Biological and molecular mechanisms behind ploidy abnormalities detected in blastocyst stage embryos using an ngs based pgt-a platform with genotyping data

Observation of two pronuclei (2PN) during in vitro fertilization is regarded as evidence of diploidy. However, pronuclei number may not always correlate with true ploidy of the embryo. 2PN assessment may therefore not be an accurate assessment for reproductive potential. In this study, a large dataset of genotyping data from a routine preimplantation genetic testing (PGT) platform was used to explore prevalence, origin and recombination patterns of ploidy alterations identified in 2PN derived blastocysts. Retrospective study including 96,660 embryos from 20,187 PGT cycles. (i) Investigate the incidence of ploidy abnormalities and potential associations with parents’ demographics and clinical history. (ii) Analysis of genotype data from 57 trios (parents and embryo) to estimate parental origin of all ploidy abnormalities and meiotic phase origin of triploidy. For this, opposite homozygous SNPs in the parents were selected as informative markers for parental origin, whereas key pericentromeric SNPs (within 5cM) were analyzed to determine meiotic phase origin. (iii) Characterization of genome-wide recombination patterns of triploid embryos by haplotype phase changes (iv) Investigate the causes of ploidy abnormalities recurrence, using a permutation analysis on available data. Prevalence of ploidy abnormalities in 2PN embryos was 1.1% (n=1.063/96.660; CI95%: 1-1,2%). Notably, the incidence of ploidy anomalies was positively correlated with maternal age (OR=1.046 per year; p<0.001). The multivariate analysis, showed no other association with ploidy abnormalities, including paternal age. Haploid fertilization was predominantly due to absence of paternal genome (n=12/14), whereas triploid embryos were exclusively maternal in origin (n=43/43) and the meiotic phase was mainly MII (72,1%; n=31/43; CI95%: 56-84%). In triploid embryos the average number of observed crossovers was of 19,8 per embryo. Interestingly, 4.65% of triploids (n=2/43) showed no genome-wide recombination events. 899 cycles had at least one embryo with a ploidy abnormality (4.4%; CI95%:4.2-4.7%). 6 (0,03%; 95%CI: 0,01-0,06%) couples were found to have recurrence of ploidy abnormalities (≥3) in the same embryonic cohort and permutation analysis revealed that this cannot be explained by the null hypothesis of a random event. This is the first study to show an increased risk of triploid conception with increasing female age (women aged >40 have twice the risk as younger women) and to corroborate prenatal diagnosis findings of patient specific risk factors for triploid conception. The addition of genotype data to a routine PGT pipeline enabled the extrapolation of recombination data which could be employed as an extra metric of patient-specific risks evaluation for chromosomal abnormalities.