title: Density gradient centrifugation before or after magnetic-activated cell sorting: which technique is more useful for clinical sperm selection?
abstract: OBJECTIVE
  Although, at present, the selection of sperm prior to ICSI is based on motility and morphology, undetectable anomalies, and more importantly damaged DNA are overlooked. In this regard, novel sperm selection procedures have gained much interest. For instance, sperm has been selected by Magnetic-Activated Cell Sorting (MACS) based on early apoptotic marker, the externalization of phosphatidylserine (EPS). Review of the literature has revealed that the efficiency of this technique has been mainly evaluated post Density Gradient Centrifugation (DGC). Therefore, there is a need to prove the efficiency of this technique independent of DGC. In addition, considering the fact that DGC induces EPS due to capacitation and acrosome reaction, therefore, the role of MACS before DGC(MACS-DGC) and MACS after DGC (DGC-MACS) should be assessed.
  
  
  METHODS
  Semen samples from fifteen infertile men were divided into three separate fractions: control, DGC, and MACS. To carry out DGC-MACS, DGC samples were further divided into two fractions and MACS was carried on the second fractions. Similarly to carry out MACS-DGC, the MACS samples were further divided into two fractions and DGC was carried on the second fractions. Percentages of sperm with normal morphology, DNA fragmentation, protamine deficiency, EPS and caspase-3 activity were determined in each fraction.
  
  
  RESULTS
  DGC is more efficient than MACS in separating intact sperm only in terms of normal morphology, DNA and chromatin integrity but not for active caspase. However, a combination of these procedures was more efficient than a single procedure to separate intact sperm for the aforementioned parameters. Comparison of the combined procedures showed only higher efficiency to separate active caspase in the MACS-DGC group.
  
  
  CONCLUSIONS
  Based on these results, we propose MACS-DGC rather than DGC-MACS to be implemented in clinical settings.
authors.affiliation: Department of Reproduction and Development, Reproductive Biomedicine Research Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Tavalaee
authors.forename: M R
authors.initials: MR
authors.lastname: Deemeh
authors.forename: M
authors.initials: M
authors.lastname: Arbabian
authors.forename: M H
authors.initials: MH
authors.lastname: Nasr-Esfahani
mesh_terms: D017209:Apoptosis / Q000235:genetics; D053148:Caspase 3 / Q000378:metabolism; D002499:Centrifugation, Density Gradient / Q000379:methods*; D004249:DNA Damage; D053938:DNA Fragmentation; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008297:Male; D010718:Phosphatidylserines / Q000378:metabolism; D011479:Protamines / Q000378:metabolism; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods; D013081:Sperm Motility; D013094:Spermatozoa / Q000166:cytology*
chemical_list: D010718:Phosphatidylserines; D011479:Protamines; D053148:Caspase 3