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 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.

• Laser-Assisted Hatching (Procedure), also known as Assisted Hatching (AH), Laser-Assisted Hatching (LAH): A standardized laser-assisted opening of the zona pellucida was performed either on Day 3 or Day 5 according to randomized group assignment. The only protocol-defined difference between study arms was the timing of assisted hatching; subsequent blastocyst culture, Day-5 assessment, and trophectoderm biopsy procedures were standardized across both groups.

Primary outcomes

• Distribution of PGT-A Genetic Classifications [Following PGT-A of blastocysts biopsied on Day 5 or Day 6 of embryo culture]: Blastocysts with a valid PGT-A result were classified into three mutually exclusive categories: euploid, mosaic, or abnormal. The distribution of these three PGT-A classifications was compared between the Day-3 and Day-5 assisted-hatching groups.
• Blastocyst Morphology at Trophectoderm Biopsy [At trophectoderm biopsy on Day 5 or Day 6 of embryo culture]: Blastocyst morphology was assessed at the time of trophectoderm biopsy using standardized criteria, including blastocyst expansion stage, inner cell mass (ICM) morphology, and trophectoderm (TE) morphology. The distributions of these morphological characteristics were compared between the Day-3 and Day-5 assisted-hatching groups.

Secondary outcomes

• Proportion of Mosaic Blastocysts [Following PGT-A of blastocysts biopsied on Day 5 or Day 6 of embryo culture]: Blastocysts with a valid PGT-A result were classified as euploid, mosaic, or abnormal. The proportion of blastocysts classified as mosaic was calculated for each assisted-hatching group, using the number of blastocysts with a valid PGT-A result as the denominator.

Other outcomes

• Association Between Blastocyst Morphology and PGT-A Classification [Blastocyst morphology at trophectoderm biopsy on Day 5 or Day 6, with subsequent PGT-A classification]: The association between blastocyst morphology at trophectoderm biopsy, including blastocyst expansion stage, inner cell mass (ICM) morphology, and trophectoderm (TE) morphology, and PGT-A classification as euploid, mosaic, or abnormal was evaluated.

Sex: Female, ages: 18 Years to any

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

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• Ngan M Hoang, MSc, Study chair, Center for Reproductive Support, 16A Ha Dong General Hospital
• Tao D Nguyen, MD, PhD, Professor, Principal investigator, Center for Reproductive Support, 16A Ha Dong General Hospital
• Ton D Nguyen, PhD, Principal investigator, Institute of Biology, Vietnam Academy of Science and Technology
• Son T Trinh, MD, PhD, Principal investigator, Vietnam Military Medical University, Ministry of National Defence