Ethics
This study was approved by the Reproductive Medical Ethics Committee of Guangzhou Women and Children’s Hospital. Written informed consent was obtained from each couple.
Embryo sources
In this study, 23 couples were undergoing PGT-A/PGT-SR in our center from March 2017 to June 2018. The indications of PGT-SR are reciprocal translocation, Robertsonian translocation carriers and chromosome inversion. Indications of PGT-A are sex chromosome aneuploidies.
In the first part of the study, 28 blastocysts from 5 couples were detected by both NGS and SNP array. In the second part, 115 blastocysts from 18 couples were only detected by NGS firstly. Among them, 39 blastocysts were normal. These blastocysts were detected by SNP array again (Fig. 1).
Embryo culture and biopsy
Intracytoplasmic sperm injection (ICSI) were performed after the oocytes were retrieved, sequence culture was enrolled by G1/G2 (Vitrolife, Sweden). Biopsy was performed on day 5 or day 6 according to the blastocyst grade [20].
All of the blastocysts were subjected to trophectoderm-cell-biopsy by laser and 5-10 TE cells were biopsied. After biopsied, blastocysts were cryopreserved as planned using vitrification technique according to the manufacturer’s protocol (ARSCI Inc, Canada) and stored in liquid nitrogen.
Whole genome amplification
The multiple displacement amplification (MDA) DNA amplification system was used for whole genome amplification (WGA). Briefly, REPLI-g Single Cell Kit (QIAGEN, Germany) was used for single cell amplification, single cell was seeding in 2.5μl phosphate-buffered saline, and then was lysised using 3μl DTT and 33μl DLB in incubator for 10 min at 65°C. After incubation, 3μl Stop Solution was added in the mix. The amplification mix was prepared and 40μl including 2μl REPLI-g DNA polymerase and 29μl REPLI-g Reaction Buffer was added to each tube. Then the mixture was incubated in incubator for 8h at 30°C following 65°C for 3 min to inactivate the REPLI-g DNA polymerase.
NGS protocol
The Illumina MiSeq platform was used in this next-generation sequencing (NGS), and the amplified genome of each blastocyst’s TE cells were sequenced at an approximate 0.01 × genome depth. An on-instrument computer performs primary and secondary data analysis to align the reads to a reference genome. We sequenced a total of approximately 36 million bases, obtaining average genome coverage of 1.2% for each blastocyst’s TE sample. PGXcloud cloud server (available at http://www.pgxcloud.com/) was used to analyze the chromosomal copy number variants (CNVs) (Jabrehoo, China). All results were examined by two independent laboratory technicians to minimize uncertainty and variable results. In the case of discrepancies in opinion, a consensus was reached after the third technician discussion. The structural variants detected is > 4Mb and the level of mosaicism detected is > 30% in our analysis.
SNP array
All the procedures were performed according the instruction of Illumina human SNP array. Briefly, DNA were hybridized with the Human Cyto‐12 BeadChip (Illumina), which contains approximately 300,000 SNPs with an average distance of 9.7 kb. The bead chips were subjected to immunostaining, followed by stringent washes to remove unhybridized and non-specifically hybridized DNA. Subsequently, the bead chips were scanned using an Illumina iScan Bead Array Reader (Illumina). The scanning results were processed using the B allele frequency and log R ratio using Illumina Genome Studio software (Illumina) to analyze the copy number of the chromosomes according the instruction of Illumina human SNP array.