Sperm DNA integrity is obviously important for normal embryo development and pregnancy outcome. Over the past 25 years, various methods have been developed to measure sperm DNA strand breaks in situ. The Sperm Chromatin Structure Assay (SCSA) treats sperm with low pH to denature DNA at the sites of DNA strand breaks, followed by acridine orange (AO) staining of green for native DNA and red for denatured DNA, as measured by flow cytometry (FCM), as well as % sperm with high DNA stainability (HDS: immature sperm with intact DNA related to decreased fertilization rates). FCM-sorted sperm from each SCSA-defined population (normal, moderate, and high DNA fragmentation and HDS sperm) show that the moderate DNA fragmentation index (DFI) population has the same image analysis characteristics as normal sperm without significant comets. Thus, an ICSI technician is not likely to differentiate between a normal and a moderate DFI sperm. The TUNEL assay uses an enzyme to add a fluorochrome-labeled base to a 3′-OH broken DNA strand. Both light microscopy and flow cytometry are used for measuring the % and extent of DNA fragmentation but cannot measure the level of HDS. For the COMET assay, sperm are suspended in an electrophoretic gel, placed on a glass microscope slide, digested with proteases and RNAse, subjected to an electric field, and then stained with a DNA dye. The % of comet positive sperm is scored, but the extent of fragmentation is difficult to define and the % HDS cannot be determined. Small pieces of fragmented DNA migrate in the gel forming a “comet.” All three methods have been used for both research and clinical diagnosis and as prognosis for livestock (bulls, boars, rams, stallions) and humans. Light microscope techniques suffer from a lack of statistical soundness needed for clinical decisions as well as present a potential bias in selection of sperm for measurements. Due to the thousands of sperm randomly selected for flow cytometry measurements, the data are statistically robust. Data from all three kinds of measurements in over a hundred manuscripts clearly show that sperm DNA fragmentation has a negative impact on embryo growth and pregnancy. Infertile animals may have nearly all of the sperm with fragmented DNA. Fertility ratings in bulls and boars are clearly related to the percent and extent of DNA fragmentation. Threshold levels for fertile/sub fertile/infertile differ for different species. Likewise different methods/laboratories have suggested various threshold levels to characterize a man with a highly fertile to low/very poor potential. The range of sperm with fragmented DNA is from ∼2% to 100%. The SCSA method has defined a 27–30% DFI as the point in which a man is placed into a statistical category of taking a longer time to achieve in vivo pregnancy, more intrauterine insemination and routine IVF cycles, or no pregnancy. Current data suggest that ICSI may help overcome the diminished pregnancy prognosis with high DFI over the other ART or natural methods.
Acridine Orange and Flow Cytometry: Which Is Better to Measure the Effect of Varicocele on Sperm DNA Integrity?
