精子冻存技术及上游法精子处理技术对精子DNA完整性的影响

目的通过对冷冻前后及精子处理前后精子DNA完整性的比较，探讨冷冻技术、冷冻时间及上游法精子处理技术对精子DNA完整性的影响。方法（1）精液常规检测正常的患者30例，手淫法取精，精液液化混匀后分4份，分别用于精子染色质扩散（SCD）实验检测精子DNA完整性、上游法处理精液、以及两组冻存实验（不加保护剂直接冻存的为冻存1组；添加蛋黄葡萄糖保护剂冻存的为冻存2组）；（2）上游法处理后的精液一部分用于检测精子动力和形态，一部分用于精子DNA完整性检测；（3）冻存1组分别于冻存第7天和第90天解冻，SCD实验检测精子DNA完整性；（4）冻存2组分别于第7天和第90天解冻，0．1ml用于精子DNA完整性检测，剩余精液应用上游法处理，检测上游处理前后精子DNA的完整性。结果（1）冻存1组中，冻存90d后解冻的精子DNA损伤率[（25．6土7．3）％]显著高于冻存7d者[（22．4±7．4）％]（P〈0．05），且均显著高于新鲜精液的精子DNA损伤率[（20．6±7．3）％]（P〈O．05）；冻存2组中，冻存90d后精子DNA损伤率显著高于冻存7d者[（25．9±7．2）％VS．（23．6土7．8）％]（P〈O．05），且均显著高于新鲜精液（P〈0．05）；而冻存7d和90d后，两个冻存组间比较，精子DNA损伤率均无显著差异（P〉0．05）。（2）新鲜精液经上游法处理后，精子DNA损伤率由处理前的（20．6±7．3）％降为（6．4±2．5）％（P〈O．05）；冻存2组中精液冻存7d和90d后复苏上游法处理后，精子DNA损伤率较未经上游法处理者均显著降低[分别为（9．38±2．8）％VS．（23．6±7．8）％和（9．7±2．6）％VS．（25．9±7．2）％]（P％0．05）。结论冻存对精子DNA有损伤，冻存时间对于精子DNA完整性有影响。不添加保护剂直接冻存和添加保护剂对精子DNA完整性的影响无显著差异。不论是新鲜精液还是冻存复苏精液，上游法处理并不会增加精子DNA的损伤，且有利于筛选出具有更好DNA完整性的精子。

Effect of cryopreservation and swim-up preparation on DNA fragmentation of human spermatozoa

Objective： To evaluate the effect of cryopreservation and swim-up preparation on DNA fragmentation of human spermatozoa. Methods： Thirty normal semen samples were collected. Each sample was divided into 3 aliquots for determining spermatozoa DNA integrity in fresh, frozen and swim-up prepared spermatozoa. Sperm DNA integritywas determined by sperm chromatin dispersion test. The aliquots for cryopreservation were divided to 2 groups. Each sample including 2 straws in group 1 was frozen in absence of cryoprotectant,and the sample in group 2 was frozen in the presence of cryoprotectant. The samples were thawed after freezing for 7 days or 90 days in the two groups. Then 0. 1 ml sample was used for determining DNA fragmentation,and the rest samples were used for swim-up and followed by determination of DNA fragmentation. Results= （1）In group 1, the DNA fragmentation index（DFI）was significantly increased in day 7 （22.4%±7.4%）and day 90 of freezing（25.6%±7.3%）compared with fresh semen（20.6%±7.3%）（P〈 0.05） ,and it was also significantly different between that in day 7 and day 90 （P〈0.05）. In group 2, the DFI was significantly increased in day 7 （23.6 %± 7.8 5 ） and day 90 （25.9 % ±7.2 %） compared with fresh semen（P〈0.05） ,and it was significantly different between that in day 7 and day 90（P〈0.05）. There was no significant difference in DFI in group 1 compared with group 2 regardless of absence or presence of cryoprotectant. （2）After swim-up preparation, the DFI in fresh semen was significantly decreased from （20.6±7.3）% to（6.4 ± 2.5）%（P〈0.05）. （3） After swim-up preparation, the DFI in frozen semen samples with cryoprotectant was significantly decreased in both day 7 group（9.38%±2.8%）and day 90 group （9.7 % ±2.6 % ） compared with that before swim-up preparation （P〈0.05）. Conclusions：The freezing-thawing procedure can cause sperm DNA damage. The longer the frozen time is,the severer sperm DNA damage appears. There is no significant difference in sperm DNA integrity in cryopreserved semen with or without cryoprotectant. Swim-up preparation is a useful method to reduce the DNA damaged sperm from fresh semen and frozen-thawed semen.