TUNEL Assay and SCSA

In their recent 2021 manual for the examination of human semen, the World Health Organization recommends the testing for sperm DNA fragmentation (SDF) in the section “Extended Examination.” SDF is an endpoint parameter for which one has to distinguish between test systems that measure real DNA damage, that is, actual strand breaks of the DNA, such as 8-hydroxydeoxyguanosine (8-OHdG), single cell gel electrophoresis (COMET), the TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL), and test systems detecting potential DNA damage such as the sperm chromatin dispersion test (SCD) or the sperm chromatin structure assay (SCSA®). The TUNEL assay and the SCSA are the two most commonly used test systems to determine SDF. The principle of the TUNEL assay is based on the binding of labelled the DNA precursor deoxyuridine triphosphate (dUTP) to 3’-OH ends of DNA, thereby labelling single- and double-strand DNA breaks. This test can be done using fluorescence microscopy or flow cytometry. Relevant cut-off values to identify infertile men have been established. The SCSA is a flow cytometric test based on the metachromatic properties of acridine orange where an orange-red fluorescence indicates single-stranded DNA and green fluorescence double-stranded DNA. While different protocols are available for the TUNEL assay, the SCSA is the most standardized test for SDF. Yet, the TUNEL assay is reportedly more predictive of male infertility than the SCSA.