A Preliminary Study On The Metabolic Profile Of D3 Embryo Culture In IVF Patients Based On Metabolomics

Objective: D3 embryos(cleavage stage embryos)are currently selected for transplantation in most reproductive centers,and the quality of the embryos is the key to the success of assisted reproductive technology.An emerging discipline developed after genomics,transcriptomics,and proteomics is called metabonomic/ metabolomics,which has become a research hotspot because of its non-invasiveness,easy access to samples,and small sample size.At present,the metabolomic analysis techniques related to the assessment of embryonic developmental potential are still being gradually improved.The value of metabolomic analysis in the selection of high-quality embryos can be fully demonstrated only when multiple components of embryo metabolites are detected and their maps are drawn.Therefore,this research by using spectroscopy and non-spectroscopy in D3 embryo culture of all the metabolites of target detection,is optimal in the embryo,carrier group and blank control group such as amino acid metabolism,fat metabolism and sugar metabolism related differences in metabolic product,through the sample collection,pretreatment,ultrahigh performance liquid chromatograph detection and embryo metabolites mapping XCMS software analysis,explore the cleavage stage embryos(D3)culture metabolites in accurately predicting the superiority of the potential of early embryonic development,provide a new perspective for the choice of high quality embryo,It also provides new ideas for selective single embryo transfer,reduction of multiple pregnancy and improvement of clinical pregnancy outcome.Methods: Patients who went to the reproductive medicine specialist hospital of the first hospital of Lan Zhou university for in vitro fertilization and embryo transfer(IVF-ET)from October 2018 to March 2019 were selected as the study subjects.Based on morphology of D3 embryos morphological evaluation,divided into the optimal embryo group(I,II)and substandard embryo group(grade III or IV),respectively the fresh embryo transfer or collect the remaining embryo culture in vitro embryo cryopreservation of embryos group 11 October as the experimental group and substandard embryo group,and with five simple embryo culture in vitro of blank control group.Untargeted detection of all metabolites in the samples of the experimental group and the blank control group was carried out by means of chromatographer-mass spectrometry,differential expression was found and analyzed,and then the metabolite map of D3 embryo culture medium was drawn.The main test steps include: sample preparation,QC sample preparation,sample LC-MS /MSZ mass spectrometry,data analysis,etc.Detection methods: metabolomics detection instrument platform GC/MS and UPLC/Q-TOF.Specific indicators: amino acid metabolism,fat metabolism,sugar metabolism and other related metabolites.Results: Results 1: D3 embryo culture medium has a typical TIC map(figure 4);Results 2: The aggregation effect and separation trend of D3 embryo culture medium in PCA analysis group were significantly different(figure 5);Results 3: PLS-DA and its substitution test analysis screened out the metabolite different between groups(figure 6);Results 4: OPLS-DA and its substitution test analysis screened out the predicted principal components between groups(figure 7);Results 5: Typical volcano plot(Volcano Plot)analysis of potential landmark metabolites between groups was significant(figure 8);Results 6: Significant differences between groups in metabolite hierarchical cluster analysis of different numbers and types(Figure 9),excellent Compared with the blank control group,the embryo group differed by 15 significant metabolites(Table 6),the poor embryo group compared with the blank control group by 19 significant metabolites(Table 7),the superior embryo group and the poor embryo group Compared with significant differences,there are 10 metabolites(Table 8).Conclusion: Metabolomics non-targeted analysis technology for the analysis of the culture medium of D3 embryos(embryos in cleavage stage)of IVF-ET patients is expected to establish an ideal database,map the metabolism,and assist in the screening of high-quality embryos due to its non-invasive nature and almost zero harm to the researcher and the embryo.Screen out the differences of metabolites,such as L-glutamine metabolic pathways involved in glucose metabolism and protein,niacinamide participates in the metabolism of lipid and glucose metabolism in the process of anaerobic glycolysis,linoleic acid,arachidonic acid and acid involved in monounsaturated fatty acid metabolism,could discern fatty acid biosynthesis pathway and center carbon metabolic pathway may be the key factor affecting the quality of embryos molecular events.On the basis of the study,from the genome,transcriptome and systems biology technology such as proteomics,further in-depth study of the molecular events marked differences metabolites KEGG pathways the response mechanism of upstream and downstream,to predict the D3 embryonic developmental potential,selective single embryo transfer,decrease the rate of multiple pregnancy,and thus improve the embryo implantation rate and improve the tire live-birth rate has important significance.