不同浓度的蔗糖溶液快速冻存人类精子的实验研究

目的:探讨使用非渗透性冷冻保护剂蔗糖快速冻存人类精子的可行性。方法:收集正常精液标本40份,经上游法处理后,收集上游精子悬液,分成6份,1份作为对照,其余5份分别用常规精子冷冻法,0.15、0.20、0.25、0.30 mol/L蔗糖溶液冷冻精子,复苏后比较6组精子活动率、前向运动精子活动率及精子质膜完整性。结果:所有冷冻组复苏后精子活动率、前向运动精子活动率及膜完整率与冷冻前上游组(96.2±1.8)%,(93.8±2.8)%,(99.0±0.8)%比较均显著下降,差异有显著性(P<0.05)。精子活动率结果:蔗糖冷冻组之间比较,0.20 mol/L组解冻后精子活动率高于0.15、0.25、0.30 mol/L组,分别为(55.5±6.3)%、(45.9±6.6)%、(50.4±9.4)%、(45.5±11.2)%,差异有显著性(P<0.05)。0.20、0.25 mol/L蔗糖冷冻组与常规慢速冷冻组(53.6±5.0)%比较,差异均无显著性(P>0.05);0.15、0.30 mol/L蔗糖组均低于常规慢速冷冻组(P<0.05)。前向运动精子活动率结果:0.20 mol/L蔗糖冷冻组与常规冷冻组比较差异无显著性(44.4±7.4)%vs(42.3±8.1)%,P>0.05];以上两组均高于0.15、0.25、0.30 mol/L蔗糖冷冻组,分别为(37.1±8.3)%、(33.1±9.2)%、(22.0±9.1)%,差异有显著性(P<0.05)。精子膜完整率结果:0.20 mol/L蔗糖冷冻组(70.1±6.9)%高于常规冷冻组、0.15、0.25、0.30 mol/L蔗糖冷冻组,分别为(63.1±6.8)%、(57.7±8.3)%、(63.5±10.7)%、(57.8±12.9)%,差异有显著性(P<0.05);常规冷冻组与0.15、0.25、0.30 mol/L蔗糖冷冻组比较差异无显著性(P>0.05)。结论:终浓度为0.20 mol/L的蔗糖溶液作为人类精子冷冻保护剂是可行的,快速冷冻方法简便、快速、经济、安全有效。

Ultra-rapid cryopreservation of human spermatozoa with different concentrations of sucrose

Objective: To explore the feasibility of ultra-rapid freezing of human spermatozoa in the cryogenic vial with different concentrations of sucrose solution.Methods: We divided 40 normal semen samples prepared with the routine swim-up technique into 6 aliquots,1 as the control and the other 5 cryopreserved with sucrose solution at the concentrations of 0.15,0.20,0.25 and 0.30 mol/L,respectively.After thawing,we determined and compared the motility,progressive motility and plasma membrane integrity of the sperm among the 6 groups.Results: The motility,progressive motility and plasma membrane integrity of the sperm were significantly lower after thawing than before cryopreservation(96.2±1.8]%,93.8±2.8]% and 99.0±0.8]%)(P<0.05).Post-thawing sperm motility was(55.5±6.3)% in the 0.20 mol/L sucrose group,significantly higher than in the 0.15,0.25 and 0.30 mol/L groups(45.9±6.6]%,50.4±9.4]% and 45.5±11.2]%)(P<0.05),and it was(53.6±5.0)% in the conventional freezing group,with no statistically significant difference from the 0.20 and 0.25 mol/L sucrose cryopreservation groups(P>0.05),but remarkably higher than in the 0.15 and 0.30 mol/L groups(P<0.05).Post-thawing progressive sperm motility exhibited no statistically significant differences between the 0.20 mol/L sucrose and conventional freezing groups(44.4±7.4]% vs 42.3±8.1]%,P>0.05),but markedly higher in both than in the 0.15,0.25 and 0.30 mol/L sucrose groups(37.1±8.3]%,33.1±9.2]% and 22.0±9.1]%)(P<0.05).Post-thawing plasma membrane integrity was significantly higher in the 0.20 mol/L sucrose cryopreservation group(70.1±6.9]%) than in either the conventional freezing group(63.1±6.8]%) or the 0.15,0.25 and 0.30 mol/L sucrose groups(57.7±8.3]%,63.5±10.7]% and 57.8±12.9]%)(P<0.05).Conclusion: As a simple,safe and effective method,ultra-rapid freezing with sucrose solution at the final concentration of 0.20 mol/L can be used for the cryopreservation of human spermatozoa.