This study aims to evaluate the feasibility and accuracy of using cfDNA analysis of spent embryo culture media as a non-invasive approach for PGD. Specifically, the objectives of the study are:

1. To collect SCM from blastocysts of good quality obtained from IVF cycles.
2. To extract cfDNA from the collected SCM using a commercially available kit.
3. To assess the chromosomal content in both cfDNA and gDNA samples via array-based comparative genomic hybridization (aCGH).
4. To compare the results obtained using cfDNA analysis to those obtained using conventional invasive PGD methods, such as blastomere biopsy.
5. To evaluate the potential advantages of using cfDNA analysis of spent embryo culture media for PGD, including reduced risk of harm to the embryo, reduced cost, and increased efficiency.

Materials and Methods:

1. 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).

2. 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).

3. 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).

4. 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).

5. 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).

6. 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).

7. 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).

8. 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).

9. 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.

10. 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

only infertile females who are subjected to ICSI and PGD are eligible for the study

Patients underwent controlled ovarian stimulation using either:

• Gonadotropin Administration:
• Ovulation Trigger:
• Oocyte Retrieval:
• Oocyte Preparation
• Insemination:
• Initial Culture
• Day 3 Processing:
• Blastocyst Culture:
• Culture Conditions:

Trophectoderm (TE) Biopsy Sample Collection and Processing DNA Extraction and Sample Preparation 7. Microarray Comparative Genomic Hybridization (aCGH)

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