Peritoneal fluid plasminogen activator activity in endometriosis and pelvic adhesive disease

FERTILITY AND STERILITY Copyright ' 1985 The American Fertility Society

Vol. 44, No.2, August 1985 Printed in U.s A.

Peritoneal fluid plasminogen activator activity in endometriosis and pelvic adhesive disease

Joel H. Batzofin, M.D.*t Steve D. Holmes, Ph.D.:j: William E. Gibbons, M.D. * Veasy C. Buttram, Jr., M.D.* Baylor College of Medicine, Houston, Texas

Plasminogen activator is a trypsin-like serine proteinase of restricted specificity, with its only known macromolecular substrate being plasminogen, which is present at high concentrations in plasma and many extracellular fluids. It has been demonstrated that when plasminogen activator activity (PAA) is reduced by at least 50% or more, fibrin cannot be cleared and permanent adhesions subsequently form. Furthermore, PAA is known to be related to mechanisms involved in ovum extrusion from follicles at the time of ovulation, and deficiencies in P AA may be related to the occurrence of luteinized unruptured follicles. In a preliminary study with uterine biopsy specimens plated on plasminogen-rich fibrin plates, Malicki suggested that patients with endometriosis may have a deficiency in PAA. The purpose of this study was the determination of whether P AA was diminished in peritoneal fluid (PF) of patients with endometriosis and/or pelvic adhesive disease. MATERIALS AND METHODS

Peritoneal fluid was aspirated from the cul-desac of 45 infertility patients at the time of lapa-

Received March 19, 1985; revised and accepted May 1, 1985. *Department of Obstetrics and Gynecology. tReprint requests: Joel H. Batzofin, M.D., Department of Obstetrics and Gynecology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. :j:Department of Urology. Vol. 44, No.2, August 1985

roscopy, which was done as part of the workup of these patients; The laparoscopy was performed at various stages of the follicular phase of the cycle in 29 patients, and in the luteal phase in 16 patients. The mean age of the patients studied was 29.8 years, with a range from 21 to 41 years; whereas the mean duration of infertility was 34 months, with a range from 3 months to 10 years. PF was aspirated with a #8 pediatric feeding tube attached to a 50"ml syringe. The aspiration was done immediately after the insertion of the suprapubic trocar, and an attempt was made to aspirate the cul-de-sac as completely as possible. The volume of fluid was noted, and the fluid was placed in plain tubes, immediately refrigerated at 4°C, and subsequently stored at - 20°C until assayed. The remainder of the laparoscopy was performed in the usual fashion. Endometriosis, when present, was classified according to the Revised American Fertility Society Classification 2 as minimal, mild, moderate, or severe. Pelvic adhesions, if present, were documented. PF was subsequently assayed for PAA by the fibrinolysis method described by Strickland and Beers 3 and quantified as percent fibrinolysis per microliter per 5 hours. Plasminogen-free fibrinogen (purchased from Miles Scientific, Naperville, IL) was iodinated with 1251 (Amersham Corporation, Arlington Heights, IL) with the use of enzymo beads (Bio-Rad Laboratories, Richmond, CA). The 1251-fibrinogen (200,000 cpm) was diluted with unlabeled fibrinogen (20 f.Lg) and dried down on multiwell culture plates (Costar, CamBatzofin et aI. Communications-in-brief

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Table 1. PAA in Patients With and Without Endometriosisa

No.

Mean PAA % fibrinolysis/f.d/5 hrs

SD

Endometriosis Mild Moderate

No endometriosis

Minimal

15 2.63

19 2.92

4 1.86

1.07

1.97

1.21

Severe

Total endometriosis

5 3.68

2 1.44

30 2.80

2.27

1.43

1.92

aThe 95% confidence limit for f.Ll-f.L2 is -2 to 1.66, where f.Ll-f.L2 is the difference between two population means. Calculations based upon the assumption of normal distribution and calculated from Student's t-test at 43 degrees of freedom.

bridge, MA). The I25I-fibrinogen in each well was converted to I25I_fibrin by incubation for 2 hours at 37°C with 0.5 ml of 10% fetal calf serum and then being washed three times with 0.5 ml of saline. The incubation was for 5 hours at 37°C and results expressed as percent fibrinolysis, compared with trypsin (0.2%). The percent fibrinolysis for samples without plasminogen was < 1.5%, equivalent to wells with buffer only. This value was subtracted from the percent fibrinolysis obtained from wells with plasminogen added. All samples were tested at two concentrations (0.5 fll and 1 fll) in duplicate, both with and without plasminogen. Values of PAA were obtained from the mean of the two samples from each patient except in those patients where the non-plasminogen-activator-dependent protease activity was high (> 1.5%), when only the lower dilution was used. The percent fibrinolysis was linear with time (2.5 to 6 hours) and directly proportional to the concentration range tested (0.5 fll to 2.5 fll).

RESULTS

Table 1 demonstrates PAA in patients with endometriosis compared with patients without evidence of this disease. No significant difference between the two major groups was noted. Furthermore, when analyzing the data with respect to increasing severity of endometriosis, we noted no discernible trend within the subgroups, or compared with the control group. Data were further analyzed according to whether pelvic adhesions were present. These results are shown in Table 2 and again demonstrate no significant difference between groups. P AA was similar from samples drawn in the follicular and luteal phases of the cycle both for patients with and without endometriosis and/or pelvic adhesions. These results are shown in Table 3. Data were also analyzed for assessment of whether 278

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Communications-in-brief

there was any difference in PAA in patients with adhesions secondary to endometriosis (a continuing insult), compared with PAA in patients with adhesions attributable to inactive pelvic inflammatory disease (presumably a one-time insult). There was no significant difference between these two groups, PAA of 2.42 ± 1.34 standard deviation (SO) versus PAA of 2.32 ± 0.94 SD for the two groups, respectively. DISCUSSION

Adhesion formation is the final common pathway of various disease processes, including pelvic infection, endometriosis, and surgical trauma; as such, a single pathogenetic mechanism is unlikely. However, it is known that the fibrinolytic system is involved in the pathogenesis of adhesion formation. 4 The concept as proposed by MalickI that patients with endometriosis may have a deficiency in PAA seemed highly tenable. A genetic basis for the development of endometriosis has been previously suggested,5 and it seemed feasible that a deficiency ofPAA may be one manifestation of an underlying genetic predisposition to develop endometriosis. In this study P AA was measured in PF rather than tissue biopsy specimens, because PF is clearly more easily obtained. It was believed that PAA measured in PF would be representative of tissue/cellular levels of this enzyme in any particular patient, because the enzyme is so ubiquitous. That this is in fact so Table 2. PAA in Patients With and Without Pelvic Adhesionsa

No.

MeanPAA % fibrinolysis/f.d/5 hrs

SD

No adhesions

Adhesions

21 2.65

24 3.26

1.74

2.62

aThe 95% confidence limit for f.Ld.L2 is - 2.13 to 0.91, based upon the assumption of normal distribution and calculated from Student's t-test at 43 degrees of freedom.

Fertility and Sterility

SUMMARY

Table 3. PAA by Phase of Menstrual Cycle AdEndo- No endometriosis metriosis hesions Follicular No. MeanPAA % fibrinolysis/ f,LI/ 5 hrs SD Luteal No. MeanPAA % fibrinolysis/ f,Ll/ 5 hrs SD

No adhesions

20 2.63

8 2.00

7 2.40

12 2.13

1.01

1.06

1.21

1.20

10 2.19

7 2.06

7 2.04

9 2.06

1.53

1.19

1.22

1.51

remains to be determined. It is noteworthy that in a report by Pattinson et al. 6 no detectable levels of PAA were discerned in samples of PF assayed. Those authors did, however, document the presence of both plasminogen and fibrinogen degradation products (FDPs) in samples of PF tested, which supports the presence of fibrinolytic activity within PF. It is possible that the inability of those authors to detect PAA reflects a difference in assay methods. Despite the fact that they were unable to detect P AA in PF, our findings of levels of PAA that were similar in patients with and without endometriosis is in direct concordance with their findings of equivalent levels of plasminogen and FDPs in patients with and without this disease, which suggests that perhaps the entire fibrinolytic mechanism in PF is similar in patients with endometriosis, compared with control patients. Because no difference in PAA was found in PF from patients with endometriosis and/or pelvic adhesive disease, compared with control patients, it is concluded that if such differences exist, they may be present in tissues, per se, but are not discernible in PF.

Vol. 44, No.2, August 1985

Plasminogen activator activity in PF was assayed by the fibrinolysis method described by Strickland and Beers. 3 In 45 patients studied, there were no discernible differences according to whether patients had endometriosis and/or pelvic adhesive disease. No differences were detected according to when in the menstrual cycle the sample of PF was obtained. These data are in concordance with a previous report and taken together suggest that there is no difference in fibrinolytic mechanisms in PF in patients with or without endometriosis and/or pelvic adhesive disease, when compared with control subjects. If such differences exist, they may be present in the tissues, per se, but are not discernible in PF. REFERENCES 1. Malick JE: The etiology of endometriosis. J Am Osteopath Assoc 81:407, 1982 2. American Fertility Society: Revised American Fertility Society Classification of Endometriosis: 1985. Fertil Steril 43:351, 1985 3. Strickland S, Beers WH: Studies on the role of plasminogen activator in ovulation. J BioI Chem 251:5694, 1976 4. Buckman RF, Woods M, Sargent L, Gervin AS: A unifying pathogenetic mechanism in the etiology of intraperitoneal adhesions. J Surg Res 20:1, 1976 5. Simpson JL, Elias S, Malinak LR, Buttram VC: Hereditable aspects of endometriosis. Am J Obstet Gynecol 137:327, 1980 6. Pattinson HA, Koninckx PR, Brosens lA, Vermylen J: Clotting and fibrinolytic activities in peritoneal fluid. Br J Obstet Gynaecol 88:160, 1981

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