Evaluation of Sperm DNA Fragmentation Using Two Different Methods: TUNEL via Fluorescence Microscopy, and Flow Cytometry.

Background and Objectives: Sperm DNA fragmentation refers to any break in one or both of the strands of DNA in the head of a sperm. The most widely used methodologies for assessing sperm DNA fragmentation are the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion assay (SCD), the single-cell gel electrophoresis assay (SCGE-comet), and the terminal-deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end labelling (TUNEL) assay. The aim of this study was to compare the efficiency and sensitivity of the analysis of sperm DNA fragmentation using TUNEL via fluorescence microscopy, and flow cytometry. Materials and Methods: Semen samples were collected and analyzed for standard characteristics using light microscopy, and for sperm DNA fragmentation using both TUNEL via fluorescence microscopy, and flow cytometry. Results: There were no significant differences in the values of the sperm DNA fragmentation index (DFI) obtained when the analysis was performed using TUNEL or flow cytometry (p = 0.543). Spearman's correlation analysis revealed a significant negative correlation between sperm motility (%) and sperm DNA fragmentation (p < 0.01), as well as between sperm concentration and sperm DNA fragmentation (p < 0.05). The Mann-Whitney U test showed no significant difference in the DFI among couples with repeated implantation failure (RIF) and miscarriages (p = 0.352). Conclusions: Both methods (TUNEL via fluorescence microscopy, and flow cytometry) have a high efficiency and sensitivity in accurately detecting sperm DNA fragmentation, and can be effectively used to assess male fertility.

Medicina (Kaunas, Lithuania). 2023 Jul 15*** epublish ***

Katerina Chatzimeletiou, Alexandra Fleva, Theodoros-Thomas Nikolopoulos, Maria Markopoulou, Glykeria Zervakakou, Kyriakos Papanikolaou, George Anifandis, Anastasia Gianakou, Grigoris Grimbizis

Unit for Human Reproduction, 1st Department of Obstetrics & Gynaecology, 'Papageorgiou' General Hospital, Aristotle University Medical School, 56403 Thessaloniki, Greece., Department of Immunology and Histocompatibility, 'Papageorgiou' General Hospital, 56403 Thessaloniki, Greece., Fertilia by Genesis, IVF Unit, 54301 Thessaloniki, Greece., Department of Obstetrics and Gynecology, School of Health Sciences, Faculty of Medicine, University of Thessaly, 41200 Larisa, Greece.