Mitochondrial DNA analysis of HVRI region in human blastocysts

Abstract

Introduction Mitochondrial DNA plays an important role in energy production, which affects chromosome segregation. Embryos with high levels of mitochondrial DNA (mtDNA) have difficulty implanting and increased aneuploidy, causing a decreased chance of pregnancy. Some haplogroups, which can be determined by sequencing the hypervariable region I (HVRI) of mtDNA, have a higher susceptibility to aneuploidy than others. The objective of this study was to determine if there are any genetic mutations within the HVRI region of mtDNA that causes a genetic predisposition to having elevated levels of mtDNA in euploid embryos. Materials and Methods The DNA used in this experiment was obtained from embryo biopsies and was whole genome amplified via SurePlex. We used polymerase chain reaction (PCR) to amplify the HVRI region of embryos from patients with two normal mtDNA euploid embryos and at least one elevated mtDNA euploid embryo. Elevated versus normal levels of mtDNA were previously determined via Mitograde testing. Three repeats of PCR were performed per embryo. Amplicons were purified by ExoSAP-IT and sequenced via Sanger sequencing. Results Gel electrophoresis of amplicons revealed the HVRI region was successfully amplified in 96.7% of embryos (264/273). Embryos from 1 out of 35 patients, labeled patient X, failed to amplify. 21 embryos from 7 patients were Sanger sequenced with an efficiency of 61.3% per amplified embryo. 11 embryos were successfully sequenced with over 675 out of 750 nucleotides readable, 8 were partially sequenced with over 350 out of 750 nucleotides readable, and 2 failed sequencing with less than 350 nucleotides readable. The two failed sequences belonged to embryos of patient X. Haplogroup analysis via HaploGrep 2.0 was successful in all 21 sequenced embryos. As expected, haplogroups were the same for all embryos from the same patient with an exception of patient X who had differing haplogroups for her elevated and normal mtDNA embryos. The failed amplification and sequencing of patient X indicate a possible mutation where the forward or reverse primer binds. Conclusions Sequencing revealed no nucleotide differences in embryos with elevated versus normal levels of mtDNA from the same patient, indicating there is no genetic predisposition caused by insertions/deletions or single nucleotide polymorphisms to having elevated levels of mtDNA encoded within the mtDNA HVRI region. 6 out of 7 patients sequenced belonged to haplogroup H, which was previously found to have lower incidences of aneuploidy. This suggests that embryos from patients within haplogroup H will have a higher percentage of euploidy but may still have fertility issues caused by elevated levels of mtDNA.