- Email: [email protected]
Surgical management of endometriosis does not affect oxidative damage in endometriotic tissue over time
compared to controls. DNA methylation arrays showed heightened methylation of this gene’s promoter region in tissues from endometriosis patients with pain compared to controls. CONCLUSIONS: Our results thus far suggest that the upregulation of polycomb proteins in endometriotic tissues modulates DNA methylation of the forkhead protein resulting in its decreased expression. This likely leads to increased growth of the endometriotic tissue in the peritoneal environment, as well as increased pain. Our results have uncovered a network of proteins that may aid in the progression of endometriotic lesions. This underlying mechanism has yet to be exploited for therapeutic value in endometriosis. Supported by: Marshall University/University of Kentucky CCTS (NIHfunded).
P-173 Tuesday, October 20, 2015 EFFICACY OF NATURAL CYCLE ENDOMETRIAL PREPARATION FOR FROZEN-THAWED EMBRYO TRANSFER IN PATIENTS WITH ENDOMETRIOSIS. H. Guo. Shanghai Ninth People’s Hospital, Shanghai, China.
P-172 Tuesday, October 20, 2015 THE EPIGENETIC ROLE OF EZH2-FOXP3 CROSSTALK IN ENDOMETRIOSIS AND ITS ASSOCIATED PAIN. K. Wright N. Santanam. Pharmacology, Physiology, and Toxicology, Marshall University, Huntington, WV. OBJECTIVE: Determine the epigenetic mechanisms involved with polycomb group proteins and associated genes in the development and progression of endometriosis and its associated pain. DESIGN: Patient tissues classified by location (eutopic or ectopic) and the presence of pain were studied to determine trends in genetic expression, interaction, and methylation. MATERIALS AND METHODS: This study involved tissues from control (non-endometriosis) subjects without pain (n¼4), as well as eutopic (nonlesion, n¼7) and endometriotic (ectopic, n¼4) tissues from patients with endometriosis and pain. All specimens were obtained from IRB-approved and consented subjects. Real-time PCR was used to analyze gene expression of polycomb repressor complex 2 (PRC2) components and Western blot was used to determine the relative protein levels. Interaction of tumor suppressor Forkhead box P3 (FoxP3) as a potential target for the PRC2 catalytic subunit, Enhancer of zeste 2 (EZH2), was also measured using chromatin immunoprecipitation and immunoblotting. Lastly, Qiagen EpiTect Methyl II Array was also used to identify methylation patterns of genes associated with inflammation and autoimmunity in endometriotic fluids compared to controls. RESULTS: Expression of EZH2 was elevated in ectopic tissues compared to controls, while lowered in eutopic tissue in endometriosis patients (fold change ¼ 3.54, 0.11, respectively). Western blots also revealed increased levels of the tri-methylation of lysine 27 on histone 3 (H3K27me3). FoxP3 gene expression was much lower in both ectopic and eutopic tissues
FERTILITY & STERILITYÒ
OBJECTIVE: To assess the efficiency of natural cycle(NC) endometrial preparation for frozen-thawed embryo transfer (FET) in women with endometriosis(EMS). DESIGN: Retrospective study MATERIALS AND METHODS: This was a retrospective study of the use of the natural cycle for endometrial preparation following FET cycles in patients with endometriosis from March, 2011 to August, 2013. The study groups included 97 patients with stage I-II endometriosis who underwent a total of 120 FET cycles(group A, included cases of mild and minimal endometriosis) and 179 patients with stage III-IV endometriosis who underwent a total of 233 FET cycles(group B, included cases of moderate and severe endometriosis). The control group included 258 patients with tubal factor infertility who underwent a total of 300 FET cycles(group C). In stage IIIIV endometriosis , 40 patients (47 FET cycles,groupB1) had endometrial cyst recurrence after laparoscopic or laparotomy treatment. All patients enrolled in the study met the following inclusion criteria: (i) normal uterine cavity as assessed by ultrasonography, hysterosalpingography or hysteroscopy; (ii) high-quality frozen embryos were transplanted. (iii)normal male semen. The exclusion criteria comprised: (i) presence of hydrosalpinges; (ii) presence of adenomyosis and (iii) past history of myomectomy;(iv) polycystic ovary syndrome(PCOS); (v)repeated failed intrauterine insemination (IUI ). For group A , B and C ,we used the natural cycle for endometrium preparation. The patients’ characteristics, including age, duration of infertility, body mass index, number of embryos transferred, average endometrial thickness of ET (mm), basal FSH, serum E2 and P of transplantation day, No. of previous failed embryo transfer cycle, the rates of basal FSH>10 were recorded. Secondary measures included clinical pregnancy rate, live birth rate, ongoing pregnancy rate, implantation rate, and pregnancy complication rate. RESULTS: There were comparable clinical pregnancy rate (43.33% stage I-II, 50.21% stageIII-IV and 45.33% tubal factor )and live birth rate(36.67% stage I-II, 41.63% stageIII-IV and 40.00% tubal factor ) among the three groups, not depending on severity of endometriosis . No differences were found in other pregnancy parameters , in terms of ongoing pregnancy rate(36.67% stage I-II, 41.63% stageIII-IVand 40.00% tubal factor ) , miscarriage rate(14.89% stage I-II, 16.24% stageIII-IV and 10.29% tubal factor ), pregnancy complication rate(7.69% stage I-II, 10.26% stageIII-IV and 12.50% tubal factor ). No congenital birth defects were found in the endometriosis groups. In addition, when high-quality embryos are transferred, the pregnancy results are not affected by endometriomas. CONCLUSIONS: Natural cycle endometrial preparation for FET obtains similar pregnancy outcomes in patients with endometriosis compared with tubal infertility, and don’t increase the risk of birth defects and other complications.
P-174 Tuesday, October 20, 2015 SURGICAL MANAGEMENT OF ENDOMETRIOSIS DOES NOT AFFECT OXIDATIVE DAMAGE IN ENDOMETRIOTIC TISSUE OVER TIME. I. Al-Aref, L. R. Goodman, R. Flyckt, A. Goyal, T. Falcone. Cleveland Clinic, Cleveland, OH. OBJECTIVE: Oxidative stress has been considered as a potential factor involved in the pathology and progression of endometriosis. The objective of this study was to evaluate the natural progression of endometriosis by
e165
comparing oxidative damage in endometriotic tissue at two distinct time points. DESIGN: Retrospective cohort trial MATERIALS AND METHODS: Women who had presented between 1999-2009 with pelvic pain and/or infertility between the ages of 18-45 years with a history of two distinct surgeries with biopsy proven endometriosis were included if there was banked blood and tissue samples available for review. Histopathochemistry was used to evaluate oxidative damage by measuring hydroxy deoxyguaninine (8OHdG) in endometriotic cells and to evaluate the cellular ability for DNA repair with 8-oxoguanine glycosylase 1 (OGG1). The markers were measured in a biopsy sample of pathology proven endometriosis for each patient at each surgery and compared to each other using Wilcoxan rank-sum tests. The pathologist was blinded to whether the samples were from the primary or secondary surgery. All surgeries were performed by the same surgeon. RESULTS: There were seven patients who met criteria and were included in the study. The average age at first surgery was 32.4 +/- 4.4 years. The average time between surgeries was 2.0 +/- 1.1 years. Stage of disease and marker intensity and presence in percentage of cells at first and second surgery are shown in table 1.
Table 1: Stage of endometriosis and markers at first and second surgery.
1 2 3 4 5 6 7
Stage
8-OHdG Intensity
8-OHdG % of cells
OGG1 Intensity
OGG1 % of cells
IV/IV IV/IV IV/IV IV/IV IV/IV IV/IV III/III
0/1 2/3 3/3 2/2 3/2 2/2 2/3
0/10 80/90 100/80 80/60 75/75 40/5 60/80
2/2 3/2 2/3 2/2 3/3 3/3 3/3
90/80 90/30 10/80 50/80 95/95 90/90 95/90
Despite complete excision of all visible endometriosis at the time of first surgery, all patients had return of the disease to the same stage at their second surgery. Temporally, there was no significant difference between perUseThumbs /Page 1 [ {Catalog} > /PUT pdfmark [ {Catalog} > ] >> >> /PUT pdfmarkcentage of cells that expressed 8-OHdG (p ¼ 0.79) or OGG1 (p ¼ 0.81) between the two surgeries. There was a correlation of 8-OHdG percent of cells and intensity (r ¼ 0.80, p < 0.01) and OGG1 percent of cells and intensity (r ¼ 0.65, p < 0.01), but no temporal relationship between surgeries. CONCLUSIONS: Overall, There was no relationship of progression of oxidative damage over time. Based on these results, it appears that baseline oxidative damage in endometriotic cells is predictive of future cell damage and that surgical excision of endometriosis has no benefit in changing the underlying pathophysiology leading to oxidative damage. P-175 Tuesday, October 20, 2015 WITHDRAWN
e166
ASRM Abstracts
P-176 Tuesday, October 20, 2015 THE RELATIONSHIP OF CASPASE-3, CASPASE-9, MMP-9 EXPRESSION AND C-1562T MMP-9 GENE POLYMORPHISM IN MENSTRUAL BLOOD AS THE ETIOPATHOGENESIS MARKER TO CLINICAL ENDOMETRIOSIS MANIFESTATION. T. Madjid. Faculty of Medicine, University of Padjadjaran, Bandung, Indonesia. OBJECTIVE: The aim of this study is to discover non-invasive diagnostic method of endo-metriosis using menstrual blood, and also to reveal clearer understanding in patho-genesis of endometriosis DESIGN: A case control study involving 149 women who visited the Reproductive Endocrinology and Fertility Clinic, FKUP/RSHS and RSHS network hospitals was performed, from February 2007 to February 2008. MATERIALS AND METHODS: Screening for suspected endometriosis was performed by history taking, physical examination, and additional examination. Diagnostic laparoscopy or laparotomy and biopsy was performed afterward. The immunocytochemical examination on caspase-3, caspase-9, MMP-9, and mmp-9 gene polymorphism of menstrual blood were performed. Based on the microscopic confir-mation of histopathological result, the relationship of endometriotic and non-endometriotic clinical manifestation with menstrual blood biomolecular marker was assessed. RESULTS: Sixty-three (42.28%) endometriosis cases and 86 (57.78%) non-endometriosis cases were found. From those subjects, 34 endometriosis cases and 48 non-endometriosis cases with complete data were enrolled in this study. The endometrial cells were successfully isolated using preservative solution, and with immunocytochemical assay, all samples from 34 endometriosis subjects could be analyzed for the expression of caspase-3, caspase-9, and MMP-9. The decreased expression of caspase-3 and caspase-9, and increased expression of MMP-9 in endometriosis group were higher than those in non-endometriosis group (82.4% vs 77.1%, p¼0.562; 97.1% vs 87.5%, pE-F¼0.129; and 85.3% vs 85.4%, p¼0.988 respectively). The frequency of allele C in -1562T region of mmp-9 gene was significantly increased in endometriosis group (p<0.039). There is a significant relationship between minimal response to pain treatment and menstrual abnormalities (menorrhagia, polymenorrhea, and menometrorrhagia) with MMP-9 biomolecular marker (p¼0.006 and 0,050 respectively). At least 4 from 12 symptoms and 1 from 5 physical signs have strong relationship with the occurrence of endometriosis (OR 3.0 and 2.65 respectively). CONCLUSIONS: The results of this study lead to a conclusion that minimal response to treatment of pain, and menstrual abnormalities (menorrhagia, polymenorrhea, and menome-trorrhagia) have diagnostic values for establishing diagnosis of endometriosis. There are no significant relationship between frequency of allele C and allele T in -1562 region of mmp-9 gene with MMP-9 expression. Immunositochemistry analysis of the menstrual blood endometrial cells can be applied as a non-invasive method for establishing diagnosis of endometriosis in daily practice.
Vol. 104, No. 3, Supplement, September 2015
P-173 Tuesday, October 20, 2015 EFFICACY OF NATURAL CYCLE ENDOMETRIAL PREPARATION FOR FROZEN-THAWED EMBRYO TRANSFER IN PATIENTS WITH ENDOMETRIOSIS. H. Guo. Shanghai Ninth People’s Hospital, Shanghai, China.
P-172 Tuesday, October 20, 2015 THE EPIGENETIC ROLE OF EZH2-FOXP3 CROSSTALK IN ENDOMETRIOSIS AND ITS ASSOCIATED PAIN. K. Wright N. Santanam. Pharmacology, Physiology, and Toxicology, Marshall University, Huntington, WV. OBJECTIVE: Determine the epigenetic mechanisms involved with polycomb group proteins and associated genes in the development and progression of endometriosis and its associated pain. DESIGN: Patient tissues classified by location (eutopic or ectopic) and the presence of pain were studied to determine trends in genetic expression, interaction, and methylation. MATERIALS AND METHODS: This study involved tissues from control (non-endometriosis) subjects without pain (n¼4), as well as eutopic (nonlesion, n¼7) and endometriotic (ectopic, n¼4) tissues from patients with endometriosis and pain. All specimens were obtained from IRB-approved and consented subjects. Real-time PCR was used to analyze gene expression of polycomb repressor complex 2 (PRC2) components and Western blot was used to determine the relative protein levels. Interaction of tumor suppressor Forkhead box P3 (FoxP3) as a potential target for the PRC2 catalytic subunit, Enhancer of zeste 2 (EZH2), was also measured using chromatin immunoprecipitation and immunoblotting. Lastly, Qiagen EpiTect Methyl II Array was also used to identify methylation patterns of genes associated with inflammation and autoimmunity in endometriotic fluids compared to controls. RESULTS: Expression of EZH2 was elevated in ectopic tissues compared to controls, while lowered in eutopic tissue in endometriosis patients (fold change ¼ 3.54, 0.11, respectively). Western blots also revealed increased levels of the tri-methylation of lysine 27 on histone 3 (H3K27me3). FoxP3 gene expression was much lower in both ectopic and eutopic tissues
FERTILITY & STERILITYÒ
OBJECTIVE: To assess the efficiency of natural cycle(NC) endometrial preparation for frozen-thawed embryo transfer (FET) in women with endometriosis(EMS). DESIGN: Retrospective study MATERIALS AND METHODS: This was a retrospective study of the use of the natural cycle for endometrial preparation following FET cycles in patients with endometriosis from March, 2011 to August, 2013. The study groups included 97 patients with stage I-II endometriosis who underwent a total of 120 FET cycles(group A, included cases of mild and minimal endometriosis) and 179 patients with stage III-IV endometriosis who underwent a total of 233 FET cycles(group B, included cases of moderate and severe endometriosis). The control group included 258 patients with tubal factor infertility who underwent a total of 300 FET cycles(group C). In stage IIIIV endometriosis , 40 patients (47 FET cycles,groupB1) had endometrial cyst recurrence after laparoscopic or laparotomy treatment. All patients enrolled in the study met the following inclusion criteria: (i) normal uterine cavity as assessed by ultrasonography, hysterosalpingography or hysteroscopy; (ii) high-quality frozen embryos were transplanted. (iii)normal male semen. The exclusion criteria comprised: (i) presence of hydrosalpinges; (ii) presence of adenomyosis and (iii) past history of myomectomy;(iv) polycystic ovary syndrome(PCOS); (v)repeated failed intrauterine insemination (IUI ). For group A , B and C ,we used the natural cycle for endometrium preparation. The patients’ characteristics, including age, duration of infertility, body mass index, number of embryos transferred, average endometrial thickness of ET (mm), basal FSH, serum E2 and P of transplantation day, No. of previous failed embryo transfer cycle, the rates of basal FSH>10 were recorded. Secondary measures included clinical pregnancy rate, live birth rate, ongoing pregnancy rate, implantation rate, and pregnancy complication rate. RESULTS: There were comparable clinical pregnancy rate (43.33% stage I-II, 50.21% stageIII-IV and 45.33% tubal factor )and live birth rate(36.67% stage I-II, 41.63% stageIII-IV and 40.00% tubal factor ) among the three groups, not depending on severity of endometriosis . No differences were found in other pregnancy parameters , in terms of ongoing pregnancy rate(36.67% stage I-II, 41.63% stageIII-IVand 40.00% tubal factor ) , miscarriage rate(14.89% stage I-II, 16.24% stageIII-IV and 10.29% tubal factor ), pregnancy complication rate(7.69% stage I-II, 10.26% stageIII-IV and 12.50% tubal factor ). No congenital birth defects were found in the endometriosis groups. In addition, when high-quality embryos are transferred, the pregnancy results are not affected by endometriomas. CONCLUSIONS: Natural cycle endometrial preparation for FET obtains similar pregnancy outcomes in patients with endometriosis compared with tubal infertility, and don’t increase the risk of birth defects and other complications.
P-174 Tuesday, October 20, 2015 SURGICAL MANAGEMENT OF ENDOMETRIOSIS DOES NOT AFFECT OXIDATIVE DAMAGE IN ENDOMETRIOTIC TISSUE OVER TIME. I. Al-Aref, L. R. Goodman, R. Flyckt, A. Goyal, T. Falcone. Cleveland Clinic, Cleveland, OH. OBJECTIVE: Oxidative stress has been considered as a potential factor involved in the pathology and progression of endometriosis. The objective of this study was to evaluate the natural progression of endometriosis by
e165
comparing oxidative damage in endometriotic tissue at two distinct time points. DESIGN: Retrospective cohort trial MATERIALS AND METHODS: Women who had presented between 1999-2009 with pelvic pain and/or infertility between the ages of 18-45 years with a history of two distinct surgeries with biopsy proven endometriosis were included if there was banked blood and tissue samples available for review. Histopathochemistry was used to evaluate oxidative damage by measuring hydroxy deoxyguaninine (8OHdG) in endometriotic cells and to evaluate the cellular ability for DNA repair with 8-oxoguanine glycosylase 1 (OGG1). The markers were measured in a biopsy sample of pathology proven endometriosis for each patient at each surgery and compared to each other using Wilcoxan rank-sum tests. The pathologist was blinded to whether the samples were from the primary or secondary surgery. All surgeries were performed by the same surgeon. RESULTS: There were seven patients who met criteria and were included in the study. The average age at first surgery was 32.4 +/- 4.4 years. The average time between surgeries was 2.0 +/- 1.1 years. Stage of disease and marker intensity and presence in percentage of cells at first and second surgery are shown in table 1.
Table 1: Stage of endometriosis and markers at first and second surgery.
1 2 3 4 5 6 7
Stage
8-OHdG Intensity
8-OHdG % of cells
OGG1 Intensity
OGG1 % of cells
IV/IV IV/IV IV/IV IV/IV IV/IV IV/IV III/III
0/1 2/3 3/3 2/2 3/2 2/2 2/3
0/10 80/90 100/80 80/60 75/75 40/5 60/80
2/2 3/2 2/3 2/2 3/3 3/3 3/3
90/80 90/30 10/80 50/80 95/95 90/90 95/90
Despite complete excision of all visible endometriosis at the time of first surgery, all patients had return of the disease to the same stage at their second surgery. Temporally, there was no significant difference between perUseThumbs /Page 1 [ {Catalog} > /PUT pdfmark [ {Catalog} > ] >> >> /PUT pdfmarkcentage of cells that expressed 8-OHdG (p ¼ 0.79) or OGG1 (p ¼ 0.81) between the two surgeries. There was a correlation of 8-OHdG percent of cells and intensity (r ¼ 0.80, p < 0.01) and OGG1 percent of cells and intensity (r ¼ 0.65, p < 0.01), but no temporal relationship between surgeries. CONCLUSIONS: Overall, There was no relationship of progression of oxidative damage over time. Based on these results, it appears that baseline oxidative damage in endometriotic cells is predictive of future cell damage and that surgical excision of endometriosis has no benefit in changing the underlying pathophysiology leading to oxidative damage. P-175 Tuesday, October 20, 2015 WITHDRAWN
e166
ASRM Abstracts
P-176 Tuesday, October 20, 2015 THE RELATIONSHIP OF CASPASE-3, CASPASE-9, MMP-9 EXPRESSION AND C-1562T MMP-9 GENE POLYMORPHISM IN MENSTRUAL BLOOD AS THE ETIOPATHOGENESIS MARKER TO CLINICAL ENDOMETRIOSIS MANIFESTATION. T. Madjid. Faculty of Medicine, University of Padjadjaran, Bandung, Indonesia. OBJECTIVE: The aim of this study is to discover non-invasive diagnostic method of endo-metriosis using menstrual blood, and also to reveal clearer understanding in patho-genesis of endometriosis DESIGN: A case control study involving 149 women who visited the Reproductive Endocrinology and Fertility Clinic, FKUP/RSHS and RSHS network hospitals was performed, from February 2007 to February 2008. MATERIALS AND METHODS: Screening for suspected endometriosis was performed by history taking, physical examination, and additional examination. Diagnostic laparoscopy or laparotomy and biopsy was performed afterward. The immunocytochemical examination on caspase-3, caspase-9, MMP-9, and mmp-9 gene polymorphism of menstrual blood were performed. Based on the microscopic confir-mation of histopathological result, the relationship of endometriotic and non-endometriotic clinical manifestation with menstrual blood biomolecular marker was assessed. RESULTS: Sixty-three (42.28%) endometriosis cases and 86 (57.78%) non-endometriosis cases were found. From those subjects, 34 endometriosis cases and 48 non-endometriosis cases with complete data were enrolled in this study. The endometrial cells were successfully isolated using preservative solution, and with immunocytochemical assay, all samples from 34 endometriosis subjects could be analyzed for the expression of caspase-3, caspase-9, and MMP-9. The decreased expression of caspase-3 and caspase-9, and increased expression of MMP-9 in endometriosis group were higher than those in non-endometriosis group (82.4% vs 77.1%, p¼0.562; 97.1% vs 87.5%, pE-F¼0.129; and 85.3% vs 85.4%, p¼0.988 respectively). The frequency of allele C in -1562T region of mmp-9 gene was significantly increased in endometriosis group (p<0.039). There is a significant relationship between minimal response to pain treatment and menstrual abnormalities (menorrhagia, polymenorrhea, and menometrorrhagia) with MMP-9 biomolecular marker (p¼0.006 and 0,050 respectively). At least 4 from 12 symptoms and 1 from 5 physical signs have strong relationship with the occurrence of endometriosis (OR 3.0 and 2.65 respectively). CONCLUSIONS: The results of this study lead to a conclusion that minimal response to treatment of pain, and menstrual abnormalities (menorrhagia, polymenorrhea, and menome-trorrhagia) have diagnostic values for establishing diagnosis of endometriosis. There are no significant relationship between frequency of allele C and allele T in -1562 region of mmp-9 gene with MMP-9 expression. Immunositochemistry analysis of the menstrual blood endometrial cells can be applied as a non-invasive method for establishing diagnosis of endometriosis in daily practice.
Vol. 104, No. 3, Supplement, September 2015