Fertilisation, both natural and by assisted reproduction techniques (ART), needs the dual support of the sperm not only for the messages it carries but also for being as a messenger cell and carrier of these messages. Nevertheless, in the era of intra-cytoplasmic sperm injection (ICSI) though, only the sperm messages are needed. These messages include the haploid genome, the centrosome necessary for the division of the cell and important factors for the development of the placenta. Sperm DNA contributes half of the offspring's genetic material, and abnormal DNA in the form of fragmented DNA – when (i.e., excessive strand breaks are present) – may lead to derangements in the reproductive process. In this perspective, it seems plausible that strand breaks in the sperm DNA may affect fertilisation and delivery of a healthy child. In mammals, sperm chromatin differs from that in somatic cells in structure and composition. In fact, sperm chromatin forms the most compact structure among all eukaryotic cells. This compact structure is important for the protection of genetic integrity during transport of the paternal genome through the male and female reproductive tracts. DNA fragmentation is characterised by single- and double-strand DNA breaks. During spermiogenesis, the maturing sperm gradually loses its ability to repair possible DNA damage. Therefore, DNA breaks occurring during or after DNA packaging may escape the corrective mechanisms and be delivered to the mature sperm. The exact mechanisms leading to DNA strand breaks are still widely elusive. The theories proposed involve defective sperm chromatin packaging, abortive apoptosis and oxidative stress. There is evidence that the mechanisms leading to DNA fragmentation are inter-related, as an abnormal chromatin packaging due to defective protaminosis makes sperm more vulnerable to environmental insults such as excessive reactive oxygen species (ROS). Some external conditions have been associated with an increase in the percentage of ejaculated spermatozoa with DNA damage, although the underlying mechanisms still remain unclear. Among them, the most important are cigarette smoking, genital tract infection, testicular cancer and Hodgkin's disease, iatrogenic damage, such as sperm preparation protocols for ART, as well as hyperthermia, exposure to pesticides and air pollution. The assays used for the assessment of sperm DNA fragmentation can be distinguished into direct and indirect. Direct assays try to detect the actual DNA breaks, whereas indirect assays quantify the susceptibility of sperm DNA to break after an external insult, such as an acid treatment. The most commonly used direct assays are terminal deoxynucleotidyl transferase-mediated nick end labelling (TUNEL), single-cell gel electrophoresis (COMET) and in situ nick translation (NT) assay. The most common indirect assays are flow cytometric acridine orange (AO) assay, AO test, DNA break detection-fluorescence in situ hybridization (DBD-FISH) and sperm chromatin dispersion (SCD) test. Despite the method of assessment, all assays attempt to determine the total amount of DNA fragmentation, irrespective of the region of the genome that occurs. It is reasonable that breaks affecting certain genes are more detrimental than others in ‘silent’ areas of the genome, although no assay can evaluate this factor yet. Thus, for the present, there is no differentiation between clinically significant and insignificant fragmentation. A recent meta-analysis showed a significant odds ratio (OR) of 6.54 [95% confidence interval (CI): 1.71–24.91] and 7.58 (95% CI: 2.54–22.67), when couples with DNA fragmentation index (DFI) of <30% and <40%, respectively, compared with couples with higher DFIs. Many studies using a variety of assays have shown statistically significant differences in sperm DNA fragmentation between fertile and infertile men. Despite these differences – referring to the mean or median – there is an extensive overlap between the values found in fertile and infertile men. In addition, clear reference values to distinguish the two groups have not yet been obtained or cannot be widely accepted without concern. It is important to note that values obtained using different assays cannot be compared. In addition, it is inappropriate to compare values obtained from different laboratories, even using the same technique, because many laboratory factors and protocol variations can significantly affect the results. In vitro fertilisation (IVF) and ICSI techniques represent a very important treatment approach, even in cases of severe male factor infertility. So far, conventional semen parameters were proven disappointing at predicting the outcome of IVF; during the last years, sperm DNA fragmentation has been promoted as a promising alternative predictive factor. Numerous studies of varying quality have examined the association between sperm DNA fragmentation and pregnancy rates after standard IVF and ICSI with conflicting results. In a recent meta-analysis of 13 studies, sperm DNA fragmentation was significantly, albeit weakly, associated with pregnancy (OR = 1.44, 95% CI: 1.03–2.03). This weak association cannot be considered clinically important neither enough adequate evidence to discriminate between couples who will conceive and who will not conceive. Thus, there is not enough evidence to support the routine use of DNA fragmentation as a prognostic factor of pregnancy after IVF or ICSI procedures.
In conclusion, DNA fragmentation is a new parameter for the evaluation of male factor infertility and a possible predictor of the outcome of ART. Although promising, there are still concerns about not only its utility usefulness as a part of the fertility evaluation predictive factor but also about what it actually measures. Protocol variations among laboratories worldwide, lack of reference values and absence of conclusive evidence about its actual value as an independent or complementary factor of male infertility still prevent its establishment in the routine diagnostic investigation of the infertile man.