This randomized controlled trial evaluates the effect of assisted hatching (AH) timing on embryo development and genetic outcomes in in vitro fertilization (IVF) cycles with preimplantation genetic testing for aneuploidy (PGT-A). Eligible patients undergoing IVF with PGT-A will have embryos randomized to receive assisted hatching either on Day-3 or Day-5 of culture, prior to trophectoderm biopsy. The primary outcome is the rate of aneuploid blastocysts. Secondary outcomes include blastocyst morphology, expansion stage, and the correlation between morphology and genetic results. The study aims to determine whether earlier or later assisted hatching affects embryo viability and the accuracy of genetic testing. The trial will provide evidence to optimize laboratory protocols in IVF centers.

This prospective randomized controlled trial compares two standardized laboratory procedures of assisted hatching (AH) timing in IVF cycles undergoing PGT-A. In Arm A, AH will be performed on Day-3 embryos; in Arm B, AH will be performed on Day-5 blastocysts prior to trophectoderm biopsy. A total of approximately 200 IVF cycles, involving around 650 embryos, will be included. Randomization will be conducted using block randomization. The primary outcome is the proportion of aneuploid blastocysts identified by next-generation sequencing (NGS). Secondary outcomes include blastocyst morphology (ICM, TE, expansion), biopsy efficiency, and correlation between morphological parameters and genetic outcomes.

All procedures are part of the routine IVF-PGT workflow, and no additional risks to patients are introduced beyond standard care. The trial has been approved by the Institutional Review Board of 16A Ha Dong General Hospital.

Inclusion Criteria:

• IVF cycles with PGT-A
• Oocyte age 20-45 years (donor cycles: donor's age)
• AMH 1-10 ng/mL within 12 months
• AFC ≥5 follicles (2-9 mm, both ovaries, day 2-5)
• Controlled ovarian stimulation: antagonist or PPOS protocols
• Stratified analysis by age (<30, 30-34, 35-39, ≥40)
• Sperm source: ejaculated samples prepared by density-gradient or swim-up
• No surgical sperm retrieval
• No advanced sperm selection (IMSI, PICSI, MACS)

Exclusion Criteria:

• History of ≥3 failed IVF cycles or ≥3 consecutive miscarriages
• Indication for PGT-M or PGT-SR (monogenic disorders, translocations)
• Severe medical/autoimmune conditions affecting oocytes/embryos (e.g. uncontrolled diabetes, active lupus)
• No MII oocytes available for ICSI
• Embryos not eligible for biopsy per SOP (early zona hatching, degeneration, poor expansion)
• Sperm from surgical retrieval or advanced sperm selection (IMSI, PICSI, MACS)
• PCOS diagnosed by Rotterdam criteria (≥2/3 features)

The plan for sharing individual participant data (IPD) has not been finalized. De-identified embryo-level data may be shared upon reasonable request for academic purposes after study completion.

The PGT-A analysts evaluating the NGS results were blinded to the assisted hatching timing.