Department of Obstetrics and Gynecology, IVF Unit, Carmel Medical Center, Haifa, Israel

Transcription

1 FERTILITY AND STERILITY VOL. 79, NO. 3, MARCH 2003 Copyright 2003 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Decreased expression of tissue inhibitor of matrix metalloproteinases in follicular fluid from women with polycystic ovaries compared with normally ovulating patients undergoing in vitro fertilization Shirly Lahav-Baratz, D.Sc., a,b Zaki Kraiem, Ph.D., b,c Hanna Shiloh, M.Sc., a Mara Koifman, M.Sc., a David Ishai, M.D., a and Martha Dirnfeld, M.D. a,c Department of Obstetrics and Gynecology, IVF Unit, Carmel Medical Center, Haifa, Israel Received March 19, 2002; revised and accepted July 26, Presented at the Annual Meeting of the Israeli Society of Fertilization Research, Tel Aviv, Israel, May 30 31, Reprint requests: Martha Dirnfeld, M.D., 7 Michal Street, Haifa, Israel (FAX: ; E- mail: dirnfeld_martha@ clalit.org.il). a Department of Obstetrics and Gynecology, IVF Unit, Carmel Medical Center. b Endocrine Research Unit, Carmel Medical Center. c Faculty of Medicine, Technion, Haifa, Israel /03/$30.00 doi: /s (02) Objective: To compare activity of matrix metalloproteinases (MMP) and expression of their tissue-specific inhibitor (TIMP) in the follicular fluid of normally ovulating women and women with the polycystic ovary syndrome (PCOS). Design: Prospective study. Setting: IVF unit and endocrine research unit. Patient(s): Fourteen patients undergoing IVF treatment (seven with normal ovulation and seven with PCOS). Main Outcome Measure(s): Activity of MMP-2 and MMP-9 and expression of MMP-1, TIMP-1, and TIMP-2 was measured in follicular fluid of the leading follicles by using gel zymography and immunoblot analysis. Result(s): The activity of MMP-2 and MMP-9 and expression of MMP-1 was similar in follicular fluid of normally ovulating patients and patients with PCOS. Significantly lower expression of TIMP-1 was found in follicular fluid of patients with PCOS women compared with normally ovulating patients. Conclusion(s): Because MMPs and TIMPs play a role in the physical and chemical structure of the follicular compartment, the decreased expression of TIMP in patients with PCOS may be part of a compensatory process to overcome the physical properties of the thick ovarian capsule. (Fertil Steril 2003;79: by American Society for Reproductive Medicine.) Key Words: Follicular fluid, matrix metalloproteinases, polycystic ovary syndrome Matrix metalloproteinases (MMPs) are a family of enzymes that are involved in extracellular matrix and basement membrane degradation and play a central role in physiologic and pathologic processes (1). Matrix metalloproteinases are inhibited by tissue-specific inhibitors (TIMPs). The TIMPs bind (in a noncovalent binding) to the active sites of MMPs at a 1:1 ratio. Matrix metalloproteinases and TIMPs are controlled by hormones, growth factors, and cytokines, and are involved in significant changes in the structure and function of reproductive organs (2). In the ovary, the extensive tissue remodeling of the extracellular matrix plays a role in the development of the preovulatory follicles, follicle rupture and luteolysis (3). Most studies showing MMP and TIMP involvement in the life span of the ovarian follicles were performed in rats (4 8) and other mammals (9 11). Few reports have been published on expression and activity of MMP and TIMP in human ovarian follicles (12 14). Expression of various TIMPs, MMP-1, MMP-2, and MMP-9 has also been demonstrated in the fetal ovary (15). The polycystic ovary syndrome (PCOS) is a common endocrine disorder that causes persistent anovulation. The condition has a spectrum of etiologies and clinical manifestations (16). Most women have oligomenorrehea, a high ratio of LH to FSH, and hyperandrogenism, and many have increased insulin resistance. Inappropriate follicle development causes the 567

2 typical cystic appearance on ultrasonography (16). Follicular growth is continuously stimulated, but failure to select the dominant follicles results in arrest of maturation and ovulation. Tissue remodeling of follicles in PCOS, and expression and activity of MMP and TIMP, are assumed to differ from that in the normal ovary. We compared expression and activity of MMP and TIMP in the follicular fluid of women with PCOS and normally ovulating women. MATERIALS AND METHODS Patient Selection and Ovarian Stimulation Follicular fluid from four or five follicles 19 to 21 mm in diameter was collected immediately after oocyte pick-up in seven women undergoing IVF who had normal ovulation and seven women in whom PCOS was diagnosed according to typical appearance of the ovaries on ultrasonography, oligomenorrhea, anovulation, and a serum LH-to-FSH ratio greater than 2 (16). In parallel, follicular fluid was collected from leading follicles of seven normally ovulating women with normal endocrine profile. These women had mechanical infertility or male factor infertility. The fluid was stored at 20 C after centrifugation for 10 minutes at 600 g. Only clear fluid without hemolysis was used. Samples were thawed in ice. Samples were loaded randomly on gels. Protein content was determined by using a protein assay kit with bovine serum albumin as the standard (Bio-Rad, Hercules, CA). Ovarian stimulation was performed in all women by using a long down-regulation protocol with the GnRH agonist buserelin (Suprefact nasal spray; Hoechst, Frankfurt, Germany), 1,000 g/d. Therapy with buserelin was continued until the day of hcg administration. When laboratory testing indicated pituitary suppression (estradiol level 40 pg/ml), treatment with i.m. hmg (Pergonal; Serono, Herzelia, Israel), 2 to 3 ampoules/d, was started. Follicular monitoring by measurement of serum estradiol, LH, and progesterone levels and serial transvaginal ultrasonography was performed as described elsewhere (17). When three or more follicles 18 mm in diameter were seen on transvaginal ultrasonography, patients received 5,000 IU of hcg i.m. Transvaginal ultrasonography guided follicular aspiration was scheduled 35 to 36 hours after hcg administration. The local ethics committee approved the study. Gel Electrophoresis and Zymography Proteolytic activity of MMPs was determined in equal volumes of clear follicular fluid from the two groups of patients by using gel zymography (18). In brief, samples were electrophorated through a 10% polyacrylamide-sodium dodecyl sulfate gel containing 0.5% gelatin as a substrate. After electrophoresis, the gels were washed twice in 2.5% Triton X-100 medium to enable enzyme renaturation. The gels were incubated for 24 hours at 37 C in50mm of Tris buffer (ph, 7.5), 0.2 M of NaCl, 5 mm of CaCl 2, and 0.02% Brij 35. The gels were stained with Coomassie Blue G in 30% methanol and 10% acetic acid for 15 minutes at room temperature on a rotary shaker. The stain was washed with water until clear bands were seen. The gels were washed in 45% methanol and 5% glycerol, and then were dried overnight between two stretched sheets of cellophane. The white band marks the MMPs activity of gelatin degradation. Gelatinase zymography standard for MMP-2 and MMP-9 was used (Chemicon International, Inc., Temecula, CA). Every sample was assayed in duplicate and every experiment was performed at least twice. Proteolitic activity wad quantified by densitometric analysis using the Bio Imaging gel documentation system (Dinco & Renium, Jerusalem, Israel) and TINA software (Raytest, Staubenhardt, Germany). Since TIMPs are separated from the MMPs during gel zymography, the total amount of active MMP enzyme that was bound or free of the inhibitor was quantified by using this method. Western Blot Analysis Equal amounts of follicular fluids (about 50 g of protein) underwent electrophoresis as described above. After electophoresis, the gels were blotted onto m nitrocellulose membranes (Scheicher & Schuel, Dassel, Germany). The membranes were incubated overnight with 20% nonfat milk and Tris-HCl buffer containing 0.01% Tween-20. The membranes were then washed twice with Tris-HCl buffer containing 0.5% Tween-20 and incubated for 1 hour with mouse antihuman MMP-1, mouse antihuman TIMP-1, and mouse antihuman TIMP-2 monoclonal antibodies (Oncogenes Science, Cambridge, MA), with 10% non-fat milk and Tris-HCl buffer containing 0.01% Tween-20. The membranes were then washed with Tris-HCl buffer containing 0.5% Tween-20 and incubated for 1 hour with horseradish peroxidase conjugated antimouse secondary antibody (Jackson ImmunoResearch Laboratories, Baltimore, PA) in 10% non-fat milk and Tris-HCl buffer containing 0.01% Tween-20. Every sample was applied on the gels in duplicate, and every experiment was performed at least twice. The final detection was performed by enhanced chemiluminescence (Biological Industries, Beit Haemek, Israel) and quantified as described above. Statistical Analysis Statistical analysis was performed by using nonparametric analysis of variance, the Kruskal Wallis test, and the Mann Whitney test. The cut-off value for significance was Lahav-Baratz et al. Tissue inhibitor of MMPs in follicular fluid Vol. 79, No. 3, March 2003

3 TABLE 1 Characteristic of study patients. Characteristic Normal patients PCOS patients P value Age (y) NS FSH level (IU/L) NS LH level (IU/L) Estradiol level on the day of hcg administration (IU/L) 5,306 2,378 8,408 4,325 NS No. of oocytes retrieved Average estradiol level per oocyte NS Note: Values are means ( SD). NS not significant; PCOS polycystic ovary syndrome. RESULTS Table 1 shows patient characteristics. Levels of LH and the number of oocytes retrieved differed significantly between patients with PCOS and normally ovulating patients. Activity of MMP-2 was similar in the follicular fluid of women with PCOS and normally ovulating women (6,059 1,673 arb units vs. 5,529 1,499 arb units) (Fig. 1B). Activity of MMP-9 varied greatly and inconsistently between patients, with no apparent difference between women with PCOS and normally ovulating women. Figure 1A shows gel zymography of the proteolytic enzymes (92 and 72 kda for MMP-9 and MMP-2, respectively). The same results were observed when the activity was calculated per g of protein. Because activity of MMP-1 on casein gel was weak and impossible to quantify, we measured expression of this enzyme by using Western blot analysis. Expression of MMP-1 was similar in follicular fluid from women with PCOS and normally ovulating women (0.11% 0.03% vs. 0.13% 0.05%, respectively). Figure 2 shows an immunoblot of MMP-1. The same results were also observed when MMP-1 expression was calculated per g protein. Expression of TIMP-1 in the follicular fluid of patients with PCOS was significantly lower than that of normally ovulating women (0.19% 0.06% and 0.3% 0.07%; P.05) (Fig. 3). Expression of TIMP-2 was also lower in the follicular fluid of patients with PCOS; this difference, however, was not statistically significant (0.21% 0.085% and 0.28% 0.08%) (Fig. 4). The same results were also observed when the inhibitor expression was calculated per g protein. Figure 5 summarizes TIMP-1 and TIMP-2 expression in the follicular fluid of patients with PCOS and normally ovulating patients. DISCUSSION We found that expression of TIMP was decreased in follicular fluid of patients with PCOS compared with normally ovulating women. Similar activity of MMP-2 and MMP-9 and expression of MMP-1 were found in follicular fluid of normally ovulating women and patients with PCOS. The observation concerning MMP-2 activity is not surprising, since MMP-2 is relatively less influenced by a FIGURE 1 (A), Gel zymography of matrix metalloproteinase (MMP)-2 and MMP-9 activity in women with the polycystic ovary syndrome (PCOS) (P) and normally ovulating women (N). St. standard molecular mass marker. (B), Activity of MMP-2 in women with PCOS (P) and normally ovulating women (N). Bars represent arbitrary units. FERTILITY & STERILITY 569

4 FIGURE 2 Expression of matrix metalloproteinase (MMP)-1 in women with the polycystic ovary syndrome (P) and normally ovulating women (N). FIGURE 4 (TIMP)-2 in women with the polycystic ovary syndrome (P) and normally ovulating women (N). MW molecular weight. specific endocrine environment in the cycling endometrium (2) and MMP-2 is the only member of its enzyme family that is not repressed by progesterone (19, 20). This can be explained by the fact that MMP-2 typically lacks the transcription factor AP-1, a key regulator in the induction of the most MMP promoters (21, 22). Matrix metalloproteinase-2 tends to be constitutively expressed, although it can eventually be regulated by post-transcriptional and post-translational processes (2). Matrix metalloproteinase-1 and MMP-9 are regulated by various hormones and cytokines, such as interleukin-1 and tumor necrosis factor- (23). The involvement of MMP-1 in ovulation has been demonstrated in several mammalian species (5, 24), but such studies have not been done in humans. Localization of MMP-1 in the fetal ovary was demonstrated by using immunohistochemistry, but its role and regulation in the human ovary has not yet been studied (15). Shalev et al. (25) reported higher levels of MMP-2 and MMP-9 in the follicular fluid of patients with PCOS who were undergoing IVF compared with normally ovulating patients. A possible explanation for the differences between those findings and ours may be that we analyzed follicular fluid only from leading follicles. This factor may be critical in patients with PCOS because of possible intrinsic differences among the different-sized follicles (26, 27). The variation in results according to follicle size may also indicate that changes in TIMP expression occur in mature leading follicles just before ovulation. Expression of TIMP is modulated by progesterone and estradiol and has been associated with steroidogenesis in granulosa cells (28). In anovulatory women with PCOS, the high level of LH may influence changes in the ratio of estradiol to progesterone, which may influence the ratio of FIGURE 3 (TIMP)-1 in women with the polycystic ovary syndrome (P) and normally ovulating women (N). MW molecular weight. FIGURE 5 (TIMP)-1 and TIMP-2 in women with the polycystic ovary syndrome (squares) and normally ovulating women (circles). *P Lahav-Baratz et al. Tissue inhibitor of MMPs in follicular fluid Vol. 79, No. 3, March 2003

5 MMP to TIMP compared with that in normally ovulating women (29, 30). In PCOS patients undergoing IVF treatment with prior hormonal stimulation, the situation can be even more complicated. Exogenous gonadotropins administered during controlled ovarian stimulation may influence levels of MMPs and TIMPs. Comparison of the response of granulosa cells to FSH in terms of estradiol production from ovaries obtained after oophorectomy demonstrated 6-fold to 10-fold more estradiol in anovulatory PCOS cells compared with normal ovarian cells. The response in cells from ovulatory PCOS was similar to the response of normal cells (26). At ovulation, an LH- and hcg-induced increase in ovarian collagenolysis leads to an increase in MMPs expression and activity (2). As this process continues, mrna of specific inhibitors (TIMP) is stimulated; the concentration of degradation products, such as collagen, decreases; and collagenolysis is attenuated (2). Since matrix components, such as -integrins, have been suggested to be involved in this control of degradation (31, 32), we might speculate that the decrease in TIMP-1 expression is part of a compensatory process to overcome the physical properties of the thick ovarian capsule in patients with PCOS. In summary, expression and activity of MMP-1, MMP-2, and MMP-9 were similar in the follicular fluid of patients with PCOS and normally ovulating patients, whereas TIMP-1 expression was decreased in the follicular fluid of patients with PCO. These changes in the ratio of MMP to TIMP are probably modulated by hormonal environment within a specific follicle. References 1. Salamonsen LA. Matrix metalloproteinases and their tissue inhibitors in endocrinology. Trends Endocrinol Metab 1996;7: Hulboy DL, Rudolph LA, Matrisian LM. Matrix metalloproteinases as mediators of reproduction function. Mol Hum Reprod 1997;3: McIntush EW, Smith MF. 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