Correction factor reveals uniform levels of mitochondrial DNA in human blastocysts irrespective of ploidy, age, or implantation potential

Abstract

To develop a mathematical formula resulting in an accurate determination of mitochondrial DNA (mtDNA) levels in human blastocysts substratified by ploidy, age, and implantation potential. Retrospective analysis of mtDNA content in human blastocysts used in preimplantation genetic diagnosis for IVF selection. 833 embryos derived from 181 patients were tested for mtDNA content by next generation sequencing (NGS), and 150 embryos derived from 96 patients were tested by quantitative polymerase chain reaction (qPCR). For each embryo, the level of mtDNA was determined from a trophectoderm biopsy by whole genome amplification followed by NGS and/or qPCR. The value was subjected to mathematical analysis tailored to the genomic DNA composition of said embryo. Grouped values were compared by Welch’s two-tailed upaired t-test. On average our quantitation method changed the conventionally determined mtDNA level of a given embryo via NGS by 1.35% +/-1.58%, with changes ranging up to 17.42%, and via qPCR by 1.33% +/-8.08%, with changes ranging up to 50.00%. Levels of mtDNA in euploid and aneuploid embryos showed a statistically insignificant difference of P=0.102 by NGS (euploid N=494, aneuploid N=339) and P=0.642 by qPCR (euploid N=100, aneuploid N=50). Blastocysts derived from younger or older patients had comparable mtDNA values, with P=0.293 by NGS (20-37 age group N=559, 38-46 age group N=274) and P=0.101 by qPCR (20-37 age group N=92, 38-46 age group N=58). Blastocysts that upon transfer resulted in implantation did not contain significantly different mtDNA levels compared to blastocysts that failed to implant, with P=0.813 by NGS (implanted N=51, non-implanted N=69) and P=0.103 by qPCR (implanted N=49, non-implanted N=51). We recommend the implementation of our correction factor to all laboratories evaluating mtDNA levels in their embryos by NGS or qPCR. Applied to our in-house data, our quantitation method reveals that overall levels of mtDNA are largely equal between human blastocysts regardless of embryo ploidy, age, or implantation potential.