AB112. Detection of human sperm DNA fragmentation by alkaline comet and neutral comet improved by research center for genetics and reproductive health (CGRH)

Abstract

Objective

To optimize the specificity and sensitivity of protocols to detect single-strand and double-strand DNA fragmentation of human sperm by using Neutral Comet and Alkaline Comet assay respectively. In both assays, the same conditions of lysis solution and gel electrophoresis were applied.

Materials and methods

Thirty samples of semen were collected to assess DNA fragmentation. The inclusion criteria were 18-40-year-old men and the exclusion criteria was azoospermia.

Results

The sample was prepared on lame at the concentration of 1×106 sperm/mL. The duration of cell lysis was successfully decrease to 30 minutes using the optimized solutions of 1.5 M NaCl and 1 mM DTT. After lysing and removing saline solutions, the gel electrophoresis was run at 20 V/70 mA in 10 minutes. The positive control sample was well-prepared by using 2% H₂O₂ to detect specificity and sensitivity of lysing and electrophoresis. In assay of Alkaline Comet, the sample was covered in alkaline solution (pH>13), 4 °C in 5 minutes after lysing, then moved into gel electrophoresis. The sample was dyed with SYBR and observed the sperm DNA fragmentation under fluorescent microscope. Four hundred of sperms were randomly counted in every sample. The images were well-captured in terms of detection of fragmented and nonfragmented sperms.

Conclusions

The optimized protocol allowed to detect the single- and double-strand DNA fragmentation in human sperms by only using the same conditions of lysis solutions and gel electrophoresis; moreover, to reduce the duration of lab performance and the cost. The protocols could be easily applied in andrology labs to provide for useful information together with semen analysis (WHO, 2010). This is the first result in Vietnam to detect DNA fragmentation by using Comet assays.