Clinical implications of zygotes showing 2.1 pronuclei: an ASPIRE Embryology Special Interest Group committee opinion

Atypical fertilisation patterns such as 2.1 pronuclei (2.1PN), defined by two normally sized pronuclei and an additional smaller pronucleus, present a clinical challenge due to inconsistent classification and uncertainty regarding their development and genetic potential. This scoping review aims to synthesise the existing evidence, identify gaps in the literature and propose a committee opinion on 2.1PN zygotes, including a working provisional definition, to guide clinical practice and future studies. A PubMed search for studies reporting 2.1PN zygotes and their developmental and clinical outcomes was conducted in May 2025. This review shows conflicting evidence regarding blastulation rates between 2.1PN and 2PN zygotes, and findings on preimplantation genetic testing and live birth rates remain inconclusive due to limited study numbers and sample sizes. Regardless, 2.1PN zygotes should be distinguished from classic 3PN zygotes, cultured to the blastocyst stage and genetically assessed if available. Based on current evidence and expert consensus, we propose a provisional working definition of the 2.1PN zygote to support standardised classification. Current evidence suggests that 2.1PN zygotes can be viable and warrant differentiation from 3PN zygotes. Future research should clarify the mechanisms underlying 2.1PN formation, determine neonatal and longer-term health outcomes following 2.1PN-derived embryo transfer and validate findings in larger cohorts.