title: Oocyte mitochondrial deletions and heteroplasmy in a bovine model of ageing and ovarian stimulation.
abstract: OBJECTIVE
  Maternal ageing and ovarian stimulation result in the accumulation of mitochondrial DNA (mtDNA) deletions and heteroplasmy in individual oocytes from a novel bovine model for human assisted reproductive technology (ART).
  
  
  RESULTS
  The levels of mtDNA deletions detected in oocytes increased with ovarian ageing. Low levels of mtDNA heteroplasmy were apparent across oocytes and no relationship was identified with respect to ovarian ageing or ovarian stimulation.
  
  
  BACKGROUND
  Oocyte quality decreases with ovarian ageing and it is postulated that the mtDNA may have a role in this decline. The impact of ovarian stimulation on oocyte quality is poorly understood. Human studies investigating these effects are often limited by the use of low quality oocytes and embryos, variation in age and ovarian stimulation regimens within the patients studied, as well as genetic and environmental variability. Further, no study has investigated mtDNA heteroplasmy in individual oocytes using next-generation sequencing (NGS), and little is known about whether the oocyte accumulates heteroplasmic mtDNA mutations following ageing or ovarian stimulation.
  
  
  METHODS
  A novel bovine model for the effect of stimulation and age in human ART was undertaken using cows generated by somatic cell nuclear transfer (SCNT) from one founder, to produce a homogeneous population with reduced genetic and environmental variability. Oocytes and somatic tissues were collected from young (3 years of age; n = 4 females) and old (10 years of age; n = 5 females) cow clones following multiple natural ovarian cycles, as well as oocytes following multiple mild (FSH only) and standard (based on human a long GnRH agonist protocol) ovarian stimulation cycles. In addition, oocytes were recovered in a natural cycle from naturally conceived cows aged 4-13.5 years (n = 10) to provide a heterogeneous cohort for mtDNA deletion studies. The presence or absence of mtDNA deletions were investigated using long-range PCR in individual oocytes (n = 62). To determine the detection threshold for mtDNA heteroplasmy levels in individual oocytes, a novel NGS methodology was validated; artificial mixtures of the Bos taurus and Bos indicus mitochondrial genome were generated at 1, 2, 5, 15 and 50% ratios to experimentally mimic different levels of heteroplasmy. This NGS methodology was then employed to determine mtDNA heteroplasmy levels in single oocytes (n = 24). Oocyte mtDNA deletion and heteroplasmy data were analysed by binary logistic regression with respect to the effects of ovarian ageing and ovarian stimulation regimens.
  
  
  RESULTS
  Ovarian ageing, but not ovarian stimulation, increased the number of oocytes exhibiting mtDNA deletions (P = 0.04). A minimum mtDNA heteroplasmy level of 2% was validated as a sensitive (97-100%) threshold for variant detection in individual oocytes using NGS. Few mtDNA heteroplasmies were detected across the individual oocytes, with only 15 oocyte-specific variants confined to two of the 24 oocytes studied. There was no relationship (P > 0.05) evident between ovarian ageing or ovarian stimulation and the presence of mtDNA heteroplasmies.
  
  
  CONCLUSIONS
  The low number of oocytes collected from the natural ovarian cycles limited the analysis. Fertilization and developmental potential of the oocytes was not assessed as the oocytes were destroyed for mtDNA deletion and heteroplasmy analysis.
  
  
  CONCLUSIONS
  If the findings of this model apply to the human, this study suggests that the incidence of mtDNA deletions increases with age, but not with degree of ovarian stimulation, while the frequency of mtDNA heteroplasmies may be low regardless of ovarian ageing or level of ovarian stimulation.
  
  
  BACKGROUND
  Funding was provided by Fertility Associates, the Nurture Foundation for Reproductive Research, the Fertility Society of Australia, and the Auckland Medical Research Foundation. J.C.P. is a shareholder of Fertility Associates and M.P.G. received a fellowship from Fertility Associates. The other authors of this manuscript declare no conflict of interest that could be perceived as prejudicing the impartiality of the reported research.
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland 1023, New Zealand.
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Hammond
authors.affiliation: AgResearch Ltd, Ruakura Research Centre, Hamilton 3240, New Zealand The Liggins Institute, University of Auckland, Auckland 1023, New Zealand School of BioSciences, University of Melbourne, Parkville, Melbourne 3010, VIC, Australia.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Green
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland 1023, New Zealand.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Shelling
authors.affiliation: AgResearch Ltd, Ruakura Research Centre, Hamilton 3240, New Zealand.
authors.forename: Martin C
authors.initials: MC
authors.lastname: Berg
authors.affiliation: Fertility Associates, Greenlane, Auckland 1051, New Zealand.
authors.forename: John C
authors.initials: JC
authors.lastname: Peek
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland 1023, New Zealand Fertility Associates, Greenlane, Auckland 1051, New Zealand l.cree@auckland.ac.nz.
authors.forename: Lynsey M
authors.initials: LM
authors.lastname: Cree
mesh_terms: D000328:Adult; D000375:Aging / Q000235:genetics* / Q000473:pathology; D000818:Animals; D002417:Cattle; D004272:DNA, Mitochondrial / Q000235:genetics*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D006801:Humans; D016015:Logistic Models; D008597:Menstrual Cycle / Q000187:drug effects / Q000235:genetics*; D008875:Middle Aged; D008928:Mitochondria / Q000235:genetics* / Q000473:pathology; D008954:Models, Biological; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D010062:Ovulation Induction
chemical_list: D004272:DNA, Mitochondrial; D007987:Gonadotropin-Releasing Hormone; D005640:Follicle Stimulating Hormone
keywords: mitochondrial DNA deletions; mitochondrial DNA heteroplasmy; next-generation sequencing; oocyte ageing; oocyte quality; ovarian stimulation