THE RATE OF REFREEZING OF THAWED SEMEN SIGNIFICANTLY AFFECTS PRESERVATION OF MOTILITY AND VIABILITY. BR Gilbert1 and TA Brown, 1North Shore University Hospital, Manhasset, NY

Objective:
Cryopreservation has provided men with severely impaired semen quality or those about to undergo treatment that will adversely affect spermatogenesis, the means to preserve their gametes. However, often the total amount stored is also limited. Therefore, refreezing thawed specimens would provide additional opportunities for conception. The purpose of this study was to evaluate the effect that the rate of refreezing has on maintenance of sperm motility and viability after repetitive freeze/thaw cycles in specimens of varying semen quality.

Design:
A prospective analysis of motility and viability of semen specimens after repetitive freeze/thaw cycles by either a slow (controlled rate freezer) or fast (vapor) refreeze method.

Methods: 22 semen specimens (4 paired and 7 unpaired specimens) were diluted with 12% glycerol, 20% egg yolk in a 1:1 ratio at room temperature. The specimens were initially cryopreserved in 1cc plastic cryovials, using a controlled rate freezer with a standard "slow" freeze cycle (-10C/min until -300C then 50C/min until -800C). All specimens were thawed using a standard thaw protocol (30 minutes at room temperature then 10 minutes at 370C). The specimen then underwent repetitive cycles of either slow (n=10; as described above) or fast (n=12; 600C/min) refreezes with the standard thaw protocol. The thaw/refreeze cycles were repeated until no motile and no viable sperm (eosin Y-nigrosin stain) remained. t-test was used for statistical evaluation.

Results:
The mean sperm concentration and motility was 41.3 million/ml (range 8.3 to 98.5 million/ml) and 45% (range 28% to 60%). Motility and viability was present in all specimens through 2 thaw/refreeze cycles with a range from 2 to 7 thaw/refreeze cycles. Mean values for motility and viability, for both paired and unpaired specimens, were significantly greater for specimens undergoing repetitive fast refreezing (p<0.01), with a linear decrease of motility and viability of 8.7%/cycle and 8.2%/cycle respectively for the slow refreeze cycles and 6.7%/cycle and 7.7%/cycle respectively for the fast refreeze cycles (R2 >0.93). A fast refreeze preserved motility for an average of 2.75 cycles longer and viability for an average of 2.0 cycles longer than a slow refreeze.

Conclusions:
Multiple thaw/refreeze cycles are possible with even markedly impaired semen specimens. A fast rate of refreezing is significantly better than a slow rate in preserving motility and viability of cryopreserved semen specimens.