Freezing Of Microquantities Of Sperm In Microcarriers Without Croypretectants

PartⅠPilot study of cryoloop for vitrifying spermatozoa without cryoprotectantsObjectiveSeeking for a suitable freezing carrier for testicular or epididymal sperm by discussing the feasibility of cryoloop for vitrifying spermatozoa without cryoprotectants.MethodsNormal sperm were frozen by conventional freezing protocol and non-cryoprotectant vitrification respectively. After thawing, compared the results of the two freezing methods according to mobilities and ultrastructures under electron microscope.ResultsMotilities were of no statistic differences between the two groups(44.5% vs 43.5%, P>0.05),but both lower evidently than control group(44.5% vs 43.5% vs 88.5%, P<0.001). At the same time, ultrastructures of those two frozen samples also changed, but structures of nucleus remained intact.Conclusions Non-cryoprotectant vitrification for sperm freezing with cryoloop is a simple,convenient and effective method;cryoloop is a hopeful freezing carrier for testicular or epididymal sperm. PartⅡFeasibility of vitrifying normospermic samples using droplets without cryoprotectansObjectiveDiscuss the feasibility of droplets to vitrify normospermic samples without cryoprotectants.MethodsNormal prepared sperm were frozen by conventional freezing protocol and non-cryoprotectant freezing in droplets respectively. Freezing in droplets means freezing sperm in every 10ul drop. After thawing, compared the two groups'sperm mobilities.ResultsMobilities were of no statistic differences between the two freezing groups(40% vs 37.5%, P>0.05),but both lower evidently than those of the control group(40% vs 37.5% vs 85%, P<0.001).ConclusionsVitrifying normospermatozoa in droplets without cryoprotectants can achieve a good result to a certainty;further studies should be carried out. PartⅢFeasibility of freezing sperm obtained from PESA using droplets without cryoprotectansObjectiveDiscuss the feasibility of droplets to freeze PESA sperm without cryoprotectants.Methods12 PESA sperm were frozen by non-cryoprotectant freezing protocol in droplets. Freezing in droplets means freezing sperm in every 10ul drop. After thawing, checked the samples'mobilities under a microscope.ResultsA sample that had〉30 mobile sperm after thawing is considered success. In general 8 samples were successfully achieved in 12 samples, that is, recovery rates of sperm after thawing were 66.7%.ConclusionsFreezing PESA sperm in droplets without cryoprotectants can achieve a good result to a certainty;droplet is an useful carrier to freeze microquantities of sperm.