The Assessment Of Blastocysts Quality And Transcriptomic Sequencing Analysis Of Spent Embryo Culture Medium Combined With Blastocoel Fluid

Background: Achieving a healthy singleton live birth is the ultimate goal in the field of assisted reproductive technology(ART).Elective single-embryo transfer(e SET)is recommended,in which single blastocyst transfer is the primary approach.Therefore,determine an evaluation criterion for blastocysts with the best developmental potential is significant to facilitate the ART treatment.At present,the most commonly used blastocyst morphology scoring method unavoidably has bias due to its subjective judgement.Moreover,the potential risk of preimplantation genetic testing for aneuploidy(PGT-A)by trophoblasts biopsy in blastocyst is also difficult to avoid.The questionable,non-invasive approach to PGT has not been widely used because of the large heterogeneity among studies and poor correlation with embryo implantation potential.Therefore,we need to further explore more accurate non-invasive or minimally invasive embryo evaluation methods to guide the embryo selection.Advances in single-cell sequencing technologies have allowed the application of RNA-sequencing techniques to low-input samples,facilitating the study of transcriptional events during early human embryonic development.Therefore,this study intends to compare the correlation between different embryo evaluation methods and pregnancy outcomes.Then explore the feasibility of a minimally invasive embryo sampling method,namely,spent embryo culture medium combined with blastocoel fluid(ECB),for cell-free RNA(cf RNA)sequencing.And transcriptomic differences among embryos with different quality and pregnancy outcomes were further compared.Purposes:1.To explore the correlation of embryo morphological parameters,ploidy properties and pregnancy outcome;2.To explore the feasibility of cf RNA sequencing in blastocyst ECB samples,and determine m RNA expression profiles in blastocyst ECB microenvironment;3.To explore differentially expressed genes(DEGs)associate with embryo quality and implantation potential in ECB based on the evidence of embryo euploidy,morphology and pregnancy outcomes after euploidy embryos transfer.Methods:1.The non-PGT-derived single frozen blastocyst transfer and the single PGT blastocyst transfer in our center from 2018 to 2021 were retrospectively analyzed.After multivariate binary logistic regression analysis and adjusting for confounding factors,whether the three morphological parameters(blastocyst expansion degree,inner cell mass(ICM)and trophectoderm(TE)grade)were independently associated with pregnancy outcomes were compared;2.We prospectively recruited patients who plan to undergo PGT-A in strict accordance with the inclusion and exclusion criteria.After informed consent,the ECB and TE cells of blastocysts formed by in vitro fertilization were collected successively.Using Smart-seq2 method combined with next-generation sequencing(NGS)for full-length RNA sequencing to construct m RNA profiles in ECB samples.TE cell samples were used for euploidy detection by whole genome amplification combined with NGS method.Further large-scale screening,sequence determination and analysis was conducted;3.The samples grouped according to embryo euploidy,morphology and pregnancy outcomes and were screened for DEGs using the DESeq2 package in R software.Subsequently,the differential transcriptomics analysis,including principal component analysis,cluster and functional enrichment analysis,and protein-protein interaction were performed to screen differential candidate genes.Results:1.In non-PGT cycles,both ICM and TE grades were significantly correlated with clinical pregnancy rate and ongoing pregnancy rate(P<0.01);the clinical pregnancy rate and the ongoing pregnancy rate of stage 4 blastocysts for expansion degree were significantly higher than stage 3 blastocysts(P<0.05).In the PGT cycle,the expansion degree,ICM and TE grades were not significantly correlated with clinical pregnancy and ongoing pregnancy outcomes(P>0.05),and the difference in the ongoing pregnancy rate between grades B and C in TE grades was the most significant(44.6% vs.35.5%;P=0.067;adjusted OR,1.75;95% CI,0.96-3.18);2.19 fresh cycles of PGT-A were finally included,with a total of 52 embryos.The average euploid rate of the cycles was 62.8% and the euploid rate of the embryos sampled for ECB was 57.7%.8 euploidy embryos were transferred and the clinical pregnancy rate was 62.5%(5/8).In trace ECB samples,amplified m RNA can account for about 17% of cf RNA,and genes with higher expression abundance are mainly involved in processes such as chromatin organization,gene transcription,nuclear transport,and cytokine signal transduction;3.The principal component analysis can effectively separate embryos with different quality and pregnancy outcomes.DEGs were significantly enriched in the process of early embryonic cell proliferation,development and adhesion.Energy metabolism pathways such as RNA metabolism and tricarboxylic acid cycle were significantly enriched.BRCA1,BRCA2 and related genes were involved in cell cycle process and regulation.USP2,USP17 L families and others were enriched in the process of protein deubiquitination.Molecules such as CTNNA1 and CTNNB1 and the WNT signaling pathway played an important role in the process of adhesions junction.Conclusions:1.In blastocyst transfer of unknown ploidy properties,prioritizing ICM and TE grades helped to select blastocysts with the highest potential of live birth,while the expansion degree is relatively weak;Among blastocysts considered as euploid after PGT detection,there was no significant difference in the pregnancy outcomes of blastocysts with different morphological scores after transfer.Relatively speaking,TE grading can be prioritized to select embryos;2.In this study,efficient RNA amplification was performed in trace ECB samples.The m RNA expression profile in the blastocyst ECB microenvironment was successfully constructed;3.The m RNA expression profiles in the ECB microenvironment showed different expression trends among embryos with different morphology,ploidy properties and pregnancy outcomes.DEGs were significantly enriched in the process of early embryonic cell proliferation,development and adhesion.