Double Zona Drilling for Trophectoderm Biopsy: A Safe Strategy When Inner Cell Mass Herniates

Background/Objectives: Laser-assisted zona pellucida (ZP) drilling on day 4 embryos is routinely performed in IVF laboratories to facilitate trophectoderm (TE) herniation for blastocyst biopsy. Nevertheless, inner cell mass (ICM) herniation through the initial ZP opening occasionally occurs and may interfere with standard TE biopsy. Methods: This retrospective study assessed the clinical and obstetric safety of a double ZP drilling strategy for TE biopsy in preimplantation genetic testing for aneuploidy (PGT-A) cycles. A total of 560 single euploid embryo transfer cycles were analyzed. Blastocysts were categorized (Groups 1-6) based on ICM/TE herniation patterns and the corresponding biopsy approach. Clinical outcomes were compared between cycles undergoing TE biopsy through a single ZP opening (TE hatching with ICM remaining within the ZP) and cycles requiring a second opening to relocate the biopsy site when the ICM herniated through the original opening or was positioned externally. Results: The single-opening approach of Group 1 accounted for 295 cycles (52.7%), with implantation, miscarriage, and live birth rates of 65.4%, 14.0%, and 56.3%, respectively. The double-opening approach of Group 3 was applied in 21 cycles (3.8%), yielding implantation, miscarriage, and live birth rates of 66.7%, 0%, and 66.7%, respectively. No significant differences were observed between the two strategies in implantation, miscarriage, or live birth rates. Obstetric and neonatal outcomes, including gestational age, birth weight, and monozygotic twinning incidence, were comparable. Fifteen healthy infants were delivered following TE biopsy using the double-opening strategy. Conclusions: These data support incorporating ICM position into TE biopsy decision-making and suggest that creating a second ZP opening to reposition the biopsy site is clinically feasible and does not compromise reproductive or obstetric outcomes in PGT-A cycles.