Study On The Mechanism Of Trimethylamine N-oxide In PCOS Metabolic Abnormalities

BackgroundPolycystic ovary syndrome(PCOS)is a common metabolic disorder that is often associated with risk factors such as abdominal obesity,insulin resistance(IR),metabolic abnormalities,and cardiovascular disease.At present,the pathogenesis is still controversial.Scholars generally believe that the interaction between genetic mutations and environmental factors leads to the occurrence of PCOS.The intestinal flora is considered the "second brain" or "second genome" of humans,is one of the most complex microbial communities,and can affect the host’s important physiology by chronically exposing the host to low levels of metabolism and decay products Function,when the intestinal microbiome-based diversity changes,it may damage the host’s metabolic health and is related to the occurrence of a variety of metabolic diseases.Trimethylamine oxide(TMAO)is an organic compound produced by intestinal microorganisms.Studies have shown that TMAO is involved in cardiovascular disease(CVD),type II diabetes,and thrombosis.So,is TMAO involved in the pathogenesis of PCOS? What about metabolic abnormalities and low-grade inflammatory responses?ObjectiveThe purpose of this study is to explore whether TMAO is involved in metabolic abnormalities and low-grade inflammatory responses in patients with PCOS,and to study the relationship between intestinal flora and its metabolites TMAO and PCOS,hoping to provide new ideas for prevention and early diagnosis and treatment of PCOS This research is divided into the following 4 parts:The first part is to study the correlation between plasma TMAO levels and PCOS patients.Part Ⅱ: Three classic modeling methods for PCOS rats(insulin + human chorionic gonadotropin,letrozole,letrozole + high-fat diet)were selected.The different groups of rats were weighed after modeling,Differences in hormone levels and intestinal microbial composition.Part Ⅲ: The mechanism of TMAO involved in metabolic disorders of PCOS rats.Part Ⅳ: Effect of TMAO on mouse oocyte maturation in vitro.MethodsPart Ⅰ: This part of the study included 41 newly diagnosed PCOS patients and 23 infertile patients due to fallopian tubes or male factors,and subgrouped them according to BMI <24 or not.The ultra-high pressure liquid chromatography-triple quadrupole mass spectrometry was used to quantitatively detect the plasma TMAO levels of patients in PCOS and control groups,and to evaluate their relationship with other biological indicators.Part Ⅱ: Twenty 9-week-old SPF female SD rats were randomly divided into Control-9w group(saline treatment group)and INS + HCG group(insulin + human chorionic gonadotropin injection),10 rats in each group.Eighteen 6-week-old SPF female SD rats were randomly divided into Control-6w group(CMC group),LTZ group(letrozole model group),and LTZ + HFD group(letrozole + high-fat diet group),6 each.Observed the ovarian changes,sex hormone changes,and glucose and lipid metabolism disturbances after modeling.The 16 S detection technology was used to statistically analyze the differences between the intestinal flora of each group of rats.Part Ⅲ: 24 female 9-week-old SPF female SD rats were randomly divided into the Control group(saline-treated group),the INS + h CG group(insulin + human chorionic gonadotropin injection),and the INS + h CG + DMB group(insulin + Human villous gonadotropin injection +DMB),8 rats in each group.Changes in sex hormones and glucose and lipid metabolism disorders were observed after treatment,and the changes of PI3 K,Akt,p-Akt,and IRS protein expression in the ovary of each group were detected by Western blot.Part Ⅳ: Add culture medium with different concentrations of TMAO to mature mouse oocytes in vitro,detect spindle and chromosome morphology,mitochondrial distribution,number and membrane potential,adenosine triphosphate(ATP)concentration,early oocyte decay detection of death explores the effects of TMAO on mouse oocyte maturation in vitro.ResultsPart Ⅰ: Plasma TMAO levels in patients with PCOS were elevated,especially in obese PCOS patients.The clinical manifestations of PCOS patients vary widely,and the levels of inflammatory factors were mostly not statistically different from those in the control group.Logistic regression analysis showed that plasma TMAO,LH / FSH,T or fasting blood glucose were independent predictors of PCOS.Higher plasma TMAO levels,LH / FSH ratios,and T or fasting blood glucose increased the risk of PCOS.Part Ⅱ: The weight increase of rats with insulin combined with h CG model was higher than that of control group,and the decrease of ovarian quality was higher than that of letrozole group.During the modeling period,the rats in the modeling group lost the regular estrous cycle,and the morphological changes of the ovary in general and under the microscope were consistent with ovarian polycystic changes.Sex hormone test results showed that T and LH of rats in INS + h CG group increased.In terms of glucose and lipid metabolism,the combined application of insulin and h CG can increase both FINS and HOMA-IR,which was more significant than the letrozole or letrozole combined with highfat diet group.Fecal 16 S test results showed that the changes in intestinal flora diversity and specific species abundance in the letrozole and letrozole combined with high-fat diet group were more significant than the insulin combined with h CG model,and the difference was statistically significant.Part Ⅲ: Establishing a rat PCOS model using insulin combined with h CG in this experiment.The results showed that the fasting insulin,HOMA-IR,LH,LH / FSH,and plasma TMAO levels in the INS + h CG group were significantly higher than those in the control group.However,rats with TMAO inhibitor DMB in drinking water can alleviate the increase of metabolic indexes caused by modeling in rats in INS + h CG group.Part Ⅳ: In this part of the study,we found that intestinal flora product TMAO can reduce the MII rate of oocyte maturation in vitro,reduce the mitochondrial content of oocytes and cause abnormal distribution,reduce ATP content,and accelerate oocytes.Early cell apoptosis.ConclusionElevated plasma TMAO levels may be involved in the occurrence of chronic lowgrade inflammation and metabolic disorders in patients with PCOS.TMAO inhibitor DMB can alleviate metabolic abnormalities in PCOS model rats.TMAO can reduce oocyte quality and development potential of PCOS mice by affecting mitochondrial function.