The effect of monocyte conditioned medium on estrogen and progesterone regulation of gene expression in cultured endometrial stromal cells

APOPTOSIS. G. M. Saed, Z. L. Jiang, N. M. Fletcher, S. Galijasevic, M. P. Diamond, H. M. Abu-Soud. The C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, MI; Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI. OBJECTIVE: We have previously identified phenotypic differences between fibroblasts isolated from normal peritoneum and adhesion tissues from the same patient(s). Specifically, adhesion fibroblasts have lower apoptosis and higher protein nitration as compared to normal fibroblasts. The adhesion phenotype can be induced by exposure of normal fibroblasts to hypoxia. The hypothesis to be tested is that increased caspase-3 nitrosylation leads to the loss of its activity which subsequently decreases apoptosis in adhesion fibroblasts. To determine whether decreased apoptosis observed in adhesion fibroblasts is caused by lower caspase-3 activity due to an increase in caspase-3 nitrosylation. DESIGN: Cell Culture Study. MATERIALS AND METHODS: Fibroblasts were treated with peroxynitrite (0.5 mM) for 24 hrs and supernatant was immunoprecipitated with anticaspase-3 polyclonal antibody conjugated with protein A/G plus agarose beads. Immunoprecipitated caspase-3 zymogen was released by boiling the beads at 95 C for 5 minutes. Biotinylated proteins were separated by SDSPAGE and detected using nitrosylation detection reagent I (HRP) according to the manufacturer’s protocol (Cayman Chemical Company, Ann Arbor, MI). Caspase-3 activity and apoptosis were measured by colormetric and TUNEL assays respectively. RESULTS: Caspase-3 nitrosylation is significantly higher in adhesion fibroblasts as compared to normal fibroblasts. This increase in nitration resulted in a 30% decrease in caspase-3 activity in adhesion fibroblasts. Peroxynitrite treatment resulted in a significant increase in caspase-3 nitrosylation, leading to a decrease in caspase-3 activity and apoptosis in normal fibroblasts. CONCLUSIONS: Our results show that nitrosylation of caspase-3 is the reason for its decreased activity and subsequent decrease in apoptosis of adhesion fibroblasts. The mechanism by which caspase-3 nitrosylation occurs is not fully understood. Although, hypoxia’s role in the formation of peroxynitrite via superoxide production suggests a possible mechanism. This may be a potential therapeutic intervention for the elimination of the adhesion fibroblasts during peritoneal healing. Supported by: NIH R01 GM 069941–01 A3.

P-303 THE EFFECT OF MONOCYTE CONDITIONED MEDIUM ON ESTROGEN AND PROGESTERONE REGULATION OF GENE EXPRESSION IN CULTURED ENDOMETRIAL STROMAL CELLS. K. M. Eyster, K. A. Hansen, O. Klinkova. Division of Basic Biomedical Sciences, Sanford School of Medicine of the University of South Dakota, Vermillion, SD; Obstetrics & Gynecology, Sanford School of Medicine of the University of South Daktoa, Sioux Falls, SD. OBJECTIVE: The objective of this project was to examine the effect of monocyte conditioned medium on the regulation of gene expression of cultured human endometrial stromal cells by estradiol and progesterone to test the hypothesis that monocytes affect endometrial cells that enter the peritoneal cavity. DESIGN: Cell culture experiments. MATERIALS AND METHODS: Telomerase-immortalized human endometrial stromal cells (T HESCs) and U937 monocytic cell line (U937) were purchased from ATCC. U937 cells were used to prepare monocyte conditioned medium (CM). T HESCs were exposed to the following treatments: vehicle control (0.1% ethanol), 108 M estradiol (E), 108 M E þ 107 M medroxyprogesterone acetate (E þ P), control (0.1%ethanol)þCM (control þ CM), 108 M E þ CM (E þ CM), and 108 M E þ 107 M P þ CM (E þ P þ CM). On day 8 of treatment, total RNA was extracted from samples. DNA microarray analysis used CodeLink Whole Human Genome Bioarrays (GE). ANOVA (GeneSpring) was used to identify differential gene expression (P%0.05). Genes showing a significant P value were further analyzed by fold-expression compared to control. Real-time RT-PCR was used to confirm differential expression of genes identified by DNA microarray and to analyze the expression of prolactin as a marker of decidualization. RESULTS: Statistical analysis identified 7288 genes to be differentially expressed in the control vs. control þ CM group with p values %0.05. Of those, 1728 were named genes. The genes were sorted into the following functional categories: signal transduction, metabolism/enzymes, cell cycle/ apoptosis related, transcription/translation regulation- related, cytoskeleton and ECM-related, receptors and unclassified genes. The expression of plas-

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minogen activator inhibitor type1 (PAI1), a decidualization marker, was markedly up-regulated in the presence of CM in all treatment groups. Growth differentiation factor 15 (GDF15) and superoxide dismutase 2 (SOD2) were found to be increased in all CM treated cells. Caspase 10 and caspase 6 were both decreased by 10-fold in control þ CM treated cells. Differential expression of PAI1 and SOD2 was confirmed by real-time RT-PCR. CONCLUSIONS: The presence of monocyte conditioned media dramatically changed gene expression in human endometrial stromal cells. These data support the hypothesis that monocytes secrete factors that modulate endometrial cell function. Supported by: This project was supported by funds from the Dept Ob/Gyn, SSOM.

P-304 POSSIBLE AGGRAVATING IMPACT OF POLYCHLORINATED BIPHENYLS, PHTHALATE ESTERS AND GLUTATHIONE S-TRANSFERASE M1 GENE NULL POLYMORPHISM IN SOUTH INDIAN WOMEN WITH ENDOMETRIOSIS. R. Rozati. Dept.of Reproductive Medicine, Baghavan Mahavir Hopital and Reaearch Center, Hyderabad, Andhrapradesh, India; Center for Fertility Management, Maternal Health and Research Trust, Hyderabad, Andhrapradesh, India. OBJECTIVE: Expression of compound metabolic enzymes is determined by polymorphism and may predispose the development of diseases aggravated by environmental factors. Glutathione S-transferase M1 (GSTM1) is an important phase II enzyme involved in the detoxification of many environmental carcinogens. GSTM1 status has been extensively studied as a risk factor for endometriosis, although inconsistent associations have been observed. To re-examine this controversy we designed a case-control study to determine the possible association between polychlorinated biphenyls (PCBs), phthalate esters (PEs) and GSTM1 null (*0/*0) mutation in the etiology of endometriosis. DESIGN: A prospective case-controlled study. MATERIALS AND METHODS: GSTM1 null genotype frequency were studied in 97 infertile laparoscopically proven women with endometriosis and 102 women without endometriosis by polymerase chain reaction (PCR) based genotyping approach. Concentrations of PCBs (PCB1, PCB5, PCB29 and PCB 98) and Pes (DnBP, BBP, DEHP and DinOP) were measured in 86/97 endometriosis and 91/102 controls due to lack of blood samples by using gas chromatography. RESULTS: Women with endometriosis showed significantly higher concentrations of PCBs and PEs compared with control group. We found that 26.8% of the cases with endometriosis and 14.7% of the controls had the GSTM1 null (*0/*0) genotype [odds ratio (OR ¼ 2.12, 95% confidence interval (CI) ¼ 1.045–4.314], which showed significant association (P¼0.03) with endometriosis. The correlation between the concentrations of PCBs, PEs, GSTM1 null genotype and different severity of endometriosis was strong and statistically significant at P<0.05. CONCLUSIONS: The study results suggest that women having higher concentration of PCBs, PEs and GSTM1 null (*0/*0) polymorphism might have an increased susceptibility of endometriosis. Supported by: None. P-305 THE LEVONORGESTREL-RELEASING INTRAUTERINE SYSTEM (LNG-IUS) AND ENDOMETRIOSIS: MOLECULAR EFFECTS ON THE TOPIC AND ON THE ECTOPIC ENDOMETRIUM. M. K. O. Gomes, R. A. Ferriani, J. C. Rosa e Silva, A. C. J. S. Rosa e Silva, C. S. Vieira, S. B. Garcia. Department of Gynecology and Obstetrics, University of Sa˜o Paulo at Ribeira˜o Preto School of Medicine, Ribeira˜o Preto, Sa˜o Paulo, Brazil; Department of Pathology, University of Sa˜o Paulo at Ribeira˜o Preto School of Medicine, Ribeira˜o Preto, Sa˜o Paulo, Brazil. OBJECTIVE: To compare the effects on proliferative and apoptosis markers from the topic and ectopic endometrium of patients with endometriosis between the LNG-IUS and the GnRH agonist(GnRHa) treatment. DESIGN: Randomized, controlled clinical trial. MATERIALS AND METHODS: Twenty-three women, with endometriosis and pelvic pain, were randomized to receive LNG-IUS (n ¼ 12) or GnRHa (n ¼ 11) for a 6 months treatment. Pre- and post-treatment endometrium and endometriosis specimens were examined immunohistochemically and changes in the expression of progesterone receptor A (PRA), estrogen receptor-a (ER-a) and Fas (a mediator of apoptotic signal) were evaluated by the H-score (a semi-quantitative microscopical method evaluating

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