Analysis of cyclooxygenase-2 (COX-2) expression in different sites of endometriosis and correlation with clinico-pathological parameters

Anna Fagotti1, Gabriella Ferrandina2, Francesco Fanfani1, Francesco Legge2, Libero Lauriola3, Marco Gessi3, Paola Castelli4, Fabrizio Barbieri5, Luca Minelli5 and Giovanni Scambia1,2,6

1 Department of Gynecological Oncology, Catholic University of the Sacred Heart, Campobasso, 2 Department of Obstetrics and Gynecology, and 3 Department of Pathology, Catholic University of the Sacred Heart, Rome and 4 Department of Pathology and 5 Department of Obstetrics and Gynecology, Sacred Heart Hospital, Negrar (VR), Italy

6 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Catholic University, L.go A.Gemelli, 8, 00168 Rome, Italy. e-mail: giovanni.scambia{at}libero.it


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Recent studies have demonstrated the overexpression of cyclooxygenase-2 (COX-2) in endometriosis. The aim of this study was to investigate the expression of COX-2 in different anatomical sites of endometriosis and its association with clinico-pathological parameters in a single institutional series of patients undergoing operative treatment. METHODS: COX-2 expression was analysed by immunohistochemistry in 136 samples of endometriotic tissue from 103 patients affected by endometriosis. RESULTS: COX-2 immunoreaction was observed in 78.5% of ovarian endometriotic cysts, in 11.1% of peritoneal implants and 13.3% of recto-vaginal nodules. COX-2 positivity was not distributed differently according to age, pre-operative serum levels of CA125 and AFS score. Moreover, COX-2 positivity did not show any significant variation according to the subjective intensity of pain, as dysmenorrhoea, chronic pelvic pain, lower urinary tract or gastrointestinal symptoms, or according to infertility. CONCLUSIONS: Increased COX-2 expression in the endometriotic ovarian cyst wall was observed with respect to other extraovarian localizations. No relevant correlations between COX-2 positivity and clinico-pathological characteristics and symptoms of patients were observed.

Key words: clinico-pathological characteristics/COX-2/endometriosis


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Endometriosis consists of the presence of endometrial tissue at sites outside the uterus. This condition is associated with the local inflammatory response induced by cytokines and growth factors (Khorram et al., 1993Go; Ryan et al., 1995Go), which can maintain a high stromal vascularization and recruit and activate peritoneal macrophages (Smith, 1995Go). It has been reported that ectopic endometrium can produce prostaglandins, contributing to the pathophysiology of endometriosis (Darke et al., 1981Go; Vermon et al., 1986Go). In fact, higher prostaglandin concentrations have been found in peritoneal fluid of patients with endometriosis or adenomyosis than in controls (Darke et al., 1981Go; Badawy et al., 1985Go).

Cyclooxygenase- 2 (COX-2), the key enzyme in the conversion of arachidonic acid to prostaglandins, is induced by growth factors, oncogenes and tumour promoters, and has been mainly associated with the inflammatory response (Williams and DuBois, 1996Go). COX-2 expression has been demonstrated in glandular and vascular endothelia of normal human endometrium (Koch et al., 1992Go; Jones et al., 1997Go), which produces a significant amount of prostaglandins (Lumsden et al., 1984Go; Smith and Kelly, 1988Go). COX-2 overexpression has been associated with inhibition of apoptosis and host immune responses, and induction of neoangiogenesis (Tsujii and DuBois, 1995Go; Tsujii et al., 1998Go; Stolina et al., 2000Go).

The immunohistochemical expression of COX-2 in eutopic and ectopic endometrium of patients affected by endometriosis and/or adenomyosis has been described recently, and significantly higher COX-2 levels in endometriomas with respect to other localizations have been reported (Ota et al., 2001Go). These results have been confirmed on COX-2 mRNA levels by RT–PCR analysis (Chishima et al., 2002Go).

To our knowledge, no data have been reported to date regarding the possible clinical significance of COX-2 in endometriosis. The aim of this study therefore has been to investigate by immunohistochemistry the expression of COX-2 and its association with clinico-pathological parameters in a single institutional series of patients undergoing operative laparoscopy for endometriosis. Moreover, a comparison between different localizations of the disease has been performed.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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Study population
All patients undergoing operative laparoscopy for endometriosis at the Department of Obstetrics and Gynecology of the ‘Sacro Cuore Don Calabria’ Hospital in Negrar (Verona, Italy) between November 1997 and November 2002 were evaluated consecutively for participation in the study. The slides were all reviewed by a dedicated pathologist and only those cases with evident endometrial epithelium and stromal component in the surgical specimens were included in the study. As a result, we retrieved the clinical records for 103 women.

Institutional review board approval was not requested because this was a retrospective study.

Pelvic pain symptoms, including complaints about the lower urinary and gastrointestinal (GI) tracts, were collected from the medical records. Patients were asked to complete a simple office-based questionnaire before surgery, which included a 10-point ranked ordinal scale asking them to rate the severity of various symptoms thought to be associated with endometriosis (from 0 = none to 10 = debilitating). This information was tabulated in the database prospectively. All women gave informed consent for their data to be used for scientific purposes. Severe dysmenorrhoea was defined as either a pain score >5 or, when the woman had not completed the scale, as a ‘severe’ rating by the woman. Deep dyspareunia was defined as regular deep dyspareunia, and chronic pelvic pain as intermittent or permanent pelvic pain not related to the menstrual cycle. We defined lower urinary tract symptoms as one or more of the following symptoms, either chronic or during menstruation: haematuria, non-microbial cystitis, recurrent urinary tract infections, pain on urinating, pollakuria and dysuria. Similarly, GI symptoms were defined as one or more of the following, either chronic or during menstruation: diarrhoea, constipation, rectal bleeding, proctitis, tenesmus and colic rectal pain. When a symptom was not specifically mentioned, it was considered to be absent.

The disease stage was scored according to the revised American Fertility Society (1985Go). Age, height, weight, body mass index (BMI) (kg/m2), infertility (failure to conceive for 1 year) and pre-operative CA125 serum levels were also recorded.

A pelvic examination and pre-operative pelvic and upper urinary tract ultrasound evaluation were routinely performed, whereas double contrast barium enema and cystoscopy were only requested when indicated by specific symptoms.

Immunohistochemistry
Endometriosis specimens were obtained from endometriomas (79 cases), peritoneal implants (27 cases) and recto-vaginal (RV) septum nodules (30 cases). Each patient was submitted to more than one biopsy on all suspicious sites of endometriosis. As expected, not all the specimens were successful in identifying endometriosis. However, only cases where endometrial epithelium and stroma could be clearly identified were considered for the study. Immunohistochemistry was performed as previously described (Ferrandina et al., 2002aGo,b). Tissues were fixed in formalin and paraffin embedded according to standard procedures. A 4 µm representative section from each case was deparaffinized in xylene, rehydrated, treated with 0.3% H2O2 in methanol for 10 min to block endogenous peroxidase activity, and subjected to heat-induced epitope retrieval in a microwave oven using the Dako ChemMate detection kit (DAKO, Glostrup, Denmark) according to the manufacturer’s instructions. Slides from all cases studied were then simultaneously processed for immunohistochemistry on the TechMate Horizon automated staining system (DAKO) using the Vectastain ABC peroxidase kit (Vector Laboratories, Burlingame, CA). Endogenous biotin was saturated by a biotin blocking kit (Vector Laboratories). Sections were incubated with normal rabbit serum for 15 min, then with rabbit polyclonal antiserum against COX-2 (Cayman, Ann Arbor, MI) diluted 1:300, for 1 h. Negative controls were performed using non-immunized rabbit serum or omitting the primary antiserum.

COX-2 expression was evaluated only in endometrial epithelium. Light microscopy analysis of all tissue sections was done by two different pathologists (L.L. and M.G.) without prior knowledge of the patient’s clinical parameters. Intensity of staining was evaluated subjectively in a range from 0 (none) through 1 (faint) to 2 (strong) and 3 (very strong). Cases in which the intensity of staining was scored ≥2 were considered positive.

Statistical analysis
Fisher’s exact test or the {chi}2 test were used to analyse the distribution of COX-2-positive cases according to several clinico-pathological features. Statistical analysis was done for each symptom separately, using a cut-off value which corresponded to the subjective score evaluation of ≥5.


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 Materials and methods
 Results
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COX-2 immunostaining
COX-2 immunostaining was observed in both nuclei and cytoplasm of endometriotic cells. Figure 1 shows a representative example of an ovarian endometriotic cyst (Figure 1A) and a RV septum nodule (Figure 1B) with intense and homogeneous COX-2 immunostaining.



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Figure 1. COX-2 immunostaining in ovarian endometriotic cyst (A) and in recto-vaginal septum endometriosis (B): an immunoreaction is located in both nuclei and cytoplasm of the epithelial layer and subjacent stromal layer. Scale bars: (A) = 75 µm; (B) = 100 µm.

 
The overall COX-2 positivity rate was 50.7%. COX-2 staining was scored as positive in 62 out of 79 specimens (78.5%) from ovarian endometriotic cysts, whereas the per centage of COX-2 positivity was 11.1% (three out of 27 cases) and 13.3% (four out of 30 cases) in peritoneal implants and RV nodules, respectively. Disagreement between the pathologists was observed in 15 of 136 COX-2 immunoreactions (11%); these cases were submitted to a second joint evaluation to obtain a consistent score.

In 14 cases, COX-2 immunostaining was performed in both peritoneal implants and RV septum nodules for the same patient, and we found that the COX-2 score was superimposable in all but two cases (percentage of agreement 85.7%).

On the basis of COX-2 positivity rates, cases were divided into two groups: group 1 (endometriomas) and group 2 (peritoneal implants and RV septum nodules). The analysis of the correlations between clinico-pathological parameters and COX-2 status followed this dichotomization.

Correlation with clinico-pathological parameters
Patient characteristics are shown in Table I. Mean age ± SD was 31.4 ± 6.03 years, while mean CA125 levels ± SD were 74.3 ± 71.2 IU/ml and mean AFS score ± SD (American Fertility Society, 1995Go) was 44.1 ± 71.2. Ninety patients (87.4%) were scored as stage III–IV according to the AFS system. Endometriosis was completely removed, leaving no residual disease in all except 10 patients (9.7%) with RV septum and/or bowel nodules, who pre-operatively decided not to accept the risk of uretheral and/or bowel lesions. Table II shows the distribution of COX-2 positivity in endometriotic ovarian cysts (group 1) according to clinico-pathological characteristics of the patients. COX-2 positivity was not distributed differently according to age, pre-operative CA125 serum levels and AFS score. Moreover, COX-2 positivity did not show any significant variation according to the subjective intensity of pain, as dysmenorrhoea, chronic pelvic pain, lower urinary tract and GI symptoms, or according to infertility. The analysis of COX-2 status in peritoneal implants and RV septum nodules (group 2) did not show any difference in any of the parameters considered (Table III).


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Table I. Women’s characteristics (n = 103)
 

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Table II. COX-2 immunohistochemical status in group 1 (endometriomas) according to clinico-pathological characteristics of the patients
 

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Table III. COX-2 immunohistochemical status in group 2 (peritoneal implants and recto-vaginal septum nodules) according to clinico-pathological characteristics of the patients
 
Similar results were obtained using different cut-off values.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study analyses COX-2 immunohistochemical status in 136 tissue samples from different endometriotic sites, categorized as ovarian endometriosis, peritoneal endometriosis and endometriosis of the RV septum (Nisolle and Donnez, 1997Go).

COX-2 positivity rates were 78.5% in the ovarian cyst wall and 11.1 and 13.3% in peritoneal implants and RV nodules, respectively. In the cases in which samples from both peritoneal implants and RV septum nodules were available, the percentage agreement of COX-2 status was ~85.7%.

These results seem to be consistent with the few data in the literature reporting the overexpression of COX-2 in endometriomas and its lower levels in adenomyosis and peritoneal endometriosis (Ota et al., 2001Go; Chishima et al., 2002Go). A similar trend in the different expression of other biological parameters in solid and cystic endometriosis was reported by Nezhat and Kalir (2002Go) who compared the expression of bcl-2, matrix metalloproteinase IX (MMP IX) and collagen IV, and showed a different behaviour of protein expression in these two entities. It is conceivable that the peculiar location of the surface ovarian cells, that are in close contact with the steroid hormones secreted by the ovarian stroma, could represent a completely different microenvironment compared with the monolayer peritoneal mesothelial cells. Furthermore, growth factors normally produced by the ovarian epithelium that would diffuse into the large potential space of the peritoneal cavity may be present at higher levels in the microenvironment of the endometriotic cyst.

Moreover, we first reported that peritoneal and RV endometriosis behave uniformly with regard to COX-2 expression. These considerations might also support a common origin for peritoneal and RV endometriosis, which could be retrograde menstruation, as suggested by Vercellini et al. (2000Go).

This is the first study analysing the association between clinico-pathological parameters and COX-2 status in a large series of patients submitted to operative laparoscopy for endometriosis. The analysis was carried out dividing patients into two groups as described in the Results. Unfortunately, we failed to demonstrate any association between COX-2 status and the following: (i) clinico-pathological characteristics; (ii) subjective intensity of pain such as dysmenorrhoea, chronic pelvic pain, lower urinary tract and GI symptoms; and (iii) infertility.

Finally, endometriosis is an ambiguous disease with no definitive medical/surgical solutions. To date, standard medical treatments have to deal with the limit of preventing spontaneous ovulation and of recurrence after discontinuation. Since the clinical profile of endometriosis is typical of inflammatory disease, therapy with anti-inflammatory drugs has been proposed (Nothnick, 2001Go). For this purpose, COX-2 inhibitors could be used in addition to surgery in adjuvant settings for the treatment and/or prevention of endometriosis-related symptoms. In this context, the ability of COX-2 inhibitors to restore immunological function and to counteract the neoangiogenetic process could also be potentially exploited.

In conclusion, this study has demonstrated a high expression of COX-2 in the endometriotic ovarian cyst wall with respect to extra-ovarian localizations. Although no relevant correlation between COX-2 positivity and clinico-pathological characteristics and symptoms was observed, our results can provide interesting cues for drawing hypotheses on endometriosis pathogenesis and treatment.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on June 25, 2003; resubmitted on August 12, 2003; accepted on October 1, 2003.