Enhanced separation of X and Y bearing sperm cells by a combined density gradient centrifugation evaluated by fluorescence in situ hybridization of the Y-chromosome

OBJECTIVES: Fluorescent in situ hybridization of decondensed sperm cells was used to evaluate the effectiveness of separating X- and Y chromosome bearing sperm cells using a combination of Percoll and NycoPrep media.

MATERIAL AND METHODS: Semen samples from seven healthy men were divided into three equal aliquots. One aliquot served as an untreated control, the other two were either applied to a seven-step Percoll gradient (100%-40%), or to a seven-step Percoll gradient (100%-40%) underlayered with 100% NycoPrep for sperm purification. After centrifugation Y-bearing sperm cells in the pellet and in the control sample was determined by fluorescent in situ hybridization.

RESULTS: A significantly reduction of Y-bearing sperm cells were observed in the pellet of the Percoll + NycoPrep centrifugation (39.3% Y-bearing sperm cells) compared to the control sample and to Percoll centrifugation without NycoPrep. The Percoll centrifugation without NycoPrep did not show a significant reduction of Y-bearing sperm cells in the pellet compared to the control sample.

CONCLUSION: By the addition of a medium with a density higher than 100% Percoll a significantly increased separation of X and Y-bearing sperm cells can be achieved. This study opens new perspectives for improving separation of X- and Y-bearing spermatozoa by simple centrifugations. The described centrifugation technique can be applied in connection with preimplantation diagnosis in order to create more pre-embryos of the desired gender.