De novo mutation burden in ART-conceived offspring: no overall increase but subgroup differences by fertilization and transfer strategy

Research question Does assisted reproductive technology (ART), particularly specific procedures such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and frozen embryo transfer (FET), affect the number of de novo mutations (DNMs) in offspring compared to natural conception (NC)? Design In this prospective cohort study, 312 ART families and 157 NC families were initially enrolled. After excluding those with low-quality DNA or kinship discrepancies, the final cohort comprised 454 families with 545 offspring, including 189 IVF (73 fresh, 116 frozen), 117 ICSI (50 fresh, 67 frozen), and 148 naturally conceived families. All subjects underwent whole-genome sequencing at an average depth of >30 × . DNMs were identified and validated using a standardized pipeline. The number of DNMs was compared across conception methods. A linear mixed model (LMM) was used to assess independent effects and interactions of ART parameters. Results No significant increase in DNM counts was observed in ART-conceived offspring compared to the NC group. In exploratory subgroup analyses, offspring conceived via frozen embryo transfer (FET) following ICSI had significantly fewer DNMs than those from fresh embryo transfer after ICSI (mean DNMs: 51.63 vs. 61.59, adjusted mean difference = 10.878, 95% CI: 2.452–19.304, P = 0.003, Bonferroni-corrected), although the ICSI-FET group did not differ significantly from the NC group. LMM analysis restricted to ART cycles identified FET (B=−6.13, P=0.015) and paternal age (B=0.74, P=0.034) as independent factors influencing DNM counts. Conclusions ART procedures do not show an overall increase DNM burden compared to naturally conceived children.; Exploratory subgroup differences by fertilization and transfer strategy were observed; however, these findings require independent replication and should be interpreted with caution. Further studies are warranted to confirm these observations and to investigate potential mechanisms