title: Effect of combined density gradient centrifugation on X- and Y- sperm separation and chromatin integrity.
abstract: BACKGROUND
  It has been claimed that by using different washing methods, the sperms can be separated according to size, motility, density, chromosomal content and surface markings and charge. These methods also reduce sperm chromatin deficiencies and screen the sperms before applying in assisted reproduction techniques.
  
  
  OBJECTIVE
  This study compared simple density gradient methods and a combined method with albumin density gradient and PureSperm separation (alb/PureSperm) for sex preselection by double fluorescence in situ hybridization (FISH) versus chromomycin A3 staining to determine chromatin integrity.
  
  
  METHODS
  30 normal semen samples were prepared with PureSperm, albumin gradients and alb/PureSperm. All samples were then stained by FISH and chromomycin A3. The results were compared with SPSS 11.5 and the Kruskal-Wallis test.
  
  
  RESULTS
  The proportion of X-bearing spermatozoa by PureSperm separation (47.58±5.67) and Y-bearing spermatozoa by albumin gradient (46.13±3.83) methods were slightly higher than in putative normal sperm samples (1:1), but there were no significant differences in the X- or Y- bearing spermatozoa counts among the three methods. Albumin gradient separation tended to underestimate abnormal spermatozoa compared to PureSperm and combined alb/PureSperm.
  
  
  CONCLUSIONS
  Routine separation methods slightly enriched X- or Y- bearing spermatozoa, but the differences were not significant for clinical purposes. The combined alb/PureSperm method had no advantages for assessing sex ratio or chromatin integrity compared to simpler gradient methods.
authors.affiliation: Department of Anatomical Sciences, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Tahereh
authors.initials: T
authors.lastname: Esmaeilpour
authors.affiliation: Department of Anatomical Sciences, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Leila
authors.initials: L
authors.lastname: Elyasi
authors.affiliation: Department of Anatomical Sciences, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Soghra
authors.initials: S
authors.lastname: Bahmanpour
authors.affiliation: Islamic Azad University, Larestan, Iran. ; Shiraz Infertility Center, Shiraz, Iran.
authors.forename: Alireza
authors.initials: A
authors.lastname: Ghannadi
authors.affiliation: Department of Pathology, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Monabbati
authors.affiliation: Department of Anatomical Sciences, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Farzaneh
authors.initials: F
authors.lastname: Dehghani
authors.affiliation: Shiraz Infertility Center, Shiraz, Iran.
authors.forename: Marjaneh
authors.initials: M
authors.lastname: Kazerooni
keywords: Albumin gradient; Double-labeled fluorescence in situ hybridization; Sex ratio; Sperm chromatin; Sperm separation