Optimizing In Vitro Maturation of Oocytes with Nitric Oxide Microbubble

Lin Qiu ¹ Qi Xu ² Yi Lin ³ Songxue Wang ¹ Huina Zhang ¹ Yi Liu ¹ Junzhao Zhao ¹ Yingzheng Zhao ³ Haitao Xi ^1*

• ¹ Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
• ² Zhejiang Zhuji People's Hospital, Zhuji, China
• ³ Wenzhou Medical University, Wenzhou, Zhejiang Province, China

Nitric oxide (NO) plays a pivotal role in female reproductive processes, yet its clinical translation is limited by its short half-life and rapid systemic clearance. In this study, we constructed lipid-based microbubbles (NO-MBs) via lyophilization to encapsulate and stabilize therapeutic NO gas, optimizing their parameters (particle count: 2.23×10 7 MBs/ml; diameter: 0.919±0.807 µm) for controlled release and prolonged circulation. Compared to conventional NO donors (e.g., SNP), NO-MBs demonstrated superior biosafety and efficacy in enhancing in vitro oocyte maturation. Specifically, NO-MBs significantly increased the maturation rate of mice immature oocytes (72.00% vs. 57.77% in controls) while improving oocyte quality, as evidenced by elevated Ca 2+ levels, mitochondrial membrane potential (MMP), and reduced intracellular ROS. In vivo, NO-MBs enhanced oocyte retrieval and fertilization rates while reducing blastocyst cell apoptosis. Mechanistically, NO-MBs uniquely activated phosphorylated ERK, suggesting ERK pathway involvement in oocyte maturation. These findings highlight NO-MBs as a novel, clinically relevant strategy for targeted NO delivery in reproductive medicine, offering potential applications in assisted reproductive technologies (ART) to improve oocyte quality and developmental outcomes. Figure 1. Schematic illustration of Nitric Oxide-loaded microbubbles (NO-MBs) preparation via lyophilization and their role in optimizing oocyte IVM outcomes.