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MP66-04 AZF GENE EXPRESSION ANALYSIS IN PERIPHERAL LEUKOCYTES AND TESTICULAR CELLS FROM IDIOPATHIC INFERTILITY.
THE JOURNAL OF UROLOGYâ
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predictive accuracy of identifying tubules with spermatogenesis vs. SCO tubules. RESULTS: Raman peak intensity changes were noted at 1000 cm-1 and 1690 cm-1 between tubules with spermatogenesis and SCO tubules. When wavelengths were utilized to predict whether seminferious tubules were SCO or had spermatogenesis, sensitivity and specificity were estimated as 96% and 100% respectively. The area under the receiver operator characteristic curve for predicting tubules with spermatogenesis was 0.98. CONCLUSIONS: RS is capable of identifying seminiferous tubules with spermatogenesis in a SCO ex-vivo rat model. Future ex-vivo studies on human testicular tissue will be necessary to confirm whether these findings can be translated into a clinical setting. Source of Funding: none
MP66-04 AZF GENE EXPRESSION ANALYSIS IN PERIPHERAL LEUKOCYTES AND TESTICULAR CELLS FROM IDIOPATHIC INFERTILITY. Ninghong Song*, Jiangsu Province, China, People’s Republic of; Huang Su, NanJing, China, People’s Republic of INTRODUCTION AND OBJECTIVES: The aim of this study was to assess the frequency of AZF microdeletions in peripheral leukocytes and testicular cells in Chinese men with idiopathic infertility. METHODS: Expression in testicular cells was also determined. In this study, we screened 62 idiopathic infertile patients, in whom karyotype, sperm count and hormonal parameters were evaluated. Genomic DNA was extracted from the peripheral leukocytes. Molecular analysis was performed by two multiplex polymerase chain reactions (PCR) using a set of eight sequence tagged sites (STS) from 3 different regions of the Y chromosome. Total cellular RNA was extracted from the testicular tissue using a Trizol-method. Reverse Transcription (RT) reactions were performed to synthesize cDNA. Amplification of DFFRY, RBM and DAZ genes was performed to analyze their expression in testicular cells. RESULTS: In this cohort, we found 12 submicroscopic deletions (12/62, 19.4%). Nine patients (9/33, 27.2%) were detected in the azoospermic group and three (3/29, 10.3%) in the severe oligozoospermic group. RT-PCR analysis from testicular cells gave normal amplifications for SRY and DFFRY mRNA in 62 idiopathic patients; two patients were negative for RBM expression; no RBM and DAZ were detected for a case; 12 patients had no expression in the AZFc region involving the DAZ gene. Of 12 cases, three patients with normal PCR analysis of DAZ gene on genomic DNA showed no RT-PCR amplification for DAZ mRNA. CONCLUSIONS: The use of RT-PCR of specific spermatid expressed genes in conjunction with examining microdeletions using peripheral leukocytes is suggested to avoid the transmission of the Y chromosomal microdeletions from a father to a son via testicular sperm aspiration (TESE), intracytoplasmic sperm injection (JCSI). Source of Funding: none
MP66-05 OXIDATIVE STRESS PROTEINS IDENTIFIED IN HUMAN SPERMATOZOA BY PROTEOMIC AND BIOINFORMATIC ANALYSIS Rakesh Sharma*, Ashok Agarwal, Damayanthi Durairajanayagam, Zhihong Cui, Ahmet Ayaz, Edmund Sabanegh, Cleveland, OH INTRODUCTION AND OBJECTIVES: Oxidative stress is associated with male infertility. Understanding the protein profile of
Vol. 191, No. 4S, Supplement, Tuesday, May 20, 2014
spermatozoa is essential for better diagnosis of male infertility. The aim of the present study was to examine the effect of high ROS production on the proteomic profile and cellular distribution of spermatozoal proteins. METHODS: Seminal ejaculates from 52 subjects (32 infertile men and 20 healthy men) were classified as ROS+ or ROS- and evaluated for their proteomic profile. Samples were pooled and subjected to LC-MS/MS analysis. Differentially (over- or under-) expressed proteins were identified and further categorized as common and unique. Functional bioinformatic analysis was done using both publicly available Gene Ontology annotations, as well as proprietary databases such as Ingenuity Pathway Analysis (IPATM) and MetacoreTM to identify the differentially affected processes, pathways, interactions and cellular distribution of the proteins. RESULTS: Of 74 identified proteins, 47 were over-expressed and 27 under-expressed compared to the ROS- group. Fifteen of the 74 proteins were 2-fold differentially expressed in the ROS+ group, of which 10 were overexpressed and 5 underexpressed. Most of the differentially expressed proteins were localized to the cytoplasmic and intracellular regions. In the ROS+ group, there was increased expression of histone cluster 1H2ba, mitochondrial malate dehydrogenase precursor, transglutaminase 4, glutathione peroxidase 4 isoform A precursor, glutamine synthetase and heat shock proteins. Androgen receptor was identified as one of the topmost regulators in the ROS+ group with 21 differentially expressed proteins interacting with the receptor. cAMP responsive element modulator (CREM) signaling, which plays an important role in male fertility, was one of the key pathways associated with the differentially expressed proteins. CONCLUSIONS: We identified various proteins that are implicated in a multitude of functions associated with response and management of oxidative stress. The increased expression of proteins in the ROS+ group suggests some of these proteins may serve as potential biomarkers of oxidative stress. Alterations reported in our study may help explain pathways leading to the altered semen phenotype, especially in men exhibiting oxidative stress. Source of Funding: Cleveland Clinic
MP66-06 MICRORNA-202-5P IS SELECTIVELY EXPRESSED BY SERTOLI CELLS IN MEN WITH NORMAL SPERMATOGENESIS Ali Dabaja*, Anna Mielnik, Peter N. Schlegel, Darius A. Paduch, New York, NY INTRODUCTION AND OBJECTIVES: MicroRNAs (miRNAs) are short non-coding RNA molecules that play a regulatory roles in expression of RNA transcripts. Recent studies suggest that miRNAs are mechanistically involved in the development of human spermatogenesis. However, little work has been done to compare the miRNA expression in fertile men and in men with Sertoli Cell Only syndrome (SCO). Our objective was to examine human microRNA (miRNA) expression in correlation with SCO and to localize micorRNAs to specific cell type. METHODS: Testicular tissues from men with confirmed SCO syndrome and normal spermatogenesis were analyzed. MicroRNA was isolated using the miRCURYÔ RNA Purification Kit’s based on spin column chromatography using a proprietary resin as the separation matrix. A miRCURY LNAÔ Universal RT microRNA PCR system was used for sensitive and accurate detection of microRNA by quantitative real-time PCR. MicroRNA localization was done by using in situ hybridizations (ISH) on formalin-fixed paraffin embedded (FFPE) tissue samples utilizing miRCURY LNAÔ microRNA ISH technology. Statistical analysis was performed by GenEx V5.0. RESULTS: microRNA expression was determined for 13 normal fertile men and 5 men with the confirmed diagnosis of diffuse SCO, using the 96 well plates of previously selected primers that are
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predictive accuracy of identifying tubules with spermatogenesis vs. SCO tubules. RESULTS: Raman peak intensity changes were noted at 1000 cm-1 and 1690 cm-1 between tubules with spermatogenesis and SCO tubules. When wavelengths were utilized to predict whether seminferious tubules were SCO or had spermatogenesis, sensitivity and specificity were estimated as 96% and 100% respectively. The area under the receiver operator characteristic curve for predicting tubules with spermatogenesis was 0.98. CONCLUSIONS: RS is capable of identifying seminiferous tubules with spermatogenesis in a SCO ex-vivo rat model. Future ex-vivo studies on human testicular tissue will be necessary to confirm whether these findings can be translated into a clinical setting. Source of Funding: none
MP66-04 AZF GENE EXPRESSION ANALYSIS IN PERIPHERAL LEUKOCYTES AND TESTICULAR CELLS FROM IDIOPATHIC INFERTILITY. Ninghong Song*, Jiangsu Province, China, People’s Republic of; Huang Su, NanJing, China, People’s Republic of INTRODUCTION AND OBJECTIVES: The aim of this study was to assess the frequency of AZF microdeletions in peripheral leukocytes and testicular cells in Chinese men with idiopathic infertility. METHODS: Expression in testicular cells was also determined. In this study, we screened 62 idiopathic infertile patients, in whom karyotype, sperm count and hormonal parameters were evaluated. Genomic DNA was extracted from the peripheral leukocytes. Molecular analysis was performed by two multiplex polymerase chain reactions (PCR) using a set of eight sequence tagged sites (STS) from 3 different regions of the Y chromosome. Total cellular RNA was extracted from the testicular tissue using a Trizol-method. Reverse Transcription (RT) reactions were performed to synthesize cDNA. Amplification of DFFRY, RBM and DAZ genes was performed to analyze their expression in testicular cells. RESULTS: In this cohort, we found 12 submicroscopic deletions (12/62, 19.4%). Nine patients (9/33, 27.2%) were detected in the azoospermic group and three (3/29, 10.3%) in the severe oligozoospermic group. RT-PCR analysis from testicular cells gave normal amplifications for SRY and DFFRY mRNA in 62 idiopathic patients; two patients were negative for RBM expression; no RBM and DAZ were detected for a case; 12 patients had no expression in the AZFc region involving the DAZ gene. Of 12 cases, three patients with normal PCR analysis of DAZ gene on genomic DNA showed no RT-PCR amplification for DAZ mRNA. CONCLUSIONS: The use of RT-PCR of specific spermatid expressed genes in conjunction with examining microdeletions using peripheral leukocytes is suggested to avoid the transmission of the Y chromosomal microdeletions from a father to a son via testicular sperm aspiration (TESE), intracytoplasmic sperm injection (JCSI). Source of Funding: none
MP66-05 OXIDATIVE STRESS PROTEINS IDENTIFIED IN HUMAN SPERMATOZOA BY PROTEOMIC AND BIOINFORMATIC ANALYSIS Rakesh Sharma*, Ashok Agarwal, Damayanthi Durairajanayagam, Zhihong Cui, Ahmet Ayaz, Edmund Sabanegh, Cleveland, OH INTRODUCTION AND OBJECTIVES: Oxidative stress is associated with male infertility. Understanding the protein profile of
Vol. 191, No. 4S, Supplement, Tuesday, May 20, 2014
spermatozoa is essential for better diagnosis of male infertility. The aim of the present study was to examine the effect of high ROS production on the proteomic profile and cellular distribution of spermatozoal proteins. METHODS: Seminal ejaculates from 52 subjects (32 infertile men and 20 healthy men) were classified as ROS+ or ROS- and evaluated for their proteomic profile. Samples were pooled and subjected to LC-MS/MS analysis. Differentially (over- or under-) expressed proteins were identified and further categorized as common and unique. Functional bioinformatic analysis was done using both publicly available Gene Ontology annotations, as well as proprietary databases such as Ingenuity Pathway Analysis (IPATM) and MetacoreTM to identify the differentially affected processes, pathways, interactions and cellular distribution of the proteins. RESULTS: Of 74 identified proteins, 47 were over-expressed and 27 under-expressed compared to the ROS- group. Fifteen of the 74 proteins were 2-fold differentially expressed in the ROS+ group, of which 10 were overexpressed and 5 underexpressed. Most of the differentially expressed proteins were localized to the cytoplasmic and intracellular regions. In the ROS+ group, there was increased expression of histone cluster 1H2ba, mitochondrial malate dehydrogenase precursor, transglutaminase 4, glutathione peroxidase 4 isoform A precursor, glutamine synthetase and heat shock proteins. Androgen receptor was identified as one of the topmost regulators in the ROS+ group with 21 differentially expressed proteins interacting with the receptor. cAMP responsive element modulator (CREM) signaling, which plays an important role in male fertility, was one of the key pathways associated with the differentially expressed proteins. CONCLUSIONS: We identified various proteins that are implicated in a multitude of functions associated with response and management of oxidative stress. The increased expression of proteins in the ROS+ group suggests some of these proteins may serve as potential biomarkers of oxidative stress. Alterations reported in our study may help explain pathways leading to the altered semen phenotype, especially in men exhibiting oxidative stress. Source of Funding: Cleveland Clinic
MP66-06 MICRORNA-202-5P IS SELECTIVELY EXPRESSED BY SERTOLI CELLS IN MEN WITH NORMAL SPERMATOGENESIS Ali Dabaja*, Anna Mielnik, Peter N. Schlegel, Darius A. Paduch, New York, NY INTRODUCTION AND OBJECTIVES: MicroRNAs (miRNAs) are short non-coding RNA molecules that play a regulatory roles in expression of RNA transcripts. Recent studies suggest that miRNAs are mechanistically involved in the development of human spermatogenesis. However, little work has been done to compare the miRNA expression in fertile men and in men with Sertoli Cell Only syndrome (SCO). Our objective was to examine human microRNA (miRNA) expression in correlation with SCO and to localize micorRNAs to specific cell type. METHODS: Testicular tissues from men with confirmed SCO syndrome and normal spermatogenesis were analyzed. MicroRNA was isolated using the miRCURYÔ RNA Purification Kit’s based on spin column chromatography using a proprietary resin as the separation matrix. A miRCURY LNAÔ Universal RT microRNA PCR system was used for sensitive and accurate detection of microRNA by quantitative real-time PCR. MicroRNA localization was done by using in situ hybridizations (ISH) on formalin-fixed paraffin embedded (FFPE) tissue samples utilizing miRCURY LNAÔ microRNA ISH technology. Statistical analysis was performed by GenEx V5.0. RESULTS: microRNA expression was determined for 13 normal fertile men and 5 men with the confirmed diagnosis of diffuse SCO, using the 96 well plates of previously selected primers that are