Sperm DNA fragmentation assessment: Is it really helpful?

Fertilization, both natural and by assisted reproduction techniques (ART), needs the dual support of the sperm not only for the messages it carries but for being as a messenger cell the and carrier of these messages as well. Nevertheless, In 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 fertilization 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 characterized by single- as well as double 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 like excessive ROS. Some external conditions factors 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, like such as in 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, while indirect assays quantify the susceptibility of sperm DNA to break after an external insult, such as acid treatment. The most commonly used direct assays are; Terminal Deoxynucleotidyl Transferase-mediated Nick End Labeling (TUNEL), Single Cell Gel Electrophoresis (COMET) and In-Situ Nick Translation (NT) assay. The most common indirect assays are; Flow flow cytometric acridine orange assay, Acridine Orange test (AO), DNA Break Detection-Fluorescence In Situ Hybridization (DBD-FISH) and Sperm Chromatin Dispertion test (SCD). Despite the method of assessment, all assays attempt to determine the total amount of DNA fragmentation, irrespectively 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, and 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 to 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 established or can not be widely accepted without concern. It is important to note that values obtained using different assays can not be compared. Sometimes in addition, it is inappropriate to compare values obtained from different laboratories, even using the same technique, as many lab factors and protocol variations can significantly affect the results. 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; and 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 thirteen studies, sperm DNA fragmentation was significantly, albeit weakly, associated with pregnancy (OR= 1.44, 95% CI; 1.03, - 2.03). This weak association can not be considered clinically important neither enough adequate evidence to discriminate between couples who will conceive or 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 regarding 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.

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Reprinted with permission of the International Society of Gynecological Endocrinology

© International Society of Gynecological Endocrinology - n. 34/ June 2009