Fertilization failure assay identifies stage-specific arrests in couples with total fertilization failure

Fertilization is the process by which sperm and oocyte recognize each other and fuse to form a zygote. Fertilization failure is a common issue encountered in ART. However, clinically, there is a lack of identification of the specific stage at which fertilization is arrested and the development of personalized improvement strategies. In this study, we conducted a fertilization failure assay (FFA) in a cohort of 445 couples undergoing IVF (n = 394) and ICSI (n = 51) who experienced total fertilization failure (TFF). In subsequent cycles, comparative analysis was performed to assess both fertilization rates and good-quality embryo rates between couples at different arrest stages undergoing either conventional ICSI or ICSI with assisted oocyte activation (ICSI-AOA). Additionally, whole-exome sequencing (WES, n = 20) was implemented to investigate potential genetic mutations associated with specific fertilization arrest stages. It found that unfertilized oocytes were categorized into four fertilization arrest phenotypes: (i) sperm penetration/fusion deficiency (SPD), (ii) histone incorporation deficiency (HID), (iii) pronuclear-formation deficiency (PFD), and (iv) mixed deficiency. SPD was the primary cause of TFF after IVF (67.8%), and this deficiency was circumvented in ICSI cycles. HID and PFD were the causes of TFF after ICSI. Couples with ≥50.0% oocytes arrested at the histone incorporation stage or ≥75.7% oocytes arrested at the pronuclear formation stage were at high risk of low fertilization rate following ICSI. Couples with ≥50.0% of oocytes arrested at the histone incorporation stage may achieve improved fertilization and embryo outcomes through ICSI-AOA. Genetic mutations associated with male-factor etiologies of fertilization failure have been identified in this patient population. In contrast, for couples with arrest at the PFD, ICSI-AOA shows no beneficial effects on fertilization or embryo development. Genetic mutations associated with female-factor etiologies of fertilization failure have been identified in this patient population. In conclusion, FFA delineates the arrest stages during fertilization failure, facilitates analysis of insemination progression, and provides clinical reference for subsequent treatment cycles.