Separating X-Bearing Human Spermatozoa Through a Discontinuous Percoll Density Gradient Proved to Be Inefficient by Double-Label Fluorescent In Situ Hybridization

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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.


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.


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.


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.

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