title: Potential importance of transition metals in the induction of DNA damage by sperm preparation media.
abstract: OBJECTIVE
  What are the mechanisms by which the preparation of spermatozoa on discontinuous density gradients leads to an increase in oxidative DNA damage?
  
  
  CONCLUSIONS
  The colloidal silicon solutions that are commonly used to prepare human spermatozoa for assisted reproduction technology (ART) purposes contain metals in concentrations that promote free radical-mediated DNA damage.
  
  
  BACKGROUND
  Sporadic reports have already appeared indicating that the use of colloidal silicon-based discontinuous density gradients for sperm preparation is occasionally associated with the induction of oxidative DNA damage. The cause of this damage is however unknown.
  
  
  METHODS
  This study comprised a series of experiments designed to: (i) confirm the induction of oxidative DNA damage in spermatozoa prepared on commercially available colloidal silicon gradients, (ii) compare the levels of damage observed with alterative sperm preparation techniques including an electrophoretic approach and (iii) determine the cause of the oxidative DNA damage and develop strategies for its prevention. The semen samples employed for this analysis involved a cohort of >50 unselected donors and at least three independent samples were used for each component of the analysis.
  
  
  METHODS
  The setting was a University biomedical science laboratory. The major techniques employed were: (i) flow cytometry to study reactive oxygen species generation, lipid peroxidation and DNA damage, (ii) computer-aided sperm analysis to measure sperm movement and (iii) inductively coupled mass spectrometry to determine the elemental composition of sperm preparation media.
  
  
  RESULTS
  Oxidative DNA damage is induced in spermatozoa prepared on PureSperm(®) discontinuous colloidal silicon gradients (P < 0.001 versus repeated centrifugation) because this medium contains metals, particularly Fe, Al and Cu, which are known to promote free radical generation in the immediate vicinity of DNA. This damage can be significantly accentuated by reducing agents, such as ascorbate (P < 0.001) and inhibited by selective chelation (P < 0.001). This problem is not confined to PureSperm(®); analysis of additional commercial sperm preparation media revealed that metal contamination is a relatively constant feature of such products.
  
  
  CONCLUSIONS
  While the presence of metals, particularly transition metals, may exacerbate the levels of oxidative DNA damage seen in human spermatozoa, the significance of such damage has not yet been tested in suitably powered clinical trials.
  
  
  CONCLUSIONS
  The results explain why the preparation of spermatozoa on discontinuous colloidal silicon gradients can result in oxidative DNA damage. The results are of immediate relevance to the development of safe, effective protocols for the preparation of spermatozoa for ART purposes.
  
  
  BACKGROUND
  The study was funded by the Australian Health and Medical Research Council. One of the authors (R.J.A.) has had a consultantship with a biotechnology company, NuSep, interested in the development of electrophoretic methods of sperm preparation. He has no current financial interest in this area. None of the other authors have a conflict of interest to declare.
authors.affiliation: Discipline of Biological Sciences and Priority Research Centre in Reproductive Science, Faculty of Science and IT, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia john.aitken@newcastle.edu.au.
authors.forename: R J
authors.initials: RJ
authors.lastname: Aitken
authors.affiliation: Discipline of Biological Sciences and Priority Research Centre in Reproductive Science, Faculty of Science and IT, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.
authors.forename: J M
authors.initials: JM
authors.lastname: Finnie
authors.affiliation: Discipline of Biological Sciences and Priority Research Centre in Reproductive Science, Faculty of Science and IT, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.
authors.forename: L
authors.initials: L
authors.lastname: Muscio
authors.affiliation: Discipline of Biological Sciences and Priority Research Centre in Reproductive Science, Faculty of Science and IT, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.
authors.forename: S
authors.initials: S
authors.lastname: Whiting
authors.affiliation: Discipline of Biological Sciences and Priority Research Centre in Reproductive Science, Faculty of Science and IT, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.
authors.forename: H S
authors.initials: HS
authors.lastname: Connaughton
authors.affiliation: Discipline of Biological Sciences and Priority Research Centre in Reproductive Science, Faculty of Science and IT, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.
authors.forename: L
authors.initials: L
authors.lastname: Kuczera
authors.affiliation: Discipline of Biological Sciences and Priority Research Centre in Reproductive Science, Faculty of Science and IT, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.
authors.forename: T B
authors.initials: TB
authors.lastname: Rothkirch
authors.affiliation: Discipline of Biological Sciences and Priority Research Centre in Reproductive Science, Faculty of Science and IT, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.
authors.forename: G N
authors.initials: GN
authors.lastname: De Iuliis
mesh_terms: D002498:Centrifugation / Q000009:adverse effects; D015331:Cohort Studies; D003102:Colloids / Q000737:chemistry; D004249:DNA Damage; D005434:Flow Cytometry; D006801:Humans; D015227:Lipid Peroxidation; D008297:Male; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D012825:Silicon / Q000494:pharmacology*; D013048:Specimen Handling / Q000009:adverse effects; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects*; D028561:Transition Elements / Q000032:analysis / Q000494:pharmacology
chemical_list: D003102:Colloids; D017382:Reactive Oxygen Species; D028561:Transition Elements; D012825:Silicon
keywords: assisted reproduction technology; colloidal silicon; sperm DNA damage; sperm preparation; transition metals