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MP76-16 IMPACT OF PRECISE MODULATION OF REACTIVE OXYGEN SPECIES LEVELS ON SPERMATOZOA PROTEINS IN INFERTILE MEN
THE JOURNAL OF UROLOGYâ
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(x2:18.0; p¼0.001), fruits (x2:25.6; p<0.001), and eggs (x2:12.1; p¼0.02), but less frequently consumed sweets (x2:9.8; p¼0.04). Conversely, no significant dietary differences were observed between fertile and infertile individuals in terms of cereals, red meat, poultry, and fish. A greater rate of fertile individuals thought that diet as a whole can significantly impact fertility (x2:7.5; p¼0.02). CONCLUSIONS: The findings of this exploratory sociological survey in a selected cohort showed that infertile Caucasian-European individuals had worse recreational and nutritional habits compared to fertile individuals. Fertile individuals reported more regular consumption of vegetables, legumes, fruits and eggs. Source of Funding: None.
MP76-16 IMPACT OF PRECISE MODULATION OF REACTIVE OXYGEN SPECIES LEVELS ON SPERMATOZOA PROTEINS IN INFERTILE MEN Rakesh Sharma*, Ashok Agarwal, Ahmet Ayaz, Edmund Sabanegh, Cleveland, OH INTRODUCTION AND OBJECTIVES: Oxidative stress is involved in the etiology of male infertility. Exposure to different amounts of reactive oxygen species (ROS) may affect the sperm quality by altering the proteins and the functions associated with these proteins. The objective was to examine differentially expressed proteins in spermatozoa from infertile men with low (<92 RLU/s), medium (>100500RLU/s) and high ROS levels (>500RLU/s) and examine the major pathways/functions affected when compared to fertile men. METHODS: In this prospective study, we examined semen samples from 12 men with low, medium and high ROS and 10 proven fertile men (control group). Protein extraction, protein estimation, gel separation of the proteins and in-gel digestion was done. The extracted peptides were injected on an LTQ-orbitrap elite hybrid mass spectrometry system. Differentially expressed proteins (DEPs) were analyzed and functional annotations carried out to identify functions, cellular localization and pathways of DEPs. RESULTS: 1035 proteins were identified in the 3 groups by global proteomic analysis. Of these 305 were differentially expressed proteins (DEP). 51 were unique to the Low ROS group, 47 Medium ROS group and 104 were unique to the High ROS group. Reactome analysis identified the DEP that were overexpressed, underexpressed or unique to each ROS group or the fertile group. 6 DEP were identified by Uniprot and DAVID that had distinct reproductive functions and were expressed only in the 3 ROS groups compared to fertile control group. CONCLUSIONS: In this novel study, we have demonstrated 6 DEP with distinct reproductive functions which are overexpressed, underexpressed or uniquely present only in men with low, medium or ROS levels. These DEP may serve as potential biomarkers of oxidative stress induced male infertility. Source of Funding: none
MP76-17 SUPPLEMENTATION OF CRYOMEDIUM WITH CATALASE AND NACETYL CYSTEINE IMPROVES HUMAN SPERM POST-THAW MOTILITY Yoshitomo Kobori*, Craig Niederberger, Gail Prins, Chicago, IL INTRODUCTION AND OBJECTIVES: Cryopreservation of human spermatozoa has now become a routine procedure in assisted reproductive technology. Cryopreservation process can lead to structural and functional alterations in spermatozoa, impairing fertilization potential. Reactive oxygen species has been suggested as a major contribution factor for cryodamage to spermatozoa. Accordingly, antioxidant supplementation has been shown to yield significantly improved quality of cryopreserve spermatozoa. We aimed to investigate effects of cryopreservation and
Vol. 193, No. 4S, Supplement, Tuesday, May 19, 2015
addition of Catalase and N-Acetyl Cysteine as antioxidants on sperm concentration, motility and DNA fragmentation in fresh spermatozoa. METHODS: Semen samples were collected from normospermic men (n¼6), freezing medium was SpermFreeze (Vitrolife), which includes 15% of glycerol. Semen samples were cryopreserved with Catalase 200U/ml, N-Acetyl Cysteine 2.5, 5, 10 mM with/without Catalase 200U/ml. Specimens were frozen for two weeks. Post-thaw semen analyses, sperm vitality and sperm DNA fragmentation (TUNEL assay) were performed. RESULTS: Sperm concentration showed significant decrease while percentage of progressive motility, sperm vitality and DNA fragmentation showed significant increase in cryopreserved samples when compared with fresh samples. There was no significant difference in sperm concentration while there was significant increase in motility and sperm vitality, DNA fragmentation showed significant decrease in samples with antioxidants. CONCLUSIONS: Catalase and N-Acetyl Cysteine supplementation during cryopreservation results in better post-thawing percentage of progressive motility and percentage of sperm vitality and improved DNA integrity. The results suggest that Catalase had the most pronounced effect in improving post-thaw quality of spermatozoa.
Source of Funding: none
MP76-18 MALE INFERTILITY AND SINGLE NUCLEOTIDE POLYMORPHISMS OF THE NOVEL SEX-LINKED TESTIS-SPECIFIC RETROTRANSPOSED PGAM4 GENE Yasushi Miyagawa*, Tetsuji Soda, Kentaro Takezawa, Shinichiro Fukuhara, Hiroshi Kiuchi, Suita, Japan; Hidenobu Okuda, Victoria, Australia; Hiromitsu Tanaka, Sasebo, Japan; Norio Nonomura, Suita, Japan INTRODUCTION AND OBJECTIVES: In man, infertility is demonstrated to be associated with various genetic factors. However, all of them are neither conclusive as the causative gene nor explain all idiopathic male infertility. Multiple studies, primarily through the use of transgenic animals, have contributed to a list of candidate genes that may affect male infertility in humans. We examined single nucleotide polymorphisms (SNPs) as a cause of male infertility in an analysis of spermatogenesis-specific genes. METHODS: Since the genomic DNA is known to be intronless, we checked the existence of single nucleotide changes in the genome of human PGAM4 in male sterile patients and compared with that of proven fertile normal volunteers by using a polymerase chain reaction technique. RT-PCR and western blot analyses were also performed to check the expression of PGAM4 in testis and sperm. Finally, We investigated whether there might be any PGAM4 genetic variants affecting male fertility by performing a case-control study of fertile and infertile men. RESULTS: Using RT-PCR and western blot analyses, we identified that PGAM4 is a functional retrogene that is expressed predominantly in the testes and is associated with male infertility. PGAM4 is
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(x2:18.0; p¼0.001), fruits (x2:25.6; p<0.001), and eggs (x2:12.1; p¼0.02), but less frequently consumed sweets (x2:9.8; p¼0.04). Conversely, no significant dietary differences were observed between fertile and infertile individuals in terms of cereals, red meat, poultry, and fish. A greater rate of fertile individuals thought that diet as a whole can significantly impact fertility (x2:7.5; p¼0.02). CONCLUSIONS: The findings of this exploratory sociological survey in a selected cohort showed that infertile Caucasian-European individuals had worse recreational and nutritional habits compared to fertile individuals. Fertile individuals reported more regular consumption of vegetables, legumes, fruits and eggs. Source of Funding: None.
MP76-16 IMPACT OF PRECISE MODULATION OF REACTIVE OXYGEN SPECIES LEVELS ON SPERMATOZOA PROTEINS IN INFERTILE MEN Rakesh Sharma*, Ashok Agarwal, Ahmet Ayaz, Edmund Sabanegh, Cleveland, OH INTRODUCTION AND OBJECTIVES: Oxidative stress is involved in the etiology of male infertility. Exposure to different amounts of reactive oxygen species (ROS) may affect the sperm quality by altering the proteins and the functions associated with these proteins. The objective was to examine differentially expressed proteins in spermatozoa from infertile men with low (<92 RLU/s), medium (>100500RLU/s) and high ROS levels (>500RLU/s) and examine the major pathways/functions affected when compared to fertile men. METHODS: In this prospective study, we examined semen samples from 12 men with low, medium and high ROS and 10 proven fertile men (control group). Protein extraction, protein estimation, gel separation of the proteins and in-gel digestion was done. The extracted peptides were injected on an LTQ-orbitrap elite hybrid mass spectrometry system. Differentially expressed proteins (DEPs) were analyzed and functional annotations carried out to identify functions, cellular localization and pathways of DEPs. RESULTS: 1035 proteins were identified in the 3 groups by global proteomic analysis. Of these 305 were differentially expressed proteins (DEP). 51 were unique to the Low ROS group, 47 Medium ROS group and 104 were unique to the High ROS group. Reactome analysis identified the DEP that were overexpressed, underexpressed or unique to each ROS group or the fertile group. 6 DEP were identified by Uniprot and DAVID that had distinct reproductive functions and were expressed only in the 3 ROS groups compared to fertile control group. CONCLUSIONS: In this novel study, we have demonstrated 6 DEP with distinct reproductive functions which are overexpressed, underexpressed or uniquely present only in men with low, medium or ROS levels. These DEP may serve as potential biomarkers of oxidative stress induced male infertility. Source of Funding: none
MP76-17 SUPPLEMENTATION OF CRYOMEDIUM WITH CATALASE AND NACETYL CYSTEINE IMPROVES HUMAN SPERM POST-THAW MOTILITY Yoshitomo Kobori*, Craig Niederberger, Gail Prins, Chicago, IL INTRODUCTION AND OBJECTIVES: Cryopreservation of human spermatozoa has now become a routine procedure in assisted reproductive technology. Cryopreservation process can lead to structural and functional alterations in spermatozoa, impairing fertilization potential. Reactive oxygen species has been suggested as a major contribution factor for cryodamage to spermatozoa. Accordingly, antioxidant supplementation has been shown to yield significantly improved quality of cryopreserve spermatozoa. We aimed to investigate effects of cryopreservation and
Vol. 193, No. 4S, Supplement, Tuesday, May 19, 2015
addition of Catalase and N-Acetyl Cysteine as antioxidants on sperm concentration, motility and DNA fragmentation in fresh spermatozoa. METHODS: Semen samples were collected from normospermic men (n¼6), freezing medium was SpermFreeze (Vitrolife), which includes 15% of glycerol. Semen samples were cryopreserved with Catalase 200U/ml, N-Acetyl Cysteine 2.5, 5, 10 mM with/without Catalase 200U/ml. Specimens were frozen for two weeks. Post-thaw semen analyses, sperm vitality and sperm DNA fragmentation (TUNEL assay) were performed. RESULTS: Sperm concentration showed significant decrease while percentage of progressive motility, sperm vitality and DNA fragmentation showed significant increase in cryopreserved samples when compared with fresh samples. There was no significant difference in sperm concentration while there was significant increase in motility and sperm vitality, DNA fragmentation showed significant decrease in samples with antioxidants. CONCLUSIONS: Catalase and N-Acetyl Cysteine supplementation during cryopreservation results in better post-thawing percentage of progressive motility and percentage of sperm vitality and improved DNA integrity. The results suggest that Catalase had the most pronounced effect in improving post-thaw quality of spermatozoa.
Source of Funding: none
MP76-18 MALE INFERTILITY AND SINGLE NUCLEOTIDE POLYMORPHISMS OF THE NOVEL SEX-LINKED TESTIS-SPECIFIC RETROTRANSPOSED PGAM4 GENE Yasushi Miyagawa*, Tetsuji Soda, Kentaro Takezawa, Shinichiro Fukuhara, Hiroshi Kiuchi, Suita, Japan; Hidenobu Okuda, Victoria, Australia; Hiromitsu Tanaka, Sasebo, Japan; Norio Nonomura, Suita, Japan INTRODUCTION AND OBJECTIVES: In man, infertility is demonstrated to be associated with various genetic factors. However, all of them are neither conclusive as the causative gene nor explain all idiopathic male infertility. Multiple studies, primarily through the use of transgenic animals, have contributed to a list of candidate genes that may affect male infertility in humans. We examined single nucleotide polymorphisms (SNPs) as a cause of male infertility in an analysis of spermatogenesis-specific genes. METHODS: Since the genomic DNA is known to be intronless, we checked the existence of single nucleotide changes in the genome of human PGAM4 in male sterile patients and compared with that of proven fertile normal volunteers by using a polymerase chain reaction technique. RT-PCR and western blot analyses were also performed to check the expression of PGAM4 in testis and sperm. Finally, We investigated whether there might be any PGAM4 genetic variants affecting male fertility by performing a case-control study of fertile and infertile men. RESULTS: Using RT-PCR and western blot analyses, we identified that PGAM4 is a functional retrogene that is expressed predominantly in the testes and is associated with male infertility. PGAM4 is