Performing Assisted Hatching on Day 4 next to the area of early cavitation reduces the chances inner cell mass (ICM) herniation and simplifies trophectoderm biopsy

Abstract Study question Can the location of the opening in the zona pellucida during Assisted Hatching reduce the herniation of cells in the ICM and simplify trophectoderm biopsy? Summary answer Performing Assisted Hatching on D4, next to an area of the zona pellucida (ZP) close to first embryo cavitation, reduces the hatching of ICM cells. What is known already Trophectoderm biopsy is performed on blastocysts to perform preimplantation genetic testing of mutations and/or aneuploidies. To simplify the excision of trophectoderm cells, Assisted Hatching (AH) is typically performed on D3 at the cleavage/morula stage by creating a hole in the ZP through which some cells will herniate. This opening is traditionally performed on a random area of the ZP and, consequently, predicting whether the trophectoderm or the ICM cells will hatch first is not possible. Should the latter be the case, the biopsy procedure would need to be adjourned until trophectoderm cells become accessible. Study design, size, duration Regular AH was performed on mouse embryos on D3 by creating a 10µm opening on a random area of their ZP. A second group of embryos was cultured until D4 and the opening of the ZP was performed in a directed way (AHD) close to where the first cavitation of the cells was observed. Both groups were compared with a third control group to which no opening was made. Participants/materials, setting, methods Thirty mouse embryos were allocated into each test group (n = 90) and cultured in an Embryoscope time-lapse incubator to better determine the first cavitation of the cells. Laser-assisted AH/AHD was performed on the Embryoslide dish. Blastocyst formation, quality and hatching rates were compared among all groups, and the belongingness of the first herniated cells to the trophectoderm/ICM was annotated individually by D5/D6. Statistical analyses were performed by Fisher’s exact test, and significance set at 5% (α = 0.05). Main results and the role of chance The blastocyst formation and hatching rates were close to 100% in all three groups with no differences among any of them, indicating that neither AH nor AHD had a detrimental effect on embryo development between D3-D6 or blastulation. In more than a third of the blastocysts of the non-treated control group, the first cells to herniate out of the ZP belonged to the ICM (38.2%), similar to what was observed in the random AH group (44.4%). By performing AHD on day 4 in the ZP next to the area of first embryo cavitation, the incidence of the ICM hatching first was significantly reduced to only 11.1% of the cases (p ≤ 0.01). Interestingly, the ratio of blastocysts that were able to completely hatch out of the ZP by day 6 was reduced both in the AH and AHD (2.8% and 5.6%, respectively) compared to the control group with no opening on the ZP (42.9%, p ≤ 0.0002). Limitations, reasons for caution The present study has been performed on the mouse model and AH/AHD timings may need to be adjusted if used in the human. Time-lapse technology has eased the recognition of early cavitation stages to perform AHD, which could result more complex in a regular culture setting. Wider implications of the findings Trophectoderm biopsy depends on the unique rate of development of each blastocyst, which complicates its integration in the laboratory workflow. The results of this study could improve the quality and simplicity of the biopsy technique by directly targeting the appropriate subgroup of cells and making them easier to be excised. Trial registration number not applicable