Nitric oxide synthesis is increased in the endometrial tissue of women with endometriosis

Ming-Yih Wu1, Kuang-Han Chao1, Jehn-Hsiahn Yang1, Tsung-Hsien Lee1, Yu-Shih Yang1 and Hong-Nerng Ho1,2,3

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Previous studies have shown that peritoneal macrophages from women with endometriosis produce excess nitric oxide (NO). This study was designed to quantify the amount of NO and determine the expression of endothelial (eNOS) and inducible NO synthases (iNOS) in women with and without endometriosis. METHODS: An enzyme-linked immunosorbent assay (ELISA) was performed on endometrial tissues obtained from controls (myoma, n = 30) and on eutopic/ectopic endometrial tissues from endometriosis patients (n = 34) to evaluate eNOS and iNOS protein concentrations in these endometrial tissues. A rapid-response chemiluminescence analyser was used to measure NO directly in fresh endometrial tissues. RESULTS: Mean (± SEM) levels of NO were significantly increased in the endometrial tissues of women with endometriosis (13.2 ± 7.8 versus 19.8 ± 12.6 nmol/g tissue; P = 0.016). Apparently higher levels of NO were found in ectopic compared with eutopic endometrium (P = 0.057). Endometrial tissues of women with endometriosis appeared to contain more iNOS than those of controls (3.6 ± 2.2 versus 8.6 ± 12.2 pg/µg protein; P = 0.06), but no significant difference was found in eNOS levels. CONCLUSIONS: Greater amounts of NO and NOS are present in the endometrial tissues of women with endometriosis, implying a possible role for NO in the pathogenesis of endometriosis.

Key words: endometriosis/nitric oxide/nitric oxide synthases


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nitric oxide (NO) is an important factor in female reproductive processes, including ovulation, menstruation, implantation, pregnancy maintenance, labour and delivery (Yallampalli et al., 1998Go; Manabe et al., 1999Go; Zervou et al., 1999Go; Chwalisz and Garfield, 2000Go; Battaglia et al., 2002Go; Ekerhovd et al., 2002Go). Women with endometriosis have a significantly higher level of activated peritoneal macrophages (PM) (Halme et al., 1983Go; Dunselman et al., 1988Go) and greater PM activation leading to increased inducible NO synthase (iNOS) expression and NO production (Weinberg, 1998Go; Osborn et al., 2002Go). High levels of NO are involved in antimicrobial and antitumour activities, and an elevation of NO can be pro-inflammatory (Weinberg, 1998Go). By contrast, low levels of NO are important for several vital physiological processes, including maintenance of smooth muscle tone, neurotransmission and modulation of apoptosis. The present authors’ group (Ho et al., 1997aGo; Wu et al., 1999Go), together with others (Lebovic et al., 2001Go; Santanam et al., 2002Go), have reported pelvic pro-inflammatory reactions in women with endometriosis.

Others have reported that increased NO concentration, with altered peritoneal immune defence reaction, was involved in the pathogenesis of endometriosis (Dong et al., 2001Go; Osborn et al., 2002Go). No difference was found in total peritoneal fluid (PF) NO in frozen samples from women with and without endometriosis (Ho et al., 1997bGo). However, the PM in endometriotic samples appeared to produce more NO after lipopolysaccharide (LPS) stimulation (Wu et al., 1999Go). A similar result, derived using fresh PM cultures, has provided further confirmation of these findings (Osborn et al., 2002Go).

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Collection of eutopic and ectopic endometrial tissue
Endometrial control tissue was obtained from myoma cases (group 1, follicular phase, n = 13; group 2, luteal phase, n = 17), while ectopic endometriotic and eutopic endometrial tissues were sampled from women with endometriosis (group 3, follicular phase, n = 17; group 4, luteal phase, n = 17) during the same period. Since the intention was to obtain ectopic tissues, only women with the peritoneal type of endometriosis were enrolled (Nisolle and Donnez, 1997Go). All of the latter samples were classified as revised American Fertility Society stage III or IV. Women were excluded from the study if they had received any hormonal treatment during the 3 months prior to surgery. Any patients with endometrial lesions such as submucosal myoma and endometrial polyps or an intrauterine device were excluded from the study.

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., 1997Go). 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 manufacturer’s 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 Mann–Whitney U-test. The Wilcoxon signed rank test was used to compare total NO between ectopic implants and uterine endometria in cases of endometriosis.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In fresh tissues, nitrite/nitrate concentrations were shown to be significantly higher in women with endometriosis (Figure 1; P = 0.033 for the follicular phase; Table I, P = 0.016 when data for both follicular and luteal phases were combined). Although statistical significance was not achieved, the NO level tended to be higher in ectopic than eutopic endometrial tissues (Figure 1, P = 0.057).



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Figure 1. Nitrite/nitrate concentrations of homogenized endometrial tissues obtained from women with and without endometriosis. Bars represent mean values (nmol/g tissue). *Concentrations were significantly higher (P = 0.033; Mann–Whitney U-test) in endometrial tissues from women with endometriosis (white circles) compared with controls (black circles). **The Wilcoxon signed rank test was used to compare results for eutopic (white circles) with ectopic tissues (white circles, x-hair) (P = 0.057).

 

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Table I. Comparisons of total NO (nitrite/nitrate) and endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in endometrial tissues from women with and without endometriosisa
 
The ELISA quantification of NOS proteins within the endometrial tissues showed no difference in eNOS concentrations between women with and without endometriosis (Table I, P = 0.657). Rather, a slight increase in endometrial iNOS protein concentration was seen in women with endometriosis when compared with that in women without endometriosis (P = 0.060).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous studies have reported that NO in PF from endometriosis cases is increased relative to that from women without endometriosis (Dong et al., 2001Go; Osborn et al., 2002Go). A significant difference in PF levels of NO was not demonstrated, however, in either a recent study using fresh PF (Khorram and Lessey, 2002Go) or the present authors’ previous report utilizing frozen PF (Ho et al., 1997bGo). Another study performed by the present authors’ group determined that PM from patients with endometriosis produced more NO after LPS stimulation compared with controls (Wu et al., 1999Go), and a similar result was reported by others (Osborn et al., 2002Go). In order to verify the difference between these two previous reports, a rapid-response chemiluminescence analyser was used in the present study to measure total NO directly in endometrial tissue, and this showed the endometriotic endometrium to contain more NO. Furthermore, the ectopic tissues contained even more NO than the eutopic tissues in affected women. Despite these findings, however, Khorram’s paracrine hypothesis of NO involvement in the pathogenesis of endometriosis could not be confirmed absolutely (Khorram and Lessey, 2002Go).

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., 1998Go; Hatazawa et al., 2000Go; Khorram and Lessey, 2002Go). Another IHC study of eNOS in endometrial tissue from endometriosis patients revealed no difference compared with that found in normal controls (Kamada et al., 2000Go), 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., 2002Go). 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., 1996Go), 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., 1997Go) 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., 2002Go) 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., 2002Go). The majority of studies, including those of the present authors (Punnonen et al., 1996Go; Harada et al., 1997Go; Ho et al., 1997aGo; Odukoya et al., 1997Go) 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.


    Acknowledgements
 
These studies were supported by grant no. NTUH 91-S027 from the National Taiwan University Hospital and grants NSC 91-2314-B-002-313 and NSC 91-2314-B-002-378 from the National Science Council.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ahmed, A., Dunk, C., Kniss, D. and Wilkes, M. (1997) Role of VEGF receptor-1 (Flt-1) in mediating calcium-dependent nitric oxide release and limiting DNA synthesis in human trophoblast cells. Lab. Invest., 76, 779–791.[ISI][Medline]

Battaglia, C., Regnani, G., Marsella, T., Facchinetti, F., Volpe, A., Venturoli, S. and Flamigni, C. (2002) Adjuvant L-arginine treatment in controlled ovarian hyperstimulation: a double-blind, randomized study. Hum. Reprod., 17, 659–665.[Abstract/Free Full Text]

Chwalisz, K. and Garfield, R.E. (2000) Role of nitric oxide in implantation and menstruation. Hum. Reprod., 15 (Suppl. 3), 96–111.

Dong, M., Shi, Y., Cheng, Q. and Hao, M. (2001) Increased nitric oxide in peritoneal fluid from women with idiopathic infertility and endometriosis. J. Reprod. Med., 46, 887–891.[ISI][Medline]

Dunselman, G.A., Hendrix, M.G., Bouckaert, P.X. and Evers, J.L. (1988) Functional aspects of peritoneal macrophages in endometriosis of women. J. Reprod. Fertil., 82, 707–710.[Abstract]

Ekerhovd, E., Weijdegard, B., Brannstrom, M., Mattsby-Baltzer, I. and Norstrom, A. (2002) Nitric oxide induced cervical ripening in the human: Involvement of cyclic guanosine monophosphate, prostaglandin F(2 alpha), and prostaglandin E(2). Am. J. Obstet. Gynecol., 186, 745–750.[CrossRef][ISI][Medline]

Halme, J., Becker, S., Hammond, M.G., Raj, M.H. and Raj, S. (1983) Increased activation of pelvic macrophages in infertile women with mild endometriosis. Am. J. Obstet. Gynecol., 145, 333–337.[ISI][Medline]

Harada, T., Yoshioka, H., Yoshida, S., Iwabe, T., Onohara, Y., Tanikawa, M. and Terakawa, N. (1997) Increased interleukin-6 levels in peritoneal fluid of infertile patients with active endometriosis. Am. J. Obstet. Gynecol., 176, 593–597.[ISI][Medline]

Hatazawa, J., Ota, H., Murata, M., Igarashi, S. and Tanaka, T. (2000) Localization of endothelial nitric oxide synthase messenger ribonucleic acid by in situ hybridization in ectopic endometrial tissue in patients with adenomyosis. Reprod. Fertil. Dev., 12, 283–287.[ISI][Medline]

Ho, H.N., Wu, M.Y., Chao, K.H. Chen, C.D., Chen, S.U. and Yang, Y.S. (1997a) Peritoneal interleukin-10 increases with decrease in activated CD4+ T lymphocytes in women with endometriosis. Hum. Reprod., 12, 2528–2533.[Abstract]

Ho, H.N., Wu, M.Y., Chen, S.U., Chao, K.H., Chen, C.D. and Yang, Y.S. (1997b) Total antioxidant status and nitric oxide do not increase in peritoneal fluids from women with endometriosis. Hum. Reprod., 12, 2810–2815.[Abstract]

Kamada, Y., Nakatsuka, M., Asagiri, K., Noguchi, S., Habara, T., Takata, M. and Kudo, T. (2000) GnRH agonist-suppressed expression of nitric oxide synthases and generation of peroxynitrite in adenomyosis. Hum. Reprod., 15, 2512–2519.[Abstract/Free Full Text]

Khorram, O. and Lessey, B.A. (2002) Alterations in expression of endometrial endothelial nitric oxide synthase and alpha(v)beta(3) integrin in women with endometriosis. Fertil. Steril., 78, 860–864.[CrossRef][ISI][Medline]

Lebovic, D.I., Mueller, M.D. and Taylor, R.N. (2001) Immunobiology of endometriosis. Fertil. Steril., 75, 1–10.[CrossRef][ISI][Medline]

Manabe, A., Hata, T., Yanagihara, T., Hashimoto, M., Yamada, Y., Irikoma, S., Aoki, S., Masumura, S. and Miyazaki, K. (1999) Nitric oxide synthesis is increased after dehydroepiandrosterone sulphate administration in term human pregnancy. Hum. Reprod., 14, 2116–2119.[Abstract/Free Full Text]

Nisolle, M. and Donnez, J. (1997) Peritoneal endometriosis, ovarian endometriosis, and adenomyotic nodules of the rectovaginal septum are three different entities. Fertil. Steril., 68, 585–596.[CrossRef][ISI][Medline]

Odukoya, O.A., Ajjan, R., Lim, K. Watson, P.F., Weetman, A.P. and Cooke, I.D. (1997) The pattern of cytokine mRNA expression in ovarian endometriomata. Mol. Hum. Reprod., 3, 393–397.[Abstract]

Osborn, B.H., Haney, A.F., Misukonis, M.A. and Weinberg, J.B. (2002) Inducible nitric oxide synthase expression by peritoneal macrophages in endometriosis-associated infertility. Fertil. Steril., 77, 46–51.[ISI][Medline]

Ota, H., Igarashi, S., Hatazawa, J. and Tanaka, T. (1998) Endothelial nitric oxide synthase in the endometrium during the menstrual cycle in patients with endometriosis and adenomyosis. Fertil. Steril., 69, 303–308.[CrossRef][ISI][Medline]

Punnonen, J., Teisala, K., Ranta, H., Bennett, B. and Punnonen, R. (1996) Increased levels of interleukin-6 and interleukin-10 in the peritoneal fluid of patients with endometriosis. Am. J. Obstet. Gynecol., 174, 1522–1526.[ISI][Medline]

Regitnig, P., Reiner, A., Dinges, H.P., Hofler, G., Muller-Holzner, E., Lax, S.F., Obrist, P., Rudas, M. and Quehenberger, F. (2002) Quality assurance for detection of estrogen and progesterone receptors by immunohistochemistry in Austrian pathology laboratories. Virchows Arch., 441, 328–334.[CrossRef][Medline]

Santanam, N., Murphy, A.A. and Parthasarathy, S. (2002) Macrophages, oxidation, and endometriosis. Ann. N. Y. Acad. Sci., 955, 183–198.[Abstract/Free Full Text]

Telfer, J.F., Irvine, G.A., Kohnen, G., Campbell, S. and Cameron, I.T. (1997) Expression of endothelial and inducible nitric oxide synthase in non-pregnant and decidualized human endometrium. Mol. Hum. Reprod., 3, 69–75.[Abstract]

Tseng, L., Zhang, J., Peresleni, T.Y. and Goligorsky, M.S. (1996) Cyclic expression of endothelial nitric oxide synthase mRNA in the epithelial glands of human endometrium. J. Soc. Gynecol. Invest., 3, 33–38.[CrossRef][ISI][Medline]

Weinberg, J.B. (1998) Nitric oxide production and nitric oxide synthase type 2 expression by human mononuclear phagocytes: a review. Mol. Med., 4, 557–591.[ISI][Medline]

Wu, M.Y., Ho, H.N., Chen, S.U., Chao, K.H., Chen, C.D. and Yang, Y.S. (1999) Increase in the production of interleukin-6, interleukin-10, and interleukin-12 by lipopolysaccharide-stimulated peritoneal macrophages from women with endometriosis. Am. J. Reprod. Immunol., 41, 106–111.[ISI][Medline]

Yallampalli, C., Dong, Y.L., Gangula, P.R. and Fang, L. (1998) Role and regulation of nitric oxide in the uterus during pregnancy and parturition. J. Soc. Gynecol. Invest., 5, 58–67.[CrossRef][ISI][Medline]

Zervou, S., Klentzeris, L.D. and Old, R.W. (1999) Nitric oxide synthase expression and steroid regulation in the uterus of women with menorrhagia. Mol. Hum. Reprod., 5, 1048–1054.[Abstract/Free Full Text]

Submitted on March 14, 2003; resubmitted on June 17, 2003; accepted on August 20, 2003.