Intracytoplasmic Sperm Injection-Associated Increased Risk of Monozygotic Twins Following Elective Single Embryo Transfer: A Retrospective Cohort Analysis

Abstract Objective: To explore the primary factors influencing monozygotic twinning (MZT) in patients undergoing assisted reproductive technology (ART) at a single center to clarify the mechanisms of MZT formation. Methods: This retrospective cohort study analyzed 2952 elective single embryo transfer (eSET) cycles conducted between December 2014 and December 2023 at the First Affiliated Hospital of Xiamen University, which resulted in 1363 clinical pregnancies. The examined variables included parental age, fertilization method, cycle type (fresh vs . frozen-thawed), and embryo stage (cleavage vs . blastocyst). Clinical pregnancy was diagnosed through human chorionic gonadotropin (hCG) levels 12–14 days post-transfer and confirmed via transvaginal ultrasound at 6–7 weeks of gestation. MZT was defined by the presence of additional gestational sacs (dichorionic) or two embryos within a single sac (monochorionic), while a monozygotic singleton (MZS) was defined as one embryo per sac. Results: The overall MZT incidence was 0.85% (25/2952) per cycle and 1.83% (25/1363) per pregnancy (1 morula, 4 cleavage-stage, and 20 blastocyst transfers). After excluding the morula case, 24 MZT pregnancies were matched 1:4 with singletons. After matching, embryo stage and cycle type (16 fresh and 8 frozen-thawed MZT cases) demonstrated no significant effect on MZT rates. When the analysis was restricted to blastocyst transfer (20 MZTs and 80 singletons), neither parental age nor cycle type showed a significant correlation with MZT occurrence, whereas the fertilization method was significantly associated with MZT frequency. Regarding embryo morphology, all 20 MZT blastocysts featured high-quality (grade A or B) inner cell masses and trophectoderms, with no grade C embryos observed. Multivariate logistic regression confirmed that the use of intracytoplasmic sperm injection (ICSI) significantly increased the risk of MZT, whereas parental age and cycle type did not. Conclusion: The fertilization method affects MZT incidence, with the ICSI procedure and sperm quality serving as key correlates. Furthermore, high-quality blastocysts may possess a higher potential for MZT, providing new insights for the development of individualized embryo transfer strategies.