Comparison of various methods of processing human cryopreserved-thawed semen samples

Abstract

We compared the efficacy of various methods of processing cryopreserved-thawed samples for the recovery of functionally adequate spermatozoa as assessed by the response to the sperm stress test (SST), an index of temperature activated sperm membrane lipid peroxidation, and immediate and delayed changes in sperm viability and motion parameters. Donor semen samples (n = 28) were cryopreserved-thawed and divided into six equal parts, one part was used as control and the remaining parts were used to compare five methods of sperm processing as follows: direct Percoll gradient processing, washing by one-step or stepwise addition of the washing medium followed by Percoll processing, and washing by one-step or stepwise addition of the washing medium. Additional samples (n = 10) were evaluated for the immediate and delayed (6 h at 37 degrees C) impact of one-step and stepwise washing (without Percoll separation). Compared with wash-only methods, samples processed using Percoll had a significantly higher SST score (P = 0.001), motility, rapid spermatozoa (>50 microm/s), curvilinear velocity and motility index (P < 0.001). Comparing various Percoll methods, direct Percoll processing resulted in the highest number of motile spermatozoa recovered (P < 0.00001) and a higher SST score based on curvilinear velocity (P = 0.001). Stepwise washing gave a significantly higher number of motile spermatozoa (P < 0.001) but with a significantly lower SST score based on the concentration of motile spermatozoa (P = 0.001), motility (P = 0.001) and motility index (P = 0.01). Sperm viability and motion parameters after 6 h of incubation showed no difference between one-step and stepwise washing. In conclusion, compared with wash-only methods, Percoll processed samples resulted in the recovery of spermatozoa with superior quality as assessed by SST and motion analysis. One-step washing of the samples gave an overall comparable recovery compared to the samples prepared stepwise. Having significantly higher SST scores, similar viability and the maintenance of motility, one-step washing may be a better method of processing thawed samples than the stepwise washing.

Superoxide dismutase content in sperm correlates with motility recovery after thawing of cryopreserved human spermatozoa

“…The method used for freezing and thawing was that described by Srisombut and co-workers (18). Briefly, the sperm suspension was slowly mixed with the freezing medium containing TES-TRIS citrate and 20% egg yolk with 12% glycerol at a final concentration of 6% (Irvine Scientific) in a dropwise fashion until a 1:1, vol/vol ratio was attained.…”

Effect of thawing temperature on the motility recovery of cryopreserved human spermatozoa

“…The method used for freezing and thawing was that described by Srisombut and coworkers (20). Briefly, the sperm suspension was slowly mixed with the freezing medium containing TES-TRIS citrate and 20% egg yolk with 12% glycerol at a final concentration of 4.5% (Irvine Scientific) in a drop-wise fashion until a 1:1 volume ratio was attained.…”

The DNA integrity of cryopreserved spermatozoa separated for use in assisted reproductive technology is unaffected by the type of cryoprotectant used but is related to the DNA integrity of the fresh separated preparation

“…Considering that subfertile men form a very large proportion of the men requiring semen cryopreservation, of most concern is the apparent increased susceptibility of spermatozoa from subfertile men to cryoinjury (6,17). Interestingly, a large number of studies examining the effectiveness of different cryoprotectants and cryopreservation methods have recruited only normospermic donors (2,(18)(19)(20). It is also important to specifically assess the quality of the separated spermatozoa that will be used for treatment, as any spermatozoon given the chance to fertilize an oocyte will come from these preparations.…”