| Background and Purpose:Mitochondria play a central role in the regulation of energy metabolism in oocytes and preimplantation embryos,and mitochondrial dysfunction is an important mechanism involved in ovarian aging.Mitochondrial casein peptidase P(ClpP)mediates the degradation of unfolded mitochondrial proteins and activates the mitochondrial unfolded protein response(UPRmt)to maintain mitochondrial protein homeostasis.UPRmt has recently been found to be related to ovarian aging,because the impaired mitochondrial stress response can lead to the accumulation of age-related damaged proteins,diminished oxidative phosphorylation and increased production of ROS,thereby inducing ovarian aging.Previous studies have shown that the ClpP mitochondrial unfolded protein response and the role of ClpP appear to be conserved in mammals.In vivo experiments have found that ClpP is necessary for the development of oocytes and embryos,as well as the function and dynamics of oocytes mitochondria.Lack of ClpP will lead to accelerated depletion of follicles and diminished ovarian reserve.Its phenotype is similar to premature reproductive failure.However,domestic and foreign related discussions mainly focus on the function of ClpP in rodent follicles,and there is a lack of research on the changes of ClpP levels in ovarian granulosa cells related to human ovarian reserve.This study aims to find out whether there is a difference in the level of ClpP protein in granulosa cells between the diminished ovarian reserve group and the normal group,so as to further study the role of the mitochondrial unfolded protein response related to ClpP in human granulosa cells in the diminished ovarian reserve.Methods:1.This study is a case-control study.2.This study included 20 assisted pregnancy patients who underwent in vitro fertilization and embryo transfer in the Center for Reproductive of the first affiliated Hospital of Zhengzhou University from October 2020 to January 2021.According to whether the ovarian function is impaired,all participates were divided into normal ovarian reserve group(10 cases)and impaired ovarian reserve group(10 cases).Collect all participants’ age,body mass index,ovulation induction program,Gn injection volume,number of eggs obtained,fertilization rate,high-quality embryo rate and other clinical data.3.After signing the informed consent form,all participants were extracted DNA from peripheral venous blood.Serum indicators of all participants were measured,such as basal follicle stimulating hormone,luteinizing hormone,testosterone,estradiol.4.After all participants signed the informed consent form,follicular fluid was collected.Extract and lyse granular cells.Elisa was used to measure the level of ClpP protein in ovarian granulosa cells.RT-qPCR was used to measure the level of ClpP mRNA in ovarian granulosa cells.5.After the ovarian granulosa cells were cultured for 24 hours,the mitochondrial function indexes were measured,such as ATP level,ROS level and mitochondrial membrane potential(ΔΨm).6.IBM SPSS Statistics 25.0 software was used for statistical analysis.Graphpad Prism 6.0 software was used for drawing pictures.The average baseline data of the hypoovarian reserve group and the normal ovarian reserve group were expressed as mean±standard deviation.The Student t test was used to compare the baseline data of the hypoovarian reserve group and the normal ovarian reserve group.Non-parametric tests was used to compare the ATP levels and ROS levels of granulosa cells from the hypoovarian reserve group and the normal ovarian reserve group.Results:1.In this study,10 impaired ovarian reserve cases and 10 normal ovarian reserve cases were included.2.There were no significant differences in Age(P=0.149),body mass index(P=0.701),Gn injection volume(P=0.948),fertilization rate(P=0.259),high-quality embryo rate(P=0.661)between hypoovarian reserve group and the normal ovarian reserve group.There were significant differences in the ovulation induction program(P=0.010)and the egg number(P<0.001)between the hypoovarian reserve group and the normal ovarian reserve group.3.There were no significant differences in the serum basal follicle stimulating hormone(P=0.140),luteinizing hormone(P=0.603),testosterone(P=0.343),and estradiol(P=0.314)between the hypoovarian reserve group and the normal ovarian reserve group.4.The mean ClpP protein concentration of granulosa cells in the two groups were:the normal ovarian reserve group(2.373± 0.349 ng/ml)and the impaired ovarian reserve group(1.870 ± 0.279 ng/ml).Compared with the normal ovarian reserve group,the ClpP protein level of granulosa cells in the hypoovarian reserve group was significantly diminished(P=0.002).5.The mean ClpP mRNA levels of granulosa cells in the two groups were:normal ovarian reserve function group(1.212 ug/ml)and hypoovarian reserve group(0.498 ug/ml).Compared with the normal ovarian reserve group,the level of ClpP mRNA in granulosa cells in the hypoovarian reserve group was significantly lower than that in the normal ovarian reserve group(P<0.05).6.The ROS level of granulosa cells in the hypoovarian reserve group was significantly higher than that in the normal ovarian reserve group,and the difference was statistically significant(P<0.05).7.The ATP level and mitochondrial membrane potential of granulosa cells in hypoovarian reserve group were significantly lower than those in the normal ovarian reserve group,and the differences were statistically significant(P<0.05).Conclusion:Compared with infertility patients with normal ovarian reserve,infertility patients with impaired ovarian reserve have diminished ClpP protein level and transcription level in granulosa cells,and mitochondrial dysfunction appears.This indicates that the ClpP-related UPRmt may play an important role in mechanism of diminished ovarian reserve. |
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