1 Department of Obstetrics and Gynecology and 2 Department of Medical Research, College of Medicine and the Hospital, National Taiwan University, Taipei, Taiwan
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, National Taiwan University Hospital, No. 7 Chung-Shan South Road, Taipei, Taiwan, 10063. e-mail: hnho{at}ha.mc.ntu.edu.tw
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Abstract |
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Key words: endometriosis/nitric oxide/nitric oxide synthases
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Introduction |
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Others have reported that increased NO concentration, with altered peritoneal immune defence reaction, was involved in the pathogenesis of endometriosis (Dong et al., 2001; Osborn et al., 2002
). No difference was found in total peritoneal fluid (PF) NO in frozen samples from women with and without endometriosis (Ho et al., 1997b
). However, the PM in endometriotic samples appeared to produce more NO after lipopolysaccharide (LPS) stimulation (Wu et al., 1999
). A similar result, derived using fresh PM cultures, has provided further confirmation of these findings (Osborn et al., 2002
).
In order to verify the source of this PF NO increase in women with endometriosis, NO levels were examined in eutopic and ectopic endometrial tissues obtained from affected women. Likewise, to compare the association between nitrite/nitrate and endothelial NO synthase (eNOS)/iNOS, the eNOS and iNOS protein concentrations for these samples were also measured.
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Materials and methods |
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The use of these tissues and the patient protocols were approved by the authors institutional review board, without any requirement for informed consent.
Measurement of nitrite/nitrate in homogenized endometrial tissue
Homogenization
The fresh endometrial tissue was weighed and carefully dissected to remove blood, mucus and/or myometrium components. The whole endometrial tissue samples were frozen in liquid nitrogen immediately after dissection and stored at 80°C until subsequent measurement of NOS activity. For measurement of NO, fresh endometrial tissue (approximate total 0.2 g) was added to 1 ml cold homogenizing buffer (20 mmol/l HEPES-KOH, pH 7.9; 25% glycerol; 420 mmol/l NaCl; 1.5 mmol/l MgCl2; 0.2 mmol/l EDTA; 0.5 mmol/l dithiothreitol; 0.2 mmol/l phenylmethylsulphonyl fluoride). The buffered tissues were homogenized in a MicrosonTM cell homogenizer (Misonix, Inc., Farmingdale, NY, USA) at maximum speed for 5 s (power consumption <4 W), and then cooled in ice-water for 30 s. This procedure was repeated five times to ensure complete tissue destruction. Dense fibrotic components of the ectopic endometrial tissues that were not easily dissolved were removed and discarded after careful weighing.
Deproteinization
Two volumes of 100% cold ethanol (Merck & Co., Inc., Whitehouse Station, NJ, USA) were added to the homogenized samples; these were then vortexed and incubated on ice for 30 min. The homogenate was centrifuged at 12 000xg for 5 min at 4°C, and the supernatant transferred to a new tube on ice for NO measurement.
Measurement of nitrite/nitrate
A rapid-response chemiluminescence analyser (NOA 280; Sievers Instruments, Boulder, CO, USA) was used to measure total gas phase NO (nitrite/nitrate). NO gas reacts with ozone, producing energy in the form of light, and the light emission is proportional to the quantity of NO present. The emission can be measured using a luminometer to determine NO concentration (Ahmed et al., 1997). The sample tube was securely connected to a Zero Gas Filter (Sievers Instruments) and room air passed through the device for 5 min. The linearity of analyser response was interpolated using four repeat calibrations (blank, 1, 10, 50, 100 and 200 µmol/l respectively; a lower limit of <1 nmol/l was demonstrated for the present instrument). The samples (10 µl) were injected into a helium-purged vessel containing 0.8% vanadium chloride in hydrochloric acid to liberate gaseous NO from the dissolved NO and nitrite. The sample gas was then exposed to the ozone in the reaction vessel to form activated nitrogen dioxide (NO2*), which was detected using a red-sensitive photomultiplier tube, and the output recorded using an integrating pen recorder. For each sample, the area under the curve was converted to NO concentration.
ELISA study of NOS
Protein preparation for NOS
Frozen tissues were homogenized using a method similar to that for NO determination. The samples were diluted 40x using wash buffer (R&D Systems, Inc., Minneapolis, MN, USA) for determination of protein concentration.
Measurement of iNOS and eNOS
A commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Inc.) was used to quantitate iNOS and eNOS levels. All samples (diluted 40x) and standards were assayed in duplicate. The iNOS and eNOS sensitivities were 50 and 25 pg/ml respectively. In accordance with the manufacturers instructions, preparations of recombinant human eNOS/iNOS/nNOS (neural NOS), and mouse eNOS/iNOS at 50 ng/ml were assayed for cross-reactivity and interference. No significant cross-reactivity or interference was observed. The NOS contents of the endometrial tissue were derived using the ELISA results and protein concentrations.
Statistical analysis
Comparisons of total NO and ELISA data of eNOS and iNOS were performed using the MannWhitney U-test. The Wilcoxon signed rank test was used to compare total NO between ectopic implants and uterine endometria in cases of endometriosis.
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Results |
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Discussion |
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Immunohistochemical (IHC) staining of iNOS and eNOS indicated that, in comparison with controls, the endometria from endometriosis patients showed a more pronounced eNOS staining in the glandular portion during both the follicular and luteal phases (data not shown). These results were similar to those reported previously (Ota et al., 1998; Hatazawa et al., 2000
; Khorram and Lessey, 2002
). Another IHC study of eNOS in endometrial tissue from endometriosis patients revealed no difference compared with that found in normal controls (Kamada et al., 2000
), although these results may have been affected by factors such as inter-laboratory staining variation, inter-observer differences in estimation of staining intensity, and the scoring system used (Regitnig et al., 2002
). Hence, the decision was made to evaluate the protein content of eNOS and iNOS directly in endometrial samples from endometriosis patients and controls. Using ELISA, an elevation in eNOS could not be demonstrated for the affected patients, and only increased quantities of iNOS were found.
An earlier Northern blot analysis of endometrial tissues showed a predominance of eNOS mRNA (Tseng et al., 1996), but only a small quantity of iNOS mRNA was identified during menstruation. A subsequent study using RT-PCR of the normal endometrium (Telfer et al., 1997
) showed that both eNOS and iNOS expression occurred in the glandular epithelial cells throughout the entire cycle, though these differences in results may have been due to variations in technique sensitivity. By using immunoblotting, one group (Osborn et al., 2002
) showed that, compared with normal controls, iNOS activity was increased in freshly isolated PM from endometriosis cases, and these results were similar to those of the present study. Furthermore, the results agreed with the ELISA findings of increased levels of iNOS protein in the endometrium of women with endometriosis.
The inducible isoform of NOS (iNOS) produces large quantities of NO, while the constitutive isoforms (nNOS and eNOS) produce lower levels (Osborn et al., 2002). The majority of studies, including those of the present authors (Punnonen et al., 1996
; Harada et al., 1997
; Ho et al., 1997a
; Odukoya et al., 1997
) have indicated that activation of PM is greater in endometriosis cases. The results from the present study demonstrated through ELISA that the iNOS isoforms were elevated in tissues from endometriosis patients compared with those from women without endometriosis.
In endometriosis, measurements of NOS and NO production may depend on the method selected, as well as the tissues targeted. In the present study, a rapid-response chemiluminescence analyser was used to measure NO production in fresh endometrial tissues, with NO overproduction in endometriosis confirmed. This was the first study to use ELISA to evaluate iNOS/eNOS protein levels in the endometriotic endometrium. An attempt to elucidate the role of NO in the pathogenesis of endometriosis may generate more specific data, though the activity of these enzymes and the associated results may not be reflected by total enzyme quantity. The present study showed there to be higher levels of NO and NOS in the eutopic/ectopic endometrial tissues of women with endometriosis. Hence, in conjunction with the results of previous studies wherein significantly higher levels of PF NO could not be demonstrated, the hypothesis is favoured that NO may produce a paracrine effect in the pathogenesis of endometriosis.
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Acknowledgements |
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References |
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Submitted on March 14, 2003; resubmitted on June 17, 2003; accepted on August 20, 2003.