Not all abnormal fertilization is equal: occurrence, developmental competence, and ploidy outcomes of 1PN, 3PN, and 2.1PN zygotes in ICSI/PGT-A cycles

To characterize the occurrence, developmental competence, and ploidy outcomes of abnormally fertilized oocyte (AFO) subtypes in ICSI/PGT-A cycles, and to assess whether maternal age independently predicts abnormal fertilization.

Prospective observational study including 1033 ICSI/PGT-A cycles (6373 inseminated oocytes). AFO occurrence was analyzed by binomial logistic regression and multivariable analysis. In 664 time-lapse cycles, pronuclear status was reassessed at 22-24 hpi (Check II) after routine evaluation at 16-18 hpi (Check I). Ploidy analysis was performed on 78 AFO-derived blastocysts by SNP genotyping using NGS technology.

AFOs accounted for 6.8% of inseminated oocytes (1PN 3.1%, 3PN 2.1%, 2.1PN 1.2%, > 3PN 0.4%). Maternal age was the only independent predictor (OR 1.04/year; p < 0.05), mainly for 3PN and 2.1PN. Blastocyst development differed across categories (1PN 11.1%, 3PN 25.7%, 2.1PN 40.5% vs. 2PN 54.4% p < 0.05), and 1PN and 2.1PN were mainly diploid (71.4% and 88.9% respectively), though both were lower than 2PN (99.9%; p < 0.001), while 3PN were mostly triploid (73.3%). The rate of blastocysts suitable for transfer calculated per zygote was 1PN 2.0%, 3PN 0.8%, and 2.1PN 8.1%, markedly lower than 2PN (22.5%; p < 0.001). Pronuclear status could change after the standard assessment window: 26% of 2.1PN-derived blastocysts were initially classified as 2PN, suggesting that single-timepoint assessment may misclassify fertilization.

AFO subtypes differed in clinical potential according to their developmental competence and ploidy status. Time-lapse monitoring further indicated that some AFOs may be misclassified in a single static pronuclear assessment. Therefore, both ploidy assessment and PGT-A should be considered before the clinical use of AFO-derived embryos.