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Effect of mifepristone on the expression of endometrial secretory leukocyte protease inhibitor in new medroxyprogesterone acetate users

2008; Elsevier BV; Volume: 90; Issue: 3 Linguagem: Inglês

10.1016/j.fertnstert.2007.01.046

1556-5653

Aimin Li, Juan C. Felix, Wangrong Yang, John K. Jain,

Endometriosis Research and Treatment

The effect of mifepristone on the expression of secretory leukocyte protease inhibitor (SLPI) in the endometrium of women using depot medroxyprogesterone acetate (DMPA) was investigated in this randomized, placebo-controlled trial. The study showed that the administration of DMPA led to a substantial inhibition of endometrial SLPI protein and mRNA, and that the addition of mifepristone to DMPA-exposed endometrium partially restored the expression of glandular SLPI. The effect of mifepristone on the expression of secretory leukocyte protease inhibitor (SLPI) in the endometrium of women using depot medroxyprogesterone acetate (DMPA) was investigated in this randomized, placebo-controlled trial. The study showed that the administration of DMPA led to a substantial inhibition of endometrial SLPI protein and mRNA, and that the addition of mifepristone to DMPA-exposed endometrium partially restored the expression of glandular SLPI. Depo-medroxyprogesterone acetate (DMPA) is a safe and effective long-acting contraceptive that is used worldwide. However, it is commonly associated with unpredictable breakthrough bleeding (BTB), particularly in the early months of use. Over the last two decades, many studies have attempted to improve bleeding performance and evaluate the changes in the endometrium exposed to progestin although the reasons for the bleeding disturbances remain unclear (1Rogers P.A. Martinez F. Girling J.E. Lederman F. Cann L. Farrell E. et al.Influence of different hormonal regimens on endometrial microvascular density and VEGF expression in women suffering from breakthrough bleeding.Hum Reprod. 2005; 20: 3341-3347Crossref PubMed Scopus (8) Google Scholar, 2Girling J.E. Heryanto B. Patel N. Rogers P.A. Effect of long-term progestin treatment on endometrial vasculature in normal cycling mice.Contraception. 2004; 70: 343-350Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 3Hickey M. 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Endometrial progesterone and estrogen receptors and bleeding disturbances in depot medroxyprogesterone acetate users.Hum Reprod. 2004; 19: 547-552Crossref PubMed Scopus (12) Google Scholar, 8Weisberg E. Brache V. Alvarez F. Massai R. Mishell Jr., D.R. Apter D. et al.Clinical performance and menstrual bleeding patterns with three dosage combinations of a Nestorone progestogen/ethinyl estradiol contraceptive vaginal ring used on a bleeding-signaled regimen.Contraception. 2005; 72: 46-52Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Mifepristone, a synthetic steroid that exerts an antiprogestational action by competing with progesterone for receptor binding (9Cadepond F. Ulmann A. Baulieu E.E. RU486 (mifepristone): mechanisms of action and clinical uses.Annu Rev Med. 1997; 48: 129-156Crossref PubMed Scopus (324) Google Scholar), was shown to decrease the incidence of BTB in Norplant users (10Glasier A.F. Wang H. Davie J.E. Kelly R.W. Critchley H.O. Administration of an antiprogesterone up-regulates estrogen receptors in the endometrium of women using Norplant: a pilot study.Fertil Steril. 2002; 77: 366-372Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 11Cheng L. Zhu H. Wang A. Ren F. Chen J. Glasier A. Once a month administration of mifepristone improves bleeding patterns in women using subdermal contraceptive implants releasing levonorgestrel.Hum Reprod. 2000; 15: 1969-1972Crossref PubMed Scopus (76) Google Scholar), Implanon users (12Weisberg E. Hickey M. Palmer D. O'Connor V. Salamonsen L.A. Findlay J.K. Fraser I.S. A pilot study to assess the effect of three short-term treatments on frequent and/or prolonged bleeding compared to placebo in women using Implanon.Hum Reprod. 2006; 21: 295-302Crossref PubMed Scopus (67) Google Scholar), and DMPA users (13Jain J.K. Nicosia A.F. Nucatola D.L. Lu J.J. Kuo J. Felix J.C. Mifepristone for the prevention of breakthrough bleeding in new starters of depo-medroxyprogesterone acetate.Steroids. 2003; 68: 1115-1119Crossref PubMed Scopus (38) Google Scholar). Inflammatory response is known to play major role in menstruation (14Salamonsen L.A. Lathbury L.J. Endometrial leukocytes and menstruation.Hum Reprod Update. 2000; 6: 16-27Crossref PubMed Scopus (237) Google Scholar). Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor and has been characterized as a critical inhibitor of neutrophil elastase in the lung (15Thompson R.C. 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: 6692-6696Crossref PubMed Scopus (430) Google Scholar). Evidence suggests that SLPI acts to inhibit various proinflammatory systems (16Zhang Y. DeWitt D.L. McNeely T.B. Wahl S.M. Wahl L.M. Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases.J Clin Invest. 1997; 99: 894-900Crossref PubMed Scopus (160) Google Scholar, 17Jin F.Y. Nathan C. Radzioch D. Ding A. Secretory leukocyte protease inhibitor: a macrophage product induced by and antagonistic to bacterial lipopolysaccharide.Cell. 1997; 88: 417-426Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). For example, SLPI decreases the production of matrix metalloproteinases (MMPs, responsible for extracellular matrix degradation) in human monocytes (16Zhang Y. DeWitt D.L. McNeely T.B. Wahl S.M. Wahl L.M. Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases.J Clin Invest. 1997; 99: 894-900Crossref PubMed Scopus (160) Google Scholar) and inhibits activation of the nuclear factor κB (NFκB) signal transduction pathway in mice macrophage cell lines (17Jin F.Y. Nathan C. Radzioch D. Ding A. Secretory leukocyte protease inhibitor: a macrophage product induced by and antagonistic to bacterial lipopolysaccharide.Cell. 1997; 88: 417-426Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Also, SLPI has been found associated with mucosal surfaces, including endometrial epithelium (18Franken C. Meijer C.J. Dijkman J.H. Tissue distribution of antileukoprotease and lysozyme in humans.J Histochem Cytochem. 1989; 37: 493-498Crossref PubMed Scopus (144) Google Scholar, 19King A.E. Critchley H.O. Kelly R.W. Presence of secretory leukocyte protease inhibitor in human endometrium and first trimester decidua suggests an antibacterial protective role.Mol Hum Reprod. 2000; 6: 191-196Crossref PubMed Scopus (108) Google Scholar). The precise function of SLPI in the endometrium has not been established; however, its role as a serine protease inhibitor in other tissues (20Hollander C. Nystrom M. Janciauskiene S. Westin U. Human mast cells decrease SLPI levels in type II–like alveolar cell model, in vitro.Cancer Cell Int. 2003; 3: 14Crossref PubMed Scopus (14) Google Scholar, 21Ashcroft G.S. Lei K. Jin W. Longenecker G. Kulkarni A.B. Greenwell-Wild T. et al.Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing.Nat Med. 2000; 6: 1147-1153Crossref PubMed Scopus (339) Google Scholar, 22Angelov N. Moutsopoulos N. Jeong M.J. Nares S. Ashcroft G. Wahl S.M. Aberrant mucosal wound repair in the absence of secretory leukocyte protease inhibitor.Thromb Haemost. 2004; 92: 288-297PubMed Google Scholar) and its hormone-regulated expression in endometrium (19King A.E. Critchley H.O. Kelly R.W. Presence of secretory leukocyte protease inhibitor in human endometrium and first trimester decidua suggests an antibacterial protective role.Mol Hum Reprod. 2000; 6: 191-196Crossref PubMed Scopus (108) Google Scholar, 23Chen D. Xu X. Cheon Y.P. Bagchi M.K. Bagchi I.C. Estrogen induces expression of secretory leukocyte protease inhibitor in rat uterus.Biol Reprod. 2004; 71: 508-514Crossref PubMed Scopus (28) Google Scholar, 24Velarde M.C. Iruthayanathan M. Eason R.R. Zhang D. Simmen F.A. Simmen R.C. Progesterone receptor transactivation of the secretory leukocyte protease inhibitor gene in Ishikawa endometrial epithelial cells involves recruitment of Kruppel-like factor 9/basic transcription element binding protein-1.Endocrinology. 2006; 147: 1969-1978Crossref PubMed Scopus (31) Google Scholar, 25Velarde M.C. Parisek S.I. Eason R.R. Simmen F.A. Simmen R.C. The secretory leukocyte protease inhibitor gene is a target of epidermal growth factor receptor action in endometrial epithelial cells.J Endocrinol. 2005; 184: 141-151Crossref PubMed Scopus (19) Google Scholar, 26Fleming D.C. King A.E. Williams A.R. Critchley H.O. Kelly R.W. Hormonal contraception can suppress natural antimicrobial gene transcription in human endometrium.Fertil Steril. 2003; 79: 856-863Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) lead us to believe that such effects may play an important role in controlling the excessive proteolysis and inflammation that has been related to the pathogenesis of BTB (27Li R. Luo X. Pan Q. Zineh I. Archer D.F. Williams R.S. Chegini N. Doxycycline alters the expression of inflammatory and immune-related cytokines and chemokines in human endometrial cells: implication in irregular uterine bleeding.Hum Reprod. 2006; 21: 2555-2563Crossref PubMed Scopus (20) Google Scholar, 28Lockwood C.J. Kumar P. Krikun G. Kadner S. Dubon P. Critchley H. Schatz F. Effects of thrombin, hypoxia, and steroids on interleukin-8 expression in decidualized human endometrial stromal cells: implications for long-term progestin-only contraceptive-induced bleeding.J Clin Endocrinol Metab. 2004; 89: 1467-1475Crossref PubMed Scopus (41) Google Scholar). In the present study, we investigated the expression of SLPI in the endometrium of DMPA users before, during, and after treatment with mifepristone using real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. Endometrial biopsy samples were obtained from a total of 50 regularly menstruating, healthy women. Each of 50 women received a DMPA injection (150 mg) on cycle day 1. On cycle day 14, two pills (25 mg each) with either mifepristone (mifepristone group) or placebo (placebo group) were given in equally randomized and double-blinded fashion (25 women in each group). The mifepristone or placebo treatments continued every 2 weeks for a total of six cycles. Four endometrial biopsy samples were obtained from each patient, one before treatment (first biopsy, secretory phase), one after the administration of DMPA for 14 days (second biopsy); an additional two biopsies were obtained to evaluate early (1 week after mifepristone or placebo, third biopsy) and late (10 weeks after mifepristone or placebo, fourth biopsy) effects of mifepristone or placebo within 1 cycle of DMPA treatment (3 months). Immunohistochemical analysis for SLPI was performed, as previously described elsewhere (29Jain J.K. Li A. Minoo P. Nucatola D.L. Felix J.C. The effect of nonoxynol-9 on human endometrium.Contraception. 2005; 71: 137-142Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 30Jain J.K. Li A. Nucatola D.L. Minoo P. Felix J.C. Nonoxynol-9 induces apoptosis of endometrial explants by both caspase-dependent and -independent apoptotic pathways.Biol Reprod. 2005; 73: 382-388Crossref PubMed Scopus (19) Google Scholar, 31Li A. Felix J.C. Hao J. Minoo P. Jain J.K. Menstrual-like breakdown and apoptosis in human endometrial explants.Hum Reprod. 2005; 20: 1709-1719Crossref PubMed Scopus (25) Google Scholar, 32Li A. Felix J.C. Minoo P. Amezcua C.A. Jain J.K. Effect of mifepristone on proliferation and apoptosis of Ishikawa endometrial adenocarcinoma cells.Fertil Steril. 2005; 84: 202-211Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), on 96 endometrial biopsy samples from 24 women (12 in each group, and four biopsies per woman) with a anti-human SLPI goat polyclonal antibody (1:100, R&D Systems, Minneapolis, MN). The immunostaining was graded as 0 (negative), 1 (trace), 2 (weak), 3 (moderate), and 4 (strong) in both luminal secretions and glandular epithelium, as noted in our previous report (31Li A. Felix J.C. Hao J. Minoo P. Jain J.K. Menstrual-like breakdown and apoptosis in human endometrial explants.Hum Reprod. 2005; 20: 1709-1719Crossref PubMed Scopus (25) Google Scholar). The average of the scores of the two compartment and two independent observers was calculated and taken as the staining index. The relative amounts of SLPI mRNA were also measured in human endometrium by RT-PCR as described previously elsewhere (29Jain J.K. Li A. Minoo P. Nucatola D.L. Felix J.C. The effect of nonoxynol-9 on human endometrium.Contraception. 2005; 71: 137-142Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 30Jain J.K. Li A. Nucatola D.L. Minoo P. Felix J.C. Nonoxynol-9 induces apoptosis of endometrial explants by both caspase-dependent and -independent apoptotic pathways.Biol Reprod. 2005; 73: 382-388Crossref PubMed Scopus (19) Google Scholar, 31Li A. Felix J.C. Hao J. Minoo P. Jain J.K. Menstrual-like breakdown and apoptosis in human endometrial explants.Hum Reprod. 2005; 20: 1709-1719Crossref PubMed Scopus (25) Google Scholar, 32Li A. Felix J.C. Minoo P. Amezcua C.A. Jain J.K. Effect of mifepristone on proliferation and apoptosis of Ishikawa endometrial adenocarcinoma cells.Fertil Steril. 2005; 84: 202-211Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) (12 women per group, and four biopsies per woman). The nucleotide sequences of the primers for SLPI were forward 5′-GCATCAAATGCCTGGATCCT-3′ and reverse 5′-TGCCCATGCAACACTTCAAG-3′. For glyceraldehyde-3-phosphate dehydrogenase (GAPDH) the sequences were forward 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse 5′-GAAGATGGTGATGGGATTTC-3′. The assay for each sample was performed in duplicate, and the amplicons were checked by agarose gel electrophoresis for a single band of the expected size with no bands in no-RT controls. Relative quantification analysis was carried out with the LightCycler software, version 4 (Roche Applied Science, Mannheim, Germany). Two ratios were compared: the ratio of SLPI to GAPDH in samples from the third and fourth biopsies with the ratio of the same two sequences in samples from the second biopsy, which served as a "calibrator" (1× sample). The results are expressed as a normalized ratio. Nonparametric, Wilcoxon's signed-rank test was chosen for comparing the immunostaining scores and relative abundance of mRNA. P<.05. was considered statistically significant. Histologic analysis revealed that all samples obtained after 14 days of DMPA (second biopsy) displayed a progestational effect and inactive glands with fewer secretions. One week after mifepristone or placebo administration (third biopsy), 15 of 20 samples (75%) in the mifepristone group showed proliferative or weakly proliferative endometrium with frequent mitosis, whereas only 2 of 20 (10%) women in the placebo group showed weakly proliferative endometrium, and no woman in the placebo group showed active proliferative endometrium. After 10 weeks of treatment (fourth biopsy), there was no appreciable difference between the two groups, with most patients showing atrophic endometrium. Immunohistochemical staining for SLPI antigen from two representative cases is shown in Figure 1. We found that SLPI was expressed in epithelial cells of glands and their secretions of all pretreated secretory endometrium, with increased expression in the middle to late secretory phase. It was virtually absent in stromal cells with the exception of some decidualized stromal cells of DMPA-treated endometrium (data not shown). After the women had received an intramuscular injection of DMPA for 14 days, SLPI expression in glandular epithelium and luminal secretions was statistically significantly decreased in the biopsy samples of the women whose initial biopsy sample had demonstrated middle (4 vs 3, P=.01) and late secretory endometrium (4 vs 1, P=.03) (see Fig. 1B, F). After 14 days of DMPA exposure, SLPI mRNA (including all tissue compartments: glandular and luminal epithelium, stroma, endothelium, vascular smooth muscle, and decidualized cells) was also significantly decreased when compared with late secretory endometrium (1 vs 0.11, P=.002), but was increased when compared with early to middle secretory endometrium (1 vs 7.9, P=.007, and 1 vs 3.84, P=.001, respectively). Administration of mifepristone to DMPA-exposed endometrium for 1 week (see Fig. 1C, mifepristone plus DMPA) had no statistically significant effect on the immunoreactivity of glandular SLPI when compared with the placebo group (see Fig. 1G, DMPA only) (1 vs 1, P=.31). However, after prolonged exposure (10 weeks) of DMPA-treated endometrium to mifepristone (see Fig. 1D), SLPI expression in glands and their secretions showed a statistically significant increase when compared with DMPA-only endometrium (see Fig. 1H) (2 vs 1, P=.002). Although the SLPI mRNA level showed a statistically significant increase after 10 weeks (1.32 vs 1, P=.001) relative to the second biopsy samples in the mifepristone group, it was statistically significantly lower than that of the placebo group (1.32 vs 17.2, P=.008). The clinical outcome of this study will be reported elsewhere that demonstrates mifepristone significantly decreased the number of bleeding intervals through the end of 6 months, and maintained a trend toward decreased BTB through 12 months, as was previously reported elsewhere (33Jain J.K. Dutton C. Harwood B. Meckstroth K.R. Mishell Jr., D.R. A prospective randomized, double-blinded, placebo-controlled trial comparing mifepristone and vaginal misoprostol to vaginal misoprostol alone for elective termination of early pregnancy.Hum Reprod. 2002; 17: 1477-1482Crossref PubMed Scopus (87) Google Scholar). In this study, we report the immunohistochemical finding that mifepristone increased glandular SLPI expression whereas DMPA decreased its expression. The function of SLPI has been implicated in the regulation of inflammation by direct inhibition of proteases. As a potent neutrophil-elastase inhibitor, it may play a role in the prevention of tissue degradation in the endometrium by decreasing the infiltration of neutrophils that contribute to the tissue breakdown (14Salamonsen L.A. Lathbury L.J. Endometrial leukocytes and menstruation.Hum Reprod Update. 2000; 6: 16-27Crossref PubMed Scopus (237) Google Scholar, 34Poropatich C. Rojas M. Silverberg S.G. Polymorphonuclear leukocytes in the endometrium during the normal menstrual cycle.Int J Gynecol Pathol. 1987; 6: 230-234Crossref PubMed Scopus (57) Google Scholar). In fact, SLPI may also play an important role in suppressing the destruction of connective tissue by blocking the signal transduction pathway, specifically a cyclooxygenase-2/prostaglandin E2/cAMP pathway leading to the production of MMPs as indicated in the monocytes (16Zhang Y. DeWitt D.L. McNeely T.B. Wahl S.M. Wahl L.M. Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases.J Clin Invest. 1997; 99: 894-900Crossref PubMed Scopus (160) Google Scholar). It may be that the decrease of SLPI in DMPA-only users results in a higher infection/inflammation rate and enhanced MMP production, leading to bleeding, and that a restoration of SLPI by mifepristone would limit infection/inflammation, reduce MMPs, and improve bleeding. Expression of SLPI appears particularly to increase in the late secretory phase around the time of decidualization and implantation and then to further increase in the decidua of pregnancy (19King A.E. Critchley H.O. Kelly R.W. Presence of secretory leukocyte protease inhibitor in human endometrium and first trimester decidua suggests an antibacterial protective role.Mol Hum Reprod. 2000; 6: 191-196Crossref PubMed Scopus (108) Google Scholar), suggesting regulation of SLPI by progesterone. In addition, expression of SLPI mRNA has been experimentally induced in the uteri of ovariectomized rats in response to estrogen, a process that was mediated via estrogen receptors (23Chen D. Xu X. Cheon Y.P. Bagchi M.K. Bagchi I.C. Estrogen induces expression of secretory leukocyte protease inhibitor in rat uterus.Biol Reprod. 2004; 71: 508-514Crossref PubMed Scopus (28) Google Scholar). These data again strongly support the hormonal regulation of this molecule, in this instance by estrogen. Because we have previously shown that DMPA plus mifepristone therapy induces both estrogen and progesterone receptors in endometrium (35Jain J.K. Li A. Yang W. Minoo P. Felix J.C. Effects of mifepristone on proliferation and apoptosis of human endometrium in new users of medroxyprogesterone acetate.Hum Reprod. 2006; 21: 798-809Crossref PubMed Scopus (17) Google Scholar), we hypothesize that the induction of SLPI highlighted in this report is mediated by the progesterone receptors or estrogen receptors or possibly both. There was a negative correlation between SLPI protein and SLPI mRNA levels in the samples from the fourth biopsy. A likely explanation for this discrepancy is that RT-PCR indiscriminately evaluates cells from all of the varied endometrial compartments. Because the total number of stromal cells greatly outnumbers those of the glandular or vascular components, we believe that these cells may preferentially influence mRNA determinations and "wash out" or even reverse the observations seen in the glandular tissue. In such a case, low levels of expression in each individual stromal cell, when examined in aggregate by RT-PCR, would yield significant levels while remaining silent when detected by immunohistochemical examination. Previous work has demonstrated that decidual cells as well as pseudo-decidualized cells express SLPI (26Fleming D.C. King A.E. Williams A.R. Critchley H.O. Kelly R.W. Hormonal contraception can suppress natural antimicrobial gene transcription in human endometrium.Fertil Steril. 2003; 79: 856-863Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). It is also possible that pseudo-decidualized stromal cells seen in our samples at 10 weeks of DMPA treatment were responsible for the increased levels of SLPI detected by RT-PCR. There are limitations to this study. Endometrial biopsy samples obtained at the end of the 3-month study contained scant endometrium. In some instances, the amount of tissue and the absence of functionalis precluded the performance of either immunohistochemical evaluation or RT-PCR. Because the endometrial tissue was at times extremely scant, the relative abundance of glandular versus stromal components may have varied significantly across such small samples. In conclusion, we report a decrease of endometrial glandular and luminal SLPI immunoreactivity in women using DMPA and a relative increase after mifepristone was added to the DMPA-exposed endometrium. We speculate that this restoration of SLPI expression levels seen with mifepristone may be mediated by estrogen and/or progesterone receptors and could play a role in the mechanism of mifepristone-reduced BTB of DMPA users.