Cryopreservation causes cryoinjury to oocytes and impairs their developmental competence. Melatonin (MLT) can improve the effect of cryopreservation in animal oocytes. However, no such studies on human oocytes have been reported. In this study, collected in vitro‐matured human oocytes were randomly divided into the following groups: fresh group, MLT‐treated cryopreservation (MC) group, and no‐MLT‐treated cryopreservation (NC) group. After vitrification and warming, viable oocytes from these three groups were assessed for their mitochondrial function, ultrastructure, permeability of oolemma, early apoptosis, developmental competence, and cryotolerance‐related gene expression. First, fluorescence staining results revealed that oocytes from the 10−9 M subgroup showed the lowest intracellular reactive oxygen species and Ca2+ levels and highest mitochondrial membrane potential among the MC subgroups (10−11, 10−9, 10−7, and 10−5 M). In subsequent experiments, oocytes from the 10−9 M‐MC group were observed to maintain the normal ultrastructural features and the permeability of the oolemma. Compared with those of the oocytes in the NC group, the early apoptosis rate significantly decreased (P < .01), whereas both the high‐quality cleavage embryo and blastocyst rates significantly increased (both P < .05) in the oocytes of the 10−9 M‐MC group. Finally, single‐cell RNA sequencing and immunofluorescence results revealed that aquaporin (AQP) 1/2/11 gene expression and AQP1 protein expression were upregulated in the MC group. Therefore, these results suggest that MLT can improve the effect of cryopreservation on human oocytes by suppressing oxidative stress and maintaining the permeability of the oolemma.

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