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Purpose: The objective of this retrospective study was to determine whether patients undergoing in vitro fertilization (IVF) benefit from reducing the gamete co-incubation time. Methods: Patients ( n = 570) were enrolled, including 281 patients in the reduced incubation time group (2-h incubation) and 289 patients in the standard IVF group (18-h incubation). Results: The observed outcomes, including the clinical pregnancy rate (CPR), implantation rate (IR), live birth rate (LBR), and miscarriage rate (MR), were similar between the two groups. When the data were divided into two subgroups based on the maternal age (≤30 and >30 years), the rates of top-quality embryos (30.83 vs. 25.89 %; p = 0.028), CPR (66.67 vs. 42.11 %; p = 0.013), and IR (41.90 vs. 31.25 %, p = 0.019) of the 2-h incubation group were significantly higher in the younger subgroup. However, for older patients, only a lower MR (7.59 vs. 20.83 %; p = 0.019) was achieved. Reducing the time of incubation still improved the CPR (OR = 1.993, 95 % CI 1.141-3.480) and MR (OR = 3.173, 95 % CI 1.013-9.936) in the younger and older subgroups, respectively, after it was adjusted for potential confounders. Conclusions: Reducing incubation time improves the clinical results of IVF, although the LBR is not statistically different between the 2- and 18-h incubation time groups. And the specific clinical outcomes of reducing incubation time varied between the >30-year-old and the ≤30-year-old. [ABSTRACT FROM AUTHOR]