The use of polarised light microscopy as a non-invasive tool for early assessment of human oocytes and embryos

The overall aim was to evaluate a non-invasive technique for the assessment of oocytes andembryos using polarized light microscopy (PolScope-LC) with the goal of improving success rates inIVF.A literature review revealed little validation of the PolScope techniques in published work. Areproducible and accurate method for measuring the zona pellucida (ZP) thickness and density involvingthe PolScope computer software was validated by achieving low coefficient of variance and smallinter/intra observer errors.Utilizing this method, 1477 oocytes from 211 stimulated cycles were analysed in this thesis.Results showed that increasing age has an adverse effect on the ZP thickness and density. Study ofextended culturing of embryos showed that the ZP starts thinning as early as day 3 and embryos tend tohave denser zonas over time.Standardisation of timing of PolScope observations in relation to the meiotic spindle was studied.Metaphase II oocytes were examined sequentially in culture from aspiration until microinjection usingthe PolScope The spindle is a highly dynamic structure that can appear and disappear over time inculture. A visible spindle was detected in 58% of the oocytes immediately after aspiration. Thispercentage increased until it stabilised at 39-40hrs post hCG and then declined significantly. Averagespindle signal intensity increased over time reaching its peak at 39-40hrs post hCG, then declinedsignificantly by 40.5hrs post hCG. The importance of spindle presence and morphology was investigated by following up embryoscreated after sperm injection at 39-40hrs post hCG. There was a significant relationship between normalmeiotic spindle shape and density and embryo quality. A higher percentage of “usable” embryos, and allof pregnancies, arose from oocytes with a normal barrel shaped spindle.Finally, the impact of two issues related to spindle formation - the type of hCG used to triggeroocyte maturation and the site of microinjection during ICSI were assessed using the PolScope. Theresults showed a biological difference in spindle formation and embryo quality between rhCG and uhCG.In a separate randomised trial embryo quality was better when injecting the sperm in the vegetal poleaway from the spindle during ICSI.The results from this thesis suggest that PolScope, if appropriately applied, may assist inimproving IVF outcome