电刺激取精术获取精子经体外培养后冻存方法研究

Sperm collection by electroejaculation and cryopreservation after in vitro culture

目的:优化电刺激取精术获取的精子处理和冷冻复苏方法,为临床不射精症的治疗和生育力保存提供新途径。方法:将20例同房不射精患者电刺激获取的精液(或尿液)样本分为两组,其中6例采用PBS洗涤并用Modified-HTF重悬(A组),另外14例用Modified-HTF直接洗涤并重悬(B组),对处理后精子行CASA,分析精子浓度和活动率;采用麦管载体和一步熏蒸法冷活动率为(1.12±0.41)%,M-HTF处理组的精子活动率为(9.23±9.43)%;冷冻复苏后,A组精子复苏后活动率和冷冻复苏率分别为(0.24±0.41)%、8.37%;B组的精子活动率和复苏率显著增加,分别为(4.11±2.33)%、35.41%。同样,电刺激尿液样本中,PBS处理组的精子活动率为(1.30±0.51)%,M-HTF处理组的精子活动率提高至(5.48±5.32)%;冷冻复苏后,A组的精子活动率和复苏率分别为(0.26±0.40)%、6.31%;B组的精子活动率和复苏率,分别为(3.02±1.64)%、45.88%。结论:本研究所建立的电刺激精子经体外培养及冷冻复苏的方法,可以显著增加精液和尿液样本中精子的活力,改善精子的冷冻复苏率,为临床不射精症的治疗和生育力保存提供新途径。

Objective： To improve the method for the cryopreservation of sperm collected by electroejaculation（ EEJ）,and find a new way for the treatment of anejaculation and preservation of fertility. Methods： Semen（ or urine） samples collected by EEJ from20 male patients with anejaculation were divided into groups A（ n = 6） and B（ n = 14）,the former washed with phosphate buffer solution（ PBS） while the latter with modified HTF,both diluted in 1 ml modified HTF,followed by CASA for sperm concentration and motility. All the samples were cryopreserved for 48 hours using one-step-vaporization with the straw. Post-thaw sperm counting was conducted directly by computer-assisted semen analysis（ CASA） in group A and after in vitro culture in rare sperm medium in group B.Results： In the semen samples collected by EEJ,sperm motility was（ 1. 12 ± 0. 41） % in group A compared with（ 9. 23 ± 9. 43） % in group B. The post-thaw sperm motility and recovery rate were（ 0. 24 ± 0. 41） % and 8. 37% in the former,and significantly increased to（ 4. 11 ± 2. 33） % and 35. 41% in the latter. In the urine samples,sperm motility was（ 1. 30 ± 0. 51） % in group A in comparison with（ 5. 48 ± 5. 32） % in group B. The post-thaw sperm motility and recovery rate were（ 0. 26 ± 0. 40） % and 6. 31% in the former and remarkably increased to（ 3. 02 ± 1. 64） % and 45. 88% in the latter. Conclusion： The method of sperm collection by electroejaculation and cryopreservation after in vitro culture can significantly improve sperm motility and sperm recovery rate in the semen or urine,which has provided a new strategy for the treatment of anejaculation and preservation of fertility.