title: Impact of vitrification on the mitochondrial activity and redox homeostasis of human oocyte.
abstract: Do the extreme conditions of vitrification affect mitochondrial health and reactive oxygen species (ROS) levels of human oocytes?
  
  
  
  Vitrification of discarded human oocytes shifts the intracellular redox potential towards oxidation but does not alter the mitochondrial potential or intracellular ROS levels.
  
  
  
  Recent studies have reflected increased ROS levels in warmed young oocytes and have highlighted the temporal dynamic loss of mitochondrial potential that could, therefore, lead to a decrease in ATP production, impairing embryo development. Mitochondrial function can also be evaluated in vivo by the FAD/NAD(P)H autofluorescence ratio, which reflects the respiratory chain activity and is considered as a marker of the intracellular redox state.
  
  
  
  A total of 629 discarded Metaphase II (MII) oocytes collected from June 2013 to April 2014 were included in this control (fresh oocytes, n= 270) versus treatment (vitrified oocytes, n= 359) study.
  
  
  
  Discarded MII oocytes were donated to research by young (<27 years old) and reproductively aged (>36 years old) women who underwent ovarian stimulation for IVF at a university-affiliated private fertility clinic. Redox state was assessed by measuring the FAD/NAD(P)H autofluorescence ratio, while ROS and mitochondrial activity were reported by in vivo labelling with carboxy-H2DCFDA and JC-1, respectively.
  
  
  
  Young and aged oocytes showed high and similar survival rates (81.8 versus 83.1%, not significant). Confocal microscopy revealed that the FAD/NAD(P)H ratio was significantly higher in vitrified oocytes than in fresh oocytes, suggesting a significant shift towards the oxidized state in oocytes after vitrification, regardless of the maternal age. Mitochondrial distribution was not affected by vitrification. Furthermore, it was not possible to resolve any difference in mitochondrial potential using JC-1 potentiometric dye or in reactive oxygen species (ROS) production (assessed with H2-DCFDA staining) between fresh and vitrified oocytes. Therefore, measurement of intracellular redox potential by autofluorescence imaging may be a more sensitive method to assess oxidative stress or mitochondrial demise in human oocytes because it showed a higher resolving power than JC-1 staining and displayed less variability than H2-DCFDA staining.
  
  
  
  Owing to sample availability, MII discarded oocytes (in vitro matured oocytes and unfertilized oocytes 20 h after ICSI) were included in the study. These discarded oocytes do not necessarily reflect the physiological condition of the MII human oocyte.
  
  
  
  Although vitrified oocytes yield comparable clinical outcomes compared with fresh oocytes, lower cleavage and blastocyst rates can be observed during in vitro culture. Data here obtained suggest that the redox state of human oocytes could be affected by vitrification. Therefore, the importance of adding protective antioxidant molecules to the vitrification solution and to the post-warming culture medium to improve embryo cleavage deserves some research.
  
  
  
  This research project was supported by the Valencian Government (Val+i+D program, M.N.-C.), INCLIVA Foundation for health research (G.S.-A.) and by the University of L'Aquila and Regione Abruzzo ('Reti per l'Alta Formazione' - P.O.F.S.E. Abruzzo 2007-2013 G.D.E.). No conflicts of interest were declared.
authors.affiliation: Instituto Universitario IVI Valencia, Valencia, Spain mar.nohales@ivi.es.
authors.forename: M
authors.initials: M
authors.lastname: Nohales-Córcoles
authors.affiliation: Instituto Universitario IVI Valencia, Valencia, Spain.
authors.forename: G
authors.initials: G
authors.lastname: Sevillano-Almerich
authors.affiliation: Department of Health, Life and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Di Emidio
authors.affiliation: Department of Health, Life and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Tatone
authors.affiliation: Instituto Universitario IVI Valencia, Valencia, Spain.
authors.forename: A C
authors.initials: AC
authors.lastname: Cobo
authors.affiliation: Centre National de la Recherche (CNRS), Laboratoire de Biologie du Développement de Villefranche-sur-mer, Observatoire Océanographique, Sorbonne Universités, UPMC Univ Paris 06, 06230 Villefranche-sur-mer, France.
authors.forename: R
authors.initials: R
authors.lastname: Dumollard
authors.affiliation: Instituto Universitario IVI Valencia, Valencia, Spain.
authors.forename: M J
authors.initials: MJ
authors.lastname: De Los Santos Molina
mesh_terms: D000328:Adult; D015925:Cryopreservation / Q000379:methods; D005260:Female; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism*; D010062:Ovulation Induction; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism*; D058989:Vitrification
chemical_list: D017382:Reactive Oxygen Species
keywords: maternal age; mitochondrial activity; oocyte vitrification; oxidative stress; redox metabolism