[Establishment and evaluation of a flow cytometry technique reflecting the severity of human sperm DNA damage].

Objective: To establish a flow cytometry (FC) technique reflecting the severity of human sperm DNA and evaluate its performance.

Methods: We analyzed the red and green fluorescence peaks of normal sperm in 1 165 human semen samples, defined the average lower limit of 5% of green fluorescence and the average upper limit of 95% of red fluorescence as the red and green fluorescence limits of the four-quadrant gate, and established an FC technique for detection of the sperm DNA fragmentation index (DFI), analysis of its repeatability and linear range and determination of the reference value of normal fertile men. We also analyzed the correlations of the sperm DFI, mild DNA damage marker (DFIm) and severe DNA damage marker (DFIs) with sperm concentration and motility in 122 men from the Infertility Clinic of Zhongda Hospital.

Results: With the established FC technique based on the four-quadrant gate, the sperm DFI, DFIm and DFIs were clearly distinguished among different groups of males, and the coefficients of variation obtained in 10 repeated examinations of the semen samples with a high, medium or low DFI using the FC technique were all lower than 5%. The sperm DFI showed a very good correlation within the range of 8.93%－3.90% (r > 0.99). With the upper limit of 95% as the range of normal reference value, the sperm DFI of 274 of the normal fertile males was ≤ 25.50%. The sperm DFI was remarkably negatively correlated with sperm motility and the percentage of progressively motile sperm (PMS) but exhibited no significant correlation with sperm concentration. The DFIs showed significantly higher related coefficients with sperm motility and PMS (r = －0.592 and －0.543) than DFIm (r = －0.323 and －0.236). Both DFIs and DFIm were markedly higher in the patients with decreased sperm motility and PMS than in the normal fertile men, the former even more significantly (P < 0.01) than the latter (P < 0.05).

Conclusions: Compared with the existing FC technique, ours can reflect the severity of sperm DNA damage and is more suitable for clinical application. DFIs may be more closely related to male fertility.