Sperm sex selection protocol.
For the study cohort, ejaculates were processed by a multilayer density gradient (Enhance-S Plus Cell Isolation Media, 100%; Vitrolife, Goteborg, Sweden) comprised of four concentrations: 20%, 40%, 60%, and 90%. After removing the seminal fluid, the resuspended sperm pellet was layered on top, and spermatozoa were able to self-select for 90 minutes at 4°C. Y-bearing spermatozoa were retrieved from the upper, 20% layer, and heavier X-bearing spermatozoa were retrieved from the bottom, 90% layer.