Purpose To investigate the factor of three pro-nuclei (3PN) incidence and its effect on pregnancy outcomes in women with good prognosis undergoing in vitro fertilization with donor sperm (IVFD). Methods We retrospectively identified all IVFD embryo transfers (IVFD-ETs) for the first time between January 2014 and January 2021 at our hospital. Women aged ≤ 35 years who received the long/ultra-long follicular phase agonist protocol were included. The patients were divided into two groups as

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... : 3PN = 0% group included patients with no 3PN zygotes and 3PN > 0% group included patients with 3PN zygotes. Results We observed that the number of retrieved oocytes was independently associated with 3PN incidence (OR: 1.078, 95% CI 1.045–1.113, p < 0.001). Both the 3PN = 0% and 3PN > 0% groups showed no significant differences in the cleavage, high-quality embryo and available embryo rates (p > 0.05). Our data indicated that the 3PN = 0% group had significantly higher ongoing pregnancy (68.50 versus 62.76%, p = 0.033) and live birth (66.93 versus 59.84%, p = 0.009) rates compared with that in the 3PN > 0% group. The miscarriage rate was significantly lower in the 3PN = 0% group than that in the 3PN > 0% group (7.78 versus 12.26%, p = 0.026). The multinucleation at two-cell stage (MN2) rate was slightly higher in the 3PN > 0% group than that in the 3PN = 0% group (23.57 versus 16.11%, p = 0.085). Conclusions The incidence of 3PN zygotes made negative influence on the pregnancy outcomes in women with good prognosis undergoing IVFD-ETs. 3PN incidence might be associated with normal embryo multinucleation at two-cell stage.