The development of a cell-based assay for quality control in human in vitro fertilisation

Quality control systems are critical to improving laboratory practices and contribute significantly to success in human in vitro fertilisation (IVF). The mouse embryo assay (MEA) is commonly used in production quality control, as well as in product research and development. Despite its popularity there are some well-known issues associated with it; these include, but are not limited to, insensitivity to sub-optimal conditions, ethical issues due to animal use and variation in end-point interpretation between operators. The aim of this thesis was to therefore develop a cell-based assay that could facilitate research and development whilst addressing the issues presented above.

Firstly, using the pluripotent P19 embryonal carcinoma cell line, 2D and 3D cell-based assays were developed and optimised, and their response to culture perturbations such as varying osmolality and energy source levels were tested. Secondly, given that mammalian cells are generally resistant to changes in culture condition, characteristic features of the mammalian cold stress response, mTOR inhibitor rapamycin and AMPK inhibitor dorsomorphin were incorporated as potential tools for decreasing cellular resistance to altering culture conditions. The P19 assay demonstrated sensitivity to changes in energy substrate availability when treated with rapamycin and dorsomorphin, and was subsequently used to systematically screen different media formulations in order to identify the ranges of pyruvate, glutamine and calcium lactate concentrations required for optimum cell growth. Lastly, the developed 2D assay and the MEA were treated with the same products and their responses were directly compared to highlight similarities, and to identify differences, between the two assays. These assays/comparisons showed that the P19 assay identified differences in human IVF culture media that were not identified by the MEA.

The P19 assay may therefore represent a sensitive alternative to the MEA that can detect discrete changes in culture conditions and indicate the extent to which different energy substrates affect cell growth and proliferation. It is important to note that the two assays differ in their robustness and the P19 assay responded differently to repeat experiments with the same treatment. Overall, this novel P19 assay may provide an effective means of validating and testing products used in IVF whilst reducing animal use in research and development