The Mechanism Of Fatty Acids And Other Metabolites Changes Cause Diminished Ovarian Reserve

Background: Diminished ovarian reserve（DOR）is defined by the depletion of oocyte quality and quantity and is one of the leading causes of female infertility today.DOR is associated with ovarian hypo-response,low pregnancy rates and high miscarriage rate in in vitro fertilization/intracytoplasmic sperm injection-embryo transfer.In recent years,the prevalence of DOR has increased year by year,becoming a major problem in assisted reproduction.During oocyte development and maturation,fatty acids and other metabolites（e.g.,cholesterol,steroid hormones,and amino acids）provided nutrients and regulatory signals.To investigate if fatty acids and other metabolites changes in granulosa cells（GCs）are the pathogenesis of DOR.Methods: First,clinical data and GCs were collected from 70 patients with normal ovarian reserve（NOR）and 70 patients with DOR.Liquid chromatography-tandem mass spectrometry（LC-MS/MS）was performed to analyze fatty acids and other metabolites（e.g.,cholesterol,steroid hormones,and amino acids）changes in GCs of DOR patients.Then,fatty acids served as the main energy source.Gas chromatographymass spectrometry（GC-MS）was utilized to accurately quantify fatty acids changes in GCs of DOR patients.In addition,the effects of fatty acids changes on GCs apoptosis and oocyte development were analyzed by mitochondrial membrane potential,ATP production,DNA damage and apoptosis.Finally,real-time fluorescence quantitative PCR was used to detect the expression of genes related to mitochondrial fatty acid β-oxidation and DNA repair.Results: Patients with DOR had significantly lower levels of antral follicle count,anti-Mullerian hormone,and higher levels of follicle-stimulating hormone compared with NOR patients（P<0.05）.The good-quality embryo rate was notably decreased in DOR patients（P<0.05）.The LC-MS/MS analysis showed that fatty acids（e.g.,oleic acid,eicosapentaenoic acid and arachidonic acid）,cholesterol（e.g.,coprostanone）,steroid hormones（e.g.,11A-acetoxyprogesterone,hydroxyprogesterone and progesterone）and amino acids（e.g.,lysine,arginine and serine）were significantly reduced in GCs of DOR patients（P<0.05）.The GC-MS analysis showed that 15 fatty acids were significantly reduced in GCs of DOR patients（P<0.05）.In addition,the mitochondrial membrane potential（1.74±0.13% vs 1.02±0.15%）and ATP content（39.07±12.89% vs 23.21±13.69%）were markedly lower in DOR patients than in NOR patients（P<0.05）.The β-oxidation genes（HADHA and ACSL）and DNA repair genes（PRKDC and RAD50）were significantly downregulated in DOR patients（P<0.05）.Compared with NOR patients,DNA damage（0.31±0.03% vs 0.87±0.07%）and apoptosis rates（6.43±2.11% vs 48.06±6.72%）in GCs of DOR patients were significantly increased（P<0.05）.Conclusions: Fatty acids and other metabolites（e.g.,cholesterol,steroid hormones,and amino acids）were associated with the development of DOR.Among them,the GCs apoptosis resulting by the decrease of fatty acids with reduced ATP production and DNA damage may be responsible for the pathogenesis of DOR.