Determination of non-invasive viability markers for human embryos in in vitro fertilization.

Abstract

The major challenge currently facing human in vitro fertilization (IVF) programs is the prevention of multiple gestation pregnancies. The replacement of multiple embryos to fertility patients is common practice to increase pregnancy rates. However this introduces a significant risk of a resultant multiple pregnancy (25-50%), with associated increased complications such as perinatal morbidity, mortality and long-term disabilities, as well as significant maternal complications. The transfer of a single embryo during IVF treatment is the only acceptable way to overcome the issues related to multiple gestation pregnancies. To encourage the replacement of only one embryo, pregnancy rates must be able to be maintained. There is therefore a great need for clinical laboratories to use markers that can identify highly viable embryos. Current laboratory embryo selection techniques involve selecting an embryo based on its appearance. However it is well established that this is only weakly linked to pregnancy success. Therefore other less subjective quantitative markers are required to select embryos that are the most viable within a patient's cohort. Any quantitative techniques which provide additional information to combine with current morphological assessment protocols must be rapid, simple, non-invasive and highly discriminating. This thesis describes the investigations of candidate non-invasive viability markers to assess the competence of the oocyte/embryo. In order to track investigations of individual oocytes and embryos, single embryo culture is a prerequisite. Mouse and human embryo development and implantation were studied as a prelude to investigations of biomarkers of human embryo viability. No differences were observed in human embryo development or viability after single or group culture. Mouse embryos cultured singly were found to have a reduced cell number, however this did not affect implantation or fetal viability. Interestingly, mouse placental weights were reduced. This and a lack of power in the human studies suggest that single embryo culture in the human needs further long-term examination. Following preliminary investigations of single embryo culture, morphological, biochemical and metabolic studies were undertaken as potential biomarkers of oocyte/embryo viability in the human. Firstly, a new morphological scoring system was described for day 4 human embryos, a stage of embryo development that has previously been overlooked. This morphological scoring system was easy to implement in the clinic and able to improve implantation rates over morphological scoring of cleavage stage embryos. It has subsequently been adopted in several clinics. Secondly, the reduction-oxidation (REDOX) state of cumulus cells surrounding oocytes retrieved for IVF was measured and retrospectively found to correlate with subsequent embryo viability. Finally, a direct measure of metabolism was also investigated in cumulus-oocyte complexes as well as cleavage stage embryos with determination of metabolic turnover of glucose, lactate, pyruvate and alanine, with pyruvate uptake by the early embryo and pyruvate: alanine turnover of the cumulus-oocyte complex being related to viability. Overall, these experiments showed that some metabolic parameters were correlated with subsequent viability. Biochemical markers in combination with current morphological measures show promise in selecting the most viable embryo for replacement. Further validations of these markers in randomized controlled trials are the next step in the introduction of these technologies to improve success rates of single embryo transfers in fertility treatment.