Custom cryopreservation of human semen

Abstract

To design a protocol to evaluate individual variability in human semen cryoprotection by native seminal plasma. Post-thaw motility from the frozen semen of pregnancy-proven donors (n = 10) and patients referred for infertility screening (n = 10) was examined in three equal aliquots (per original ejaculate) that comprised varying ratios of native seminal plasma to TES and Tris (TEST)-yolk buffer (Irvine Scientific, Irvine, CA) in a dose-titration curve format. All aliquots from the same ejaculate contained final vol/vol 6% glycerol, had equal sperm density, and had undergone centrifugation for 5 minutes at 600 x g before buffer:semen ratio adjustment and standard precooling protocol for submersion in liquid nitrogen. Post-thaw measurement of percent original motility preserved (post-thaw percent motility/original percent motility x 100) was used for standardization of results. In 14 of 20 specimens (70%), the maximal yield of original motility was obtained in 50% seminal plasma, with an average post-thaw motile yield of 50%. In 6 of 20 specimens (30%), the best preservation of original motility was noted at 100% seminal plasma, with an average post-thaw motile yield of 58%. No specimen had a greatest percent motility preserved at 0% seminal plasma. Donor specimens have equal preference for either 50% or 100% seminal plasma, whereas patient specimens have a preference for 50% seminal plasma (P < 0.05). Native seminal plasma has variable cryoprotectant qualities for which custom cryopreservation can compensate. A simple two-point dose-titration test of cryopreservation buffer:seminal plasma ratio (i.e., 50:50 versus 0:100) can determine the optimal mixture for cryopreservation of a given individual's semen.