Concentration of soluble intercellular adhesion molecule-1 in serum samples from patients with endometriosis collected during the luteal phase of the menstrual cycle

A.-M. Steff, D. Gagné, M. Pagé, P. Hugo and D. Gosselin1

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Soluble intercellular adhesion molecule-1 (sICAM-1), released by endometriotic lesions, is involved in the regulation of cytotoxic processes. Altered levels of sICAM-1 in the circulation could parallel its deregulation in the peritoneal cavity. We therefore investigated whether sICAM-1 could represent a serum marker for endometriosis. METHODS: sICAM-1 levels were measured by enzyme-linked immunosorbent assay in serum samples from 176 subjects with surgically confirmed endometriosis (134 patients with stage I–II and 42 patients with stage III–IV) and 198 controls with no surgical evidence of the disease. All serum samples were collected during the luteal phase of the menstrual cycle. Detailed information about demographics, symptoms and clinical profile were collected. RESULTS: Mean levels of sICAM-1 appeared significantly reduced in patients with stage III–IV endometriosis in a crude comparison of means. However, when means were adjusted for potential confounders such as the pre-operative indication or fertility status, no significant difference between cases with stage III–IV disease and controls was observed. CONCLUSIONS: Serum levels of sICAM-1 during the luteal phase of the cycle are not able to discriminate women suffering from endometriosis from controls when confounders are taken into account. These results underline the importance of careful identification of confounders, based on patients’ demographic and clinical data in studies aiming at discovering diagnostic markers for endometriosis.

Key words: clinical profile/confounders/diagnostic/inflammation/serum markers


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Endometriosis is a disease characterized by the presence of endometrial tissue at ectopic sites. The prevalent hypothesis for the aetiology of this disease is the dissemination of endometrial cells in the peritoneal cavity through retrograde menstruation (Sampson, 1927Go). In women prone to developing the disease, refluxed endometrial cells, which would normally be eliminated by the immune system, implant and grow on various peritoneal surfaces, thus forming endometriotic lesions. In turn, the persistence of ectopic endometrial cells within the peritoneal cavity induces an inflammatory state. This is mainly evidenced by increased levels of inflammatory cytokines in the peritoneal cavity and infiltration of leukocytes at the site of endometriotic implants (Lebovic et al., 2001Go). For these reasons, endometriosis is also considered as an immune-related condition and efforts have been made to better understand the role of inflammatory molecules in the pathogenesis of this disease.

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, 1996Go). 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., 1995Go). 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., 1989Go). 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, 1990Go). 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., 1995Go). Alterations in levels of circulating sICAM-1 are found in several pathologies (Gearing and Newman, 1993Go), 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., 1991Go), NK cells (Oosterlynck et al., 1991Go) or macrophages (Braun et al., 1998Go). 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., 1998Go) could prevent their proper elimination by cytotoxic cells, such as NK cells (Busacca et al., 1994Go; Somigliana et al., 1996Go; Fukaya et al., 1999Go). 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., 1996Go; Vigano et al., 2000Go; Maeda et al., 2002Go; Prefumo et al., 2002Go) and elevated levels of sICAM-1 were noted in the peritoneal cavity of patients with endometriosis (Kupker et al., 1998Go; Fukaya et al., 1999Go; Calhaz-Jorge et al., 2003Go).

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 I–IV endometriosis in three studies (Wu et al., 1998Go; Daniel et al., 2000Go; Matalliotakis et al., 2001Go). In three other studies, no difference in sICAM-1 levels was found between cases and controls (De Placido et al., 1998Go; Somigliana et al., 2002Go; Hammadeh et al., 2003Go). In contrast, another study highlighted a decrease (Barrier and Sharpe-Timms, 2002Go) in serum sICAM-1 levels in women with stage III–IV 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. (2000Go) and Matalliotakis et al. (2001Go) 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, 2002Go).


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Table I. Summary of case–control studies measuring soluble intercellular adhesion molecule-1 (sICAM-1) in the serum of patients with endometriosis
 
Because the detection of altered levels of sICAM-1 in the serum of individuals could nonetheless have a clear application in the diagnostic of endometriosis, we have undertaken an extensive study in which sICAM-1 levels were compared in the serum of 374 individuals with or without endometriosis. Only serum samples collected in the luteal phase of the menstrual cycle were studied because comparison of sICAM-1 in controls and patients with endometriosis had never been, to our knowledge, strictly performed for this phase of the cycle before. Furthermore, histological dating, which allows more accurate control for potential variations in sICAM-1 related to the day of the menstrual cycle, can only be performed during the luteal phase (i.e. not in the follicular phase). The relatively large number of patients in our study allowed us to identify potential confounders, such as the indication for surgery, the presence of symptoms associated with endometriosis or the presence of leiomyomas. Mean serum sICAM-1 levels adjusted for these potential confounders were then compared between cases and controls.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study subjects
Blood samples were collected from 374 subjects who underwent laparoscopy or laparotomy for different indications including diagnostic evaluation, tubal ligation/reanastomosis, or hysterectomy/ovariectomy (including cyst removal). All subjects were of premenopausal age, not currently menstruating, had regular menstrual cycles (between 21 and 35 days), had no acute salpingitis, had not been pregnant for the last 3 months, had not been under hormonal treatment, nor using intrauterine device for the last 3 months. Only subjects who were in the luteal phase of the menstrual cycle, based on histological dating, were included in the study. A written consent, approved by Ethics Review Boards from the 10 collaborating medical institutions, was obtained from all subjects enrolled in the study. At the time of surgery, subjects were assigned to the control group or to the endometriosis group, by skilled surgeons, on the basis of the absence or the presence of endometriotic lesions respectively. In the latter case, the disease was scored as minimal/mild endometriosis (stages I–II) or moderate/severe endometriosis (stages III–IV) according to the classification of the American Society of Reproductive Medicine (1997Go). The clinical investigators also completed a questionnaire cumulating data on clinical profile, personal habits and menstrual characteristics. A computerized database was constituted with the information collected from these questionnaires. A total of 198 patients fell in the control group and 176 patients in the endometriosis group (134 patients stage I–II and 42 patients stage III–IV). The level of participation among eligible subjects was >90%.

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 (1975Go).

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 Pearson’s {chi}2-test. The effect of these parameters on sICAM-1 levels was examined either by Student’s t-test or by one-way analyses of variance. The association of sICAM-1 levels and stage of the disease was evaluated by bivariate Spearman’s 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 Student’s 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The optimal diagnostic marker for endometriosis should most importantly predict for the presence of the disease regardless of the type or the severity of the symptoms, or yet the initial indication for surgery. Therefore, it is important to include patients suffering from endometriosis who underwent surgery for several different indications (e.g. tubal ligation, hysterectomy or diagnostic laparoscopy), and then to control for potential confounders specific to the categories of patients studied. Therefore, we have (i) compared the demographic and clinical variables among the control and the case subjects, (ii) evaluated the effect of such parameters on the serum levels of sICAM-1, and (iii) compared such levels between controls and cases whilst adjusting for potential confounders.

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 15–19), mid (days 20–24) or late (days 25–28) luteal phase was similar between cases and controls.


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Table II. Percentage distribution of demographics and clinical profile by study status
 
Subjects included in the present study underwent surgery for several different indications (i.e. tubal ligation, diagnostic laparoscopy, hysterectomy). While the majority of controls underwent surgery for tubal ligation or tubal reanastomosis, cases most often underwent diagnostic laparoscopy or hysterectomy/ovariectomy. Of note, and as expected, only 5% of stage III–IV patients underwent surgery for tubal ligation or reanastomosis, a proportion that is significantly different from controls (Table II).

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, 2002Go). 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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Table III. Soluble intercellular adhesion molecule-1 (sICAM-1) serum level according to demographics and clinical profile
 
Importantly, levels of sICAM-1 were significantly different according to the indication for surgery. Indeed, women undergoing hysterectomy or diagnostic laparoscopy showed reduced levels of the molecule compared with women undergoing tubal ligation or reanastomosis. Regarding symptoms related to endometriosis, sICAM-1 concentrations appeared to be modulated in infertile patients, but not in those with pelvic pain. The presence or the absence of leiomyomas did not influence sICAM-1 levels. This indicates that sICAM-1 levels may correlate with the different disease manifestations or the presence of symptoms related to endometriosis. Based on results from Tables II and III, pre-operative indication and fertility status represent confounders in our study.

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 I–IV) 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 I–II and III–IV 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 III–IV patients (216.6 ± 51.6 ng/ml), as no significant difference was observed between controls and stage I–II 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 III–IV 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 I–II or III–IV endometriosis.


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Table IV. Proportions (%) of subjects included in the study with symptoms related to endometriosis or leiomyoma according to primary indication for surgery
 

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Table V. Comparison of sICAM-1 levels (ng/ml) between controls and cases in crude and adjusted models
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several studies have demonstrated that expression of ICAM-1 is altered in ectopic or eutopic endometrial cells of patients with endometriosis (Somigliana et al., 1996Go; Vigano et al., 1998Go; Prefumo et al., 2002Go). In addition, increased levels of the soluble form of the molecule (sICAM-1) have been found in the peritoneal fluid of patients with endometriosis (Kupker et al., 1998Go; Fukaya et al., 1999Go; Calhaz-Jorge et al., 2003Go). If such local alterations of sICAM-1 levels in endometriosis were reflected at the systemic level, this molecule could certainly represent an interesting diagnostic marker for the disease. Some previous studies had documented the levels of sICAM-1 in the serum of patients with endometriosis (summarized in Table I) with discordant conclusions. We therefore undertook a comparison of sICAM-1 levels in 374 serum samples from patients with endometriosis and controls collected during the luteal phase of the cycle. None of the previous reports had selected patients exclusively in the luteal phase; therefore our results are peculiar to this phase of the cycle. In addition, because we had a much larger number of patients than in previous studies, we were able to categorize our data and to adjust means according to potential confounders.

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., 1998Go; Somigliana et al., 2002Go; Hammadeh et al., 2003Go). However, in other studies, either an increase (Wu et al., 1998; Matalliotakis et al., 2001Go) or a decrease (Barrier and Sharpe-Timms, 2002Go) 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., 1990Go; Balasch et al., 1996Go; Walter et al., 2001Go). 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., 1990Go; Redwine and Yocom 1990Go; Balasch et al., 1996Go). 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., 1994Go; Gruppo Italiano per lo studio dell’endometriosi, 1994Go). 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, 2002Go), 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.


    Acknowledgements
 
We would like to thank Julie Poliquin-Fleury and the team of technicians for their assistance; we are also grateful to the clinicians for their collaboration in sample and data collection. This study would not have been possible without the generous contribution of all participating women. This work was sponsored by the private company Procrea/Metriogene and at the time the research work reported in this paper was carried out, all the authors were employees of the company.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ajossa S, Mais V, Guerriero S, Paoletti AM, Caffiero A, Murgia C and Melis GB (1994) The prevalence of endometriosis in premenopausal women undergoing gynecological surgery. Clin Exp Obstet Gynecol 21,195–197.[Medline]

American Society for Reproductive Medicine (1997) Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil Steril 67,817–821.[CrossRef][ISI][Medline]

Balasch J, Creus M, Fabregues F, Carmona F, Ordi J, Martinez-Roman S and Vanrell JA (1996) Visible and non-visible endometriosis at laparoscopy in fertile and infertile women and in patients with chronic pelvic pain: a prospective study. Hum Reprod 11,387–391.[Abstract]

Barrier BF and Sharpe-Timms KL (2002) Expression of soluble adhesion molecules in sera of women with stage III and IV endometriosis. J Soc Gynecol Invest 9,98–101.[CrossRef][ISI][Medline]

Bonello N and Norman RJ (2002) Soluble adhesion molecules in serum throughout the menstrual cycle. Hum Reprod 17,2272–2278.[Abstract/Free Full Text]

Braun DP, Gebel H, Rana N and Dmowski WP (1998) Cytolysis of eutopic and ectopic endometrial cells by peripheral blood monocytes and peritoneal macrophages in women with endometriosis. Fertil Steril 69,1103–1108.[CrossRef][ISI][Medline]

Busacca M, Vigano P, Magri B and Vignali M (1994) The adhesion molecules on human endometrial stromal cells. Immunological implications. Ann NY Acad Sci 734,43–46.[ISI][Medline]

Calhaz-Jorge C, Costa AP, Santos MC and Palma-Carlos ML (2003) Soluble intercellular adhesion molecule 1 in the peritoneal fluid of patients with endometriosis correlates with the extension of peritoneal implants. Eur J Obstet Gynecol Reprod Biol 106,170–174.[CrossRef][ISI][Medline]

Daniel Y, Geva E, Amit A, Eshed-Englender T, Baram A, Fait G and Lessing JB (2000) Do soluble cell adhesion molecules play a role in endometriosis? Am J Reprod Immunol 43,160–166.[CrossRef][ISI][Medline]

De Placido G, Alviggi C, Di Palma G, Carravetta C, Matarese G, Landino G and Racioppi L (1998) Serum concentrations of soluble human leukocyte class I antigens and of the soluble intercellular adhesion molecule-1 in endometriosis: relationship with stage and non-pigmented peritoneal lesions. Hum Reprod 13,3206–3210.[Abstract]

Fukaya T, Sugawara J, Yoshida H, Murakami T and Yajima A (1999) Intercellular adhesion molecule-1 and hepatocyte growth factor in human endometriosis: original investigation and a review of literature. Gynecol Obstet Invest 47 (Suppl. 1),11–16.[CrossRef][ISI][Medline]

Gearing AJ and Newman W (1993) Circulating adhesion molecules in disease. Immunol Today 14,506–512.[CrossRef][ISI][Medline]

Gruppo Italiano per lo studio dell’endometriosi (1994) Prevalence and anatomical distribution of endometriosis in women with selected gynaecological conditions: results from a multicentric Italian study. Hum Reprod 9,1158–1162.[Abstract]

Hammadeh ME, Fishcer-Hammedeh C, Hoffmeister H, Huebner U, Georg T, Rosenbaum P and Schmidt W (2003) Fibroblast growth factor (FGF), intracellular adhesion molecule (sICAM-1) level in serum and follicular fluid of infertile women with polycystic ovarian syndrome, endometriosis and tubal damage, and their effect on ICSI outcome. Am J Reprod Immunol 50,124–130.[CrossRef][ISI][Medline]

Kupker W, Schultze-Mosgau A and Diedrich K (1998) Paracrine changes in the peritoneal environment of women with endometriosis. Hum Reprod Update 4,719–723.[Abstract/Free Full Text]

Languino LR, Duperray A, Joganic, KJ, Fornaro M, Thornton GB and Altieri DC (1995) Regulation of leukocyte–endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-fibrinogen recognition. Proc Natl Acad Sci USA 92,1505–1509.[Abstract]

Lebovic DI, Mueller MD and Taylor RN (2001) Immunobiology of endometriosis. Fertil Steril 75,1–10.[CrossRef][ISI][Medline]

Maeda N, Izumiya C, Oguri H, Kusume T, Yamamoto Y and Fukaya T (2002) Aberrant expression of intercellular adhesion molecule-1 and killer inhibitory receptors induces immune tolerance in women with pelvic endometriosis. Fertil Steril 77,679–683.[CrossRef][ISI][Medline]

Mahutte NG and Arici A (2002) New advances in the understanding of endometriosis related infertility. J Reprod Immunol 55,73–83.[CrossRef][ISI][Medline]

Makgoba MW, Sanders ME and Shaw S (1989) The CD2-LFA-3 and LFA-1-ICAM pathways: relevance to T-cell recognition. Immunol Today 10,417–422.[CrossRef][ISI][Medline]

Matalliotakis IM, Vassiliadis S, Goumenou AG, Athanassakis I, Koumantakis, GE, Neonaki MA and Koumantakis EE (2001) Soluble ICAM-1 levels in the serum of endometriotic patients appear to be independent of medical treatment. J Reprod Immunol 51,9–19.[CrossRef][ISI][Medline]

Meyer DM, Dustin ML and Carron CP (1995) Characterization of intercellular adhesion molecule-1 ectodomain (sICAM-1) as an inhibitor of lymphocyte function-associated molecule-1 interaction with ICAM-1. J Immunol 155,3578–3584.[Abstract]

Nisolle M, Paindaveine B, Bourdon A, Berliere M, Casanas-Roux F and Donnez J (1990) Histologic study of peritoneal endometriosis in infertile women. Fertil Steril 53,984–988.[ISI][Medline]

Norris DA (1990) Cytokine modulation of adhesion molecules in the regulation of immunologic cytotoxicity of epidermal targets. J Invest Dermatol 95,111S–120S.[Abstract]

Noyes RW, Hertig AT and Rock J (1975) Dating the endometrial biopsy. Am J Obstet Gynecol 122,262–263.[Medline]

Oosterlynck DJ, Cornillie FJ, Waer M, Vandeputte M and Koninckx PR (1991) Women with endometriosis show a defect in natural killer activity resulting in a decreased cytotoxicity to autologous endometrium. Fertil Steril 56,45–51.[ISI][Medline]

Prefumo F, Semino C, Melioli G and Venturini PL (2002) A defective expression of ICAM-1 (CD54) on secretory endometrial cells is associated with endometriosis. Immunol Lett 80,49–53.[CrossRef][ISI][Medline]

Redwine DB and Yocom LB (1990) A serial section study of visually normal pelvic peritoneum in patients with endometriosis. Fertil Steril 54,648–651.[ISI][Medline]

Sampson J (1927) Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol 14,422–469.

Somigliana E, Vigano P, Gaffuri B, Guarneri D, Busacca M and Vignali M (1996) Human endometrial stromal cells as a source of soluble intercellular adhesion molecule (ICAM)-1 molecules. Hum Reprod 11,1190–1194.[Abstract]

Somigliana E, Vigano P, Candiani M, Felicetta I, Di Blasio AM and Vignali M (2002) Use of serum-soluble intercellular adhesion molecule-1 as a new marker of endometriosis. Fertil Steril 77,1028–1031.[CrossRef][ISI][Medline]

Van de Stolpe A and van der Saag PT (1996) Intercellular adhesion molecule-1. J Mol Med 74,13–33.[ISI][Medline]

Vigano P, Vercellini P, Di Blasio AM, Colombo A, Candiani GB and Vignali M (1991) Deficient antiendometrium lymphocyte-mediated cytotoxicity in patients with endometriosis. Fertil Steril 56,894–899.[ISI][Medline]

Vigano P, Gaffuri B, Somigliana E, Busacca M, Di Blasio AM and Vignali M (1998) Expression of intercellular adhesion molecule (ICAM)-1 mRNA and protein is enhanced in endometriosis versus endometrial stromal cells in culture. Mol Hum Reprod 4,1150–1156.[Abstract]

Vigano P, Somigliana E, Gaffuri B, Santorsola R, Busacca M and Vignali M (2000) Endometrial release of soluble intercellular adhesion molecule 1 and endometriosis: relationship to the extent of the disease. Obstet Gynecol 95,115–118.[Abstract/Free Full Text]

Walter AJ, Hentz JG, Magtibay PM, Cornella JL and Magrina JF (2001) Endometriosis: correlation between histologic and visual findings at laparoscopy. Am J Obstet Gynecol 184,1407–1411.[CrossRef][ISI][Medline]

Wu MH, Yang BC, Hsu CC, Lee YC and Huang KE (1998) The expression of soluble intercellular adhesion molecule-1 in endometriosis. Fertil Steril 70,1139–1142.[CrossRef][ISI][Medline]

Submitted on June 18, 2003; resubmitted on August 4, 2003; accepted on September 16, 2003.