Testing sperm DNA damage by tunel assay in specific cases of male infertility

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OBJECTIVE: The TUNEL (terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling) assay is a direct assay measuring single and double strand DNA breaks. Our objective was to standardize the TUNEL assay by: 1) establishing inter- intra observer variability, inter assay variability, cutoff value, sensitivity, specificity of the assay and 2) study the distribution of the DNA damage in a population of infertile men referred to the clinical andrology laboratory.DESIGN: Sperm DNA damage by TUNEL and flow cytometry.MATERIALS AND METHODS: Seminal ejaculates from 43 controls and 114 infertile men referred to andrology lab were tested for sperm DNA damage by TUNEL assay using Apo-Direct kit. Negative and positive controls were included in each run. Flow cytometric analysis was performed and percentage of cells positive for TUNEL was calculated using the flow cytometer software. Receiver operating characteristic (ROC) curve, cutoff value, sensitivity and specificity was calculated.RESULTS: Inter- and intra-observer and interassay variability was <10%. Higher DNA damage was seen in patients (29.5% ± 18.7% compared with controls (11.9% ± 6.8%; P<0.001). The cutoff value of 19.3% showed 64.9% sensitivity and 100 % specificity. When the DNA damage was categorized into 0-10%, 10-20%, 20-30%, 30-40% and >40%, in the patients, 14.9% (29 of 194) had DNA damage between 0-10%; 22.7% (44 of 194) between 10-20%, 8.8% (17 of 194) between 20-30%, 17.5% (34 of 194) between 30-40% and 27.3% (53 of 194) had TUNEL values >40%.CONCLUSION: We report a detailed stepwise standardization of the TUNEL assay for clinical use. A cutoff of 19.5% with 100% specificity can differentiate infertile men with DNA damage from healthy men. High sensitivity and 100% specificity in our program makes it an ideal test. Each program must establish their threshold values. This test can be offered to infertile men who are idiopathic or who have oxidative stress and are considering assisted reproductive options. OBJECTIVE: The TUNEL (terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling) assay is a direct assay measuring single and double strand DNA breaks. Our objective was to standardize the TUNEL assay by: 1) establishing inter- intra observer variability, inter assay variability, cutoff value, sensitivity, specificity of the assay and 2) study the distribution of the DNA damage in a population of infertile men referred to the clinical andrology laboratory. DESIGN: Sperm DNA damage by TUNEL and flow cytometry. MATERIALS AND METHODS: Seminal ejaculates from 43 controls and 114 infertile men referred to andrology lab were tested for sperm DNA damage by TUNEL assay using Apo-Direct kit. Negative and positive controls were included in each run. Flow cytometric analysis was performed and percentage of cells positive for TUNEL was calculated using the flow cytometer software. Receiver operating characteristic (ROC) curve, cutoff value, sensitivity and specificity was calculated. RESULTS: Inter- and intra-observer and interassay variability was <10%. Higher DNA damage was seen in patients (29.5% ± 18.7% compared with controls (11.9% ± 6.8%; P<0.001). The cutoff value of 19.3% showed 64.9% sensitivity and 100 % specificity. When the DNA damage was categorized into 0-10%, 10-20%, 20-30%, 30-40% and >40%, in the patients, 14.9% (29 of 194) had DNA damage between 0-10%; 22.7% (44 of 194) between 10-20%, 8.8% (17 of 194) between 20-30%, 17.5% (34 of 194) between 30-40% and 27.3% (53 of 194) had TUNEL values >40%. CONCLUSION: We report a detailed stepwise standardization of the TUNEL assay for clinical use. A cutoff of 19.5% with 100% specificity can differentiate infertile men with DNA damage from healthy men. High sensitivity and 100% specificity in our program makes it an ideal test. Each program must establish their threshold values. This test can be offered to infertile men who are idiopathic or who have oxidative stress and are considering assisted reproductive options.