Sperm Chromatin Structure Assay (SCSA)

The SCSA^� is the pioneering assay for the detection of damaged sperm DNA and altered proteins in sperm nuclei via flow cytometry of acridine orange (AO) stained sperm. The SCSA^� is considered to be the most precise and repeatable test providing very unique, dual parameter data (red vs. green fluorescence) on a 1,024 � 1,024 channel scale, not only on DNA fragmentation but also on abnormal sperm characterized by lack of normal exchange of histones to protamines. Raw semen/sperm aliquots or purified sperm can be flash frozen, placed in a box with dry ice and shipped by overnight courier to an experienced SCSA^� lab. The samples are individually thawed, prepared, and analyzed in ∼10 min. Of significance, data on 5,000 individual sperm are recorded on a 1,024 � 1,024 dot plot of green (native DNA) and red (broken DNA) fluorescence. Repeat measurements have virtually identical dot plot patterns demonstrating that the low pH treatment that opens up the DNA strands at the sites of breaks and staining by acridine orange (AO) are highly precise and repeatable (CVs of 1–3%) and the same between fresh and frozen samples. SCSAsoft^� software transforms the X -Y data to total DNA stainability versus red/red + green fluoresence (DFI) providing a more accurate determination of % DFI as well as the more sensitive value of standard deviation of DFI (SD DFI) as demonstrated by animal fertility and dose–response toxicology studies. The current established clinical threshold is 25% DFI for placing a man into a statistical probability of the following: (a) longer time to natural pregnancy, (b) low odds of IUI pregnancy, (c) more miscarriages, or (d) no pregnancy. Changes in lifestyle as well as medical intervention can lower the %DFI to increase the probability of natural pregnancy. Couples of men with >25% DFI are counseled to try ICSI and when in the >50% range may consider TESE/ICSI. The SCSA^� simultaneously determines the % of sperm with high DNA stainability (%HDS) related to retained nuclear histones consistent with immature sperm; high HDS values are predictive of pregnancy failure.