Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, Seoul, Korea

FERTILITY AND STERILITY VOL. 73, NO. 5, MAY 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP-3 protease activity in the peritoneal fluid of patients with and without endometriosis Jung Gu Kim, M.D., Ph.D., Chang Seok Suh, M.D., Ph.D., Seok Hyun Kim, M.D., Ph.D., Young Min Choi, M.D., Ph.D., Shin Yong Moon, M.D., Ph.D., and Jin Yong Lee, M.D., Ph.D. Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, Seoul, Korea Received September 1, 1999; accepted November 29, 1999. Supported by grant no. 04-97-021 from Seoul National University Hospital Research Fund and the Nondirected Research Fund (no. 1997-001-F00194), Korea Research Foundation, Seoul, Korea. Presented in part at the 16th World Congress on Fertility and Sterility/54th Annual Meeting of the American Society for Reproductive Medicine, San Francisco, California, October 1998. Reprint requests: Jung Gu Kim, M.D., Ph.D., Department of Obstetrics and Gynecology, Seoul National University Hospital, 28 Yeungun-Dong, Chongno-Ku, Seoul 110-744, Korea (FAX: 82-2- 762-3599; E-mail: kimjg @plaza.snu.ac.kr). 0015-0282/00/$20.00 PII S0015-0282(00)00493-3 Objective: To compare levels of insulin-like growth factor (IGF) components in the peritoneal fluid of patients with and without endometriosis. Design: Patients with endometriosis were compared with control patients. Setting: Seoul National University Hospital, Korea. Patient(s): Forty-three patients with endometriosis and 20 patients without endometriosis. Intervention(s): Peritoneal fluid specimens were collected. Main Outcome Measure(s): Insulin-like growth factors, IGF binding protein (IGFBP) profiles and IGFBP-3 protease activity. Result(s): The IGF-I levels in peritoneal fluid were significantly higher in patients with endometriosis than in control patients, while the IGFBP-3 levels and the relative proportion of IGFBP-2 in peritoneal fluid were significantly lower in patients with endometriosis than in control patients. However, IGF-II levels, IGFBP-4 profiles, and IGFBP-3 protease activity did not differ significantly between the two groups. No correlation between these IGF components in peritoneal fluid and the stage of endometriosis was noted. Conclusion(s): The profiles of IGF components in peritoneal fluid of patients with pelvic endometriosis may play an important role in the growth of ectopic endometrium and endometriosis-induced infertility. (Fertil Steril 2000;73:996 1000. 2000 by American Society for Reproductive Medicine.) Key Words: Endometriosis, IGF components, peritoneal fluid Endometriosis, characterized by the presence and growth of ectopic endometrial tissue outside the endometrial cavity, is one of most common gynecologic diseases in women of reproductive age. The causes and factors involved in the growth of ectopic endometrium in pelvic endometriosis are still not clear. The most common location of endometriosis is the pelvis. The pelvic structures are continuously bathed in peritoneal fluid, and it has been demonstrated that peritoneal fluid from patients with endometriosis causes the proliferation of endometrial stromal cells in vitro (1). Therefore, it has been suggested that soluble components in peritoneal fluid may be involved in the growth of ectopic endometrium in pelvic endometriosis. Recent studies (2 4) have shown that the concentrations of steroid hormones, epidermal growth factor, and basic fibroblastic growth factor in peritoneal fluid from women with and those without endometriosis do not differ significantly. On the other hand, it is well known that insulin-like growth factor (IGF) is another potent mitogenic peptide. Its possible role as one of several possible mediators of estrogen and other growth factors in various body tissues has been proposed (5, 6). Insulin-like growth factor has been found to be complexed with a high 996

affinity to a family of IGF binding proteins (IGFBPs) in the circulation and in other body fluids (7). Six IGFBPs have been characterized, and their cdnas have been cloned and sequenced (8). These IGFBPs are believed to modulate the availability of IGF and to mediate its action in their target cells. The functions of IGFBP are further modulated by a specific IGFBP protease (9). Recently, the presence of an IGF system was reported in human peritoneal fluid (10, 11). The IGF peptides and their receptors have also been demonstrated immunohistochemically in ectopic endometrial tissues (12). In addition, it has been demonstrated that IGFs are mitogenic to endometrial stromal cells cultured in vitro (10). However, so far only a few studies (13, 14) have compared IGF and IGFBP levels in peritoneal fluid from patients with and those without endometriosis, and their results have been controversial. Furthermore, IGFBP-3 protease activity in peritoneal fluid of patients with endometriosis has not yet been compared with that in women without endometriosis. The purpose of this study was to evaluate whether IGF components may be involved in the growth of ectopic endometrium by determining and comparing the levels of IGF-I, IGF-II, and IGFBPs and IGFBP-3 protease activity in peritoneal fluid of patients with and those without endometriosis. MATERIALS AND METHODS Patients Forty-three patients aged 30 to 40 years who had endometriosis diagnosed by laparoscopy during the infertility workup were recruited for this study. Twenty additional patients undergoing laparoscopic tubal ligation, who did not have endometriosis, served as the control group. All participants had regular menstrual cycles ranging from 28 to 32 days, and none received any medication for 3 months before the study. Laparoscopy was performed during the late proliferative phase of the menstrual cycle, determined on the basis of the last menstrual period. Patients with any other pathologic condition of the pelvis, such as myoma, were excluded. This study was approved by the Institutional Review Board of Seoul National University Hospital. According to the revised American Fertility Society classification (15), 10 patients had stage I endometriosis, 15 had stage II disease, 10 had stage III disease, and 8 had stage IV disease. Eighteen normal, healthy, nonpregnant women of reproductive age were also included. Serum and peritoneal fluid samples were collected in accordance with the guidelines of the Declaration of Helsinki. Peritoneal fluid specimens were aspirated from the cul-de-sac before any manipulation during laparoscopy, and gross bloody peritoneal fluid samples were excluded. Peritoneal fluid was centrifuged at 800 g for 10 minutes to separate the cells from the supernatant, which was then divided into aliquots and frozen at 70 C until assayed. Blood specimens from the 18 normal, healthy, nonpregnant women were processed, pooled, and stored similarly to the peritoneal fluid samples until their use as controls in Western ligand blotting experiments. Assay Methods Body mass index was calculated by dividing body weight in kg by body weight in m 2. After separating the IGFs from the IGFBPs in peritoneal fluid by using the Bio-Spin P-10 method as described elsewhere (16), IGF-I and IGF-II were measured by using immunoradiometric assay kits (Diagnostic Systems Laboratories Inc., Webster, TX). In brief, a mixture of peritoneal fluid, 1.25 M/L of acetic acid, and 125 mm/l of NaCl was applied to a Bio-Spin P-10 column and centrifuged at 1250 g for 5 minutes four times. The final eluant from each Bio-Spin P-10 column was lyophilized, and before immunoradiometric assay, these samples were reconstituted with an assay buffer at a dilution ratio of 1:30 for IGF-I measurements and 1:96 for IGF-II measurements. The minimum detection limits were 0.8 ng/ml for IGF-I and 12 ng/ml for IGF-II. Respective intraassay and interassay variations were 1.5% and 3.7% for IGF-I and 4.3% and 10.4% for IGF-II. The IGFBPs were measured by using Western ligand blotting and immunoprecipitation with each antihuman IGFBP-specific antibody. Immunoprecipitation was performed as described elsewhere (17). In brief, IGFBPs were immunoprecipitated from peritoneal fluid with protein-a (Pansorbin, Sigma, St. Louis, MO) purified anti-igfbp-1 monoclonal antibody and anti-igfbp-2 IGFBP-4 polyclonal antibodies. The immunoprecipitates were then solubilized and visualized by Western ligand blotting. For the Western ligand blotting, nonreduced samples of peritoneal fluid or the immunoprecipitated samples were subjected to 12% sodium dodecylpolyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto nitrocellulose, and incubated with [ 125 I]IGF-I (10 6 cpm), as described elsewhere (17). Autoradiograms were obtained by exposing the filter to an imaging plate (Fuji Photo Film Co. Ltd., Tokyo, Japan) and scanned by using a Bio-Imaging Analyzer system (BAS 2500, Fuji Photo Film Co. Ltd.) and the Tina 2.0 program (Fuji Photo Film Co. Ltd.). The integrated intensity per unit area for each band with background correction (PSL-Bkg/ mm 2 ) was measured. The level of each IGFBP was calculated relative to the intensity of the same IGFBP band in sera pooled from the 18 normal, healthy, nonpregnant women of reproductive age (normalized to nonpregnant women sera 1.0 for each IGFBP band). The relative proportion of each specific IGFBP was calculated by dividing the band intensity of the specific IGFBP fraction by the total band intensities of all IGFBP fractions and were expressed as the IGFBP ratio. The interassay coefficient of variation was 7.5% for IGFBP-2, 5.1% for IGFBP-3, and 6.4% for IGFBP-4. Proteolytic activity in the peritoneal fluid directed against FERTILITY & STERILITY 997

TABLE 1 IGF levels and IGFBP-3 protease activity in peritoneal fluid of patients with endometriosis and control patients. Group No. of participants IGF-I level (ng/ml) IGF-II level (ng/ml) IGFBP-3 protease activity (%) Control 20 46.12 7.53 331.82 25.28 25.07 2.79 Endometriosis 43 75.68 6.31* 369.07 18.11 26.70 3.43 Stage I or II 25 81.16 9.99* 365.65 26.90 25.14 2.56 Stage III or IV 18 68.08 5.81* 373.81 22.61 26.92 5.61 Note: Values are expressed as means SE. IGF insulin-like growth factor; IGFBP insulin-like growth factor binding protein. * P.05 vs. controls. Kim. IGF components in endometriosis. Fertil Steril 2000. 125 I-labeled recombinant glycosylated IGFBP-3 (Austral Biologicals, San Roman, CA) was measured as described elsewhere (18). Briefly, 30,000 cpm of either 125 I-labeled recombinant IGFBP-3 alone or 125 I-labeled recombinant IGFBP-3 incubated with peritoneal fluid for 5 hours at 37 C was subjected to 12% SDS-PAGE under nonreducing conditions. The gels were dried and autoradiographed. The intensity of the cleavage fragments appearing as a result of proteolytic activity was calculated and expressed as a percentage of the total density of all bands in each lane to give the percentage of protease activity. Statistical Analysis Data were expressed as means SE. Statistical significance was determined by using the Student s t-test and analysis of variance. A P value.05 was considered significant. As shown in Table 1, IGF-I levels in peritoneal fluid were significantly higher in patients with endometriosis than in controls (P.05). However, no difference in IGF-II levels in peritoneal fluid was noted between the two groups. The IGFs levels in peritoneal fluid did not significantly differ between early-stage (I and II) endometriosis and late-stage (III and IV) endometriosis. No differences between the groups in age or body mass index were noted. Four IGFBP bands with apparent molecular weights of 43/37 kilodalton (kda), 31 kda, 26 kda, and 24 kda were detected by Western ligand blotting in the peritoneal fluid of all the patients. The 43/37 kda, 31 kda, and 24 kda bands were confirmed as IGFBP-3, IGFBP-2, and IGFBP-4, respectively, by immunoprecipitation techniques using each antihuman IGFBP-specific antibody. The 26-kDa IGFBP was not precipitated by the antihuman IGFBP-1 monoclonal antibody. The relative levels of IGFBP-3 in peritoneal fluid were significantly lower in patients with endometriosis than in controls (P.05). However, IGFBP-2 and IGFBP-4 levels did not significantly differ between the two groups (Fig. 1A). In addition, patients with endometriosis showed a lower IGFBP-2 ratio (IGFBP-2 intensity to total IGFBPs intensity) in peritoneal fluid than did controls (P.05) (Fig. 1B). Incubation of iodinated IGFBP-3 with peritoneal fluid of all the patients resulted in a reduction in the amount of intact IGFBP-3 and an increase in the intensity of proteolytic fragments, such as 31-kDa, 21-kDa, and 18-kDa bands. The IGFBP-3 protease activity did not significantly differ between patients with endometriosis and control patients (Table 1). The intensity of each proteolytic fragment showed a similar trend. Changes in the IGFBP profiles and IGFBP-3 protease activity by stage of endometriosis were not noted. DISCUSSION In the current study, IGF-I levels in peritoneal fluid were higher in patients with endometriosis than in control patients. FIGURE 1 Relative levels (A) and proportions (B) of IGFBPs (means SE) in peritoneal fluid of patients with endometriosis and control patients., : P.005;, : P.05. RESULTS Kim. IGF components in endometriosis. Fertil Steril 2000. 998 Kim et al. IGF components in endometriosis Vol. 73, No. 5, May 2000

In contrast to the results of Gurgan et al. (14), who reported higher IGF-I levels in patients with late-stage endometriosis compared with early-stage endometriosis, no difference was noted according to stage of endometriosis. A possible reason for this discrepancy is that Gurgan et al. used an acid ethanol extraction, whereas we used Bio-Spin P-10 separation. It has been reported that more than 30% of the IGFBPs remain unextracted from serum samples after acid ethanol extraction, and complete removal of the IGFBPs is accomplished when Bio-Spin P-10 separation is used (16). Incomplete separation of the IGFBPs produces serious artifacts in the IGF assay. The IGF-I receptor has been demonstrated in both endometriotic and endometrial tissue (12). It has been reported that IGF-I has a mitogenic effect in vitro on cultured endometrial cells obtained from patients without endometriosis (10). Our results, in conjunction with these reports, further support the involvement of IGF-I in the growth of ectopic endometrium in pelvic endometriosis. A recent study of rats demonstrated that uterine IGF-I is involved in an increase of early embryonic loss due to superovulation (19). Similarly, high IGF-I levels in the peritoneal fluid of patients with endometriosis may be responsible for clinically undetectable embryonic loss, which would lead to infertility. Using Western ligand blotting, we detected four types of IGFBP in the peritoneal fluid of all the patients. The relative levels of IGFBP-3 among these IGFBPs in peritoneal fluid were significantly lower in patients with endometriosis than in controls. This is contrary to the results reported by Taskin et al. (13), who found that patients with endometriosis had significantly higher radioimmunoassayable IGFBP-3 during the luteal phase than the control group. It is also contrary to the results of Gurgan et al. (14), who reported no difference in the IGFBP-3 levels between patients with endometriosis and control patients. Taskin et al. (13) also reported that the level of 27-kDa IGFBP in peritoneal fluid was significantly lower in patients with endometriosis than in control patients. There are several methodologic differences between our study and theirs. First, although they used similar Western ligand blotting to determine the IGFBP, they did not consider the possible variability in the transfer of protein during the blotting process, whereas we standardized the measurement of IGFBP intensity by using a calculated ratio relative to the intensity of the same IGFBP band in the sera pooled from normal, healthy, nonpregnant women. Second, they included only patients with early-stage endometriosis. Third, they did not confirm the type of IGFBP by immunoprecipitation, whereas we did. Since no immunoprecipitable band was detected by using the antihuman IGFBP-1-specific antibody, which is known to immunoprecipitate IGFBP-1 specifically in human amniotic fluid samples (17), the 26-kDa IGFBP is not an IGFBP-1. Further characterization of this IGFBP is needed. The variations in the relative proportion of each IGFBP might be more important than the absolute levels in determining the bioavailability of IGFs in the tissue. Patients with endometriosis showed a lower proportion of IGFBP-2 in peritoneal fluid than control patients. It has been shown that most IGFs in biologic fluid are bound to plasma IGFBP-3 and that IGFBP-2 blocks IGF-I action in bone (7, 20). Therefore, a decrease in both the IGFBP-3 level and the relative proportion of IGFBP-2 might further increase the bioavailability of IGFs in peritoneal fluid. Insulin-like growth factor binding protein protease has recently been introduced as one component of the IGF system. Proteolysis of IGFBP-3 is known to induce a structural change in IGFBP-3, resulting in reduction of its binding affinity for IGFs (9). To our knowledge, this is the first report in which IGFBP-3 protease activity and the intensity of each proteolytic fragment in patients with endometriosis have not differed from those in control patients. Although it has been suggested that proteolytic fragments are involved in the mitogenic effect of peritoneal fluid (11), our data may indicate that changes in the IGF-I and IGFBP profiles in peritoneal fluid are more important than IGFBP-3 protease activity in patients with endometriosis. The exact source of IGF components in peritoneal fluid is not known, but possibilities include serum exudate; sloughed endometrium; follicular fluid; endometriotic tissues; and products of the peritoneal inflammatory cells, such as macrophages (21). The levels of IGF components in peritoneal fluid have been reported to be approximately 50% 65% of serum levels (10). 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