Zilos tk laser

All embryos were cultured in sequential media (G1 and G2, Vitrolife; Goteborg, Sweden) to the blastocyst stage. Approximately 3-8 trophectoderm (TE) cells were aspirated using a biopsy pipette (internal diameter, 30 mm) and dissected with a Zilos TK laser (Hamilton Thorne, MA). Biopsied TE cells were washed in G-MOPS medium (Vitrolife) and PGT-A was conducted using next-generation sequencing using a previously described method (3) at the Beijing Genomics Institute.

On the morning of day 6, all blastocysts of the patient were biopsied simultaneously. Blastocysts were placed in a drop of G-MOPS (Vitrolife). The blastocyst was positioned using the holding pipette to locate the herniating TE at the 3 o'clock position. A piece of TE away from the inner cell mass was aspirated with a biopsy pipette (internal diameter, 30 mm) and dissected with a Zilos TK laser (Hamilton Thorne).
Biopsied TE cells were prepared for FISH, and the blastocysts were vitrified within 1-2 hours after TE biopsy using a Kitazato vitrification kit (Kitazato Biopharma) in combination with closed High Security Vitrification Straws (Cryo Bio System). The vitrification procedure was performed according to the manufacturer's protocols. Each blastocyst was stored in an individual straw.

Cleavage-stage biopsy was executed on day 3, and only one blastomere was biopsied. Briefly, one blastomere was collected by aspiration with a biopsy pipette (internal diameter, 40 mm) from a hole drilled by laser on the zona pellucida. Removal of a cell without lysis, rendering it available for fixation and subsequent analysis, was considered a successful biopsy (31) . The blastomeres were used for fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR), and the biopsied embryos were cultured continuously for blastocyst transfer.
For the blastocyst biopsy, an 18-mm hole was made in the zona pellucida of all embryos on day 4. On day 5 or day 6, trophoblasts that had herniated out of the zona pellucida were chosen for biopsy. Depending on the actual situation of blastocysts, approximately 4-15 trophoblast cells were aspirated with a biopsy pipette (internal diameter, 30 mm) and dissected with Zilos TK laser (Hamilton Thorne) (32) . The trophoblast cells were washed 3 times in G-MOPS medium (Vitrolife) and detected using different methods such as FISH, PCR, comparative genomic hybridization, or single-nucleotide polymorphism (SNP) array analysis.

Trophectoderm biopsies of good-quality blastocysts were performed on day 6 after fertilization (up to 6 pm). Approximately 5 to 8 trophectoderm cells were aspirated using a biopsy pipette (internal diameter 30 mm) and dissected with a Zilos TK laser (Hamilton Thorne) as described elsewhere (12) . Biopsied trophectoderm cells were then subjected to whole genome amplification (WGA) following the manufacturer's instructions (Sigma-Aldrich).
The NGS analysis was performed to identify the normal/balanced blastocysts as described previously elsewhere (12, 13) . In brief, after omitting the initial 20 base pairs (bp), the read sequences were aligned to a human reference genome (Hg19; NCBI Build37) using the Short Oligonucleotide Analysis Package 2 (SOAP2). After alignment, we used an in-house developed algorithm for chromosome copy number analysis. The copy number variations larger than 1 megabase (>1M) were reported. For mosaicism detection, we first validated the feasibility using mixing experiments with different ratios of euploid and aneuploid samples to mimic chromosomal mosaicisms. The authenticity of aneuploidy and mosaicism identified by NGS were confirmed by fluorescence in situ hybridization or single-nucleotide polymorphism array as described in our previous studies (12, 13) .