[1]梁晓东,冯敏怡,莫淦文,等.流式细胞术检测精子HSPA2方法初步建立及对体外受精低受精率的预测价值研究[J].现代检验医学杂志,2025,40(05):149-152.[doi:10.3969/j.issn.1671-7414.2025.05.028]
　LIANG Xiaodong,FENG Minyi,MO Ganwen,et al.Preliminary Establishment of a Flow Cytometry Method for Detecting Sperm HSPA2 and Its Predictive Value for Low Fertilization Rate in IVF[J].Journal of Modern Laboratory Medicine,2025,40(05):149-152.[doi:10.3969/j.issn.1671-7414.2025.05.028]
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流式细胞术检测精子HSPA2方法初步建立及对体外受精低受精率的预测价值研究()

《现代检验医学杂志》[ISSN:/CN:]

卷:
第40卷
期数:
2025年05期
页码:
149-152
栏目:
论著
出版日期:
2025-09-15

文章信息/Info

Title:
Preliminary Establishment of a Flow Cytometry Method for Detecting Sperm HSPA2 and Its Predictive Value for Low Fertilization Rate in IVF
文章编号:
1671-7414（2025）05-149-04
作者:
梁晓东，冯敏怡，莫淦文，纪 鹏，骆强翔，郭江华
江门市中心医院生殖医学中心，广东江门 529030
Author(s):
LIANG Xiaodong，FENG Minyi，MO Ganwen，JI Peng，LUO Qiangxiang，GUO Jianghua
Department of Reproductive Medicine Center, Jiangmen Central Hospital, Guangdong Jiangmen 529030, China
关键词:
热休克蛋白A2; 流式细胞术; 体外受精; 低受精率; 精子
分类号:
R446.19；Q503
DOI:
10.3969/j.issn.1671-7414.2025.05.028
文献标志码:
A
摘要:
目的建立精子热休克蛋白A2（HSPA2）流式细胞检测法，并探索精子HSPA2的表达水平在预测体外受精（IVF）低受精率的作用。方法采用间接免疫荧光法（IIF）原理对精子HSPA2进行荧光染色，精子样本经透膜和封闭后，依次加入兔抗人HSPA2抗体（一抗）和异硫氰酸荧光素（FITC）标记的山羊抗兔IgG抗体（二抗）作为检测管，同时设立不加一抗的样本作为对照管，并用流式细胞仪（FCM）分别测定两管的阳性率，计算检测管的阳性率与对照管的比值（阳性率比值）。使用棋盘法对最适合检测的精子数、一抗及二抗最适工作稀释度进行探索。在最优条件下，分别评估方法的重复性、线性范围和参考值范围，从而初步建立流式细胞术检测精子HSPA2表达水平的方法。方法建立后，进一步对2023年在江门市中心医院生殖医学中心的IVF患者夫妇的精子样本共85例进行初步检测，比较IVF受精成功组（n=63）与低受精率组（n=22）患者的HSPA2阳性率比值，用受试者工作特征（ROC）曲线分析界值。结果对照管中的HSPA2阳性率较低，显示出较低的背景信号，而检测管荧光信号明显增强，提示此法能有效检测HSPA2。棋盘法确定的最适合检测的精子数为2×106，一抗及二抗最适工作稀释度分别为1∶300和1∶400。重复性及线性范围评估显示方法学性能较好。对IVF受精成功组和低受精率组的对比分析显示，低受精率组的精子HSPA2阳性率比值（6.19±4.07）低于受精成功组（10.69±8.26），差异具有统计学意义（t=2.446，P<0.05）。ROC曲线和Youden指数显示，阳性率比值的界值为5.5067时达到最佳预测效能，此时的敏感度和特异度分别为71.4%，55.5%。结论成功建立了精子HSPA2的流式细胞术检测法，通过此法检测的精子HSPA2表达水平提示其对IVF低受精率有预测价值，为今后临床科学选择受精方式提供了依据。
Abstract:
Objective To establish a flow cytometric assay for detecting heat shock protein A2 (HSPA2) in sperm and explore the role of HSPA2 expression levels in predicting low fertilization rates in in vitro fertilization (IVF). Methods The principle of in-direct immunofluorescence(IIF) was used to fluorescently stain sperm HSPA2. After the sperm sample was permeabilized and sealed, rabbit anti- human HSPA2 antibody (primary antibody) and fluorescein isothiocyanate (FITC) labeled goat anti- rabbit IgG antibody (secondary antibody) were sequentially added as detection tubes. At the same time, a sample without primary anti-body was set up as a control tube, and the positive rates of the two tubes were measured by flow cytometer. The ratio of the posi-tive rate of the detection tube to the control tube (positive rate ratio) was calculated. The optimal number of sperm for detection and the optimal working dilutions of primary and secondary antibodies were explored using the chessboard method. Under the optimal conditions, the repeatability, linear range and reference range of the method were evaluated separately, in order to estab-lish a preliminary method for detecting sperm HSPA2 expression levels using flow cytometry. After the establishment of the method, preliminary testing was conducted on a total of 85 sperm samples from couples who underwent IVF at the Reproductive Medicine Center of Jiangmen Central Hospital in 2023. The ratio of HSPA2 positivity rates between the group with IVF success-ful (n=63) and the group with low fertilization rate (n=22) was compared, and the receiver operating characteristic (ROC) curve was used to analyze the threshold. Results The positive rate of HSPA2 in the control tube was relatively low, showing a low background signal, while the fluorescence signal of the detection tube was significantly enhanced, indicating that this method can effectively detect HSPA2. The optimal number of sperm samples for detection determined by the chessboard method was 2×106, and the optimal working dilutions for primary and secondary antibodies were 1∶300 and 1∶400, respectively. Evaluation of repeatability and linear range showed good methodological performance. Comparative analysis between the group with IVF success-ful and the group with low fertilization rate showed that the ratio of sperm HSPA2 positivity rate in the group with low fertilization rate (6.19±4.07) was lower than successful fertilization group (10.69±8.26), the difference was statistically significant (t=2.446, P<0.05). The ROC curve and Youden index showed that the best predictive power was achieved when the cutoff value for the ratio of positivity rate was 5.5067, with a sensitivity and a specificity of 71.4%, 55.5%, respectively. Conclusion A flow cytometric method for detecting HSPA2 in sperm is successfully established. The expression level of sperm HSPA2 detected by this method suggests its predictive value for low fertilization rate in IVF, providing a basis for future clinical scientific selection of fertilization methods.

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备注/Memo

备注/Memo:
基金项目：江门市科学技术局医疗卫生领域科技计划项目（编号：2022YL01025）。
作者简介：梁晓东（1983-），男，硕士研究生，副主任技师，研究方向：体外受精失败相关的精子因素，E-mail：229290147@qq.com。
更新日期/Last Update: 2025-09-15