O-063 Accurate mitochondrial DNA quantification clarifies the clinical value of measuring mtDNA in trophectoderm biopsy specimens

Abstract

Study question Can accurate mitochondrial DNA (mtDNA) quantification of trophectoderm (TE) biopsy specimens provide insights into the biology and viability of blastocyst-stage human embryos? Summary answer mtDNA quantity in TE cells is correlated with embryo morphology and shows alterations associated with aneuploidy. However, measurement does not significantly improve embryo viability assessment. What is known already Mitochondria are essential organelles, responsible for producing ATP. Changes in the amount of mtDNA in blastocysts biopsy specimens have been reported in association with embryo implantation potential, leading to proposals that mtDNA quantification might serve as a useful biomarker of embryo viability. However, results from clinical studies to explore this possibility have yielded contradictory data, due in part to deficiencies of the molecular methods used for mtDNA measurement. We sought to clarify what quantification of mtDNA can tell us about embryo biology and viability by developing and applying a method that we believe to be the most accurate ever devised. Study design, size, duration This study involved the analysis of samples collected during the course of routine preimplantation genetic testing for aneuploidy (PGT-A). The IVF treatments and embryo biopsies were undertaken at two different clinics, while chromosomal analyses were carried out at a single reference laboratory. Mitochondrial data was subsequently analysed in a university setting. The embryos analysed were derived from a broad population of patients referred for PGT-A (average age 38.7 years; range 25-47). Participants/materials, setting, methods 651 blastocysts from 133 couples underwent trophectoderm biopsy on day-5 or day-6. The specimens were analysed using a highly validated real-time PCR method, which was used to quantify three distinct sites in the mtDNA and 198 loci in the nuclear genome. The measurement of multiple independent loci provided outstanding sensitivity and accuracy. The nuclear loci were used to normalise the mtDNA data, adjusting for differences in the number of cells in the biopsy specimens. Main results and the role of chance The method developed displayed extraordinary sensitivity and accuracy when quantifying mtDNA. Lower mtDNA quantities were associated with day-6 biopsy (p < 0.0001), extent of blastocyst expansion (p < 0.0001), and superior TE morphology, although the latter was not statistically significant (p = 0.09). mtDNA levels were higher in aneuploid embryos (p < 0.0001), independent on patient age. However, the difference was not sufficient to be considered diagnostic. There was no correlation between mtDNA level and the chances of blastocyst implantation in this dataset. Lower mtDNA levels, previously reported to be associated with higher probabilities of embryo implantation, were most often observed in embryos of excellent morphological grade and likely reflect the increased TE cell numbers of such embryos. Little if any mtDNA replication occurs during preimplantation development and consequently the mtDNA content is divided amongst an ever-growing number of cells, meaning less mtDNA per cell. In this context, mtDNA quantification of blastocyst biopsy specimens provides a highly sensitive measure of TE cellularity, but probably provides little additional benefit for embryo selection beyond conventional morphological grading. However, the fact that higher mtDNA quantities were observed in aneuploid embryos, may indicate that subtle differences in TE cellularity exist in abnormal embryos, which are not fully captured by traditional morphological assessment. Limitations, reasons for caution Previous studies suggested that some blastocysts have greatly elevated mtDNA levels and that such embryos are not viable. In this study, only 5% of embryos were considered outliers in terms of mtDNA quantity. Unfortunately, none of these embryos were transferred, so the potential of these embryos could not be assessed. Wider implications of the findings The quantification of mtDNA in trophectoderm biopsies has sometimes been used for the prioritisation of embryos for transfer. While our results confirm existence of biologically interesting associations between mtDNA and aneuploidy, and a relationship with certain aspects of embryo morphology, measurement of mtDNA seems unlikely to significantly improve embryo selection. Trial registration number not applicable