TUNEL labeling with BrdUTP/anti-BrdUTP greatly underestimates the level of sperm DNA fragmentation in semen evaluation

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.

Fig 1. Example of dot plots obtained and strategy followed in TUNEL-FC analysis.

A) direct labeling and B) indirect labeling. a) FSC/SSC plots and flame shaped region (red) including spermatozoa and semen apoptotic bodies; b) PI/FSC plots and region (red) including PI-positive events (i.e. all spermatozoa) and excluding M540 bodies. Note the presence of two sperm populations with different PI staining intensity (PI brighter and PI dimmer); c) PI/FITC plots of negative control (without TdT enzyme), where quadrants are set; d) PI/FITC plots of a test sample, where TUNEL-positivity is calculated. Percentage of spermatozoa in UR quadrants reveals TUNEL-positivity of the PI-brighter population. Percentage of spermatozoa in LR quadrants reveals TUNEL-positivity of the PI-dimmer population. FSC-forward scatter, SSC-side scatter, PI- propidium iodide fluorescence, FITC-fluorescein isothiocyanate fluorescence, UL-upper left quadrant, UR-upper right quadrant, LL-lower left quadrant, LR-lower right quadrant. PI-dimmer and PI-brighter populations are indicated.

Fig 2. TUNEL-LIVE/DEAD data analysis.

A) FSC/SSC plots and flame shaped region containing spermatozoa and semen apoptotic bodies. B) PI/FSC plots showing the gates for selection of all spermatozoa (black ellipse) or PI-brighter (blue) and PI-dimmer (red) populations. C) DEAD/ALIVE/FSC plot with the dead cells showing positive staining for LIVE/DEAD dye (upper region). D) and E) LIVE/DEAD/ FITC plots for direct and indirect staining, respectively. The UR quadrants show dead spermatozoa staining for TUNEL and LR quadrants show live spermatozoa staining for TUNEL. By selecting, in plot B, only the PI-brighter population (in blue) or the PI-dimmer population (in red) the Accuri C6 software can quantify separately the SDF in the distinct dead populations. UL-upper left quadrant, UR-upper right quadrant, LL-lower left quadrant, LR-lower right quadrant.

Fig 3. Comparison of TUNEL results obtained with the two kits.

(A) Total percentage of TUNEL-positive spermatozoa; (B) Percentage of PI-dimmer population positive for TUNEL; (C) Percentage of PI-brighter population positive for TUNEL. Direct labeling: grey columns; Indirect labeling: white columns. For each semen sample the percentage of immotile spermatozoa calculated in the conventional semen analysis and the percentage of PI-dimmer population obtained after PI-staining during TUNEL assay are indicated in the X-axis, in the first sample, 38/20 corresponds to a sample with 38% of immotile spermatozoa and 20% of PI dimmer spermatozoa. The non-normozoospermic men (2010 WHO reference values) are indicated with an asterisk (S1 Table). CI-confidence interval, p-Wilcoxon Signed Rank p.

Fig 4. Flow chart showing the strategy of exchange of kits components.

Fig 5. Example of dot plots obtained in TUNEL analysis with the indirect labeling system after decondensation of spermatozoa populations.

A) PI-dimmer; B)-PI- brighter. a) negative controls of non decondensed samples, b) stained non decondensed samples, c) negative controls of decondensed samples, d) stained decondensed samples. PI- propidium iodide fluorescence, FITC-fluorescein isothiocyanate fluorescence; UL-upper left quadrant, UR-upper right quadrant, LL-lower left quadrant, LR-lower right quadrant.

Fig 6. Comparison of TUNEL-LIVE/DEAD results.

(A) Percentage of live population positive for TUNEL; (B) Percentage of total dead population positive for TUNEL; (C) Percentage of dead PI-dimmer and dead PI-brighter populations positive for TUNEL. Direct labeling: filled or stripped grey columns. Indirect labeling: filled or stripped white columns. For each semen sample the total percentage of dead spermatozoa stained with LIVE/DEAD kit and the percentage of PI-dimmer population are indicated in the X-axis. The non-normozoospermic men (2010 WHO reference values) are indicated with an asterisk. CI-confidence interval, p- Wilcoxon Signed Rank p.