Date of Award

2014

Document Type

Thesis

Publisher

Edith Cowan University

Degree Name

Master of Science

School

School of Medical Sciences

Faculty

Faculty of Health, Engineering and Science

First Supervisor

Dr Peter Roberts

Second Supervisor

Dr Phillip Matson

Abstract

Male infertility is typically diagnosed upon routine semen analysis following the World Health Organisation’s (WHO) semen analysis manual. Recent editions of the manual have essentially changed the diagnosis of a semen sample, prompting debate between experts as to which edition should be followed. Deoxyribonucleic Acid (DNA) integrity analysis is proving to be a useful adjunct to semen analysis as 15% of infertile men have a normal semen analysis but they have an increased DNA fragmentation level (DFL) which has been associated with increased disease incidence in any resultant offspring. However, such tests are not endorsed by the WHO, possibly due to a lack of standardisation in the implementation, analysis and clinical interpretation of methods used to evaluate DNA integrity. Improved efficiency of testing is achieved by batch testing or sending samples to a central laboratory for analysis, requiring an effective storage system. Most current protocols for semen storage and related DNA integrity testing are complex, expensive and require specialised equipment. Nevertheless, since the Halosperm® G2 Kit, requires only standard laboratory equipment, a simple, convenient and stable storage method for the purpose of testing sperm DNA fragmentation would be advantageous. DNA has been successfully extracted from air‐dried semen and one particular study has investigated the use of air‐dried semen slides as a method of storage prior to DNA fragmentation testing, however, the effects of time and temperature on the integrity of spermatozoa DNA has not been considered.

The first objective of this present study was to investigate the relationship between sperm DNA fragmentation (using the Halosperm® G2 Test Kit) and semen analysis results (measured according to the 4th and 5th Edition WHO semenanalysis manuals) to determine the clinical utility of the Halosperm assay. The second objective was to consider extrinsic effects on the DNA integrity of air‐dried semen in order to develop an alternative storage method for semen prior to DNA fragmentation testing using the Halosperm assay.

A retrospective analysis was carried out on 905 consecutive semen samples with 4th and 5th Edition semen analysis and Halosperm result. Pearson correlations, analysis by ANOVA and post‐hoc testing by Tukey’s HSD were used for statistical analysis. Multiple aliquots of semen samples were prepared to achieve fresh, snap frozen and air‐dried samples. Samples were sequentially assessed for sperm DNA fragmentation using the Halosperm® G2 kit (Halotech DNA SL, Spain) and scored against 300 sperm, with fragmentation results ≥30% considered positive. Fragmentation levels were compared between the different protocols. Multiple aliquots of semen samples were then air‐dried to test the fragmentation levels between different slide types, reconstituting fluids, times and temperatures. Pearson’s correlation coefficient and paired t‐tests were used for statistical analysis.

In summary, whilst significant associations exist between sperm DNA fragmentation and sexual abstinence, volume of the ejaculate, sperm concentration, normal sperm morphology and sperm motility, the Halosperm assay may provide an explanation for infertility where semen analysis cannot. Furthermore, air‐drying semen is a simple and stable storage method, for up to one month at ‐22 degrees, prior to DNA fragmentation testing with the Halosperm® G2 kit.

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