Zona opening size by laser-assisted hatching and monozygotic multiple pregnancy after frozen-thawed single blastocyst transfer

Summary Manipulation of the zona pellucida, including laser-assisted hatching (LAH), has been associated with an increased risk of monozygotic multiple pregnancy (MMP). A leading hypothesis suggests that embryonic splitting could be triggered by the division of the inner cell mass as the blastocyst herniates through the zona pellucida. The present study aimed to investigate whether the size of the zona pellucida opening created by LAH influences the prevalence of MMP in frozen-thawed single blastocyst transfer (SBT) cycles. This retrospective observational study included 9452 pregnancies resulting from frozen-thawed SBT, of 13,271 clinical pregnancies between January 2020 and October 2024. MMP was defined as the presence of ≥2 gestational sacs and/or ≥2 fetuses after SBT. Between January and May 2021, the LAH opening size was enlarged from 30° to 90°. Prevalence of MMP was compared between 30° and 90° zona openings. Among the 9452 pregnancies analysed, 175 (1.85%) were MMP cases, including 169 twin and 6 triplet pregnancies. No significant differences in maternal age, fertilization method, blastocyst grade, culture duration or embryo biopsy rate were observed between singleton and MMP cases. In univariate analysis, a small zona opening was associated with an increased risk of MMP (odds ratio [OR] 1.51, 95% confidence interval [CI] 1.01–2.29, p = 0.047); however, this association was not significant after adjustment for confounders (OR 1.47, 95% CI 0.98–2.22, p = 0.06). A small zona pellucida opening created by LAH showed a trend towards an increased risk of MMP and warrants further investigation.