Investigation Of Effects And Potential Mechanisms Of Mito-TEMPO On Oocytes During In Vitro Maturation

Objective: The maturity rate of oocytes may have an impact on the clinical outcome of patients receiving intracytoplasmic sperm injection(ICSI)treatment.This study aims to evaluate whether low oocyte maturity rate affects clinical outcomes,the role of in vitro maturation(IVM)of oocytes in such patients,and to propose optimized treatment strategies for IVM.Mito-TEMPO may positively affect the IVM process of oocytes,but its effective concentration,specific effects,and underlying mechanisms are unclear.This study will explore the effects of Mito-TEMPO at the optimal concentration on the quality and developmental potential of mouse oocytes after IVM,and investigate possible mechanisms,providing a scientific basis for subsequent clinical applications and improving treatment outcomes for patients.Methods: Collect patients who underwent their first ICSI treatment in our center from January 2016 to November 2020.Divide them into low maturity rate group and normal maturity rate group based on whether the oocyte maturity rate was ≤50%,and perform a 1:3 propensity score matching to obtain the case group and the control group,and compare the clinical outcomes of the two groups.Subgroup clinical outcome analysis of the case group was conducted based on whether they underwent rescue-IVM(R-IVM).Collect prophase oocytes from female ICR mice and use culture media supplemented with different concentrations(100n M to 50μM)of Mito-TEMPO for IVM.Determine the optimal concentration by comparing the rates of germinal vesicle breakdown and oocyte maturation.Then,compare the quality of IVM oocytes between the optimal concentration treatment group and the control group,including chromosome alignment,spindle assembly,mitochondrial membrane potential,and oxidative damage indicators(reactive oxygen species levels(ROS)and mitochondrial superoxide relative levels).To further explore the mechanism of action of Mito-TEMPO,compare the oxidative stress levels(ROS,mitochondrial superoxide,and malondialdehyde(MDA)),antioxidant capacity(glutathione(GSH)level),and DNA damage levels(γH2AX)between the treatment group and the control group.Evaluate the effect of Nrf2 inhibitor on the action of MitoTEMPO,and compare and analyze the expression levels of Nrf2 pathway-related genes,mitochondrial assembly-related genes,spindle assembly-related genes,DNA damage and repair-related genes,apoptosis-related genes,and other oxidative stress-related genes to determine the regulation of IVM process by Mito-TEMPO.Results: The ICSI fresh cycle outcomes of patients with low oocyte maturation rates did not decrease,but the cumulative live birth rate was significantly lower than that of the control group.For patients with low maturity rates,IVM treatment significantly improved clinical outcomes.The effect of Mito-TEMPO on mouse oocyte IVM is closely related to its concentration.5μM is the optimal concentration of Mito-TEMPO for oocyte IVM,which increases oocyte maturation rate,improves mitochondrial function,reduces oxidative damage,and significantly increases the rate of blastocyst formation in subsequent culture compared to the control group.Further mechanistic studies revealed that the ROS and MDA levels of matured oocytes in the Mito-TEMPO treatment group after IVM significantly decreased,while the GSH level significantly increased.Meanwhile,the expression levels of mitochondrial assembly-related genes MFN1 and MFN2 significantly increased,and the Nrf2 pathway was upregulated.The expression levels of downstream genes of Nrf2,such as HO-1,NQO1,other antioxidant-related genes PGC-1α,and DNA damage repair gene BRCA1 were significantly higher compared to the control group.Nrf2 inhibitors significantly reduced the effect of Mito-TEMPO.Conclusions: The maturity rate of oocytes is closely related to the ICSI treatment outcome,and for patients with low maturity rates,R-IVM treatment can improve clinical outcomes by increasing the number of mature oocytes.The use of 5μM Mito-TEMPO can significantly improve the quality and developmental potential of mouse IVM oocytes,possibly by upregulating the Nrf2 pathway,enhancing antioxidant capacity and DNA repair ability,thereby reducing oxidative stress and DNA damage,and improving the IVM outcomes.Part Ⅰ: The impact of R-IVM on the clinical outcomes of ICSI in patients with a low oocyte maturity rateObjective: To explore the association between oocyte maturity rate and ICSI clinical outcomes and to investigate the treatment effect of R-IVM for patients with low oocyte maturity rates.Methods: This retrospective study included patients who underwent their first ICSI treatment at Tongji Hospital Reproductive Center from January 2016 to November 2020.The patients were divided into a low maturity ratio group(≤50%)and a control group(>50%)according to the oocyte maturity rate,and a 1:3 propensity score matching was performed.Laboratory outcomes(fertilization rate,blastocyst formation rate,and available blastocyst rate),ICSI pregnancy outcomes(clinical pregnancy rate,fresh cycle live birth rate,and cumulative live birth rate),and perinatal outcomes(incidence of pregnancy complications,preterm delivery rate,etc.)were compared.Subgroups were created for patients with low maturity rates based on whether R-IVM was used,and the above outcomes were compared.Results: A total of 1239 low-maturity patients and 3266 control patients were included.Compared with controls,there were no significant differences in fresh cycle outcomes(clinical pregnancy rate,live birth rate,etc.)and perinatal outcomes(pregnancy complications,preterm delivery,etc.);laboratory outcomes(normal fertilization rate,blastocyst formation rate,usable blastocyst rate)and cumulative live birth rate were reduced in patients with low maturation rates.R-IVM was used in 978 of the patients with low maturation rates and not used in 261 patients(Non-IVM).The differences in fresh cycle outcomes(live birth rate,miscarriage rate)were not statistically significant and the cumulative live birth rate was significantly higher in R-IVM patients compared to Non-IVM patients.Conclusions: Fresh cycle clinical pregnancy and live birth rates were not decreased in ICSI patients with low oocyte maturation rates,but cumulative live birth rates were significantly lower.Treatment of patients with low maturation rates with R-IVM improved their pregnancy outcomes and,once pregnant,resulted in perinatal outcomes similar to those of the general population treated with ICSI.Part Ⅱ: A study on the effect of Mito-TEMPO on in vitro maturation of mouse oocytesObjective: Mito-TEMPO may have a positive impact on the IVM process of oocytes,but its effective concentration and specific effects are not clear.This section will compare the differences in quality between in vivo matured and in vitro matured oocytes,and explore the optimal concentration of Mito-TEMPO and its effects on the quality and developmental potential of mouse IVM oocytes at the optimal concentration.Methods: The mature oocytes obtained from control superovulated female ICR mice were collected.Immature oocytes from young and old female ICR mice were subjected to IVM with the addition of different concentrations of Mito-TEMPO in the culture medium to compare the maturation rates and select the optimal concentration.Subsequently,the optimal concentration of Mito-TEMPO was added to the culture medium of the treatment group,and the ICSI outcomes(fertilization rate,cleavage rate,blastocyst formation rate),chromosome alignment,spindle assembly,mitochondrial membrane potential,and ROS level were compared between the control group,treatment group,and in vivo matured oocytes.Results: The quality of in vitro matured oocytes is significantly lower than that of oocytes obtained by controlled ovarian hyperstimulation.A similar reduction in IVM oocytes occurred in aged mice compared to younger mice.The effect of Mito-TEMPO on the outcome of oocyte IVM is closely related to its concentration,with concentrations above 50μM inhibiting oocyte maturation and concentrations between 0.1-10μM showing varying degrees of promoting oocyte maturation.Among them,the 5μM treatment group had the highest maturation rate after IVM and was therefore selected as the optimal concentration.For IVM oocytes from young mice,5μM Mito-TEMPO significantly increased ICSI cleavage and blastocyst formation rates,increased mitochondrial membrane potential,and reduced ROS levels and mitochondrial superoxide levels,reaching levels similar to those of in vivo matured oocytes.For IVM oocytes from aged mice,5μM Mito-TEMPO significantly improved IVM maturation rate,ICSI blastocyst formation rate,reduced ROS levels,and mitochondrial superoxide levels,reaching levels similar to those of IVM oocytes from young mice.Conclusions: 5μM of Mito-TEMPO can enhance mitochondrial activity,reduce oxidative damage,and decrease mitochondrial superoxide deposition,thus improving the quality and developmental potential of IVM oocytes from young and aged mice.Part III: Exploration of the mechanism underlying the improvement of in vitro matured oocyte quality by Mito-TEMPOObjective: Exploring the possible mechanisms of Mito-TEMPO and investigating the role of the Nrf2 pathway in improving the quality of IVM oocytes by Mito-TEMPO.Methods: Collect immature oocytes from young female ICR mice and set up four groups: control group(adding 0.1% DMSO),MT group(adding 5μM Mito-TEMPO),ML group(adding 1.9μM Nrf2-specific inhibitor ML385),and Mix group(adding 5μM Mito-TEMPO and 1.9μM ML385).Compare the m RNA expression levels of related genes in the ML group and control group.Compare the oocyte maturation rates,oxidative damage levels(ROS,mitochondrial superoxide,MDA),DNA damage levels(γH2AX level),and antioxidant levels(reduced glutathione content)among the four groups.Results: In the MT group,the expression of Nrf2 and its downstream genes HO-1 and NQO1 were upregulated,while the expression of Keap1 was downregulated;the expression of mitochondrial fusion genes MFN1 and MFN2 was upregulated;the expression of antioxidant gene PGC-1α and DNA repair gene BRCA1 was upregulated.The maturity rate and antioxidant level were increased in the MT group.The addition of an Nrf2 inhibitor suppressed this effect.Conclusions: Treatment with 5μM Mito-TEMPO upregulates the Nrf2 pathway,reduces oxidative stress,enhances mitochondrial function,upregulates the expression of DNA repairrelated gene BRCA1,reduces DNA damage,and consequently improves the quality and developmental potential of oocytes.