- Email: [email protected]
The presence of endometrial cells in the peritoneal cavity initiates a mesothelial inflammatory response
Objective: Endometriosis affects 5-10% of reproductive-aged women in the US with chronic pain, dysmenorrhea, and infertility. Its etiology remains uncertain, but family history of endometriosis and exposures indicating high circulating estrogen levels are both associated with increased disease risk. GSTM1 is largely a detoxification enzyme that reduces xenobiotic toxicity by conjugating hydrophobic xenobiotics with glutathione to form hydrophilic and more readily excretable substances, and that also functions as an intracellular drug- and hormone-binding protein. GSTM1 appears in two active allele variants and one null-allele without mRNA or protein product. COMT is involved in the clearance of exogenous and endogenous estrogen via O-methylation. A transition of guanine to adenosine at nucleotide 544 of the COMT gene results in a change of amino acid 158 from valine (val) to methionine (met). The met/met (LL) and val/met (HL) COMT genotypes are associated, respectively, with low and intermediate levels of catechol substrate methylation relative to the val/val (HH)-genotype. The purpose of this study was to determine whether the risk of endometriosis is associated with the null GSTM1 genotype (GSTM1 0/0) or the low activity COMT genotype (COMT-LL). Design: Population-based case-control study of women enrolled in a large health-maintenance organization. Materials and Methods: Study subjects were female enrollees of Group Health Cooperative of Puget Sound (GHC). Cases were 281 women aged 18-49 first diagnosed with endometriosis by surgery or ultrasound between April 1, 1996 and March 31, 2001. Controls were 581 subjects randomly selected from a list of women enrolled in GHC during the same time period and frequency matched on age to the cases. Genomic DNA was isolated using salt precipitation. Genotypes were determined using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Results: The frequency of GSTM1 0/0 in the cases was 53.7%, and in the controls was 46.8%. Compared with other GSTM1 genotypes, GSTM1 0/0 genotype was associated with an odds ratio (OR) of 1.32 (95% CI, 0.991.76). The frequencies of COMT-LL, -LH, and HH among cases and among controls, respectively, were 23.1%, 53.7%; 23.1%, and 24.4%; and 50.1% and 25.5%. Compared with the COMT-HH, COMT-LL was associated with an OR of 0.96 (95% CI, 0.63-1.45), and COMT-LH was associated with an OR of 1.13 (95% CI, 0.80-1.61). Women with GSTM1 null genotype and COMT-LL or-LH genotypes had an OR of 1.33 (95% CI, 0.99-1.78). The Hardy-Weinberg distribution of GSTM1 alleles could not be confirmed because the homozygous null state was measured dichotomously, but the COMT alleles were found to be in Hardy-Weinberg equilibrium (p⬎0.05). Conclusion: Prior research on polymorphisms of genes that may play a role in endometriosis risk has focused most on GSTM1, with mixed findings. In this population, GSTM1 null genotype appears to be associated with a modestly increased risk of endometriosis and does not interact significantly with COMT. COMT genotype alone does not appear to affect endometriosis risk.
P-316 Interobserver variability in the diagnosis of minimal and mild endometriosis using the revised American Society of Reproductive Medicine Classification of Endometriosis. Olaf Buchweitz, Ludwig Kiesel, Klaus Diedrich, Eduard Malik. Univ of Muenster, Muenster, Germany; Univ of Luebeck, Luebeck, Germany; Univ of Aachen, Aachen, Germany. Objective: The laparoscopy under white light illumination and the rASRM classification of endometriosis are the international standard for diagnosis of endometriosis. Despite its popularity the reproducibility of this system has not been established yet. The purpose of this study was to evaluate the interobserver variability in endometriosis diagnosing using the rASRM classification. Design: Videotapes of laparoscopies of 3 patients with endometriosis⬘s typical troubles were scored by 108 observers. Materials and Methods: Three digital videotapes showing diagnostic laparoscopy on patients with endometriosis⬘s typical troubles (rASRM I, II and no endometriosis) were demonstrated to 108 gynaecologists who participated in the workshop on endometriosis and fibroids in Hamburg, Germany 2002. They were asked to determine lesions in a standardized surgical outline which they would define as endometriosis and to score the pelvis according to the rASRM classification. Statistical evaluation is done by Kendall W coefficient. Results: There was a considerable variability in number and localisation of endometriotic lesions assigned to each videotape. The interobserver
FERTILITY & STERILITY威
agreement concerning the number of lesions ranges between 18% (rASRM II) and 30%. (rASRM I). There was marginal agreement regarding the localisation of endometriotic lesions. Kendall W coefficient ranged from 0,14 (rASRM II) to 0,44 (rASRM I) (p⬍0,001). Only 13% (rASRM II) to 22% (rASRM I) of physicians used the correct endometriotic classification. None of the participants denoted the percentage of surface involvement of each peritoneal implant type as demanded. Conclusion: The classification of the pelvis seems to be subjective. Histological confirmation of peritoneal implants could help to make diagnosis observer⬘s independent In the majority of the cases the rASRM classification is not applied correctly. Particularly the different phenotypes of endometriotic implants and therefore its biological activity is not taken into account. The activity of the disease should be more emphasised in future revisions of ASRM classification.
P-317 The presence of endometrial cells in the peritoneal cavity initiates a mesothelial inflammatory response. Xuan Cao, Ming Qing Song, Sampath Parthasarathy. Emory Univ, Atlanta, GA. Objective: Increased presence of peritoneal macrophages has been observed in women with endometriosis. As retrograde menstruation been suggested to account for the presence of endometrial cells in the peritoneal cavity of women with endometriosis, we proposed that the presence of endometrial cells together with mesothelial cells might induce an inflammatory response resulting in increased monocyte recruitment. Accordingly, we observed that the co-culture of endometrial cells with mesothelial cells resulted in several fold higher production of monocyte chemotactic protein-1 (MCP-1) as compared to either of these cell types alone (Song M. et al. Fertility & Sterility. 79: 789-94, 2003). The objective of the present study is to determine whether the presence of endometrial cells in the peritoneal cavity would lead to an induction of MCP-1 gene expression and to increased recruitment of monocytes in vivo. Design: The presence of mononuclear cells in the peritoneal cavity and the expression of monocyte chemotactic protein-1 (MCP-1) were evaluated in mice after the intraperitoneal administration of endometrial cells from donor mice. Materials and Methods: In vivo studies were performed with female Swiss-Webster mice (immune intact) and endometrial epithelial and stromal cells from donor mice. The number of monocyte/macrophages, the quantity of MCP-1, and the mesothelial expression of MCP-1 (JE) gene were determined. Results: The administration of mouse endometrial epithelial and stromal cells resulted in an increased number of macrophages in the peritoneal cavity of recipient mice. The nature of the cells was established by the presence of functional scavenger receptor activity. Epithelial cells were more effective than stromal cells. Maximum MCP-1 levels in the peritoneal lavage fluid were observed at 4 hours after injection of the cells, after which the levels were drastically reduced. Maximum macrophage numbers in the peritoneal lavage fluid were observed at 24 hours. The expression of MCP-1 gene in the mesothelium was also similarly activated at 4 hours. Conclusion: These results suggest that retrograde menstruation could account for the presence of mononuclear cells in the peritoneal cavity of women with endometriosis. As the expression of MCP-1 gene and protein was affected very early, the results also would seem to suggest that the increased presence of MCP-1 in the peritoneal cavity of women with endometriosis might signify the presence of recent and persistent activation of MCP-1 gene.
P-318 Extracellular matrix components and proteoglycans inhibit adherence of peritoneal macrophage. Nalini Santanam, Sonia A. Karabina, Ana A. Murphy, Sampath Parthasarathy. Emory Univ, Atlanta, GA. Objective: To study the effect of extracellular matrix components (ECM) and proteoglycans (PG) in the peritoneal fluid (PF) of women with endometriosis on the adherence of macrophages. Design: In vitro cell-culture study. Materials and Methods: Mouse peritoneal macrophages (MPM) were isolated by lavaging the peritoneum. The isolated macrophages were cul-
S227
P-316 Interobserver variability in the diagnosis of minimal and mild endometriosis using the revised American Society of Reproductive Medicine Classification of Endometriosis. Olaf Buchweitz, Ludwig Kiesel, Klaus Diedrich, Eduard Malik. Univ of Muenster, Muenster, Germany; Univ of Luebeck, Luebeck, Germany; Univ of Aachen, Aachen, Germany. Objective: The laparoscopy under white light illumination and the rASRM classification of endometriosis are the international standard for diagnosis of endometriosis. Despite its popularity the reproducibility of this system has not been established yet. The purpose of this study was to evaluate the interobserver variability in endometriosis diagnosing using the rASRM classification. Design: Videotapes of laparoscopies of 3 patients with endometriosis⬘s typical troubles were scored by 108 observers. Materials and Methods: Three digital videotapes showing diagnostic laparoscopy on patients with endometriosis⬘s typical troubles (rASRM I, II and no endometriosis) were demonstrated to 108 gynaecologists who participated in the workshop on endometriosis and fibroids in Hamburg, Germany 2002. They were asked to determine lesions in a standardized surgical outline which they would define as endometriosis and to score the pelvis according to the rASRM classification. Statistical evaluation is done by Kendall W coefficient. Results: There was a considerable variability in number and localisation of endometriotic lesions assigned to each videotape. The interobserver
FERTILITY & STERILITY威
agreement concerning the number of lesions ranges between 18% (rASRM II) and 30%. (rASRM I). There was marginal agreement regarding the localisation of endometriotic lesions. Kendall W coefficient ranged from 0,14 (rASRM II) to 0,44 (rASRM I) (p⬍0,001). Only 13% (rASRM II) to 22% (rASRM I) of physicians used the correct endometriotic classification. None of the participants denoted the percentage of surface involvement of each peritoneal implant type as demanded. Conclusion: The classification of the pelvis seems to be subjective. Histological confirmation of peritoneal implants could help to make diagnosis observer⬘s independent In the majority of the cases the rASRM classification is not applied correctly. Particularly the different phenotypes of endometriotic implants and therefore its biological activity is not taken into account. The activity of the disease should be more emphasised in future revisions of ASRM classification.
P-317 The presence of endometrial cells in the peritoneal cavity initiates a mesothelial inflammatory response. Xuan Cao, Ming Qing Song, Sampath Parthasarathy. Emory Univ, Atlanta, GA. Objective: Increased presence of peritoneal macrophages has been observed in women with endometriosis. As retrograde menstruation been suggested to account for the presence of endometrial cells in the peritoneal cavity of women with endometriosis, we proposed that the presence of endometrial cells together with mesothelial cells might induce an inflammatory response resulting in increased monocyte recruitment. Accordingly, we observed that the co-culture of endometrial cells with mesothelial cells resulted in several fold higher production of monocyte chemotactic protein-1 (MCP-1) as compared to either of these cell types alone (Song M. et al. Fertility & Sterility. 79: 789-94, 2003). The objective of the present study is to determine whether the presence of endometrial cells in the peritoneal cavity would lead to an induction of MCP-1 gene expression and to increased recruitment of monocytes in vivo. Design: The presence of mononuclear cells in the peritoneal cavity and the expression of monocyte chemotactic protein-1 (MCP-1) were evaluated in mice after the intraperitoneal administration of endometrial cells from donor mice. Materials and Methods: In vivo studies were performed with female Swiss-Webster mice (immune intact) and endometrial epithelial and stromal cells from donor mice. The number of monocyte/macrophages, the quantity of MCP-1, and the mesothelial expression of MCP-1 (JE) gene were determined. Results: The administration of mouse endometrial epithelial and stromal cells resulted in an increased number of macrophages in the peritoneal cavity of recipient mice. The nature of the cells was established by the presence of functional scavenger receptor activity. Epithelial cells were more effective than stromal cells. Maximum MCP-1 levels in the peritoneal lavage fluid were observed at 4 hours after injection of the cells, after which the levels were drastically reduced. Maximum macrophage numbers in the peritoneal lavage fluid were observed at 24 hours. The expression of MCP-1 gene in the mesothelium was also similarly activated at 4 hours. Conclusion: These results suggest that retrograde menstruation could account for the presence of mononuclear cells in the peritoneal cavity of women with endometriosis. As the expression of MCP-1 gene and protein was affected very early, the results also would seem to suggest that the increased presence of MCP-1 in the peritoneal cavity of women with endometriosis might signify the presence of recent and persistent activation of MCP-1 gene.
P-318 Extracellular matrix components and proteoglycans inhibit adherence of peritoneal macrophage. Nalini Santanam, Sonia A. Karabina, Ana A. Murphy, Sampath Parthasarathy. Emory Univ, Atlanta, GA. Objective: To study the effect of extracellular matrix components (ECM) and proteoglycans (PG) in the peritoneal fluid (PF) of women with endometriosis on the adherence of macrophages. Design: In vitro cell-culture study. Materials and Methods: Mouse peritoneal macrophages (MPM) were isolated by lavaging the peritoneum. The isolated macrophages were cul-
S227