A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay

Abstract

Baseline DNA damage in spermatozoa from fertile and infertile men was compared using a modified alkali single cell gel electrophoresis (comet) assay. Semen from normozoospermic fertile, normozoospermic infertile and asthenozoospermic infertile (World Health Organization criteria, 1992) samples were studied. No significant difference was observed in levels of baseline damage between the three groups. A median value for baseline damage of approximately 20% (80% head DNA) was obtained in all samples. Irradiation with X-rays (5-30 Gy) produced no additional damage in fertile samples when median values were examined. However, irradiation with 30 Gy X-rays produced significant damage in both infertile groups. Hydrogen peroxide (40 microM) treatment induced significant damage in the asthenozoospermic group, whereas 100 microM H2O2 was required to cause significant damage in the normozoospermic fertile and infertile samples. Within the fertile population a subgroup in which percentage head DNA was greater than 80% was observed in both treated and untreated specimens. This subgroup significantly decreased with treatment in both infertile groups. We conclude that the asthenozoospermic infertile group is more susceptible to damage than the normozoospermic infertile group, which in turn is more susceptible than the fertile group. The fertile group contains a resistant subpopulation of spermatozoa with relatively intact DNA.

Shortening of alkaline DNA unwinding time does not interfere with detecting DNA damage to mouse and human spermatozoa in the comet assay

“…As previously reported by Hughes et al, 11 background DNA damage in human spermatozoa is higher (11.2%, Figure 5a; 19.1%, Figure 5b) than that in mouse spermatozoa (3.53%, Figure 1; 4.40%, Figure 3; 10.5%, Figure 4), being similar to the baseline in somatic cells.…”

Fluctuation of “sperm DNA integrity” in accordance with semen parameters, and it’s relationship with infertility

“…Many studies have shown that the levels of sperm DNA damage in fertile and infertile males were considerably different [22]. In fact, the sperm of infertile individuals has been found to be more sensitive to agents like H 2 O 2 and radiation which cause damage to DNA [21]. If the sperm DNA damage exceeds 30 % as detected by the SCSA [23] or 20 % by TUNEL [24], the in vivo fertilization rate seems to be close to zero.…”

“…The alkaline COMET assay was performed as previously described methods [20,21] with some modifications. Briefly, the fully frosted microscope slides were covered with 100 μl of 0.75 % normal melting-point agarose (NMPA; Sigma A9539, USA) in PBS and then dried at room temperature.…”

Semen analysis and sperm function testing

“…The most common types of identified sperm DNA damage are: (i) single and double DNA strand breaks; (ii) the chemical modification of a base by, for example, oxidation or alkylation; (iii) inter-or intrastrand crosslinkage; and (iv) DNA-protein crosslinks. 57,58 A variety of tests have been introduced including terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling or TUNEL, 59 comet, 60 in situ nick translation, 61 DNA breakage detection-fluorescence in situ hybridisation, 62 sperm chromatin dispersion test 63 and sperm chromatin structure assay. 64 Some of these tests measure DNA damage directly, such as TUNEL, or comet at neutral pH; others measure DNA damage after denaturation steps, such as the sperm chromatin structure assay, sperm chromatin dispersion and comet at acid or alkaline pH.…”