- Email: [email protected]
Proteomic analysis of seminal plasma in adolescents with and without varicocele
CONCLUSION: DE success rates with vitrified oocytes are significantly better than those with NDE cycles and similar to those with fresh donor oocytes as reported by SART. DE programs should give serious consideration to establishing oocyte banks to improve flexibility and patient convenience. Supported by: IFP
O-294 Wednesday, October 19, 2011 12:45 PM DERIVATION OF NOVEL GENETICALLY DIVERSE HUMAN EMBRYONIC STEM CELL LINES. C. Hansis, R. Lehmann. Obstetrics and Gynecology/REI, NYU School of Medicine, New York, NY; Cell Biology (Skirball), NYU School of Medicine, New York, NY. OBJECTIVE: Since many of the human embryonic stem cell (hESC) lines derived to date are poorly characterized, unavailable or do not represent desired traits, we generated twelve novel genetically diverse hESC lines with IRB approval and performed in-depth characterization of their molecular and cellular features. DESIGN: Experimental MATERIALS AND METHODS: Zona-free human blastocysts or inner cell masses (ICMs) (n ¼ 60), some of them previously assessed by preimplantation genetic diagnosis (PGD) for genetic conditions, were plated onto feeder cells and cultured in DMEM-based media. Differentiation of hESCs was achieved by colony overgrowth or embryoid body formation. Embryonic and control cells were subjected to marker gene, protein and chromosome analysis for pluripotency, differentiation, DNA fingerprinting and mutation examination by reverse transcription and genomic PCR, immunofluorescence, fluorescence in situ hybridization (FISH) and microarray analysis, respectively. RESULTS: Colonies (n ¼ 21; 35% of plated blastocysts/ICMs) and hESC lines (n ¼ 12; 57% of colonies) could be established, seven of which carrying disease-specific mutations (one each of trisomy 14, trisomy 17, trisomy X, 18, 21, 22, alpha-thalassemia X-linked mental retardation syndrome, Zellweger syndrome and two of unbalanced translocation long arm of chromosomes 8, 15) and one derived from an inconclusively tested embryo (BRCA1). HESC lines harbored diverse ethnic heritage as well as linkage to conditions with a genetic component such as asthma or poor sperm morphology. Genetic abnormalities, gender, X chromosome activation status and DNA fingerprint and growth pattern could be assessed. Furthermore, hESCs could be differentiated into a variety of cell types, including the tissues most affected by the conditions. CONCLUSION: Our detailed analyses showed that we could establish novel genetically diverse hESC lines. Those lines will provide new tools for basic research, disease modeling, regenerative medicine, drug discovery and toxicity testing.
MALE REPRODUCTION AND UROLOGY: RESEARCH
O-295 Wednesday, October 19, 2011 03:45 PM PROTEOMIC ANALYSIS OF SEMINAL PLASMA IN ADOLESCENTS WITH AND WITHOUT VARICOCELE. D. S. Zylbersztejn, L. Borsari, P. T. Del Giudice, G. M. Souza, D. M. Spaine, R. Fraietta. Human Reproduction Seccion, Universidade Federal de S~ao Paulo, S~ao Paulo, Brazil; Waters Corporation, Mass Spectrometry Applications Research and Development Laboratory, S~ao Paulo, Brazil. OBJECTIVE: Varicocele is the leading treatable cause of male infertility. This study aims to compare the proteomic profile of seminal plasma of adolescents with varicocele and alteration in seminal quality, adolescents with varicocele and normal semen and adolescents without varicocele. DESIGN: A transversal study. MATERIALS AND METHODS: One hundred and fifty six adolescents were initially evaluated from a school from S~ao Paulo and 67 adolescents were recruited for the study. The adolescents were divided into three groups: Control group (without varicocele and normal seminal analysis) with 21 individuals, normal semen varicocele group (NSV) with 28 individuals and altered sperm varicocele group (ASV) with 18 individuals. Two semen samples of each adolescent with one week interval between them were obtained and analyzed according to World Health Organization criteria of 1999 and sperm morphology by Kruger. The seminal plasma proteomic profile initially was analyzed by two-dimensional electrophoresis, and the final proteins of inter-
FERTILITY & STERILITYÒ
est were identified by mass spectrometry and bioinformatics database. The data were analyzed by ANOVA test and a LSD (post hoc) test. RESULTS: Two specific proteins for apoptosis (SMG1_HUMAN and IBP3_HUMAN) were found exclusively in ASV group. The NSV group was the only to show the BRE1B_HUMAN, a protein related to spermatogenesis. The control group detected the greater number of proteins related to sperm motility (SEMG1_HUMAN, SEMG2_HUMAN, KLK3_HUMAN) and sperm capacitation (PPAP_HUMAN, LRMP_HUMAN and NPC2_HUMAN). CONCLUSION: Clinical varicocele with or without seminal alterations cause alterations in the protein profile of seminal plasma. The differences between the protein expressions in seminal plasma of adolescents with and without varicocele should be studied for better understanding of their functions, promoting the acquisition of early biomarkers of damage to sperm function.
O-296 Wednesday, October 19, 2011 04:00 PM EXPRESSION AND FUNCTIONAL SIGNIFICANCE OF THE ENDOCANNABINOID SYSTEM IN HUMAN SPERMATOZOA. A. A. Amoako, T. H. Marczylo, E. L. Marczylo, C. Boes, J. M. Willets, J. C. Konje. Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, Leicestershire, United Kingdom; MRC Toxicology Unit, University of Leicester, Leicester, Leicestershire, United Kingdom. OBJECTIVE: D9-Tetrahydrocannabinol disrupts endocannabinoid signalling through the cannabinoid receptor 1 (CB1) which accounts for the adverse reproductive consequences observed in marijuana users. How deregulation of the endogenous signalling pathway affects human sperm functions remains largely unexplored. The purpose of this study was to elucidate the pathophysiological role of the endocannabinoid system (ECS) in male fertility. DESIGN: Prospective laboratory based clinical study. MATERIALS AND METHODS: Seminal plasma anandamide (AEA) was quantified by Ultra high performance liquid chromatography-tandem mass spectrometry from 90 semen samples provided by men with normal and abnormal semen parameters. Using qRT-PCR, CB1, CB2, NAPEPLD and FAAH mRNA expression profiles were characterized in human spermatozoa from men with normal and abnormal semen parameters. We evaluated in vitro, the effects of methanadamide (Met-AEA), a non-hydrolyzable analog of AEA, on sperm motility and mitochondrial membrane potential. RESULTS: AEA concentration was lower in asthenozoospermics (P¼0.002), oligoasthenoteratozoospermics (P¼0.001) and azoospermics (P¼0.01) compared to normozoospermic controls. CB1 mRNA was decreased in asthenozoospermics (p < 0.0001) and oligoasthenoteratozoospermics (P < 0.0001). There were no significant differences in the expression of CB2, NAPEPLD or FAAH mRNA in abnormal human spermatozoa. Exposure of human spermatozoa to Met-AEA significantly decreased sperm motility through inhibition of mitochondrial membrane potential in a dose dependent manner. The inhibitory effect of Met-AEA was attenuated by the CB1 antagonist AM 251. CONCLUSION: Normal regulation of the ECS may be necessary for the preservation of normal sperm function and male fertility. These observations highlight a potential toxicity mechanism for exocannabinoids (marijuana) and identifies the ECS as a potential target for treatment of male fertility. Supported by: This work was supported by Perkin Elmer and University Hospitals of Leicester NHS Trust
O-297 Wednesday, October 19, 2011 04:15 PM HYDRODISSECTION FOR IMPROVED MICROSURGICAL DENERVATION OF THE SPERMATIC CORD: PROSPECTIVE BLINDED RANDOMIZED CONTROL TRIAL IN A RAT MODEL. A. Gudeoglu, Z. Iqbal, S. J. Parekattil, A. C. Groth, K. B. Priola, R. W. Allen. Urology, Winter Haven Hospital & University of Florida, Winter Haven, FL. OBJECTIVE: Microsurgical denervation of the spermatic cord (MDSC) may provide a 70-85% success rate in eliminating pain in patients with chronic orchialgia who have failed conservative treatment options. Failures in MDSC could be due to small diameter nerves (%1mm) left behind on the arteries and veins in the cord based on previous studies. Our goal was
S87
O-294 Wednesday, October 19, 2011 12:45 PM DERIVATION OF NOVEL GENETICALLY DIVERSE HUMAN EMBRYONIC STEM CELL LINES. C. Hansis, R. Lehmann. Obstetrics and Gynecology/REI, NYU School of Medicine, New York, NY; Cell Biology (Skirball), NYU School of Medicine, New York, NY. OBJECTIVE: Since many of the human embryonic stem cell (hESC) lines derived to date are poorly characterized, unavailable or do not represent desired traits, we generated twelve novel genetically diverse hESC lines with IRB approval and performed in-depth characterization of their molecular and cellular features. DESIGN: Experimental MATERIALS AND METHODS: Zona-free human blastocysts or inner cell masses (ICMs) (n ¼ 60), some of them previously assessed by preimplantation genetic diagnosis (PGD) for genetic conditions, were plated onto feeder cells and cultured in DMEM-based media. Differentiation of hESCs was achieved by colony overgrowth or embryoid body formation. Embryonic and control cells were subjected to marker gene, protein and chromosome analysis for pluripotency, differentiation, DNA fingerprinting and mutation examination by reverse transcription and genomic PCR, immunofluorescence, fluorescence in situ hybridization (FISH) and microarray analysis, respectively. RESULTS: Colonies (n ¼ 21; 35% of plated blastocysts/ICMs) and hESC lines (n ¼ 12; 57% of colonies) could be established, seven of which carrying disease-specific mutations (one each of trisomy 14, trisomy 17, trisomy X, 18, 21, 22, alpha-thalassemia X-linked mental retardation syndrome, Zellweger syndrome and two of unbalanced translocation long arm of chromosomes 8, 15) and one derived from an inconclusively tested embryo (BRCA1). HESC lines harbored diverse ethnic heritage as well as linkage to conditions with a genetic component such as asthma or poor sperm morphology. Genetic abnormalities, gender, X chromosome activation status and DNA fingerprint and growth pattern could be assessed. Furthermore, hESCs could be differentiated into a variety of cell types, including the tissues most affected by the conditions. CONCLUSION: Our detailed analyses showed that we could establish novel genetically diverse hESC lines. Those lines will provide new tools for basic research, disease modeling, regenerative medicine, drug discovery and toxicity testing.
MALE REPRODUCTION AND UROLOGY: RESEARCH
O-295 Wednesday, October 19, 2011 03:45 PM PROTEOMIC ANALYSIS OF SEMINAL PLASMA IN ADOLESCENTS WITH AND WITHOUT VARICOCELE. D. S. Zylbersztejn, L. Borsari, P. T. Del Giudice, G. M. Souza, D. M. Spaine, R. Fraietta. Human Reproduction Seccion, Universidade Federal de S~ao Paulo, S~ao Paulo, Brazil; Waters Corporation, Mass Spectrometry Applications Research and Development Laboratory, S~ao Paulo, Brazil. OBJECTIVE: Varicocele is the leading treatable cause of male infertility. This study aims to compare the proteomic profile of seminal plasma of adolescents with varicocele and alteration in seminal quality, adolescents with varicocele and normal semen and adolescents without varicocele. DESIGN: A transversal study. MATERIALS AND METHODS: One hundred and fifty six adolescents were initially evaluated from a school from S~ao Paulo and 67 adolescents were recruited for the study. The adolescents were divided into three groups: Control group (without varicocele and normal seminal analysis) with 21 individuals, normal semen varicocele group (NSV) with 28 individuals and altered sperm varicocele group (ASV) with 18 individuals. Two semen samples of each adolescent with one week interval between them were obtained and analyzed according to World Health Organization criteria of 1999 and sperm morphology by Kruger. The seminal plasma proteomic profile initially was analyzed by two-dimensional electrophoresis, and the final proteins of inter-
FERTILITY & STERILITYÒ
est were identified by mass spectrometry and bioinformatics database. The data were analyzed by ANOVA test and a LSD (post hoc) test. RESULTS: Two specific proteins for apoptosis (SMG1_HUMAN and IBP3_HUMAN) were found exclusively in ASV group. The NSV group was the only to show the BRE1B_HUMAN, a protein related to spermatogenesis. The control group detected the greater number of proteins related to sperm motility (SEMG1_HUMAN, SEMG2_HUMAN, KLK3_HUMAN) and sperm capacitation (PPAP_HUMAN, LRMP_HUMAN and NPC2_HUMAN). CONCLUSION: Clinical varicocele with or without seminal alterations cause alterations in the protein profile of seminal plasma. The differences between the protein expressions in seminal plasma of adolescents with and without varicocele should be studied for better understanding of their functions, promoting the acquisition of early biomarkers of damage to sperm function.
O-296 Wednesday, October 19, 2011 04:00 PM EXPRESSION AND FUNCTIONAL SIGNIFICANCE OF THE ENDOCANNABINOID SYSTEM IN HUMAN SPERMATOZOA. A. A. Amoako, T. H. Marczylo, E. L. Marczylo, C. Boes, J. M. Willets, J. C. Konje. Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, Leicestershire, United Kingdom; MRC Toxicology Unit, University of Leicester, Leicester, Leicestershire, United Kingdom. OBJECTIVE: D9-Tetrahydrocannabinol disrupts endocannabinoid signalling through the cannabinoid receptor 1 (CB1) which accounts for the adverse reproductive consequences observed in marijuana users. How deregulation of the endogenous signalling pathway affects human sperm functions remains largely unexplored. The purpose of this study was to elucidate the pathophysiological role of the endocannabinoid system (ECS) in male fertility. DESIGN: Prospective laboratory based clinical study. MATERIALS AND METHODS: Seminal plasma anandamide (AEA) was quantified by Ultra high performance liquid chromatography-tandem mass spectrometry from 90 semen samples provided by men with normal and abnormal semen parameters. Using qRT-PCR, CB1, CB2, NAPEPLD and FAAH mRNA expression profiles were characterized in human spermatozoa from men with normal and abnormal semen parameters. We evaluated in vitro, the effects of methanadamide (Met-AEA), a non-hydrolyzable analog of AEA, on sperm motility and mitochondrial membrane potential. RESULTS: AEA concentration was lower in asthenozoospermics (P¼0.002), oligoasthenoteratozoospermics (P¼0.001) and azoospermics (P¼0.01) compared to normozoospermic controls. CB1 mRNA was decreased in asthenozoospermics (p < 0.0001) and oligoasthenoteratozoospermics (P < 0.0001). There were no significant differences in the expression of CB2, NAPEPLD or FAAH mRNA in abnormal human spermatozoa. Exposure of human spermatozoa to Met-AEA significantly decreased sperm motility through inhibition of mitochondrial membrane potential in a dose dependent manner. The inhibitory effect of Met-AEA was attenuated by the CB1 antagonist AM 251. CONCLUSION: Normal regulation of the ECS may be necessary for the preservation of normal sperm function and male fertility. These observations highlight a potential toxicity mechanism for exocannabinoids (marijuana) and identifies the ECS as a potential target for treatment of male fertility. Supported by: This work was supported by Perkin Elmer and University Hospitals of Leicester NHS Trust
O-297 Wednesday, October 19, 2011 04:15 PM HYDRODISSECTION FOR IMPROVED MICROSURGICAL DENERVATION OF THE SPERMATIC CORD: PROSPECTIVE BLINDED RANDOMIZED CONTROL TRIAL IN A RAT MODEL. A. Gudeoglu, Z. Iqbal, S. J. Parekattil, A. C. Groth, K. B. Priola, R. W. Allen. Urology, Winter Haven Hospital & University of Florida, Winter Haven, FL. OBJECTIVE: Microsurgical denervation of the spermatic cord (MDSC) may provide a 70-85% success rate in eliminating pain in patients with chronic orchialgia who have failed conservative treatment options. Failures in MDSC could be due to small diameter nerves (%1mm) left behind on the arteries and veins in the cord based on previous studies. Our goal was
S87