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Beck, Andrea (2014): Analysis of early bovine embryogenesis after in vitro and in vivo oocyte maturation by time-lapse imaging and 3-D confocal microscopy. Dissertation, LMU München: Tierärztliche Fakultät



In the in vitro production of embryos in humans and animals it is aimed to produce embryos of good quality in order to reach a high pregnancy rate after the transfer on a recipient. Nevertheless, data until 2007 show that in Europe the pregnancy rate after the transfer of human IVF embryos was only 33% (de Mouzon et al., 2012). Recently time-lapse imaging of early embryonic cleavage was found to be a helpful and non-invasive tool to predict the developmental capacity of embryos and select embryos of good quality (Wong et al., 2010, Sugimura et al., 2012). As the morphokinetic parameters of the first and second cleavage were the most predictive values, the fate of an embryo seems to be, at least partly, already determined very early in embryogenesis. This determination of the developmental competence might even go back further namely until oocyte maturation. In this experiment we used bovine embryos as an animal model to study the reasons for success and failure of mitotic cleavage during early embryo development. First a live monitoring system for the observation of bovine embryos was applied in order to search for the values that are highly predictable for the developmental competence to the blastocyst stage. To consider the effect of the oocyte maturation onto the further development, we observed embryos from oocytes after in vitro (n=398) versus in vivo maturation (n=143). In average embryos that developed to the blastocyst stage showed an earlier timing of the first, second and third cleavage than embryos that arrested (p<0.001). Nevertheless if the first cleavage occurred too early, the blastocyst rate also decreased. The reason for this observation is unclear and it was not described specifically for bovine embryos yet. Due to a high frequency of image acquisition, we could include the synchronicity of the second cleavage into our analyses. As already shown for human embryos we confirmed a decreasing blastocyst rate with increasing asynchronicity of the second cleavage in bovine embryos. Furthermore we observed three types of abnormal morphology during the first mitotic cleavage: 1) cleavage into two unequal sized blastomeres, 2) direct cleavage into VI. Summary 90 3- or for cells, 3) cleavage with fragments in the perivitelline space. Abnormal morphology of the first cleavage had a negative effect on the blastocyst rate as it was decreased about 25% compared to the blastocyst rate after normal first cleavage (p<0.001). In vitro versus in vivo maturation had no effect on the speed or morphology of the first and second cleavage. Nevertheless, from the third cleavage on until the development to the blastocyst stage we found embryos after in vivo maturation to be faster in development compared to the in vitro matured ones (p<0.01). Also the hatching rate was higher in blastocysts after in vivo maturation compared to in vitro maturation (p=0.001). Due to a logistic regression model the variables with the highest prediction rate were identified and based on two selected regression models the fate of an embryo regarding the blastocyst stage could be correctly classified in up to 76%. To get more insights into the normal and abnormal process of the first mitotic cleavage division we fixated embryos at defined time points and analyzed them with a confocal laser scanning microscope. 3-D images of embryos showed that fertilization with more than one sperm was a main cause for abnormal cleavage. Polyspermy could result in different cleavage patterns and could partly be associated with events observed under live observation. Accordingly too early timing of the first cleavage and abnormal morphology of the first cleavage are probably mainly caused by polyspermic fertilization. Accordingly time-lapse imaging was successfully applied to find values be able to predict the developmental competence of an embryo. In vivo maturation of oocytes had a positive effect on embryo development although this was only present from the third cleavage on. After 3-D confocal microscopic analysis, polyspermic fertilization seems to be a cause for abnormalities in timing and morphology of the first cleavage.