IL-1, IL-6 and TNF-{alpha} concentrations in the peritoneal fluid of women with pelvic adhesions

Y.C. Cheong1, J.B. Shelton2, S.M. Laird2, M. Richmond1, G. Kudesia3, T.C. Li1,4 and W.L. Ledger1

1 The Jessop Wing, Central Sheffield University Hospitals, Tree Root Walk, Sheffield, S10 2PS, 2 Division of Biomedical Sciences/BMRC, Sheffield Hallam University, City Campus, Sheffield S1 1WB and 3 Sheffield Virology Service, Sheffield Teaching Hospital Trust, Northern General Hospital, Herries Road, Sheffield S5 7AU, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Pelvic adhesions are a significant cause of morbidity and are associated with infertility and pain. The three pro-inflammatory cytokines interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF)-{alpha} are involved in adhesion formation/reformation. METHODS: The concentration of these three cytokines was examined in the peritoneal fluid of women undergoing laparoscopy, in order to examine the factors affecting their concentrations, with specific reference to the presence or absence of adhesions. RESULTS: We found that the concentration of TNF-{alpha} in the peritoneal fluid was significantly correlated to the menstrual cycle day (P < 0.01), with increasing concentration as the menstrual cycle progressed from the follicular to the luteal phase. In contrast, IL-1 and IL-6 levels did not show any variation throughout the menstrual cycle. Increased TNF-{alpha} was found in patients with pelvic adhesions compared with those with normal pelvis; the concentration of TNF-{alpha} was highest in mild compared with severe adhesions. In contrast, IL-1 concentration was higher in the presence of severe adhesions. IL-6 levels were significantly correlated with the grade of endometriosis (P < 0.05), but there were no significant correlations of either TNF-{alpha} or IL-1 concentrations with the various grades of endometriosis. CONCLUSION: The exact role of TNF-{alpha} and IL-1 in adhesion formation is still unknown, but the results from this study suggest that their concentration in the peritoneal fluid is associated with the degree of adhesions present.

Key words: endometriosis/interleukin/menstrual cycle/peritoneal fluid/TNF-{alpha}


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pelvic adhesions are a significant cause of morbidity (Rosenthal et al., 1984Go; Stout et al., 1991Go; Menzies, 1993Go) and adhesiolysis is an increasingly common surgical treatment for pelvic adhesions in our reproductive surgery unit (Cheong et al., 2001bGo). A study has shown that 33% of patients who had previous surgery would be re-admitted to hospital on an average of twice for adhesion-related complications (Ellis et al., 1999Go).

Several studies have shown that adhesion formation involves three important components: (i) acute inflammatory response; (ii) fibrinolysis; (iii) metalloproteinases and their tissue inhibitors (Mori et al., 1991Go; Saba et al., 1998Go; Sharpe-Timms et al., 1998Go; Holmdahl, 1999Go). Cellular mediators within the peritoneal fluid can potentially modulate inflammatory responses over a large surface area due to the liquid nature of the peritoneal fluid. There are three important pro-inflammatory cytokines involved in adhesion formation/reformation: interleukin (IL)-1 and tumour necrosis factor (TNF)-{alpha} are both pro-inflammatory cytokines important in the early phase of wound healing (Lowry, 1993Go) and are produced by activated macrophages in the peritoneal fluid (Halme, 1986Go; Mori et al., 1991Go), whereas IL-6 is expressed by macrophages and its production is up-regulated by IL-1 during the inflammatory process (Hirano, 1998Go). Both IL-1 and TNF-{alpha} are potent inducers of IL-6 (Bauer et al., 1988Go). These cytokines are thought to be important as they interact extensively with the fibrinolytic pathway and contribute directly or indirectly to the remodelling of the extracellular matrix (Cheong et al., 2001aGo).

Current understanding of the function of these three pro-inflammatory cytokines suggests that their concentrations are elevated in women with adhesions. However, previous studies have shown that IL-1 concentrations are not elevated in women with adhesions (Chegini et al., 1999Go). The two studies on TNF-{alpha} have shown conflicting results. In one study, the investigators showed elevated TNF-{alpha} concentrations in peritoneal fluid of women with pelvic adhesions (Guerra-Infante et al., 1999Go), but other investigators found no difference in the TNF-{alpha} levels in women with or without pelvic adhesions (Chegini et al., 1999Go). As far as IL-6 is concerned, all the published studies relate to endometriosis, with none directly examining the relation of adhesions alone to the cytokine concentration in the peritoneal fluid (Buyalos et al., 1992Go; Mahneke et al., 2000Go).

A review of the literature on the concentration of various cellular mediators of the peritoneal fluid suggests that a number of factors can affect their concentrations, including the stage of the menstrual cycle, the underlying pathology and the volume of peritoneal fluid present (Cheong et al., 2001aGo). Attempts have been made to stratify results of peritoneal cytokine assays according to the stage of the menstrual cycle into the proliferative and luteal phase, but detailed data on the effects of ovarian hormones on peritoneal cytokines is still lacking.

In this study, we have measured the concentration of three pro-inflammatory cytokines, IL-1, IL-6 and TNF-{alpha}, in the peritoneal fluid of women undergoing laparoscopy in order to examine the factors affecting the concentration of these cytokines, with a specific reference to the presence or absence of pelvic adhesions.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
Women undergoing laparoscopy for benign gynaecological conditions were prospectively recruited into the study. Women with a normal menstrual cycle (25–35 days) and aged 18–45 years were included in the study. Subjects were excluded from the study if they had fibroids, dermoid cysts or any gynaecological malignancy. They were also excluded if they were on the oral contraceptive pill, hormone replacement therapy or had a clinical history or laboratory evidence of pelvic inflammatory disease. None of the patients included in this study had acute abdominal symptoms indicative of acute pelvic pathology.

Ethical issues
The study was carried out with the approval of the local ethics committee. Patient information leaflets were given and written consent was obtained prior to the surgery.

Peritoneal fluid collection
Peritoneal fluid was collected during laparoscopy using a follicular aspiration needle under direct vision at the beginning of the procedure, prior to the commencement of any pelvic surgery. In some cases, a palpator was used to move away the bowel to expose the pouch of Douglas, from where the peritoneal fluid could then be collected. Care was taken not to contaminate the peritoneal fluid with blood. The fluid was collected, centrifuged and the supernatant was immediately stored in liquid nitrogen at –70°C.

Clinical details
The findings at surgery were recorded on a standardized operation sheet. During laparoscopy, the pelvis was inspected in detail for signs of pelvic inflammatory disease. Patients with perihepatic adhesions indicative of Fitz Hugh Curtis Syndrome were excluded from the study. The last menstrual period (LMP), the volume of peritoneal fluid collected (ml), the colour of the peritoneal fluid collected (straw, dark old blood or fresh red blood), the presence or absence of adhesions and the grade of adhesions (if present), scored according to a well-validated adhesion scoring method (MCASM) (Adhesion Scoring Group, 1994Go), were recorded. Patients with adhesions were divided into three groups according to their adhesion scores: (i) mild (adhesion score 0–46), (ii) moderate (47–92) and (iii) severe (93–138). The extent of endometriosis, when present, was scored according to the American Fertility Society Classification System (American Fertility Society, 1985Go). Patients with endometriosis were subdivided into four groups: (i) minimal, (ii) mild, (iii) moderate and (iv) severe endometriosis.

Analysing peritoneal fluid for Chlamydia infection
The peritoneal fluid samples collected were tested for C.trachomatis by a nucleic acid amplification technique, namely Strand Displacement Assay (SDA).

Menstrual cycle
Ovulation detection
Detailed menstrual history was taken from the women recruited into the study and only women with regular menstrual cycles were included into the study. In addition, during laparoscopy, the visualization of a corpus luteum or follicular cysts was noted on a standard proforma.

Menstrual cycle correction
In this study, we used the onset of the last menstrual period as a reference point and have made adjustments for women whose menstrual cycle were not 28 days, according to the recommendations of Lenton et al. (Lenton et al., 1984Go). The assumption is that the luteal phase is much more constant than the follicular phase and is usually ~14 days. For example, in women with a 35-day cycle, the likelihood is that the follicular phase is 21 days and the luteal phase 14 days (Lenton et al., 1984Go).

Laboratory assays
IL-1 and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA) using duoset kits (R&D Systems Ltd, Abingdon, Oxon, UK) according to the manufacturer's instructions. Samples and standards were assayed in duplicate. TNF-{alpha} was measured by bioassay using L929 cells (Aarons and Borish, 1993Go; Gaines Das and Meager, 1995). These cells were split the day before the start of the assay to ensure that they were in log-phase growth. Cells were plated into all wells of a 96 well plate at a density of 2.2x105 cells per ml (100 µl per well) and grown overnight at 37°C in an atmosphere of 5% CO2. The next day, 100 µl of sample or standard TNF-{alpha} (8–500 pg/ml) (R&D Systems Ltd) was added and the cells were incubated as before for a further 24 h. Cells were then washed twice in phosphate-buffered saline (PBS), fixed in methanol for 15 min and stained using crystal violet. After staining, the cells were solubilized using 10% acetic acid and the absorbance at 570 nm was read. All samples and standards were assayed in triplicate. The sensitivity of the assays was 1, 1 and 2 pg/ml for IL-1, IL-6 and TNF-{alpha} respectively. The inter- and intra-assay variation was 10 and 5% for the IL-6 and IL-1 immunoassay, and 15 and 5% for the TNF-{alpha} bioassay. To ensure that the other constituents of the peritoneal fluid were not interfering with the assays, known amounts of each cytokine were added to a single peritoneal sample and assayed in parallel to the unspiked sample. The protein concentrations in the samples were measured using the Bicinchoninic Acid method with bovine serum albumin as a standard.

C.trachomatis was tested by SDA (Probe TecTM ET, Becton Dickinson). Briefly, 100 µl of peritoneal fluid was added to 2 ml of sample diluent and then tested according to the manufacturer's instructions. Positive and negative controls were included in each batch.

Statistical analysis
Data obtained were entered into a database created on Statistical Package for Social Sciences-PC (Sheffield University, Sheffield, UK). The concentrations of IL-1, IL-6 and TNF-{alpha} in women with adhesions were compared with those without adhesions using the Kruskal–Wallis test. Spearman's rank order correlation coefficient was used to analyse the relationship between the concentration of IL-1, IL-6 and TNF-{alpha} and the following variables: (i) the volume of peritoneal fluid collected; (ii) the colour of the peritoneal fluid; (iii) the protein concentration; (iv) the corrected menstrual cycle day; and (v) the degree of adhesions. Univariate analysis of variance (ANOVA) was used to control these variables when they were found to significantly affect the concentrations of IL-1, IL-6 or TNF-{alpha}.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In total, 175 women were recruited into the study. For 22 subjects, there were insufficient amounts of peritoneal fluid in the pouch of Douglas for sampling. In the remaining 153 subjects, samples of peritoneal fluid were successfully collected. The mean age of the subjects was 28 ± 0.7 years. Among the 153 subjects, 59 (38.5%) women had a history of infertility, 86 (56.2%) had a history of pelvic pain and eight (5.2%) had laparoscopic sterilization. None of the patients with infertility had anovulation. The laparoscopic findings included pelvic adhesion: 71 (46.4%), endometriosis: 53 (34.6%) and normal pelvis, i.e. no pathology: 37 (24.1%). All of the eight subjects who had sterilization had normal pelvis. Eight (5.2%) of the subjects had both pelvic adhesions and endometriosis. All women had a normal menstrual cycle of 28–32 days. Among the 71 women with adhesions, the degree of adhesions were graded as mild: 25 (35.2%), moderate: 25 (35.2%) and severe 21 (29.6%). All of these patients had had one or more previous surgery (laparoscopy or laparotomy). Of the 53 patients with endometriosis, 21 (39.6%), 10 (18.8%), 11 (20.8%) and 11 (20.8%) had grades I, II, III and IV endometriosis respectively, according to the American Fertility Society scoring system (American Fertility Society, 1985Go).

The majority of women in the luteal phase had physiological evidence of ovulation; we observed the presence of the corpus luteum in 98% (70/72) of the patients who had laparoscopy after day 14 of the menstrual cycle. In two patients who had laparoscopy on day 15 and 16, both had a follicle visualized at laparoscopy.

TNF-{alpha} bioassay
The lack of interference from serum and/or protein in the peritoneal fluid was ensured, as the amount of cell killing from incubation with some peritoneal fluid samples was the same as the zero control samples where cells were incubated with either cell culture medium or PBS. This then results in a zero value obtained for a concentration of TNF-{alpha}. This is unlikely to be true if there was something in the fluid that caused cell death. The addition of known amounts of TNF-{alpha} to a peritoneal fluid sample also gave the expected values when assayed, suggesting further that components of the peritoneal fluid did not interfere with the assay.

Characteristics of the peritoneal fluid
Colour
Among the 153 peritoneal fluid samples collected, the colour of the fluid was divided into straw-coloured: 71 (46%), brown blood-stained: 54 (35%), fresh red blood-stained: 19 (12%).

Volume
The mean (± SEM, range) volume of peritoneal fluid collected was 6.2 ml (± 4.6, 0.8–19). The mean (± SEM) volume collected from women with no adhesions, mild, moderate and severe adhesions was 6.5 (± 0.8), 7.0 (± 0.5), 5.2 (± 0.4) and 4.7 ml (± 0.4) respectively. There was no difference in the volume of peritoneal fluid collected in relation to the degree of adhesions. The mean (± SEM) volume collected in minimal, mild, moderate and severe endometriosis was 6.8 (± 0.71), 5.7 (± 0.40), 5.2 (± 0.41) and 6 ml (± 0.37) respectively. There was no difference in the volume collected in relation to the severity of endometriosis.

Protein concentration
The mean (± SEM) protein concentration in the peritoneal fluid was 67 (± 2.1) mg/ml. The protein concentration did not vary with the menstrual cycle. The mean (± SEM) protein concentration in the peritoneal fluid of women with no adhesions, mild, moderate and severe adhesions was 68.1 (± 2.9), 65.9 (± 4.2), 68.8 (± 5.3) and 65.0 mg/ml (± 6.0) respectively. There was no difference in the protein concentration of peritoneal fluid collected in relation to the degree of adhesions. The mean (± SEM) protein concentration in the peritoneal fluid collected in minimal, mild, moderate and severe endometriosis was 59.8 (± 2.1), 71.7 (± 7.0), 78.5 (± 11.6) and 70.9 mg/ml (± 9.8) respectively. There was no difference in the protein concentration of the peritoneal fluid collected in relation to the severity of the endometriosis.

Exclusion of pelvic infection
In total, there was sufficient peritoneal fluid in 99 patients for analysis of Chlamydia infection; these all tested negative for Chlamydia infection. Another 39 women in the study had previous negative Chlamydia serology (range: 2–6 months). However, there was no Chlamydia status obtained in 15 patients. All of these 15 patients were from the endometriosis group.

In the group of patients with adhesions and without endometriosis, the underlying cause of adhesions probably results from previous surgical trauma and not infection for the following reasons: (i) all the patients included in the study had a history of previous pelvic/abdominal surgery; (ii) none of the patients had a history of pelvic inflammatory disease; (iii) none of the patients have acute abdominal symptoms. They all had chronic symptoms; (iv) on laparoscopy, routine inspection for perihepatic adhesions indicative of Fitz Hugh Curtis Syndrome was performed. None of the patients had perihepatic adhesions.

The variation of the cytokine concentrations throughout the menstrual cycle
The concentration of IL-1 (0–8 pg/ml) was considerably lower than that of TNF-{alpha} (0–300 pg/ml) and IL-6 (0–320 pg/ml). The concentration of TNF-{alpha} in the peritoneal fluid was significantly correlated with the menstrual cycle day (n = 0.64, P < 0.01), with increasing concentration as the menstrual cycle progressed from the follicular to the luteal phase (Figure 1Go). In contrast, IL-1 and IL-6 levels did not show any variation throughout the menstrual cycle in women with normal pelvis, adhesions or endometriosis. There was no correlation between IL-1, IL-6 and TNF-{alpha} concentrations and the volume, total concentration of protein or colour of peritoneal fluid.



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Figure 1. TNF-{alpha} concentration in women with normal pelvis throughout the menstrual cycle (standardized to 28 days).Lines = mean and 95% confidence intervals.

 
Comparison between women with and without adhesions
In women with adhesions, the mean (± SEM) concentrations of the three pro-inflammatory cytokines were TNF-{alpha}: 111 (± 7.4), IL-1: 2.7 (± 0.7) and IL-6: 41 pg/ml (± 7.5). In women with normal pelvis, the mean (± SEM) concentrations of the cytokines were TNF-{alpha}: 138 (± 9.3), IL-1: 1.91 (± 0.1) and IL-6: 27.9 pg/ml (± 4.2). There was no difference in the concentration of IL-1 and IL-6 between women with or without adhesions. Because the concentration of TNF-{alpha} was significantly correlated to the stage of the menstrual cycle, univariate analysis (general linear model) was used to take into account the effect of the stage of the menstrual cycle on the results. The concentration of TNF-{alpha}, after correction for the effect of the menstrual cycle, was significantly different in women with pelvic adhesions compared with women with normal pelvis (P < 0.05) (Figure 2Go).



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Figure 2. The mean concentrations of TNF-{alpha} in the peritoneal fluid of women with normal pelvis and women with mild, moderate and severe adhesions respectively.

 
The impact of the degree of adhesions
The mean (± SEM) concentration of TNF-{alpha} in women with mild, moderate and severe adhesions was 173 (± 6.4), 69 (± 3.6) and 12 (± 0.4) pg/ml respectively. After correcting for the stage of the menstrual cycle, TNF-{alpha} concentrations varied significantly according to the degree of the adhesions (P < 0.05), with more TNF-{alpha} present in women with mild adhesions and less present in women with severe adhesions (Figure 3Go).



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Figure 3. Concentrations of TNF-{alpha} in the peritoneal fluid of women with mild, moderate and severe adhesions.{diamondsuit} = mean; whiskers = SEM. *P < 0.05.

 
Mean (± SEM) peritoneal fluid concentrations of IL-1 in women with mild, moderate and severe adhesions were 1.24 (± 0.1), 3.5 (± 0.6) and 7.7 pg/ml (± 0.7) respectively. There was a significant correlation between IL-1 concentration and the severity of adhesions, with more IL-1 present in women with more severe adhesions (P < 0.05) (Figure 4Go).



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Figure 4. Concentrations of IL-1 in the peritoneal fluid of women with mild, moderate and severe adhesions. {diamondsuit} = mean;whiskers - SEM. *P < 0.05.

 
The mean (± SEM) concentrations of IL-6 in the peritoneal fluid from women with mild, moderate and severe adhesions were 27.5 (± 2.6), 51.0 (± 4.0) and 77.8 (± 8.0) pg/ml respectively. In contrast with IL-1 and TNF-{alpha}, IL-6 concentrations did not vary significantly according to the degree of the adhesions.

Endometriosis
Table IGo shows the concentration of IL-1, IL-6 and TNF-{alpha} in peritoneal fluid of women with endometriosis. IL-6 concentrations correlated significantly with the grade of endometriosis (P < 0.05), but there were no significant correlations for TNF-{alpha} and IL-1 concentrations between the various grades of endometriosis.


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Table I. Concentration of IL-1, IL-6 and TNF-{alpha} in the peritoneal fluid of women with different grades of endometriosis
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, we measured the concentrations of the three pro-inflammatory cytokines; TNF-{alpha}, IL-1 and IL-6 in the peritoneal fluid of women with pelvic adhesions, endometriosis and normal pelvis. Our results showed that in women with normal pelvis, the concentration of TNF-{alpha} depended on the stage of the menstrual cycle, with an increase in concentration observed from the follicular to the luteal phase. In contrast, the concentration of IL-1 and IL-6 in the peritoneal fluid did not vary with the menstrual cycle. In addition, we have shown that TNF-{alpha}, but not IL-1 or IL-6 concentrations, were increased in women with adhesions compared with women with normal pelvis. In women with adhesions, the levels of TNF-{alpha} were inversely correlated with the severity of the adhesions; in contrast, the concentration of IL-1 was positively correlated with the severity of adhesions. In women with endometriosis, IL-6 concentrations positively correlated with the grade of endometriosis.

Although a number of previous studies have examined the concentration of cytokines in the peritoneal fluid, many of them have one or more of the following criticisms: (i) small sample size (n<100) (Mori et al., 1991Go; Buyalos et al., 1992Go; Chegini et al., 1999Go); (ii) failure to account for the variability of results throughout the cycle (Guerra-Infante et al., 1999Go); and (iii) lack of detailed grading of adhesions (Buyalos et al., 1992Go; Chegini et al., 1999Go; Guerra-Infante et al., 1999Go). In this study, we examined in detail the possible confounding factors affecting the concentrations of the cytokines in the peritoneal fluid, including the age of the subjects, the volume of the peritoneal fluid and the effect of the stage of the cycle.

Volume of the peritoneal fluid
Many factors could potentially influence the peritoneal fluid volume, for example, ovulation could release ~5 ml of follicular fluid into the peritoneal cavity. The volume of peritoneal fluid present in the pelvis would generally be small, and thus its impact on the concentration of cytokines would likely be significant. To analyse the cytokine data in a meaningful way, the volume of the fluid present in the peritoneal cavity should be measured so that the impact of dilution could be evaluated. However, in this study, the volume of the peritoneal fluid collected did not significantly affect the results, suggesting that cytokine and fluid production parallel each other.

Variation of the menstrual cycle
Previous studies on the measurement of cytokines in the peritoneal fluids had taken little account of the day of the menstrual cycle when the samples were taken. Studies have suggested that the production of IL-1, IL-6 and TNF-{alpha} by the endometrium and ovary is menstrual cycle-dependent (Philippeaux and Piguet, 1993Go; Norman and Brannstrom, 1996Go; Tabibzadeh and Kothapalli, 1996Go; Terranova and Rice, 1997Go). The exact cellular source of the peritoneal cytokines is unknown, but both the ovary and endometrium might contribute to peritoneal fluid cytokine levels and thus variation in the cycle might be expected. The cyclic variation in cytokine production by the endometrium and ovary suggests that cytokine production could be influenced by steroid hormones. This might also be true for cytokine production from other cells such as macrophages and mesothelial cells, which would also contribute to peritoneal fluid cytokine concentrations. Therefore, the timing of the collection of the fluid in relation to the menstrual cycle would be of critical importance. We found that TNF-{alpha} varied significantly with the menstrual cycle and was highest during the luteal phase. Previous studies have shown that ovarian and endometrial TNF-{alpha} levels are also increased during this period of the cycle (Cianci et al., 1996Go; Laird et al., 1996Go).

Impact of the degree of adhesions
Compared with patients with no adhesions, the altered cytokine profile found in patients with existing, well-established adhesions during surgery could either be a consequence of the presence of adhesions, or it could represent an altered environment that predisposed to the formation of adhesions in the first instance. After accounting for the effect of the menstrual cycle, we found that TNF-{alpha} concentrations varied significantly and inversely with the degree of adhesions, i.e. TNF-{alpha} concentrations were highest in mild adhesions and lowest in severe adhesions. TNF-{alpha} is a multi-functional pro-inflammatory cytokine implicated in the pathogenesis of many diseases. We have therefore speculated that it might play a dual role in the pathogenesis of adhesion formation; similar to that in brain injury where it has a potential effect in both damage and repair, depending on the timing and extent of activation of the cytokine (Shohami et al., 1999Go; Galasso et al., 2000Go). In other words, a higher concentration of TNF-{alpha} in the peritoneal fluid could be protective, and a lower concentration harmful for adhesion formation. Another possible explanation for the TNF-{alpha} concentration being elevated in the peritoneal fluid of patients with milder adhesions could be our inability to distinguish macroscopically ongoing disease processes such as indolent pelvic inflammatory disease or microscopic endometriosis. Clearly at an acute phase of these diseases, the inflammatory markers would be elevated. However, all women with adhesions were tested negative for C.Trachomatis infection in the peritoneal fluid. Lastly, elevated TNF-{alpha} concentrations in patients with milder adhesions compared with those with severe adhesions may be due to the events being recent. However, since the diagnosis of adhesions is a retrospective one, these can only be speculated explanations of the results.

IL-1 is a pro-inflammatory cytokine which can exert a paradoxical anti-inflammatory effect in chronic inflammation (Dawson et al., 1991Go). We are unable to fully explain the increased IL-1 concentration in severe well-established adhesions compared with mild well-established adhesions. It is unknown if this was a cause or an effect of the adhesions present.

Endometriosis
Previous studies have shown an altered peritoneal fluid cytokine concentration in women with endometriosis. Thus, in our study, we analysed the cytokine concentrations in women with endometriosis separately from those with adhesions alone. Our study confirmed that peritoneal fluid IL-6 concentrations were increased in women with endometriosis and more importantly, that IL-6 concentration increased with the severity of the disease. In this respect, our study findings agree with those of a number of other investigators (Mahneke et al., 2000Go; Pellicer et al., 2000Go). The fact that we showed increased IL-6 concentrations in peritoneal fluid of women with endometriosis and not in those with adhesions would suggest that the pathology of the two conditions are different and therefore future studies should not consider them as a single group.

In conclusion, we found increased TNF-{alpha} in patients with pelvic adhesions compared with those with normal pelvis; and the concentration of TNF-{alpha} is highest in mild compared with severe adhesions. In contrast, IL-1 concentration is higher in the presence of severe adhesions. We showed in our study the importance of taking into account variables such as the day of menstrual cycle when studying peritoneal fluid constituents. We also highlighted the importance of systematic methodology when researching into peritoneal fluid cytokines. Future studies on peritoneal fluid biochemistry should take into consideration these potential sources of variability/error mentioned in our study.

Although this study showed some interesting results, it did have some limitations: (i) the adhesions were pre-established rather than in the process of being formed, and thus the alteration of the cytokines was not a true reflection of the adhesion formation process. Thus, the alteration of cytokines found in this study might imply an association with adhesions, but by no means implied a causation; (ii) as with the measurement of cytokines in blood, there was a large variation in the concentrations of cytokines between different individuals; and (iii) differences from the previous investigators in the method of laboratory analysis as well as sample collection could have accounted for the discrepancies in the results obtained, e.g. the use of bioassays for TNF-{alpha} rather than immunoassays. Future studies should attempt to examine adhesions in the process of formation, although this might be a logistically challenging task to undertake in practise.


    Notes
 
4 To whom correspondence should be addressed at: The Jessop Wing, Royal Hallamshire Hospital, Tree Root Walk, Sheffield S10 2SF, UK. E-mail: yingcheong{at}hotmail.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aarons, A. and Borish, L. (1993) Cytokines and Inflamation. Immunomethods, 3, 3–12.

Adhesion Scoring Group (1994) Improvement of interobserver reproducibility of adhesion scoring system. Fertil. Steril., 62, 984–988.[ISI][Medline]

American Fertility Society (1985) Revised classification of endometriosis. Fertil. Steril., 48, 1062–1063.

Bauer, J., Ganter, U., Geiger, T. et al. (1988) Regulation of IL-6 expression in cultured human blood monocytes and monocyte-derived macrophages. Blood, 72, 1134–1140.[Abstract]

Buyalos, R.P., Funari, V.A., Azzizz, R. et al. (1992) Elevated interleukin-6 levels in peritoneal fluid of patients with pelvic pathology. Fertil. Steril., 58, 302–306.[ISI][Medline]

Chegini, N., Rong, H., Bennett, B. and Stone, I. (1999) Peritoneal fluid cytokine and eicosanoid levels and their relation to the incidence of peritoneal adhesion. J. Soc. Gynecol. Invest., 6, 153–157.[ISI][Medline]

Cheong, Y.C., Laird, S.M., Li, T.C., et al. (2001a) Peritoneal healing and adhesion formation/reformation. Hum. Reprod. Update, 7, 556–566.[Abstract/Free Full Text]

Cheong, Y.C., Tucker, D., Wong, Y.M. et al. (2001b) The changing nature of elective laparosocpic surgery: a review over a 7 year period in a reproductive surgery unit. Hum. Fertil., 4, 31–36.

Cianci, A., Calogero, A.E., Palumbo, M.A. et al. (1996) Relationship between tumour necrosis factor alpha and sex steriod concentrations in follicular fluid of women with immunological infertility. Hum. Reprod., 11, 265–268.[Abstract]

Dawson, J., Edwards, J., Sedgwick, A. and Lees, P. (1991) IL-1 alpha inhibits lymphocyte migration into a site of chronic inflammation. Immunol. Lett., 30, 319–323.[ISI][Medline]

Ellis, H., Moran, B., Thompson, J. et al. (1999) Adhesion-related hospital readmissions after abdominal and pelvic surgery: a retrospective cohort study. Lancet, 353, 1476–1480.[ISI][Medline]

Gaines Das, R. and Meager, A. (1995) Evaluation of assays using microtitre plates: results of a study of in vitro bioassays and immunoassays for tumour necrosis factor (TNF). Biologicals, 23, 285–297.[ISI][Medline]

Galasso, J., Wang, P., Martin, D. and Silverstein, F. (2000) Inhibition of TNF-alpha can attentuate or exacerbate excitotoxic injury in rat brain. Neuroreport, 11, 231–235.[ISI][Medline]

Guerra-Infante, F.M., Flores-Medina, S., Lopex-Hurtado, M. et al. (1999) Tumour necrosis factor in peritoneal fluid from asymptomatic infertile women. Arch. Med. Res., 30, 138–143.[ISI][Medline]

Halme, J. (1986) Release of tumour necrosis factor-alpha by human peritoneal macrophages in vivo and in vitro. Am J. Obstet. Gynecol., 61, 1718–1725.

Hirano, T. (1998) IL-6 and its receptor: ten years later. Int. Rev. Immuno., 16, 249–284.

Holmdahl, L. (1999) The plasmin system, a marker of the propensity to develop adhesions. In DiZerega, G. (ed.) Peritoneal Surgery, Springer-Verlag, New York, pp. 117–131.

Laird, S.M., Tuckerman, E., Saravelos, H. and Li, T.C. (1996) The production of TNF-alpha by human endometrial cells in culture. Hum. Reprod., 11, 1318–1323.[Abstract]

Lenton, E.A., Landgren, B.M. and Sexton L. (1984) Normal variation in the length of the luteal phase of the menstrual cycle: identification of the short luteal phase. Br. J. Obstet. Gynaecol., 91, 685–689.[ISI][Medline]

Lowry, S. (1993) Cytokines mediators of immunity and inflammation. Arch. Surg., 128, 1235–1241.[Abstract]

Mahneke, J.L., Dawood, Y. and Huang, J.C. (2000) Vascular endothelial growth factor and interleukin-6 in peritoneal fluid of women with endometriosis. Fertil. Steril., 73, 166–170.[ISI][Medline]

Menzies, D. (1993) Postoperative adhesions: their treatment and relevance in clinical practice. Ann. R. Coll. Surg. Engl., 72, 147–153.

Mori, H., Sawairi, M., Nakagawa, M. et al. (1991) Peritoneal fluid IL-1 beta and TNF in patients with benign gynaecological disease. Am. J. Reprod. Immun., 26, 62–67.[ISI][Medline]

Norman, R. and Brännström, M. (1996) Cytokines in the ovary: pathophysiology and potential for pharmacological intervention. Pharmacol. Ther., 69, 219–236.[ISI][Medline]

Pellicer, A., Albert, C., Garrido, N. et al. (2000) The pathophysiology of endometriosis-associated infertility: follicular and embryo quality. J. Reprod. Fertil. Steril., 55, 109–119.

Philippeaux, M. and Piguet, P. (1993) Expression of tumour necrosis factor-alpha and its mRNA in the endometrial mucosa during the menstrual cycle. Am. J. Pathol., 143, 480–486.[Abstract]

Rosenthal, R., Ling, F., Rosenthal, T. and McNeeley, S. (1984) Chronic pelvic pain: psychological features and laparoscopic findings. Psychosomatics, 25, 833–841.[ISI][Medline]

Saba, A.A., Godziachvili, V., Mavani, A.K. and Silva, Y.J. (1998) Serum levels of interleukin 1 and tumour necrosis factor alpha correlate with peritoneal adhesion grades in human after major abdominal surgery. Am. Surg., 8, 734–737.

Sharpe-Timms, K., Keisier, L.W., McIntush, E.W. and Keisuer, D.H. (1998) Tissue inhibitor of metalloproteinase-1 concentrations are attenuated in peritoneal fluid and sera of women with endometriosis and restored in sera by gonadotrophin-releasing hormone agonist therapy. Fertil. Steril., 69, 1128–1134.[ISI][Medline]

Shohami, E., Ginia, I. and Hallenbeck, J. (1999) Dual role of tumour necrosis factor in brian injury. Cytokine Growth Factor Rev., 10, 119–130.[ISI][Medline]

Stout, A., Steege, J., Dodson, W. and Hughes, C. (1991) Relationship of laparoscopic findings to self-report of pelvic pain. Am. J. Obstet. Gynecol., 46, 39–73.

Tabibzadeh, S. and Kothapalli, R. (1996) From steriod signals to local regulatory factors involved in endometrial bleeding. Eur. J. Obstet. Gynecol. Reprod. Biol., 70, 25–27.[ISI][Medline]

Terranova, P. and Rice, V. (1997) Review: cytokine involvment in ovarian processes. Am. J. Reprod. Immunol., 37, 50–63.[ISI][Medline]

Submitted on August 9, 2001; accepted on September 17, 2001.