Incidence and risk factors of monozygotic twinning following ART: analysis of 154 671 live births resulting from single embryo transfer.

study questionWhat is the incidence of and risk factors for monozygotic twinning (MZT) following single embryo transfer (SET) in ART cycles in Australia and New Zealand? SUMMARY ANSWER: MZT occurred in 1.5% of live births following SET, with blastocyst transfer and fresh embryo transfer identified as key risk factors, while vitrified-thaw transfers were associated with a lower MZT risk. WHAT IS KNOWN ALREADY: ART has been associated with a higher incidence of MZT compared to natural conception. Previous studies have suggested younger maternal age, blastocyst culture, fresh embryo transfer, and certain ART techniques, such as assisted hatching and preimplantation genetic testing may elevate MZT risk. However, findings have been inconsistent, and with many prior studies underpowered and few reflecting contemporary ART practices. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study analyzed data from 590 441 SET cycles conducted between 2009 and 2021 in Australia and New Zealand. The analysis included 154 671 live births following autologous SET cycles recorded in the Australian and New Zealand Assisted Reproductive Technology Database (ANZARD). PARTICIPANTS/MATERIALS, SETTING,

methodsThe study focused on autologous fresh and thawed SET cycles. MZT incidence was estimated by applying Weinberg's differential rule, which assumes a 1:1 ratio of sex-concordant and sex-discordant dizygotic twins in the population of twins born following SET cycles. A multivariable logistic regression model with generalized estimating equations was used to identify risk factors for MZT, adjusting for potential misclassification of zygosity due to the absence of DNA confirmation. MAIN RESULTS AND THE ROLE OF CHANCE: The MZT rate was 1.5% among live births following SET. Blastocyst transfer was associated with a nearly 2-fold increase in MZT risk compared to cleavage-stage transfer (adjusted odds ratio [aOR] = 1.99, 95% CI: 1.71-2.31), and vitrified-thaw transfers had a lower MZT risk than fresh transfers (aOR = 0.87, 95% CI: 0.79-0.95). Sensitivity analyses supported these findings, with consistent MZT risk patterns across subgroups by maternal age, fertilization technique, and embryo transfer type (fresh/frozen). LIMITATIONS, REASONS FOR CAUTION: Zygosity estimation was based on Weinberg's differential rule rather than DNA testing, which could lead to some misclassification. Additionally, the study lacked data on embryo quality, a variable with potential influence on MZT risk, and was limited to a retrospective design, potentially introducing treatment and information biases. WIDER IMPLICATIONS OF THE

findingsThis large-scale study identifies blastocyst transfer and fresh embryo transfer as significant MZT risk factor in ART, with potential implications for patient counseling and obstetric care. Future research should further investigate the mechanisms underlying these associations. STUDY FUNDING/COMPETING INTEREST(S): Funding was received from the Ferring Pharmaceuticals Pty Ltd as part of the Ferring FSANZ Leaders in Fertility Research and Education grant (to R.C.P.). The sponsors had no role in the design and conduct of the study; data collection, management, analysis, and interpretation; manuscript preparation, review, or approval; or the decision to submit for publication. FSANZ contracts National Perinatal Epidemiology and Statistics Unit (NPESU) of the University of New South Wales (UNSW) to prepare annual reports and benchmarking reports from the ANZARD: one of those datasets is used in this study. R.C.P. is a Research Fellow of the NPESU, UNSW; C.A.V. is affiliated with the NPESU, UNSW; G.M.C. is an employee of the UNSW and is the Director of the NPESU, UNSW. C.A.V., based at Aristotle University of Thessaloniki (Greece), is a member of the Executive Board of the Hellenic Society of Fertility and Sterility and serves as Senior Deputy of the Steering Committee for the SIG Reproductive Endocrinology of ESHRE. C.A.V. also reports lecture and advisory roles from Merck Ltd, Merck Sharpe & Dohme, Ferring, Organon, Gedeon-Richter, IBSA, Vianex, and Sonapharm; travel support from Merck Ltd, Merck Sharpe & Dohme, Ferring, Organon, Gedeon-Richter, and Vianex; and holds stock or stock options in Virtus Health Ltd, all outside the submitted work. O.F. reports funding from Ferring Pharmaceuticals Pty Ltd, unrelated to this study. TRIAL REGISTRATION NUMBER: N/A.