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DNA microarray analysis of gene expression of spermatogenesis in mouse
modeled outcomes using neural computational techniques. Neural computation employs computer programs that implement mathematical models based loosely on the function of networks of biological neurons. Statistical feature extraction, allowing examination of the significance of individual features such as sperm source, was employed using Wilk’s Generalized Likelihood Ratio Test (GLRT). Design: Neural computational modeling of retrospective data. Materials/Methods: We modeled fertilization outcomes from ICSI from technician (7 different technicians performed the procedure,) sperm source (testis extraction, MESA, EEJ,) number of ova retrieved and maternal age using neUROn2⫹⫹, a set of C⫹⫹ programs we developed to implement neurocomputational algorithms, discriminant function and regression techniques using the Cygwin (Red Hat) GNU C⫹⫹ port for Windows (Microsoft) and Visual C⫹⫹ (Microsoft) distributed across Pentium (Intel) platforms. Wilk’s GLRT was implemented in neUROn2⫹⫹ for statistical feature extraction. A dataset of 1070 unique ICSI outcomes was randomized into a training set of 1349 exemplars and 449 test exemplars. N1/N2 cross-validation was employed. Results: Overall model accuracy was ROC AUC 0.922 (sensitivity 86.9%, specificity 79.0%) in the training set, and 0.897 (sensitivity 82.8%, specificity 75.1%) in the test set. Feature extraction using Wilk’s GLRT in reverse regression is shown in the table. All features were significant to the model (all p ⬍1e-5), however, with p ⬍1e-106, technician was highly significant.
the plasma membrane of apoptotic spermatozoa. The percent apoptosis was determined by epifluorescent microscopy. Western Blot technique was used to analyze for p65, p50 and IB proteins using rabbit polyclonal anti p65, p50 and IB antibodies. After treating with secondary antibody conjugated to horseradish peroxidase, antigen-antibody reaction was visualized by an enhanced chemiluminescence assay. Bands were quantitated using the Alpha Fluorochem analyzer and Actin counter staining was done on the blots to ensure that equal amounts of protein were loaded. Results: Using univariate regression analyses, sperm concentration was directly correlated with levels of p65 (P ⫽ 0.01), p50 (P ⫽ 0.05), and IB (P ⫽ 0.01). Patients with poor sperm concentration had significantly lower levels of NFKB as compared to patients with normal sperm concentration. Infertile men had significantly higher levels of apoptosis (p ⫽ 0.02) and lower levels of NFKB (p ⬍0.001) as compared to normal donors. Levels of p50 was significantly lower in varicocele patients when compared to patients with no clinical varicocele (p ⫽ 0.03). Conclusions: These results demonstrate significant correlation between low sperm concentration and low levels of NFKB expression. We speculate that low expression of NFKB is related at least in part to poor sperm production in infertile men. Supported by: None.
Regression analysis of neural computational model to predict ICSI outcomes. Feature
p-value
Technician No. ova retrieved Testis extraction Maternal age MESA EEJ
1.04e-107 9.84e-45 3.81e-21 7.70e-20 2.33e-10 1.52e-06
p-values indicate significance of the individual feature to the likelihood of fertilization with ICSI. Conclusions: We modeled ICSI outcome with high accuracy (ROC AUC 0.897 in the test set) using a neural computational approach. In order to determine the significance of individual features to the model’s outcome, regression based on Wilk’s GLRT was performed. All features examined (sperm source, number of ova retrieved, maternal age and which technician performed the procedure) were statistically significant to the model’s outcome. Supported by: NIH P01 HD36289 to Dolores J Lamb, Larry I Lipshultz and Craig Niederberger.
Wednesday, October 16, 2002 3:45 P.M. O-250 Correlation of nuclear factor kappa B (NFKB) with sperm quality and clinical diagnoses in infertile men. Pavithra Ranganathan, Namita Kattal, Mohamed H. Moustafa, Rakesh K. Sharma, Anthony J. Thomas Jr., Ashok Agarwal. Cleveland Clin Fdn, Cleveland, OH. Objective: NFKB (p65, p50 and I B) plays a major role in regulating apoptosis. Its role in the pathogenesis of male infertility has never been studied. The objectives of our study were to examine 1) the relationship of NFKB with semen quality (sperm concentration, motility and morphology) and the clinical diagnoses of male infertility, and 2) correlate levels of NFKB and apoptosis in ejaculated human spermatozoa. Design: Prospective experimental design. Materials/Methods: Semen samples from 12 donors and 40 infertile men (varicocele, n ⫽ 13; and others, n ⫽ 27) were used to probe for levels of p65, p50 and IB molecules. Semen samples were stratified into patients with poor sperm concentration (⬍20 ⫻ 106/mL; n ⫽ 11) and patients with normal sperm concentration (⬎20 ⫻ 106/mL; n ⫽ 29). Levels of apoptosis were detected using Annexin-V staining in neat semen specimens. The assay detects externalization of phosphatidylserine to the outer surface of
FERTILITY & STERILITY威
Wednesday, October 16, 2002 4:00 P.M. O-251 DNA microarray analysis of gene expression of spermatogenesis in mouse. Weixiong Li. National Research Institute for Family Planning, Beijing, China. Objective: Spermatogenesis is a complex and productive process that originates from stem cell spermatogonia and ultimately results in formation of mature spermatozoa. Recent studies have suggested that more than 100 genes involved this process. In the past, the investigation of gene expression was limited by the inability to study more than one gene at time. The introduction of DNA microarray technology has made it possible to examine the expression of thousands of genes at a time. For understanding the effect of multi-gene interaction on each stage of spermatogenesis. The gene expression was study by DNA microarray. Design: The critical stages of spermatogenesis are at 8, 21 and 58 days after birth in mouse. In this study the gene expression been compared in 8,21 and 58 days old testis by DNA microarray in mouse. Materials/Methods: Total RNA was extracted from the testis of 8, 21 and 58 days old mouse. AtlasTM mouse cancer 1.2 Array were obtained from CLONTECH. There is 1185 mouse genes array on the membrane. Total RNA 5ug were reverse transcribed to cDNA in present of 32Plabelled dATP. The hybridization reaction was carrying out overnight at 42 C. Washed membranes were analysis on Phosphoimage Stome 840 ( Pharmacia Co.). The Image Quanta 5.0 software was been used for result analysis. Results: Of 1185 genes on the membrane, 198 genes were expression in the mouse testis. There are 49 genes expression only on the 8 days old testis. The function of these genes are involved in cell cycle, growth factor and its receptors, signal transduction, and protease and its inhibitor. There are 55 genes expression only on 58days old testis. The function of these genes are connected with transcription, stress, signal transduction, cell cycle, and protease and its inhibitor. There are not find any gene expression uniquely on 21 days old testis. There are 36 genes expression both on 8 days and 58 days old testis. When gene expression in 8 days old was compared with 58 days old testis, 30 genes are down-regulation, one gene is up-regulation and 5 genes expression are unchanged. Conclusions: The data show that the DNA microarray is an effect tool for the identification of gene expressed during the different stages of spermatogenesis. Further study the impact of multi-gene interaction on spermatogenesis will expand our understanding of the mechanism of spermatogenesis. Supported by: This research supported by The special funds for major state basic research project of China (CG199055901).
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the plasma membrane of apoptotic spermatozoa. The percent apoptosis was determined by epifluorescent microscopy. Western Blot technique was used to analyze for p65, p50 and IB proteins using rabbit polyclonal anti p65, p50 and IB antibodies. After treating with secondary antibody conjugated to horseradish peroxidase, antigen-antibody reaction was visualized by an enhanced chemiluminescence assay. Bands were quantitated using the Alpha Fluorochem analyzer and Actin counter staining was done on the blots to ensure that equal amounts of protein were loaded. Results: Using univariate regression analyses, sperm concentration was directly correlated with levels of p65 (P ⫽ 0.01), p50 (P ⫽ 0.05), and IB (P ⫽ 0.01). Patients with poor sperm concentration had significantly lower levels of NFKB as compared to patients with normal sperm concentration. Infertile men had significantly higher levels of apoptosis (p ⫽ 0.02) and lower levels of NFKB (p ⬍0.001) as compared to normal donors. Levels of p50 was significantly lower in varicocele patients when compared to patients with no clinical varicocele (p ⫽ 0.03). Conclusions: These results demonstrate significant correlation between low sperm concentration and low levels of NFKB expression. We speculate that low expression of NFKB is related at least in part to poor sperm production in infertile men. Supported by: None.
Regression analysis of neural computational model to predict ICSI outcomes. Feature
p-value
Technician No. ova retrieved Testis extraction Maternal age MESA EEJ
1.04e-107 9.84e-45 3.81e-21 7.70e-20 2.33e-10 1.52e-06
p-values indicate significance of the individual feature to the likelihood of fertilization with ICSI. Conclusions: We modeled ICSI outcome with high accuracy (ROC AUC 0.897 in the test set) using a neural computational approach. In order to determine the significance of individual features to the model’s outcome, regression based on Wilk’s GLRT was performed. All features examined (sperm source, number of ova retrieved, maternal age and which technician performed the procedure) were statistically significant to the model’s outcome. Supported by: NIH P01 HD36289 to Dolores J Lamb, Larry I Lipshultz and Craig Niederberger.
Wednesday, October 16, 2002 3:45 P.M. O-250 Correlation of nuclear factor kappa B (NFKB) with sperm quality and clinical diagnoses in infertile men. Pavithra Ranganathan, Namita Kattal, Mohamed H. Moustafa, Rakesh K. Sharma, Anthony J. Thomas Jr., Ashok Agarwal. Cleveland Clin Fdn, Cleveland, OH. Objective: NFKB (p65, p50 and I B) plays a major role in regulating apoptosis. Its role in the pathogenesis of male infertility has never been studied. The objectives of our study were to examine 1) the relationship of NFKB with semen quality (sperm concentration, motility and morphology) and the clinical diagnoses of male infertility, and 2) correlate levels of NFKB and apoptosis in ejaculated human spermatozoa. Design: Prospective experimental design. Materials/Methods: Semen samples from 12 donors and 40 infertile men (varicocele, n ⫽ 13; and others, n ⫽ 27) were used to probe for levels of p65, p50 and IB molecules. Semen samples were stratified into patients with poor sperm concentration (⬍20 ⫻ 106/mL; n ⫽ 11) and patients with normal sperm concentration (⬎20 ⫻ 106/mL; n ⫽ 29). Levels of apoptosis were detected using Annexin-V staining in neat semen specimens. The assay detects externalization of phosphatidylserine to the outer surface of
FERTILITY & STERILITY威
Wednesday, October 16, 2002 4:00 P.M. O-251 DNA microarray analysis of gene expression of spermatogenesis in mouse. Weixiong Li. National Research Institute for Family Planning, Beijing, China. Objective: Spermatogenesis is a complex and productive process that originates from stem cell spermatogonia and ultimately results in formation of mature spermatozoa. Recent studies have suggested that more than 100 genes involved this process. In the past, the investigation of gene expression was limited by the inability to study more than one gene at time. The introduction of DNA microarray technology has made it possible to examine the expression of thousands of genes at a time. For understanding the effect of multi-gene interaction on each stage of spermatogenesis. The gene expression was study by DNA microarray. Design: The critical stages of spermatogenesis are at 8, 21 and 58 days after birth in mouse. In this study the gene expression been compared in 8,21 and 58 days old testis by DNA microarray in mouse. Materials/Methods: Total RNA was extracted from the testis of 8, 21 and 58 days old mouse. AtlasTM mouse cancer 1.2 Array were obtained from CLONTECH. There is 1185 mouse genes array on the membrane. Total RNA 5ug were reverse transcribed to cDNA in present of 32Plabelled dATP. The hybridization reaction was carrying out overnight at 42 C. Washed membranes were analysis on Phosphoimage Stome 840 ( Pharmacia Co.). The Image Quanta 5.0 software was been used for result analysis. Results: Of 1185 genes on the membrane, 198 genes were expression in the mouse testis. There are 49 genes expression only on the 8 days old testis. The function of these genes are involved in cell cycle, growth factor and its receptors, signal transduction, and protease and its inhibitor. There are 55 genes expression only on 58days old testis. The function of these genes are connected with transcription, stress, signal transduction, cell cycle, and protease and its inhibitor. There are not find any gene expression uniquely on 21 days old testis. There are 36 genes expression both on 8 days and 58 days old testis. When gene expression in 8 days old was compared with 58 days old testis, 30 genes are down-regulation, one gene is up-regulation and 5 genes expression are unchanged. Conclusions: The data show that the DNA microarray is an effect tool for the identification of gene expressed during the different stages of spermatogenesis. Further study the impact of multi-gene interaction on spermatogenesis will expand our understanding of the mechanism of spermatogenesis. Supported by: This research supported by The special funds for major state basic research project of China (CG199055901).
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