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Updated: Oct 9, 2025
Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects
Published on: April 21, 2022
[Standardization and quality control for detection of sperm DNA damage by flow cytometry: A preliminary
Jin-Chun Lu1, Zhen-Bo Wu2, Shan-Shan Tang1
1Center for Reproductive Medicine, Zhongda Hospital, Southeast University, Nanjing, Jiangsu 210037, China.
Objective:
To investigate preliminarily the standardization and quality control for the detection of sperm DNA damage by flow cytometry.
Methods:
Semen samples were randomly selected and observed for the effects of acid denaturation time, acridine orange (AO) staining time, semen sample refrigeration, freezing and repeated freezing-thawing on the results of sperm DNA fragmentation index (DFI).
Results:
Sperm DFI increased gradually with the prolongation of acid denaturation time, significantly at 2 minutes in comparison with that at 30 seconds (P<0.05). There was no significant difference in sperm DFI after AO staining for 5, 20, 40, 60, 100 and 140 minutes. Sperm DFI also exhibited an evident increase with the prolongation of the refrigeration of the semen samples at 2℃－8℃, significantly at 2 days. The semen samples could be frozen directly at －20℃, and three times of repeated freezing and thawing produced little effect on the results of sperm DFI, except for some inadequate stability. Based on the data obtained from freezing the semen samples after sub-packed and tested 2 days for 1 month and simulation of inter-laboratory quality control, the calculated CV value was 7.13%.
Conclusions:
In detection of sperm DFI, it is very important to ensure the accuracy of acid denaturation time, which cannot exceed 1 minute at most. The time of or after AO staining does not significantly affect the results of sperm DFI. The samples for detection of sperm DFI should be fresh and not exceed 1 day in case of refrigeration. Directly frozen semen samples can be used as the materials for inter-laboratory quality control for detection of sperm DFI. Whether cryoprotectants can make frozen semen samples more stable and how to prepare and transport the materials for inter-laboratory quality control are the key problems to be solved in the future.
