优化精液处理中的密度梯度离心法以提高受精结局的探讨  

Exploration of Optimizing Density Gradient Centrifugation in Semen Processing to Improve Fertilization Outcomes

作　　者：孙桂花 张志强 陈文秋[1] 李婷婷[1] SUN Guihua;ZHANG Zhiqiang;CHEN Wenqiu;LI Tingting(Reproductive Medicine Center of the Sixth Affiliated Hospital of Sun Yat-sen University,Guangdong Fertility Conservation Engineering Technology Research Center,Guangzhou Huangpu District Zhongliu Biomedical Innovation Research Institute,Guangzhou 510655,China)

机构地区：[1]中山大学附属第六医院生殖医学中心、广东省生育力保存工程技术研究中心、广州市黄埔区中六生物医学创新研究院,广东广州510655

出　　处：《中山大学学报（医学科学版）》2024年第4期657-665,共9页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：北京健康促进会2023年生殖医学中青年医生研究项目(BJHPA-2023-SZHYXZHQN-001)。

摘　　要：【目的】本研究旨在改进生殖男科领域现有的精液处理方法,特别是针对双层密度梯度法中的300×g 20 min处理条件,以提高受精结局。【方法】收集2020年7月和9月以及2022年3月和5月在中山大学附属第六医院生殖医学中心进行辅助生殖助孕的1623例患者的精液标本进行实验。预实验中,比较四种不同的双层密度梯度方法(200×g 10 min、200×g 20 min、300×g 10 min和300×g 20 min)处理后标本的精子DNA碎片率和回收率。然后,筛选出一种最优的方法作为新方法,并与目前在用的旧方法(300×g 20 min双层梯度法)进行对比,检验受精率是否有统计学差异。在新方法的基础上,进一步优化为单层密度梯度法,并与双层密度梯度法进行对比,检验是否存在统计学差异。实验过程中严格控制温度、离心速度和离心时间,同时记录每组样本的数量和处理条件。【结果】在保证足够的精子回收率的基础上,发现四种双层密度梯度法中300×g 10 min的精子DNA碎片率低于300×g 20 min。因此,选择300×g 10 min作为新方法进行试验。结果表明,新方法300×g 10 min的总受精率、二核体(2pn)受精率均高于300×g 20 min,且差异有统计学意义(P<0.05);300×g 10 min的卵裂率也略高于300×g 20 min,但差异没有统计学意义(P>0.05)。单层密度梯度法的总受精率、2pn受精率均高于双层密度梯度法,但差异没有统计学意义(P>0.05);单层密度梯度法的卵裂率高于双层密度梯度法,囊胚形成率低于双层密度梯度法,且差异均有统计学意义(P<0.05)。【结论】相对于现有的300×g 20 min双层梯度法,300×g 10 min双层梯度法成功提高了总受精率、2pn受精率、卵裂率,缩短了精液优化处理的时间;而单层密度梯度法虽提高了卵裂率、节约了试剂成本和操作时间,但其囊胚形成率却出现了下降的情况。这些发现为生殖男科领域的精液处理方法提供了有益的指导和启�【Objective】This study aimed to improve the existing semen processing methods in the field of reproductive male medicine,particularly focusing on the 300×g 20 min treatment condition in the double-layer density gradient method,to enhance fertilization outcome.【Methods】Semen specimens from 1623 patients undergoing assisted reproductive techniques at the Reproductive Medicine Center of the Sixth Affiliated Hospital of Sun Yat-sen University from July and September 2020 and March and May 2022 were collected for preliminary experiments.Four different double layer density gradient methods(200×g 10 min,200×g 20 min,300×g 10 min,and 300×g 20 min)were compared for sperm DNA fragmentation rates and recovery rates after processing.Subsequently,the optimal method was selected as the new approach and compared with the current method in use(300×g 20 min double-layer gradient method)to assess any statistical differences in fertilization rates.Further optimization to a single-layer density gradient method was performed based on the new method and compared with the double-layer density gradient method to determine any statistical differences.Experimental conditions were strictly controlled for temperature,centrifugation speed,and duration,with the quantity and processing conditions of each sample recorded.【Results】Among the four double-layer density gradient methods,the sperm DNA fragmentation rate was lower with the 300×g 10 min treatment compared to 300×g 20 min while ensuring sufficient sperm recovery rates.Consequently,the 300×g 10 min method was selected as the new approach for experimentation.Results indicated that the total fertilization rate and 2 pronuclei(2 PN)fertilization rate with the new 300×g 10 min method were higher than with the 300×g 20 min method,the difference was statistically significant(P<0.05).Although the cleavage rate with 300×g 10 min was slightly higher than 300×g 20 min,the difference was not statistically significant(P>0.05).The total fertilization rate and 2 PN fertilizatio

关 键 词：精子DNA碎片率 双层密度梯度法 单层密度梯度法 受精率 卵裂率 

分 类 号：R493[医药卫生—康复医学]