YANG Zhi-yong,XING Ya-xin,YANG Yan-nan,et al.A novel cryopreservation method for microsample of human spermatozoa in assisted reproductive procedure[J].同济大学学报（医学版）,2017,38(6):41-45. [点击复制]
Objective To evaluate the feasibility of a novel cryopreservation method for microsample of human sperm in assisted reproductive procedure. Methods Fifty glass denuding pipette (inner diameter 130μm) as frozen carrier undertook 10 repeated freeze-thaw cycle tests, the wreck rate was calculated in different freeze-thaw cycle. Fifty qualified semen sample according to WHO 5th edition criterion were diluted to (0.5-1)×106/ml, and then mixed with sperm cryoprotectant (1∶1 volume) for pre-equilibrium for 10min. Each sample was loaded in two denuding pipettes, the consuming time was calculated. One was quickly put into the liquid nitrogen as quick freezing group, and another was exposed to liquid nitrogen vapor for 30min, then subjected into the liquid nitrogen preservation. The recovery rate after thawing was compared between two groups. Results Thirty six of 50 frozen carriers were intact after bearing 10 successive frozen-thaw cycles with a wreck rate of 28.0%, 78.6% of the damage were caused by the clamping contact of metal tweezers. Denuding pipettes were tolerated at least 4 repeated frozen-thaw cycles, a spontaneous breakage occurred in 4%- 6% of the pipettes. The mean time for loading was (112.3±94.1)s with a maximum of 398s and a minimum of 45s. Before cryopreservation, the sperm motility was 65.6%±9.9%, and it was 36.64%±8.7% in quick freezing group and 23.48%±6.2% in vapor group after thawing. The recovery rate was 55.9%±11.0% in quick freezing group, and was 36.3%±9.8% in vapor group (F=18.31, P<0.01). Conclusion Using denuding pipette as the frozen carrier and combined with vitrification is a safe, effective and simple method for cryopreservation of microsample human sperm, it is suitable for clinical application.
