不同浓度的蔗糖溶液快速冻存人类精子的实验研究

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

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。

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 signifi- cantly 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, O. 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 conven- tional freezing group, with no statistically significant difference from the O. 20 and O. 25 mol/L sucrose cryopreservation groups （P 〉 O. 05 ）, but remarkably higher than in the 0.15 and O. 30 mol/L groups （ P 〈 O. 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 suerose 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 su- crose eryopreservation group （ [ 70.1 ± 6.9 ] % ） than in either the conventional freezing group （ [ 63.1 ± 6.8 ] % ） or the 0.15, 0.25 and0.30 mol/L sucrose groups （[57.7±8.31%, [63.5±10.71% and [57.8±12.91%） （P〈0.05）. Conclusion： Asasim- ple, 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.