title: Sequential comprehensive chromosome analysis on polar bodies, blastomeres and trophoblast: insights into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development.
abstract: OBJECTIVE
  What is the optimal stage from oocyte through preimplantation embryo development for biopsy and preimplantation genetic screening (PGS) to detect abnormal chromosome segregation patterns in eggs or embryos from advanced maternal age (AMA) patients?
  
  
  CONCLUSIONS
  Testing at the polar body (PB) stage was the least accurate mainly due to the high incidence of post-zygotic events. This suggests that postponing the time of biopsy to the blastocyst stage of preimplantation embryo development may provide the most reliable results for PGS.
  
  
  BACKGROUND
  In the PGS field there is an ongoing debate about the optimal biopsy stage for PGS. This is a result of the lack of understanding of how aneuploidy arises in the human embryo. To date, most of the cytogenetic data obtained during PGS investigations have been derived through the analysis of cells at isolated points in the preimplantation window, thus potentially missing critical information on chromosomal segregation. Understanding the chromosome segregation patterns during preimplantation development holds the potential to significantly increase the success rates of IVF. In this study, a sequential comprehensive chromosome analysis of both the PBs and the corresponding embryos at both the cleavage and the blastocyst stages is presented.
  
  
  METHODS
  This is a prospective longitudinal cohort study performed between October 2009 and August 2011 involving 9 infertile couples and 21 sets of complete comprehensive chromosomal screening data, including PB1, PB2, corresponding blastomeres and trophectoderm (TE) samples.
  
  
  METHODS
  Infertile couples undergoing IVF cycles with PGS where the female partner was older than 40 years and with a good response to controlled ovarian stimulation (>10 MII oocytes retrieved) were enrolled into the study. The exclusion criteria were (i) patients presenting with abnormal karyotype; (ii) specific ovarian pathologies including polycystic ovary syndrome, endometriosis grade III or higher and premature ovarian failure and (iii) severe male factor infertility (motile sperm count of <500 000/ml after preparation of a fresh ejaculate). The PBs, blastomere and TE samples were sequentially biopsied and analyzed by array comparative genomic hybridization (aCGH). The analysis of chromosome segregation patterns was performed to infer the origin of aneuploidy and to investigate the diagnostic accuracy of both PB and cleavage-stage PGS strategies.
  
  
  RESULTS
  Twenty-one sets of complete data (PB1/PB2/blastomere/TE) including 84 aCGH experiments showed a pattern of multiple meiotic errors typically caused by sister chromatid separation errors and predominantly arising in the second meiotic division. Twenty-two of the 24 (91.7%) errors in the first meiotic division arose as a consequence of premature sister chromatid predivision. In half of these cases, the second meiotic division resulted in a balancing chromosome segregation event producing a normal female complement for that chromosome in the resulting embryo. Overall, only 62 out of 78 (79.5%) of the abnormal meiotic segregations had errors in the either one or both PBs consistent with the aneuploidies observed in their resulting embryos. Ten of the 21 (47.6%) embryos had aneuploidies other than female meiotic-derived ones, most of which detected on Day 3 and confirmed on Day 5 or 6 of embryo development (20/25) with chromosomal loss being three times more frequent than gains. Notably, as high as 20% of female-derived aneuploidies detected on PBs and confirmed on Day 3 were rescued at the blastocyst stage, mainly as a result of diploidization of trisomic chromosomes. On a per chromosome basis, the sensitivity in predicting blastocyst chromosomal complement was significantly lower for PB approach, 61.7%, compared with blastomeres analysis, 86.4% (P < 0.01).
  
  
  CONCLUSIONS
  The study was limited to the analysis of oocytes and embryos from AMA patients. Thus, these findings apply only to this patient group. Comparisons with other patient populations including patients with different indications for PGS should be made in future research. In addition, higher resolution and/or more accurate chromosomal screening tests could be used in future studies to corroborate the current findings.
  
  
  CONCLUSIONS
  These findings provide critical insights into the mechanisms causing errors during female meiosis and the preimplantation embryo development period to improve the design and treatment outcome of PGS.
authors.affiliation: Centre for Reproductive Medicine, GENERA, Clinica Valle Giulia, Via G. De Notaris 2b, Rome 00197, Italy. capalbo@generaroma.it
authors.forename: Antonio
authors.initials: A
authors.lastname: Capalbo
authors.forename: Sara
authors.initials: S
authors.lastname: Bono
authors.forename: Letizia
authors.initials: L
authors.lastname: Spizzichino
authors.forename: Anil
authors.initials: A
authors.lastname: Biricik
authors.forename: Marina
authors.initials: M
authors.lastname: Baldi
authors.forename: Silvia
authors.initials: S
authors.lastname: Colamaria
authors.forename: Filippo Maria
authors.initials: FM
authors.lastname: Ubaldi
authors.forename: Laura
authors.initials: L
authors.lastname: Rienzi
authors.forename: Francesco
authors.initials: F
authors.lastname: Fiorentino
mesh_terms: D000328:Adult; D000782:Aneuploidy; D001757:Blastomeres / Q000166:cytology*; D020090:Chromosome Segregation / Q000502:physiology*; D055028:Comparative Genomic Hybridization; D020732:Cytogenetic Analysis; D047108:Embryonic Development / Q000235:genetics*; D005260:Female; D006801:Humans; D059785:Karyotype; D008137:Longitudinal Studies; D008423:Maternal Age; D008540:Meiosis / Q000502:physiology*; D018895:Microsatellite Repeats; D008875:Middle Aged; D059705:Polar Bodies / Q000166:cytology*; D019836:Preimplantation Diagnosis / Q000379:methods*; D014327:Trophoblasts / Q000166:cytology*