An inexpensive method for gender pre-selection

Abstract

To test a simple, reliable, and inexpensive method to attempt selection of gender specific spermatozoa. The only available technique today is flow cytometry, however, it is expensive and requires DNA staining with a fluorochrome coadjuvated by laser beam excitation. We assessed multilayer discontinuous density gradient (DG) to measure which layer is capable of enriching X- or Y-bearing spermatozoa. Semen specimens were processed by various discontinuous DG: 1) 60-90% (double), 2) 40-60-90% (triple), 3) 20-40-60-90% (quad). Putatively X-bearing spermatozoa were searched in 90% fraction while Y-bearing spermatozoa were identified from the lightest DG fraction. Sperm suspensions were smeared on slides for FISH analysis using centromeric probes for chromosomes 18, X, and Y. The ratio of X- to Y- chromosome bearing spermatozoa was blindly assessed as a percentage on at least 200 cells per slide. Aneuploid cells and those without signals were omitted. Unselected fractions of each sample served as controls. A total of 6 samples with a concentration of 48.3 ± 17 x 106/ml, motility of 50.9 ± 6%, and normal morphology of 2.4 ± 1% were included. The overall proportion of gender specific spermatozoa in unselected samples was 50.1% for X and 49.9% for Y. When selecting for X, a two-layer gave 63.2%, a triple 75.5%, and the quad 80.3%. This provided a direct correlation with the increasing number of layers (P = 0.0001). When assessing for Y, a double layer yielded 62.7%, triple 75.5%, and quad 78.5%. As the density gradient became lighter, the proportion of Y-bearing spermatozoa increased (P = 0.0001). Selection of spermatozoa is an ethically accepted method for gender pre-selection. The only commercially available method to gender select spermatozoa is Microsort®. We were able, with an inexpensive, safe, and reproducible way, to enrich gender specific spermatozoa at a comparable level with Microsort®.