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

Infertility; In Vitro Fertilization; Embryo Development

Assisted hatching, Day-3 vs Day-5, Blastocyst morphology, Trophectoderm biopsy, PGT-A, Aneuploidy, Mosaicism

This randomized controlled trial compared assisted hatching (AH) performed on Day 3 versus Day 5 in patients undergoing IVF-ICSI with preimplantation genetic testing for aneuploidy (PGT-A). Eligible IVF-ICSI cycles were randomly assigned to one of the two AH strategies, and all embryos from each randomized cycle followed the assigned strategy. The study evaluated whether the timing of AH was associated with differences in blastocyst development, blastocyst morphology, and PGT-A outcomes. The findings are intended to help optimize the timing of assisted hatching in IVF laboratory practice.

This prospective randomized controlled trial compared two standardized assisted-hatching strategies in IVF-ICSI cycles undergoing preimplantation genetic testing for aneuploidy (PGT-A). Eligible IVF-ICSI cycles 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 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. Women who underwent more than one eligible IVF-ICSI cycle could contribute more than one randomized cycle, with each eligible cycle assigned according to the randomization sequence. 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 141 women contributed 158 randomized IVF-ICSI cycles to the study, with 79 cycles allocated to Day-3 assisted hatching and 79 cycles allocated to Day-5 assisted hatching. Sixteen women contributed more than one IVF-ICSI cycle: 15 women contributed two cycles and one woman contributed three cycles. Statistical analyses accounted for within-participant correlation arising from repeated IVF-ICSI cycles and multiple embryos contributed by the same woman, where appropriate. 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.

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