Hormone-independent ovarian influence on adhesion development

FERTILITY AND STERILITY威 VOL. 78, NO. 2, AUGUST 2002 Copyright ©2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.

Hormone-independent ovarian influence on adhesion development Michael L. Freeman, M.D., Ghassan M. Saed, Ph.D., and Michael P. Diamond, M.D. Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Detroit Medical Center, Wayne State University, Detroit, Michigan

Objective: To determine which ovarian sex steroid(s), when removed from an intact organism, reduce(s) postoperative adhesion development. Design: Randomized, prospective, blinded study. Setting: University vivarium. Patient(s): One hundred twenty sexually mature female Sprague-Dawley rats, 226 –250 g. Intervention(s): Day 0, sham ovariectomy or bilateral ovariectomy, accompanied by continuous-release sex steroid replacement of either no steroids (control), 17␤-E2, natural P (P4), or combined E2/P4. Day 7, standardized cecal abrasion; day 14, necropsy with assessment of adhesion presence or absence. Main Outcome Measure(s): Adhesion formation. Result(s): Three rats died because of anesthesia or surgical complications, and 117 rats reached necropsy. The ovary-intact (sham) rats adhesion incidence was 60.9%; ovariectomized control rats, 20.8%; E2, 28.6%; P4, 33.3%; and combined E2/P4, 24.0%. Despite differing sex steroid replacement, two-tailed ␹2 testing with correction for multiple comparisons showed no statistical difference in adhesion incidence among the four ovariectomy groups. A statistically significant lower adhesion incidence was noted between the ovary-intact sham cohort and the collective ovariectomy groups and between the sham and ovariectomized control cohorts. Conclusion(s): Ovarian presence or absence at the time of surgical wounding, and not the 17␤-E2 or P milieu, modulates adhesion development. This implicates other ovarian factor(s) in postoperative adhesion development. (Fertil Steril威 2002;78:340 – 6. ©2002 by American Society for Reproductive Medicine.) Key Words: Adhesions, cecum, rat, ovariectomy, pellets, estradiol, progesterone, abrasion Received October 4, 2001; revised and accepted February 4, 2002. Presented in part at the annual meeting of the Society of Gynecologic Investigation, which was held in Toronto, Ontario, Canada, on March 22–24, 2001. Supported in part by a grant from ACOG/Ethicon’s 2001–2002 Research Award for Innovations in Gynecologic Surgery. Reprint Requests: Michael L. Freeman, M.D., Division of Reproductive Endocrinology and Infertility, Hutzel Hospital, 4707 Saint Antoine Boulevard, Detroit, MI 48084 (FAX: 313-745-7693; E-mail: mfreeman@med. wayne.edu). 0015-0282/02/$22.00 PII S0015-0282(02)03237-5

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Postoperative adhesion development is the nemesis of both surgeon and patient. Its potential sequelae have been well described, with infertility (1, 2), pain (3–5), bowel obstruction (6, 7), and difficult reoperation (8, 9) being the most notable. In an effort to prevent these occurrences, different strategies to decrease postsurgical adhesions have been examined. These attempts have met with varying success and include manipulation of the hormonal environment at the time of surgery, placement of surgical membranes on operated surfaces (10, 11), perioperative glucocorticoid steroids (12), and laparoscopic technique (13), among others. Several investigators have reported that establishing a hypoestrogenic environment at the time of reproductive tissue wounding results in decreased adhesion development (14 –16). Creating the hypoestrogenism in these studies in-

volved down-regulation of the normal steroidogenic output of the ovary through chemical means. A GnRH agonist (GnRH-a), leuprolide acetate (Lupron; TAP Pharmaceuticals, Deerfield, IL), has been used, resulting in reduced adhesion outcome (15). Other studies used medroxyprogesterone acetate (MPA) to produce hypoestrogenism (14), or even an antiprogestin with noncompetitive antiestrogenic properties, mifespristone (RU-486; Roussel Uclaf, Romainevelle, France) (17), and observed diminished postsurgical adhesions. Both the hypoestrogenism itself and the potentially immunosuppressive effect of progestins have been suggested as the mechanism behind the diminished adhesiogenesis seen in these studies (14, 18 –20). Although a common end point in the previously mentioned studies was production of hypoestrogenism, the manner in which this was

achieved differed. Use of leuprolide acetate reduces both P and estrogen to menopausal levels after an initial transient rise in both (the so-called flare effect). MPA prevents folliculogenesis, thereby reducing estrogen, but a circulating progestin remains present. Noncompetitive estrogen inhibition will prevent normal estrogen receptor function, but endogenous estrogen levels remain normal. Therefore, to better characterize the individual effects of estrogen and P on adhesion development, we produced a hypoestrogenic, hypoprogestogenic environment in the female rat via ovariectomy, and then replaced the sex steroids in question in animals undergoing a standardized surgical injury.

MATERIALS AND METHODS Four groups of 30 female, 226 –250 g, Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were evaluated over a period of 3 months. Each group was allowed to acclimate to 12-hour light-cycle days with ad-lib rat chow and water for a minimum of 5 days before use. Institutional approval for the procedure as outlined was obtained from our institution’s Animal Investigation Committee. Separately and sequentially, each set of 30 rats underwent the following procedures, which were performed from day 0 to day 14 (start to finish) before we began again with the next set of rats. On day 0, each rat was anesthetized IM in the thigh with a combination of ketamine 85 mg/kg and rompun 6 mg/kg. This route was chosen over intraperitoneal administration to avoid any potential effect of the anesthesia itself on the eventual intraperitoneal wound sites. After anesthesia was adequate, each rat was ear-tagged for identification. Each cohort of 30 rats was randomized using a table into five treatment groups of six rats each: [1] ovariectomies alone (control), [2] ovariectomies with 17␤-E2 replacement, [3] ovariectomies with natural P replacement, [4] ovariectomies with combined E2 and P replacement, and [5] skin incisions without ovary removal (sham). Steroid hormone replacement was effected by SC placement of matrix-driven drug delivery pellets (Innovative Research, Sarasota, FL). These pellets, implanted SC in the test subjects, achieve systemic steady state levels of the matrixed drug within 24 – 48 hours after placement. A 21-day drug delivery formulation was used. Each rat had its flanks and neck shaved, with the flanks also receiving betadine cleansing followed by 70% isopropyl alcohol wiping. The neck was not treated because this was the site of steroid pellet placement and the presence of liquid might potentially coat the pellets as they were placed and impair subsequent drug uptake. The ovariectomies were performed bilaterally through the flanks via stab incisions. The ovaries were exposed, with division/ligation occurring proximal to the ovary, on the ipsilateral distal uterine horn to ensure complete ovarian tissue removal. Steroid pellet placement followed the ovariectomies with two separate, laterally located 0.5-mm stab FERTILITY & STERILITY威

incisions made on the nape of the neck of each rat, with an SC pocket produced with a hemostat to accept the pellet(s). The 17␤-E2 and P pellets were 0.85- and 200-mg dosages, respectively. Steroid pellets were placed SC according to the rat’s ear tag number and previously devised randomization table. Rats in the 17␤-E2 cohort received one 0.85-mg pellet, rats in the P cohort received four 200-mg P pellets distributed in the two SC pockets, and the combined estrogen/P rats received both 0.85 mg of 17␤-E2 and four 200-mg P pellets. These weight-based dosages were selected using the pellet manufacturer’s internal data for achieving our desired target serum levels—to match or slightly exceed peak the E2 and P levels that endogenously occur during the rat estrus cycle. Control rats received no steroid replacement after their ovariectomies. Assignment to the sham group was made known to the surgeon after creating the flank skin incisions; assignment to the other groups was made only after ovariectomies and skin closure. The skin incisions were closed with metal surgical clips. The sham cohort had both flank and neck incisions and closure only, without pellet placement. All rats concluded the ovariectomy-pellet placement phase of the study with identical incisions and wound closures. The rats were observed as they recovered and were kept in separate cages for the remainder of the study. Access to rat chow and water was ad-lib. The ovariectomies, cecal abrasions, and serum collection at necropsy were not timed to the rat estrus cycle.

Cecal Abrasion On day 7, standardized cecal abrasions were carried out in a fashion that has been previously described and standardized (21, 22). Anesthesia again consisted of IM ketamine and rompun at the aforementioned dosages. The animal’s abdomen was shaved, prepped with betadine, and then cleansed with 70% isopropanol. Individual sterile drapes were placed on the subject with exposure of only its abdomen. The surgeon was unaware of assigned animal grouping. An approximately 3-cm midline laparotomy adequately exposed the cecum. No adhesions from the prior ovariectomy were noted upon entering the abdomen, and the ovariectomy sites were dependent on the cecum and not visible at the time of cecal abrasion. The cecum was then gently externalized using sterile cotton swabs (Harwood Products, Guilford, ME) and placed onto a raised platform. A sterile polyvinyl chloride (PVC) mat containing a 1.9-cm diameter hole was placed over the exposed cecum. To create the abrasions, an abrading device that has been used in prior rat cecal abrasion studies (21, 22), which employs a rotating, motor-driven, constant-force, 70-g metal spline shaft, was used. The shaft had attached at its tip a rubber septum containing a bound, stretched, flat sterile 8-ply all-cotton gauze surface (Johnson & Johnson, Arlington, TX). The shaft with attached cotton gauze abrading surface was lowered onto, and contacted, the cecum through the 1.9-cm diameter hole in the PVC matting. A researcher 341

blinded to rat grouping assignment operated the abrading device for all animals. The rats were abraded in random order, not based on groupings. Thirty revolutions in two separate cecal locations, the midportion of both the ventral and dorsal cecal surfaces, produced the standardized lesions. The cecum was then returned to the abdomen. The muscle was closed with 3-0 vicryl (Ethicon, Somerville, NJ), and the skin was closed with metal surgical clips. The rats were observed as they recovered and were returned to their respective cages. Rat chow and water was again ad-lib. Powder-free gloves were used throughout all procedures.

FIGURE 1 Adhesion development by grouping. Percentages were calculated by dividing the number of rats within a group with adhesions at necropsy by the total number of rats in that group reaching necropsy.

Necropsy Seven days after abrasion, on day 14, necropsies were performed and adhesions were scored. CO2 asphyxiation was not used, as we wanted to collect fresh tissue specimens for snap freezing and later molecular analysis and to perform phlebotomy for confirmation of adequate sex steroid replacement. Instead, ketamine and rompun were administered IM, with the doses increased to 100 and 7 mg/kg, respectively. Cardiac puncture with a 21-gauge needle followed, which allowed on average 4 – 6 mL of whole blood to be collected per rat. The blood was collected in red plain top clot tubes, spun down, serum separated, and then stored at ⫺20°C until analysis. Immediately after phlebotomy, the abdomen was opened in the midline above the prior incision, the diaphragm visualized, and bilateral pneumothoraceses created. A blinded investigator assessed adhesion formation for either presence or absence, and tissue was collected if appropriate. Only adhesions directly involving the abraded areas of the cecum were included.

Radioimmunoassay Sex steroid serum levels were measured to document adequate hormone replacement. P levels were run using the Coat-a-Count P radioimmunoassay (RIA; Diagnostic Products Corporation, Los Angeles). This assay has been validated in the rat and has a sensitivity down to 0.02 ng/mL (0.06 nmol/L) (15, 23, 24). The 17␤-E2 levels were analyzed with the E2 Double Antibody RIA kit from Diagnostic Products. This test is highly specific for E2 and has a low limit of sensitivity of 1.4 pg/mL (25). Both assays were run without ether extraction and assayed in duplicate, serum permitting.

Freeman. Ovarian influence on adhesion development. Fertil Steril 2002.

plications (two from the estrogen-only group and one sham). One rat that was assigned to the estrogen-only cohort received both estrogen and P pellets. This was noted from an incorrect remaining pellet count at the conclusion of that particular ovariectomy pellet placement session and confirmed by subsequent serum steroid level testing and was included in the combined E2/P group for data analysis. RIA testing revealed no differences in the replaced sex steroid serum levels between the adhesion-producing and non–adhesion-producing rats of each particular treatment subset (Figs. 1 and 2). Replaced sex steroid levels were as desired, being physiological or slightly supraphysiological

FIGURE 2 Comparison of the mean 17␤-E2 levels in adhesion-present and adhesion-free rats of the same cohort at the time of necropsy. Open bars represent the adhesion-present rats; filled bars represent the adhesion-free rats. Y-error bars are ⫾ SE. E2 levels were measured in duplicate, and all samples were assayed simultaneously using a double-antibody RIA.

Statistical Analysis Adhesion scores following cecal abrasion were considered to be categorical variables, and two-tailed ␹2 testing with Holm modification of the Bonferroni correction for multiple comparisons was used. Statistical significance for alpha was set at P⬍.05, and this was adjusted downward based on the number of comparisons made. Analysis was performed using SPSS 9.0 for Windows (Chicago, IL).

RESULTS One hundred seventeen of the 120 total rats reached necropsy; three died from either anesthesia or surgical com342

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TABLE 1 Incidence of adhesions by treatment group vs. sham. Group

No. of adhesions/total

%

Observed P-valuea

Significant P-valueb

Sham Control E2 P4 E2/P4

14/23 5/24 6/21 8/24 6/25

60.9 20.8 28.6 33.3 24.0

— .008 .04 .082 .018

— .0125 .025 .05 .017

Two-tailed ␹2 testing. As determined by the Holm modification of the Bonferroni correction for multiple comparisons.

a

b

Freeman. Ovarian influence on adhesion development. Fertil Steril 2002.

for the replaced groups. After correction for outliers, t-tests were performed among the adhesion-present and adhesionfree rats for each cohort, and no statistically significant differences were noted (see Table 1). The estrogen interassay coefficient of variance was 3.12%, and the P interassay coefficient of variance was 9.37%. All rats reaching necropsy were healthy, eating and grooming normally, and without wound infection. By grossly examining the data (Fig. 3), one can appreciate that the incidence of adhesions was lower in all groups that had undergone ovariectomy (i.e., treatment rats) before cecal abrasion versus those rats that had their ovaries remaining in situ at the time of abrasion (the sham rats). When comparisons were made between the four treatment groups, estrogen, P, combined estrogen/P, and control, no statistically significant difference in adhesion incidence was noted (P⫽.778). With this lack of statistical difference noted between ovari-

FIGURE 3 Comparison of the mean P levels in adhesion-present and adhesion-free rats of the same cohort at the time of necropsy. Open bars represent the adhesion-present rats; filled bars represent the adhesion-free rats. Y-error bars are ⫾ SE. P levels were measured in duplicate, and all samples were assayed simultaneously using a validating RIA.

Freeman. Ovarian influence on adhesion development. Fertil Steril 2002.

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ectomized groups, we collapsed the four separate treatment groups into one larger cohort and compared this against the sham (nonovariectomized) group. Incidence of adhesions in the sham subjects was 60.9% versus 26.6% in the combined treatment cohort (P⫽.003). Comparing the treatment groups individually against the sham group involved making multiple comparisons; therefore, the Holm modification of the Bonferroni correction was used to determine the cutoff P-values for two-tailed significance for our observations. The Holm modification produces a rank order of ascending P-values for significance by taking the initially desired alpha level (0.05) and dividing it by k, the number of contrasts in question. The smallest P-value from the ␹2 analysis is then compared against alpha/k. If the smallest P-value is not less than alpha/k, there is no statistical significance and no further comparisons are made. If the P-value is less than alpha/k, then the second smallest P-value is compared against alpha/(k ⫺ 1), and if this is made, then the third smallest is compared against alpha/(k ⫺ 2). The denominator is reduced by 1 each time, until nonsignificance occurs (26). For our purposes, we had four contrasts (groups), which produced ascending alpha cutoff values of 0.05/4 ⫽ 0.0125; 0.05/3 ⫽ 0.017; 0.05/2 ⫽ 0.025; and 0.05, which our observed P-values would have to meet to achieve statistical significance. Statistical significance was observed between the control (ovariectomized, no replacement) group and the sham rats. The control rats had a decreased incidence of adhesions of 20.8%, compared with the sham incidence of 60.9% (P⫽.008). With the Holm modification, significance for the first observation was set and met at Pⱕ.0125. The combined estrogen/P rate of adhesion formation was 24.0%, compared with the sham rate of 60.9% (P⫽.018). This was just short of significance, as significance for the second observation was set at Pⱕ.017. The two remaining individual comparisons of estrogen versus sham and P versus sham were not statistically significant, although they did demonstrate a lower incidence of adhesions at 28.6% and 33.3%, respectively.

DISCUSSION To facilitate making comparisons between treatment groups, we attempted to minimize the variability of the surgical injury by using an animal model that is standardized, has been previously validated and used extensively, and is known to create reproducible adhesions (21, 22). In addition, as the cecum is separate from the subject’s reproductive organs and site of ovariectomy, both contact of the ovariectomy site to the healing cecum and inference of the assigned grouping from visualizing the reproductive organs were minimized. Replaced sex steroid levels were adequate. The data are rather intriguing. This study showed an ovarian effect of adhesion development upon a nonreproductive tract tissue, the cecum. Prior studies evaluating sex 343

steroid effects on adhesion development centered on adhesiogenic surgical insults of reproductive tract tissues, such as uterine horn (14, 15, 20, 27, 28). Additionally, instead of observing a direct, dose-response relationship between individual ovarian sex steroids and adhesion formation, the identified difference in adhesion formation centered on whether at the time of cecal abrasion and wound healing, the ovaries were in situ or previously removed. Adhesion incidence in the four treatment groups of E2, P, combined E2 and P, and control did not differ statistically. In contrast, the ovary intact (sham) rats demonstrated a statistically significant increased incidence of adhesion development compared with the four ovariectomized groups taken collectively. When compared with treatment groups individually, the sham rats had statistically significant increased adhesive disease with respect to the control rats and were very close to achieving the same results with respect to the combined E2/P-replaced rats. When acting alone as the solely replaced hormone, estrogen did not exacerbate adhesive disease as might have been expected based on the hypoestrogenism premise of prior studies (14 –16). Likewise, P did not demonstrate an ability to decrease postoperative adhesions while acting alone, a test of its potential inherent immunosuppressive property (18, 19, 29, 30). Furthermore, since adhesion development in the E2-alone and combined E2-P rats did not differ, it is unlikely that the estrogen-antagonistic action of P as a mechanism for attenuating postoperative adhesions is significant. These findings thus raise the question of what might be accounting for the difference in adhesion development between the ovary-intact and ovary-removed rats? Based on our results, and the fact that both leuprolide acetate and MPA inhibit folliculogenesis and had similar adhesion-reducing effects in the prior studies, the likely candidate would be a previously unconsidered, ovarian follicle–produced factor. Such possibilities include T, activin, and inhibin. In men, leuprolide acetate reduces T to hypogonadic levels and, as such, has been used in the treatment of prostate cancer (31, 32). In women, the ovary contributes approximately two-thirds of the total serum T level and this drops accordingly with leuprolide acetate therapy (33, 34). Although there is little literature that directly compares postsurgical adhesion formation in men and women, in a 1996 prospective, randomized, double-blind study assessing a modified sodium hyaluronate/carboxymethylcellulose-based bioresorbable membrane for postoperative adhesion reduction, males had greater adhesion development than females in both the treatment and control groups (35). This study provides a clinical corollary for the hypothesis that T is a putative element for promoting adhesion development. Certainly, future study isolating T replacement and adhesion outcome in an animal model is warranted. Inhibin and activin are both members of the transforming growth factor-beta (TGF-␤) superfamily, and structurally 344

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they are closely related peptides composed of two disulfidebridged subunits. Inhibin consists of an ␣-subunit and one of two highly homologous ␤-subunits (A or B), which combine with it to produce either inhibin A (␣-␤A) or inhibin B (␣-␤B) (36). Activin is a homo- or heterodimer of the same ␤-subunits of inhibin, generating activin-A (␤A-␤A), activin-AB (␤A-␤B), and activin-B (␤B-␤B). Activin has two classes of receptors, type I (actRI) and type II (actRII). ActRI binds activin with high affinity, but the signaling peptide actRII must be present and bound to actRI for signaling to occur through its serine-threonine kinase (36). No known receptor has been positively identified for inhibin, but indirect evidence is mounting (37). Activin is produced by the ovary and various other tissues, including the placenta, brain, and bone marrow. Activin has an important role in cell-cell adhesion during organogenesis, and in the adult organism it participates in tissue repair, cellular proliferation, and differentiation (38 – 42). Serum levels of activin remain stable throughout the menstrual cycle (36) and are not down-regulated greatly by GnRH-a (43) and thus fail to fulfill our requirements for the unknown ovarian factor. Given its known functions, widespread tissue distribution, and nonfluctuating serum levels, activin’s role in adhesiogenesis is likely centered on local growth factor activities rather than on behaving as a traditional long-range hormone. The ovary also produces inhibin, like activin, and its diverse functions throughout the body are only beginning to be appreciated. Unlike activin, however, inhibin’s serum levels fluctuate during the varying phases of the menstrual cycle (44, 45), are responsive to leuprolide acetate administration (43), and drop rapidly after castration (46, 47). Inhibin thus acts in a more traditional long-range endocrine hormonal fashion. While the search is on for a cellular inhibin receptor, inhibin’s main mode of action is currently believed to be through inhibition of activin’s actRII receptor. The drop in inhibin levels after ovariectomy potentially allows increased activin activity at the local cellular level to drive decreased postoperative adhesiogenesis, as activin has been demonstrated to blunt various inflammatory responses (48 –50). Lastly, it is also possible that ovariectomy impacts postoperative adhesion development in an indirect rather than a direct manner. The most proximate cause of adhesiogenesis may be part of a secondary disruption in the hypothalamicpituitary-ovarian axis resulting from the ovariectomy. This could come in the form of elevated FSH or decreased hepatic production of sex hormone– binding globulin. One human clinical trial suggested that use of GnRH-a preoperatively does not reduce post– open-myomectomy adhesion formation (11). The main objective of this particular study was to assess the efficacy of Seprafilm Bioresorbable Membrane (Genzyme Corporation, Cambridge, MA) in reducing the development of uterine adhesions after myomec-

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tomy. The resulting adhesions were evaluated via secondlook laparoscopy 7–70 days after the open myomectomy. As an afterthought, regression analysis was performed to determine if there was a difference in adhesion development between the GnRH-a users and nonusers in their respective Seprafilm-treated and -nontreated groups. Patients were considered to be GnRH-a users if they had received their GnRH-a within 31 days before the myomectomy surgery. The adhesion quality parameters of extent and area were not statistically different between the GnRH-a users and nonusers within their respective Seprafilm-treated and -nontreated groups. Adhesion severity was noted to be statistically slightly increased in the GnRH-a users compared with nonusers within the nontreatment Seprafilm group, but no such difference was observed within the Seprafilm-treated group. Given the design of the study and the incidental nature of the GnRH-a analysis, no strong statements from this study regarding the impact of GnRH-a on postoperative adhesions can truly be made, as the investigators themselves pointed out. Sex steroid levels were not determined perioperatively, and a significant number of those patients who received GnRH-a within 31 days of their myomectomy may very well have been in the flare effect stage of GnRH-a administration, and thus have had elevated sex steroid levels. This rather straightforward study has put a new twist on some old observations. Additional studies are certainly necessary to [1] confirm our findings and [2] investigate directly other ovarian follicle–produced factors for their effects on postoperative adhesion development. References 1. Caspi E, Halperin Y, Bukovsky I. The importance of peri-adnexal adhesions in tubal reconstructive surgery for infertility. Fertil Steril 1979;31:296 –300. 2. Rock JA, Katayama KP, Martin EJ, Woodruff JD, Jones HW Jr. Factors influencing the success of salpingostomy techniques for distal fimbrial obstruction. Obstet Gynecol 1978;52:591– 6. 3. Kresch AJ, Seifer DB, Sachs LB, Barrese I. Laparoscopy in 100 women with chronic pelvic pain. Obstet Gynecol 1984;64:672– 4. 4. Daniell JF. Laparoscopic enterolysis for chronic abdominal pain. J Gynecol Surg 1989;5:61– 6. 5. Steege JF, Stout AL. Resolution of chronic pelvic pain after laparoscopic lysis of adhesions. Am J Obstet Gynecol 1991;165:278 – 83. 6. Ellis H. The clinical significance of adhesions: focus on intestinal obstruction. [Review] (29 references). Eur J Surg Suppl 1997;577:5–9. 7. Menzies D. Postoperative adhesions: their treatment and relevance in clinical practice [review]. Ann R Col Surg Engl 1993;75:147–53. 8. van Goor H. Complications in re-operated patients. Prevention and treatment of adhesive complications in colorectal surgery. Paper presented at the Annual Scientific Meeting of the Association of Surgeons of Great Britain and Ireland, June 7–11, 1998, Malmo, Sweden. 9. Moran BJ. The workload from adhesions in re-operative surgery. Clinical and epidemiological perspectives on post-operative adhesions. Paper presented at XVIIth Biennial Congress of the International Society of University Colon and Rectal Surgeons, May 13–15, 1998, Scotland, UK. 10. Moreira H Jr., Wexner SD, Yamaguchi T, Pikarsky AJ, Choi JS, Weiss EG, et al. Use of bioresorbable membrane (sodium hyaluronate ⫹ carboxymethylcellulose) after controlled bowel injuries in a rabbit model. Dis Colon Rectum 2000;43(2):182–7. 11. Diamond MP. Reduction of adhesions after uterine myomectomy by Seprafilm membrane (HAL-F): a blinded, prospective, randomized, multicenter clinical study. Seprafilm Adhesion Study Group. Fertil Steril 1996;66:904 –10. 12. Jansen RP. Failure of intraperitoneal adjuncts to improve the outcome of pelvic operations in young women. Am J Obstet Gynecol 1985;153: 363–71.

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