Genomic fragmentation and extrachromosomal telomeric repeats impact assessment of telomere length in human spermatozoa: quantitative experiments and systematic review.

STUDY QUESTION: Can differences in DNA isolation alter assessment of sperm telomere length (spTL) and do they account for conflicting results in the literature on spTL and male fertility? SUMMARY ANSWER: DNA isolation methods preferentially include or exclude short, extrachromosomal (EC) telomere-specific sequences that alter spTL measurements, and are responsible for a proportion of the disparity observed between investigations. WHAT IS KNOWN ALREADY: The relationship between spTL and male fertility has become an active area of research. The results across investigations, however, have been discordant, generating a need to critically evaluate the existing body of knowledge to guide future investigations. STUDY DESIGN, SIZE, DURATION: Quantitative experiments determined the effect of DNA isolation on the integrity of sperm DNA and measures of spTL, while a systematic analysis of the current literature evaluated the effect of DNA isolation and study design on experimental outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two DNA isolation methods were compared: Genomic Tips which isolate 'High Molecular Weight' (HMW) DNA exclusively, and QIAamp® DNA Mini which isolates 'Total' genomic DNA irrespective of size. DNA quality was assessed via field inversion gel electrophoresis (FIGE) and spTL was measured via terminal restriction fragment analysis. In addition, major databases in medicine, health and the life sciences were subject to a targeted search, and results were independently screened according to defined exclusion/inclusion criterion. Findings from primary articles were analyzed for concordance and study designs were compared across six moderator variables (sample size, participant age, fertility status, semen fraction, telomere population and type of analysis). MAIN RESULTS AND THE ROLE OF CHANCE: HMW DNA spTL was significantly longer than spTL measured from total DNA (P < 0.01), indicating that Total DNA contained short, EC telomeric repeats that shifted downstream assessment towards shorter spTL. HMW DNA spTL reflected the length of intact, chromosomal telomeres. Major findings on spTL showed the greatest concordance amongst studies that implemented HMW DNA isolation prior to spTL assessment. Studies that utilized Total DNA varied in concordance, but outcomes were similar if (i) a comparative analysis was applied or (ii) a sample size threshold of 81 was achieved for correlative analysis. LIMITATIONS, REASONS FOR CAUTION: Chromosomal and EC telomeric DNA were distinguished based on outcomes of HMW DNA isolation and size. Further experiments are required to determine the nature and function of these two types of telomeric sequences. WIDER IMPLICATIONS OF THE FINDINGS: This study reveals a dramatic impact of upstream DNA processing and study design on measurements of spTL, which accounts for conflicting results in the literature. Future assessments of spTL should incorporate independent detection of chromosomal and EC telomeric DNA and specific experimental planning. STUDY FUNDING/COMPETING INTERESTS: This study was funded by CReATe Fertility Centre, Toronto, Ontario, Canada. The authors have declared no conflict of interest. REGISTRATION NUMBER: N/A.

Sperm survival: relationship to age-related sperm DNA integrity in infertile men.

The purpose of our study was to evaluate the effect of age and sperm DNA integrity on sperm survival. Semen samples from fifty six unselected patients undergoing infertility evaluation were assessed in terms of standard semen parameters, DNA integrity, and sperm survival after 6-24 h of incubation. Prolonged incubation of density gradient selected sperm adversely effects sperm survival in older patients and patients with extensive sperm DNA damage. Immediate preparation of such samples prior to use for assisted reproductive technology (ART) may overcome the negative effect of extended incubation.

Sperm DNA integrity: correlation with sperm plasma membrane integrity in semen evaluated for male infertility.

Previous independent studies have indicated that abnormally low parameters of sperm DNA integrity and sperm membrane integrity correlate to reduced fertility due in part to implantation disorders. The purpose of our study was to evaluate the relationship between sperm plasma membrane functional integrity assessed by the hypo-osmotic swelling test (HOST) and sperm DNA integriy test assesed by DNA fragmentation index (DFI). Semen samples from 102 random patients were evaluated in terms of standard semen parameters and assessed by DFI and HOST. Both tests showed a significant correlation to standard semen parameters (p < .05). In addition, patients with abnormal HOST results had a higher likehood of a subnormal or abnormal DFI result (p < .001). Our results suggest that a common sublethal insult may manifest as abnormalities in both the nucleus and the plasma membrane that act at the implantation and/or subsequent levels of development rather than at the fertilization stage.