Acridine Orange Test for Assessment of Human Sperm DNA Integrity

Abstract

Acridine orange test (AOT) is a simple microscopic procedure based on acid conditions to denaturant DNA followed by staining with acridine orange. The AOT measures the metachromatic shift of AO fluorescence from green (native DNA) to red (denatured DNA). Acridine Orange flu- oresce green when it binds to native DNA and red when it binds to the fragmented DNA. Many authors observed that ≥50% green fluorescence in sample is a normal cut-off value for AOT in sample from fertile donors. AOT using fluorescence microscopy provides a general picture of the status of DNA denaturation. Similarly, the sperm chromatin structure assay (SCSA) using cytometry and SCSA Software measures the intensity of Acridine Orange (AO) fluo- rescence. The ratio of red/red + green yields the percentage of DNA fragmentation, referred to as a DNA fragmentation index (DFI%). The semen samples with SCSA value of less than or equal 15% DFI represent low level, greater than 15% to less than or equal 30% DFI values represent moderate, and more than or equal 30% DFI values represent high levels of DNA fragmentation. In the present article the history, principle, mechanism, technique and troubleshooting points and the clinical significance of Acridine Orange Test are thoroughly discussed.