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 cel ls was used to evaluate the effectiveness of separating X- and Y chrom osome 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 gr adient (100%-40%), or to a seven-step Percoll gradient (100%-40%) unde rlayered with 100% NycoPrep for sperm purification. After centrifugati on Y-bearing sperm cells in the pellet and in the control sample was d etermined by fluorescent in situ hybridization. Results. A significant ly reduction of Y-bearing sperm cells were observed in the pellet of t he Percoll+NycoPrep centrifugation (39.3% Y-bearing sperm cells) compa red to the control sample and to Percoll centrifugation without NycoPr ep. The Percoll centrifugation without NycoPrep did not show a signifi cant 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 an d Y-bearing sperm cells can be achieved. This study opens new perspect ives for improving separation of X- and Y-bearing spermatozoa by simpl e centrifugations. The described centrifugation technique can be appli ed in connection with preimplantation diagnosis in order to create mor e pre-embryos of the desired gender.