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Glycodelin mediates vascular endothelial growth factor (VEGF) production during oxidative stress of endometrial epithelial cells
normozoospermic males with semen parameters exceeding WHO (1999) reference ranges for the normal fertile population. Following liquefaction, mature spermatozoa were separated on a double density gradient centrifugation and resuspended in Biggers, Whitten and Whittingham media. Spermatozoa were incubated with 100, 300, 400, and 500 pg/mL human recombinant TNF-␣ (rTNF-␣, R & D Systems, Minneapolis, MN) for 0, 2, 6, 12 and 24 hours. Each aliquot had its corresponding control without TNF-␣. All aliquots were tested for sperm motility, plasma membrane integrity by hypoosmotic swelling (HOS) test and DNA damage by TUNEL assay at each time point. RESULTS: Significant reduction in sperm motility was observed following incubation for 24 hours with both 400 pg/mL and 500 pg/mL TNF-␣ compared to controls (400 pg/mL: 26.4 ⫾ 9.28 vs. 42.8 ⫾ 6.26, P ⫽ 0.005; 500 pg/mL: 20.4 ⫾ 7.4 vs. 35 ⫾ 6.67, P ⫽ 0.009). The lowest concentration of TNF-␣ that affected sperm motility after 24h was 300 pg/mL (P ⫽ 0.003). Sperm exposed to 500 pg/mL had significantly lower HOS values compared to controls after 12 h (P ⫽ 0.03). A significant increase in percentage of sperm with DNA fragmentation was seen following incubation for 24 hours with 400 pg/mL and 500 pg/mL of TNF-␣ compared to controls (400 pg/mL: 31.71% ⫾ 9.12% vs. 14.8% ⫾ 7.0%, P ⫽ 0.01; 500 pg/mL: 30.08% ⫾ 15.07% vs. 18.12% ⫾ 9.21%, P ⫽ 0.02). CONCLUSION: Exposure of spermatozoa to pathological concentrations of TNF-␣, similar to what may be present in endometriosis patients, can significantly result in loss of sperm motility, plasma membrane functional integrity as well as DNA damage. This may be one of the causes of infertility in cases of endometriosis. Future research should be directed towards evaluating the benefits of TNF-␣ inhibitors for treatment of infertile patients with endometriosis. Supported by: None
P-66 Reversibility of tumor necrosis factor (TNF)-␣ induced toxic effects by infliximab in human spermatozoa. T. M. Said, A. Agarwal, R. K. Sharma, M. A. Bedaiwy, T. Falcone. Cleveland Clinic Foundation, Cleveland, OH; Assuit University, Assuit, Egypt. OBJECTIVES: Patients with endometriosis present with elevated levels of TNF-␣. The presence of these pathological levels of TNF-␣ in the female reproductive tract may have toxic effects on spermatozoa. Infliximab a monoclonal antibody binds both soluble and membrane forms of TNF-␣ and neutralizes its toxic effects. The objective of our study was to evaluate the ability of Infliximab to counteract the toxic effects induced by TNF-␣ in human spermatozoa. DESIGN: Prospective-controlled study. MATERIALS AND METHODS: Semen samples were collected from 6 normozoospermic males. Following liquefaction, spermatozoa were separated on a double density gradient and resuspended in BWW media. Four aliquots of sperm suspension were incubated at 37°C for 6 hours with: 1) human recombinant TNF-␣ (rTNF-␣, 2.5 g/mL, R & D systems, Minneapolis, MN); 2) Infliximab (400 g/mL, Centocor, Malvern, PA); 3) TNF-␣ (2.5 g/mL) ⫹ Infliximab (400 g/mL); and 4) BWW alone (control). Concentration of TNF-␣, Infliximab and incubation were selected based on results from our pilot experiment. Sperm motility, functional integrity of plasma membrane (hypoosmotic swelling test, HOS) and DNA damage (TUNEL assay) were evaluated before and after the incubation period. RESULTS: Spermatozoa quality declined significantly (P⬍0.05) following 6 hours of incubation with TNF-␣ alone (percentage motility, plasma membrane integrity and DNA damage) compared to controls. The percentage of spermatozoa with DNA fragmentation was lower (P⬍0.05) in samples incubated with TNF-␣ ⫹ Infliximab compared to samples treated with TNF-␣ only. Infliximab was able to reverse the toxic effects of TNF-␣ after incubation for 6 h (Table). Similarly, sperm motility and membrane integrity was significantly improved (P⬍0.05) and was comparable both with controls and Infliximab alone (Table). CONCLUSIONS: TNF-␣ in pathological levels exerts toxic effects on human spermatozoa. Infliximab plays a significant role in reversing and conferring protection against TNF-␣ induced toxic effects. Infliximab may be a potential candidate in the management of infertility patient with endometriosis who present with elevated levels of TNF-␣. Supported by: None
FERTILITY & STERILITY威
Effect of TNF-␣, Infliximab and TNF-␣ ⫹ Infliximab on sperm motility, membrane integrity and DNA damage, following 6 hours of incubation.
P-67 Glycodelin mediates vascular endothelial growth factor (VEGF) production during oxidative stress of endometrial epithelial cells. J. K. Park, M. Q. Song, S. Parthsarathy, C. E. Dominguez, A. A. Murphy. Emory University School of Medicine, Atlanta, GA; Louisiana State University, New Orleans, LA. OBJECTIVE: The purpose of this study was to determine the relationship between glycodelin and vascular endothelial growth factor (VEGF) produced by endometrial epithelial cells while subjected to oxidative stress. We hypothesized that glycodelin, produced during oxidative stress, promotes VEGF expression in endometrial epithelial cells. DESIGN: In vitro study. MATERIALS AND METHODS: Cultures of EM42 cells (human endometrial epithelial cell line) were grown at a concentration of 1 ⫻ 103/ml. The EM42 cells were subjected to oxidative stress in the form of minimally oxidized LDL (mLDL) and extensively oxidized LDL (oxLDL). Cells were grown for 24 hrs with media containing either no LDL, 20 g/ml of native LDL (nLDL), 20 g/ml of mLDL, or 20 g/ml of oxLDL. Each condition was grown with and without 50 g of glycodelin antibody. Cell culture supernatant was removed after 24 hrs and ELISA assays were performed for glycodelin and VEGF. RESULTS: Glycodelin concentrations were significantly higher in EM42 cells grown with nLDL (P⬍0.05), mLDL (P⬍0.01) and oxLDL (P⬍0.01) compared to controls. Maximal glycodelin concentration was from cells grown with mLDL and was significantly higher compared to cells grown with nLDL (P⬍0.05). Concentration of VEGF was significantly increased for EM42 cells treated with mLDL (P⬍0.01) and oxLDL (P⬍0.01) compared to controls. Maximal VEGF concentration was found from cells grown with mLDL, which was significantly higher compared to cells grown with oxLDL (P⬍0.05). The addition of glycodelin antibody resulted in a significant reduction in VEGF concentration for cells grown with mLDL (P⬍0.01) and oxLDL (P⬍0.05). Statistical analysis was performed by ANOVA followed by the t test using the Bonferroni correction for multiple comparisons. CONCLUSION: This study demonstrates that oxidative stress in the form of mLDL and oxLDL increases EM42 cell production of glycodelin and VEGF. These angiogenic proteins may contribute to ectopic endometrial cell neovascularization as a result of oxidative stress. In addition, this study demonstrates that VEGF regulation is at least partly mediated by the glycodelin produced by EM42 cells during oxidative stress. Supported by: Financial support was provided by a grant from NIH/ NICHD #K24HD01471– 01.
P-68 High prevalence of progesterone receptor gene polymorphism (PROGINS) found in women with endometriosis. C. V. De Carvalho, P. D’Amora, E. Schor, H. Sato, M. J. C. Gira˜ o, I. D. G. Da Silva. Molecular Ginecology Laboratory, Department of Gynecology, Federal University of Sa˜ o Paulo and Centro Universita´ rio Fundac¸ a˜ o Santo Andre´ , Sa˜ o Paulo, Brazil. OBJECTIVE: The aim of the study was to investigate whether mutant PROGINS allele could serve as a marker of susceptibility to endometriosis. DESIGN: Case control study. MATERIALS AND METHODS: We have genotyped in a population of
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P-66 Reversibility of tumor necrosis factor (TNF)-␣ induced toxic effects by infliximab in human spermatozoa. T. M. Said, A. Agarwal, R. K. Sharma, M. A. Bedaiwy, T. Falcone. Cleveland Clinic Foundation, Cleveland, OH; Assuit University, Assuit, Egypt. OBJECTIVES: Patients with endometriosis present with elevated levels of TNF-␣. The presence of these pathological levels of TNF-␣ in the female reproductive tract may have toxic effects on spermatozoa. Infliximab a monoclonal antibody binds both soluble and membrane forms of TNF-␣ and neutralizes its toxic effects. The objective of our study was to evaluate the ability of Infliximab to counteract the toxic effects induced by TNF-␣ in human spermatozoa. DESIGN: Prospective-controlled study. MATERIALS AND METHODS: Semen samples were collected from 6 normozoospermic males. Following liquefaction, spermatozoa were separated on a double density gradient and resuspended in BWW media. Four aliquots of sperm suspension were incubated at 37°C for 6 hours with: 1) human recombinant TNF-␣ (rTNF-␣, 2.5 g/mL, R & D systems, Minneapolis, MN); 2) Infliximab (400 g/mL, Centocor, Malvern, PA); 3) TNF-␣ (2.5 g/mL) ⫹ Infliximab (400 g/mL); and 4) BWW alone (control). Concentration of TNF-␣, Infliximab and incubation were selected based on results from our pilot experiment. Sperm motility, functional integrity of plasma membrane (hypoosmotic swelling test, HOS) and DNA damage (TUNEL assay) were evaluated before and after the incubation period. RESULTS: Spermatozoa quality declined significantly (P⬍0.05) following 6 hours of incubation with TNF-␣ alone (percentage motility, plasma membrane integrity and DNA damage) compared to controls. The percentage of spermatozoa with DNA fragmentation was lower (P⬍0.05) in samples incubated with TNF-␣ ⫹ Infliximab compared to samples treated with TNF-␣ only. Infliximab was able to reverse the toxic effects of TNF-␣ after incubation for 6 h (Table). Similarly, sperm motility and membrane integrity was significantly improved (P⬍0.05) and was comparable both with controls and Infliximab alone (Table). CONCLUSIONS: TNF-␣ in pathological levels exerts toxic effects on human spermatozoa. Infliximab plays a significant role in reversing and conferring protection against TNF-␣ induced toxic effects. Infliximab may be a potential candidate in the management of infertility patient with endometriosis who present with elevated levels of TNF-␣. Supported by: None
FERTILITY & STERILITY威
Effect of TNF-␣, Infliximab and TNF-␣ ⫹ Infliximab on sperm motility, membrane integrity and DNA damage, following 6 hours of incubation.
P-67 Glycodelin mediates vascular endothelial growth factor (VEGF) production during oxidative stress of endometrial epithelial cells. J. K. Park, M. Q. Song, S. Parthsarathy, C. E. Dominguez, A. A. Murphy. Emory University School of Medicine, Atlanta, GA; Louisiana State University, New Orleans, LA. OBJECTIVE: The purpose of this study was to determine the relationship between glycodelin and vascular endothelial growth factor (VEGF) produced by endometrial epithelial cells while subjected to oxidative stress. We hypothesized that glycodelin, produced during oxidative stress, promotes VEGF expression in endometrial epithelial cells. DESIGN: In vitro study. MATERIALS AND METHODS: Cultures of EM42 cells (human endometrial epithelial cell line) were grown at a concentration of 1 ⫻ 103/ml. The EM42 cells were subjected to oxidative stress in the form of minimally oxidized LDL (mLDL) and extensively oxidized LDL (oxLDL). Cells were grown for 24 hrs with media containing either no LDL, 20 g/ml of native LDL (nLDL), 20 g/ml of mLDL, or 20 g/ml of oxLDL. Each condition was grown with and without 50 g of glycodelin antibody. Cell culture supernatant was removed after 24 hrs and ELISA assays were performed for glycodelin and VEGF. RESULTS: Glycodelin concentrations were significantly higher in EM42 cells grown with nLDL (P⬍0.05), mLDL (P⬍0.01) and oxLDL (P⬍0.01) compared to controls. Maximal glycodelin concentration was from cells grown with mLDL and was significantly higher compared to cells grown with nLDL (P⬍0.05). Concentration of VEGF was significantly increased for EM42 cells treated with mLDL (P⬍0.01) and oxLDL (P⬍0.01) compared to controls. Maximal VEGF concentration was found from cells grown with mLDL, which was significantly higher compared to cells grown with oxLDL (P⬍0.05). The addition of glycodelin antibody resulted in a significant reduction in VEGF concentration for cells grown with mLDL (P⬍0.01) and oxLDL (P⬍0.05). Statistical analysis was performed by ANOVA followed by the t test using the Bonferroni correction for multiple comparisons. CONCLUSION: This study demonstrates that oxidative stress in the form of mLDL and oxLDL increases EM42 cell production of glycodelin and VEGF. These angiogenic proteins may contribute to ectopic endometrial cell neovascularization as a result of oxidative stress. In addition, this study demonstrates that VEGF regulation is at least partly mediated by the glycodelin produced by EM42 cells during oxidative stress. Supported by: Financial support was provided by a grant from NIH/ NICHD #K24HD01471– 01.
P-68 High prevalence of progesterone receptor gene polymorphism (PROGINS) found in women with endometriosis. C. V. De Carvalho, P. D’Amora, E. Schor, H. Sato, M. J. C. Gira˜ o, I. D. G. Da Silva. Molecular Ginecology Laboratory, Department of Gynecology, Federal University of Sa˜ o Paulo and Centro Universita´ rio Fundac¸ a˜ o Santo Andre´ , Sa˜ o Paulo, Brazil. OBJECTIVE: The aim of the study was to investigate whether mutant PROGINS allele could serve as a marker of susceptibility to endometriosis. DESIGN: Case control study. MATERIALS AND METHODS: We have genotyped in a population of
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