人精子活力试验在IVF实验室质量控制中的应用

Application Study of Human Sperm Motility Bioassay in IVF Laboratory Quality Control

目的:评价人精子活力试验作为体外授精-胚胎移植(IVF)实验室质量控制方法的敏感性及应用价值。方法:实验1实验组HTF培养液中加入0.25%、0.75%的甲醛,对照组不加甲醛;所有HTF培养液均加入0.3%牛血清白蛋白、矿物油或者两者都有,了解人精子活力在这些培养条件下的变化。实验2采用人精子活力试验检测3种组织培养管的胚胎毒性。在HTF培养液中加入牛血清白蛋白、矿物油或两者都加,对照组不加任何附加物,了解这些附加物对精子有无保护作用。结果:实验1人精子在含0.25%甲醛的HTF培养液中培养24 h,活力指数为0.594±0.331,高于含0.75%甲醛培养液的0.450±0.284,差异有统计学意义(P<0.01);在含0.25%和0.75%甲醛的胚胎培养液中加入0.3%牛血清蛋白、矿物油,精子活力指数分别为0.683±0.334、0.527±0.345,高于不含牛血清白蛋白及矿物油的0.394±0.311、0.424±0.311,差异有统计学意义(P<0.01)。实验2同样的培养时间与培养条件,丹麦Nunc lom公司7 m l组织培养管中的精子活力指数为0.677±0.335,高于美国Falcon公司的14 m l培养管的0.596±0.327和美国Corn ing公司15 m l组织培养管的0.551±0.317,差异有统计学意义(P<0.01);在培养液中加入0.3%牛血清白蛋白、矿物油,精子活力指数为0.821±0.259、0.645±0.335,高于不含牛血清白蛋白及矿物油的0.571±0.321与0.395±0.245,差异有统计学意义(P<0.01)。结论:精子对培养环境中的微量毒性成分十分敏感,其活力在短时间内明显下降。人精子活力生物试验是一种敏感的生物测试方法;丹麦Nunc lom公司7 m l组织培养管对精子活力影响最小,适合于人胚胎的培养;培养液中加入牛血清白蛋白及矿物油可以减少培养环境中的毒素影响,对精子及其活力有保护作用。

Objective： To investigate the sensitivity of human sperm survival bioassay to using known concentrations of potential toxin of formalin and to elevate the application value of human sperm motility assay as a quality control method in detecting the components used in IVF program. Methods： Fresh semen was obtained from health males at andrology laboratory by masturbation. Sperm was processed on a gradient column of isolate medium and PBS medium. In experiment 1, the medium with O. 25%, O. 75% concentration of formalin and control medium were added to the Falcon culture tubes containing HTF medium with or without O. 3% bovine albumin serum and with or without light mineral oil. In experiment 2, in 3 types of culture tubes containing HTF medium with or without 0.3% bovine albumin serum and with or without light mineral oil, the sperm was exposed to each culture tube and cultured for 24 and 48 hrs at room temperature, and the motile sperms were counted under the microscope. Results： The average sperm motility index in the HTF medium with 0.25% formalin at 24 hrs was 0. 594±0.331, significantly higher than in the HTF medium with 0.75% formalin （0.450±0. 284） （P 〈0.01 ）. In the medium containing 0.25% and 0.75% formalin with 0.3% bovine albumin serum and light mineral oil, the average sperm survival indexes were 0.683 ±0.334 and 0.527 ±0.345, respectively, higher than without bovine albumin serum and light mineral oil （0.394 ±0.311 and 0.424 ±0.311 ）. The average sperm index of 7 ml tissue culture tube made in Denmark was 0.677 ±0.335, higher than the other two types of culture tubes made in the USA （0.551 ±0.317 and 0.596 ±0.327） （P 〈0.001 ）. When the sperm cultured in the medium with 0.3% bovine albumin serum and light mineral oil, the average sperm survival indexes were 0. 821 ±0.259 and 0.645 ±0.335, respectively, higher than without bovine albumin serum or light mineral oil （0.571±0.321 and 0.395±0.245 ） （P 〈 0.01 ）. Conclusion： The sperm survival bioassay is a sensitivity quality control method to detect the components in the IVF laboratory. The 7 ml tissue culture tube made in Denmark is most suitable for culturing human embryos. Sperm can be protected when cultured in the medium with 0.3% albumin bovine serum and light mineral oil.