Abstract
Purpose: A retrospective study of 89 hypo-fertile male patients attending for in vitro fertilization was undertaken in order to evaluate possible correlations among sperm DNA damage, sperm analysis parameters and pregnancies. Methods: Sperm parameters (concentration, normal morphology and multiple anomalies) were evaluated according to the World Health Organization guidelines. DNA damages were simultaneously evaluated on each sperm sample by i) sperm chromatin decondensation test; ii) sperm DNA fragmentation evaluated by Sperm Chromatin Dispersion and Halo sperm Kit; iii) sperm DNA fragmentation evaluated by the Terminal Uridine Nick-End Labelling procedure. Results: Statistical analysis was performed by using analysis of variance and least squares regression. The sperm chromatin dispersion and fragmentation assays showed a statistically significant positive correlation (P=0.0017) in all the samples confirming the good efficacy of either of the two tests in detecting sperm DNA damage. Both the two test negatively correlated with normal sperm morphology (P=0.008), however only by using Halo sperm test we obtained a significant correlation with multiple sperm pathological morphologies (P=0.029) and an inverse correlation with pregnancies outcome (P=0.013). No correlation was detected among DNA damages, sperm concentration and chromatin decondensation. Conclusions: These data suggest a similar efficacy of sperm chromatin dispersion and Terminal Uridine Nick-End Labelling in detecting sperm DNA integrity. Due to the higher sensitivity of Halo test, its prognostic role in the diagnosis of fertilizing ability of a semen sample and possible pregnancy rate is discussed
Highlights
It has been documented that almost 50% of the infertility problems are at present attributed to male factors
Each semen sample was allowed to liquefy for 30 minutes, after that the sample was mixed carefully, divided into four aliquots and simultaneously processed as follows: i) sperm concentration and morphology; ii) nuclear chromatin decondensation test; iii) DNA fragmentation test evaluated by the TUNEL; iv) DNA fragmentation test evaluated by Halo test
According to Fernandez et al [22] using the Halo test kit, four types of halos were evaluated by using their own nucleoid as a reference as follows: i) Big halos: those whose halo width is similar or higher than the minor diameter of the core; ii) Medium-size halos: the halo size is between those with high and with very small halo; iii) Small-size halo: the halo width is similar or smaller than one third of the minor diameter of the core and; iv) No halo
Summary
It has been documented that almost 50% of the infertility problems are at present attributed to male factors. In IVF centres, potential infertile men are screened for the sperm quality by semen analysis, in particular investigating concentration, motility and morphology according to WHO guidelines [1]. At present, these parameters appear to be poor predictors of the fertilization success since infertile men may often show normal semen analysis [2]. For these reasons, it is claimed that, due to poor diagnostic methods and no fully effective infertility treatments, the use of sperm functionality test should be mandatory and routinely associated to conventional semen analysis [3,4,5]. Due to the diffuse application of ICSI, it has been stressed that the injection of DNA-damaged spermatozoa may introduce damaged genome into the oocytes with dangerous drawbacks on fertilization, embryonic, foetal and post-natal development [7,8]
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