title: Significant enrichment of Y-bearing chromosome human spermatozoa using a modified centrifugation technique.
abstract: The effective separation of X- and Y-bearing chromosome spermatozoa has been a topic of major attraction to a number of scientific disciplines. Approaches have typically been based upon either the kinetic or the physical characteristics of spermatozoa. Much of the information available to date has either suggested conflicting evidence between different approaches or a lack of repeatability, while other robust and reproducible techniques require expensive equipment and are being questioned with relation to their safety in clinical applications. This study describes a safe and efficient method for the successful enrichment of the Y-bearing chromosome spermatozoa cells from their X counterparts in the human male using a simple approach based on centrifugation. Five donor candidates with normal semen profiles and proven fertility were recruited. In total, 20 semen specimens were processed using conventional swim-up. During each attempt, half of the swim-up product was subjected to the enrichment technique and the other half served as control. Parameters important for successfully skewing sex ratios included the relative centrifugal force, the duration of centrifugal separation and the number of centrifugation rounds. Assessment of samples following the separation technique was effected by a three-colour-labelled fluorescent in situ hybridization. More than 1000 spermatozoa for each donor specimen were assessed for the presence of an X or Y chromosome. The enrichment technique produced a significantly higher (p < 0.001) overall frequency of 85.5% for the Y-bearing chromosome spermatozoa in the experimental group (3606 X-bearing/21, 209 Y-bearing) compared with a frequency of 50.1% in the control group (11,801 X-bearing/11,269 Y-bearing), where there was no statistically significant difference in the number of either X- or Y-chromosome-bearing spermatozoa. In conclusion, successful skewing of human Y-bearing chromosome spermatozoa can be reproducibly achieved by the use of simple swim-up followed by a meticulous centrifugation protocol.
authors.affiliation: Genesis Centre for Fertility and Human Pre-implantation Genetics, Limassol, Cyprus.
authors.forename: S
authors.initials: S
authors.lastname: Koundouros
authors.affiliation: and the Institute of Medical Research, Monash University, Melbourne, Victoria, Australia.
authors.forename: P
authors.initials: P
authors.lastname: Verma
mesh_terms: D002498:Centrifugation / Q000379:methods*; D041322:Chromosomes, Human, Y; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D013094:Spermatozoa / Q000648:ultrastructure*