临床微量精子标本冷冻方法的研究

A novel cryopreservation method for microsample of human spermatozoa in assisted reproductive procedure

关键词:  精子  微量  冷冻保存  冷冻载体  复苏率

Key words:  spermatozoa  microsample  cryopreservation  frozen carrier  recovery rate

摘要:

目的 探索一种安全、便捷、有效的用于微量精子冷冻的方法。方法 选择内径130μm的玻璃剥卵针50支作为冷冻载体,进行10个周期的反复冻融,检测其完整性,计算不同冷冻周期中载体折损率。选取50例经检测合格的精液标本,将密度调整至(0.5～1)×10⁶/ml,使用精子冷冻保护剂按照1∶1体积混合预平衡10min,每份标本装载2支载杆,计算装载时间,其中1支载杆作为快速冷冻组,迅速投入液氮中冷冻保存,另1支载杆采用液氮熏蒸30min后再投入液氮中保存,比较两种方法解冻后精子的复苏率。结果 50支冷冻载体中,其中有36支完成全部10轮的冷冻-复苏周期测试,折损率为28.0%,损坏原因中78.6%为金属镊子直接夹持导致玻璃剥卵针破裂。玻璃剥卵针至少可耐受4个周期反复冻融,但之后约4%～6%的会出现自发破损。100支冷冻载体装载精子时间为45～398s,平均为(112.3±94.1)s。冷冻前平均精子活力为65.6%±9.9%,快速冷冻复苏后平均精子活力为36.64%±8.7%,熏蒸法冷冻复苏后平均精子活力为23.48%±6.2%。快速冷冻法精子复苏率为55.9%±11.0%,熏蒸冷冻法精子复苏率为36.3%±9.8%,两者比较,差异具有统计学意义(F=18.31,P＜0.01)。结论 采用剥卵针作为冷冻载体,并配合快速冷冻法,冷冻保存微量精子是一种安全、有效、简便的方法,适合临床广泛开展。

Abstract:

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 5^th 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.