改良快速冷冻载体冻融人类稀少精子的实验研究

目的探讨人类稀少精子冷冻改良快速冷冻载体的实验研究,为重度少弱精子症患者的精子冷冻保存提供参考。方法2018年12月至2019年8月,在联勤保障部队第九〇〇医院生殖中心就诊的男性不育症患者:根据精液质量分为正常精液组和少弱精液组,每个实验样本选取3份精液分别进行充分混合,将混合精液稀释至浓度为(1~2)×10~6/ml,实验共计选取81份正常精液标本混匀稀释形成27个正常精液样本,选取54份少弱精液标本混匀稀释形成18个少弱精子样本。根据不同的冷冻载体各分3组进行精子冷冻:薄片精子冷冻管组(A组),麦管微量体积冷冻组(B组),改良快速冷冻载体组(C组),均采用商品化精子冷冻试剂1∶1混匀,熏蒸法后快速冷冻。比较3种载体冷冻复苏后,精子的活动力、存活率、DNA碎片指数和正常形态变化、精子顶体酶活性等指标变化,以评估改良快速冷冻载体的冷冻效率及安全性。统计学方法采用t检验、ANOVA分析、LSD多重比较。结果正常精液组的3种冷冻方法都会造成精子质量下降。正常精液组的C组的复苏后精子活动力、存活率均高于A组和B组,差异有统计学意义(P<0.05)。正常精液组:C组复苏精子DNA碎片指数略低于A和B组,但差异无统计学意义(P>0.05);A、B、C 3组复苏后精子正常形态率均低于冷冻前,差异有统计学意义(P<0.05)。少弱精液组:A、B两组复苏后精子DNA碎片指数均高于冷冻前,差异有统计学意义(P<0.01);C组精子DNA碎片指数高于冷冻前,但差异无统计学意义(P=0.068),C组复苏精子DNA碎片指数略低于A和B组,但差异无统计学意义(P>0.05),A、B、C 3组冷冻精子复苏后正常形态率均低于冷冻前,差异无统计学意义(P>0.05)。正常精液组:冷冻前精子顶体酶活性为(123.6±12.8)IU/10~6个精子,复苏后A、B、C 3组顶体酶活性均有下降,分别为(74.7±15.6)、(84.7±13.5)、(91.2±16.2)IU/10~6个精子,差异有统计学意义(F=37.896,P<0.001),从对于精子顶体影响看,B、C组优于A组,差异有统计学意义(P=0.043、P=0.001)。结论改良快速冷冻载体对于稀少精子冷冻保存有一定优势,值得进一步探讨研究。

Investigation of experiment in improving of rapid cryopreservation carrier on scarce human spermatozoa

Objective To explore the investigation of experiment in improving of rapid cryopreservation carrier on scarce human spermatozoa, and provide reference for cryopreservation of sperm in patients with severe oligo-asthenozoospermia. Methods From December 2018 to August 2019, male infertility treated in center of Reproductive Medicine, 900 Hospital of the Joint Logistics Team were divided into the normal sperm group and oligo-asthenospermic group through their semen quality. Three samples of sperm were selected from each experimental sample and were mixed and diluted to concentration(1-2)×10~6/ml, 27 mixed cases were selected from 81 samples of normal semen, 18 mixed cases were selected from 54 oligoasthenic sperm samples. According to different frozen carrier spermatozoon were frozen in 3 groups: thin slice sperm tube(group A), wheat tube micro-volume freezing group(group B), modified rapid freezing carrier group(group C). All of them used commercialized sperm freezing reagent 1 ∶ 1 to mix, rapid cryopreservation after fumigation. Survival rate, DNA fragmentation index, acrosome enzyme activity and normal morphology rate after cryopreservation of three scarce sperm cryopreservation carrier were compared to evaluate the freezing efficiency and safety of rapid cryopreservation carrier. Statistical methods were carried out by t-test, anova analysis and LSD multiple comparison. Results Sperm quality was decreased in normal sperm group, three cryopreservation methods were used. Group C were higher than that in group A and group B in the survival rate of resuscitation sperm motility for the normal sperm group with statistical significance(P<0.05). Group C was slightly lower than that in group A and group B in DNA fragment index of resuscitated sperm for the normal sperm group with no statistical significance(P>0.05). Normal morphologic rate after cryopreservation in the group A, B and C was lower than that before cryopreserved with statistical significance(P<0.05). Oligo-asthenospermic group: DNA fragment index of resuscitated sperm in both groups A and B was higher than that before cryopreservation with statistical significance(P<0.01). DNA fragment index of resuscitated sperm in group C was higher than that before cryopreservation but with no statistical significance(P=0.068). DNA fragment index of resuscitated sperm in group C was slightly lower than that in group A and group B with no statistical significance(P>0.01). The normal morphology rate of frozen sperm in group A, B and C was lower than that before freezing with no statistical significance(P>0.05). Normal sperm group: the acrosomal enzyme activity of sperm was(123.6±12.8) IU/10~6 sperm before freezing, and decreased in group A(74.7±15.6), B(84.7±13.5) and C(91.2±16.2) IU/10~6 after resuscitation with statistical significance(F=37.896,P<0.001). Group B and C were superior to group A in acrosomal influence of sperm with statistical significance(P=0.043, P=0.001). Conclusion The improved rapid cryopreservation carrier has certain advantages for scarce human spermatozoa, which is worthy of further study.