Abstract
Abstract Study question Are there cell lineage-related differences in the apoptotic rates of human blastocysts classified as euploid, mosaic and aneuploid after preimplantation genetic testing for aneuploidy (PGT-A)? Summary answer In human blastocysts, apoptosis is more frequent in trophectoderm (TE) cells and is associated with the presence of mosaic and uniform chromosomal alterations. What is known already Embryos diagnosed as mosaic after PGT-A can develop into healthy infants. However, the reason why these embryos achieve reproductive competence needs further research. One hypothesis is that aneuploid cells are negatively selected during embryo development through cell competition mechanisms, such as apoptosis or differential proliferation. While these mechanisms have been demonstrated in mouse embryos, in which apoptosis occurs more frequently in the inner cell mass (ICM), evidence in human embryos is scarce. In fact, only one previous study has shown an association between mosaicism and aneuploidy after PGT-A and increased levels of apoptosis, which was more frequent in TE cells. Study design, size, duration Prospective cohort study performing colocalization of cell-lineage and apoptotic expression markers by immunofluorescence (IF). A total of 53 vitrified human blastocysts with a previous PGT-A diagnosis on day 5 (D5) or day 6 (D6) of development were analysed: n = 13 euploid embryos (n = 11 on D5; n = 2 on D6), n = 22 mosaic embryos (n = 16 on D5; n = 6 on D6), and n = 18 aneuploid embryos (n = 11 on D5; n = 7 on D6). All embryos were donated for research purposes. Participants/materials, setting, methods Following warming and re-expansion, blastocysts were fixed in 4% paraformaldehyde, processed for IF and imaged by confocal microscopy. Primary antibodies: 1:80 goat anti-human Nanog (AF1997, R&D-Systems), 1:100 mouse anti-human Gata3 (MAB6330, R&D-Systems) and 1:1000 rabbit anti-human Caspase-3 (NB10056113, Novus Biologicals). 165 μM DAPI was used for nuclei staining. Total cells: DAPI+ cells. TE: Gata3+ cells. ICM: Gata3-/Nanog+ cells. Incidence of apoptosis: % Caspase-3+ cells (per embryo). Data were analysed using Chi-square or ANOVA (p < 0.05 significant). Main results and the role of chance Total cell number was similar among euploid embryos (59.2±20.6 on D5; 78.5±0.7 on D6), mosaic embryos (49.6±15 on D5; 58.8±16.9 on D6) and aneuploid embryos (48.6±14.2 on D5; 59.6±23.5 on D6) (P > 0.05). Nanog and Gata3 expression evidenced the establishment of ICM and TE cell lineages during the blastocyst stage: while the proportion of Nanog+ cells decreased from 38.2% (755/1979) on D5 to 20.3% (188/927) on D6 (P < 0.0001), the proportion of Gata3+ cells increased from 77.8% (1540/1979) on D5 to 83.1% (770/927) on D6 (P < 0.01). The persistence of Nanog expression in TE cells was associated with the chromosomal status of the embryo, as evidenced by the significantly higher proportion of Gata3+/Nanog+ cells found in aneuploid embryos (27.9%=149/534 on D5; 4.6%=19/417 on D6) compared to mosaic embryos (13.1%=104/794 on D5; 3.4%=12/353 on D6) and euploid embryos (9.7%=63/651 on D5; 0%=0/157 on D6) (P < 0.05). The incidence of apoptosis in the TE was significantly higher in aneuploid embryos (45.9%±16.1 on D5; 49%±15.1 on D6) and mosaic embryos (44.1%±19.6 on D5; 43%±16.8 on D6), compared to euploid embryos (26.6%±16.6 on D5; 17.5%±14.8 on D6) (P < 0.05). Conversely, apoptosis was much less frequent in the ICM (2.2%±7.7), and no differences were found among the study groups (P > 0.05). Limitations, reasons for caution This is a descriptive, single-centre study with a limited sample size. The karyotype concordance between ICM and TE cells was not tested. Wider implications of the findings Our findings demonstrate that apoptosis is common in human blastocysts, particularly in TE cells. The increased apoptotic rates found in the TE of embryos with chromosomal abnormalities support its role as a mechanism to eliminate aneuploid cells. This mechanism may indeed be related to the reproductive potential of mosaic embryos. Trial registration number Not applicable
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