Bluefuse multi analysis software

Trophectoderm biopsy involved making a hole in the zona pellucida by using diode laser (RI Saturn 3, England) on day 3 of embryonic development, which allowed the developing trophectoderm cells to protrude after blastulation, facilitating the biopsy. On day 5 or 6 after fertilization, between five and eight cells were excised using laser energy, without loss of inner cell mass. If the embryo was on day 6 or if the hatching part of the embryo had excessive trophectoderm cells, both laser and mechanic techniques were used. A mechanical cut was performed using a pipette with a 30 mm inner diameter (Origio, Denmark).
If a small number of cells protruded or if the trophectoderm score was B, then detachment was done by mechanical cutting only.
Two PGT-A techniques were used to study biopsy material: NGS and in a minority of cases aCGH. aCGH was performed between 2011 and 2016 using 24Sure kit (Illumina, USA) following standard procedures in the provided manual. Analyses were done using BlueFuse Multi Analysis Software (Illumina, USA), illustrating the chromosome copy numbers in a biopsy sample. NGS was performed between 2017 and 2020 using ReproSeq kit (ThermoFisher, USA) and initially PGM (Ion Personal Genome Machine, ThermoFisher, USA) and latterly S5 (ThermoFisher, USA). Analyses were performed on Ion Reporter software suite v5.2 and v5.6 (ThermoFisher, USA).

In the scope of this study, two PGT-A techniques were used, aCGH (array comparative genomic hybridization) and NGS (next generation sequencing).
aCGH was performed between 2011 and 2016 using 24Sure kit (Illumina, USA) following standard procedures on the provided manual. Analyses were done using BlueFuse Multi Analysis Software (Illumina, USA), illustrating the chromosome copy numbers in a biopsy sample.
NGS was performed between 2017 and 2018 using ReproSeq kit (ThermoFisher, USA) and initially PGM (Ion Personal Genome Machine, ThermoFisher, USA) and latterly S5 (ThermoFisher, USA). Analyses were performed on Ion Reporter software suit v5.2 and v5.6 (ThermoFisher, USA).
Mosaicism was only determined in NGS-tested samples; the range of mosaicism was identified between 20–80% as stated in Preimplantation Genetic Diagnosis International Society (PGDIS) guidelines (http://pgdis.org/docs/newsletter_071816.html). The copy number detection resolution of both aCGH and NGS techniques is similar in terms of base pairs. However, as NGS is based on numerical counting of reads and not logarithmic comparison, it can identify mosaicism in biopsies. Beyond mosaicism, there is no difference between these techniques in aneuploidy detection. Mosaicism was reported only when NGS was performed.

Each chromosomal copy number was comprehensively estimated using the Bluefuse Multi Analysis Software (Illumina). This platform was demonstrated to have a detection performance of around 10 Mb of segmental aneuploidy and 20% of mosaicism. The range of mosaicism was defined as 20%‐80%, which complied with Preimplantation Genetic Diagnosis International Society guidelines.¹¹, ¹² Statistical analysis was performed using Fisher's exact test with the Holm correction for multiple testing, and differences were considered significant at P < .05.