title: Human embryos commonly form abnormal nuclei during development: a mechanism of DNA damage, embryonic aneuploidy, and developmental arrest.
abstract: OBJECTIVE
  What is the prevalence and developmental significance of morphologic nuclear abnormalities in human preimplantation embryos?
  
  
  CONCLUSIONS
  Nuclear abnormalities are commonly found in human IVF embryos and are associated with DNA damage, aneuploidy, and decreased developmental potential.
  
  
  BACKGROUND
  Early human embryonic development is complicated by genomic errors that occur after fertilization. The appearance of extra-nuclear DNA, which has been observed in IVF, may be a result of such errors. However, the mechanism by which abnormal nuclei form and the impact on DNA integrity and embryonic development is not understood.
  
  
  METHODS
  Cryopreserved human cleavage-stage embryos (n = 150) and cryopreserved blastocysts (n = 105) from clinical IVF cycles performed between 1997 and 2008 were donated for research. Fresh embryos (n = 60) of poor quality that were slated for discard were also used. Immunohistochemical, microscopic and cytogenetic analyses at different developmental stages and morphologic grades were performed.
  
  
  METHODS
  Embryos were fixed and stained for DNA, centromeres, mitotic activity and DNA damage and imaged using confocal microscopy. Rates of abnormal nuclear formation were compared between morphologically normal cleavage-stage embryos, morphologically normal blastocysts, and poor quality embryos. To control for clinical and IVF history of oocytes donors, and quality of frozen embryos within our sample, cleavage-stage embryos (n = 52) were thawed and fixed at different stages of development and then analyzed microscopically. Cleavage-stage embryos (n = 9) were thawed and all blastomeres (n = 62) were disaggregated, imaged and analyzed for karyotype. Correlations were made between microscopic and cytogenetic findings of individual blastomeres and whole embryos.
  
  
  RESULTS
  The frequency of microscopic nuclear abnormalities was lower in blastocysts (5%; 177/3737 cells) than in cleavage-stage embryos (16%, 103/640 blastomeres, P < 0.05) and highest in arrested embryos (65%; 44/68 blastomeres, P < 0.05). DNA damage was significantly higher in cells with microscopic nuclear abnormalities (γH2AX (phosphorylated (Ser139) histone H2A.X): 87.1%, 74/85; replication protein A: 72.9%, 62/85) relative to cells with normal nuclear morphology (γH2AX: 9.3%, 60/642; RPA: 5.6%, 36/642) (P < 0.05). Blastomeres containing nuclear abnormalities were strongly associated with aneuploidy (Fisher exact test, two-tailed, P < 0.01).
  
  
  CONCLUSIONS
  The embryos used were de-identified, and the clinical and IVF history was unknown.
  
  
  CONCLUSIONS
  This study explores a mechanism of abnormal embryonic development post-fertilization. While most of the current data have explored abnormal meiotic chromosome segregation in oocytes as a primary mechanism of reproductive failure, abnormal nuclear formation during early mitotic cell division in IVF embryos also plays a significant role. The detection of abnormal nuclear formation may have clinical application in noninvasive embryo selection during IVF.
  
  
  BACKGROUND
  The study was supported by Columbia University and the New York Stem Cell Foundation. Authors declare no competing interest.
authors.affiliation: Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Columbia University College of Physicians and Surgeons, 622 W. 168th Street, PH-16, New York, NY 10032, USA Present address: Monmouth Medical Center, Damien Fertility Partners, Shrewsbury, NJ 07702, USA.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Kort
authors.affiliation: Department of Pediatrics, Columbia University of Physicians and Surgeons, 1150 St. Nicholas Avenue, New York, NY 10032, USA.
authors.forename: Gloryn
authors.initials: G
authors.lastname: Chia
authors.affiliation: Reproductive Medicine Associates, New Jersey, 140 Allen Road, Basking Ridge, NJ 07920, USA Department of Microbiology and Molecular Genetics, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635, USA.
authors.forename: Nathan R
authors.initials: NR
authors.lastname: Treff
authors.affiliation: Department of Pediatrics, Columbia University of Physicians and Surgeons, 1150 St. Nicholas Avenue, New York, NY 10032, USA.
authors.forename: Akemi J
authors.initials: AJ
authors.lastname: Tanaka
authors.affiliation: Department of Microbiology and Molecular Genetics, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635, USA.
authors.forename: Tongji
authors.initials: T
authors.lastname: Xing
authors.affiliation: The New York Stem Cell Foundation Research Institute, New York, NY 10032, USA.
authors.forename: Lauren Bauer
authors.initials: LB
authors.lastname: Vensand
authors.affiliation: Department of Pediatrics, Columbia University of Physicians and Surgeons, 1150 St. Nicholas Avenue, New York, NY 10032, USA.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Micucci
authors.affiliation: Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Columbia University College of Physicians and Surgeons, 622 W. 168th Street, PH-16, New York, NY 10032, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Prosser
authors.affiliation: Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Columbia University College of Physicians and Surgeons, 622 W. 168th Street, PH-16, New York, NY 10032, USA.
authors.forename: Roger A
authors.initials: RA
authors.lastname: Lobo
authors.affiliation: Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Columbia University College of Physicians and Surgeons, 622 W. 168th Street, PH-16, New York, NY 10032, USA.
authors.forename: Mark V
authors.initials: MV
authors.lastname: Sauer
authors.affiliation: Department of Pediatrics, Columbia University of Physicians and Surgeons, 1150 St. Nicholas Avenue, New York, NY 10032, USA The New York Stem Cell Foundation Research Institute, New York, NY 10032, USA de2220@cumc.columbia.edu.
authors.forename: Dieter
authors.initials: D
authors.lastname: Egli
mesh_terms: D000782:Aneuploidy; D001755:Blastocyst / Q000166:cytology* / Q000648:ultrastructure; D002467:Cell Nucleus / Q000648:ultrastructure; D004249:DNA Damage; D047108:Embryonic Development; D006801:Humans; D007150:Immunohistochemistry
keywords: DNA damage; micronucleation; mitosis; mosaicism; preimplantation development