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

Materials and Methods:

• Study Design

• Type of Study: This is a retrospective study.
• Location: The study was conducted at the Assisted Reproductive Technology Unit Life Zena Center, Baghdad, Baghdad Governorate, Iraq.
• Duration: The study was conducted from October 2023 to August 2024.
• Patient Criteria:

• Included: All patients referred for PGT-A.
• Excluded: Patients who had no blastocyst for biopsy were excluded.
• Ethical Approval: The study was approved by the Committee for the Scientific Research Ethics of Sohag University (CSRE-16-24).
• Ovarian Stimulation and Oocyte Retrieval

• Protocols: Patients underwent controlled ovarian stimulation using either:

o A long down-regulation protocol with buserelin nasal spray (Suprecur, Hoechst, Germany) (Ravhon et al., 2000).

A short protocol incorporating a gonadotropin-releasing hormone (GnRH) antagonist (Cetrotide, Merck Serono, Germany) (Hohmann et al., 2003).

• Gonadotropin Administration: Daily administration included highly purified human menopausal gonadotropin (Menopur, Ferring, USA) or recombinant FSH (rFSH; Gonal-F, Serono, Switzerland, or Puregon, MSD, USA). Doses ranged from 150 to 450 IU (Bosch et al., 2024).
• Ovulation Trigger: Ovulation was triggered when at least three follicles reached ≥18 mm in diameter. The trigger agents used were either 5,000 IU of hCG (Pregnyl, MSD, USA) or 0.2 mg triptorelin (Decapeptyl, Ferring, Sweden) (Schachter et al., 2008)).
• Oocyte Retrieval: Transvaginal oocyte retrieval was performed 36 hours post-trigger, followed by intracytoplasmic sperm injection (ICSI).
• Fertilization and Embryo Culture

• Oocyte Preparation: Collected oocytes were denuded using hyaluronidase (Ref. 10017, Vitrolife, Goteborg, Sweden). They were then incubated for a minimum of one hour before insemination.
• Insemination: Oocytes were inseminated via ICSI after 4-6 hours of recovery.
• Initial Culture: Fertilized oocytes were cultured in G-1 medium (Ref. 10127, Vitrolife, Goteborg, Sweden) supplemented with 10% serum substitute supplement (SSS; Ref. 99193, Irvine Scientific, USA).
• Day 3 Processing: On day 3 of development, each embryo was gently washed and rinsed using a 200 µm pipette (K-FPIP-1170-10, Cook Medical, USA). A ~10 µm hole was drilled in the zona pellucida using a non-contact laser (Saturn 5 Active Laser System, Research Instrument, UK) (Desai et al., 2020).
• Blastocyst Culture: Embryos were then cultured individually to the blastocyst stage in G-2 medium (Ref. 10131, Vitrolife, Goteborg, Sweden) with 10% SSS.
• Culture Conditions: The sequential culture was carried out in 30 µl microdrops of media under oil (Ref. 10029, Vitrolife, Goteborg, Sweden). Incubation was performed at 37.0°C in a gas environment of 5% CO₂ and 5% O₂ balanced with N₂ (Zeng et al., 2024).
• Trophectoderm (TE) Biopsy

• 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).
• Sample Collection and Processing

• DNA Contamination Minimization:

• Embryos and culture media were managed under stringent sterile conditions.
• Laboratory personnel received comprehensive training in embryo handling protocols and consistently utilized personal protective equipment (masks, caps, gloves).
• All laboratory materials and equipment were exclusively dedicated to the study to prevent cross-contamination.
• Meticulous removal of surrounding cumulus cells was performed prior to microinjection in ICSI cycles or at the time of fertilization in conventional IVF cycles to reduce maternal DNA contamination.
• These practices align with established guidelines for good laboratory practices in IVF settings (Krasic et al., 2021).
• Sample Collection: Paired samples were obtained from 50 embryos that had reached the Day-5 blastocyst stage. From each embryo, we collected the SCM in which it was developing. Subsequently, a corresponding biopsy of the TE was performed and the tissue was collected. All embryos were sourced from a cohort of 20 patients.

• SCM was harvested after embryo culture with care taken not to include cellular material.
• TE biopsies were performed according to standard protocols under strict aseptic conditions.
• Sample Storage: Samples were stored immediately at -80°C until further processing to prevent degradation of DNA.
• Processing for Genetic Analysis: TE samples were processed directly for genetic analysis (Magli et al., 2008).
• DNA Extraction and Sample Preparation

• cfDNA Extraction: cfDNA was extracted from SCM using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany). This kit includes a centrifugation step, protein digestion, and silica membrane-based DNA binding optimized for low-yield samples.
• gDNA Extraction: Genomic DNA (gDNA) was concurrently isolated from the TE biopsy samples using the DNeasy Blood and Tissue Kit (Qiagen), following the manufacturer's instructions (Rubio et al., 2020).
• Microarray Comparative Genomic Hybridization (aCGH)

• Chromosomal Assessment: Chromosomal content was assessed in both cfDNA and gDNA samples via array-based comparative genomic hybridization (aCGH).
• BAC-chip Slides: MACArray Karyo 1440 BAC-chip slides were employed to enable high-resolution, whole-genome profiling.
• Labeling and Hybridization:

• Extracted DNA was fluorescently labeled: cfDNA with Cy3 (green) and TE DNA with Cy5 (red).
• Labeled DNA was hybridized onto oligonucleotide microarrays containing probes across all 23 chromosomes (Mertzanidou et al., 2013).
• Scanning and Data Acquisition:

• Post-hybridization, arrays were scanned using an Agilent SureScan microarray scanner (Agilent Technologies, Santa Clara, USA).
• Signal intensities were quantified, and log₂ ratios of Cy3 to Cy5 fluorescence were calculated to identify chromosomal gains and losses.
• Data Processing and Analysis:

• Chromosomal imbalances were analyzed using Agilent CytoGenomics and Genomic Workbench v7.0.4.0 software.
• Signal data were converted into log₁₀ ratios to determine DNA copy number variations (CNVs).
• Statistical thresholds were applied to minimize false positives and negatives, regarding established concordance frameworks (Yatsenko et al., 2009).
• Validation and Quality Control

• Robust quality control measures included:
• Negative controls during DNA extraction to detect contamination.
• Evaluation of hybridization efficiency via reference DNA consistency.
• Exclusion of results with poor signal-to-noise ratios (Rubio et al., 2019).
• Statistical Analysis

• Comparison of Profiles: Chromosomal profiles of cfDNA and TE samples were compared using Chi-square tests.
• Statistical Significance: P-values <0.05 were considered statistically significant.
• Software Tools

• Statistical Analyses: All statistical analyses were performed using SPSS.
• Visual Data Representations: Visual data representations were generated using GraphPad Prism.

Inclusion criteria

All patients referred for PGT-A.

Exclusion criteria

Patients who had no blastocyst for biopsy were excluded