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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
LI Tingting; E-mail: litt33@mail.sysu.edu.cn
Published:20 July 2024,
Received:17 April 2024,
Accepted:30 May 2024
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孙桂花,张志强,陈文秋等.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[J].中山大学学报(医学科学版),2024,45(04):657-665.
SUN Guihua,ZHANG Zhiqiang,CHEN Wenqiu,et al.Exploration of Optimizing Density Gradient Centrifugation in Semen Processing to Improve Fertilization Outcomes[J].Journal of Sun Yat-sen University(Medical Sciences),2024,45(04):657-665.
孙桂花,张志强,陈文秋等.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[J].中山大学学报(医学科学版),2024,45(04):657-665. DOI: 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20240617.005.
SUN Guihua,ZHANG Zhiqiang,CHEN Wenqiu,et al.Exploration of Optimizing Density Gradient Centrifugation in Semen Processing to Improve Fertilization Outcomes[J].Journal of Sun Yat-sen University(Medical Sciences),2024,45(04):657-665. DOI: 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20240617.005.
目的
2
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
2
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1 623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
2
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
2
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示。
Objective
2
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
2
Semen specimens from 1 623 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 quan
tity and processing conditions of each sample recorded.
Results
2
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 (2PN) 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 2PN fertilization rate were slightly higher with the single-layer density gradient method compared to the double-layer density gradient method, but the difference was not statistically significant (
P
>
0.05). The cleavage rate with the single-layer density gradient method was higher than the double-layer density gradient method, and the blastocyst formation rate is lower than that of the double-layer density gradient method, and the differences are statistically significant (
P
<
0.05).
Conclusion
2
The 300 ×
g
10 min double-layer density gradient method successfully improved total fertilization rates, 2PN fertilization rates, and cleavage rates compared to the existing 300 ×
g
20 min method, while reducing the time required for semen optimization processing. Although the single-layer density gradient method improves the cleavage rate, and saves reagent costs and operation time, its blastocyst formation rate has decreased. These findings provide valuable guidance and ins
ights for semen processing methods in the field of reproductive andrology.
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
sperm DNA fragmentation indexdouble-layer density gradient centrifugationsingle-layer density gradient centrifugationfertilization ratecleavage rate
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