title: Ultrastructural Changes and Methylation of Human Oocytes Vitrified at the Germinal Vesicle Stage and Matured in vitro after Thawing.
abstract: OBJECTIVE
  The study aimed to assess the effect of in vitro vitrification and maturation on the nuclear configuration, cytoplasmic maturation and global DNA methylation pattern of human germinal vesicle (GV) stage oocytes.
  
  
  METHODS
  Human oocytes from infertile women were randomly assigned to one of 3 groups: (i) metaphase II (MII) oocytes matured in vivo (vivo-MII, n = 56) as controls; (ii) MII oocytes matured in vitro (vitro-MII, n = 106); and (iii) MII oocytes that were vitrified at the GV stage, warmed and matured in vitro (cryo-MII, n = 122). All MII oocytes were fixed and immunofluorescence staining for spindle, chromosome, mitochondrion and cortical granules (CGs) were performed; examination was done using immunofluorescence laser scanning confocal microscope. The expression of 5-methycytosine in these MII oocytes was also assessed.
  
  
  RESULTS
  No significant difference was observed between vitro-MII and cryo-MII groups with respect to oocyte maturation rate (72.4 vs. 78.3%). No significant differences were observed in the distribution of mitochondria, migration of CG and global DNA methylation pattern among the 3 study groups. However, the abnormal configuration of spindle and chromosome was significantly higher in cryo-MII group (78.9 and 84.2%) as compared to that in the vitro-MII (45.0 and 50.0%, p < 0.05) and vivo-MII groups (27.3 and 36.4%, p < 0.05).
  
  
  CONCLUSIONS
  GV oocytes appeared to resist vitrification and retain their potential for maturation to MII stage oocytes after thawing. However, GV oocytes vitrification combined with in vitro maturation could affect the organization of spindle and chromosome.
authors.affiliation: Medical Center for Human Reproduction, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, PR China.
authors.forename: Ming-Hui
authors.initials: MH
authors.lastname: Liu
authors.forename: Wen-Hui
authors.initials: WH
authors.lastname: Zhou
authors.forename: Da-Peng
authors.initials: DP
authors.lastname: Chu
authors.forename: Lei
authors.initials: L
authors.lastname: Fu
authors.forename: Wei
authors.initials: W
authors.lastname: Sha
authors.forename: Yuan
authors.initials: Y
authors.lastname: Li
mesh_terms: D002467:Cell Nucleus; D015925:Cryopreservation / Q000379:methods*; D019175:DNA Methylation; D005260:Female; D006358:Hot Temperature; D006801:Humans; D059471:In Vitro Oocyte Maturation Techniques; D007247:Infertility, Female; D008677:Metaphase; D009865:Oocytes / Q000737:chemistry* / Q000648:ultrastructure*
keywords: Human oocytes; In vitro maturation; Ultrastructure; Vitrification