A large retrospective study on 1PN embryo transfer supports the need for updated harmonized best practice guidelines.

study questionDo the genetic and clinical outcomes of monopronuclear blastocysts (MPBs) differ between standard insemination and intracytoplasmic sperm injection cycles, and what do these differences imply for risk stratification and individualized clinical decision-making? SUMMARY ANSWER: IVF-derived MPBs demonstrate significantly lower rates of uniparental inheritance than ICSI-derived MPBs (96.9% vs 65.9%) biparental inheritance (P < 0.001), with comparable clinical and neonatal outcomes to 2PN blastocysts following transfer of euploid biparental embryos. WHAT IS KNOWN ALREADY: A proportion of monopronuclear (1PN) zygotes can develop into euploid blastocysts and, following transfer, result in healthy live births, yet these embryos are widely discarded following fertilization check due to atypical pronucleation. The mechanisms underlying 1PN formation are varied and include asynchronous pronuclear formation, early pronuclear fusion, and premature pronuclear breakdown, meaning a subset may represent normally fertilized diploid zygotes missed at static assessment. STUDY DESIGN, SIZE, DURATION: Retrospective cohort study of 1PN embryos (N = 13 203) derived from IVF (n = 5266) or ICSI (n = 5464) inseminations across 10 730 cycles performed at multiple Australian clinics between January 2010 and December 2023. PARTICIPANTS/MATERIALS, SETTING,

methodsEmbryos were defined as 1PN by the appearance of a single pronucleus at fertilization check 16-18 h post-insemination. Time-lapse footage was reviewed on Day 3 to identify late appearing 1PNs and exclude late second pronucleus appearance. Suitable blastocysts underwent trophectoderm biopsy for pre-implantation genetic testing for aneuploidy (PGT-A) and short tandem repeat (STR)-based biparental inheritance testing; only euploid embryos with confirmed biparental inheritance were available for frozen embryo transfer. Outcomes assessed included ploidy, biparental inheritance, blastocyst development, utilization, morphokinetics, pregnancy, live birth, and maternal and neonatal outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: IVF-derived MPBs had similar aneuploidy rates to two pronuclei (2PN) embryos (37.3% vs 33.9%) and 440/454 (96.9%) demonstrated biparental inheritance. ICSI-derived MPBs had higher aneuploidy rates (45.5% vs 31.5%, P < 0.05) and only 108/164 (65.9%) had biparental inheritance. Uniparental inheritance was predominantly maternal (IVF 92.8%; ICSI 94.6%). Both IVF and ICSI MPBs were less likely to reach blastocyst stage by Day 5 than 2PN embryos (IVF 19.3% vs 63.3%; ICSI 9.7% vs 60.6%, P < 0.05), and biparental IVF-1PN zygotes were more likely to have more nucleoli compared with uniparental IVF-1PN zygotes (P = 0.008). For embryos with confirmed biparental inheritance, there was no significant difference in clinical pregnancy, ongoing pregnancy, live birth rates, or neonatal outcomes compared with 2PN blastocysts. In approximately one in six cycles containing a 1PN embryo, no utilizable 2PN embryo were available (IVF 15.6%; ICSI 16.7%), with the 1PN embryo representing the sole option for embryo utilization. LIMITATIONS, REASONS FOR CAUTION: Retrospective single-entity design introduces potential selection bias and limits generalizability. Uniform protocols across sites preclude the level of evidence required for formal guideline revision. Differential use of time-lapse imaging for ICSI versus static assessment for IVF embryos may contribute to differences in 1PN identification rates between fertilization methods. The STR-based biparental classification platform has not been validated against an orthogonal technology for parental origin calling in 1PN embryos, and the possibility of triploid misclassification or absorption into unreported inconclusive outcomes cannot be excluded. WIDER IMPLICATIONS OF THE

findingsThese findings support a risk-stratified approach to MPB management based on fertilization method. IVF-derived MPBs meeting specific morphological and developmental criteria demonstrate a low-risk profile that warrants reconsideration of genetic testing requirements and may inform individualized consent discussions, particularly where 2PN embryos are unavailable. ICSI-derived MPBs carry a substantially higher risk of uniparental inheritance and comprehensive genetic testing remains indicated. STUDY FUNDING/COMPETING INTEREST(S): No funding was attached to this study. The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.