RBMOnline - Vol 14. No 5. 2007 620-625 Reproductive BioMedicine Online; www.rbmonline.com/article/2742 on web 19 March 2007 Article Effect of ovarian involvement on peritoneal fluid cytokine concentrations in patients Dr Mohamed Bedaiwy graduated from Assuit School of Medicine, Egypt in 1992, where he remains on the staff. His training in obstetrics and gynaecology has taken him to several institutions in USA, notably the Cleveland Clinic Foundation, Ohio. He became expert in reproductive biology and minimally invasive surgery. Currently he is engaged on a reproductive endocrinology and IVF fellowship at the University of Toronto, Canada. Dr Bedaiwy has published extensively and has been honoured with awards from the Society of Reproductive Surgeons (2001), the Pacific Coast Reproductive Society (Serono In-Training Award, 2002), and the ASRM (Best Video Award, 2001 2003). His special interests include and ovarian tissue cryopreservation. Dr Mohamed Ali Bedaiwy Mohamed Ali Bedaiwy 1,2,3, Sherif A El-Nashar 4, Rakesh K Sharma 1,2, Tommaso Falcone 1,2,5 1 Reproductive Research Center, Cleveland Clinic, Cleveland, OH 44195, USA; 2 Minimally Invasive Surgery Center, Cleveland Clinic; 3 Department of Obstetrics Gynecology, Cleveland Clinic; 4 Department of Obstetrics and Gynecology, Assiut University, Assiut, Egypt 5 Correspondence: Tel: +11 216 444 1758; Fax: +11 216 445 5526; e-mail: falcont@ccf.org Abstract Peritoneal fluid cytokines are important for initiation and progression of. The objective of this study was to compare a group of five cytokines (interleukins IL-1β, IL-6, IL-8, IL-13 and tumour necrosis factor α; TNFα) in peritoneal fluid of patients with ovarian involvement (Group I, n = 17) to those in patients without ovarian involvement (Group II, n = 33) and to a reference group without (Group III, n = 25). All three groups were comparable regarding age, parity and body mass index. IL-8 concentrations were significantly higher in groups I and II compared with the reference group (P = 0.01 and 0.02, respectively). Similarly, TNFα concentrations were significantly higher in groups I and II compared with the reference group (P < 0.0001 and 0.0004, respectively). All other cytokines were comparable in the three groups. No significant differences were found between groups I and II with respect to the cytokines measured. In conclusion, peritoneal fluid IL-8 and TNFα concentrations are significantly higher in. Ovarian involvement does not alter the pattern of cytokines. It appears that the inflammatory mediators of are similar with and without ovarian involvement. Keywords: cytokines, endometrioma, pathogenesis, peritoneal fluid Introduction 620 Endometriosis is one of the most common benign gynaecological disorders and is present in >10% of women of reproductive age in the USA. In spite of extensive basic and clinical research to unveil the aetiology of this disease, little is known. The current consensus is that is associated with a local pelvic inflammatory process with altered function of the local and systemic immune responses (Bedaiwy and Falcone, 2004). Increased production of pro-inflammatory cytokines, such as the interleukins IL-1, IL-8, tumour necrosis factor α (TNFα) (Arici et al., 1997) and vascular endothelial growth factor have been consistently demonstrated in the peritoneal fluid (PF) of patients (Bedaiwy et al., 2002; Bedaiwy and Falcone, 2004). In addition, previous studies have shown that cytokines in PF and the endometrium affect fertilization, implantation and future pregnancy outcome (Bulletti et al., 2005; Esfandiari et al., 2005; Norwitz, 2006). Since retrograde menstruation is observed in almost all cycling women, is postulated to develop as a result of the coexistence of a defect in clearance of endometrial cells in the menstrual efflux from pelvic peritoneal surfaces. One popular hypothesis is that women who are liable to get have a defective or abnormal immune system that prevents timely clearance of deposited endometrial cells (Lamb and Nichols, 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
1986; Lebovic et al., 2001). However, several investigators have demonstrated that the immunological perturbations in nonhuman primates with induced pelvic are secondary to the presence of the ectopic endometrium, rather than being a possible aetiopathogenic factor. It is likely that molecular changes such as immunological alterations are secondary to the presence of ectopic endometrium (Gashaw et al., 2006). While ovarian is a common site of pelvic, the ovary, under physiological conditions, is a site for inflammatory reactions mediated by numerous cytokines, particularly around the time of ovulation, and so the ovary is the single most important source for PF (Maathuis et al., 1978; Syrop and Halme, 1987). It is unclear if the presence of ovarian contributes in a unique way to the inflammatory environment of the peritoneum. The specific aims of this study were: (i) to compare a group of cytokines in the PF of patients with ovarian involvement to those in patients without ovarian involvement; and (ii) to compare the concentrations of different cytokines in both cohorts to a reference group without. Materials and methods The Institutional Review Board of the Cleveland Clinic Foundation approved this study. The study included a cohort of 75 consecutive women who underwent laparoscopy from January 1998 to December 2003 at the Minimally Invasive Surgery unit in the Cleveland Clinic Foundation. The indications for laparoscopy included chronic pelvic pain, infertility, tubal ligation, or sterilization reversal. All patients included in this study had no significant co-morbidities except for the primary indication of surgery. After obtaining their informed consent, intraoperative peritoneal samples were collected. From 121 women who underwent laparoscopy in the period of study, 46 were excluded because of blood-contaminated PF and no patients were excluded for frozen pelvis (Figure 1). All patients included in the study had general anaesthesia using the same approach. In the patients with, the involvement of the ovary was reported and the severity of the disease was graded according to the revised four-stage American Fertility Society scoring system (American Society for Reproductive Medicine, 1997). After surgery and pathological examination, the included cohort was subdivided into three subgroups which Figure 1. Flow chart showing the composition of the three study groups. 621
were predetermined a priori. The three groups were: women with involving one or both ovaries (Group I; n = 17); women with without ovarian involvement (Group II; n = 33); and women who did not have (reference Group III, n = 25). Some patients in this trial were included in a previously published trial (Bedaiwy et al., 2002). Preparation of the peritoneal fluid During laparoscopy, PF was aspirated from the peritoneal cavity from an abdominal port. The cellular constituents of the PF were removed by centrifugation at 300 g for 20 min. PF supernatants were then collected and stored in aliquots at 70 C until the cytokine concentrations were determined. Detection of cytokines in the peritoneal fluid Concentrations of IL-1β, IL-6, IL-8, IL-13, and TNFα were measured in the PF supernatants using commercially available cytokine-specific enzyme-linked immunosorbent assays (ELISA) (R and D Systems, Inc., Minneapolis, MN, USA). The frozen samples were thawed and analysed. Samples from each patient group were always measured in parallel and in duplicate to avoid interassay variance. The sensitivities of the IL-1β, IL-6, IL-8, IL-13, and TNFα ELISA were 1, 0.7, 10, 32, and 4.4 pg/ml, respectively, with standard curve ranges of 3.9 250, 3.12 300, 31.2 2000, 62.5 4000, and 15.6 1000 pg/ml, respectively. Statistical analysis The baseline and demographic data were presented as proportions for categorical variables and as medians and interquartile ranges (IQR: 25 75% centiles) for continuous variables. The demographic variables, serum measurements and PF measurements were compared across the three study groups. Chi-squared test and Fisher s exact tests (for categorical variables) and Kruskal Wallis tests (for continuous variables) were used for comparing different demographic characteristics and outcomes. Pair-wise comparisons between the groups were performed with the chi-squared test or Wilcoxon (Mann Whitney) rank-sum test, accordingly. A sample size of 15 in each group was determined to have 80% power to detect a probability of 20% that an observation in one group is less than an observation in another group using a Wilcoxon ranksum test with a 0.05 two-sided significance level (nquery Advisor, version 4.0, Statistical Solutions, Saugus, MA, USA). Statistical computations were performed with SAS version 8.1 (SAS Institute, Cary, NC, USA), and statistical significance was assessed using two-tailed tests and an alpha level of P < 0.05. Results Baseline characteristics and demographic variables Adequate PF was obtained from 75 women who gave their consent. The final analysis included 75 women: 17 (23%) women were diagnosed with with one or both ovaries involved (Group I); 33 (44%) had diagnosis without ovarian involvement (Group II); and 25 (33%) women did not have (Group III) (Figure 1). The median age of the study cohort was 33 years (IQR: 29 39 years), body mass index (BMI) was 24 kg/m 2 (IQR: 21 28 kg/m 2 ) and parity was 1 (IQR: 0 2). No significant differences were seen in age, parity and BMI between the three groups of patients. Each group included comparable patients in the proliferative and luteal phase of the menstrual cycle. Of the 50 patients with, 29/50 (58%) had early disease (stage I and II) and 21/50 (42%) had advanced disease (stages III and IV) (Table 1). Peritoneal fluid cytokines in the different groups Details of the peritoneal fluid cytokine concentrations in the three study groups are given in Table 2. PF IL-8 concentrations were significantly higher in groups I and II compared with the reference group (P = 0.02 and 0.01, respectively) (Figure 2a). Likewise, PF TNFα concentrations were significantly higher in groups I and II compared with the reference group (P < 0.004 and 0.001, respectively) (Figure 2b). However, there was no difference in the PF concentrations of both cytokines in women with and without ovarian involvement. All other PF cytokines were comparable in the three groups. No significant differences were found between groups I and II with respect to the cytokines measured. Cytokine concentrations were also not affected by the stage of the disease or by the phase of the menstrual cycle. Table 1. Demographic characteristics of the three study groups. Characteristic Group I (ovarian Group II (non-ovarian Group III (reference) ) ) 622 Age (years) 35.0 (30.0 37.0) 32.0 (29.0 38.0) 34.0 (29.0 39.0) Gravidity 0.0 (0.0 2.0) 0.5 (0.0 1.5) 2.5 (1.0 3.0) Parity 0.0 (0.0 1.0) 0.0 (0.0 1.0) 2.0 (1.0 2.0) Weight (kg) 62.0 (55.0 70.0) 62.4 (57.6 75.0) 72.0 (65.7 79.0) Height (m) 1.7 (1.6 1.7) 1.6 (1.6 1.7) 1.6 (1.6 1.7) Body Mass Index (kg/m 2 ) 22.0 (21.7 24.2) 23.8 (23.8 26.0) 26.8 (26.4 29.0) Values are median (interquartile range). There were no statistically significant differences between the groups.
Table 2. Peritoneal fluid cytokine concentrations (pg/ml) in the three study groups. PF cytokine Group I (ovarian Group II (non-ovarian Group III (reference) P-value a ) ) IL-1β 2.53 (0 64.36) 3.71 (1.59 33.34) 0.00 (0.00 8.14) NS IL-6 38.74 (21.63 92.64) 31.01 (12.52 50.27) 21.94 (15.29 34.02) NS IL-8 28.8 (1.03 226.18) 14.59 (4.18 14.59) 1.04 (0.00 9.02) 0.01 IL-13 1.2 (0 21.51) 0.60 (0.00 15.54) 0.00 (0.00 0.00) NS TNFα 47.57 (24.36 62.50) 54.83 (43.75 64.42) 0.00 (0.00 0.00) <0.0001 Values are median (interquartile range). IL = interleukin; PF = peritoneal fluid; TNFα = tumour necrosis factor α. a Kruskal Wallis test 4 a P = 0.02 Log of peritoneal fluid interleukin-8 3 2 1 P = 0.29 P = 0.01 0 Ovarian Non-ovarian Reference 80 b P = 0.004 Peritoneal fluid tumour necrosis factor-α 70 60 50 40 30 20 10 P = 0.59 P = 0.001 0 Ovarian Non-ovarian Reference Figure 2. Analysis of the peritoneal fluid of the three study groups. (a) Interleukin 8; (b) tumour necrosis factor α. P < 0.05 was considered statistically significant. 623
624 Discussion These results indicate that ovarian involvement in patients with is not associated with a specific profile of cytokines compared with other patients without ovarian involvement. This study evaluated the effect of the involvement of the ovary in on the concentrations of cytokines in the PF. In agreement with previous reports, both PF IL-8 and TNFα concentrations were significantly higher in women with compared with patients without (Awadalla et al., 1987; Taketani et al., 1992). In addition, PF IL-8 was related to the stage and severity and was proposed as a marker for (Gazvani et al., 1998; Garcia-Velasco and Arici, 1999; Maas et al., 2001; Bedaiwy et al., 2002). On the other hand, TNFα concentration is significantly higher in the PF of women with, and it has been related to the stage, severity and the activity of the disease (Maas et al., 2001; Calhaz-Jorge et al., 2003; Yamauchi et al., 2004). Moreover, small molecules that can inhibit the activity of TNFα have been proposed as a potential effective treatment of (Said et al., 2005; Falconer et al., 2006). The PF environment is host to the processes of ovulation, gamete transportation, fertilization and early embryonic development. The cellular and acellular constituents of this dynamic fluid are in a constantly interactive state, being influenced by the physiological events of the menstrual cycle and pelvic disease processes; these constituents probably influence disease manifestation and reproduction. In a study to evaluate cyclic changes of PF parameters in normal and infertile patients, it was shown that cyclic and post-ovulatory differences in PF volume support proposed pathophysiological roles (Syrop and Halme, 1987). As is one of the commonest benign disorders changing the homeostasis of the PF, involvement of the different intraperitoneal structures may modulate the fluid dynamics. The central role of the ovary in the pathogenesis of and the early anatomical involvement suggest a strong relation with. If the previously observed defective follicular development and lower quality of ova retrieved from women with is taken into consideration, it is interesting to see whether these effects are inherent during the process of oogenesis or acquired during the passage through the peritoneal cavity. Since the ovary is a major source of PF, it was proposed that a difference in PF cytokine concentrations would be observed if the ovaries become affected by the disease (Maathuis et al., 1978; Syrop and Halme, 1987). In addition, a recent study showed that ovarian steroids, TNFα and transforming growth factor β act as key regulators of endometrial IL-13 and IL-15 expression (Roberts et al., 2005). Consequently, there may be a possible modulatory effect of ovarian steroids on PF cytokines concentrations. Herein, it is postulated that if these changes were organ-specific, patients with ovarian involvement would be more likely to have unique or perhaps higher concentrations of PF cytokines. In the current study, no differences were seen in the concentrations of all five studied cytokines, including IL-8 and TNFα which were previously reported to be influential in the pathogenesis and good markers of disease progression. While these results suggest the exclusion of a possible anatomical role of the ovary in, nevertheless, the pathophysiological impact of ovarian steroids and cytokines on initiation and severity still needs to be tested. The current study was limited by the relatively small numbers included in each group. However, using these small samples, a difference in the cytokine profile between the patient and patients without was detected. In addition, using the available sample size, the study was able to detect a difference of 10 pg/ml or more between the two groups. This is a reasonable difference below which any detected difference would not provide a significant laboratory difference given the observed wide range in the measured IL-8 and TNFα in the study population. In addition, the authors appreciate the possibility of measurement bias from the heterogeneity in the clinical presentation in the group, including differences in the clinical presentation and in the stage of menstruation at laparoscopy. Both might limit the internal validity of the results. Nevertheless, including cases presenting with pelvic pain and/or infertility increases the external validity of the study by including the wide array of presentation of. Subgroup analyses based on the different presentations and different stages are beyond the scope of this study. Another possible limitation may be the large number of samples excluded as a result of blood contamination. This might affect the external validity of the study results, but a trade off between internal and external validity always exists and should be taken into consideration during design and interpretation of the results of patient-oriented research. Despite these limitations, as far as is known, this is the first study to address the issue of ovarian involvement and its relation to PF cytokines. In conclusion, PF IL-8 and TNFα were significantly higher in patients. However the cytokine concentrations were comparable between women who had ovarian involvement and those who did not. Ovarian involvement does not alter the pattern of PF cytokines in these patients. Acknowledgements This study was supported by a research grant from the Minimally Invasive Surgery Centre (MISC) of the Cleveland Clinic Foundation (RPC#2156). The authors are grateful to David Nelson, MS, Department of Quantitative Health, Cleveland Clinic Foundation for biostatistical support. 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