Sperm DNA fragmentation: the evolution of guidelines for patient testing and management

Sperm DNA fragmentation (SDF) represents a perplexing biological phenomenon that can, at an elevated level, interfere with either initiating and/or maintaining pregnancy. Although we are in our third decade of studying this problem, few investigators have attempted to assess the value of the various available assays for SDF with the specific aim of amalgamating them into practice guidelines for managing SDF. Agarwal, in collaboration with urological experts from North and South America, has done just that, employing a number of clinical scenarios involving SDF to produce some evidence-based, practical guidelines for patient management (1). But if in our third decade of investigation, why are management guidelines so difficult to come by for patients with elevated SDF? First, it should be emphasized that SDF is an exceedingly complex process involving not one, but a number of causative mechanisms that can generate a variety of insults to the integrity of sperm DNA. Sakkas and Alvarez (2) have described six main mechanisms that can damage both sperm nuclear as well as mitochondrial DNA. Included in this list are testicular apoptotic processes during spermatogenesis, aberrations during the events of chromatin re-modeling, induction by exogenous caspases and nucleases plus damage by chemo- and radiotherapy as well as environmental toxicants. Perhaps the most robust events occur in the epididymis in response to insult by reactive oxygen species. That epididymal and ejaculated sperm have higher amounts of DNA fragmentation than testicular sperm is now well documented (3-5). What is not known are the testicular events that render sperm labile to chemical radicals traversing the epididymis. Secondly, the insults can produce single-stranded breaks, double-stranded breaks and/or nucleotide damage. Not all are detected by the same tests, the different types of damage are repaired to different degrees by processes resident in the oocyte and thus, do not have the same prognosis. Likely though, it is the tests used to detect and measure SDF, along with their varying levels of complexity, that introduce the bulk of the conflicting data and conclusions common in the SDF literature.

As Agarwal et al. demonstrate in concise tabular form, there are at least eight tests for SDF, each with attendant advantages and disadvantages. The predictive value of these, assuming proper execution and quality control, depends upon the type of DNA damage, the percent of sperm with fragmentation, the extent of damage per sperm, whether there is combined fragmentation and nucleotide damage, whether the lesions affect exons or introns and the effectiveness of the oocytes in repairing DNA damage in zygote genomic DNA contributed by the fertilizing sperm (2). Of interest, SDF fails to affect the fertilizing capacity of sperm (6), so testing is performed to either visualize the state of sperm DNA via differential staining of fragmented versus intact DNA, to reveal the physical restrictions fragmentation places upon DNA dispersion, to measure differential electrophoretic mobility of intact versus fragmented DNA or measure nucleotide incorporation into DNA nicks. A very recent review and meta-analysis by Cissen et al. (7) examined the value of SDF measures in predicting the chance of ongoing pregnancy with in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Of 658 studies reviewed, 30 had data incorporated into the meta-analysis. In general, their results supported the conclusion that SDF tests had reasonable to good sensitivity but limited to low specificity, acknowledging that a wide variety of other factors may also affect IVF/ICSI production of ongoing pregnancy. The predictive value of sperm chromatin structure assay (SCSA) was determined from the analysis of 14 studies, of which 11 were prospective, whereas five were included for sperm chromatin dispersion test (SCD), nine for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and four for COMET, all of which were prospective. Whereas the TUNEL and comet assays had fair discriminatory capacity, SCSA and SCD had poor predictive value. Furthermore, meta-regression indicated that TUNEL, SCD and comet assays showed no predictive value between IVF and ICSI cases, a finding that differed from SCSD, which did. They conclude that SDF tests have limited capacity to predict the chance of pregnancy in assisted reproductive technology (ART) and are therefore not recommended for routine application to couples undergoing IVF/ICSI. Other studies are in conflict with Cissen et al. For example, Zhao et al. (8) saw a negative influence of SDF upon patients undergoing IVF but not ICSI in a meta-analysis that included nearly 3,000 couples.

Despite these controversial findings, SDF testing decidedly has its place in ART. It can direct the appropriate patient towards ICSI or testicular biopsy and testicular sperm extraction (TESE). It can well shed light upon the path for successful continued management in fertility therapy; that SDF can adversely influence embryogenesis, even in euploid embryos, is known (9-11). Agarwal et al. have been judiciously conservative in making their recommendations and have included only clinical scenarios where evidence allows. Additionally, they have not relied on just one test or measure of SDF. This is important in that different tests measure different aspects of DNA damage such as single-strand breaks, double-strand breaks and protamine loss. Although the strength of the evidence leading to the suggested guidelines is moderate to low, it is the complicated nature of the DNA fragmenting process and fragmented state, as well as the SDF testing, that makes it so and to date, it remains the best evidence available. Is there a clinical presentation where the urologist or andrologist would rely solely upon the results of SDF testing? Not according to these guidelines or other conclusions from the current literature. As the authors suggest, it is an additional tool, nothing more.