R John Aitken1, Adrienne R Hanson, Lesley Kuczera. 1. Discipline of Biological Sciences and Priority Research Centre for Reproductive Science, Faculty of Science and IT, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia. john.aitken@newcastle.edu.au
Abstract
BACKGROUND: The purpose of this study was to optimize the electrophoretic conditions that should be used for the effective isolation of functional human spermatozoa and to determine whether this method of isolating cells was associated with oxidative stress and DNA damage. METHODS: Human spermatozoa were prepared by repeated centrifugation, discontinuous density gradient centrifugation and electrophoresis followed by assessments of sperm quality. RESULTS: Systematic analysis of optimal electrophoresis conditions demonstrated that field strength was positively correlated with sperm recovery rates but negatively correlated with sperm movement, irrespective of whether the current or the voltage was held constant. This loss of functionality observed at high power settings was not associated with a major increase in superoxide generation or the induction of oxidative DNA damage. In contrast, discontinuous Percoll gradient centrifugation was shown to produce a significant rise in oxidative DNA base adduct expression in live cells (P < 0.05). As a result of these analyses, optimized electrophoretic conditions were defined that permitted sperm recovery rates of around 20%. These electrophoretically isolated cells were not only free of oxidative stress but exhibited significantly enhanced motility (P < 0.01) and vitality (P < 0.001) compared with the original samples. CONCLUSIONS: We conclude that while field strength is positively correlated with sperm recovery rates; it is negatively associated with sperm motility. Optimized conditions are described that represent a balance between these opposing forces and permit the isolation of highly motile, vital sperm populations, free from the oxidative DNA damage associated with conventional density gradient centrifugation technologies.
BACKGROUND: The purpose of this study was to optimize the electrophoretic conditions that should be used for the effective isolation of functional human spermatozoa and to determine whether this method of isolating cells was associated with oxidative stress and DNA damage. METHODS:Human spermatozoa were prepared by repeated centrifugation, discontinuous density gradient centrifugation and electrophoresis followed by assessments of sperm quality. RESULTS: Systematic analysis of optimal electrophoresis conditions demonstrated that field strength was positively correlated with sperm recovery rates but negatively correlated with sperm movement, irrespective of whether the current or the voltage was held constant. This loss of functionality observed at high power settings was not associated with a major increase in superoxide generation or the induction of oxidative DNA damage. In contrast, discontinuous Percoll gradient centrifugation was shown to produce a significant rise in oxidative DNA base adduct expression in live cells (P < 0.05). As a result of these analyses, optimized electrophoretic conditions were defined that permitted sperm recovery rates of around 20%. These electrophoretically isolated cells were not only free of oxidative stress but exhibited significantly enhanced motility (P < 0.01) and vitality (P < 0.001) compared with the original samples. CONCLUSIONS: We conclude that while field strength is positively correlated with sperm recovery rates; it is negatively associated with sperm motility. Optimized conditions are described that represent a balance between these opposing forces and permit the isolation of highly motile, vital sperm populations, free from the oxidative DNA damage associated with conventional density gradient centrifugation technologies.
Authors: Luke Simon; Kristin Murphy; Kenneth I Aston; Benjamin R Emery; James M Hotaling; Douglas T Carrell Journal: J Assist Reprod Genet Date: 2016-03-23 Impact factor: 3.412