| Background and PurposePolycystic ovary syndrome(PCOS)is a highly heterogeneous ovarian dysfunction syndrome that affects 5%to 10%of women of reproductive age and is the main cause of anovulatory infertility.Clinical features of polycystic ovary syndrome usually include irregular menstruation,hyperandrogenism and obesity,as well as insulin resistance and elevated serum LH levels.Metabonomics is an emerging discipline that combines genomics,transcriptomics,and proteomics,providing a more comprehensive understanding of many small molecule endogenous metabolites and their associated metabolic pathways.In recent years,liquid chromatography-mass spectrometry(LC-MS)has been widely used in the study of metabolomics.Compared with other analytical techniques,LC-MS offers a wider mass range,greater analytical fluxes and superior versatility.Follicular fluid(FF)is derived from plasma and its secretions and contains metabolites essential for oocyte growth and development.One potential method for evaluating embryo fertility using a rapid,noninvasive,sensitive,and clinically applicable platform is metabolic profiling of embryo media.Studies have shown that changes in medium composition are correlated with differences in embryo quality and reproductive potential.Metabonomics analysis of the correlation between embryo culture medium and embryo development potential in vitro shows that metabonomics is more objective,comprehensive,accurate and non-invasive in predicting embryo development potential than embryo morphology.The purpose of this study was to analyze the different metabolites and related metabolic pathways in follicular fluid and embryo culture fluid of PCOS and non-PCOS patients.Furthermore,the relationship between follicular fluid and embryo culture fluid metabolism was analyzed from the perspective of metabonomics,and the potential markers related to metabolic disorders in PCOS patients were further explored by combining the embryonic laboratory data and clinical outcomes.Methods1.30 infertile patients diagnosed as PCOS by Rotterdam standard were selected,and 30 control patients who underwent IVF-ET only for female tubal infertility were selected.Nontargeted UHPLC-QE-MS analysis was performed on the metabolites in the first tube follicular fluid of all patients at the time of oocyte pick up and the waste embryo culture medium after D3 high-quality embryo transplant.2.Univariate and multivariate statistical analysis was performed on the original data obtained by mass spectrometry to identify and screen the different metabolites.KEGG and Metaboanalyst were used to analyze the related pathways,and further topology analysis and enrichment analysis were performed on the screened related pathways.3.Combined with clinical data,Pearson correlation analysis was conducted on the differential metabolites in follicular fluid and the indicators of embryonic development potential in laboratory to study the ability of differential metabolites in follicular fluid and embryo culture fluid to predict clinical outcomes.Results1.The analysis results of the baseline data of the patients included in the metabolic analysis showed that the levels of luteinizing hormone(R=0.48,P=0.0027;R=0.47,P=0.0086;R=0.53,P=0.0026),testosterone(0.47±0.30 ng/ml vs 0.25±0.15 ng/ml)and the number of years of infertility(4.66±3.03 y vs 3.67±1.97 y)in the PCOS group were higher than those in the non-PCOS group all P<0.05),and there were no statistical differences in the other baseline data.There were no statistical differences in the number of oocyte retrieved,2PN fertilization rate,2PN cleavage rate,high-quality embryo rate on D3,blastocyst formation rate and embryo utilization rate between PCOS group and non-PCOS group(P>0.05).There were no statistical differences in the average number of embryos transferred,implantation rate,pregnancy rate,delivery rate,live birth rate and abortion rate between the two groups(P>0.05).2.PCA and OPLS-DA analysis based on metabonomics results showed that the separation trend of follicular fluid and embryo culture fluid of PCOS patients and non-PCOS patients was obvious.OPLS-DA replacement test results showed that the model was robust,and there was no over-fitting phenomenon.3.After univariate statistical analysis(UVA)screening,there were 11 significant difference metabolites in follicular fluid between PCOS patients and non-PCOS patients,among which 7 metabolites were down-regulated,respectively L-Palmitoyl carnitine,LysoPE(16:0/0:0),Linoleyl carnitine,trans-Hexadec-2-enoyl carnitine,1-Arachidonoylglycerophosphoinosito,2-propylpentanoic acid and LysoPA(18:1(9Z)/0:0).There were 4 up-regulated differential metabolites,respectively DG(15:0/18:3(6Z,9Z,12Z)/0:0),DG(18:2(9Z,12Z)/15:0/0:0),Androsterone sulfate and L-Erythrulose.4.There was a significant difference of 56 metabolites in embryo culture medium,including 32 kinds of lipids and organic acids,respectively L-Alloisoleucine,D-Proline,L-Valin,Taurine,Creatine,beta-Alanine,Ureidopropionic acid,4Guanidinobutanoic acid,D-Alanine,Allantoic acid,L-Phenylalanine,Glycerophos phocholine,beta-Santalal,L-Asparagine,Elaidic carnitine,Phosphatidylcholine O-34:2,Glycine,Isobutyric acid,Pelargonic acid,Tridecanoic acid,Undecanoic acid,12-Hydroxydodecanoic acid,L-Serine,Ketoleucine,D-Glutamine,3-Hydroxycapric acid,2-Hydroxyethanesulfonate,2-Hydroxy-3-methylbutyric acid,L-Allothreonine,Undecylenic acid and(R)-3-Hydroxy-tetradecanoic acid.5.KEGG annotation and comprehensive analysis(including enrichment analysis and topology analysis)of the different metabolites showed that the metabolic pathways related to the difference in ovarian fluid metabolism between PCOS patients and non-PCOS patients were mainly Glycerolipid metabolism,Glycerophospholipid metabolism,Arginine and proline metabolism and Drug metabolization-cytochrome P450.Pantothenate and CoA biosynthesis and Glycine,serine and threonine metabolism were the key pathways with the highest correlation with metabolite differences in embryo culture medium.In addition,Glycerophospholipid metabolism was also enriched in embryo culture medium,which was the same as that in follicular fluid analysis.6.Pearson correlation analysis showed that there was a correlation between different lipid metabolites in follicular fluid and laboratory indicators.DG(15:0/18:3(6Z,9Z,12Z)/0:0)in PCOS group was positively correlated with fertilization rate,cleavage rate and 2PN fertilization rate.L-palmitylcarnitine was negatively correlated with blastocyst formation rate,embryos top-quality rate on day3 and high scoring blastocyst formation rate in the control group.There was a negative correlation between Linoleyl carnitine and blastocyst formation rate in the control group.In the control group,trans-Hexadec-2-enoyl carnitine was negatively correlated with the blastocyst formation rate and the blastocyst formation rate of embryos top-quality.In the control group,Androsterone sulfate was negatively correlated with the blastocyst formation rate and the blastocyst formation rate of embryos top-quality.DG(18:2(9Z,12Z)/15:0/0:0)was positively correlated with the blastocyst formation rate of Class III embryos in the control group.7.LysoPE(16:0/0:0),DG(18:2(9Z,12Z)/15:0/0:0),Linoleyl carnitine and androsterone sulfate in follicular fluid of PCOS group had better predictive ability for abortion rate,with AUC of 0.824,0.706,0.706 and 0.941,respectively.DG(15:0/18:3(6Z,9Z,12Z)/0:0)and LysoPA(18:1(9Z)/0:0)were good indicators for predicting the live birth rate and delivery rate in PCOS group,with AUC of 0.7 and 0.88.LysoPA(18:1(9Z)/0:0)was a good predictor of pregnancy rate in PCOS group,with an AUC of 0.89.In the follicular fluid of non-PCOS group,LysoPE(16:0/0:0)and DG(18:2(9Z,12Z)/15:0/0:0)had a better ability to predict pregnancy rate,delivery rate and live birth rate,with an AUC of 0.733.Glycerophosphocholine,(R)-3-Hydroxy-tetradecanoic acid and Elaidic carnitine in embryo culture medium of PCOS group were important indicators for predicting clinical pregnancy rate,with AUC of 0.933,0.767 and 0.933,respectively.Pelargonic acid and Elaidic carnitine had better predictive effect on pregnancy rate in PCOS group,with AUC of 0.771 and 0.757,respectively.Pelargonic acid in the PCOS group was better in predicting the birth rate and live birth rate,and the AUC was 0.764.Beta-Santalal and(R)-3Hydroxy-tetradecanoic acid in embryo culture medium of non-PCOS group were good indicators for predicting live birth rate,delivery rate and pregnancy rate,with AUC of 0.798 and 0.726,respectively.Conclusion1.The metabolism of follicular fluid and embryo culture fluid in PCOS and non-PCOS patients were abnormal,and the metabolites of the difference were mainly a variety of lipids.2.Different metabolites in the follicular fluid and embryo culture fluid of PCOS and non-PCOS patients are related to a variety of metabolic pathways,among which the glycerol phospholipid metabolic pathway is enriched in both the follicular fluid and embryo culture fluid,and abnormal lipid metabolism of PCOS patients has been manifested in early embryo metabolism.3.Various lipid differential metabolites are correlated with embryonic development potential and can predict clinical outcomes to a certain extent... |
PDF Full Text Request