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 cutoffvalue 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.

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 cutoffvalue 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.