(69) Mitoquinol Enhances Embryo Development by Reducing Oxidative Stress During Ionomycin-Induced Parthenogenesis and Intracytoplasmic Sperm Injection in Mice

Mitoquinol Enhances Embryo Development by Reducing Oxidative Stress During Ionomycin-Induced Parthenogenesis and Intracytoplasmic Sperm Injection in Mice

Mingxiang Zhang, PhD¹, Alexander Lagunov, MS², William B Schoolcraft, MD¹, Jason  E. Swain, PhD¹,  Ye Yuan, PhD¹;

¹CCRM Fertility of Lone Tree, CO; ²CCRM Fertility of Miami.

Abstract Text:

One of the primary causes of total fertilization failure (TFF) is the inability of sperm to activate the oocytes. In this scenario, artificial oocyte activation (AOA) has emerged as an effective treatments. Ionomycin, a calcium ionophore, has been proven to increase fertilization rates in both mouse models and humans. Despite the promise of improving IVF outcomes, the safety of using ionomycin remains elusive. Notably, a mice study has reported that ionomycin generated excessive oxidative stress, leading to a dramatic increase of DNA damage. Here, we investigated the efficiency of AOA using ionomycin in conjunction with mitoquinol (MitoQ), a mitochondria-targeted antioxidant in both parthenogenetic activation (PA) and intracytoplasmic sperm injection (ICSI)-based oocyte-activation-deficient mouse model. In vivo-matured oocytes from Swiss Webster mice were used for PA experiments, while CF-1 mouse oocytes were used for ICSI. For PA, the oocytes were exposed to 10 µM ionomycin ± 25 nM MitoQ followed by a 5-h incubation with 5 µg/ml cytochalasin D (CD). In ICSI experiments, sperm were sonicated to remove the tails, followed by chemical inactivation with 0.1 M Na₂CO₃ (PH = 11.5) and injected to the oocytes using piezo-ICSI. Without AOA treatment, oocytes displayed no cleavage, mimicking TFF in human IVF. One hour post injection, oocytes were exposed to 10 µM ionomycin ±25 nM MitoQ for 15 minutes. Presumptive zygotes were cultured for 120h under 6.0% CO₂ and 5.0% O₂ at 37 °C. The developmental assessment including cleavage rate, day 4 blastocyst rate, day 5 hatching rate as well cell count and mechanistic assessments including reactive oxygen species (ROS) staining and mitochondrial membrane potential (MMP) analysis via JC-1 staining. Cleavage, blastocyst, and hatching rates were analyzed using Fisher’s exact test (PA) and paired t-test (ICSI), with p < 0.05 considered significant. Cell counts, ROS, and JC-1 data were analyzed using Student’s t-test (PA), and ROS staining data in ICSI were analyzed by one-way ANOVA. MitoQ supplementation significantly improved day 4 blastocyst formation (PA: 80.14% vs. 68.85%; ICSI: 68.99 ± 2.32% vs. 57.56 ± 2.75%) and day 5 hatching rates (PA: 74.52% vs. 53.4%; ICSI: 63.74 ± 3.83% vs. 52.61 ± 2.71%). No significant differences were observed in total, ICM, or TE cell numbers in PA-derived blastocysts. Mechanistically, MitoQ significantly reduced ROS levels and enhanced MMP during AOA in the PA model, with similar trends observed in the ICSI model. In conclusion, MitoQ supplementation to the ionomycin activation medium improved the embryonic development and reduced oxidative stress in mouse models, suggesting a role in maintaining proper redox balance during AOA, which offer potential to further optimize the treatment for human IVF.