| Objective:1.Our group independently developed a rare spermatozoa freezing carrier named Micro-straw.The purpose of this study was to further explore the optimal freeze-thaw protocol of Micro-straw carrier to improve the quality of sperm after cryopreservation.2.The freezing effects of Micro-straw,LSL and Cryoplus carriers were compared,to obtain the carrier with the best cryopreservation effect,which could be used to guide the cryopreservation of human rare spermatozoa in clinical practice.Method:This study involves ninety-three samples from healthy sperm donors and twenty samples collected from patients diagnosed with oligospermia,asthenospermia,or oligoasthenospermia,and 20 samples of epididymal sperm collected from obstructive azoospermia sufferers undergoing PESA.1.According to the temperature curve,different freezing and thawing protocols were set,the sperm quality after freezed by Micro-straw carrier was compared,to obtain the optimal freezing and thawing protocol of Micro-straw carrier.2.The sperm survival rate,normal morphology rate,acrosome integrity and DFI of Micro-straw,LSL and Cryoplus were compared,and the freeze-thaw effects of the three carriers were evaluated.3.We verified the effects of freezing using these carriers by comparing the qualities of post-thaw spermatozoa from patients.Results:1.Comparing the survival rate of spermatozoa after gradient centrifugation and untreated,we found that spermatozoa after gradient centrifugation had a higher PR survival rate after freezing and thawing(P<0.01).Comparing the sperm quality of Micro-straw carrier under different freezing and thawing protocols,it was found that Micro-straw carrier was placed at 1 cm of liquid nitrogen surface for fumigation for 70 sec,and rewarmed in 42-℃ water bath for 40 sec,which had a higher sperm PR recovery rate(67.7%)and TM recovery rate(74.9%),while the normal morphological rate,acrosome integrity and DFI of sperm were not significantly changed under different freezing protocols.2.The cooling rate of Micro-straw and Cryoplus was faster than that of LSL,while the thawing rate of Micro-straw was slower than that of Cryoplus and LSL.By cryopreserving the donor semen samples,we found that there were no significant differences in the PR survival rate,acrosome integrity and DFI of sperm after freezing and thawing among the three carriers,but cryopreservation by Micro-straw resulted in a higher normal sperm morphology rate than that by Cryoplus(8.7% vs7.8%,P<0.05),and the TM survival rates for the Micro-straw and LSL carriers are significantly different(68.4% vs 63.5%,P<0.01).3.20 ejaculate sperm samples of patients with oligospermia,asthenozoospermia or oligoasthenospermia were cryopreserved by the three carriers,we found that the TM survival rate of the Micro-straw carrier was higher than that of LSL and Cryoplus(50.07% vs 39.84% vs46.5%),with no significantly difference.Another 20 epididymal sperm samples were freezed,we also found that there was no significant difference in the TM survival rate between Micro-straw,LSL and Cryoplus(51.12% vs 49.7% vs 56.72%).4.A total of 10 male infertile patients used the optimized Micro-straw carrier for rare spermatozoa cryopreservation,among which3 patients used the thawed spermatozoa for ICSI cycle,the average fertilization rate was 79.3%,and the cleavage rate was 95.7%,The high-quality embryo rate was 59.1%.Conclusion:1.The optimal cryopreservation method for the Micro-straw carrier is cryopreservation at 1 cm above LN2 for 70 sec and thawing in a water-bath at 42-°C for 40 sec.2.Before rare spermatozoa cryopreservation,gradient centrifugation of semen samples can effectively improve the quality of sperm after freezing and thawing.3.Micro-straw,Cryoplus,and LSL carriers are all efficient means of freezing a small number of human spermatozoa.However,the Micro-straw carrier is more economical,safe,user-friendly and has a higher cryoprotective effect. |
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