Date of Award

2018

Document Type

Thesis

Publisher

Edith Cowan University

Degree Name

Master of Science (Medical Science)

School

School of Medical and Health Sciences

First Supervisor

Associate Professor Peter Roberts

Second Supervisor

Professor Phillip Matson

Abstract

The cryopreservation of human semen is a vital asset in assisted reproductive technologies (ART). Although advances have been in the freezing of sperm, further refinement is both necessary and ongoing. Computer-assisted semen analysis (CASA) has been increasingly utilised in both research and diagnostic however there are a range of variables that must first be controlled in order to produce reliable measurements. Following thawing, sperm must be isolated from both the original seminal plasma and the cryoprotectants; the two most used isolation methods include density gradient centrifugation (DGC) and the swim-up method.

The present thesis sought to investigate the following areas (i) a technical validation phase investigating variables that can influence CASA measurements (ii) the effect of neat glycerol and a commercial cryoprotective medium (CPM) upon sperm motility prior to cryopreservation, and subsequent effects of diluting these samples (iii) the effectiveness of neat glycerol versus a CPM in the post-thaw recovery of motile sperm, and (iv) the effectiveness of DGC, a direct swim-up procedure and a commercial device that utilises the swim-up procedure.

Several variables were identified in the measuring of semen samples in conjunction with CASA software. Firstly the use of a capillary-loading chamber was found to result in decreased levels of total and progressive motility, as well as reduced kinematic parameters when compared to a droplet-loaded configuration. The time between the loading of a sample was found to be stable at the 2 minute time interval, and as such this was set for all measurements in the study. Finally, operator-corrections were discovered to be crucial in not only accurately measuring sperm concentration, but also sperm motility.

The commercial CPM containing glycerol had the least toxic effect on sperm motility pre-cryopreservation. There was a linear relationship between decreased sperm motility and increased presence of glycerol, as demonstrated by 10% v/v glycerol addition. The further dilution of glycerol-containing semen samples with two common gamete handling media were found to cause a further significant reduction in sperm motility, whereas in contrast seminal plasma was not found to reduce sperm motility in these samples. The CPM was found to have the greatest yield of cryopreserved motile sperm post-thaw when compared to glycerol at both 5 and 10% v/v.

Finally, DGC yielded increased concentrations of sperm post-isolation, but with a reduced level of motility (10.2M/ml and 20% progressive motility respectively), whereas both the swim-up methods had reduced levels of concentration (1.8M/ml for the standard swim-up, and 1.5M/ml for the commercial device) but with increased levels of progressive motility (39.1% and 42.8% respectively for the standard swim-up and commercial device).

In summary, CASA software is able to provide reliable results given the chamber type is controlled and that operator-corrections are applied. Secondly, glycerol has a complex relationship with the cryosurvivability of spermatozoa and the toxic effects it exerts on them. Glycerol toxicity appeared dose-dependent, with decreased sperm motility with increased glycerol presence, both pre and post-cryopreservation/thawing. Finally, the most effective isolation technique for frozen-thawed sperm is dependent on what ART procedure is to be undertaken.

Access Note

Access to Chapter 8: The appendices, of this thesis is not available.

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