O-102 The utility of human two plus one small pronucleated zygotes (2.1pn) based on clinical outcomes and novel ploidy analysis

Study question Are human embryos arising from two plus one small pronucleated zygotes, referred to as 2.1 pronuclei (PN), clinically useful? Summary answer Five healthy babies were born from vitrified-warmed 2.1PN blastocysts. In ploidy analysis, most of the 2.1PN were biparental diploid, indicating clinical usability. What is known already Embryos identified as 2.1PN, which have two normal-sized PN with one additional small pronucleus, unlike 3PN embryos, are considered a type of atypical pronuclear zygote. Their clinical usefulness has been discussed, but the number of subjects in each study has been small and their characteristics are still unknown. Study design, size, duration This study includes a retrospective embryo cohort study and a prospective experimental study. In a total of 490 cycles, 2,231 2PN zygotes and 514 2.1PN zygotes were obtained between August 2018 and July 2024. Twenty-seven 2.1PN-derived blastocysts, which were—at the patient’s request—discarded and donated for research, were subjected to biopsy and ploidy analysis as a prospective experimental study. Participants/materials, setting, methods 2.1PN was defined as two normal-sized PN with one additional small pronucleus no larger than half the normal size and with only one nucleolus precursor body. We compared the embryo development rates and clinical results between 2PN and 2.1PN embryos in the sibling embryo study. For ploidy analysis, we analyzed all chromosomes by a multiplex polymerase chain reaction–based target sequence method. The reference allele and alternative allele of each single-nucleotide polymorphism were counted. Main results and the role of chance Of the 514 2.1PN embryos, the average diameter of two normal-sized PN in 2.1PN embryos was 24.4±3.6µm, which was significantly smaller than 25.3±2.9µm in 2PN embryos (P < 0.05). The average diameter of small PN in 2.1PN embryos was 10.8±2.6µm. Evaluation of the embryonic development of 2.1PN embryos showed the blastocyst development rate was 35.3% (176/499), which was significantly lower than the 55.3% (1195/2162) of 2PN embryos (P < 0.05). Furthermore, the good blastocyst rate (ICM and TE grade 1 or 2) for 2.1PN embryos was 18.4% (92/499), significantly lower than the 29.0% (626/2162) for 2PN embryos (P < 0.05). Comparison of the clinical results of frozen-thawed blastocyst transfer showed the clinical pregnancy rate was 22.6% (7/31), which was significantly lower than the 46.4% (209/450) of 2PN embryos (P < 0.05). No congenital anomalies were found in the five babies derived from 2.1PN embryos. In the results of the ploidy analysis, 96.1% (25/26) of the analyzed 2.1PN embryos were diploid, while one embryo (3.8%, 1/26) was triploid. One embryo could not be analyzed. The diameter of the small PN of the triploid embryo was 15.0 µm, which was larger than the average for diploid embryos (9.8±1.8 µm). Limitations, reasons for caution The number of 2.1PN embryos used for embryo transfer and ploidy testing was limited. Further research is needed to clarify the impact of embryos derived from 2.1PN on neonates. Additionally, because this is a sibling oocyte study, the influence of case characteristics on the occurrence of 2.1PN is unknown. Wider implications of the findings Our results suggest 2.1PN embryos have lower embryonic developmental and pregnancy potential than 2PN embryos. Most of the 2.1PN were diploid, suggesting clinical usability, despite these usually being discarded. However, since triploid embryo was also observed, it’s recommended to perform embryo transfer following a combination of PGT-A and ploidy analysis. Trial registration number No