Objective <p>To determine the most effective insemination protocol for patients undergoing emergency oocyte retrieval in natural cycles, comparing immediate denudation and intracytoplasmic sperm injection (ICSI) versus overnight culture before ICSI.</p> Design <p>Retrospective study comparing two insemination protocols for patients undergoing emergency oocyte retrieval in natural cycles.</p> Subjects <p>Reproductive Medicine Department, Affiliated Reproductive Hospital of Shandong University, from 2015 to 2021.Women undergoing natural cycle IVF treatment with emergency oocyte retrieval due to declining estradiol (E2) levels.</p> Exposure (for observational studies) <p>Two groups were formed based on denudation and ICSI timing. Group E underwent immediate denudation on the day of oocyte retrieval, while Group L underwent overnight culture before ICSI.</p> Main outcome measures <p>Fertilization rate, high-quality embryo rate, oocyte utilization rate, and live birth rate from fresh and frozen embryo transfers.</p> Results <p>In this study, no significant differences were found in overall embryonic development or clinical outcomes between Groups E and Group L. However, Group L exhibited a higher fertilization rate (87.87% vs. 77.12%, P = 0.002), and Group E achieved more higher-quality embryos (52.82% vs. 40.65%, P = 0.036). The oocyte utilization rates were comparable between groups (33.61% vs. 31.98%, P = 0.702). The overall live birth rate was also similar between groups (13.33% vs. 12.5%, P = 0.88).</p> Conclusions <p>These two protocols are feasible for patients in the natural cycle undergoing emergency oocyte retrieval. Group E achieved better results for fresh cycle transplantation. Group L showed a higher fertilization rate with reduced manipulation. To select protocols, clinicians should carefully consider patient-specific factors, including oocyte maturity and the clinical context.</p>