Separating X-bearing human spermatozoa through a discontinuous Percoll density gradient proved to be inefficient by double-label fluorescent in situ hybridization.
OBJECTIVE
Double-label fluorescence in situ hybridization (FISH) was used to evaluate the efficiency of separating X- and Y-chromosome-bearing spermatozoa through 12-step discontinuous Percoll gradients.


METHODS
Liquefied normal semen samples from 10 healthy donors were overlaid onto 25% Percoll and centrifuged. Parts of the sperm pellet were saved as control, while the remaining portion was separated by 12-step Percoll gradient. After centrifugation, the spermatozoa in the 80% Percoll layer were collected. The X:Y ratio of the control and separated spermatozoa was verified by double-label FISH (CEP SOX/SGY probes) and scored blindly by one observer. Differences in the X:Y ratios between matched groups were analyzed by paired t tests.


RESULTS
The overall average labeling efficiency was 99.2%. A significant enrichment (P = 0.02) of X-bearing spermatozoa was obtained in Percoll separated fractions (mean X:Y ratio = 52.2:46.4) compared with the control group (X:Y ratio = 49.5:48.4). Discontinuous Percoll gradients also decreased the proportion of aneuploid spermatozoa (from 1.0 to 0.8%), but the differences were nonsignificant.


CONCLUSIONS
Discontinuous Percoll separation did increase the X:Y ratio significantly, but the enrichment of X-bearing spermatozoa is insufficient for clinical use in preconceptional sex selection.
Lin
S P
SP
Department of Medical Research, Mackay Memorial Hospital, Tamshui, Taipei, Taiwan.
Lee
R K
RK
Tsai
Y J
YJ
Hwu
Y M
YM
Lin
M H
MH
D002499:Centrifugation, Density Gradient / Q000592:standards; D002843:Chromatin / Q000737:chemistry; D003102:Colloids / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008856:Microscopy, Fluorescence; D011205:Povidone / Q000737:chemistry; D012743:Sex Preselection / Q000379:methods*; D012822:Silicon Dioxide / Q000737:chemistry; D013094:Spermatozoa / Q000502:physiology*; D014960:X Chromosome / Q000737:chemistry*; D014998:Y Chromosome / Q000737:chemistry*
D002843:Chromatin; D003102:Colloids; D015342:DNA Probes; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone