title: Correlation between aneuploidy, standard morphology evaluation and morphokinetic development in 1730 biopsied blastocysts: a consecutive case series study.
abstract: Are there correlations among human blastocyst ploidy status, standard morphology evaluation and time-lapse kinetics?
  
  
  
  Correlations were observed, in that euploid human blastocysts showed a higher percentage with top quality inner cell mass (ICM) and trophectoderm (TE), higher expansion grades and shorter time to start of blastulation, expansion and hatching, compared to aneuploid ones.
  
  
  
  Embryo quality has always been considered an important predictor of successful implantation and pregnancy. Nevertheless, knowledge of the relative impact of each morphological parameter at the blastocyst stage needs to be increased. Recently, with the introduction of time-lapse technology, morphokinetic parameters can also be evaluated. However, a large number of studies has reported conflicting outcomes.
  
  
  
  This was a consecutive case series study. The morphology of 1730 blastocysts obtained in 530 PGS cycles performed from September 2012 to April 2014 that underwent TE biopsy and array comparative genomic hybridization was analyzed retrospectively. A total of 928 blastocysts were cultured in a time-lapse incubator allowing morphokinetic parameters to be analyzed.
  
  
  
  Mean female age was 36.8 ± 4.24 years. Four hunderd fifty-four couples were enrolled in the study: 384, 64 and 6 of them performed single, double or triple PGS cycles, respectively. In standard morphology evaluation, the expansion grade, and quality of the ICM and TE were analyzed. The morphokinetic parameters observed were second polar body extrusion, appearance of two pronuclei, pronuclear fading, onset of two- to eight-cell divisions, time between the two- and three-cell (cc2) and three- and four-cell (s2) stages, morulae formation time, starting blastulation, full blastocyst stage, expansion and hatching timing.
  
  
  
  Of the 1730 biopsied blastocysts, 603 were euploid and 1127 aneuploid. We observed that 47.2% of euploid and 32.8% of aneuploid blastocysts showed top quality ICM (P < 0.001), and 17.1% of euploid and 28.5% of aneuploid blastocysts showed poor quality ICM (P < 0.001). Top quality TE was present in 46.5% of euploid and 31.1% of aneuploid blastocysts (P < 0.001), while 26.6% of euploid and 38.1% of aneuploid blastocysts showed poor quality TE (P < 0.001). Regarding expansion grade, 81.1% of euploid and 72.4% of aneuploid blastocysts were fully expanded (Grade 5-6; P < 0.001). The timing of cleavage from the three- to four-cell stage, of reaching four-cell stage, of starting blastulation, reaching full blastocyst stage, blastocyst expansion and hatching were 2.6 (95% confidence interval (CI): 1.7-3.5), 40.0 (95% CI: 39.3-40.6), 103.4 (95% CI: 102.2-104.6), 110.2 (95% CI: 108.8-111.5), 118.7 (95% CI: 117.0-120.5) and 133.2 (95% CI: 131.2-135.2) hours in euploid blastocysts, and 4.2 (95% CI: 3.6-4.8), 41.1 (95% CI: 40.6-41.6), 105.0 (95% CI: 104.0-106.0), 112.8 (95% CI: 111.7-113.9), 122.1 (95% CI: 120.7-123.4) and 137.4 (95% CI: 135.7-139.1) hours in aneuploid blastocysts (P < 0.05 for early and P < 0.0001 for later stages of development), respectively. No statistically significant differences were found between euploid and aneuploid blastocysts for the remaining morphokinetic parameters.A total of 407 embryo transfers were performed (155 fresh, 252 frozen-thawed blastocysts). Higher clinical pregnancy, implantation and live birth rates were obtained in frozen-thawed compared to fresh embryo transfers (P = 0.0104, 0.0091 and 0.0148, respectively). The miscarriage rate was 16.1% and 19.6% in cryopreserved and fresh embryo transfer, respectively. The mean female age was lower in the euploid compared to aneuploid groups (35.0 ± 3.78 versus 36.7 ± 4.13 years, respectively), We found an increasing probability for aneuploidy with female age of 10% per year (odds ratio (OR) = 1.1, 95% CI: 1.1-1.2, P < 0.001).
  
  
  
  The main limitation of morphology assessment is that it is a static system and can be operator-dependent. In this study, eight embryologists performed morphology assessments. The main limitation of the time-lapse technology is that it is impossible to rotate the embryos making it very difficult to observe them in case of blastomere overlapping or increased cytoplasmic fragmentation.
  
  
  
  Although there seems to be a relationship between the ploidy status and blastocyst morphology/development dynamics, the evaluation of morphological and morphokinetic parameters cannot currently be improved upon, and therefore replace, PGS. Our results on ongoing pregnancy and miscarriage rates suggest that embryo evaluation by PGS or time-lapse imaging may not improve IVF outcome. However, time-lapse monitoring could be used in conjunction with PGS to choose, within a cohort, the blastocysts to analyze or, when more than one euploid blastocyst is available, to select which one should be transferred.
  
  
  
  No specific funding was obtained for this study. None of the authors have any competing interests to declare.
authors: Centre for Reproductive Medicine, European Hospital, Via Portuense 700, Rome 00149, Italy mg.minasi@gmail.com.
authors: Maria Giulia
authors: MG
authors: Minasi
authors: Centre for Reproductive Medicine, European Hospital, Via Portuense 700, Rome 00149, Italy.
authors: Alessandro
authors: A
authors: Colasante
authors: Centre for Reproductive Medicine, European Hospital, Via Portuense 700, Rome 00149, Italy.
authors: Teresa
authors: T
authors: Riccio
authors: Centre for Reproductive Medicine, European Hospital, Via Portuense 700, Rome 00149, Italy.
authors: Alessandra
authors: A
authors: Ruberti
authors: Centre for Reproductive Medicine, European Hospital, Via Portuense 700, Rome 00149, Italy.
authors: Valentina
authors: V
authors: Casciani
authors: Centre for Reproductive Medicine, European Hospital, Via Portuense 700, Rome 00149, Italy.
authors: Filomena
authors: F
authors: Scarselli
authors: Molecular Genetics Laboratory, "GENOMA", Via di Castel Giubileo 11, Rome 00138, Italy.
authors: Francesca
authors: F
authors: Spinella
authors: Molecular Genetics Laboratory, "GENOMA", Via di Castel Giubileo 11, Rome 00138, Italy.
authors: Francesco
authors: F
authors: Fiorentino
authors: Centre for Reproductive Medicine, European Hospital, Via Portuense 700, Rome 00149, Italy.
authors: Maria Teresa
authors: MT
authors: Varricchio
authors: Centre for Reproductive Medicine, European Hospital, Via Portuense 700, Rome 00149, Italy.
authors: Ermanno
authors: E
authors: Greco
mesh_terms: D000328:Adult; D000782:Aneuploidy; D001755:Blastocyst / Q000502:physiology*; D055028:Comparative Genomic Hybridization; D046149:Embryo Culture Techniques; D010064:Embryo Implantation / Q000502:physiology*; D047108:Embryonic Development / Q000502:physiology*; D005260:Female; D006801:Humans; D011247:Pregnancy; D012189:Retrospective Studies
keywords: PGS; aneuploidy; blastocyst biopsy; cryopreserved embryo transfer; embryo morphology; fresh embryo transfer; morphokinetic; time-lapse