G2 medium

Embryos were cultured in G-series media (Vitrolife, Västra Frölunda, Sweden) in low oxygen atmosphere. Embryos were classified according to the scale of Cummins et al. [34 (link)]: good—6–8 cells, A-B class (equal size and fragmentation of blastomers 0–10%), poor—6–8 cells, C class (differences in blastomers size, fragmentation 11–25%). Only embryos graded as A or B were included in the present study.
Biopsies were performed on day-3 post fertilization when the embryos reached the 6–8 cells stage by selecting a single blastomere that underwent three mitotic divisions. Laser technology (telecomunnication’s laser Anritsu 1488 nm in Saturn 3 RI) was used to create an opening in the zona pellucida that encapsulates each embryo. Single blastomere was gently aspirated. After the biopsy each embryo was washed, transferred to G2 medium (Vitrolife, Västra Frölunda, Sweden) and cultured for two more days. In each case embryos were transferred to the uterus on day-5 during the same cycle (fresh transfer).

Remaining or warmed day 3 embryos were cultured in G2 medium (Vitrolife, Sweden) at 37°C with 5% O₂ and 6% CO₂ in incubators until day 5. The blastocyst score was determined according to Gardner’s grading system. Blastocysts reaching the expanded or hatching stage and earning a score above grade CC (inner cell mass/trophectoderm) were cryopreserved by vitrification. All blastocysts were shrunk by laser-assisted hatching to ensure that vitrification was effective.

Excess cleavage-stage embryos were cryopreserved, or cultured to the blastocyst stage and then cryopreserved for subsequent FET. The decision regarding whether to perform extended culture or not was made by the doctor and patient together. If a patient was concerned about having no or very few available embryos after extended culture, typically two day 3 embryos were frozen, while the other embryos were further cultured. G2 medium (Vitrolife, Sweden) was used for blastocyst culture. CO₂, O₂, and N₂ were maintained at 6, 5, and 89% in an incubator (CO₂ incubator C60, Labotect, Germany, or K-MINC-1000, Cook, Australia). The embryos were further cultured at 37°C until day 5 or 6. The blastocysts were scored using Gardner and Schoolcraft’s grading system (15 (link)).

The oocytes were placed in G-1 medium (Vitrolife) in an incubator with 5% O₂, 6% CO₂, and 89% N₂ after fertilization until day 3. All day 3 embryos were transferred to G-2 medium (Vitrolife) to be cultured for another 3-4 days. Zygotes were observed 16–18 h after fertilization. Only 2PN-derived embryos were included in this study. Embryos were observed and scored according to the Istanbul consensus on day 2 and day 3 [23 (link)]. The morphology scoring system was based mainly on cell number and fragmentation. Blastocysts were scored according to Gardner and the Schoolcraft system [24 (link)]. At our center, blastocysts with a score above 3 BC were defined as available blastocysts. Approximately 5–10 TE cells were biopsied using the laser-assisted method from available and expanded blastocysts on days 5–7 after insemination. The blastocysts were vitrified after they were biopsied.

Collected embryos were cultured singularly at 37 °C and 6% CO₂ in 50 μl microdrops containing G2 medium (Vitrolife) supplemented with 6% HSA. On the morning of Day 5 or 6 of culture, embryos were examined using an inverted microscope and scored according to the Gardner blastocyst Scoring system19 (link) (Supplementary Figure S2). High-quality blastocysts represent those with grades equal or higher than 3BB (Day 5) or 4BB (Day 6). Only those with obvious blastocoel cavities were biopsied using a micromanipulation system (Narashige, Japan) that had been fitted onto the inverted microscope (Nikon). This approach allowed us to distinguish trophoblast cells. After laser-assisted zona drilling (ZILOS-tk, Hamilton Thorne), 3–5 trophoblast cells (TE) from Day 5 or Day 6 blastocysts were retrieved for SNP analysis using biopsy needles. For non-blastocysts, 3–5 cells with clear cell morphologies were retrieved using biopsy needles after laser-assisted zona drilling. These cells were also subjected to SNP analysis.
In addition, a total of 111 blastocyst-related SNP results from 54 couples were collected from the clinical PGD samples that were used in this study. The biopsy procedure used to retrieve cells from PGD samples was consistent with that used for clinically discarded embryos.

The oocytes and embryos for this study were donated from female volunteers who provided informed consent. After ICSI (intracytoplasmic sperm injection), embryos were cultured in G1.3 medium (Vitrolife, Sweden) covered with mineral oil (Sigma, 6 % CO₂). Oocytes, zygotes, and 2-cell-stage embryos were collected at the appropriate time during embryonic development.
Embryos at the 4-cell and 8-cell stages were thawed immediately after removal from liquid nitrogen as described previously [1 ]. The embryos were cultured in G2 medium (Vitrolife) continuously to obtain morulae and early blastocysts, blastocysts, and hatched blastocysts.
Each selected oocyte or embryo was transferred drop wise to the acidic solution to remove the zona pellucida by mouth pipette. Then, the embryo or oocyte was washed gently several times before being transferred to lysis buffer.
To obtain ICM and TE transcriptome information, we isolated these compartments from each other by laser cutting. This process was executed carefully to retain all cells in the ICM with minimal laser damage.