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Add-Back Therapy in the Treatment of Endometriosis Associated Pain
microscopy and a computerized cell counter. Invasion of endometrial cells was evaluated in the presence and absence of the phorbol ester, phorbol 12-myristate 13-acetate (PMA) (50 ng/ml), an activator of PKC, which was added at the time that the endometrial cells were placed on the PMC monolayer. The number of invaded cells, in the presence and absence of PMA, was compared using a paired Student’s t-test. RESULTS: CLSM confirmed that the PMCs grow as a monolayer on the Matrigel®-coated 8 micron pore membranes. Endometrial invasion through the PMC monolayer was seen within 6 hours. Invasion through the 8 micron pores and into the underlying adherent, contracted collagen was demonstrated within 12 hours. By 24 hours, PMCs proliferated over sites of endometrial cell invasion. ESCs, EECs, including EM42 cells, invade into the modeled peritoneum. There was a trend towards greater invasion of EECs, including the EM42 cells, compared with ESCs. The addition of PMA to cultures lead to a greater than 2- fold increase in the number of invaded ESCs (p ⬍ 0.005). CONCLUSIONS: This modification of our novel assay of the early endometriotic lesion allows for rapid quantitation of endometrial cell invasion into modeled human peritoneum. We demonstrated that activation of PKC-dependent signaling pathways leads to significant increased invasion of endometrial cells into modeled peritoneum. This suggests that these pathways may contribute to the genesis of the early endometriotic lesion. We hypothesize that cytokines and growth factors, present in the peritoneal fluid of women with endometriosis, mediate the development of new lesions through PKC signaling pathways. 1. Fertil Steril. 2004;82(sup 2): S72, O-179 Supported by: None
P-104 Add-Back Therapy in the Treatment of Endometriosis Associated Pain. E. Zupi, M. Sbracia, D. Marconi, F. Zullo, G. Sorrenti. Universita` Tor Vergata, Rome, Italy; CERM, Rome, Italy; San Giuseppe Hospital, Rome, Italy; Universita` Magna Grecia, Catanzaro, Italy; Policlinico Tor Vergata, Rome, Italy. OBJECTIVE: To determine the efficacy of GnRH analogue plus addback therapy compared to GnRH analogue alone and estroprogestin in patients with relapse of endometriosis associated pain DESIGN: Randomized controlled study MATERIALS AND METHODS: 133 women with relapse of endometriosis-related pain after previous endometriosis surgery were included in the study.They were randomly assigned to three study groups: 46 women were treated with GnRH analogue plus add-back therapy, 44 women were given GnRH analogue alone and 43 women received estroprogestin, for 12 months long. The parameters evaluated were: pain levels, quality of life, adverse effect occurrence, loss of bone density. Pain was evaluated by a visual analogue scale, quality of life in treated patients using the SF36 questionnaire, and occurrence of adverse effects including bone mass density loss, at pre-treatment, after six months of treatment, at the end of treatment, and six months after discontinuation of treatment. RESULTS: Patients treated either with GnRH analogue alone and plus add-back therapy showed a higher reduction of pelvic pain, dysmenorrhea, and dyspareunia then ones treated with oral contraceptive, whereas patients treated with add-back therapy showed a better quality of life, assessed by the SF-36 questionnaire, and adverse effects rate, then the other two groups. CONCLUSIONS: Add-back therapy allows treating women with relapse of endometriosis-associated pain for a longer period reducing bone mineral density loss, with a good control of pain symptoms, compared to GnRH analogue alone or oral contraceptive, and a better patient’s quality of life, who have a better general perception of well-being then is experienced with the other two treatments. Supported by: None
P-105 Effects of Pre-Operative Gonadotrophin-Releasing Hormone Agonist (GnRHa) and Continuous Progestin Treatments on Expression of Potential Candidate Genes Involved in Endometriosis Pathophysiology. S. Matsuzaki, M. Canis, J. Pouly, B. Rabischong, R. Botchorischvili, G. Mage. Polyclinique de l’Hotel-Dieu, CHU, Clermont-Ferrand, France.
FERTILITY & STERILITY威
OBJECTIVE: Our recent DNA microarray analysis using laser capture microdissection (LCM) identified several potential candidate genes that might be involved in endometriosis pathopysiology. Heat shock protein 90 alpha (Hsp90A), which actively participates in steroid-induced signal transduction, was up-regulated and two potential negative regulators of aromatase expression, chicken ovalbumin upstream promoter transcription factor 2 (COUP-TF2) and prostaglandin E2 receptor subtype EP3 (PGE2EP3), were down-regulated in endometriosis epithelial cells. Plateletderived growth factor receptor alpha (PDGFRA) and members of the downstream RAS/RAF/MAPK signaling pathway were differentially expressed in stromal cells between endometriosis and matched eutopic endometrium (Up-regulation: PDGFRA, PKC beta1, JAK1; Down-regulation: sprouty2, MKK7). Studies to date have shown that both gonadotrophinreleasing hormone agonists (GnRHa) and continuous progestins are effective treatment regimens in patients with endometriosis in terms of reduction in rAFS score and relief of symptoms. However, these medical treatments are ineffective in eliminating the disease in most patients. Studies to investigate the changes in expression levels of the potential candidate genes during these medical treatments may well provide a better understanding of the pathophysiology and give rise to novel therapeutic strategies for this disease. In the present study, we further investigated mRNA expression levels of the potential candidate genes in deep endometriotic tissues during preoperative GnRHa or continuous oral progestin (P) treatments, using LCM and real-time RT-PCR techniques. DESIGN: Prospective study MATERIALS AND METHODS: Samples of 1) DIE (n⫽16) and matched eutopic endometrium (EE) (n⫽16) without pre-operative treatment, 2) pre-operative GnRHa-treated DIE (n⫽6) and 3) pre-operative P-treated DIE (n⫽5), were obtained during laparoscopic surgery. Glandular epithelial cells and stromal cells from endometriotic tissues were microdissected using LCM. Total RNA (10 ng) from microdissected tissues was subjected to an RT reaction and quantitative real-time PCR was performed in a Light Cycler System. The Mann-Whitney U test or the Kruskal-Wallis test was applied to compare results from different groups. The wilcoxon sign rank test was performed to compare differences in paired samples. Statistical significance was defined as p ⬍ 0.05. RESULTS: Expression levels of Hsp90A in epithelial cells were significantly lower in GnRHa-treated DIE (P⬍.007) than those in untreated DIE. There was no significant difference in Hsp90A expression between P-treated and untreated DIE. Sprouty 2 expression levels in stromal cells were significantly higher in both GnRHa- (P⬍.04) and P-treated (P⬍.009) DIE than those in untreated DIE. However, no significant difference was detected in expression levels of the remaining 6 genes, COUP-TF2 and PGE2EP3 in epithelial cells, and PDGFRA, PKC beta1, JAK and MKK7 in stromal cells among untreated, GnRHaand P-treated DIE. CONCLUSIONS: The present findings could partly explain why current medical treatment regimes are ineffective in eliminating the disease in most patients. Supported by: Supported in part by grants PHRC 2003 and PHRC 2004 of CHU Clermont-Ferrand
P-106 Plasminogen Activator Inhibitor (PAI) and Thrombin Activatable Fibrinolysis Inhibitor (TAFI) Polymorphisms in Women With Endometriosis. M. A. Bedaiwy, T. Falcone, R. F. Casper. Mount Sinai Hospital, Toronto, ON, Canada; The Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: Although most women experience retrograde menses during their reproductive life, endometriosis develops only in a small percentage. It has been shown that cells from endometrial explants can proliferate and invade a 3-dimensional fibrin matrix in vitro resulting in the formation of typical endometriotic tissue. We hypothesized that persistence of a fibrin matrix in peritoneal pockets could allow menstrually deposited endometrial fragments to initiate endometriosis in vivo. Polymorphisms in the genes of the fibrinolytic system have been shown to result in inhibition of fibrinolysis and maintenance of fibrin clots in vivo. Variants of the plasminogen activator inhibitor-1 (PAI-1) gene, containing at least one 4G allele, are associated with higher PAI-1 activity than the 5G/5G genotype. The Thr325Ile mutation in the TAFI gene has also been shown to be associated with increased antifibrinolytic
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P-104 Add-Back Therapy in the Treatment of Endometriosis Associated Pain. E. Zupi, M. Sbracia, D. Marconi, F. Zullo, G. Sorrenti. Universita` Tor Vergata, Rome, Italy; CERM, Rome, Italy; San Giuseppe Hospital, Rome, Italy; Universita` Magna Grecia, Catanzaro, Italy; Policlinico Tor Vergata, Rome, Italy. OBJECTIVE: To determine the efficacy of GnRH analogue plus addback therapy compared to GnRH analogue alone and estroprogestin in patients with relapse of endometriosis associated pain DESIGN: Randomized controlled study MATERIALS AND METHODS: 133 women with relapse of endometriosis-related pain after previous endometriosis surgery were included in the study.They were randomly assigned to three study groups: 46 women were treated with GnRH analogue plus add-back therapy, 44 women were given GnRH analogue alone and 43 women received estroprogestin, for 12 months long. The parameters evaluated were: pain levels, quality of life, adverse effect occurrence, loss of bone density. Pain was evaluated by a visual analogue scale, quality of life in treated patients using the SF36 questionnaire, and occurrence of adverse effects including bone mass density loss, at pre-treatment, after six months of treatment, at the end of treatment, and six months after discontinuation of treatment. RESULTS: Patients treated either with GnRH analogue alone and plus add-back therapy showed a higher reduction of pelvic pain, dysmenorrhea, and dyspareunia then ones treated with oral contraceptive, whereas patients treated with add-back therapy showed a better quality of life, assessed by the SF-36 questionnaire, and adverse effects rate, then the other two groups. CONCLUSIONS: Add-back therapy allows treating women with relapse of endometriosis-associated pain for a longer period reducing bone mineral density loss, with a good control of pain symptoms, compared to GnRH analogue alone or oral contraceptive, and a better patient’s quality of life, who have a better general perception of well-being then is experienced with the other two treatments. Supported by: None
P-105 Effects of Pre-Operative Gonadotrophin-Releasing Hormone Agonist (GnRHa) and Continuous Progestin Treatments on Expression of Potential Candidate Genes Involved in Endometriosis Pathophysiology. S. Matsuzaki, M. Canis, J. Pouly, B. Rabischong, R. Botchorischvili, G. Mage. Polyclinique de l’Hotel-Dieu, CHU, Clermont-Ferrand, France.
FERTILITY & STERILITY威
OBJECTIVE: Our recent DNA microarray analysis using laser capture microdissection (LCM) identified several potential candidate genes that might be involved in endometriosis pathopysiology. Heat shock protein 90 alpha (Hsp90A), which actively participates in steroid-induced signal transduction, was up-regulated and two potential negative regulators of aromatase expression, chicken ovalbumin upstream promoter transcription factor 2 (COUP-TF2) and prostaglandin E2 receptor subtype EP3 (PGE2EP3), were down-regulated in endometriosis epithelial cells. Plateletderived growth factor receptor alpha (PDGFRA) and members of the downstream RAS/RAF/MAPK signaling pathway were differentially expressed in stromal cells between endometriosis and matched eutopic endometrium (Up-regulation: PDGFRA, PKC beta1, JAK1; Down-regulation: sprouty2, MKK7). Studies to date have shown that both gonadotrophinreleasing hormone agonists (GnRHa) and continuous progestins are effective treatment regimens in patients with endometriosis in terms of reduction in rAFS score and relief of symptoms. However, these medical treatments are ineffective in eliminating the disease in most patients. Studies to investigate the changes in expression levels of the potential candidate genes during these medical treatments may well provide a better understanding of the pathophysiology and give rise to novel therapeutic strategies for this disease. In the present study, we further investigated mRNA expression levels of the potential candidate genes in deep endometriotic tissues during preoperative GnRHa or continuous oral progestin (P) treatments, using LCM and real-time RT-PCR techniques. DESIGN: Prospective study MATERIALS AND METHODS: Samples of 1) DIE (n⫽16) and matched eutopic endometrium (EE) (n⫽16) without pre-operative treatment, 2) pre-operative GnRHa-treated DIE (n⫽6) and 3) pre-operative P-treated DIE (n⫽5), were obtained during laparoscopic surgery. Glandular epithelial cells and stromal cells from endometriotic tissues were microdissected using LCM. Total RNA (10 ng) from microdissected tissues was subjected to an RT reaction and quantitative real-time PCR was performed in a Light Cycler System. The Mann-Whitney U test or the Kruskal-Wallis test was applied to compare results from different groups. The wilcoxon sign rank test was performed to compare differences in paired samples. Statistical significance was defined as p ⬍ 0.05. RESULTS: Expression levels of Hsp90A in epithelial cells were significantly lower in GnRHa-treated DIE (P⬍.007) than those in untreated DIE. There was no significant difference in Hsp90A expression between P-treated and untreated DIE. Sprouty 2 expression levels in stromal cells were significantly higher in both GnRHa- (P⬍.04) and P-treated (P⬍.009) DIE than those in untreated DIE. However, no significant difference was detected in expression levels of the remaining 6 genes, COUP-TF2 and PGE2EP3 in epithelial cells, and PDGFRA, PKC beta1, JAK and MKK7 in stromal cells among untreated, GnRHaand P-treated DIE. CONCLUSIONS: The present findings could partly explain why current medical treatment regimes are ineffective in eliminating the disease in most patients. Supported by: Supported in part by grants PHRC 2003 and PHRC 2004 of CHU Clermont-Ferrand
P-106 Plasminogen Activator Inhibitor (PAI) and Thrombin Activatable Fibrinolysis Inhibitor (TAFI) Polymorphisms in Women With Endometriosis. M. A. Bedaiwy, T. Falcone, R. F. Casper. Mount Sinai Hospital, Toronto, ON, Canada; The Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: Although most women experience retrograde menses during their reproductive life, endometriosis develops only in a small percentage. It has been shown that cells from endometrial explants can proliferate and invade a 3-dimensional fibrin matrix in vitro resulting in the formation of typical endometriotic tissue. We hypothesized that persistence of a fibrin matrix in peritoneal pockets could allow menstrually deposited endometrial fragments to initiate endometriosis in vivo. Polymorphisms in the genes of the fibrinolytic system have been shown to result in inhibition of fibrinolysis and maintenance of fibrin clots in vivo. Variants of the plasminogen activator inhibitor-1 (PAI-1) gene, containing at least one 4G allele, are associated with higher PAI-1 activity than the 5G/5G genotype. The Thr325Ile mutation in the TAFI gene has also been shown to be associated with increased antifibrinolytic
S189