Institutional approval and informed consent
Embryos classified as chromosomally abnormal by PGT-A were used in this study. The application of human blastocysts in this study was approved by the institutional ethics committee of First Affiliated Hospital, Nanjing Medical University. All methods of this study were performed in accordance with the relevant guidelines and regulations.Each participant was over 18 years old and signed a written informed consent.
Embryo origin and culture
We used high-quality blastocysts donated after PGT-A in the Reproductive Medicine Center of the First Affiliated Hospital of Nanjing Medical University, and a total of 24 embryos from 13 patients (mother's age 29±7) were included in the study. All embryos were subjected to a standard in vitro fertilization intracytoplasmic sperm injection cycle and cultured and incubated at 37°C, 5% CO2, and saturated humidity.
Establishment of embryonic stem cell lines
For embryos detected as A by PGT-A, the embryonic trophectoderm and inner cell mass were carefully separated with a glass needle. The cells were cultured in an incubator with 5% CO2 and saturated humidity for 12 days and shed cells were harvested with a pipette every day and stored in an EP tube at −20°C. The medium was replaced with a fresh culture medium, and the cell growth state was observed. After 10-12 days, 6 of the 24 ICMs seeded on the dish formed stem cell clones. Some cells were picked with glass needles for a subculture to verify the pluripotency of the ES cells, and the remaining outgrowths were subjected to next-generation sequencing, and the results were compared with the original diagnosis (Fig.1).
Immunofluorescence detection of stem cell pluripotency
A small number of digested clones from human embryonic stem cells (hESCs) were seeded onto coverslips in pre-prepared 24-well plates. The cells were cultured for 4 days, fixed with 4% paraformaldehyde at room temperature for 20 minutes, and washed three times with Dulbecco's phosphate-buffered saline (DPBS). After fixation, the cells were emersed in 100 µl 0.4% TritonX-100/DPBS, broken up on ice for 10 minutes, and washed with DPBS. We added 5% BSA (200 µl/well) and placed the plates in a 37°C incubator for 30 minutes of blocking. The primary antibody was added and the cells were labeled overnight at 4°C. The primary antibodies used were rabbit anti-OCT4 (1:200 Abcam), rabbit anti-Nanog (1:200 Cell Signaling Technology), mouse anti-SSEA4 (1:200 Abcam), and mouse anti-TRA-1-60(1:200 Cell Signaling Technology). The primary antibody was washed away three times using DPBS, and the cells were labeled with the secondary antibody by incubating at room temperature for 1 hour in the dark. The secondary antibody used was donkey anti-rabbit IgG (H+L)(1:1000 Thermo Fisher Scientific), and donkey anti-mouse IgG (H+L)(1:1000 Thermo Fisher Scientific). After dropping 20 µl of mounting/blocking solution onto the glass slide, the small disc was lifted with a curved needle and flipped so it lay upside down on the glass slide. A fluorescent inverted microscope was used to observe and record micrographs of the cells.
Detection of pluripotency of stem cells by flow cytometry
The cells were collected, and their density was adjusted to 1.0 × 105/10 µl, followed by the addition of 0.1 ml of freshly prepared Fixation Buffer (#420801, Biolegend). After mixing gently, the cells were fixed for 30-60 minutes at room temperature in the dark. Freshly prepared Permeabilization Wash Buffer (#421002, Biolegend) was added to each tube and left at room temperature for 3 minutes to rupture the cell membranes, and then the treatment was repeated. For primary antibody (same as used for immunofluorescence) labeling, 0.1 ml/tube Permeabilization Wash Buffer was used to resuspend the cells, then 3 µl primary antibody was added to each tube, mixed gently, and incubated overnight at 4°C in the dark. Permeabilization Wash Buffer 1.4 ml was added to wash away the primary antibody. For the secondary antibody (same as used for immunofluorescence) labeling, 0.1 ml/tube Permeabilization Wash Buffer was added to each tube to resuspend the cells, followed by 1 µl of secondary antibody, and incubated at room temperature for at least 1 hour in the dark. The cells were washed to remove the secondary antibody, and a 200 µl staining solution was added before the cells were placed into the flow cytometry machine for detection.
In vitro differentiation assay
Collagenase IV and neutral protease were used to digest the hESCs, and the cell clones were collected and placed in an ultra-low adhesion culture dish for suspension culture. A small globular mass of cells called an embryoid body (EB) formed. Gradually, the EB increased in size and the surface became smoother with the passing of culture days EBs were collected on days 0, 3, 7, and 14 of differentiation, and a 1:1 mix of 0.25% trypsin and collagenase II was added and incubated in a water bath at 37°C for 5 minutes, with mixing every 1 minute during the incubation. Cell adhesion molecules were digested to give single cells, which were seeded onto Matrigel-coated discs, cultured in an incubator for 24 hours, fixed with 4% PFA, and detected by immunofluorescence.
Whole-genome amplification of stem cell gDNA and next-generation sequencing
The multiple-annealing SurePlex DNA Amplification Kit (Illumina: catalog # F1006000900) was used to amplify gDNA from the stem cells according to the manufacturer’s instructions. Following the protocol for the Veriseq DNA Library Prep Kit (Illumina; F1006001002, F1006001002), sequencing libraries for NGS were constructed for sequencing on an Illumina MiSeq platform (Illumina, San Diego, CA, USA).
Identifying karyotype and copy number variations by NGS genetic screening of stem cells
The read numbers were counted along the 24 chromosomes with a bin size of 4 MB and normalized to the mean of the corresponding bin in all samples. Our earlier pilot data showed that an even distribution of reads along the chromosomes indicates the targeting of chromosomal contents and the cell karyotypes. The pipeline for chromosomal copy number analysis was conducted as described previously[11].