E to measure DNAChamproux et al. Basic and Clinical Andrology (2016) 26:Page
E to measure DNAChamproux et al. Basic and Clinical Andrology (2016) 26:Page 16 ofbreaks in individual sperm. During this procedure, sperm cells are embedded in a thin layer of agarose on a microscope slide and lysed with detergent under high salt concentration conditions. This process removes protamines and histones allowing the nucleus to form a nucleoid-like structure containing supercoiled loops of DNA. Alkaline pH conditions relaxing double-stranded DNA, and subsequent electrophoresis result in the migration of Dalfopristin biological activity broken strands towards the anode, forming a comet tail, when observed under fluorescence microscopy. The amount of DNA in the head and tail is reflected by its fluorescent intensity. The relative fluorescence in the tail compared with its head serves as a measure of the level of fragmented DNA [202]. ?SCD or sperm chromatin dispersion assay. This assay is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins [203]. ?DBD-FISH or DNA breakage detection fluorescence in situ hybridization [204]. This assay allows in situ detection and quantification of DNA breaks in single cells. Cells embedded within an agarose matrix on a slide are exposed to an alkaline unwinding solution that transforms DNA strand breaks into ssDNA (single strand DNA) motifs. After neutralizing and protein removal, ssDNA are accessible to hybridization with whole genome or specific DNA probes. The probes highlight the chromatin area to be analyzed. As DNA breaks increase in the targeted region, more ssDNA are produced by the alkaline solution and more probes hybridize, resulting in an increase in the fluorescence intensity and in the surface area of the FISH signal. ?8-OHdG assay. One of the latest assays reported is the evaluation of the level of oxidized guanine residue that gives an indication as to the degree of sperm DNA oxidative damage. To date it is essentially used for research purpose and has yet to be developed for clinical routine testing. Sperm 8OHdG evaluation is of interest because it is known to be associated with the level of de novo mutation in the germline [205]. The present current minimum standard is assessment of seminal plasma by volume, appearance and liquefaction of the ejaculate, and, for spermatozoa, measurement of concentration, motility and morphology [206]. The above mentioned techniques are rarely used in routine clinical evaluation because the tested spermatozoa are unsuitable for clinical purpose afterwards. However, for some of them they do allow the establishment of a DNAfragmentation rate, a useful marker in the prediction of fertility. Studies have shown that the chance of spontaneous conception starts to decline at sperm DNA damage (DFI) values above 20 and are close to zero for readings over 30?0 [207]. In another study using the Comet assay, the authors also showed that there was a strong correlation between sperm DNA fragmentation and fertility status of men [134]. Thus, there is robust evidence from all the DNA fragmentation assays that the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27741243 chance of spontaneous pregnancy is reduced when sperm DNA damage is excessive.Conclusions Thus, it appears that sperm defective nuclear condensation and sperm nuclear fragmentation are nowadays easy situations to assess with trustable/reliable direct and/or indirect assays (see above). Since Evenson et al. [.