investigation on the roles of NO and peroxynitrite in adenomyosis and endometriosis is necessary.


    Notes
 
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Okayama University Medical School, 2-5-1 Shikata, Okayama-City, Okayama, 700-8558, Japan.E-mail: mikiya{at}cc.okayama-u.ac.jp Back


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 Materials and methods
 Results
 Discussion
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Submitted on January 4, 2000; accepted on August 15, 2000.