Comprehensive mitochondrial DNA analysis and IVF outcome

Abstract

STUDY QUESTION Do mitochondrial DNA (mtDNA) copy number and heteroplasmy in human embryos affect the ongoing pregnancy rate? SUMMARY ANSWER Our study suggests that mtDNA copy number above a specific threshold is associated with the ongoing pregnancy rate. WHAT IS KNOWN ALREADY Mitochondria play a vital role in cell function. Recently, there has been increasing research on mtDNA as a biomarker of embryo implantation. Although reports showed that high levels of mtDNA in the blastocyst are associated with low implantation potential, other publications were unable to confirm this. Confounding factors may influence the mtDNA copy number in euploid embryos. On the other hand it has been speculated that both mtDNA heteroplasmy and copy number contribute to mitochondrial function. Next generation sequencing (NGS) allows us to study in depth mtDNA heteroplasmy and copy number simultaneously. STUDY DESIGN, SIZE, DURATION A prospective non-selection study was performed. We included 159 blastocyst biopsies from 142 couples who attended our clinic for preimplantation genetic testing for aneuploidies (PGT-A), from January 2017 to December 2017. All embryos were biopsied on Day 5 or Day 6. The aneuploid testing was performed by NGS. All blastocysts were diagnosed as euploid non-mosaic and were transferred. The mtDNA analysis was performed once the embryo diagnosis was known. PARTICIPANTS/MATERIALS, SETTING, METHODS Sequencing reads mapping to the mtDNA genome were extracted from indexed bam files to identify copy number and heteroplasmy. The relative measure of mtDNA copy number was calculated by dividing the mtDNA reads by the nuclear DNA value to normalize for technical variants and the number of cells collected at the biopsy. All the results were subjected to a mathematical correction factor according to the embryo genome. Heteroplasmy was assigned by MitoSeek. MAIN RESULTS AND THE ROLE OF CHANCE The mean average copy number and SD of mtDNA per genome was 0.0016 ± 0.0012. Regarding heteroplasmy, 40 embryos were heteroplasmy carriers (26.32%). MtDNA variants were detected in coding and non-coding regions and the highest number of variants in an embryo was eight. With respect to IVF outcome for mtDNA copy number analysis, we set a threshold of 0.003 for the following analysis. The vast majority of the embryos were below the threshold (142/159, 89.31%) and 17 embryos were classified as having higher mtDNA levels. We showed a reduction in ongoing pregnancy rate associated with elevated mtDNA copy number (42.96% versus 17.65%, P < 0.05). This result was independent of maternal age and day of the biopsy: these factors were included as confounding factors because mtDNA copy number was negatively correlated with female age (25 –30 y: 0.0017 ± 0.0011, 30 –35 y: 0.0012 ± 0.0007, 35 –40 y: 0.0016 ± 0.0009, over 40 y: 0.0024 + 0.001...

Impact of Oxidative Stress on Age-Associated Decline in Oocyte Developmental Competence

“…Point mutations and deletions in the mtDNA in oocytes are also found with maternal aging (23–25). Moreover, embryos obtained from elderly women have an abnormally high copy number of mtDNA and such embryos do not implant (26–28). It can be presumed that a vicarious increase in mtDNA copy number via mitochondrial biogenesis may effectively compensate for heteroplasmic mtDNA mutations and mitochondrial dysfunction (15).…”

Preimplantation Genetic Testing for Chromosomal Abnormalities: Aneuploidy, Mosaicism, and Structural Rearrangements

“…Since then, the field of mtDNA quantitation in embryology has been turbulent and equivocal. Some studies have supported the initial reports [176][177][178], but many others have refuted them [179][180][181][182][183], showing no predictive power of mtDNA quantitation in TE biopsies regarding clinical outcome. An interesting observation to come out of this debate is that some clinics produce no embryos with substantially elevated mtDNA levels (about half of the 37 participating clinics in one study), whereas some clinics generate upwards of 20% of blastocysts with very high mtDNA copy number [176].…”

Qualitative and Quantitative Ovarian and Peripheral Blood Mitochondrial DNA (mtDNA) Alterations: Mechanisms and Implications for Female Fertility

“…Different authors have examined the association between human blastocyst mtDNA content, female age, embryo chromosome status, viability, and implantation potential [ 9 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 ]. For the sake of clarity, we reported their results in Table 1 .…”