Increased killer inhibitory receptor expression among natural killer cells in women with pelvic endometriosis

P-69 The expression of ER, PR, PR-B, PS2 in adenomyosis. L. Wei, Y. Zhao, J. Wang. Dept of Gynecology, People’s Hosp, Peking Univ, Beijing, China. Objective: Adenomyosis is a common disease in women, but its pathogenesis is still uncertain. Studies showed high level estrogen has closed relationship with adenomyosis, so we studied the expression of ER, PR, PR-B, and PS2 in adenomyosis. Design: Basic study. Materials/Methods: The expression of ER, PR, PR-B, PS2 were detected by immunohistochemical in 25 cases ectopic and 14 cases eutopic endometrium from adenomyosis. Results: 1. In uterus endometrium, the positive expression rate of ER, PR, PR-B, PS2 is 42.9%, 57.1%, 35.7%, 42.9% respectively; in ectopic endometrium, the rate is 64%, 56%, 44%, 56% respectively. 2. The expression of ER, PR, PR-B, PS2 have no significant difference in two groups. 3. Significant relationship was found between PS2 and ER. 4. The expression of PR-B is mainly located in glands. Conclusions: Clinical evidences showed sex hormone has various effect on endometrium in different location, accordingly, although both eutopic and ectopic endometrium express the ER, PR, PR-B and PS2, maybe sex hormone regulate them in different ways. Besides this, receptor isoforms may have diverse biological characters. Supported By: National Natural Science Foundation of China.

P-70 The function of extracellular and their receptors in the pathogenesis of endometriosis. P. Klemmt, J. Carver, P. Koninckx, S. Kennedy, D. Barlow, H. Mardon. Nuffield Dept of Obstetrics and Gynaecology, Univ of Oxford, The Women’s Ctr, Headley Way, Oxford, UK. Objectives: The development of an endometriotic lesion involves cell adhesion, proliferation, differentiation and invasion, all of which can be modulated by the interaction of extracellular matrix (ECM) components and integrin receptors. Our aim was to determine the expression and function of ECM molecules and integrin receptors in an in vitro model of endometriosis. Materials/Methods: Endometriotic tissues comprising endometrioma, surface lesions (nodules) and deeply infiltrating endometriosis (deep nodules) were obtained from women undergoing laparoscopy for endometriosis, infertility or benign indications. Stromal cells were isolated by digestion with collagenase and DNase, and separated using a Percoll gradient. Endometriotic stromal cells were maintained in vitro and expression of stromalspecific markers assessed. The levels of integrin subunits on freshly isolated and cultured cells were examined by quantitative immunohistochemistry. The function of specific ECM components was determined in assays for adhesion, proliferation, differentiation. The data were evaluated according to the type of lesion. Results: We found differences in the levels of different integrin subunits in freshly isolated and cultured endometriotic stromal cells, depending upon the source of the lesion. The adhesive capacity of endometriotic stromal cells on different ECM components also was dependent upon the source of the lesion. The adhesion of cultured endometrioma cells to ECM components was 2.5-fold higher, and cultured nodule cells 1.5-fold higher, than the adhesion of cells derived from endometrium. All cell types exhibited higher adhesive capacity on collagens IV and I compared to fibronectin. The influence of ECM on the proliferative and invasive capacities of endometriotic cells are being determined. Conclusions: Cells derived from nodules and deep nodules respond differently to the extracellular matrix. Modulation of ECM components therefore may determine the extent of invasion of endometriotic lesions. Supported By: None reported.

P-71 Concentration of matrix metalloproteinase-1, tissue inhibitor of matrix metalloproteinase-1 and its complex in peritoneal fluid in patients with endometriosis. M. Gogacz1, J. Kotarski2, P. Skorupski1, J. Jakowicki1, T. Rechberger1. 12nd Dept of Obstet and Gynecol, Univ Sch of Medicine, Lublin, Poland; 21st Dept of Obstet and Gynecol, Lublin, Poland.

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Abstracts

Objectives: Many studies on endometriosis are focused on peritoneal fluid and immune cells regarding it the most frequent localization in peritoneal cavity. Immune cells potentially posses ability to produce matrix metalloproteinases (MMPs). MMPs belong to zinc dependent lytic enzymes that are responsible for degradation of collagen. This effect can enhance the process of implantation and progression of endometriosis. Tissue inhibitor of matrix metaloproteinase-1 (TIMP-1) is responsible for natural inactivation of MMP-1 by forming inactive complex MMP-1/TIMP-1. Design: In our study we measured MMP-1, TIMP-1 and its complex MMP-1/TIMP-1 in peritoneal fluid in patients with endometriosis. Materials/Methods: The study group consist of 23 patients with endometriosis who underwent laparoscopy. Stage I of endometriosis was diagnosed in 10 patients, stage II in 8 patients and 5 patients had stage III/IV of the disease. 12 patients with presence of benign non inflammatory adnexal tumor served as a reference group. Peritoneal fluid was stored until analysis at ⫺700C. MMP-1, TIMP-1 and MMP-1/TIMP-1 complex were measured using ELISA (Amersham). Results: MMP-1 concentration was significantly higher (p ⬍ 0.05) in patients with endometriosis comparing to reference group: 3.75 vs 3.45 ng/ml. MMP-1 concentration was significantly higher in all stages of endometriosis in comparison to reference group. MMP-1 concentration was significantly higher (p ⬍ 0.05) in patients with stage III/IV of endometriosis in comparison to stage I and II of the disease. TIMP concentration was significantly higher (p ⬍ 0.05) in the reference group comparing to endometriotic patients: 218.75 vs 202.04 ng/ml. It is worth to notify that TIMP-1 was higher in patient with stage II and III of endometriosis in comparison to stage I of endometriosis. There was no difference in MMP-1/TIMP-1 concentration (p ⬎ 0.05) both between studied groups of patients 7.15 vs 5.98 ng/ml and among patients with endometriosis. Conclusions: Observed changes suggest that progression of the disease may differ depending on the stage of the disease. TIMP may be an important factor responsible for limiting the process of endometriosis. Better understanding the role of collagenolytic enzymes and its inhibitors may serve as background for the improvement of the classification and/or treatment of the disease. Supported By: None reported.

CLINICAL RESEARCH Tuesday, February 26, 2002 P-73 Increased killer inhibitory receptor expression among natural killer cells in women with pelvic endometriosis. T. Kusume, C. Izumiya, N. Maeda, T. Fukaya. Obstetrics and gynecology, Kochi Medical Sch, Kochi, Japan. Objective: To investigate the host immunologic response to endometriosis in terms of killer inhibitory receptor (KIR) expression by natural killer (NK) cells. Design: Case-control study of immunologic markers. Materials/Methods: We compared cells from 42 Japanese women laparoscopically diagnosed with endometriosis to cells from 40 women without endometriosis (control). Peripheral blood (PB) was collected before laparoscopy, and in 12 subjects with endometriosis, also 1 month after laparoscopic surgery. Peritoneal fluid (PF) was collected at laparoscopy. We examined the percentage of KIR-expressing (KIR ⫹) NK cells among NK cells in PF and PB. Relationships between percentage of KIR ⫹ NK cells and clinical severity in women with endometriosis were examined. The percentage of KIR ⫹ NK cells in PB was reexamined after laparoscopic removal of endometriotic lesions. KIR expression by NK cells was assessed by flow cytometry. Informed consent for obtaining PF and PB samples was obtained before the procedure. The Kruskal-Wallis test and the MannWhitney U-test was used to test KIR levels among the groups. Results: In women with endometriosis, percentage of KIR ⫹ NK cells in both PF and PB was significantly higher than in control subjects (PF, p ⫽ .017; PB, p ⫽ .0008). Percentages of KIR ⫹ NK in PF correlated with American Society for Reproductive Medicine (r-ASRM) scores (p ⫽ .009), however, percentages of KIR ⫹ NK in PB did not. No significant differences in proportion of KIR ⫹ NK cells were identified between PB sampled before and 1 month after laparoscopic surgery. Conclusions: The percentage of KIR ⫹ NK cells were increased in PB

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from women with endometriosis, the difference likely to be related to immunosuppression of NK cells in endometriosis. This increase in KIR ⫹ NK cells may represent a pathogenetic factor in endometriosis. Supported By: No support.

P-74 Determination of matrix metalloproteinase-2 and sex hormone in sera and peritoneal fluid of women with endometriosis and infertility of endometriosis. Y. Tan. The Second Hosp of Yunnan Province, Kunming, China. Objective: To study concentration variation and function of matrix metalloproteinase-2 (MMP-2) and estradiol (E2) and progesterone (P) of serum and peritoneal fluid of women with endometriosis and infertility of endometriosis. Design: Cross-sectional study with control groups. Materials/Methods: 47 cases with infertility of endometriosis diagnosed by laparoscopic surgery were divided into two groups: 31 cases in mild group (I, II stage) and 16 cases in severe group (III, IV stage). 18 women with normal pelvic cavity were used as control. MMP-2, E2 and P were determined by ELISA and RIA. Results: The concentration of MMP-2 and E2 of sera and peritoneal fluid of endometriosis groups were higher than that of control. The level in follicular phase were higher than that in the luteal phase (p ⬍ 0.05). The concentration of MMP-2 and E2 in peritoneal fluid were remarkably higher than that in sera (p ⬍ 0.01). The level of the two groups of endometriosis were not different (p ⬎ 0.05), but the concentration of E2 of severe endometriosis group was higher than that of mild one (p ⬍ 0.05). The concentrations of P in follicular phase of both endometriosis groups and control group were not different (P ⬎ 0.05). The concentration of P of serum and peritoneal fluid in the luteal phase of control were higher than that of endometriosis group (p ⬍ 0.05). The MMP-2 concentration in serum and peritoneal fluid were significantly correlated with E2 concentrations (r ⫽ 0.678 and 0.679, P ⫽ 0.001 and 0.002, respectively), and was negatively correlated with P concentration (r ⫽ ⫺0.518 and 0.788, P ⫽ 0.023 and 0.002, respectively). Conclusions: MMP-2, E2 and P participate in the forming and developing of endometriosis. To reduce the level of MMP-2 in pelvic cavity and improve inner environment of reproduction may have action for increasing cure rate of infertility of endometriosis. Supported By: Research fund from the Second Hospital of Yunnan Province.

P-75 Identification of differentially expressed markers for endometriosis. K. Shazand, B. Malette, E. Cherry, S. Baban, C. Prive´ , P. Hugo. PROCREA Biosciences, Montreal, PQ, Canada. Objective: Identify differentially expressed genes in the endometrium of diseased women as potential diagnostic markers. Design: All patients were selected in the study according to the following criteria: subjected to laparoscopy or laparatomy, have a regular menstrual cycle (between 21 to 35 days), no acute salpingitis, no hormonal treatments and no intra-uterine device in the last three months. The study was based on the comparison of two groups: an endometriosis group (endo) composed of all four stages of the disease, and a control group composed of women who were surgically confirmed not to have endometriotic lesions in the peritoneal cavity. The half of the latter group consisted in gynecologically healthy subjects, while the other half was composed of patients with various gynecological disorder other than endometriosis, such as fibroma or cysts. Materials/Methods: Endometrial biopsies from patients were used to extract total RNA for analysis of the gene expression pattern by the mean of different technical approaches such as differential display, cDNA arrays and real-time PCR. Results: We have identified a large array of genes exhibiting abnormal expression at the transcriptional level in the endometrium of diseased women. A first step of identification has been carried out on up to 50 samples in each experimental group (control and endo). A second step of validation consisted in measuring the mRNA levels of the candidate genes in 170 samples in the control group and 110 samples in the endo group.

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Statistical analysis of the final data showed that several genes were differentially transcribed in the endo group in comparison to the control group and that the difference was significant on a rather high number of samples. Conclusions: The identified genes are targeted to be used as markers to diagnose the presence of endometriosis in patients presenting various symptoms associated with the disease. Further analyses remain to be done in order to define the predictive value of individual as well as combinations of such markers. Supported By: No support.

P-76 Transvaginal ultrasonography combined with three-dimensional ultrasonography in the diagnosis of endometrioma. S. Chii-Shinn. Chang Gung Memorial Hosp, Taipei, Taiwan. Objective: To assess the diagnostic accuracy of transvaginal ultrasonography alone and combined with three-dimensional ultrasonography in differentiating ovarian endometrioma from other nonendometriotic mass. Design: nil. Materials/Methods: A series of 56 consecutive patients diagnosed as having an adnexal masses (three patients had bilateral masses) and scheduled for surgery. CA-125 plasma level were measured in all patients. Ultrasound diagnosis was compared with definitive histopathological diagnosis. The presence of a round-shaped homogenous cyst with low-level echoes (Fig. 1) was considered as transvaginal ultrasonographic diagnosis of ovarian endometriomas. The presence of irregular thickened wall (Fig. 2), incomplete formated septum (Fig. 3), mural hyperechoic echogenecity without acoustic shadow (Fig. 4) was considered as three-dimensional ultrasonographic diagnosis of ovarian endometriomas. The sensitivity, specificity, and positive and negative predictive values were calculated for transvaginal ultrasonography alone and combined with three-dimensional ultrasonographic imaging. Results: Twenty-two (39.2%) of the 56 masses were proven to be ovarian endometriomas. Transvaginal morphological assessment diagnosed correctly 20 (86.3%) of 22 endometriomas. Typical three-dimensional architectures was present in 90.1% of endometriomas. The sensitivity, specificity, and positive and negative predictive values of transvaginal ultrasonography alone and combined with three-dimensional ultrasonography were 86.3%, 88.2%, 82.6%, 90.9%, and 91%, 91.2%, 86.9%, and 93,9%, respectively. Conclusions: The use of three-dimensional ultrasonography improve the diagnostic accuracy of transvaginal ultrasonography alone in the diagnosis of ovarian endometriomas and may help in the diagnosis of other gynecologic disorders with given criteria. Supported By: No support.

P-77 Comparative study of patients with endometriosis in the United States and in the United Kingdom. W. Kuohung1, S. Kennedy2, D. W. Cramer1, M. D. Hornstein1. 1Brigham and Women’s Hosp, Boston, MA; 2John Radcliffe Hosp, Oxford, UK. Objective: To investigate differences in characteristics of patients with endometriosis in the United States and in the United Kingdom. Design: Consecutive patients at two university-based hospitals and reproductive endocrinology referral centers, one in the U.S. and one in the U.K., were asked to complete a questionnaire on life events related to their endometriosis. Materials/Methods: Patients undergoing follow-up for previously documented endometriosis were surveyed. Of the American patients, all recruited except for one agreed to participate in the study. Results: Seventy-nine American women and 103 British women participated. Mean age of the U.S. patients was 33.0 years; mean age of the U.K. patients was 32.8. The majority of subjects in both groups were white. Other demographics were similar, including body mass index before and after diagnosis, menstrual characteristics, pregnancy history, and the identification of endometriosis as a primary cause of infertility. There were several differences in demographics: American patients were more likely to have completed college or graduate school, and British patients were more likely to be married. Symptoms leading to diagnosis were similar in both groups

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