Context: N-acetyl-cysteine (NAC) is a potent antioxidant that can be used for many gynecological diseases such as polycystic ovary syndrome and endometriosis. Controlled ovarian hyperstimulation (COH) is a critical step in infertility treatment. Our previous clinical studies have shown that repeated COH led to oxidative stress in follicle fluid and ovarian granulosa cells. Aims: In this study, we investigated whether NAC could inhibit oxidative stress in mice caused by repeated COH and improve the mitochondrial function of oocytes. Methods: Female Institute of Cancer Research (ICR) mice were randomly assigned into three groups: normal group, model (repeated COH) group, NAC group. We examined the morphology, number and quality of mitochondria. The mechanism of regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) by NAC to ameliorate oxidative stress was also investigated. Key results: Repeated COH caused oxidative damage in ovaries and oocytes and decreased oocyte quality, while NAC prevented oxidative damage and increased oocyte mitochondrial function. In in vitro experiments, it was verified that NAC can promote the nuclear translocation of Nrf2, which transcriptionally activates the expression of superoxide dismutase and glutathione peroxidase, which removed excessive reactive oxygen species that causes mitochondria damage. Conclusions: The results suggest that NAC raises mitochondrial function in oocytes and improves oocyte quality through decreasing oxidative stress in mice with repeated COH. The underlying mechanism is related to the regulation of the Nrf2 signaling pathway. Implication: This study provides a meaningful foundation for the future clinical application of NAC during repeated COH.

Vascular endothelial growth factor receptor-2 (VEGFR-2) regulates the mitogen-activated protein kinase (MAPK) signaling pathway and plays an important role in angiogenesis. Bu Shen Zhu Yun decoction (BSZYD) can improve endometrial receptivity and embryo implantation rates in patients undergoing in vitro fertilization. However, whether BSZYD improves endometrial receptivity via angiogenesis remains unclear. Here, we investigated the effects of BSZYD on the proliferation, migration, and angiogenesis of human endometrial microvascular endothelial cells (HEMECs) and found that BSZYD upregulated the expression of cyclin D1, matrix metalloproteinase 9 (MMP9), and proliferating cell nuclear antigen (PCNA) in HEMECs. Cell Counting Kit 8 assay, scratch-wound assay, and Tube Formation Assay results showed that BSZYD promoted the proliferation, migration, and angiogenesis of HEMECs. Western blot analysis results revealed the activation of the MAPK signaling pathway by BSZYD through the upregulation of VEGF and VEGFR-2 expression. Together, these findings highlight the novel mechanism underlying BSZYD-mediated improvement in endometrial receptivity through the MAPK signaling pathway.

Morroniside is the main ingredient of Cornus officinalis and has a variety of biological activities including antioxidative effects. Ovarian granulosa cells (GCs) are responsible for regulating the development and atresia of follicles, which are susceptible to oxidative stress. In this study, we determined whether morroniside can inhibit the oxidative stress of GCs induced by hydrogen peroxide (H₂O₂), leading to improved oocyte quality. The oxidative damage and apoptosis of ovarian GCs cultured in vitro were induced by the addition of H₂O₂. After pretreatment with morroniside, the levels of ROS, MDA, and 8-OHdG in ovarian GCs were significantly decreased. Morroniside significantly upregulated p-Nrf2 and promoted the nuclear translocation of Nrf2, which transcriptionally activated antioxidant SOD and NQO1. In addition, morroniside significantly regulated the levels of apoptosis-related proteins Bax, Bcl-2, cleaved caspase-9, and cleaved caspase-3 via the p38 and JNK pathways. These results suggest that morroniside can reduce the oxidative damage and apoptosis of ovarian GCs induced by H₂O₂.

Endometrial angiogenesis is necessary for good endometrial receptivity. Krüppel-like factor 4 (KLF4) is a transcription factor that is essential for regulating angiogenesis. Here we found that vascular endothelial growth factor A (VEGFA) can form a positive feedback loop with KLF4 to promote the proliferation and migration of human endometrial microvascular endothelial cells (HEMECs) and inhibit cell apoptosis. General control non-derepressible 5 (GCN5) is also time-dependent on VEGFA and participates in the KLF4-VEGFA loop. In addition, we found that GCN5 is a succinyltransferase that modulates the succinylation of histones and nonhistones. GCN5 interacts with KLF4 and is recruited to the KLF4-binding site of the VEGFA promoter to succinylate H3K79, which initiates gene transcription epigenetically. For nonhistones, GCN5 succinylates KLF4 that is activated by ERK signaling in HEMECs treated with VEGFA to increase its transcription activity. These results demonstrate KLF4-VEGFA positive feedback loop is regulated by epigenetics, which contributes to endometrial angiogenesis.