title: Double-strand DNA breaks and repair response in human immature oocytes and their relevance to meiotic resumption.
abstract: OBJECTIVE
  Only 50-60 % of immature human oocytes attain the mature stage in vitro. Such a deficiency may be a reflection of inadequate conditions of in vitro maturation (IVM) or a manifestation of intrinsic oocyte defects. In the present study, we explored the possibility that the DNA of immature oocytes may be damaged and that such a condition, or inability to trigger a repair action, is associated to germinal vesicle (GV) arrest.
  
  
  METHODS
  Immature oocytes (GV-stage oocytes) were obtained from women undergoing stimulated (Stim-C) or IVM (IVM-C) cycles. GV oocytes obtained from stimulated cycles were fixed for successive analysis either after recovery (T0) or following 30 h (T30) of culture if still arrested at the GV stage. Oocytes retrieved in IVM cycles were used only if they were found arrested at the GV stage after 30 h (T30) of culture. All oocytes were fixed and stained to detect chromatin and actin. They were also assessed for positivity to γH2AX and Rad51, markers revealing the presence of double-strand DNA breaks and the activation of a DNA repair response, respectively. Labelled oocytes were analysed using a Leica TCS SP2 laser scanning confocal microscope.
  
  
  RESULTS
  In Stim-C oocytes, γH2AX positivity was 47.5 and 81.5 % in the T0 and T30 groups, respectively (P = 0.003), while γH2AX-positive oocytes were 58.3 % in the IVM-C T30 group (Stim-C T0 vs. IVM-C T30, P = 0.178; Stim-C T30 vs. IVM-C T30, P = 0.035). Positivity for nuclear staining to Rad51 occurred in 42.1 and 74.1 % of Stim-C in the T0 and T30 subgroups, respectively (T = 0.006), while 66.7 % of IVM-C T30 oocytes resulted positive for a DNA repair response (Stim-C T0 vs. IVM-C T30, P = 0.010; Stim-C T30 vs. IVM-C T30, P = 0.345).
  
  
  CONCLUSIONS
  The present data document the existence of double-strand DNA breaks (DSBs) in human immature oocytes. Also, they are consistent with the hypothesis that insults to DNA integrity may be an important factor affecting meiotic resumption.
authors.affiliation: Biogenesi Reproductive Medicine Centre, Istituti Clinici Zucchi, Via Zucchi, 24, Monza, Italy. coticchio.biogenesi@grupposandonato.it.
authors.forename: Giovanni
authors.identifier: http://orcid.org/0000-0003-1635-9205
authors.initials: G
authors.lastname: Coticchio
authors.affiliation: Biogenesi Reproductive Medicine Centre, Istituti Clinici Zucchi, Via Zucchi, 24, Monza, Italy.
authors.forename: Mariabeatrice
authors.initials: M
authors.lastname: Dal Canto
authors.affiliation: Biogenesi Reproductive Medicine Centre, Istituti Clinici Zucchi, Via Zucchi, 24, Monza, Italy.
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Guglielmo
authors.affiliation: Center for Human Reproduction, New York, NY, USA.|Department of Molecular and Integrative Physiology, University of Kansas, Kansas City, USA.
authors.forename: David F
authors.initials: DF
authors.lastname: Albertini
authors.affiliation: Biogenesi Reproductive Medicine Centre, Istituti Clinici Zucchi, Via Zucchi, 24, Monza, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Mignini Renzini
authors.affiliation: Biogenesi Reproductive Medicine Centre, Istituti Clinici Zucchi, Via Zucchi, 24, Monza, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Merola
authors.affiliation: Biogenesi Reproductive Medicine Centre, Istituti Clinici Zucchi, Via Zucchi, 24, Monza, Italy.
authors.forename: Monia
authors.initials: M
authors.lastname: Lain
authors.affiliation: Biogenesi Reproductive Medicine Centre, Istituti Clinici Zucchi, Via Zucchi, 24, Monza, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Sottocornola
authors.affiliation: Department of Medical Physics, San Gerardo Hospital, Monza, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: De Ponti
authors.affiliation: Biogenesi Reproductive Medicine Centre, Istituti Clinici Zucchi, Via Zucchi, 24, Monza, Italy.
authors.forename: Rubens
authors.initials: R
authors.lastname: Fadini
mesh_terms: D000328:Adult; D002478:Cells, Cultured; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D005260:Female; D006657:Histones / Q000378:metabolism; D006801:Humans; D059471:In Vitro Oocyte Maturation Techniques / Q000379:methods; D008423:Maternal Age; D008540:Meiosis; D009865:Oocytes / Q000502:physiology*; D051135:Rad51 Recombinase / Q000378:metabolism
chemical_list: C468783:H2AX protein, human; D006657:Histones; D051135:Rad51 Recombinase
keywords: DNA damage; DNA repair; Germinal vesicle; In vitro maturation; Oocytes