The overall aim was to evaluate a non-invasive technique for the assessment of oocytes and embryos using polarized light microscopy (PolScope-LC) with the goal of improving success rates in IVF. A literature review revealed little validation of the PolScope techniques in published work. A reproducible and accurate method for measuring the zona pellucida (ZP) thickness and density involving the PolScope computer software was validated by achieving low coefficient of variance and small inter/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 of extended culturing of embryos showed that the ZP starts thinning as early as day 3 and embryos tend to have 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 using the PolScope The spindle is a highly dynamic structure that can appear and disappear over time in culture. A visible spindle was detected in 58% of the oocytes immediately after aspiration. This percentage increased until it stabilised at 39-40hrs post hCG and then declined significantly. Average spindle signal intensity increased over time reaching its peak at 39-40hrs post hCG, then declined significantly by 40.5hrs post hCG. The importance of spindle presence and morphology was investigated by following up embryos created after sperm injection at 39-40hrs post hCG. There was a significant relationship between normal meiotic spindle shape and density and embryo quality. A higher percentage of ???usable??? embryos, and all of 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 trigger oocyte maturation and the site of microinjection during ICSI were assessed using the PolScope. The results 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 pole away from the spindle during ICSI. The results from this thesis suggest that PolScope, if appropriately applied, may assist in improving IVF outcome.

Thesis--Auburn University, 2009. / Abstract. Includes bibliographic records (p.49-55).

No description available.

The overall aim was to evaluate a non-invasive technique for the assessment of oocytes and embryos using polarized light microscopy (PolScope-LC) with the goal of improving success rates in IVF. A literature review revealed little validation of the PolScope techniques in published work. A reproducible and accurate method for measuring the zona pellucida (ZP) thickness and density involving the PolScope computer software was validated by achieving low coefficient of variance and small inter/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 of extended culturing of embryos showed that the ZP starts thinning as early as day 3 and embryos tend to have 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 using the PolScope The spindle is a highly dynamic structure that can appear and disappear over time in culture. A visible spindle was detected in 58% of the oocytes immediately after aspiration. This percentage increased until it stabilised at 39-40hrs post hCG and then declined significantly. Average spindle signal intensity increased over time reaching its peak at 39-40hrs post hCG, then declined significantly by 40.5hrs post hCG. The importance of spindle presence and morphology was investigated by following up embryos created after sperm injection at 39-40hrs post hCG. There was a significant relationship between normal meiotic spindle shape and density and embryo quality. A higher percentage of ???usable??? embryos, and all of 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 trigger oocyte maturation and the site of microinjection during ICSI were assessed using the PolScope. The results 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 pole away from the spindle during ICSI. The results from this thesis suggest that PolScope, if appropriately applied, may assist in improving IVF outcome.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1995. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

published_or_final_version / Medicine / Master / Doctor of Medicine

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Loss of germ cells that entered meiosis at different developmental stages was compared. Mice were injected with BrdU at 13.3, 14.3 or 15.3 days post coitum (dpc) and sacrificed either 3 days after BrdU injection or 4 days post partum (dpp). BrdU-labeled germ cells were detected in ovarian sections through double immunofluorescent staining for BrdU and either GCNA-1 or MVH as a germ cell marker. The results show that the loss of germ cells that entered meiosis at 13.3 or 15.3 dpc was excessive compared to the loss of total germ cells. Such preferential elimination was not found for germ cells that entered meiosis at 14.3 dpc. We conclude that oocyte loss during meiotic prophase is influenced by the timing of meiotic onset. / The mechanism of germ cell loss during ovarian development was tested by the presence of markers for apoptosis. Mouse ovaries were isolated at 12.5 dpc, 18.5 dpc and 2 dpp and cultured with doxorubicin (DXR) to induce cell death. Ovarian histological sections were double immunofluorescent stained for GCNA-1 and cleaved caspase-3 or PARP-1. The results suggest that caspase-3 is not activated in germ cells throughout ovarian development whereas PARP-1 is activated in germ cells at 12.5 dpc and 2 dpp but not at 18.5 dpc. Thus, no evidence has yet been provided to support the hypothesis that oocyte death during the meiotic prophase is mediated by apooptosis.