多级梯度离心后X/Y精子浓度分析

Analysis of X/Y Sperm Concentration after Multistage Gradient Centrifugation

目的在辅助生殖中,利用多层密度梯度离心的方法来分选X/Y精子,以检验常规IVF-ET中,X/Y精子比例是否失衡.方法使用Sperm Grad TM(Vitrolife,Sweden)与Sperm Rinse TM(Vitrolife,Sweden)配制10层梯度液,分别为100%、95%、90%、85%、80%、75%、70%、65%、60%、55%.在离心管中制成多层梯度后,每层梯度间分界明显.选择液化正常、浓度约50×106/mL、活动率约50%,畸形率正常的5人精液混合后,去除个体差异取3 mL进行梯度离心.分别收集每层梯度底部精子,利用双色X/Y FISH探针(Vysis)进行FISH检测,统计X/Y精子比例.结果 100%、95%、90%、85%、80%五层梯度中X精子含量比例分别为:51.41%、51.41%、52.19%、50.99%、54.10%.结论每层梯度液中X/Y精子含量比例虽有不同,但差异并不明显.现在辅助生殖技术中,利用商品化精子梯度分离液,即使使用多层梯度进行离心分离,也不会造成性别比例失衡.

[Abstract] Objective In assisted reproduction, the X/Y sperm was sorted by multi density gradientcentrifugation to examine whether the proportion of X/Y sperm was unbalanced in conventional IVF-ET.MethodThe use of SpermGrad （Vitrolife, Sweden） and SpermRinse （Vitrolife, Sweden） to prepare 10 gradientliquid layer,were 100%,95%,90%, 85%, 80%, 75%, 70%,65%,60%,55%. After the multilayergradient was made in the centrifuge tube, the boundary between each gradient was obvious. Choose liquefactionnormal,concentration of about 50×106/mL, activity rate of about 50%, low deformity rate of 5 people, aftermixing semen,removing individual differences,and take 3mL mixture gradient centrifugation. Collect each gradientbottom sperm, using two-color X/Y FISH probe（Vysis ） for detection, and statistics X/Y sperm percentage.Results 100%, 95%, 90%, 85%, 80%,The proportion of X/Y sperm in each gradient was： 51.41%,51.41%,52.19%,50.99%,54.10%.Conclusions Although the proportion of X/Y sperm in each gradient wasdifferent,the difference was not obvious. In assisted reproductive technology, the use of commercialized spermwashing fluid,even if the use of multi-layer gradient centrifugation, will not cause gender imbalance.