LAUSR

May the mosaicism ratio be influenced by the time of blastulation in preimplantation genetic testing for aneuploidies (PGT-A)? The mosaicism ratio is significantly higher in day–6 blastocysts when compared to day–5 when transferable embryos are considered only (euploids and mosaics). Conventionally, PGT-A has classified preimplantation embryos as either euploid or aneuploid. Yet, a major improvement in PGT-A methodology, with the introduction of high sensitivity diagnostic Next Generation Sequencing (NGS) technology, has allowed the identification of a third embryo category: the mosaic (Coll et al., 2021). Embryonic mosaicism can be defined as the presence of karyotypically distinct cell lines within an embryo and can be detected by NGS at a rate between 20–80%. In the absence of euploid embryos, mosaic embryos, when transferred, have been shown to deliver healthy live births (PGDIS, 2019). This retrospective study was based on 9828 trophectoderm biopsies performed in a single ART clinic between January 2017 and October 2020 for PGT-A cycles with more than one blastocyst. PGT-A cycles with only one blastocyst were excluded because in these cycles’ day–5/day–6 biopsy percentage cannot be calculated. A total number of 8398 and 1430 blastocysts were biopsied on day–5 and day–6, respectively for PGT-A by ReproSeq on Ion Torrent S5 (Thermo Fisher Scientific). Three categories were defined in the PGT-A group with >1 blastocyst biopsied to compare the rate of mosaicism: C1:cycles in which blastocysts were only biopsied on day–5 (n = 1872), C2:99–60% of blastocysts were biopsied on day–5 (n = 483) and C3:0–60% of blastocysts biopsied on day–5 (n = 411). The mean female age (C1:36.0±5.2; C2:35.7±4.8; C3:37.1±4.9) and metaphase II oocytes punctured (C1:9.8±6.5; C2:10.4±5.2; C3:8.4±5.4) were similar and statistically non-significant in all groups. T-test and Chi-square tests were used where appropriate. Overall, from the blastocysts biopsied on day–5 and 6, 35.4% and 25.5% were euploid, 13.7% and 14.7% were mosaic, 50.9% and 59.8% were aneuploid, respectively (p < 0.0001, p = 0.32, p < 0.0001), the mosaicism rate being not statistically different. However, when only transferable blastocysts (euploids and mosaics) were considered (aneuploids being discarded), the rate of mosaic embryos was significantly higher in day–6 when compared to day–5 blastocysts (36.6% vs. 28.0%; p < 0.0001). Morphological blastocyst grading was then investigated: from the day–5 and 6 blastocysts biopsied, 51.5% and 30.8% were of top-quality and 48.5% and 69.2% were of good-quality, respectively. Looking deeper into the categories defined, the euploidy rate was found to be higher in C1 (35.1%) when compared to C2 (34.9%) and C3 (27.7%) (p < 0.0001). The mosaic embryo rate was found to be non-significant when all blastocysts (euploids, aneuploids, mosaics) were considered (C1: 13.7%; C2: 14.1%; C3: 14.2%; p = 0.6492). However, when transferable blastocysts were considered (euploids and mosaics only), the mosaic embryo rate was significantly higher in C3 (33.9%) when compared to C1 (28.1%) and C2 (28.8%) (p = 0.02). For morphological blastocyst grading, regardless of blastulation day, mosaicism was higher for good-quality embryos both when all biopsies and only transferrable embryos were considered (p = 0.0018, p < 0.0001, respectively). This study focused on blastocyst formation day and morphological blastocyst grading. Extrinsic factors have also been reported to induce mosaicism: ovarian stimulation, culture media, laboratory and culture conditions, technical issues during the biopsy and sample processing (Munné and Wells, 2017; Katz-Jaffe et al., 2018, Fragouli et al., 2010, 2019). Wider implications of the findings: Mitotic errors during cleavage stage causing mosaicism may lead to lower morphological grade and late blastulation as some cells in those embryos are not diploid thus leading to higher mosaicism for blastocysts that reach blastulation on day6 and/or yield only good-quality embryos. Not applicable