1 Center for Advanced Research in Human Reproduction, Infertility, and Sexual Function, 2 Minimally Invasive Surgery Center, 3 Department of Obstetrics-Gynecology, 4 Urological Institute, 5 Department of Biostatistics and Epidemiology, The Cleveland Clinic Foundation, Cleveland, Ohio, USA
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Abstract |
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Key words: cytokines/endometriosis/non-surgical diagnosis/reactive oxygen species
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Introduction |
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The identification of such a non-surgical approach has led researchers to consider why endometriosis occurs. There is ample evidence to suggest that the immune system may be responsible, and in fact it is considered by some that altered immune responsiveness explains why some women develop endometriosis, whereas others do not (Ramey and Archer, 1993). Moreover, several studies have found that the immunological components in the peritoneal fluid (PF) of patients with endometriosis play an essential role in the pathogenesis and progression of the disease (Ramey and Archer, 1993
). Increased macrophage numbers and activity in the PF also have been documented (Hill et al., 1988
).
The endometriosis-associated inflammatory response, tissue repair and neovascularization are dependent on the PF macrophages and their secretory products (cytokines) (Calhaz-Jorge et al., 2000). Cytokines are diverse proteins that play a central role in regulating cell proliferation, activation, motility, adhesion, chemotaxis and morphogenesis. The cytokinesinterleukin (IL)-1, IL-2, IL-6, IL-8, IL-10 and tumour necrosis factor (TNF)-
have been implicated in the pathogenesis of endometriosis (Keenan et al., 1995
; Arici et al., 1996
; Punnonen et al., 1996
; Ho et al., 1997
; McLaren et al., 1997
; Gazvani et al., 1998
; Koninckx et al., 1998
; Küpker et al., 1998
; Zeyneloglu et al., 1998
; Garcia-Velasco and Arici, 1999
; Iwabe et al., 2000
; Shimoya et al., 2000
; Mass et al., 2001). Other components such as reactive oxygen species (ROS) have been detected in the PF of endometriosis patients (Wang et al., 1997
), though their role in the disease progression has yet to be determined. The immunological alterations associated with endometriosis are not only local, but also systemic.
Other studies (Keenan et al., 1995; Arici et al., 1996
; Punnonen et al., 1996
; Ho et al., 1997
; McLaren et al., 1997
; Wang et al., 1997
; Gazvani et al., 1998
; Zeyneloglu et al., 1998
; Garcia-Velasco and Arici, 1999
; Calhaz-Jorge et al., 2000
; Iwabe et al., 2000
; Koga et al., 2000
) have analysed the potential link between cytokines and endometriosis, but were limited in that only one or two cytokines were evaluated, either in the PF or in the serum for a given cohort of patients in an uncontrolled fashion. In addition, none of the studies analysed the inter-relationships between local and systemic cytokine production for the same group of endometriosis patients. Finally, whether these cytokines have any predictive value in terms of non-surgical diagnosis of endometriosis was not determined.
The present study was conducted in an attempt to overcome the limitations of the previous studies and to assess whether endometriosis can be diagnosed non-surgically. A non-surgical screening test would enable physicians to prescribe medical treatment without their patients having to undergo laparoscopy in order to establish a diagnosis. The objectives of this study were to: (i) evaluate a group of cytokines both in the PF and serum of endometriosis patients and in control patients without endometriosis throughout the menstrual cycle; (ii) evaluate ROS in the PF of endometriosis patients and controls; and (iii) assess whether any of these markers can, non-surgically, discriminate between patients with endometriosis and those without.
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Materials and methods |
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In patients with endometriosis, the severity of the disease was graded according to the revised four-stage American Fertility Society scoring system (American Society for Reproductive Medicine, 1996).
Preparation of serum
Venous blood was withdrawn aseptically into sterile 10 ml tubes containing 0.2 ml heparin at a concentration of 1000 IU/ml. The collected blood samples were centrifuged at 300 g for 7 min, and the clear serum was stored at 70° C until taken for analysis.
Preparation of PF
PF was aspirated from the peritoneal cavity from an abdominal port during laparoscopy. ROS levels were measured in the unprocessed sample. The cellular constituents of the PF were removed by centrifugation at 300 g for 20 min, after which the supernatants were removed and stored in aliquots at 70° C until cytokine concentrations were determined.
Measurement of ROS levels
ROS levels were measured in the PF using a Berthold luminometer (Autolumat LB 953; Wallac Inc., Gaithersburg, MD, USA). Aliquots of 400 µl of unprocessed specimens were prepared in duplicate along with a blank and a control. ROS levels were determined with a chemiluminescence assay using luminol (5-amino-2,3 dehydro-1,4 phthalazinedione; Sigma Chemical Co., St Louis, MO, USA) as the probe (Wang et al., 1997). Following the addition of 10 µl of luminol [5 mmol/l; dissolved in dimethyl sulphoxide (Sigma)] to the specimens, measurements were recorded for 15 min in the integration mode. Results were expressed as x104 counted photons per minute (c.p.m.).
Measurement of serum and PF cytokines
Concentrations of IL-1ß IL-6, IL-8 (PF only), IL-12, IL-13 and TNF- were measured in serum PF using commercially available, cytokine-specific, enzyme-linked immunosorbent assays (ELISA) (R&D Systems, Inc., Minneapolis, MN, USA). Frozen serum PF samples were thawed and then analysed. Samples from each patient group were always measured in parallel and in duplicate in order to avoid inter-assay variance. The sensitivities of the IL-1ß, IL-6, IL-8, IL-12, IL-13 and TNF-
ELISAs were 1.0, 0.7, 10.0, 5.0, 32.0 and 4.4 pg/ml respectively, with standard curve ranges of 3.9250, 3.12300, 31.22000, 7.8500, 62.54000 and 15.61000 pg/ml respectively.
Statistical analysis
The demographic variables, serum measurements and PF measurements were compared across patient groups with KruskalWallis tests. Pair-wise comparisons between groups were performed using the Wilcoxon's rank-sum test. Results of these analyses were reported using the median and interquartile range (IQR: 2575th percentile). Comparisons of percentages among the groups were performed with Fisher's Exact tests.
In order to determine which serum marker could best distinguish the patients with endometriosis from those without, a multivariate logistic regression was performed. Sensitivity and specificity were assessed with receiver operating characteristic (ROC) curves. The area under the curve was approximately the percentage correctly classified if the test was used as a diagnostic tool. The same analyses were performed with the PF measures. Positive (sensitivity/100 specificity) and negative (100 sensitivity/specificity) likelihood ratios (LRs) were also calculated. 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.
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Results |
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Serum cytokines
Serum IL-6 levels were significantly higher in the endometriosis group than in the control group (P = 0.001), and approached significance when compared with the idiopathic infertility group (P = 0.06) (Table I). There were no significant differences in the percentage of patients with insufficient serum to obtain a serum IL-6 among the three groups. Serum IL-1ß, IL-12 and IL-13 levels were similar in all three groups. Patients in the endometriosis group had higher serum TNF-
levels than the control group (P = 0.003), but had comparable levels with the idiopathic infertility group. There was no significant difference in cytokine levels between the follicular and luteal phases of the menstrual cycle. For the endometriosis group, the stage of the disease (early versus late) did not influence cytokine levels (Table I
).
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PF ROS concentrations
ROS levels in the PF of endometriosis patients were not significantly higher than those of the control group, but were significantly higher in the idiopathic infertility group compared with controls (P = 0.02). ROS levels were stable throughout the menstrual cycle in all groups. For the endometriosis group, ROS levels did not correlate with the stage of the disease (early versus late) (Table I).
Serum and PF cytokines in predicting endometriosis
Among the serum markers, significant differences were observed with IL-6 and TNF-. The endometriosis group had significantly higher serum IL-6 (P = 0.001) and TNF-
(P = 0.003) than the control group. Among the PF measures, endometriosis patients had higher TNF-
levels than both the idiopathic group (P < 0.001) and the control group (P < 0.001). In addition, the idiopathic group had significantly higher PF TNF-
levels than the control group (P = 0.02). Moreover, endometriosis patients had significantly higher PF IL-8 levels (P = 0.002) than did controls.
When the idiopathic and control patients were combined (non-endometriosis group; n = 35), patients with endometriosis had significantly higher serum IL-6 (P <0.001) and TNF- (P = 0.002), and PF TNF-
(P < 0.001) and IL-8 (P = 0.01) levels (Table II
). In multivariate analyses using all the serum measures, only IL-6 levels could be used to discriminate between patients with endometriosis and those without. None of the other serum measures improved this discrimination. The distribution of serum IL-6 levels among the patient groups is shown in Figure 1
. Among the PF measures, multivariate logistic regression analysis showed that only TNF-
could be used to discriminate between patients with endometriosis and those without. The PF levels of TNF-
in the three patient groups is shown in Figure 2
.
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Discussion |
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IL-6 is a pleiotropic cytokine that is produced by a variety of cell types, including monocytes, lymphocytes, fibroblasts, endothelial cells, keratinocytes and mesangial cells (Ray et al., 1997). The cytokine, which appears to mediate numerous physiological and pathogenic processes, acts on a wide variety of cells and regulates immune responses, acute-phase responses of the liver, haematopoiesis, neuronal functions and osteoclastogenesis (Gorospe et al., 1992
). The cytokine may also have important functions in reproductive physiology, including the regulation of ovarian steroid production, folliculogenesis and early events related to implantation (Jacobs et al., 1992
; Akoum et al., 1996
). Previous studies (Akoum et al., 1996
; Tseng et al., 1996
) have indicated that both eutopic and ectopic endometrium produce IL-6, and that this may limit the value of serum IL-6 as an independent tool for predicting the presence of endometriosis.
TNF- is secreted by activated macrophages (Halme, 1989
), and has potent inflammatory, cytotoxic and angiogenic properties (Mori et al., 1991
). It may also play a role in the progression of endometriosis and its associated infertility. Several studies have shown that TNF-
levels are increased in the PF of women with endometriosis (Eisermann et al., 1988
; Mori et al., 1991
; Taketani et al., 1992
; Overton et al., 1996
; Rana et al., 1996
). However, other studies have been unable to confirm whether differences occur in TNF-
levels in women with and without endometriosis (Vercellini et al., 1993
; Harada et al., 1997
). These apparently conflicting results may be due to: (i) differences in the techniques used to assess TNF-
(earlier studies used bioassays based on low-sensitivity kits); (ii) variations in the definition of the studied populations; and (iii) the heterogeneity of the endometriosis disease itself.
In the present study, a marked elevation in TNF- level was apparent in the PF of patients with endometriosis. The positive and negative LRs were so good that it is possible that ultrasonographically guided transvaginal aspiration of the PF from the cul-de-sac might serve as a basis for the non-surgical diagnosis of endometriosis.
ROS exert their cytotoxic effects mainly by causing peroxidation of membrane phospholipids, which in turn leads to increased membrane permeability, degraded membrane integrity, inactivated enzymes and structural damage of the DNA; cell death rapidly follows (Halliwell, 1994). ROS levels were not significantly elevated in patients with endometriosis compared with the control and idiopathic infertility groups. In addition, the levels of ROS produced by the PF varied widely, being significantly higher in idiopathic infertility patients than in controls. This difference indicated that high levels of ROS may contribute to infertility in patients with idiopathic infertility, and consequently, ROS cannot be used as a marker to predict endometriosis.
The present study had two main limitations. First, although it was not possible to obtain sufficient serum and PF to measure all cytokines (including IL-6 and TNF-) in all of the study population, the complete range of target cytokines was measured in a sufficient number of patients. Second, all bloody PF samples were excluded because cytokine levels might have been affected by blood contamination; consequently, the present findings are not applicable to patients with such PF.
In summary, serum IL-6 and PF TNF- may be good markers of endometriosis, and permit non-surgical diagnosis. However, such findings must be verified in a larger group of patients and controls before being applied within the clinical situation.
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Acknowledgements |
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Notes |
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References |
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accepted on September 28, 2001.