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Identification of differential gene expression in human endometriosis by subtractive hybridization and gene expression profiling with real-time PCR
steroid hormone in reproductive women, steroid hormone have to act through receptor in or out target tissue. Study have showed that their difference in the location or concentration of estrogen progesterone receptors and their isoforms in endometriosis, they may involve in the pathogenesis of endometriosis. Supported By: National Natural Science Foundation of China.
Tuesday, February 26, 2002 5:00 P.M. O-47 Identification of differential gene expression in human endometriosis by subtractive hybridization and gene expression profiling with real-time PCR. W. P. Hu, S. K. Tay. Singapore Gen Hosp, Singapore. Objective: Endometriosis is one of the most common diseases in women at reproductive age. It is also one of the major causes of infertility. There is yet no conclusion on the etiology and pathogenesis of endometriosis. It is hypothesized in this study that specific genetic alterations and/or aberrant expression of specific genes in endometrial cells is the underlying molecular mechanism for successful implantation and growth of the disseminated endometrial cells to form endometriosis. This project focused on the differential gene expression in human endometriosis compared with the paired normal uterine endometrium. Design: By making comparisons of gene expression in endometrisis against normal tissue counterparts from the same patients, we took advantage of the ‘subtractive’ effect to minimize the influence of different genetic and physiological conditions on gene expression patterns, and focused on genes that distinguished normal and ectopic tissues. Materials/Methods: Subtractive hybridization using cDNAs from the ectopic and normal uterine endometrial tissues was employed to screen the differentially expressed genes in endometriosis. The subtracted cDNA was globally amplified by PCR, and cloned into a vector to generate a subtractive library. Some 750 colonies were random-selected from the libraries for differential screening with colony-lift and/or DNA dot blot hybridization. Northern blot analysis was used to confirm the differential gene expression. Real-time PCR was applied to transcriptional profiling of the identified candidate genes in multiple tissue samples. Results: Dozens of cDNA clones were isolated and analyzed with DNA sequencing. About 20 clones were confirmed to be differentially expressed by Northern blot analysis. To identify the deregulated genes contributing to the pathogenesis of endometriosis from these potential candidates, gene expression profiling in 15 pairs of clinical tissue biopsies has been performed by using Real-time PCR. The preliminary data obtained from about 30 genes are analyzed by hierarchical clustering algorithm. The genes and experimental samples are grouped on the basis of similarities of gene expression patterns. On the other hand, gene expression profile analysis has confirmed the prevalence of differential expression of several candidate genes in the endometriotic tissues, including a protooncogene and a homeobox gene. Conclusions: Further study on the roles and mechanism of these identified genes in the development and progression of human endometriosis are warranted. Supported By: National Medical Research Council, Ministry of Health, Singapore (NMRC/0311/1998).
Tuesday, February 26, 2002 5:15 P.M. O-48 NAT2 and GSTM1 DNA polymorphisms: increased GSTM1 (active) genotypes in endometriosis. F. Z. Bischoff, D. Marquez-Do, D. Dang, S. A. Carson, J. E. Buster, J. L. Simpson. Baylor Coll of Medicine, Houston, TX. Objective: Although there is evidence of genetic predisposition and environmental factors contributing to the overall incidence of endometriosis, causative gene(s) and mechanism remain elusive. Given that endometriosis displays features similar to cancer and given that heritable differences in drug-metabolizing enzymes (DMEs) often influence cancer development
FERTILITY & STERILITY威
and/or progression, we sought to determine whether a genotype association exists between polymorphic DME genes and endometriosis. Because dioxin and dioxin-like compounds have been implicated as factors involved in the development of endometriosis, two polymorphic DMEs that are induced by dioxin—N-acetyltransferase 2 (NAT2) and Glutathione S Transferase M1 (GSTM1)—were selected for analysis. Design: Using genomic blood DNA, frequencies of the polymorphic NAT2 and GSTM1 genotypes were determined among endometriosis patients and normal controls. Materials/Methods: IRB approval and informed consent were obtained prior to initiation of the study. Control blood DNA was obtained from women surgically and histologically confirmed not to have endometriotic implants (n ⫽ 37). Blood DNA from cases with endometriosis was also collected for amplification by polymerase chain reaction (PCR) of the NAT2 (n ⫽ 111) and GSTM1 (n ⫽ 62) loci. For NAT2, restriction digestion was used to detect the wildtype (W) and mutant (M) alleles, enabling classification into slow (M/M) or rapid (W/W; W/M)-acetylation genotypes. For GSTM1, allele-specific PCR was used to distinguish active (⫹/⫺ or ⫹/⫹) from null (⫺/⫺) genotypes. Results: No difference was observed between the control and endometriosis patients for the NAT2 genotypes (Table). However, the frequency of GSTM1 active genotypes was nearly two-fold greater among endometriosis cases. Frequency of observed NAT2 and GSTM1 genotypes among patients and controls.
NAT2/slow
NAT2/rapid
GSTM1 active
GSTM1 null
Endometriosis 45/111 (41%) 66/111 (59%) 49/62 (79%) 13/62 (21%) Controls 15/37 (41%) 22/37 (59%) 16/36 (44%) 20/36 (56%) Conclusions: We found an association between endometriosis and the GSTM1 active genotypes. The GSTM1 protein functions as a phase II foreign compound metabolizing enzyme and is believed to be important in the detoxification of the oxidative stress products produced during the repair of the ovarian epithelium. That the active form is more frequent in endometriosis could reflect either production of excess toxic metabolic intermediates or interactions with other cellular components that affect the expression of causative genes or protein binding. Supported By: Work supported by the Endometriosis Association.
BASIC RESEARCH: GROWTH/DIFFERENTIATION Tuesday, February 26, 2002 2:00 P.M. O-49 Activity of extracellular superoxide dismutase in peritoneal fluid of infertile women. G. Polak, M. Koziol-Montewka, R. Tarkowski, J. Kotarski. Univ Sch of Medicine, Lublin, Poland. Objective: It has been suggested that oxidative stress in the peritoneal fluid environment may play a role in the pathogenesis of endometriosis as well as in the pathophysiology of endometriosis-associated infertility. Superoxide dismutases are important components of the defense mechanisms against oxygen toxicity. Design: We decided to assess the activity of extracellular superoxide dismutase (EC SOD) in peritoneal fluid (PF) obtained from infertile women. Materials/Methods: 59 infertile women were enrolled including 10 patients suffering from minimal and mild endometriosis (I and II rAFS), 11 women with PCOS, 14 patients with tubal infertility and 24 women diagnosed as unexplained infertility. Peritoneal fluid EC SOD activity was measured using a commercially available spectrophotometric assay (Calbiochem-Novabiochem Corp.). Results: Mean (⫾ SEM) peritoneal fluid EC SOD activity was 0.83 ⫾ 0.23 U/ml in endometriotic patients, 0.99 ⫾ 0.24 U/ml in women with PCOS, 0.76 ⫾ 0.2 U/ml in patients with tubal infertility and 0.86 ⫾ 0.12 U/ml in women with idiopathic infertility and did not differ significantly between the studied groups. Activity of ECSOD was undetectable in PF
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Tuesday, February 26, 2002 5:00 P.M. O-47 Identification of differential gene expression in human endometriosis by subtractive hybridization and gene expression profiling with real-time PCR. W. P. Hu, S. K. Tay. Singapore Gen Hosp, Singapore. Objective: Endometriosis is one of the most common diseases in women at reproductive age. It is also one of the major causes of infertility. There is yet no conclusion on the etiology and pathogenesis of endometriosis. It is hypothesized in this study that specific genetic alterations and/or aberrant expression of specific genes in endometrial cells is the underlying molecular mechanism for successful implantation and growth of the disseminated endometrial cells to form endometriosis. This project focused on the differential gene expression in human endometriosis compared with the paired normal uterine endometrium. Design: By making comparisons of gene expression in endometrisis against normal tissue counterparts from the same patients, we took advantage of the ‘subtractive’ effect to minimize the influence of different genetic and physiological conditions on gene expression patterns, and focused on genes that distinguished normal and ectopic tissues. Materials/Methods: Subtractive hybridization using cDNAs from the ectopic and normal uterine endometrial tissues was employed to screen the differentially expressed genes in endometriosis. The subtracted cDNA was globally amplified by PCR, and cloned into a vector to generate a subtractive library. Some 750 colonies were random-selected from the libraries for differential screening with colony-lift and/or DNA dot blot hybridization. Northern blot analysis was used to confirm the differential gene expression. Real-time PCR was applied to transcriptional profiling of the identified candidate genes in multiple tissue samples. Results: Dozens of cDNA clones were isolated and analyzed with DNA sequencing. About 20 clones were confirmed to be differentially expressed by Northern blot analysis. To identify the deregulated genes contributing to the pathogenesis of endometriosis from these potential candidates, gene expression profiling in 15 pairs of clinical tissue biopsies has been performed by using Real-time PCR. The preliminary data obtained from about 30 genes are analyzed by hierarchical clustering algorithm. The genes and experimental samples are grouped on the basis of similarities of gene expression patterns. On the other hand, gene expression profile analysis has confirmed the prevalence of differential expression of several candidate genes in the endometriotic tissues, including a protooncogene and a homeobox gene. Conclusions: Further study on the roles and mechanism of these identified genes in the development and progression of human endometriosis are warranted. Supported By: National Medical Research Council, Ministry of Health, Singapore (NMRC/0311/1998).
Tuesday, February 26, 2002 5:15 P.M. O-48 NAT2 and GSTM1 DNA polymorphisms: increased GSTM1 (active) genotypes in endometriosis. F. Z. Bischoff, D. Marquez-Do, D. Dang, S. A. Carson, J. E. Buster, J. L. Simpson. Baylor Coll of Medicine, Houston, TX. Objective: Although there is evidence of genetic predisposition and environmental factors contributing to the overall incidence of endometriosis, causative gene(s) and mechanism remain elusive. Given that endometriosis displays features similar to cancer and given that heritable differences in drug-metabolizing enzymes (DMEs) often influence cancer development
FERTILITY & STERILITY威
and/or progression, we sought to determine whether a genotype association exists between polymorphic DME genes and endometriosis. Because dioxin and dioxin-like compounds have been implicated as factors involved in the development of endometriosis, two polymorphic DMEs that are induced by dioxin—N-acetyltransferase 2 (NAT2) and Glutathione S Transferase M1 (GSTM1)—were selected for analysis. Design: Using genomic blood DNA, frequencies of the polymorphic NAT2 and GSTM1 genotypes were determined among endometriosis patients and normal controls. Materials/Methods: IRB approval and informed consent were obtained prior to initiation of the study. Control blood DNA was obtained from women surgically and histologically confirmed not to have endometriotic implants (n ⫽ 37). Blood DNA from cases with endometriosis was also collected for amplification by polymerase chain reaction (PCR) of the NAT2 (n ⫽ 111) and GSTM1 (n ⫽ 62) loci. For NAT2, restriction digestion was used to detect the wildtype (W) and mutant (M) alleles, enabling classification into slow (M/M) or rapid (W/W; W/M)-acetylation genotypes. For GSTM1, allele-specific PCR was used to distinguish active (⫹/⫺ or ⫹/⫹) from null (⫺/⫺) genotypes. Results: No difference was observed between the control and endometriosis patients for the NAT2 genotypes (Table). However, the frequency of GSTM1 active genotypes was nearly two-fold greater among endometriosis cases. Frequency of observed NAT2 and GSTM1 genotypes among patients and controls.
NAT2/slow
NAT2/rapid
GSTM1 active
GSTM1 null
Endometriosis 45/111 (41%) 66/111 (59%) 49/62 (79%) 13/62 (21%) Controls 15/37 (41%) 22/37 (59%) 16/36 (44%) 20/36 (56%) Conclusions: We found an association between endometriosis and the GSTM1 active genotypes. The GSTM1 protein functions as a phase II foreign compound metabolizing enzyme and is believed to be important in the detoxification of the oxidative stress products produced during the repair of the ovarian epithelium. That the active form is more frequent in endometriosis could reflect either production of excess toxic metabolic intermediates or interactions with other cellular components that affect the expression of causative genes or protein binding. Supported By: Work supported by the Endometriosis Association.
BASIC RESEARCH: GROWTH/DIFFERENTIATION Tuesday, February 26, 2002 2:00 P.M. O-49 Activity of extracellular superoxide dismutase in peritoneal fluid of infertile women. G. Polak, M. Koziol-Montewka, R. Tarkowski, J. Kotarski. Univ Sch of Medicine, Lublin, Poland. Objective: It has been suggested that oxidative stress in the peritoneal fluid environment may play a role in the pathogenesis of endometriosis as well as in the pathophysiology of endometriosis-associated infertility. Superoxide dismutases are important components of the defense mechanisms against oxygen toxicity. Design: We decided to assess the activity of extracellular superoxide dismutase (EC SOD) in peritoneal fluid (PF) obtained from infertile women. Materials/Methods: 59 infertile women were enrolled including 10 patients suffering from minimal and mild endometriosis (I and II rAFS), 11 women with PCOS, 14 patients with tubal infertility and 24 women diagnosed as unexplained infertility. Peritoneal fluid EC SOD activity was measured using a commercially available spectrophotometric assay (Calbiochem-Novabiochem Corp.). Results: Mean (⫾ SEM) peritoneal fluid EC SOD activity was 0.83 ⫾ 0.23 U/ml in endometriotic patients, 0.99 ⫾ 0.24 U/ml in women with PCOS, 0.76 ⫾ 0.2 U/ml in patients with tubal infertility and 0.86 ⫾ 0.12 U/ml in women with idiopathic infertility and did not differ significantly between the studied groups. Activity of ECSOD was undetectable in PF
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