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.

Telomere length and telomerase activity in bovine pre-implantation embryos in vitro.

Telomeres are specialized structures that cap the ends of chromosomes and help to maintain genomic integrity and stability. Telomeres undergo dynamic changes during embryo development, which also represents an important stage for telomere elongation through telomerase enzyme activity. The objectives of this study were to examine changes in telomere length and telomerase activity from the early oocyte, through to the blastocysts stage of development, and the expression of factors with the potential to directly regulate telomeres. In vitro-produced bovine embryos were lysed and analysed for either relative telomere length, or telomerase activity using quantitative real-time PCR protocols. Our results reveal that relative telomere length is the shortest in the presumptive zygote stage of development and gradually increases to the blastocyst stage. We also demonstrate that differences between the mean telomere lengths throughout these stages are statistically significant (p < 0.05). Telomerase activity in the stages examined appears relatively constant until the blastocyst, where the highest level of activity is detected, leading to a significant difference in telomerase activity across embryonic stages (p < 0.005). Bovine telomerase RNA component (bTERC) expression levels were highest in the blastocyst, TERF1 transcripts showed little change in expression, and TERF2 expression decreased in the blastocysts (p < 0.05). Our results suggest that a complex integration of telomere-related RNA and proteins influences the regulatory mechanisms involved in 'reprogramming' of telomeres during early embryonic stages.