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.

Melatonin (MT) regulates reproductive performance as a potent antioxidant; however, its beneficial effects on oocyte development remain largely unknown, especially in human oocytes. The collected 193 immature oocytes from the controlled ovarian hyperstimulation (COH) cycle underwent in vitro maturation (IVM) in IVM medium contained 10 μmol/L MT (n = 105, M group) and no MT (n = 88, NM group), followed by insemination and embryo culture. The results showed that the high-quality blastocyst formation rate in the M group (28.4%) was significantly higher than that in the NM group (2.0%) (P = .0001), and the aneuploidy rate was low (15.8%). In the subsequent clinical trials, three healthy infants were delivered. Next, single-cell RNA-seq data revealed 1026 differentially expressed genes (DEGs) between the two groups, KEGG enrichment analysis revealed that the majority of DEGs involved in oxidative phosphorylation pathway, which associated with ATP generation, was upregulated in the M group. Finally, confocal fluorescence staining results revealed that the mitochondrial membrane potential in the oocytes significantly increased and intracellular ROS and Ca 2+ levels greatly decreased in the M group. Melatonin can promote the development of immature human oocytes retrieved from the COH cycle into healthy offspring by protecting mitochondrial function.

Research funding is provided by the Ministry of Science and Technology of China [2012CB944704; 2012CB966702], the National Natural Science Foundation of China [Grant number: 31171429] and Beijing Advanced Innovation Center for Structural Biology. The authors declare no conflict of interest.