Abstract
Objective The recent advent of flow cytometry (FCM), coupled with fluorescent dyes, has been successfully applied to assess mitochondrial function. The aim of this study was to investigate the feasibility and clinical significance of detecting sperm mitochondrial function and to evaluate sperm mitochondrial function by using Rhodamine 123/propidium (Rh123/PI) dual fluorescent staining and FCM in asthenospermia and oligoasthenozoospermia. Methods Twenty-five fertile men (with normal sperm parameters) and 230 infertile patients were examined. Fifty-five patients of the above 230 patients were selected for idiopathic infertility samples and were divided into two groups: asthenospermia ( n = 30) and oligoasthenozoospermia ( n = 25). Rh123/PI dual fluorescent staining and FCM were carried out to examine sperm mitochondrial function. Results Significant differences were found between the normal and abnormal semen samples ( P < 0.05) when Rh123 +/PI−, Rh123−/PI + and Rh123−/PI-sperm were examined by FCM, but there was no significant difference between the asthenospermia ( P = 0.469) and oligoasthenozoospermia group ( P = 0.950) when Rh123 +/PI-and Rh123−/PI + sperm were then examined; however, a significant difference was found between the 2 groups ( P = 0.003) when Rh123-/PI-sperm were examined. There was no correlation between Rh123-/PI-sperm and semen parameters in the normal group, but there was a significant negative correlation between the sperm concentration and Rh123-/PI-sperm in asthenospermia and oligoasthenozoospermia patients ( r = −0.509, −0.660; P = 0.018, 0.038). Conclusion Rh123/PI dual fluorescent staining and FCM can provide reliable information to assess the quality of sperm and reveal differences in mitochondrial membrane potential in asthenospermia and oligoasthenozoospermia.
Highlights
The structural and functional integrity of the plasmaZou.TJ et al / Journal of Biomedical Research, 2010, 24(5): 404-410 morphology and motility
Cell membrane integrity can be evaluated by staining sperm with propidium iodide (PI), a fluorescent dye that binds with DNA
Sperm stained by double fluorescent dyes Rh123/PI were marked by a green body or a green body and a red head (Fig. 1B)
Summary
Zou.TJ et al / Journal of Biomedical Research, 2010, 24(5): 404-410 morphology and motility. This conventional analysis of semen provides considerable information, it is limited in predicting male fertility. Cell membrane integrity can be evaluated by staining sperm with propidium iodide (PI), a fluorescent dye that binds with DNA. Damaged membrane of dead/moribund sperm allows PI to enter and bind with DNA to produce red fluorescence. Dual fluorescent staining (Rh123/PI) combined with FCM has been developed for simultaneous determination of mitochondrial membrane potential (MMP) and membrane integrity of sperm[2,9,10,11]. In other assays mainly based upon the use of DNA intercalating fluorescent compounds, acridine orange and ethidium bromide have been used to evaluate the presence and the number of such cells by FCM[12,13]
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