title: Optimization of microelectrophoresis to select highly negatively charged sperm.
abstract: OBJECTIVE
  The sperm membrane undergoes extensive surface remodeling as it matures in the epididymis. During this process, the sperm is encapsulated in an extensive glycocalyx layer, which provides the membrane with its characteristic negative electrostatic charge. In this study, we develop a method of microelectrophoresis and standardize the protocol to isolate sperm with high negative membrane charge.
  
  
  METHODS
  Under an electric field, the percentage of positively charged sperm (PCS), negatively charged sperm (NCS), and neutrally charged sperm was determined for each ejaculate prior to and following density gradient centrifugation (DGC), and evaluated for sperm DNA damage, and histone retention. Subsequently, PCS, NCS, and neutrally charged sperm were selected using an ICSI needle and directly analyzed for DNA damage.
  
  
  RESULTS
  When raw semen was analyzed using microelectrophoresis, 94 % were NCS. In contrast, DGC completely or partially stripped the negative membrane charge from sperm resulting PCS and neutrally charged sperm, while the charged sperm populations are increased with an increase in electrophoretic current. Following DGC, high sperm DNA damage and abnormal histone retention were inversely correlated with percentage NCS and directly correlated with percentage PCS. NCS exhibited significantly lower DNA damage when compared with control (P < 0.05) and PCS (P < 0.05). When the charged sperm population was corrected for neutrally charged sperm, sperm DNA damage was strongly associated with NCS at a lower electrophoretic current.
  
  
  CONCLUSIONS
  The results suggest that selection of NCS at lower current may be an important biomarker to select healthy sperm for assisted reproductive treatment.
authors.affiliation: Andrology and IVF Laboratory, Department of Surgery (Urology), Salt Lake City, Utah, USA.
authors.forename: Luke
authors.initials: L
authors.lastname: Simon
authors.affiliation: Andrology and IVF Laboratory, Department of Surgery (Urology), Salt Lake City, Utah, USA.
authors.forename: Kristin
authors.initials: K
authors.lastname: Murphy
authors.affiliation: Andrology and IVF Laboratory, Department of Surgery (Urology), Salt Lake City, Utah, USA.
authors.forename: Kenneth I
authors.initials: KI
authors.lastname: Aston
authors.affiliation: Andrology and IVF Laboratory, Department of Surgery (Urology), Salt Lake City, Utah, USA.
authors.forename: Benjamin R
authors.initials: BR
authors.lastname: Emery
authors.affiliation: Andrology and IVF Laboratory, Department of Surgery (Urology), Salt Lake City, Utah, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Hotaling
authors.affiliation: Andrology and IVF Laboratory, Department of Surgery (Urology), Salt Lake City, Utah, USA. douglas.carrell@hsc.utah.edu.|Department of Obstetrics and Gynecology, Salt Lake City, Utah, USA. douglas.carrell@hsc.utah.edu.|Department of Human Genetics, University of Utah, 675 Arapeen Drive, Suite 201, Salt Lake City, Utah, 84109, USA. douglas.carrell@hsc.utah.edu.
authors.forename: Douglas T
authors.initials: DT
authors.lastname: Carrell
mesh_terms: D015415:Biomarkers; D004249:DNA Damage; D004586:Electrophoresis / Q000379:methods*; D019276:Glycocalyx / Q000737:chemistry*; D006801:Humans; D008297:Male; D055101:Semen Analysis / Q000379:methods*; D013094:Spermatozoa / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D013499:Surface Properties
chemical_list: D015415:Biomarkers
keywords: Membrane glycocalyx; Micro-electrophoresis; Sperm DNA damage; Sperm membrane charge; Sperm selection