Cell-free DNA Analysis of Spent Embryo Culture Media as a Non-invasive Approach for Preimplantation Genetic Diagnosis

"* Blastocyst Grading: Blastocyst grading was based on criteria that classified embryos as good, fair, or poor using the simplified SART embryo scoring system (Heitmann et al., 2013):~ * Good: AA or AB.~ * Fair: BA, BB, BC.~ * Poor: CB or CC.~* Biopsy Criteria: TE biopsy was performed when an embryo had at least one grade B or better for either the ICM or TE on day 5 of development. When no good-quality blastocysts were available in the same cohort, CC grade blastocysts were biopsied.~* Biopsy Procedure: 5-8 cells were laser-biopsied from the TE. These cells were then rinsed and tubed for PGT.~* Cryopreservation: The biopsied embryo was cryopreserved by vitrification (Ref. 90133, Vit Kit-Freeze, Irvine Scientific, Santa Ana, USA) (Richardson et al., 2015)."

"* DNA Contamination Minimization:~ o SCM was harvested after embryo culture with care taken not to include cellular material.~* Sample Storage:~* Processing for Genetic Analysis:~ 6\. DNA Extraction and Sample Preparation 7. Microarray Comparative Genomic Hybridization (aCGH)~* Chromosomal Assessment:~* BAC-chip Slides:~* Labeling and Hybridization:~* Scanning and Data Acquisition:~* Data Processing and Analysis:~ 8\. Validation and Quality Control~* Robust quality control measures included:"