What is the chromosomal constitution of blastomeres excluded from the formation of a morphologically normal blastocyst

Introduction Blastomeres that have arisen at any time throughout cleavage-stage are sometimes excluded from the formation of the blastocyst and are sequestered either between the zona pellucida and the perivitelline space or remain internally during blastocyst formation and are sequestered to the blastocoel cavity. In both ways, once isolated, those cells do not take further part in preimplantation development. It has been demonstrated that excluded cells have poor gap junction communication with the embryo ( Hardy et al., 1996 ). Material and Methods This observational study was based on 40 good or top-quality blastocysts (at least 4BB) with blastomeres excluded from the formation of the blastocyst and sequestered between the zona pellucida and the perivitelline space. They were belonging to 33 patients (mean age: 33.15) applying for preimplantation genetic screening (PGS) due to advanced maternal age (>37). Patients with structural abnormalities or a history of abnormal fetal karyotype were not included in the analysis. The 40 trophectoderm biopsies were prospectively analyzed and the corresponding excluded blastomeres (2- 5/ blastocyst) samples were in parallel diagnosed either by next generation sequencing (NGS) (PGM platform, ThermoFisher) or by array Comparative Genomic Hybridization (aCGH, Illumina). Assisted hatching was done on day-3 and blastocysts having excluded blastomeres in the axis of the zona opening were included in the analysis to decrease manipulation and most importantly, not to harm the blastocyst. Results The PGS analysis of the trophectoderm biopsies revealed 17 chromosomes involved in monosomies, 37 in trisomies, 7 in mosaics and 13 euploids, showing that 32.5% of the blastocysts with excluded blastomeres were euploid. In 13 blastocysts one chromosome was involved in the aneuploidy whereas for 9 blastocysts two chromosomes were implicated. Only three blastocysts were aneuploid for three chromosomes. None but one of the excluded blastomeres had the same diagnosis with the corresponding embryo (trisomy 4). However, in nearly half of the embryos (15/40) at least one chromosomal aneuploidy was shared by both samples; the excluded blastomeres having a more complex diagnosis with a mean of four chromosomes involved in the aneuploidy [for example, a diagnosis of monosomy 7, 14 for the blastocyst and a diagnosis of trisomy 1q, monosomy (3, 4, 7, 14, 16, 19) for the corresponding excluded blastomeres]. Only two samples of excluded blastomeres were euploid although the corresponding blastocyst was aneuploid. Conclusions Morphologically normal blastocysts with excluded blastomeres were diagnosed with aneuploidy affecting a mean of 1.4 chromosomes, whereas excluded blastomeres were chromosomally chaotic (mean of 4.35 aneuploid chromosomes). It may be hypothesized that either the embryo attempts to exclude blastomeres with chaotic aneuploidies from the blastocyst or that the excluded blastomeres have become chromosomally chaotic because of apoptotic cell death mechanisms that have been already initiated in the aneuploid embryo.