Abstract
Many studies have now confirmed that sperm DNA fragmentation (SDF) is associated with a poorer outcome of some forms of assisted reproduction technology. For this reason, SDF is an important parameter to evaluate in male fertility assessment. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay coupled to flow cytometry is one of the most promising methods for SDF quantification. Several kits for the detection of DNA fragmentation are currently available on the market and all are recommended as equally appropriate to quantify SDF. In this work we compared for the first time the efficacy of two different types of TUNEL kits for SDF quantification: one using an indirect antibody-based labeling system (BrdUTP/fluorescein-anti-BrdUTP) and another using a direct labeling system (fluorescein-dUTP). We demonstrated that TUNEL indirect labeling system largely underestimates SDF when compared with the direct labeling, the differences ranging from 19.2% to 85.3% (p<0.05, n = 22). We observed that these differences were most pronounced among dead spermatozoa where indirect labeling stained 40.1% [23.6%, 58.2%] and the direct system 65.7% [36.5%, 90.9%] (n = 10, p<0.05). Interestingly, we found that both systems stained the living spermatozoa with the same efficiency. We showed that the differences are due to the steric hindrance of the antibody during its binding to the BrdUTP. Indeed, after sperm DNA decondensation, the percentages of TUNEL positivity increased significantly from 46.3% [31.8%, 61.7%] to 97.5% [96.1%, 98.8%] (p<0.05, n = 5). Our results are important for future use of TUNEL in clinical practice. Laboratories relying on the use of an antibody-based system heavily underestimate SDF, most particularly in infertile patients with reduced sperm motility. As a consequence, the kit using BrdUTP/fluorescein-anti-BrdUTP should not be recommended as a method to assay DNA damage in semen. This study represents one further step in the standardization of TUNEL among laboratories.
Highlights
Ever since the publication of the first reports [1, 2] the role of sperm DNA fragmentation (SDF) in understanding male infertility has steadily grown
Comparison between transferase dUTP Nick End Labeling (TUNEL) results obtained with indirect (BrdUTP/ fluorescein isothiocyanate (FITC)-anti-BrdUTP) and direct (FITC-dUTP) labeling
TUNEL-FC results are given as the subpopulation of spermatozoa that simultaneously stain positive for propidium iodide (PI) and for the fluorophore used to label the DNA strand breaks (Fig 1)
Summary
Ever since the publication of the first reports [1, 2] the role of sperm DNA fragmentation (SDF) in understanding male infertility has steadily grown. Numerous studies have confirmed its adverse effects on reproductive outcome [3,4,5,6,7,8,9,10,11]. Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) ranks among the most promising assays for the quantification of SDF. TUNEL measures both single and double DNA-strand breaks through the enzymatic incorporation of a modified dUTP at the 3’-OH terminal end of a DNA strand. TUNEL directly quantifies DNA damage, which in comparison to all other methods, constitutes one of its advantages [16]. Other widely used tests such as the Sperm Chromatin Structure Assay (SCSA) [17], Sperm Chromatin Dispersion (SCD) [18] or alkaline Comet assay [19], which assess the chromatin structural integrity of spermatozoa by testing its susceptibility to acid or alkaline -induced denaturation and thereby assess DNA damage indirectly
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