| Objective: To investigate whether there are biomarkers affecting normal fertilization and abnormal fertilization of human cumulus cells in(CCS)gene transcriptional group.Method:(1)Screening patients with infertility due to fallopian tube factors.The routine program was used to promote ovulation.Under the guidance of transvaginal ultrasound,the cumulus-oocyte complex(COC)was obtained.Half an hour before fertilization,the peripheral cumulus cells of the subjects were cut by mechanical method,and 20 to 30 cumulus cell clusters were obtained.The cumulus cell masses were digested with hyaluronidase and stored in aseptic tubes.The rest of the egg structure is fertilized with microdroplets(one egg per drop).20 hours after fertilization,the remaining cumulus cells around the egg were removed.The fertilized zygotes were observed under 200 × inverted microscope and microdroplet culture was carried out.The cumulus cells of single pronucleus and multi-pronucleus fertilized eggs of the same subject were mixed as abnormal fertilization group.The cumulus cells of the normal fertilized(double pronuclear)eggs of the same subject were mixed as the normal fertilization group.(2)A total of 3 repeats were set up in this experiment.Each repeat was divided into cumulus cells in normal fertilization group and cumulus cells in abnormal fertilization group.Total m RNA of cumulus cells was extracted separately to construct c DNA library.Then reverse transcriptional group sequencing was performed.(3)The FPKM(Fragments Per Kilobase Million)is sequenced on the Illumina Hi Seq platform,and the data is mapped.The obtained data were analyzed by RNA-Seq correlation test to analyze the correlation of gene expression level among samples,and the overall quality control and filtering achieved data standardization and correction.(4)the selected differentially expressed genes were analyzed by Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis.Results The differentially expressed gene analysis shows a wealth of gene types and cellular pathways.In the first subject,there was a significant difference in the expression of 163 genes between normal fertilization(MNF)and abnormal fertilization(MAF)(up-regulation of 30 genes and down-regulation of 133genes).In the second subject,493 genes were significantly different between normal fertilized(WNF)and abnormal fertilized(WAF)(up-regulated 379 genes and down-regulated 114genes).In the third subject,there was a significant difference in the expression of 200 genes between normal fertilization(LNF)and abnormal fertilization(LAF)(up-regulation of 113 genes and down-regulation of 87 genes).Compared with normal fertilization,40 genes were significantly up-regulated and 11 enes were significantly down-regulated in the abnormal fertilization group of the three subjects.KEGG analysis showed that the up-regulated genes were related to oocyte growth,maturation,proliferation,differentiation,survival,apoptosis,bioadhesion,hormone secretion,immunity,tumorigenesis,mitosis and fertilization.The down-regulated genes are related to cellular substance metabolism and endocrine regulation.Conclusions It is find that there are four differential genes: BMP2,SESN3,CDK6 and GRLF1(ARHGAP35)may related to abnormal fertilization.The expression of these genes were significantly up-regulated.No obvious expression related to abnormal fertilization was found in the down-regulated genes.Through the analysis of experimental data and the review of a large number of related literatures,we found that SESN3 may have a potential mechanism for regulating abnormal fertilization of oocytes,and the effect of SEN3 on abnormal fertilization needs to be further verified by QRT-PCR. |
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