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Seminal plasma lipid fingerprinting and its association to semen oxidative stress and sperm nuclear dna fragmentation
MALE REPRODUCTION AND UROLOGY - RESEARCH O-403 Wednesday, October 16, 2013 04:00 PM THE NOVEL GENETIC BIOMARKERS DLX5 AND GATA4 MAY PLAY A ROLE IN THE REGULATION OF MALE INFERTILITY. J. R. Kovac, J. Addai, L. I. Lipshultz, D. J. Lamb. Scott Department of Urology, Center for Reproductive Medicine, Baylor College of Medicine, Houston, TX. OBJECTIVE: Spermatogenic failure in men with non-obstructive azoospermia (NOA) involves mechanisms that are not well understood. This reflects the complexity of the gene signaling pathways required for normal spermatogenesis. Gene expression microarrays allow for an in-depth analysis of these pathways and a comprehensive assessment of altered cellular function. Thus, they aid in the discovery of new gene defects causing non-obstructive spermatogenesis. DESIGN: Tissues obtained from men undergoing testis biopsy for nonobstructive azoospermia (NOA; n¼16) were compared with vasectomy controls (n¼5). MATERIALS AND METHODS: Gene-expression microarray (Agilent Sureprint G3) screened for genetic variations with data evaluated via heatmaps, clustering & statistical analysis. Ingenuity Pathway Analysis (IPA) software using False Discovery Rates at 5% highlighted candidate genes and pathways involved. Quantitative PCR validated microarray data. RESULTS: Microarray data followed by IPA analysis identified DLX5 as one of the top upregulated genes in NOA men. The relative expression of both DLX5 (1.30.1 vs. 3.50.4, p<0.01) and GATA 4 (5.61.7 vs. 8.30.1, p<0.01) were significantly increased in men with NOA. qPCR confirmed NOA men exhibited a 7.3 fold increase in DLX5 expression. CONCLUSION: Increased levels of DLX5 and GATA4 expression were found in men with NOA suggesting a role in normal spermatogenesis. Since GATA4 is known to interact with DLX5 while regulating Sertoli cell function, it is tempting to speculate that altered signaling of these pathways might be associated with male infertility. Supported by: A Male Reproductive Health Research Career (MHRH) Development Physician Scientist Award (K12) (HD073917-01) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Program and by NIH grants P01HD36289 from the Eunice Kennedy Shriver NICHD and 1R01DK078121 from the National Institute of Kidney and Digestive Diseases.
O-404 Wednesday, October 16, 2013 04:15 PM SEMINAL PLASMA LIPID FINGERPRINTING AND ITS ASSOCIATION TO SEMEN OXIDATIVE STRESS AND SPERM NUCLEAR C. Bruna de Lima,a DNA FRAGMENTATION. P. Intasqui,a b c a M. Nichi, E. J. Pilau, E. G. Lo Turco, R. P. Bertolla.a aDepartment of Surgery, Division of Urology, Human Reproduction Section, Sao Paulo Federal University, Sao Paulo, SP, Brazil; bDepartment of Animal Reproduction, School of Veterinary Medicine, University of Sao Paulo, Sao Paulo, Brazil; c Center for Exact Sciences, Deparment of Chemistry, Maringa State University, Maringa, Parana, Brazil. OBJECTIVE: To investigate the association between the seminal plasma lipid fingerprint, semen oxidative stress, and sperm nuclear DNA fragmentation. DESIGN: Cross sectional study including 133 normozoospermic patients. MATERIALS AND METHODS: Oxidative stress levels were assessed by Thiobarbituric acid-reactive substances (TBARS) method and sperm nuclear DNA integrity was evaluated by an alkaline Comet assay. Patients were subdivided into groups according to (i) LPO levels (Low or High), and (ii) sperm DNA fragmentation (Low or High). Seminal plasma lipids were extracted using a Bligh and Dyer protocol, and lipid fingerprints were produced using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Spectral counts of lipids were analyzed by both univariate (Mann-Whitney or Student’s T test, according to normality of distribution, a¼5%) and multivariate data analysis (Principal Component Regression). Lipids were analyzed with a maximum mass error of 50 ppm using a Lipid Maps database. RESULTS: 24 ions were significantly different (p<0.05) between Low and High LPO levels groups and 130 ions between Low and High sperm DNA fragmentation groups. The main lipid subclasses overrepresented in both
FERTILITY & STERILITYÒ
cases were acid glycosphingolipids, glycerophosphates and triradylglycerol. The logistic regression models demonstrated total predictive values of 83.9% and 93.8%, respectively. CONCLUSION: The lipid content of seminal plasma demonstrated to be sensitive to its redox state, with important differences between patients with high levels of LPO and sperm DNA fragmentation. Because we identified sperm membrane lipids in the seminal plasma of these patients, we suggest that even small changes in lipid fingerprinting may be indicative of important physiological alterations in spermatozoa. Supported by: FAPESP (2011/00385-4), and CNPq (Brazil). O-405 Wednesday, October 16, 2013 04:30 PM PHYSICAL AND SEDENTARY ACTIVITIES IN RELATION TO SEMEN QUALITY. A. J. Gaskins,a M. Afeiche,a C. Tanrikut,b J. C. Petrozza,b R. Hauser,c J. E. Chavarro.a aDepartment of Nutrition, Harvard School of Public Health, Boston, MA; bMassachusetts General Hospital Fertility Center, Boston, MA; cDepartment of Environmental Health, Harvard School of Public Health, Boston, MA. OBJECTIVE: To examine the relationship between physical and sedentary activities and semen quality. DESIGN: Cross-sectional study. MATERIALS AND METHODS: Male partners of couples presenting to the Massachusetts General Hospital Fertility Center between 2006 and 2012 were invited to participate in the EARTH Study. Average time per week spent in physical and sedentary activities over the preceding year was assessed using a previously validated questionnaire. The dataset included 137 men who contributed a total of 337 semen samples. Men’s activity levels were classified into quartiles of moderate-to-vigorous and sedentary activity. Linear mixed models, accounting for multiple semen samples per man, were used to analyze the association of activity levels with semen parameters (sperm concentration, motility, morphology, and count) adjusted for abstinence interval, age, body mass index (BMI), and smoking status. RESULTS: Among the 137 men (median age: 36 years, 85% Caucasian, median BMI: 27 kg/m2, 40% ever smokers), the median time spent on moderate to vigorous activities was 3.2 hours per week (interquartile range: 1.0-6.4 hours per week). Sperm concentration was directly and significantly related to physical activity. Men in the highest quartile of moderate-to-vigorous activity had 48.2% (95% CI 14.0-92.8%) higher sperm concentrations than men in the lowest quartile (p-trend, 0.04). Physical activity was not significantly related to sperm motility or morphology. Of the activities contributing to physical activity, increasing time spent weight lifting and in outdoor activities (e.g. yard work or gardening) was associated with higher sperm concentrations (p-trend, 0.02 and 0.02). Jogging and bicycling were not related to sperm concentrations. Increased sedentary activity was not significantly related to semen parameters. CONCLUSION: In this population of men, higher physical activity was significantly associated with higher sperm concentrations. Supported by: Grants ES009718 and ES000002 from NIEHS, DK46200 from NIDDK, and T32DK007703-16 from NIH. O-406 Wednesday, October 16, 2013 04:45 PM PROSPECTIVE COMPARISON OF FLEXIBLE FIBEROPTIC CO2 LASER AND STANDARD MONOPOLAR CAUTERY FOR ROBOTIC MICROSURGICAL DENERVATION OF THE SPERMATIC CORD PROCEDURE. A. Gudeloglu, J. Brahmbhatt, S. Parekattil. Urology, University of Florida & Winter Haven Hospital, Winter Haven, FL. OBJECTIVE: Microsurgical denervation of the spermatic cord (MSDC) is a treatment option for patients with chronic orchialgia. The procedure requires precise tissue dissection to ensure ablation of small diameter nerve fibers while preserving surrounding structures. Due to inherent precision and minimal energy scatter, laser ablation provides targeted dissection while limiting collateral peripheral tissue injury. Our group sought to evaluate the extent of peripheral tissue damage sustained with the CO2 laser and compare against standard monopolar cautery dissection during robotic MDSC (RMDSC). DESIGN: A cadaveric model was utilized to perform RMDSC on one spermatic cord using monopolar cautery and CO2 laser dissection on the contralateral spermatic cord.
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O-404 Wednesday, October 16, 2013 04:15 PM SEMINAL PLASMA LIPID FINGERPRINTING AND ITS ASSOCIATION TO SEMEN OXIDATIVE STRESS AND SPERM NUCLEAR C. Bruna de Lima,a DNA FRAGMENTATION. P. Intasqui,a b c a M. Nichi, E. J. Pilau, E. G. Lo Turco, R. P. Bertolla.a aDepartment of Surgery, Division of Urology, Human Reproduction Section, Sao Paulo Federal University, Sao Paulo, SP, Brazil; bDepartment of Animal Reproduction, School of Veterinary Medicine, University of Sao Paulo, Sao Paulo, Brazil; c Center for Exact Sciences, Deparment of Chemistry, Maringa State University, Maringa, Parana, Brazil. OBJECTIVE: To investigate the association between the seminal plasma lipid fingerprint, semen oxidative stress, and sperm nuclear DNA fragmentation. DESIGN: Cross sectional study including 133 normozoospermic patients. MATERIALS AND METHODS: Oxidative stress levels were assessed by Thiobarbituric acid-reactive substances (TBARS) method and sperm nuclear DNA integrity was evaluated by an alkaline Comet assay. Patients were subdivided into groups according to (i) LPO levels (Low or High), and (ii) sperm DNA fragmentation (Low or High). Seminal plasma lipids were extracted using a Bligh and Dyer protocol, and lipid fingerprints were produced using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Spectral counts of lipids were analyzed by both univariate (Mann-Whitney or Student’s T test, according to normality of distribution, a¼5%) and multivariate data analysis (Principal Component Regression). Lipids were analyzed with a maximum mass error of 50 ppm using a Lipid Maps database. RESULTS: 24 ions were significantly different (p<0.05) between Low and High LPO levels groups and 130 ions between Low and High sperm DNA fragmentation groups. The main lipid subclasses overrepresented in both
FERTILITY & STERILITYÒ
cases were acid glycosphingolipids, glycerophosphates and triradylglycerol. The logistic regression models demonstrated total predictive values of 83.9% and 93.8%, respectively. CONCLUSION: The lipid content of seminal plasma demonstrated to be sensitive to its redox state, with important differences between patients with high levels of LPO and sperm DNA fragmentation. Because we identified sperm membrane lipids in the seminal plasma of these patients, we suggest that even small changes in lipid fingerprinting may be indicative of important physiological alterations in spermatozoa. Supported by: FAPESP (2011/00385-4), and CNPq (Brazil). O-405 Wednesday, October 16, 2013 04:30 PM PHYSICAL AND SEDENTARY ACTIVITIES IN RELATION TO SEMEN QUALITY. A. J. Gaskins,a M. Afeiche,a C. Tanrikut,b J. C. Petrozza,b R. Hauser,c J. E. Chavarro.a aDepartment of Nutrition, Harvard School of Public Health, Boston, MA; bMassachusetts General Hospital Fertility Center, Boston, MA; cDepartment of Environmental Health, Harvard School of Public Health, Boston, MA. OBJECTIVE: To examine the relationship between physical and sedentary activities and semen quality. DESIGN: Cross-sectional study. MATERIALS AND METHODS: Male partners of couples presenting to the Massachusetts General Hospital Fertility Center between 2006 and 2012 were invited to participate in the EARTH Study. Average time per week spent in physical and sedentary activities over the preceding year was assessed using a previously validated questionnaire. The dataset included 137 men who contributed a total of 337 semen samples. Men’s activity levels were classified into quartiles of moderate-to-vigorous and sedentary activity. Linear mixed models, accounting for multiple semen samples per man, were used to analyze the association of activity levels with semen parameters (sperm concentration, motility, morphology, and count) adjusted for abstinence interval, age, body mass index (BMI), and smoking status. RESULTS: Among the 137 men (median age: 36 years, 85% Caucasian, median BMI: 27 kg/m2, 40% ever smokers), the median time spent on moderate to vigorous activities was 3.2 hours per week (interquartile range: 1.0-6.4 hours per week). Sperm concentration was directly and significantly related to physical activity. Men in the highest quartile of moderate-to-vigorous activity had 48.2% (95% CI 14.0-92.8%) higher sperm concentrations than men in the lowest quartile (p-trend, 0.04). Physical activity was not significantly related to sperm motility or morphology. Of the activities contributing to physical activity, increasing time spent weight lifting and in outdoor activities (e.g. yard work or gardening) was associated with higher sperm concentrations (p-trend, 0.02 and 0.02). Jogging and bicycling were not related to sperm concentrations. Increased sedentary activity was not significantly related to semen parameters. CONCLUSION: In this population of men, higher physical activity was significantly associated with higher sperm concentrations. Supported by: Grants ES009718 and ES000002 from NIEHS, DK46200 from NIDDK, and T32DK007703-16 from NIH. O-406 Wednesday, October 16, 2013 04:45 PM PROSPECTIVE COMPARISON OF FLEXIBLE FIBEROPTIC CO2 LASER AND STANDARD MONOPOLAR CAUTERY FOR ROBOTIC MICROSURGICAL DENERVATION OF THE SPERMATIC CORD PROCEDURE. A. Gudeloglu, J. Brahmbhatt, S. Parekattil. Urology, University of Florida & Winter Haven Hospital, Winter Haven, FL. OBJECTIVE: Microsurgical denervation of the spermatic cord (MSDC) is a treatment option for patients with chronic orchialgia. The procedure requires precise tissue dissection to ensure ablation of small diameter nerve fibers while preserving surrounding structures. Due to inherent precision and minimal energy scatter, laser ablation provides targeted dissection while limiting collateral peripheral tissue injury. Our group sought to evaluate the extent of peripheral tissue damage sustained with the CO2 laser and compare against standard monopolar cautery dissection during robotic MDSC (RMDSC). DESIGN: A cadaveric model was utilized to perform RMDSC on one spermatic cord using monopolar cautery and CO2 laser dissection on the contralateral spermatic cord.
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