DNA fragmentation of human spermatozoa: Simple assessment of single‐ and double‐strand DNA breaks and their respective dynamic behavioral response

Abstract

Background Procedures to detect sperm DNA fragmentation (SDF), like the sperm chromatin dispersion (SCD) test, determine the “global” SDF without discriminating between spermatozoa with single‐strand DNA breaks only (SDF‐SSBs) and those containing double‐strand DNA breaks (SDF‐DSBs). Objectives (a) To validate a test to distinguish human spermatozoa with massive DSBs (DSB‐SCD assay), (b) to study the baseline SDF‐SSBs and SDF‐DSBs, and (c) to assess their dynamics in vitro. Materials and methods (a) SDF‐DSBs were determined by visualization of diffused DNA fragments from spermatozoa lysed under non‐denaturing conditions. This was validated by in vitro incubation with DNase I and the comet assay. (b) Baseline SDF‐DSBs and SDF‐SSBs were determined in ejaculates from 95 males. (c) Their dynamic appearance was studied in samples untreated or exposed to hyperthermia, acidic pH, nitric oxide released by sodium nitroprusside (SNP), and the metabolic energy inhibitors 2‐deoxy‐D‐glucose and antimycin A. Results (a) DNase I and comet assay experiments confirmed that the assay successfully determined SDF‐DSBs. (b) The higher the SDF of the semen sample, the higher the frequency of SSBs, whereas DSBs behaved independently. Abnormal samples showed higher SDF than normozoospermic, the difference being only significant for SDF‐SSBs. (c) During the first hours of incubation, the linear rate of increase in SDF‐SSBs was 3.7 X higher than that of SDF‐DSBs. All hazardous agents accelerated the SDF rate when compared to untreated spermatozoa, primarily being associated with SDF‐SSBs. SNP treatment was the most damaging, rapidly inducing spermatozoa with SSBs which progressively evolved to DSBs. Remarkably, this phenomenon was also evidenced after acute SNP exposure, revealing cryptic sperm damage. Conclusion The DSBs‐SCD is an easy complement for SDF assessment. The dynamic study of SSBs and DSBs may improve the evaluation of sperm quality in clinical settings, particularly “unmasking” the presence of non‐specific cryptic sperm damage that might otherwise go undetected.

Sperm DNA damage in men with spinal cord injury: the relative incidence of single‐ and double‐strand DNA breaks

“…The relative proportion of DSBs/SSBs observed in men with SCI compared to that of men with normozoospermic samples in the current study was approximately 3 to 1. A recent study reported by Timermans et al 17 . combined the assessment of global SDF obtained by the standard sperm chromatin dispersion (SCD) test with that of a new variant of the assay for determining the proportion of DSBs specifically, and found that in a sample of normozoospermic subjects, the proportion of sperm with fragmented DNA by SSBs and by DSBs was very similar; a finding which is further supported by our observations of normozoospermic ejaculates in the current study.…”

Antibiotic toxicity on human spermatozoa assessed using the sperm DNA fragmentation dynamic assay

“…The dynamic progression of SSBs to DSBs was not observed either with the low dose of ciprofloxacin or with the high dose of doxycycline. This confirms that, under the dynamic assay, the quantification of the intensity of the adverse effect on spermatozoa can not only be derived from the acceleration level of r-SDF but also from the estimation of the type of DNA breakage, either SSBs or DSBs, and how they evolve (Tímermans et al, 2020).…”

“…Furthermore, as usual in the apoptotic response, it can be triggered after the damage of one or several different cell targets. Under the dynamic assay, spermatozoa that initially did not present fragmented DNA may develop it faster than normal after in vitro incubation, therefore revealing previous cryptic damage, at one or several targets, that is not detectable on initial evaluation (Santiso et al, 2012; Tímermans et al, 2020).…”

“…DNA breakage may affect one strand, known as single‐strand DNA breaks (SSBs) or both complementary strands, that is double‐strand DNA breaks (DSBs). Recently, the evolution of SDF‐SSBs and SDF‐DSBs has been documented (Tímermans et al, 2020), allowing for a more informative SDF‐dynamic assay that can better differentiate the pathology of DNA damage in terms of its relative effect on SSBs and DSBs.…”

The influence of paternal overweight on sperm chromatin integrity, fertilization rate and pregnancy outcome among males attending fertility clinic for IVF/ICSI treatment

“…With the development of new predictive tools to identify male fertility potential. Sperm deoxyribonucleic acid fragmentation index is a commonly used technique involving methods such as Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, Comet assay, Sperm chromatin structure assay (SCSA), acridine Orange test, and sperm chromatin dispersion (SCD) assay [14,16]. Identification of DFI through SCD assays is cheaper yet equally reliable when compared with TUNEL assay which is expensive and utilizes advanced equipment [17][18][19].…”