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
Cy. Andersen et Ag. Byskov, 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, Acta obstetricia et gynecologica Scandinavica, 76(2), 1997, pp. 131-134
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.