人类稀少精子细管法超快速冷冻的实验研究

目的研究人类稀少精子超快速冻存方法,寻求稀少精子冷冻保存的最优方式。方法选取20份在上海市人类精子库捐精的20名健康志愿者的精液。将精液稀释后密度调至(3～5)×106/mL(每个高倍视野3～5条精子),按照不同冷冻方式分为四组进行冷冻:A组应用0.5 mL麦管和精子冷冻剂,以程序降温方式冷冻;B组应用毛细玻璃管和精子冷冻剂,以程序降温方式冷冻;C组应用毛细玻璃管和改良甘油-卵黄-枸橼酸钠(GYC)型冷冻保护剂,以程序降温方式冷冻;D组应用毛细玻璃管和改良GYC型冷冻保护剂,以超快速降温方式冷冻。比较四种方法复苏后精子活率和正常形态率。结果冷冻前精子活率为(68.6±21.3)%;A、B、C、D组复苏后活率分别为(32.2±17.2)%、(41.5±18.4)%、(44.9±18.0)%和(50.2±19.7)%,B、C、D各组复苏后活率高于A组(P<0.05),D组高于其他三组(P<0.05)。冷冻后A、B、C、D组精子正常形态率分别为(14.2±4.1)%、(15.0±4.7)%、(14.5±5.2)%和(15.9±4.9)%,差异无统计学意义(P>0.05)。结论应用超快速降温冷冻模式冷冻稀少精子,具有较高的冷冻复苏率。改良GYC冷冻保护剂具有与精子冷冻剂相似的冷冻保护效果。

Research on ultra-rapid cryopreservation of small numbers of human spermatozoa in capillary

Objective To explore sperm cryopreservation in capillary by ultra-rapid procedure,and seek an optimal method to cryopreserve small numbers of human spermatozoa.Methods Twenty semen samples from 20 healthy donors were selected from Shanghai Human Sperm Bank.Each sample was divided into 4 groups for cryopreservation after dilution to the density of(3-5)×106/mL(3-5/high power filed).In group A,samples mixed with sperm freezing medium in 0.5 mL straw were frozen by program procedure.In group B,samples mixed with sperm freezing medium in capillary were frozen by program procedure.In group C,samples mixed with modified glycerol-yolk-citrate(GYC) in capillary were frozen by program procedure.In group D,samples mixed with modified GYC in capillary were frozen by direct liquid nitrogen vapor method with ultra-rapid speed.The post-thaw viability rates and normal morphology rates were compared among groups.Results The pre-thaw viability rate of the 4 groups was(68.6±21.3)%,and the post-thaw viability rates of group A,B,C and D were(32.2±17.2)%,(41.5±18.4)%,(44.9±18.0)% and(50.2±19.7)% respectively.The post-thaw viability rates of group B,C and D were significantly higher than that of group A(P<0.05),and the post-thaw viability rate of group D was significantly higher than those of the other 3 groups(P<0.05).There was no significant difference in the post-thaw normal morphology rates among group A,B,C and D [(14.2±4.1)%,(15.0±4.7)%,(14.5±5.2)% and(15.9±4.9)% respectively,P>0.05].Conclusion Ultra-rapid freezing with direct liquid nitrogen vapor in capillary is an optical method for cryopreservation of small numbers of human spermatozoa,which may yield high recovery rate.Modified GYC has the same cryoprotecting effect as sperm freezing medium.