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Journal of Assisted Reproduction and Genetics

, Volume 32, Issue 10, pp 1509–1516 | Cite as

Double-strand DNA breaks and repair response in human immature oocytes and their relevance to meiotic resumption

  • Giovanni Coticchio
  • Mariabeatrice Dal Canto
  • Maria Cristina Guglielmo
  • David F. Albertini
  • Mario Mignini Renzini
  • Maria Merola
  • Monia Lain
  • Manuela Sottocornola
  • Elena De Ponti
  • Rubens Fadini
Gamete Biology

Abstract

Purpose

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.

Keywords

Oocytes DNA damage DNA repair In vitro maturation Germinal vesicle 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Giovanni Coticchio
    • 1
  • Mariabeatrice Dal Canto
    • 1
  • Maria Cristina Guglielmo
    • 1
  • David F. Albertini
    • 2
    • 3
  • Mario Mignini Renzini
    • 1
  • Maria Merola
    • 1
  • Monia Lain
    • 1
  • Manuela Sottocornola
    • 1
  • Elena De Ponti
    • 4
  • Rubens Fadini
    • 1
  1. 1.Biogenesi Reproductive Medicine CentreIstituti Clinici ZucchiMonzaItaly
  2. 2.Center for Human ReproductionNew YorkUSA
  3. 3.Department of Molecular and Integrative PhysiologyUniversity of KansasKansas CityUSA
  4. 4.Department of Medical PhysicsSan Gerardo HospitalMonzaItaly

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