Exploration Of A Novel IVM Technology System Specific To The Culture Of Human Immature Oocytes In COH Cycles

Objective:Human oocytes are extremely scarce reproductive resources.If immature oocytes from controlled ovarian hyperstimulation（COH）cycles can be cultured to maturity using in vitro maturation（IVM）technology and effectively utilized,it is expected to provide an egg source for patients with special needs.However,the quality of mature oocytes obtained by current IVM technology is significantly inferior to that of normal oocytes matured in vivo,resulting in lower blastocyst rates and clinical pregnancy rates.Therefore,improving the quality of IVM oocytes is an urgent problem to be solved in assisted reproductive technology（ART）.This study is the first to attempt to use nicotinamide adenine dinucleotide（NADH）as an additive in the IVM culture medium,aiming to explore whether NADH can promote the in vitro maturation of human immature oocytes,improve the fertilization and development capabilities of oocytes,and analyze the key role mechanism it plays in this process.This research will accumulate experience and theoretical foundation for the development of a safe and efficient human oocyte IVM technology system.Methods:Human immature oocytes discarded during the COH cycles were collected and randomly assigned to IVM medium containing 0,10^-4,10^-5,10^-6,10^-7,10^-8mol/L（M）concentrations of NADH.The optimal NADH concentration is selected based on IVM,intracytoplasmic sperm injection（ICSI）,and in vitro embryo culture results.Further analysis is conducted on the maturation,development,blastocyst aneuploidy rate,mitochondrial function,and the expression and regulation of key molecules related to oocyte maturation in IVM oocytes cultured with the optimal NADH concentration,using technologies such as array-comparative genomic hybridization（array CGH）,laser scanning confocal microscopy,and single-cell RNA sequencing（sc RNA-seq）.Results:1.In the study,the rates of maturation,fertilization,cleavage,high-quality embryo,blastocyst and high-quality blastocyst were the highest in the 10^-6M NADH group compared with other concentrations,and the aneuploidy rate of high-quality blastocysts was 33.33%,so 10^-6M was determined to be the optimal concentration for human oocytes.2.The levels of adenosine triphosphate（ATP）（P<0.05）,mitochondrial membrane potential（P<0.01）and glutathione（P<0.001）in mature ovum cultured with 10^-6M NADH（NADH group）were significantly higher than those in mature ovum cultured without NADH（Control group）.There were no significant differences in calcium ion,mitochondrial content,reactive oxygen species,apoptosis and the protein expressions of key molecules（NDUFV1,NDUFS7,DAP13 and ND1）of enzyme complexⅠin oxidative phosphorylation chain（P>0.05）.3.Sc RNA-seq showed that 260 genes were up-regulated and 246 genes were down-regulated in the NADH group.Among the differentially expressed genes,6up-regulated genes related to oocyte maturation were screened out（P<0.001）,which were ranked as FIGN,FAM9B,GAS6,C14orf39,DMC1,CDK2 according to the differences in descending order.4.A novel IVM medium formula specific to the culture of immature oocytes from COH cycles has been developed and patented.5.A novel IVM technology system has been established specifically to the culture of human immature oocytes from COH cycles with the new IVM medium as the core.Conclusions:NADH effectively improves the IVM effect of human immature oocytes from COH cycles.Specifically,NADH significantly enhances the mitochondrial function,increases intracellular ATP,mitochondrial membrane potential and glutathione levels,protects the meiosis and promotes cytoplasmic maturation of IVM oocytes,thus obtaining more high-quality IVM oocytes.This improvement was concentration dependent,with 10^-6M being the optimal concentration.