Protein, progesterone, and protease inhibitors in uterine and peritoneal fluids of women with endometriosis*

Transcription

1 FERTILITY AND STERILITY Vol. 47, No.2, February 1987 Copyright 1987 The American Fertility Society Printed in U.8A. Protein, progesterone, and protease inhibitors in uterine and peritoneal fluids of women with endometriosis* Asgerally T. Fazleabas, Ph.D. t Firyal S. Khan-Dawood, Ph.D. M. Yusoff Dawood, M.D. Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Illinois College of Medicine at Chicago, Illinois This study was undertaken to determine whether women with endometriosis have altered protein, progesterone (P), and protease inhibitor concentrations in their uterine fluid and peritoneal fluid (PF) compared with controls at different phases of the menstrual cycle. Uterine flushings (UFs), PF, and blood were obtained during the follicular and luteal phases of the cycle from 29 normal women and 16 women who were diagnosed as having endometriosis. Protein content in UF did not change significantly throughout the cycle in either group. However, PF protein in patients with endometriosis was significantly (P < 0.05) higher than in controls during the luteal phase. Total UF P was significantly (P < 0.05) reduced in women with endometriosis during the late luteal phase. During the early luteal phase, trypsin inhibitory activity in UF from normal women was significantly (P < 0.05) higher than at any other phase of the cycle, whereas inhibitory activity in UF from patients with endometriosis remained relatively constant. Patients with endometriosis had significantly (P < 0.05) higher total activity in PF during the early luteal phase than did controls. These results indicate that women suffering from endometriosis have (1) significantly lower levels of P and less protease inhibitor within their uterine cavity during the luteal phase of the cycle, and (2) significantly higher concentrations of protein and protease inhibitor in PF during the luteal phase. Fertil Steril 47:218, 1987 Endometriosis is a major cause of infertility, but its mechanism is not always readily explicable. The incidence of endometriosis among infertile women may be as high as 30% to 40%. With severe endometriosis, mechanical obstructions of the reproductive organs could be a cause, Received June 9, 1986; revised and accepted October 10, *Presented in part at the Forty-First Annual Meeting of The American Fertility Society, Chicago, Illinois, September 28 to October 2, treprint requests: A. T. Fazleabas, Ph.D., Department of Obstetrics and Gynecology, University of Illinois, 840 South Wood Street, Chicago, Illinois but the mechanisms by which mild or moderate disease induce infertility are still unclear. Several studies have examined the prostanoid, l steroid,2 and enzymatic composition 3 of peritoneal fluid (PF) from patients with endometriosis, but they were not compared with secretions of the uterine cavity from the same patients. Comparative studies of this nature are important because alterations in the uterine environment due to the disease may have adverse effects on blastocyst development and implantation. Because progesterone (P) has been shown to have a stimulatory. effect on protein secretion by the human endometrium in vitro4, 5 and plasma protease inhibitors have been implicated to be associated with in- 218 Fazleabas et ai. Uterine and peritoneal fluid composition Fertility and Sterility

2 flammation and proteolytic equilibrium during implantation,6 this study was undertaken to determine whether endometriosis alters these parameters. Specifically we wanted to determine whether endometriosis affects (1) the amount of protein present in uterine fluid and PF; (2) the P content of uterine fluid and PF; (3) the concentration of protease inhibitors, particularly ai-antitrypsin, in uterine fluid and PF; and (4) the concentration of protease inhibitors in these fluids when compared with serum. MATERIALS AND METHODS EXPERIMENTAL SUBJECTS Uterine flushing (UF), PF, and blood were obtained with prior consent from a total of 45 patients who underwent diagnostic lapa~oscopy for infertility or laparoscopic tubal ligation. Twentynine patients were normal (tubal ligation), and 16 were diagnosed as having endometriosis. In the endometriosis group, 10 had stage 1 (mild), 4 had stage 2 (moderate), and 2 had stage 3 (severe) forms of the disease according to The American Fertility Society classification for endometriosis. 7 The laparoscopies were performed at various stages of the menstrual cycle, and the fluid samples obtained were classified as being from the early follicular (days 0 to 8: normal, n = 2; endometriosis, n = 2); late follicular (days 9 to 14: normal, n = 10; endometriosis, n = 5); early luteal (days 15 to 22: normal, n = 9; endometriosis, n = 5); and late luteal (days 23 to 30: normal, n = 8; endometriosis, n = 4) phases of the cycle, respecti vely. COLLECTION OF FLUIDS PF was aspirated from the cul-de-sac with a 35-ml syringe attached to a suction cannula before any surgical manipulation, and the volume was recorded. The fluid was centrifuged (500 g x 10 minutes x 4 C) and the supernatant frozen at -70 C. UFs were obtained under sterile conditions with a double-lumen stainless steel cannula as previously described in detail elsewhere.s The anterior lip of the cervix was grasped with a tenaculum and the uterine cavity gently sounded for uterine direction. For avoidance of loss of lavage fluid, the cervix was not dilated. The cannula was then assembled, and the taps connected to the injecting and aspirating syringes were checked. The injecting syringe was filled with 10 ml normal saline and the length ofthe cannula "primed" with saline from the injecting syringe to remove air and avoid air-fluid bubbling and "locking" during injection into the uterus. The cannula was inserted through the cervical canal into the uterine cavity in the direction of the uterine axis as determined by the uterine sounding. The metal cone of the cannula sits flush with the exocervix to form a good seal. The saline was injected slowly into the uterine cavity, and after 1 to 2 ml had been injected, aspiration was initiated into the collecting syringe. This continued until all the saline had been injected. Once the collection syringe was filled, the tap leading to it was shut, the UF transferred into a container on ice, and the syringe connected back to its previous site on the cannula. About 10 ml air was then injected into the uterine cavity through the injecting syringe to clear out all the residuallavage fluid from the uterine cavity while the aspiration was applied on the collection syringe. The volume of both aspirates was determined, the flushings were centrifuged (10,000 g x 10 minutes x 4 C), and the supernatants were stored at -70 C. Blood (10 ml) was obtained by venipuncture into heparinized tubes. The plasma was separated by centrifugation (10,000 g x 10 minutes x 4 C) and stored at -70 C until analyzed. ASSAYS P levels in PF, UF, and plasma were determined by a specific radioimmunoassay.9 Protein content in the fluids was determined according to the method of Lowry et al.,lo with the use of bovine serum albumin as a standard. Protease inhibitor activity (arantitrypsin) was determined with the l25i_fibrin plate assayy Briefly, the fluids to be tested for inhibitory activity were added at 4 C at a range of concentrations to the microtiter wells containing the l25i-fibrin substrate. Trypsin was then added, and the plates were transferred to an incubator at 37 C for 3 hours. Inhibition was determined by comparing the release of 1251 with control values of trypsin tested in the absence of inhibitor. Inhibition curves were derived by testing 25 to 100 f.lg fluid protein against 2.5 jj.-g trypsin in phosphate buffered saline (ph, 7.4). Specific and total inhibitory activities were calculated from these curves by de- Vo!' 47, No.2, February 1987 Fazleabas et al. Uterine and peritoneal fluid composition 219

3 termination of the amount of protein required for 30% inhibition of fibrinolytic activityy PERITONEAL FLUID PROGESTERONE NG/Ml 80 1 STATISTICAL ANALYSIS Data were analyzed by analysis of variance for unequal treatment groups, and significance was determined with the Newman-Keuls multiple range test. Differences between treatment groups were analyzed with the Student's t-test for unpaired samples. 12 PROTEIN RESULTS The protein concentrations in uterine fluid and PF of normal women and patients with endometriosis are shown in Table 1. There was no significant change in the amount of protein in UF throughout the cycle, and the protein content in UF of controls compared with patients with endometriosis were not significantly different. In contrast, the total amount of protein in PF increased in the luteal phase compared with the follicular phase, and patients with endometriosis had a significantly (P < 0.05) higher amount of protein in both the early and late luteal phases of the cycle compared with the controls. PROGESTERONE Total P content in UF and peripheral plasma P levels are shown in Table 2. There was considerable variation in P levels in UF at all phases of 20 Figure 1 P concentration in PF from both groups of women. The P concentration in PF was not significantly different in either group. The data in this and all other figures are expressed as mean ± standard error of the mean. Normal (open bars), endometriosis (shaded bars). EF, early follicular; LF, late follicular; EL, early luteal; LL, late luteal. the cycle. During the late luteal phase, the P content in UF from patients with endometriosis was significantly (P < 0.05) lower than in normal women. Plasma P levels were consistent with the phase of the cycle in both controls and patients with endometriosis and were significantly higher in both groups during the luteal phase. PF P levels (Fig. 1) were very low during the follicular phase of the cycle and increased during the luteal phase in both groups. These levels, however, were not significantly different in either group at any phase of the cycle. PROTEASE INHIBITOR Trypsin inhibitory activity at each phase of the cycle was determined in UF and PF of patients with endometriosis and controls with the use of the 125I_fibrin plate assay. There was signifi- Table 1. Protein Content of UF and PF Diagnosis Total protein (mg)a Early follicular Late follicular Early luteal Late luteal UF Normal 1.14± ± 1.23 (0.12 ± 0.36)6 (0.30 ± 0.39) Endometriosis 2.30 ± ± 1.48 (0.23 ± 0.20) (0.15 ± 0.40) PF Normal (78.1) ± 22.6 (73.8 ± 3.7) Endometriosis (72.9) ± 14.2 (79.9 ± 2.7) a All values expressed as mean ± standard error of the mean. 6Figures in parentheses are concentrations in milligrams per milliliter. cp < 0.05 compared with controls ± ± 1.06 (0.12 ± 0.28) (0.19 ± 0.38) 0.99 ± ± 0.78 (0.10 ± 0.40) (0.12 ± 0.30) ± ± 30.7 (84.5 ± 3.5) (81.2 ± 5.8) ± 26.3 c 1913 ± 33.3 c (81.1 ± 1.7) (89.2 ± 1.8) 220 Fazleabas et al. Uterine and peritoneal fluid composition Fertility and Sterility

4 Table 2. P Concentration in UFs and Peripheral Plasma a Stage of cycle Early follicular Late follicular Early luteal Late luteal Normal Endometriosis UF Plasma UF Plasma ng 2.59 b 0.76 b 1.89 ± ± ± 0.90 nglml 2.1b 1.9 b 1.6 ± 0.7 c 12.7 ± 2.9 d 10.5 ± 2.2d ng 1.33 b 0.80 b 1.83 ± ± ± 0.6st aall values expressed as mean ± standard error of the mean. bindividual values for each of the women in both groups. c versus dfollicular versus luteal phase plasma P levels are significantly different (P < 0.05). versus fp < nglml 2.7 b 3.4 b 2.2 ± 1.2 c 7.5 ± 2.9 d 11.1 ± 3.7 d cantly (P < 0.05) higher total and specific protease inhibitory activity in UF of normal women in the early luteal phase of the cycle than in the late follicular or late luteal phases (Fig. 2). Because of the limited number of samples from the early follicular phase, statistical significance was not determined. In contrast with the normal women, protease inhibitor activity in UF ofwomen with endometriosis remained relatively constant throughout the cycle but was not significantly different from controls at any given phase of the menstrual cycle. Specific trypsin inhibitory activity in PF was similar and increased gradually in both groups from the follicular to the luteal phases. However, when total inhibitory activity was determined, patients with endometriosis had significantly (P < 0.05) higher amounts of inhibitor during the early luteal phase than did the controls (Fig. 3). Unlike in UF, neither the specific nor total inhibitory activity in PF changed significantly at different phases of the cycle in either group. To determine whether the phase of the cycle or disease influenced the selective transudation of circulating protease inhibitors, UF and PF from four patients in each group were compared with plasma on an equivalent protein basis at the follicular and luteal phases of the cycle. Protease inhibitory activity in controls and patients with endometriosis was significantly higher in UF than in plasma at both phases of the cycle (Fig. 4A). In contrast, only patients with endometriosis had significantly higher levels of protease inhibitors in their PF than their plasma (P < 0.01). The inhibitory activity in.luteal phase PF from this group of patients with endometriosis was also significantly (P < 0.05) higher than in the controls (Fig. 4B). DISCUSSION This is the first study that directly comparee the composition of proteins and steroids in UF and PF of patients with endometriosis. Our data indicate that the protein content in UF from either group did not change significantly throughout the cycle. Previous studies 13, 14 have also demonstrated that uterine fluid proteins in normal women are composed primarily of serum transudate and do not change markedly through UTERfoE FUJI) PROTEASE _OR SPECFIC ACTIVITY IMG PROTEIj...,~...._.----T~O~T~~AC~W~rr~y----, Figure 2 Specific and total protease inhibitory activity in UF from controls (open bars) and women with endometriosis (shaded bars). Inhibitory activity was calculated from inhibition curves derived by testing 25 to 100 jj.g UF protein against 2.5 jj.g trypsin. Trypsin inhibitor activity in controls was significantly (P < 0.05) higher at the early luteal phase of the cycle. Vol. 47, No.2, February 1987 Fazleabas et al. Uterine and peritoneal fluid composition 221

5 0 0 0 PERITONEAL FLUID PROTEASE INHBITDR SPECIFIC ACTIVITY IMG PROTEt.I I,..Ie" Figure 3 Specific and total protease inhibitor activity in PF from controls (open bars) and women with endometriosis (shaded bars). Inhibitory activity was calculated as described in Figure 2. Specific activity in both groups was not significantly different at any phase of the cycle, but patients with endometriosis had significantly (P < 0.05) higher total activity at the early luteal phase than did controls. " " "."'.. " TOTAL ACTIVITY I ~,-l."'..,-l " " " " conception, in vitro studies have demonstrated that P has a direct effect on protein synthesis by the human endometrium in culture. 4, 5 In species other than the human, ovarian steroids control either the synthesis or secretion of numerous proteins and enzymes present in uterine fluid during the period immediately before implantation. 20 Therefore, this decrease in luminal P in women with endometriosis may have further implications by altering the synthesis or sequestering of uterine proteins, which may adversely affect the embryo at a critical stage of development. The absence of any significant change in the P content of PF in the patients with endometriosis further substantiates our previous findings2 and supports the concept that luteal phase defects are usually not the causative factor of infertility in these patients.21 Our findings of a significant increase in protease inhibitors in UF during the luteal phase of the cycle of normal women confirm an earlier re- out the cycle. In contrast, PF protein increased throughout the cycle. Patients with endometriosis had significantly higher PF protein than normal controls during the luteal phase which confirms an earlier preliminary report15 and is most likely due to the increase in PF volume during the luteal phase.2 Alternatively, this increase in PF protein could also be the result of increased macrophage invasion16 or retrograde menstruation, particularly during the late luteal phase around the peri menstrual period. 17 P content in UF was significantly reduced during the late luteal phase in patients with endometriosis. Although peripheral plasma P levels during the luteal phase are not significantly different in patients with endometriosis18 and in this study, the decrease within the uterine cavity may reflect more subtle hormonal or endometrial changes in situ. Previous studies have determined that the uterine luminal P/estrogen ratio in normal women during the peri implantation period may be as high as 2000/1. 19 Although there is no direct evidence to suggest that alterations in uterine luminal P alter the likelihood of DlA.GNOSIS INHSITORY ACTIVITY ~ PERITONEAL FLUID COMPARED WITH PLASMA B DlA.GNOSIS Figure 4 (A), Inhibitory activity in UF compared with plasma (closed bars) of four patients from each group at the follicular (open bars) and luteal (shaded bars) phases of the cycle. The ability of an equivalent amount (50 ILg) of UF and plasma protein to inhibit 2.5 ILg trypsin was tested with the 125I_fibrin plate assay, and the results are expressed as percent inhibition compared with the trypsin control. (B), Inhibitory activity in PF compared with plasma from the same four patients in each group shown in A. Unlike in the UF, in which inhibitory activity was always significantly.higher than in plasma, only PF from endometriosis patients had significantly (P < 0.01) higher inhibitory activity than that present in plasma. 222 Fazleabas et al. Uterine and peritoneal fluid composition Fertility and Sterility

6 ,; I, port. 6 Because plasma protease inhibitors in UF may help balance proteases associated with the intrauterine stages ofreproduction,22 the decreae in inhibitor activity in UF of women with endqmetriosis during the early luteal phase may disrupt the intrauterine proteolytic equilibrium, resulting in a failure to implant or defective implantation. Although this concept has yet to be experimentally demonstrated in the human being, studies with mice, rabbits, and pigs have suggested that proteases and protease inhibitors play essential interactive roles within the uterine lumen during the process of implantation. 11, 23, 24 In addition to maintaining proteolytic equilibrium, protease inhibitors, particularly ai-antitrypsin and a2-macroglobulin, are also acute phase reactants. The increase in total inhibitory activity associated with the significant increase in protein content in the PF of patients with endometriosis during the early luteal phase observed in our studies is indicative of a local inflammatory reaction. Increased plasminogen activator production by macrophages25 is also usually associated with the inflammatory process, and increased macrophage invasion into the peritoneal cavity has been demonstrated in patients with endometriosis.1 6 However, a recent study3 failed to show evidence for increased activity of plasminogen activator in the PF of these patients. Our data would suggest that this lack of detectable increase in plasminogen activator activity in PF may be due to the inhibition of plasmin in their assay system by protease inhibitors in the PF of women with endometriosis. The fact that PF from our patients with endometriosis had significantly higher protease inhibitory activity when compared with their plasma further suggests that, in response to the inflammatory reaction caused by this disease, there is a selective transudation of protease inhibitors into the peritoneal cavity from the circulation (Fig. 4B). In summary, this is the first report of a comprehensive study on the protein, P, and protease inhibitor composition of uterine fluid and PF from women with endometriosis. On the basis of our findings, it would appear that women withendometriosis have lower levels of P and protease inhibitor within the uterine lumen and significantly higher amounts of protease inhibitory activity within the peritoneal cavity. We suggest that these alterations within the uterine lumen and peritoneal cavity of these women may contribute to their inability to conceive. REFERENCES 1. 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7 20. Caudle MR, Scott JR: Maternal-fetal interactions in early pregnancy. Semin Reprod Endocrinol1:259, Pittaway DE, Maxson W, Daniell J, Herbert C, Wentz AC: Luteal phase defects in infertility patients with endometriosis. Fertil Steril 39:712, Casslen B, Ohlsson K: Oll-antitrypsin-complexation and inactivation in uterine fluid of IUD-users. Acta Obstet Gynecol Scand 60:103, Dabich D, Andary TJ: Prevention of blastocyst implantation in mice with proteinase inhibitors. Fertil Steril 25:954, Morton DB: The occurrence and function of proteolytic enzymes in the reproductive tract of mammals. In Proteinases in Mammalian Cells and Tissues, Edited by AJ Barret. Amsterdam, Elsevier-North Holland Biomedical Press, 1977, p Mullins DE, Rohrlich ST: The role ofproteinases in cellular invasiveness. Biochem Biophys Acta 695:177, Fazleabas et al. Uterine and peritoneal fluid composition Fertility and Sterility