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ORIGINAL RESEARCH |
From the Department of Obstetrics and Gynecology, Nagoya City University Medical School, Nagoya; the Department of Anatomy (2), Fukui Medical University, Fukui, Japan; and the Department of Pathology, Baylor College of Medicine, Houston, Texas.
Address reprint requests to: Nobuhiro Suzumori, MD Department of Pathology Baylor College of Medicine One Baylor Plaza Houston, TX 77030 E-mail: suzumori{at}bcm.tmc.edu
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Abstract |
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Methods: In 32 women with endometriomas (17 untreated and 15 treated with GnRH analogue) and 21 with ovarian cystadenomas, we examined the expression of secretory leukocyte protease inhibitor messenger RNA (mRNA) by Northern blot analysis; protein distribution was measured immunohistochemically. Concentrations of secretory leukocyte protease inhibitor in peritoneal fluid were measured by enzyme-linked immunosorbent assay. Expression of secretory leukocyte protease inhibitor in endometrioma explants in vitro was also studied with and without the GnRH analogue treatment.
Results: Secretory leukocyte protease inhibitor mRNA expression was identified only in untreated endometriomas. In the GnRH agonist-treated endometriomas, the semiquantitative H-score for secretory leukocyte protease inhibitor immunostaining was significantly lower than that for untreated endometriomas (P < .001). The peritoneal fluid of the GnRH agonist-treated women also contained significantly lower concentrations of secretory leukocyte protease inhibitor (median 76 ng/mL, interquartile range 51–131 ng/mL; P < .001) than untreated women (124 ng/mL, 70–186 ng/mL).Secretory leukocyte protease inhibitor in endometrioma explants in vitro was significantly inhibited by the GnRH analogue (P < .05).
Conclusion: Expression of secretory leukocyte protease inhibitor in tissue and peritoneal fluid of women with ovarian endometriomas was decreased by GnRH agonist treatment.
Endometriosis is relatively common, mostly affecting the extrauterine pelvic cavity. Transcripts of secretory leukocyte protease inhibitor have been localized in the epithelium with ovarian, peritoneal, and rectovaginal endometriosis by in situ hybridization.1 Our immuno-histochemical studies found that secretory leukocyte protease inhibitor localized in epithelial cells of ovarian endometriomas.1.
A molecule sized 12 kDa, secretory leukocyte protease inhibitor inhibits elastase, cathepsin G, trypsin, chymotrypsin, and mast cell chymase.2–4 It is contained in human cervical, nasal, and bronchial mucus under pathophysiologic conditions5,6 and helps protect mucosas from injury associated with inflammation.3 Recent findings suggested that secretory leukocyte protease inhibitor production by cervical tissues is regulated by ovarian hormones.7
Although data concerning the relation between endometriomas and secretory leukocyte protease inhibitor are limited, our previous findings suggested that secretory leukocyte protease inhibitor may have a great influence on the pathogenesis of ovarian endometriomas,1 which grow progressively and invasively in an estrogen-dependent manner.8 In most affected women, induction of hypoestrogenism using a GnRH agonist results generally in temporary involution, but not complete regression, of endometriotic implants.9,10 It also has been reported that more than half of ovarian endometriomas are decreased in size by treatment with drainage and GnRH agonist.11 The induced hypogonadal state causes regression, which allows for symptomatic improvement.12
The effects of GnRH agonist therapy on the production of secretory leukocyte protease inhibitor and the significance of secretory leukocyte protease inhibitor expression in endometriomas are unclear. Secretion of secretory leukocyte protease inhibitor under the influence of GnRH analogues and estradiol (E2) in vitro has yet to be elucidated. The present study was designed to measure secretory leukocyte protease inhibitor production by ovarian endometriomas in women with or without GnRH agonist treatment.
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Materials and Methods |
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Tissue was stained with hematoxylineosin and confirmed morphologic diagnoses of ovarian endometrioma based on the microscopic features, endometrialtype epithelium, endometrial stroma, and hemosidelinladen macrophages. Tissue and peritoneal fluid samples taken from women with ovarian endometriomas and ovarian cystadenomas were collected during laparoscopy or laparotomy. No evidence of inflammation or malignancy was found in any cases.
Protocols for the Northern blot and immunohisto-chemical analyses were based on published methods.1 The full length of the cloned secretory leukocyte protease inhibitor cDNA fragment and ß-actin cDNA (Nippon Gene, Osaka, Japan) were labeled using a Dig High prime DNA labeling and detection kit (Boehringer Mannheim Biochemica, Mannheim, Germany) for use as probes. Membranes were used to expose x-ray film (Fuji Photo Film, Tokyo, Japan) with intensifying screens. The resultant autoradiograms were scanned using a laser densitometer (Lumi-Imager; Boehringer Mannheim Biochemica).
Human ovarian tissues were immunohistochemically examined with an antisecretory leukocyte protease inhibitor polyclonal antibody (R&D Systems, Minneapolis, MN). To compare the intensity of immunostaining of the positive epithelial cells, we applied H-scores,13 calculated as H-score = Pi x (i + 1), where i is optical intensity, from 0 (negative cells) to 3 (high staining intensity), and Pi is the percentage of stained cells for each given i (from 0% to 100%). This method is a modification of the semiquantitative H-score analysis described by McCarty et al.14
The immunoassay protocol for concentrations of secretory leukocyte protease inhibitor in peritoneal fluid was based on a published method15 with secretory leukocyte protease inhibitor (SLPI)-Immunoassay kit (R&D Systems). Samples from each woman were measured in parallel and duplicate to avoid interassay variance. Sensitivity of the secretory leukocyte protease inhibitor enzyme-linked immunosorbent assay (ELISA) was <32 pg/mL, and the standard curve range was 62.5–4000 pg/mL.
Biopsy specimens from epithelial tissues of ovarian endometriomas were taken from five women without GnRH agonist therapy (three were tested in the proliferative and two in the secretory phase). We used the tissue culture method described by Denison et al.15 The surface epithelia were scraped and the pieces were placed in phenol red-free N-2-hydroxyethylpiperazine-N’-2 ethanesulfonic acid-buffered Dulbecco’s modified Eagle medium/Ham’s F-12 culture medium (Gibco BRL, Gaithersburg, MD). Explants from endometrioma samples were treated in quadruplicate with 10-7 mol/L 17ßE2 (Research Biochemicals Inc., Natick, MA) or 10-7 mol/L GnRH analogue (leuprolide acetate depot; Takeda Seiyaku) and incubated for 24, 48, and 72 hours at 37C. Conditional medium from each explant was analyzed by ELISA. Calculated values were expressed with median and interquartile ranges. Comparisons were made with Kruskal–Wallis and Mann–Whitney tests, P < .05 being regarded as statistically significant.
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Results |
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). The same membranes were reblotted using a ß-actin oligonucleotide probe as a control and the absorbance values for secretory leukocyte protease inhibitor mRNA transcripts were normalized to values for the ß-actin mRNA. A significant difference was found in levels of secretory leukocyte protease inhibitor mRNA between the untreated endometriomas (n = 10), the GnRH agonist-treated endo-metriomas (n = 10), and the cystadenomas (n = 12; seven women were tested in the proliferative and five in the secretory phase), P < .01 determined by Kruskal–Wallis test (Figure 2A).
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). An asymmetric secretory leukocyte protease inhibitor distribution in the supranuclear cytoplasm of epithelial cells was observed with diffuse apical staining. In contrast, no apparent secretory leukocyte protease inhibitor staining was noted in the same endometrioma sample after 10-7 mol/L GnRH analogue treatment for 48 hours in vitro (Figure 3B).
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). In endometriomas from the GnRH analogue-treated women (n = 15), H-scores were between 0 and 120 (median 20, P < .001, compared with 17 untreated endometrioma samples by Mann–Whitney test). We also found that secretory leukocyte protease inhibitor immunostaining was almost undetectable in cystadenomas (n = 21) with H-scores between 0 and 20 (median 0). Among the three groups, the significant difference was determined by Kruskal–Wallis test (P < .001).
Concentrations of secretory leukocyte protease inhibitor in the peritoneal fluid were detected by ELISA in all women sampled. The peritoneal fluid of the GnRH agonist-treated women also contained significantly lower concentrations of secretory leukocyte protease inhibitor (n = 15; median 76 ng/mL, interquartile range 51–131 ng/mL; P < .001) than untreated women (n = 17; 124 ng/mL, 70–186 ng/mL), determined by Mann–Whitney test. The significant difference was noted among untreated women (n = 17), GnRH agonist-treated women with endometriomas (n = 15), and women with cystadenomas (n = 21; 70 ng/mL, 48–127 ng/mL), P < .001, determined by Kruskal–Wallis test (Figure 2C).
To evaluate whether secretory leukocyte protease inhibitor shedding could be modulated by GnRH analogue and E2, ovarian endometrioma explants were grown in 17ß -E2–free medium then treated for 24, 48, and 72 hours with test agents. After culture, 24-, 48-, and 72-hour–conditioned medium was collected from the endometrioma explants of five different women with treatments in quadruplicate. All samples contained detectable concentrations of secretory leukocyte protease inhibitor (Table 1 and Figure 4). In 48- and 72-hour–conditioned medium, release of secretory leukocyte protease inhibitor was significantly inhibited by 10-7 mol/L GnRH analogue (P < .05 by Mann–Whitney test). In contrast, 10-7 mol/L E2 had no effect.
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Discussion |
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Secretory leukocyte protease inhibitor is a secreted molecule that inhibits leukocyte proteases, targeting extracellular matrix proteins,16 so it is likely that its expression is related to proteolysis and remodeling of extracellular matrices. Recent findings suggest that GnRH agonist treatment in rats might alter fibrinolysis and extracellular matrix remodeling, and that those alterations might affect the mechanism of GnRH agonist-induced reduction in adhesion formation.17 Therefore, it is possible that this process is mediated in part by suppression of secretory leukocyte protease inhibitor.
Decreased secretory leukocyte protease inhibitor expression could be associated with a hypogonadal state because in cervical tissues secretory leukocyte protease inhibitor production is regulated by ovarian hormones.7 However, our in vitro data suggest that secretory leukocyte protease inhibitor production by the endometrioma is not influenced by 17ß–E2. Donnez et al18 reported that 3 months of preoperative GnRH agonist treatment reduced pelvic vascularity and endometriotic cyst size. Our findings for endometrioma explants with or without GnRH analogue treatment suggest that the GnRH agonists exert a direct influence on secretory leukocyte protease inhibitor production. With respect to the mechanism of the suppression by the GnRH analogue, functional GnRH receptor mRNA has been described in ectopic endometrium in an abstract (Meltz HL, Feng J, Yalcinkaya TY. Identification of a functional gonadotropin releasing hormone receptor in human endometrial tissues. J Soc Gynecol Invest 1999;3:141A), so GnRH analogues could bind19 and suppress their secretory leukocyte protease inhibitor production, as shown in the long term. GnRH agonists have a long biologic half-life, which might cause a loss of GnRH receptors and down-regulation of GnRH activity.
Extrauterine endometriotic cells and peritoneal macrophages are the two possible major sources of secretory leukocyte protease inhibitor in the peritoneal fluid.1 No apparent endometriotic lesions were found in cystadenoma patients, and it is difficult to obtain peritoneal fluid samples from disease-free women, so we substituted cystadenoma women for controls. Thus, it could be estimated that the secretory leukocyte protease inhibitor levels in the peritoneal fluid is normally attributable to macrophages (median 70 ng/mL). In the untreated endometrioma women the level was significantly higher (median 124 ng/mL), the difference (median 54 ng/mL, untreated women 2 cystoadenoma women) presumably derived from active endometriotic lesions and activated macrophages. Our findings also suggest that the secretory leukocyte protease inhibitor produced by endometriomas is transferred into peritoneal fluid. The fact that the amount of secretory leukocyte protease inhibitor in the GnRH agonist-treated women (median 76 ng/mL) was similar to that in the controls indicated that little secretory leukocyte protease inhibitor is produced in their endometriomas, in line with mRNA data.
Among macrophage-derived growth-promoting and angiogenic factors, concentrations of transforming growth factor-beta, intercellular adhesion molecule-1, and vascular endothelial growth factor in peritoneal fluid were significantly decreased after a 4-month GnRH agonist treatment in endometriosis patients.20 Our present data also indicated that the GnRH analogue might suppress activation of peritoneal macrophages in the endometriosis patients and the secretion of secretory leukocyte protease inhibitor from them might be reduced by the GnRH analogue. Our tissue culture findings showed direct depression of endometrioma activity by GnRH analogue without a toxic effect on the endometrioma cells, so it is likely that the secretory leukocyte protease inhibitor is an important marker of GnRH analogue action.
A component in uterine cervical mucus capable of binding immunoglobulins was identified as secretory leukocyte protease inhibitor, and was involved in pathogenesis of immunologic infertility by trapping sperm.21 It would be of interest to study whether suppression of secretory leukocyte protease inhibitor by GnRH agonist directly improved infertility in patients with endometriosis.
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Footnotes |
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Received July 5, 2000. Received in revised form December 6, 2000. Accepted December 7, 2000.
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2. Thompson RC, Ohlsson K. Isolation, properties, and complete amino acid sequence of human secretory leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase. Proc Natl Acad Sci USA 1986;83:6692–6.
3. Eisenberg SP, Hale KK, Heimdal P, Thompson RC. Location of the protease-inhibitory region of secretory leukocyte protease inhibitor. J Biol Chem 1990;265:7976–81.
4. Hubbard RC, Crystal RG. Antiproteases. In: The lung: Scientific foundations. Crystal RG, West JB, Barners F, eds. New York: Raven Press, 1991:1775–88.
5. Abe T, Kobayashi N, Yoshimura K, Trapnell BC, Kim H, Hubbard RC, et al. Expression of the secretory leukoprotease inhibitor gene in epithelial cells. J Clin Invest 1991;87:2207–15.
6. Jin F, Nathan C, Radzioch D, Ding A. Secretory leukocyte protease inhibitor: A macrophage product induced by and antagonistic to bacterial lipopolysaccharide. Cell 1997;88:417–26.[Medline]
7. Moriyama A, Shimoya K, Ogata I, Kimura T, Nakamura T, Wada H, et al. Secretory leukocyte protease inhibitor (SLPI) concentrations in cervical mucus of women with normal menstrual cycles. Mol Hum Reprod 1999;5:656–61.
8. Rock JA, Markham SM. Pathogenesis of endometriosis. Lancet 1992;340:1264–71.[Medline]
9. Meldrum DR. Management of endometriosis with gonadotropin-releasing hormone agonists. Fertil Steril 1985;44:581–2.[Medline]
10. Nisolle M, Donnez J. Peritoneal endometriosis, ovarian endometriosis, and adenomyotic nodules of the rectovaginal septum are three different entities. Fertil Steril 1997;68:585–96.[Medline]
11. Donnez J, Nisolle M, Gillet N, Smets M, Bassil S, Casanas-Roux F.Large ovarian endometriomas. Hum Reprod 1996;11:641–6.
12. Sharpe-Timms KL, Keisler LW, McIntush EW, Keisler DH. Tissue inhibitor of metalloproteinase-1 concentrations are attenuated in peritoneal fluid and sera of women with endometriosis and restored in sera by gonadotropin-releasing hormone agonist therapy. Fertil Steril 1998;69:1128–34.[Medline]
13. Nisolle M, Casanas-Roux F, Donnez J. Coexpression of cytokeratin and vimentin in eutopic endometrium and endometriosis throughout the menstrual cycle: Evaluation by a computerized method.Fertil Steril 1995;64:69–75.[Medline]
14. McCarty KS Jr, Miller LS, Cox EB, Konrath J, McCarty KS Sr.Estrogen receptor analysis: Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies. Arch Pathol Lab Med 1985;109:716–21.[Medline]
15. Denison FC, Calder AA, Kelly RW. The action of prostaglandin E2 on the human cervix: Stimulation of interleukin 8 and inhibition of secretory leukocyte protease inhibitor. Am J Obstet Gynecol 1999; 180:614–20.[Medline]
16. Owen CA, Cambell MA, Sannes PL, Boukedes SS, Cambell EJ. Cell surface-bound elastase and caathepsin G on human neutrophils: A novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. J Cell Biol 1995; 131:775–89.
17. Sharpe-Timms KL, Zimmer BS, Jollif WJ, Wright JA, Nothnick WB, Curry TE. Gonadotropin-releasing hormone agonist (GnRH-a) therapy alters activity of plasminogen activators, matrix metalloproteinases, and their inhibitors in rat models for adhesion formation and endometriosis: Potential GnRH-a-regulated mechanisms reducing adhesion formation. Fertil Steril 1998;69:916–23.[Medline]
18. Donnez J, Nisolle M, Gillerot S, Anaf V, Clerckx-Braun F, Casanas-Roux F. Ovarian endometrial cyst: The role of gonadotropin-releasing hormone agonist and/or drainage. Fertil Steril 1994;62: 63–6.[Medline]
19. Hus C-C, Lin Y-S, Wang S-T, Huang K-E. Immunomodulation in women with endometriosis receiving GnRH agonist. Obstet Gynecol 1997;89:993–8.[Abstract]
20. Kupker W, Schultze-Mosgau A, Diedrich K. Paracrine changes in the peritoneal environment of women with endometriosis. Hum Reprod Update 1998;4:719–23.
21. Hirano M, Kamada M, Maegawa M, Gima H, Aono T. Binding of human secretory leukocyte protease inhibitor in uterine cervical mucus to immunoglobulins: Pathophysiology in immunologic infertility and local immune defense. Fertil Steril 1999;71:1108–14.[Medline]
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