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

This prospective randomized controlled trial compared two standardized assisted-hatching strategies in IVF-ICSI cycles undergoing preimplantation genetic testing for aneuploidy (PGT-A). Eligible participants were randomly allocated in a 1:1 ratio to Day-3 assisted hatching (Arm A) or Day-5 assisted hatching (Arm B). Randomization was performed at the participant/IVF-ICSI cycle level using fixed block randomization with a block size of 4. Randomization was not stratified by maternal age, anti-Müllerian hormone level, or other clinical characteristics. All embryos derived from each randomized cycle followed the assigned assisted-hatching strategy.

In Arm A, laser-assisted hatching was performed on Day-3 embryos. In Arm B, laser-assisted hatching was performed on Day-5 blastocysts. In both groups, blastocysts were evaluated on Day 5 and subsequently cultured for approximately 6-7 additional hours before trophectoderm biopsy. This interval was standardized across both study arms and was consistent with the routine laboratory workflow, while allowing additional time for blastocyst expansion and hatching before biopsy. Blastocyst morphology used for the primary morphological analyses was assessed at the time of trophectoderm biopsy according to standardized criteria, including blastocyst expansion stage, inner cell mass (ICM) morphology, and trophectoderm (TE) morphology.

Blastocysts meeting biopsy criteria underwent trophectoderm biopsy followed by PGT-A using next-generation sequencing. PGT-A was performed using paired-end sequencing with the EmbryoMap™ Kit (Vitrolife) on the Illumina MiSeq sequencing system. PGT-A testing and interpretation were conducted by a separate genetics laboratory whose analysts were blinded to the assisted-hatching allocation.

A total of 158 participants/IVF-ICSI cycles were randomized, with 79 allocated to each study arm. The study evaluated blastocyst development and morphology and PGT-A outcomes according to the timing of assisted hatching, as well as the relationship between blastocyst morphology and genetic findings.

Participants and treating clinicians were unaware of the assigned assisted-hatching timing. The embryologist performing assisted hatching could not be blinded at the time of the intervention because knowledge of the assigned timing was required to perform the procedure.

Inclusion criteria

• Female participants undergoing IVF-ICSI with an indication for PGT-A
• Age ≥18 years; no prespecified upper age limit. Age was recorded for subsequent analyses
• Anti-Müllerian hormone (AMH) level ≥1.0 ng/mL
• Antral follicle count (AFC) ≥5 follicles
• Controlled ovarian stimulation using an antagonist or progestin-primed ovarian stimulation (PPOS) protocol
• Use of ejaculated sperm prepared by density-gradient centrifugation or swim-up
• Written informed consent for participation

Exclusion criteria

• History of ≥3 failed IVF cycles or ≥3 consecutive miscarriages
• Indication for PGT-M or PGT-SR, including monogenic disorders or structural chromosomal rearrangements
• Severe uncontrolled medical or autoimmune conditions considered likely to affect oocyte or embryo development
• No metaphase II (MII) oocytes available for ICSI
• Use of surgically retrieved sperm
• Use of advanced sperm-selection techniques, including IMSI, PICSI, or MACS
• Polycystic ovary syndrome diagnosed according to the Rotterdam criteria