The Effects Of Metabolites In Human Follicular Fluid On Embryo Development Potentials And Pregnancy Outcome Of PCOS Patients Who Received IVF

Part One:Metabolic changes in the follicle fluid of PCOS patients by NMR and its effects on embryo developmental potentialsObjectivesPCOS is one of the major reasons that cause female infertility of reproductive age.FF sample of PCOS patients and control group were collected when they received IVF-ET or ICSI treatment.The potential metabolites and their concentration in the FF were detected by NMR,which could decipher the local microenvironment difference of oocytes development between PCOS and control group and provide a new method to investigate the etiology of PCOS and improve their pregnancy outcomes.Methods1.Subjects:A total of 32 subjects(control group,n=36;PCOS group,n=15)who went to infertility clinic of Renmin Hospital of Wuhan University in 2014 were involved in this study.2.Ovarian stimulation:A standard long protocol was applied to all subjects.The follicle size and serum hormone levels were monitored until HCG injection.The dose of drugs was adjusted according to the follicle size and serum hormone levels.3.FF sampling and oocyte collection:The induction of ovulation was performed with 10000 IU HCG when at least two leading follicles had reached a diameter of 18mm under transvaginal ultrasound examination.Oocytes were retrieved transvaginal under ultrasound guidance 34-36 hours after HCG administration.Oocytes were collected and incubated in a special media and FF were collected in EP tubes for further analysis.Subsequently,the follicular fluid were centrifuged twice under the condition of 12000 rpm,4 ? for 10 min and the resultant supernatant were obtained as FF samples and stored at-80 ? until further analysis.4.Fertilization protocol and embryo examination:Insemination was conducted 3 hours after oocytes retrieval.Subsequently,oocytes were transferred to a fresh medium and the occurrence of 2 PN was observed and calculated.The embryo qualities were assessed on 3 days after cleavage.5.FF sample preparing:Each frozen serum sample was thawed,vortexed,centrifuged and then 530 ?L was transferred to 5-mm NMR tubes.A co-axial capillary containing 60 ?L TSP(0.53 mmol)in D20 was placed into the NMR tube to serve as chemical shift and quantitative reference.6.NMR data collection:All NMR experiments were carried out at a 25 ? on a Bruker DRX 500-MHz NMR spectrometer.NMR data for each sample was acquired using CPMG pulse sequences.Water was suppressed using a presaturation pulse.For each spectrum,128 transients were collected and 16K data points were acquired using a spectral width of 6000 Hz.An exponential weighting function corresponding to 0.5 Hz line broadening was applied to the free-induced decay before Fourier transformation.7.NMR data analysis:The datasets were aligned with reference to the alanine peak at 1.46 ppm using MestReNova version 6.6.1.Each CPMG NMR spectrum was binned to 4k frequency buckets of equal size(0.003 ppm).The spectra were then normalized with reference to the total spectra integral(? 10.0?0.40 ppm),excluding the residual water and urea region ? 4.7?5.2 ppm.The spectral data were initially mean-centered and subjected to PCA,PLS,OSC-PLS to discover the difference between PCOS patients and control group.8.Identification of difference metabolites in PCOS FF:According to previous articles and databases,we further make identification and integration of NMR characteristic peaks.TheNMR characteristic peaks were identified by t-test and multiple calibrations and a significant test was carried out in the identified characteristic peaks.And p value less than 0.05 or 0.1 were found to be significant.Results1.Metabolic Differences at a Glance:The NMR CPMG spectra averaged over follicular fluids from PCOS patients and healthy controls,together with the average difference spectrum,are suggested.The two population cohorts were kind of separated in PLS score plot.An OSC-PLS score plot exhibited clearer separated clusters.The 59-peaks model providing the total accuracy of 0.68 ± 0.06,sensitivity of 0.62 ± 0.10,specificity of 0.71 ± 0.08,AUROC of 0.77 ± 0.08.2.Biomarker Selection using Filter Methods:he PLS loadings illustrated that acetic acid,choline,glycerol and lipid region,glycoprotein,and glutamate were the top 5 metabolites contributed most to the scores separation on LV1.The OSC-PLS loadings indicated that acetic acid,hydroxybutyrate,acetoacetic acid,glycerol and lipid region,and isoleucine have the highest loading weights.A total of 9 metabolite regions with p<0.05 were selected from 59 metabolite regions,including increased intensity of glycerol and lipid region,glycoprotein,acetate,cholesterol and decreased levels of lactic acid,glutamine,pyruvate,and alanine.The PLS-DA model(p-value selected model)comprised of these 9 metabolite regions was constructed,producing the sensitivity of 0.73,specificity of 0.86 and AUROC of 0.88 for the cross-validation result.The model was further evaluated by MCCV.The average sensitivity and specificity are 0.72 ± 0.08 and 0.81 ± 0.04.3.Biomarker Selection using L-1 Penalized Logistic Regression:The L1-penalized logistic regression was employed on whole sample population using the 59 integrated regions.The technique automatically retained the metabolites that were mostly helpful for discrimination.Five metabolite regions were glutamine,pyruvate,acetate and cholesterol.Glutamine and pyruvate was down-regulated in PCOS,while cholesterol and acetic acid were upregulated.Especially,the percentage change of acetic acid was as high as 41%.A PLS-DA model using all these five metabolite regions was generated.The cross-validation gave the sensitivity of 0.73,specificity of 0.83,and AUROC of 0.86.4.Follicular Fluid Metabolic Pathways in PCOS:All the metabolites with p-value<0.05 between two sample cohorts were imported into MetPA.The pathways include pyruvate metabolism,glycolysis,alanine,aspartate and glutamate metabolism were found to be changed in PCOS FF.5.The correlation between the metabolites and oocytes development potentials:It is suggested that the concentration of creatine is negative correlated with good embryo rate.The histidine in PCOS FF is significantly correlated with good embryo rate.Moreover,formate,P-glucose and hydroxybutyrate in PCOS FF were found to significantly correlated with cleavage rate and fertilization rate.Conclusions1.Both the score plots and this 59-pks PLS-DA model indicate the clear difference between the two groups,and bring a dawn of discrimination of the two groups from a set of these metabolites.2.The PLS-DA model(p-value selected model)comprised of these 9 metabolite,from any point of view,has a significant improvement from the model based on 59 metabolite regions.3.The PLS-DA model was again built based on the selected 3 metabolites including pyruvate,acetic acid,and cholesterol has a better discrimination ability.4.The pathways include pyruvate metabolism,glycolysis,alanine,aspartate and glutamate metabolism were found to be different in PCOS FF.5.Histidine,formate,?-glucose and hydroxybutyrate in PCOS FF were closely associated with oocytes development potentials.Part Two:Concentration of ribonucleotides in human follicular fluid and its effects on embryo development potentials and pregnancy outcomesObjectivesIn female reproductive area,PCOS is one of the common reasons of infertility.FF was collected in PCOS patients and control group when they received IVF-ET or ICSI treatment.LC-MRM-MS/MS was used to identify the existence and determine the concentration of ribonucleotides.And we further carried out correlation analysis between the concentrations of ribonucleotides and oocytes development potentials and pregnancy outcomes aiming at finding out new method to understand the etiology and improve pregnancy outcomes of PCOS patients.Methods1.Subjects:A total of 32 participants(control group,n=16;PCOS group,n=16)who went to infertility clinic of Renmin Hospital of Wuhan University in 2014 were involved in the present study.2.Ovarian stimulation:A standard long protocol was applied to all subjects.The follicle size and serum hormone levels were monitored until HCG injection.The dose of drugs was adjusted according to the follicle size and serum hormone levels.3.FF sampling and oocyte collection:The induction of ovulation was performed with 10000 IU HCG when at least two leading follicles had reached a diameter of 18mm under transvaginal ultrasound examination.Oocytes were retrieved transvaginal under ultrasound guidance 34-36 hours after HCG administration.Oocytes were collected and incubated in a special media and FF were collected in EP tubes for further analysis.Subsequently,the follicular fluid were centrifuged twice under the condition of 12000 rpm,4 ? for 10 min and the resultant supernatant were obtained as FF samples and stored at-80 ? until further analysis.4.Fertilization protocol and embryo examination:Insemination was conducted 3 hours after oocyte retrieval.Subsequently,oocytes were transferred to a fresh medium and the occurrence of 2 PN was observed and calculated.The embryo qualities were assessed on 3 days after cleavage.5.Statistical analysis:The statistical data were expressed by mean ± SD and analyzed with SPSS 19.0 software.The normality distribution of statistics were processed by Kolmogorov-Smirnov test followed by independent t test for which distribution was normal and Kruskal-Wallis test for which distribution was not normal.A ROC curve was performed to test the discriminatory power of 5'-Deoxyadenos ine,5-Methylcytidine and 5-Methoxycarbonylmethyl-2'-thiouridine for pregnancy.p<0.05 were considered to have statistical significance.Results1.The general characteristics of the participants:As we can see,subjects in control group were on average 29.5±2.45 years old and were 28.6±2.68 in PCOS group,no significance was observed.Due to the special clinical manifestation of PCOS,participants in PCOS groups had a higher BMI(23.49±4.06 vs 20.39±2.10,p=0.01),increased LH/FSH(1.08±0.62 vs 0.70±0.41,p=0.48)and increased AMH level(4.21 ±0.29 vs 2.26±0.26,p<0.001).IVF-ET was performed in the majority of subjects(53%),the others were treated with ICSI.Conclusively,41%achieved a clinical pregnancy.2.The ART record in the control group and the PCOS group:People in control group and PCOS group showed different ovarian stimulation outcomes,manifested as increased follicle count(p=0.001),retrieved oocytes number(p=0.001),2PN fertilization count(p=0.04)and good embryo count(p=0.02)in the PCOS group compared with the control group,whereas the 2PN cleavage count(p=0.052)was not obviously changed between the two groups.3.The concentrations of 5'-deoxyadenosine,5-methylcytidine and 5-methoxycar-bonylmethyl-2'-thiouridine were changed in the FF of the PCOS patients compared with the control group:The concentration of 5'-deoxyadenosine(p=0.001)and 5-methoxycarbonylmethyl-2'-thiouridine(p=0.005)were increased in the FF of the PCOS group compared with that of the control group.And the concentration of 5-methylcytidine also found to be decreased in the PCOS group(p=0.002).Despite of the relative big error bars partly owning to the extremely small content,significant difference were observed in all three substances.4.The concentrations of 5'-deoxyadenosine and 5-methoxycarbonylmethyl-2'-thiouridine were positive correlated with the oocytes developmental competence:The concentration of 5'-deoxyadenosine was slightly positive correlated with the oocytes developmental potentials,demonstrated by a correlation coefficient of 0.389(p=0.028)with the number of retrieved oocytes,0.527(p=0.002)with the number of 2PN fertilization,0.520(p=0.002)with the 2PN cleavage and 0.506(p=0.003)with the number of top quality embryos in all subjects.And a similar correlation was found between the concentration of 5'-deoxyadenosine and the oocyte quality in the PCOS group,indicated by a correlation coefficient of 0.521(p=0.039)with the number of 2PN fertilization,0.513(p=0.042)with the number of 2PN cleavage and 0.570(p=0.021)with the number of top quality embryos.However,no significant correlation was observed in the control group.The analysis between the concentration of 5-methylcytidine and the oocytes developmental competence was also conducted,but no statistical correlation was detected overall.It is indicated that a moderate positive correlation was exhibited between the concentrations of 5-methoxycarbonylmethyl-2'-thiouridine,demonstrated a correlation coefficient of 0.572(p=0.001)with the number of retrieved oocytes,0.679(p<0.001)with the number of 2PN fertilization,0.684(p<0.001)with the 2PN cleavage and 0.816(p<0.001)with the number of top quality embryos in all subjects.And similar trends were also observed in the PCOS group,manifested as a correlation coefficient of 0.516(p=0.041)with the number of retrieved oocytes,0.685(p=0.003)with the number of 2PN fertilization,0.695(p=0.003)with the 2PN cleavage and 0.816(p<0.001)with the number of top quality embryos.However,no significant correlation was found in the control group.5.The correlation between the concentration of 5'-deoxyadenosine,5-methylcytidine,5-methoxycarbonylmethyl-2'-thiouridine and 2PN fertilization rate:the concentration of 5'-deoxyadenosine was positively correlated with 2PN fertilization rate with a correlation coefficient of 0.226(p=0.213)in all participants,0.499(p=0.049)in the control group and 0.530(p=0.035)in the PCOS group.Meanwhile the concentration of methoxycarbonylmethyl-2'-thiouridine showed a relative high positive correlation with the 2PN fertilization rate,demonstrated by a correlation coefficient of 0.627(p=0.009).Moreover,no significant correlation was found in other analysis.6.The concentration of 5-methoxycarbonylmethyl-2'-thiouridine was a good predictor for pregnancy:We can easily draw the conclusion that the concentration of 5-methoxycarbonylmethyl-2'-thiouridine was a relative good predictor to the outcome of ART,with a 95%AUC of 0.942.We also tested the predictive value of 5-methoxycarbonylmethyl-2-thiouridine in PCOS group,and a similar result was detected,however with no statistical significance.Conclusions1.Compared with the control group,PCOS patients have different ART outcomes,manifested as increased follicle count,retrieved oocytes number,2 PN fertilization count and good embryo count,whereas the 2PN cleavage count was not obviously changed between the two groups.2.The concentration of 5'-deoxyadenosine and 5-methoxycarbonylmethyl-2'-thiouridine were increased in the FF of the PCOS group compared with that of the control group.And the concentration of 5-methylcytidine also found to be decreased in the PCOS group.3.The concentration of 5'-deoxyadenosine was positively correlated with oocytes development potentials;the concentration of 5-methoxycarbonylmethyl-2'-thiouridine was moderate positive correlated with oocytes development potentials.4.The concentration of 5-methoxycarbonylmethyl-2'-thiouridine in the FF was a good predictor for pregnancy outcomes.