title: Accurate quantitation of mitochondrial DNA reveals uniform levels in human blastocysts irrespective of ploidy, age, or implantation potential.
abstract: To accurately determine mitochondrial DNA (mtDNA) levels in human blastocysts.
  
  
  
  Retrospective analysis.
  
  
  
  IVF clinic.
  
  
  
  A total of 1,396 embryos derived from 259 patients.
  
  
  
  Blastocyst-derived trophectoderm biopsies were tested by next-generation sequencing (NGS) and quantitative real-time polymerase chain reaction (qPCR).
  
  
  
  For each sample the mtDNA value was divided by the nuclear DNA value, and the result was further subjected to mathematical analysis tailored to the genetic makeup of the source embryo.
  
  
  
  On average the mathematical correction factor changed the conventionally determined mtDNA score of a given blastocyst via NGS by 1.43% ± 1.59% (n = 1,396), with maximal adjustments of 17.42%, and via qPCR by 1.33% ± 8.08% (n = 150), with maximal adjustments of 50.00%. Levels of mtDNA in euploid and aneuploid embryos showed a statistically insignificant difference by NGS (euploids n = 775, aneuploids n = 621) and by qPCR (euploids n = 100, aneuploids n = 50). Blastocysts derived from younger or older patients had comparable mtDNA levels by NGS ("young" age group n = 874, "advanced" age group n = 514) and by qPCR ("young" age group n = 92, "advanced" age group n = 58). Viable blastocysts did not contain significantly different mtDNA levels compared with unviable blastocysts when analyzed by NGS (implanted n = 101, nonimplanted n = 140) and by qPCR (implanted n = 49, nonimplanted n = 51).
  
  
  
  We recommend implementation of the correction factor calculation to laboratories evaluating mtDNA levels in embryos by NGS or qPCR. When applied to our in-house data, the calculation reveals that overall levels of mtDNA are largely equal between blastocysts stratified by ploidy, age, or implantation potential.
authors: Zouves Fertility Center, Foster City, California. Electronic address: andrea@goivf.com.
authors: Andrea R
authors: AR
authors: Victor
authors: Zouves Fertility Center, Foster City, California.
authors: Alan J
authors: AJ
authors: Brake
authors: Zouves Fertility Center, Foster City, California.
authors: Jack C
authors: JC
authors: Tyndall
authors: School of Biosciences, University of Kent, Canterbury, United Kingdom.
authors: Darren K
authors: DK
authors: Griffin
authors: Zouves Fertility Center, Foster City, California.
authors: Christo G
authors: CG
authors: Zouves
authors: Zouves Fertility Center, Foster City, California.
authors: Frank L
authors: FL
authors: Barnes
authors: Zouves Fertility Center, Foster City, California.
authors: Manuel
authors: M
authors: Viotti
mesh_terms: D001706:Biopsy; D001755:Blastocyst / Q000378:metabolism* / Q000473:pathology; D004272:DNA, Mitochondrial / Q000235:genetics*; D046149:Embryo Culture Techniques; D010064:Embryo Implantation; D004624:Embryo Transfer; D005260:Female; D005307:Fertilization in Vitro / Q000009:adverse effects; D005819:Genetic Markers; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D008423:Maternal Age; D054315:Oocyte Retrieval; D011003:Ploidies; D011237:Predictive Value of Tests; D011247:Pregnancy; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012189:Retrospective Studies; D012307:Risk Factors; D014022:Tissue Survival; D016896:Treatment Outcome
chemical_list: D004272:DNA, Mitochondrial; D005819:Genetic Markers
keywords: Blastocyst mtDNA; IVF biomarker; PGS/PGD; correction factor; embryonic mitochondria