- Email: [email protected]
Combined Effect of Oxidative Stress and Tumor Necrosis Factor-α on Mouse Oocyte Spindle Structure
looks at the symptoms chosen and where there is a pattern of symptoms we look for consistently high or low improvement scores. Over three months, 93 per cent of patients showed an improvement in normalised MYMOP scores. The study has allowed us to follow-up a cohort of patients who have undergone nutritional counselling and therapy. The paper presents the results of these patients over three months and six months of treatment, giving an insight into the benefits of sound nutrition to patients with endometriosis. CONCLUSIONS: Nutritional counselling and therapy alongside orthodox medical treatments provides a cost effective method in the reduction of pain and improvements in the quality of life of women with endometriosis. The Endometriosis and Fertility Clinic is committed to working towards the provision of complementary medicine at the point of need. We conclude that further collaborative projects, which include patients, practitioners and researchers, should be carried out in the field of nutrition. Supported by: None.
P-119 Quantitative Expression of Growth Regulatory Genes in Matched, Eutopic and Ectopic Endometrial Cells from Women with Endometriosis. D. P. Braun, J. Ding, F. Shaheen, N. Rana, J. Willey, W. Dmowski. Medical College of Ohio, Toledo, OH; Institute for the Study and Treatment of Endometriosis, Chicago, IL; Institute for the Study and Treatment of Endometriosis and Rush Medical College, Chicago, IL. OBJECTIVE: Endometriosis (Endo) arises as a consequence of ectopic endometrial cells (EC) exhibiting the capacity to evade spontaneous and induced apoptosis, and to exploit inflammatory molecules within the peritoneal cavity to promote cell survival, implantation, and growth. We hypothesize that quantitative differences in the expression of genes that regulate cell survival in eutopic vs. ectopic endometrial cells, will reflect key adaptive mechanisms which favor cell survival and establishment of endometriosis within the peritoneal environment. DESIGN: The quantitative expression of various growth regulatory genes were compared in matched, eutopic and ectopic EC from women with Endo. MATERIALS AND METHODS: Paired samples of eutopic and ectopic endometrium were obtained from women with endometriosis (n⫽4); RAFS stage II (3), III (1). Eutopic endometrium was collected with Novak’s curette while ectopic endometrium was obtained from implants laparoscopically. EC suspensions were prepared by digestion of the tissues with collagenase (0.014%) and DNAse (0.01%). Thereafter, EC were cultured for 24 hours, non-adherent cells removed, total RNA extracted from adherent cells with RNAzol-B, cDNA prepared, and mRNA for different genes quantitated by the StaRT-PCR® method with results expressed as # molecules mRNA per 1 million molecules of -Actin. Expression levels ⱖ 3 fold were considered significant. The expression of various anti-apoptotic, proapoptotic, cytokine, oxidative and eicosanoid genes was evaluated. RESULTS: With respect to the anti-apoptotic genes, expression of BCL2 and the BCLxL: BCLxS ratio in ectopic EC was significantly greater than in matched eutopic EC in 2/4 and 3/4 samples respectively. In contrast, there were no significant differences in expression of the DAD-1 gene. With respect to pro-apoptotic genes, expression of BAK-1 in ectopic EC was reduced significantly in 3/4 cases while reduced expression of P53, Catalase-1, DAP-1, and PCD10 genes was detected in only 1/4 cases. In the case of cytokine-mediated growth regulatory genes, ectopic EC expressed significantly increased amounts of TRAIL compared to matched eutopic EC in 3/4 cases. In contrast, significant downregulation of TNFr1 expression and upregulation of TGFr2 expression in ectopic EC was detected in only 1/4 paired specimens. The expression of genes for modulation of oxidation, such as superoxide dismutase and catalase were significantly different in only 1/4 cases with increased expression for SOD but reduced expression for catalase in the same specimen. With respect to genes that metabolize arachidonic acid to produce eicosanoids, significantly increased expression of COX-2 and 5-Lipoxygenase in ectopic vs. eutopic EC was seen in 2/4 cases. CONCLUSIONS: In conclusion, adaptation to the peritoneal environment by EC from women with endometriosis appeared to involve increased expression of selected “self-protective” genes including BCL2/BCLxL, TRAIL, and arachidonic acid metabolizing genes. Adaptation through reduction in expression of pro-apoptotic genes appeared to be involved primarily with BAK1. This sort of analysis with increased numbers of specimens may uncover opportunities to develop targeted therapies for
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management of endometriosis. In individual cases, this approach may facilitate selection of individualized treatment modalities. Supported by: None P-120 Atherosclerosis in Women With Endometriosis. S. Ferrero, S. Pretta, V. Remorgida, M. Del Sette, C. Gandolfo, N. Ragni. San Martino Hos..pital, University of Genoa, Genoa, Italy; Department of Neurosciences, Ophthalmology and Genetics, University of Genoa, Genoa, Italy. OBJECTIVE: Patients with chronic inflammatory states (i.e. rheumatoid arthritis, systemic lupus erythematosus, Crohn’s disease) may show acceleration of atherosclerosis. Oxidative stress, which is known to be associated with atherosclerosis, has recently been proved to play a role in the development and progression of endometriosis. In addition, activated macrophages, production of inflammatory cytokines, and mildly oxidized LDL play similar roles in both atherosclerosis and endometriosis. The purpose of the current study is to determine whether women with endometriosis have greater subclinical atherosclerosis than the general population. DESIGN: Cross sectional study. MATERIALS AND METHODS: This study included 66 women with endometriosis and 66 age-matched controls (⫾ 3 years). All subjects were ⱖ 35 years old and were regularly menstruating. Exclusion criteria were diabetes, hypertension, hypercholesterolemia, renal or metabolic diseases. Control group subjects had no detectable endometriotic lesions at surgical examination and had not previously undergone surgical treatment for endometriosis; they underwent surgery for uterine myomas (n ⫽ 33), ovarian cysts (n ⫽ 21), and pelvic pain (n ⫽ 12). Before laparoscopy, all patients underwent carotid scanning. Intima-media thickness (IMT) was measured on the far wall of the distal portion of the common carotid artery on each side 1cm proximal to the bulb. The arterial wall distensibility coefficient (DC) was calculated by dividing the relative stroke change in the diameter of the arterial wall through the pulse pressure, defined as systolic blood pressure minus diastolic blood pressure. Transcranial Doppler was used to evaluate cerebrovascular reactivity to hypercapnia, the breathholding index (BHI) was obtained by dividing the percent increase in mean flow velocity occurring during breath-holding by the time (seconds) in which the subjects held their breath after a normal inspiration. Before surgery after at least 10 hours of fasting and not smoking blood samples were taken to determine the levels of LDL, HDL, cholesterol, triglycerides, fibrinogen, C-reactive protein, homocysteine, fasting glycemia, antithrombin III, plasminogen, protein C, protein S, and activated protein C resistance. Univariate comparisons of cases and controls were conducted by Mann-Whitney U test for continuous data and 2 test for categorical data. RESULTS: No significant difference was detected in mean (⫾ SD) age between study and control group (41.0 ⫾ 3.7 and 40.8 ⫾ 4.4 years; p ⫽ 0.287); body mass index and smoking status were similar in the two groups. All the biochemical parameters evaluated had similar levels in women with and without endometriosis. IMT was similar in women with endometriosis and in controls both on left (mean ⫾ SEM, 0.444 ⫾ 0.021 and 0.484 ⫾ 0.020 mm, respectively; p ⫽ 0.330) and right (0.456 ⫾ 0.025 and 0.473 ⫾ 0.015 mm, respectively; p ⫽ 0.648) carotid artery. Similarly, no significant difference was observed in the DC between women with endometriosis and controls both on left (mean ⫾ SEM, 4.738 ⫾ 2.111 and 4.374 ⫾ 2.246 ⫻ 10-3 /mmHg; respectively; p ⫽ 0.539) and right (4.939 ⫾ 0.287 and 5.132 ⫾ 0.241 ⫻ 10-3 /mmHg, respectively; p ⫽ 0.178) carotid artery. No significant difference was observed in the BHI between women with and without endometriosis. CONCLUSIONS: Our observations indicate that patients with endometriosis do not have more subclinical atherosclerosis than the general population. Supported by: None P-121 Combined Effect of Oxidative Stress and Tumor Necrosis Factor-␣ on Mouse Oocyte Spindle Structure. W. Choi, J. Banerjee, A. Agarwal, W. Paik, T. Falcone, R. K. Sharma. Cleveland Clinic Foundation, Cleveland, OH; Gyeongsang National University, Jinju, Republic of Korea. OBJECTIVE: Normal spindle structure consisting of microtubule and chromosomes is associated with successful cell division and subsequently
Vol. 84, Suppl 1, September 2005
embryo development. Numerous studies have identified tumor necrosis factor (TNF)- ␣ and reactive oxygen species to be increased in peritoneal fluid of women with endometriosis. The objective of the study was to examine the individual and combined effect of exogenous exposure to hydrogen peroxide (H2O2) and TNF-␣ during the in-vitro culture system on the oocyte spindle structure. DESIGN: Prospective in vitro study MATERIALS AND METHODS: Previous studies from our group have shown that 25 M concentrations of H2O2 and 200 ng/mL TNF-␣ affect the oocyte spindle structure. Mouse metaphase II oocytes (Embryotech Laboratories Inc. Wilmington, MA) were incubated after thawing at 37°C for 30 mins. Oocytes were divided into 4 groups: group I: Control incubated with human tubal fluid (HTF) alone, group II: 25 M H2O2, group III: 200 ng/mL TNF-␣, and group IV: H2O2 (25 M) ⫹ TNF-␣ (200 ng/mL). Following incubation for 30 min in 37°C, immunohistochemical staining was done to evaluate the effect on oocyte microtubule and chromosome alignment. Fixed oocytes were incubated with anti-␣-tubulin monoclonal antibody followed by incubation with FITC labeled anti-mouse IgG antibody. For chromosome staining, oocytes were incubated with propidium iodide. Microtubule morphology and chromosome alignment was scored and a score of 1-2 was considered normal, whereas 3-4 abnormal (modified from Saunders and Parks, 1999). RESULTS: Higher scores for changes in microtubule morphology and alteration in chromosome alignment were seen following exposure to H2O2, TNF-␣ and H2O2 ⫹ TNF-␣ compared with control (P ⬍ 0.05, Fig. 1). Significantly higher incidence of abnormal microtubule morphology was seen in H2O2 alone group compared with TNF-␣ alone group (3.16 ⫾ 0.78 vs. 2.71 ⫾ 0.52, P ⫽ 0.009). Also, compared with both TNF-␣ alone group, and H2O2 ⫹ TNF-␣ group, a significantly higher score was seen for microtubule morphology in H2O2 ⫹ TNF-␣ group (3.19 ⫾ 0.63 vs. 2.71 ⫾ 0.52, P ⫽ 0.003, Fig. 1). Changes in chromosomal alignment were comparable between the 3 groups.
OBJECTIVE: It is believed that the extracellular matrix (ECM) remodeling is relevant to the progression of endometriosis. The physiological changes in endometriosis involving in multiple steps of matrix remodeling include abnormal tissue growth, invasion, and adhesion phenomena. The changes of surrounding environment including the ECM expressions may affect the attachment and de-attachment of the cells. This study aims to investigate the expression pattern of various ECM molecules surrounding stromal and vascular tissues of eutopic and ectopic endometrium in patients with endometriosis. The correlation of altered expressions of matrix molecules with the progression of endometriosis was studied and discussed. DESIGN: In vitro study in a university based laboratory. MATERIALS AND METHODS: Biopsies of eutopic and ectopic endometrium from patients with adenomyosis and ovarian endometriosis were collected at the University Hospital. The expressions of various matrix molecules were analyzed at both mRNA and protein levels. In addition, their distribution was studied by immunohistochemistry. RESULTS: The matrix molecules including collagen type I, aggrecan, and small leucine rich proteoglycans (SLRPs) were up-regulated at both mRNA and protein levels in the ectopic endometrium compared to that in the eutopic endometrium of endometriosis. Immunohistochemistry indicated that most of the matrix molecules were expressed by stromal cells whereas the SLRP biglycan was stained intensively in the epithelium. The levels of both SLRP decorin and biglycan were significantly increased at follicular phase and to the maximum at ovulation phase and dramatically decreased in luteal phase during the menstrual cycle. However, the expressions of both decorin and biglycan remained without affected by the hormone regulation. Additionally, the expressions of the matrix degradation enzymes and their inhibitors were altered where increased expression of TIMP-1 was found in patients with endometriosis. CONCLUSIONS: Our results suggested that the matrix integrity was indeed altered and might be associated with the progression of endometriosis. The ECM remodeling in stroma and epithelium might be affected by several molecular factors including proteolytic enzymes and their inhibitors, ovarian steroids, various growth factors, cytokines, and chemotaxis. This study provides more information for understanding the pathogenesis of endometriosis and the mechanism of invasion and adhesion of the endometrium with endometriosis. Supported by: National Science Council in Taiwan, NSC93-2314-B-038029.
P-123 Proteomic Pattern Profiling of the Eutopic Endometrial and Ovarian Endometriotic Tissues in Same Patients Using SELDI-TOF Mass Spectroscopy. M. Chung, H. Kwon, D. Lee, S. Lee, K. Cha, T. Yoon. Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University, Seoul, Republic of Korea.
Figure 1. Effect of exogenous exposure to H2O2 and TNF-␣ alone and in combination on alterations in microtubule (MT) structure and chromosomal alignment (CH).
CONCLUSIONS: Oxidative stress as a result of exposure to hydrogen peroxide or TNF-␣ results in alterations in microtubule and chromosomal alignment. Compared to TNF-␣ hydrogen peroxide alone or in combination with TNF-␣ has more deleterious effects on the microtubule structure. This may explain the poor oocyte quality seen in many women with endometriosis where elevated TNF-␣ levels may be seen in response to oxidative stress. Supported by: None P-122 Altered Extracellular Matrix in Eutopic and Ectopic Endometrium of Endometriosis. W. Yang, Y. Huang, H. Chen, C. Chen, P. Chen, C. Tzeng. Taipei Medical University, Taipei, Taiwan, Republic of China; National Yang Ming University, Taipei, Taiwan, Republic of China; Department of Obstetrics and Gynecology, Taipei Medical University Hospital, Taipei, Taiwan, Republic of China.
FERTILITY & STERILITY威
OBJECTIVE: Endometriosis is thought to be a major factor for female infertility at reproductive ages. Despite of improvements in diagnosis and therapy, the treatment rate is poor so far. For a better understanding of the molecular mechanisms behind the process of endometriosis, we have analyzed the changes of protein expression pattern between the eutopic endometrial and matched ovarian endometriotic tissues in same patients by using a protein chip surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy. DESIGN: Comparative study between the eutopic and matched ectopic noncystic ovarian lesions and ovarian endometriotic cysts in same patients by proteomic pattern profiling analysis. MATERIALS AND METHODS: Endometrial tissues obtained from eutopic endometrium, noncystic ovarian lesions and ovarian endometriotic cysts in the same patients (n⫽4, age: 31-40) were stained by H&E to confirm the tissue identification. Therefore, the tissues were analyzed on ProteinChip arrays. Total lysates were analyzed using a strong anion exchange (Q10) Protein Chip array (Ciphergen Biosystems). The samples per spot were incubated and washed with a binding buffer (50 mM Tris-HCl, pH 9.0, containing 0.05% Triton X-100). The EAM (sinapinic acid) was applied to each spot. The Q10 ProteinChip was placed in a PBS-II mass spectrometer and time-of-flight spectra were generated at laser intensities of 150⬃250 and a detector sensitivity of seven. Each spot was analyzed by averaging 55 laser shots from various regions of the spot surfaces. The peaks were analyzed by ProteinChip software ver. 3.0. RESULTS: In this study, the derived mass spectrometry profiles exhib-
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P-119 Quantitative Expression of Growth Regulatory Genes in Matched, Eutopic and Ectopic Endometrial Cells from Women with Endometriosis. D. P. Braun, J. Ding, F. Shaheen, N. Rana, J. Willey, W. Dmowski. Medical College of Ohio, Toledo, OH; Institute for the Study and Treatment of Endometriosis, Chicago, IL; Institute for the Study and Treatment of Endometriosis and Rush Medical College, Chicago, IL. OBJECTIVE: Endometriosis (Endo) arises as a consequence of ectopic endometrial cells (EC) exhibiting the capacity to evade spontaneous and induced apoptosis, and to exploit inflammatory molecules within the peritoneal cavity to promote cell survival, implantation, and growth. We hypothesize that quantitative differences in the expression of genes that regulate cell survival in eutopic vs. ectopic endometrial cells, will reflect key adaptive mechanisms which favor cell survival and establishment of endometriosis within the peritoneal environment. DESIGN: The quantitative expression of various growth regulatory genes were compared in matched, eutopic and ectopic EC from women with Endo. MATERIALS AND METHODS: Paired samples of eutopic and ectopic endometrium were obtained from women with endometriosis (n⫽4); RAFS stage II (3), III (1). Eutopic endometrium was collected with Novak’s curette while ectopic endometrium was obtained from implants laparoscopically. EC suspensions were prepared by digestion of the tissues with collagenase (0.014%) and DNAse (0.01%). Thereafter, EC were cultured for 24 hours, non-adherent cells removed, total RNA extracted from adherent cells with RNAzol-B, cDNA prepared, and mRNA for different genes quantitated by the StaRT-PCR® method with results expressed as # molecules mRNA per 1 million molecules of -Actin. Expression levels ⱖ 3 fold were considered significant. The expression of various anti-apoptotic, proapoptotic, cytokine, oxidative and eicosanoid genes was evaluated. RESULTS: With respect to the anti-apoptotic genes, expression of BCL2 and the BCLxL: BCLxS ratio in ectopic EC was significantly greater than in matched eutopic EC in 2/4 and 3/4 samples respectively. In contrast, there were no significant differences in expression of the DAD-1 gene. With respect to pro-apoptotic genes, expression of BAK-1 in ectopic EC was reduced significantly in 3/4 cases while reduced expression of P53, Catalase-1, DAP-1, and PCD10 genes was detected in only 1/4 cases. In the case of cytokine-mediated growth regulatory genes, ectopic EC expressed significantly increased amounts of TRAIL compared to matched eutopic EC in 3/4 cases. In contrast, significant downregulation of TNFr1 expression and upregulation of TGFr2 expression in ectopic EC was detected in only 1/4 paired specimens. The expression of genes for modulation of oxidation, such as superoxide dismutase and catalase were significantly different in only 1/4 cases with increased expression for SOD but reduced expression for catalase in the same specimen. With respect to genes that metabolize arachidonic acid to produce eicosanoids, significantly increased expression of COX-2 and 5-Lipoxygenase in ectopic vs. eutopic EC was seen in 2/4 cases. CONCLUSIONS: In conclusion, adaptation to the peritoneal environment by EC from women with endometriosis appeared to involve increased expression of selected “self-protective” genes including BCL2/BCLxL, TRAIL, and arachidonic acid metabolizing genes. Adaptation through reduction in expression of pro-apoptotic genes appeared to be involved primarily with BAK1. This sort of analysis with increased numbers of specimens may uncover opportunities to develop targeted therapies for
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Abstracts
management of endometriosis. In individual cases, this approach may facilitate selection of individualized treatment modalities. Supported by: None P-120 Atherosclerosis in Women With Endometriosis. S. Ferrero, S. Pretta, V. Remorgida, M. Del Sette, C. Gandolfo, N. Ragni. San Martino Hos..pital, University of Genoa, Genoa, Italy; Department of Neurosciences, Ophthalmology and Genetics, University of Genoa, Genoa, Italy. OBJECTIVE: Patients with chronic inflammatory states (i.e. rheumatoid arthritis, systemic lupus erythematosus, Crohn’s disease) may show acceleration of atherosclerosis. Oxidative stress, which is known to be associated with atherosclerosis, has recently been proved to play a role in the development and progression of endometriosis. In addition, activated macrophages, production of inflammatory cytokines, and mildly oxidized LDL play similar roles in both atherosclerosis and endometriosis. The purpose of the current study is to determine whether women with endometriosis have greater subclinical atherosclerosis than the general population. DESIGN: Cross sectional study. MATERIALS AND METHODS: This study included 66 women with endometriosis and 66 age-matched controls (⫾ 3 years). All subjects were ⱖ 35 years old and were regularly menstruating. Exclusion criteria were diabetes, hypertension, hypercholesterolemia, renal or metabolic diseases. Control group subjects had no detectable endometriotic lesions at surgical examination and had not previously undergone surgical treatment for endometriosis; they underwent surgery for uterine myomas (n ⫽ 33), ovarian cysts (n ⫽ 21), and pelvic pain (n ⫽ 12). Before laparoscopy, all patients underwent carotid scanning. Intima-media thickness (IMT) was measured on the far wall of the distal portion of the common carotid artery on each side 1cm proximal to the bulb. The arterial wall distensibility coefficient (DC) was calculated by dividing the relative stroke change in the diameter of the arterial wall through the pulse pressure, defined as systolic blood pressure minus diastolic blood pressure. Transcranial Doppler was used to evaluate cerebrovascular reactivity to hypercapnia, the breathholding index (BHI) was obtained by dividing the percent increase in mean flow velocity occurring during breath-holding by the time (seconds) in which the subjects held their breath after a normal inspiration. Before surgery after at least 10 hours of fasting and not smoking blood samples were taken to determine the levels of LDL, HDL, cholesterol, triglycerides, fibrinogen, C-reactive protein, homocysteine, fasting glycemia, antithrombin III, plasminogen, protein C, protein S, and activated protein C resistance. Univariate comparisons of cases and controls were conducted by Mann-Whitney U test for continuous data and 2 test for categorical data. RESULTS: No significant difference was detected in mean (⫾ SD) age between study and control group (41.0 ⫾ 3.7 and 40.8 ⫾ 4.4 years; p ⫽ 0.287); body mass index and smoking status were similar in the two groups. All the biochemical parameters evaluated had similar levels in women with and without endometriosis. IMT was similar in women with endometriosis and in controls both on left (mean ⫾ SEM, 0.444 ⫾ 0.021 and 0.484 ⫾ 0.020 mm, respectively; p ⫽ 0.330) and right (0.456 ⫾ 0.025 and 0.473 ⫾ 0.015 mm, respectively; p ⫽ 0.648) carotid artery. Similarly, no significant difference was observed in the DC between women with endometriosis and controls both on left (mean ⫾ SEM, 4.738 ⫾ 2.111 and 4.374 ⫾ 2.246 ⫻ 10-3 /mmHg; respectively; p ⫽ 0.539) and right (4.939 ⫾ 0.287 and 5.132 ⫾ 0.241 ⫻ 10-3 /mmHg, respectively; p ⫽ 0.178) carotid artery. No significant difference was observed in the BHI between women with and without endometriosis. CONCLUSIONS: Our observations indicate that patients with endometriosis do not have more subclinical atherosclerosis than the general population. Supported by: None P-121 Combined Effect of Oxidative Stress and Tumor Necrosis Factor-␣ on Mouse Oocyte Spindle Structure. W. Choi, J. Banerjee, A. Agarwal, W. Paik, T. Falcone, R. K. Sharma. Cleveland Clinic Foundation, Cleveland, OH; Gyeongsang National University, Jinju, Republic of Korea. OBJECTIVE: Normal spindle structure consisting of microtubule and chromosomes is associated with successful cell division and subsequently
Vol. 84, Suppl 1, September 2005
embryo development. Numerous studies have identified tumor necrosis factor (TNF)- ␣ and reactive oxygen species to be increased in peritoneal fluid of women with endometriosis. The objective of the study was to examine the individual and combined effect of exogenous exposure to hydrogen peroxide (H2O2) and TNF-␣ during the in-vitro culture system on the oocyte spindle structure. DESIGN: Prospective in vitro study MATERIALS AND METHODS: Previous studies from our group have shown that 25 M concentrations of H2O2 and 200 ng/mL TNF-␣ affect the oocyte spindle structure. Mouse metaphase II oocytes (Embryotech Laboratories Inc. Wilmington, MA) were incubated after thawing at 37°C for 30 mins. Oocytes were divided into 4 groups: group I: Control incubated with human tubal fluid (HTF) alone, group II: 25 M H2O2, group III: 200 ng/mL TNF-␣, and group IV: H2O2 (25 M) ⫹ TNF-␣ (200 ng/mL). Following incubation for 30 min in 37°C, immunohistochemical staining was done to evaluate the effect on oocyte microtubule and chromosome alignment. Fixed oocytes were incubated with anti-␣-tubulin monoclonal antibody followed by incubation with FITC labeled anti-mouse IgG antibody. For chromosome staining, oocytes were incubated with propidium iodide. Microtubule morphology and chromosome alignment was scored and a score of 1-2 was considered normal, whereas 3-4 abnormal (modified from Saunders and Parks, 1999). RESULTS: Higher scores for changes in microtubule morphology and alteration in chromosome alignment were seen following exposure to H2O2, TNF-␣ and H2O2 ⫹ TNF-␣ compared with control (P ⬍ 0.05, Fig. 1). Significantly higher incidence of abnormal microtubule morphology was seen in H2O2 alone group compared with TNF-␣ alone group (3.16 ⫾ 0.78 vs. 2.71 ⫾ 0.52, P ⫽ 0.009). Also, compared with both TNF-␣ alone group, and H2O2 ⫹ TNF-␣ group, a significantly higher score was seen for microtubule morphology in H2O2 ⫹ TNF-␣ group (3.19 ⫾ 0.63 vs. 2.71 ⫾ 0.52, P ⫽ 0.003, Fig. 1). Changes in chromosomal alignment were comparable between the 3 groups.
OBJECTIVE: It is believed that the extracellular matrix (ECM) remodeling is relevant to the progression of endometriosis. The physiological changes in endometriosis involving in multiple steps of matrix remodeling include abnormal tissue growth, invasion, and adhesion phenomena. The changes of surrounding environment including the ECM expressions may affect the attachment and de-attachment of the cells. This study aims to investigate the expression pattern of various ECM molecules surrounding stromal and vascular tissues of eutopic and ectopic endometrium in patients with endometriosis. The correlation of altered expressions of matrix molecules with the progression of endometriosis was studied and discussed. DESIGN: In vitro study in a university based laboratory. MATERIALS AND METHODS: Biopsies of eutopic and ectopic endometrium from patients with adenomyosis and ovarian endometriosis were collected at the University Hospital. The expressions of various matrix molecules were analyzed at both mRNA and protein levels. In addition, their distribution was studied by immunohistochemistry. RESULTS: The matrix molecules including collagen type I, aggrecan, and small leucine rich proteoglycans (SLRPs) were up-regulated at both mRNA and protein levels in the ectopic endometrium compared to that in the eutopic endometrium of endometriosis. Immunohistochemistry indicated that most of the matrix molecules were expressed by stromal cells whereas the SLRP biglycan was stained intensively in the epithelium. The levels of both SLRP decorin and biglycan were significantly increased at follicular phase and to the maximum at ovulation phase and dramatically decreased in luteal phase during the menstrual cycle. However, the expressions of both decorin and biglycan remained without affected by the hormone regulation. Additionally, the expressions of the matrix degradation enzymes and their inhibitors were altered where increased expression of TIMP-1 was found in patients with endometriosis. CONCLUSIONS: Our results suggested that the matrix integrity was indeed altered and might be associated with the progression of endometriosis. The ECM remodeling in stroma and epithelium might be affected by several molecular factors including proteolytic enzymes and their inhibitors, ovarian steroids, various growth factors, cytokines, and chemotaxis. This study provides more information for understanding the pathogenesis of endometriosis and the mechanism of invasion and adhesion of the endometrium with endometriosis. Supported by: National Science Council in Taiwan, NSC93-2314-B-038029.
P-123 Proteomic Pattern Profiling of the Eutopic Endometrial and Ovarian Endometriotic Tissues in Same Patients Using SELDI-TOF Mass Spectroscopy. M. Chung, H. Kwon, D. Lee, S. Lee, K. Cha, T. Yoon. Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University, Seoul, Republic of Korea.
Figure 1. Effect of exogenous exposure to H2O2 and TNF-␣ alone and in combination on alterations in microtubule (MT) structure and chromosomal alignment (CH).
CONCLUSIONS: Oxidative stress as a result of exposure to hydrogen peroxide or TNF-␣ results in alterations in microtubule and chromosomal alignment. Compared to TNF-␣ hydrogen peroxide alone or in combination with TNF-␣ has more deleterious effects on the microtubule structure. This may explain the poor oocyte quality seen in many women with endometriosis where elevated TNF-␣ levels may be seen in response to oxidative stress. Supported by: None P-122 Altered Extracellular Matrix in Eutopic and Ectopic Endometrium of Endometriosis. W. Yang, Y. Huang, H. Chen, C. Chen, P. Chen, C. Tzeng. Taipei Medical University, Taipei, Taiwan, Republic of China; National Yang Ming University, Taipei, Taiwan, Republic of China; Department of Obstetrics and Gynecology, Taipei Medical University Hospital, Taipei, Taiwan, Republic of China.
FERTILITY & STERILITY威
OBJECTIVE: Endometriosis is thought to be a major factor for female infertility at reproductive ages. Despite of improvements in diagnosis and therapy, the treatment rate is poor so far. For a better understanding of the molecular mechanisms behind the process of endometriosis, we have analyzed the changes of protein expression pattern between the eutopic endometrial and matched ovarian endometriotic tissues in same patients by using a protein chip surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy. DESIGN: Comparative study between the eutopic and matched ectopic noncystic ovarian lesions and ovarian endometriotic cysts in same patients by proteomic pattern profiling analysis. MATERIALS AND METHODS: Endometrial tissues obtained from eutopic endometrium, noncystic ovarian lesions and ovarian endometriotic cysts in the same patients (n⫽4, age: 31-40) were stained by H&E to confirm the tissue identification. Therefore, the tissues were analyzed on ProteinChip arrays. Total lysates were analyzed using a strong anion exchange (Q10) Protein Chip array (Ciphergen Biosystems). The samples per spot were incubated and washed with a binding buffer (50 mM Tris-HCl, pH 9.0, containing 0.05% Triton X-100). The EAM (sinapinic acid) was applied to each spot. The Q10 ProteinChip was placed in a PBS-II mass spectrometer and time-of-flight spectra were generated at laser intensities of 150⬃250 and a detector sensitivity of seven. Each spot was analyzed by averaging 55 laser shots from various regions of the spot surfaces. The peaks were analyzed by ProteinChip software ver. 3.0. RESULTS: In this study, the derived mass spectrometry profiles exhib-
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