MetrioGene BioSciences, Inc. (a subsidiary of Procrea BioSciences, Inc.), 6100 Royalmount Avenue, Montreal (Quebec) H4P 2R2, Canada
1 To whom correspondence should be addressed. e-mail: dgosselin{at}metriogene.com
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Abstract |
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Key words: clinical profile/confounders/diagnostic/inflammation/serum markers
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Introduction |
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Intercellular adhesion molecule-1 (ICAM-1 or CD54) is a member of the immunoglobulin superfamily, constitutively expressed at the surface of numerous cells, including endometrial cells, endothelial cells and leukocytes, and its expression is modulated by cytokines (van de Stolpe and van der Saag, 1996). ICAM-1 plays different roles, all related to immune processes, depending on the cell type where it is expressed. For instance, ICAM-1 on endothelial cells triggers the migration of leukocytes to sites of inflammation (Languino et al., 1995
). On the other hand, the binding of ICAM-1, expressed on antigen-presenting cells, to one of its counter receptors, lymphocyte function-associated antigen-1 (LFA-1 or CD11b/18) expressed on leukocytes, is an important co-activation process during antigen recognition by T cells (Makgoba et al., 1989
). In addition, the interaction between LFA-1 and ICAM-1 plays a major role in many cytotoxic mechanisms, including the lysis of target cells by natural killer (NK) cells (Norris, 1990
). Interestingly, a soluble form of ICAM-1 (sICAM-1) is generated by shedding of the extracellular portion of the molecule and the shed form is thought, during inflammation, to compete with ICAM-1 for the binding to LFA-1, thus playing an inhibitory role on ICAM-1 functions (Meyer et al., 1995
). Alterations in levels of circulating sICAM-1 are found in several pathologies (Gearing and Newman, 1993
), and in particular in inflammatory and autoimmune diseases including acute infections, lupus erythematosus and active multiple sclerosis (van de Stolpe and van der Saag, 1996, and references therein).
It has been suggested that persistence of endometrial cells at ectopic sites could be linked to a lack in their proper elimination by cytotoxic mechanisms mediated either by lymphocytes (Vigano et al., 1991), NK cells (Oosterlynck et al., 1991
) or macrophages (Braun et al., 1998
). Due to the pivotal role of ICAM-1 in regulating cytotoxic processes, it has been postulated that altered expression of ICAM-1 by ectopic endometrial cells (Vigano et al., 1998
) could prevent their proper elimination by cytotoxic cells, such as NK cells (Busacca et al., 1994
; Somigliana et al., 1996
; Fukaya et al., 1999
). An association between levels of sICAM-1 produced by eutopic endometrial cells in culture and stage of the disease has been demonstrated (Somigliana et al., 1996
; Vigano et al., 2000
; Maeda et al., 2002
; Prefumo et al., 2002
) and elevated levels of sICAM-1 were noted in the peritoneal cavity of patients with endometriosis (Kupker et al., 1998
; Fukaya et al., 1999
; Calhaz-Jorge et al., 2003
).
It is possible that local deregulation of sICAM-1 levels is reflected by altered levels of the molecule in the serum of women suffering from endometriosis. Several investigators compared serum levels of sICAM-1 in women with the disease and controls, but these studies led to different conclusions (Table I). An increase in sICAM-1 levels was observed for patients with stage IIV endometriosis in three studies (Wu et al., 1998; Daniel et al., 2000
; Matalliotakis et al., 2001
). In three other studies, no difference in sICAM-1 levels was found between cases and controls (De Placido et al., 1998
; Somigliana et al., 2002
; Hammadeh et al., 2003
). In contrast, another study highlighted a decrease (Barrier and Sharpe-Timms, 2002
) in serum sICAM-1 levels in women with stage IIIIV endometriosis. Such apparent inconsistencies could be explained by the fact that these studies have been undertaken in different clinical set-ups, where cases and controls could present with very different symptoms or clinical profiles, thus introducing different variables that could influence sICAM-1 levels. Moreover, the studies of Daniel et al. (2000
) and Matalliotakis et al. (2001
) have mainly focused on the follicular phase of the cycle, whereas the other studies did not take the phase of the cycle into consideration, despite the fact that it is known that local and serum sICAM-1 concentrations vary during the menstrual cycle (Bonello and Norman, 2002
).
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Materials and methods |
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Blood collection and serum preparation
Blood samples were collected before anaesthesia into 10 ml sterile tubes containing no additives. Subjects abstained from food for 8 h. Samples were kept at room temperature for
5 h, until centrifugation at 150 g for 10 min was performed. Serum aliquots were frozen at 80°C until measurements were done.
Measurement of sICAM-1 serum levels
A commercially available enzyme-linked immunosorbent assay kit was used for the determination of sICAM-1 (Quantikine®; R&D Systems, USA). Serum samples (diluted 1/20) and standards were incubated in microplates coated with antibodies against sICAM-1. A second set of peroxidase-linked antibodies directed against sICAM-1 was then added to the wells. Tetramethylbenzidine substrate was added after a series of washings to remove unbound antibodies. Absorbance was measured at 450 nm, with a correction at 650 nm, using a Vmax microplate reader and Soft MAX Pro software (Molecular Devices, USA). All samples were run in duplicate and a standard curve was established for each assay. The minimal detectable dose of sICAM-1 is typically <0.35 ng/ml. Intra- and inter-assay coefficients of variation were <10%.
Histological dating
Endometrial tissue specimens were collected under anaesthesia with a suction curette. Phase of the menstrual cycle and dating were determined by histopathology from a section of formalin-fixed endometrial tissue according to the method of Noyes (1975).
Statistical analysis
Statistical analysis was performed using the SPSS software (SPSS Inc., USA). Comparisons of cases and controls with respect to demographics and clinical profile were done with Pearsons 2-test. The effect of these parameters on sICAM-1 levels was examined either by Students t-test or by one-way analyses of variance. The association of sICAM-1 levels and stage of the disease was evaluated by bivariate Spearmans correlation in a two-tailed significance test. Any variables that were significantly modulated between cases and controls and/or that were shown to correlate significantly with the serum levels of sICAM-1 were considered as potential confounders in the present study. Mean serum levels of sICAM-1 were compared between cases and controls with Students t-test for independent samples. Mean sICAM-1 serum levels were also adjusted for potential confounders using a univariate general linear model and were compared between cases and controls. P < 0.05 was considered statistically different.
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Results |
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Comparison of demographic variables and clinical data among cases and controls
The great majority (i.e. 97%) of the subjects enrolled in this study were Caucasians. No significant difference was observed between cases and controls regarding age and body mass index (data not shown). In order to minimize cycle-specific variations in serum sICAM-1 levels, all samples were collected during the luteal phase of the menstrual cycle because histological confirmation of the dating is possible only during this period. Table II shows that the proportion of subjects in early (days 1519), mid (days 2024) or late (days 2528) luteal phase was similar between cases and controls.
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Also as expected, the proportion of patients experiencing symptoms related to endometriosis, such as pelvic pain or infertility, was significantly higher for cases compared with controls (Table II). In addition, as we previously observed that the proportion of subjects with leiomyoma is significantly increased in patients with endometriosis (R.Hemmings, M.Rivard, D.H.Olive, J.Poliquin-Fleury, D.Gagné, P.Hugo and D.Gosselin, submitted), we verified whether this was also the case in the present cohort. Indeed, a significantly higher proportion of women had leiomyoma among patients with endometriosis compared with controls (Table II).
Influence of demographic and clinical characteristics on sICAM-1 serum levels
We next verified whether sICAM-1 levels varied according to demographic or clinical factors (Table III). Age and body mass index were not associated with variations in sICAM-1 levels. Serum levels of sICAM-1 have been shown to differ according to the stage of the luteal phase of the menstrual cycle (Bonello and Norman, 2002). Although all serum samples included in this study were collected in the luteal phase of the cycle, we verified whether differences in sICAM-1 levels could be found between early, mid or late luteal phase of the menstrual cycle. This did not appear to be the case in our cohort because no significant variations in sICAM-1 levels were found across the luteal phase (Table III). These results together with those from Table II thus demonstrate that stage of the luteal phase does not represent a potential confounder in our study.
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Comparison of sICAM-1 concentration in the serum of cases and controls
Mean serum sICAM-1 levels were compared between 176 patients with minimal to severe (stage IIV) endometriosis and 198 controls. A significant reduction in sICAM-1 levels was observed in patients with endometriosis compared with controls (228.2 ± 55.5 ng/ml for cases compared with 241.7 ± 66.6 ng/ml for controls). In fact, when sICAM-1 levels were evaluated separately in stage III and IIIIV patients, it became apparent that the overall difference observed between cases and controls was mainly due to a pronounced decrease in sICAM-1 levels in stage IIIIV patients (216.6 ± 51.6 ng/ml), as no significant difference was observed between controls and stage III patients (231.8 ± 56.4 ng/ml).
Variations in sICAM-1 levels associated with the pre-operative indication (Table III) could correlate with different disease manifestations. For instance, the proportion of subjects included in the study exhibiting symptoms of endometriosis, such as pelvic pain or infertility, or associated pathologies such as leiomyoma, was significantly different between the three categories of pre-operative indications (Table IV). Together with the indication for surgery, symptoms and the presence of leiomyoma may thus represent potential confounders in our study. Therefore, in order to have a more accurate estimation of sICAM-1 levels, which would take into account the overall disease manifestations, we compared levels of sICAM-1 between cases and controls after adjustment for either one or all of the following variables: pre-operative indication, pelvic pain, infertility and presence of leiomyoma (Table V). A significant difference could still be observed between stage IIIIV patients and controls when means were adjusted for either pelvic pain or the presence of leiomyoma. This is in accordance with results from Table III that show that these variables do not correlate with sICAM-1 levels. However, adjustment for either pre-operative indication or infertility obliterated all significant differences between cases and controls. This was also the case when the pre-operative indication and the presence of pelvic pain, infertility and leiomyoma were controlled simultaneously. In conclusion, when carefully controlling for confounders, no difference in sICAM-1 levels between controls and patients with endometriosis can be observed, including those suffering from either stage III or IIIIV endometriosis.
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Discussion |
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In an overall comparison between cases and controls, following adjustment for possible confounding factors, we observed no significant difference in sICAM-1 levels between cases and controls. These results are in accordance with three studies reporting no changes in sICAM-1 levels between cases and controls (Table I) (De Placido et al., 1998; Somigliana et al., 2002
; Hammadeh et al., 2003
). However, in other studies, either an increase (Wu et al., 1998; Matalliotakis et al., 2001
) or a decrease (Barrier and Sharpe-Timms, 2002
) in sICAM-1 serum levels was reported in patients with endometriosis compared with controls. Differences in study design, criteria for the selection of subjects, phase of the menstrual cycle or, in some cases, low numbers of samples tested, might explain these apparent discrepancies. Furthermore, some studies have used histology to confirm the diagnosis of endometriosis, while others, including ours, have relied on visual examination. One must take into consideration that, although histological confirmation may appear appealing, it is also subjected to interpretation. In fact, confusing and ambiguous results have been reported in the literature, and the rate of histological confirmation of peritoneal endometriosis is extremely variable, ranging from 3 to 100% of cases depending on the study (Nisolle et al., 1990
; Balasch et al., 1996
; Walter et al., 2001
). Even more puzzling is the fact that histological evidences of endometriosis have been found in normal peritoneum with no evidence of disease (Nisolle et al., 1990
; Redwine and Yocom 1990
; Balasch et al., 1996
). In addition, to our knowledge, no study has yet examined the degree of inter-observer reproducibility of histological findings.
In our study, subjects were scheduled for laparoscopy or laparotomy for different indications, and our results clearly show that disease manifestations among subjects are very different according to the pre-operative indication. This is in accordance with previous studies showing that prevalence of endometriosis is higher in patients undergoing diagnostic laparoscopy versus tubal ligation or hysterectomy (Ajossa et al., 1994; Gruppo Italiano per lo studio dellendometriosi, 1994
). Patients undergoing tubal ligation/reanastomosis, diagnostic laparoscopy or hysterectomy may thus represent different subgroups in terms of disease manifestations, symptoms or associated pathologies, such as leiomyoma. It is therefore crucial to take the pre-operative indication into account when comparing sICAM-1 levels between cases and controls because this variable represents a major confounder.
Another confounder identified in our study is infertility, because fertility status is associated with different serum sICAM-1 levels. Furthermore, when controlling for this parameter, the difference in sICAM-1 levels between cases and controls is no longer significant. The reason why infertility could be associated with variations in levels of sICAM-1 is not known. The mechanisms leading to infertility in a high proportion of patients with endometriosis are not well understood, but it has been hypothesized that infertility in these patients is linked to an abnormal immune response (Mahutte and Arici, 2002), that could lead to sICAM-1 deregulation.
Endometriosis is a heterogeneous and complex disease, with significant environmental and genetic influences. It is also a disease with a wide variety of symptoms, appearances or even definitions. This study emphasizes the need to gather detailed information about patients clinical profiles in order to identify potential confounders. Only this approach allowed us to demonstrate that no significant difference in serum sICAM-1 levels could be observed between cases and controls. Concerning this cytokine, the fact that clinical data has not necessarily been taken into account or was not consistent among studies may explain why discrepant results were obtained among different teams. Because serum concentrations of cytokines can also be altered in a number of inflammatory states or pathological conditions, we believe that the discovery of blood markers of interest in the diagnosis of endometriosis will be possible only if comparative studies are carried out on large numbers of individuals, with well-characterized clinical profiles, allowing the adjustment for potential confounders.
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Acknowledgements |
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References |
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Submitted on June 18, 2003; resubmitted on August 4, 2003; accepted on September 16, 2003.