Assessment of the Impact Induced by Different Incubation Time, Storage Time, Storage Medium and Thawing Methods on Sperm DNA Fragmentation Assay: A Before–After Study

BACKGROUND: The sperm DNA fragmentation has been considered an important index in the field of male infertility. AIMS: Our study aims to evaluate the impact of different factors, including incubation time, storage time, storage medium and method of thawing, on DNA fragmentation of semen samples. SETTINGS AND DESIGN: This study was designed as a before–after study in five experiments. MATERIALS AND METHODS: Experiment 1 was conducted to assess the effect of storage time in liquid nitrogen on 15 semen samples. In experiment 2, DNA fragmentation was performed on 10 semen samples with different incubation times before freezing. In experiments 3, 4, two different storage media and thawing methods were applied respectively in two separate groups, each containing 30 samples and the DNA fragmentation index (DFI) was measured using the sperm chromatin structure assay method. STATISTICAL ANALYSIS: Data were analysed using Stata version 11. RESULTS: There was a significant increase in sperm DNA fragmentation of samples stored in liquid nitrogen for 1 month. This increase occurred in the first 2 weeks. Furthermore, our results showed a significant increase in the DFI after 120 min of incubation at room temperature (RT) and also thawing in RT separately. CONCLUSION: It is better to use fresh samples to measure DNA fragmentation up to 2 h after ejaculation to achieve more accurate results. Furthermore, if sperm freezing is inevitable, the use of a water bath (37°C) to thaw will be the most appropriate option, as it can lead to less DNA damage.