Day-3 vs Day-5 Assisted Hatching: Impact on Blastocyst Morphology and PGT-A Outcomes

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)