Effects Of Low Expression Of MiR-320-3p On Embryonic Development In Polycystic Ovary Syndrome

BackgroundPolycystic ovary syndrome（PCOS）is a familiar and complex reproductive endocrine disorder with clinical manifestations such as oligo-ovulation or anovulation,hirsutism,acne,obesity,etc.Current research shows that the quality of oocytes is a key factor affecting embryonic development.Micro RNAs（miRNAs）,as a branch of the field of epigenetics,can indirectly regulate gene expression and play an indispensable role in regulating development.Several studies have shown that miR-320（miR-320a）is abnormally expressed in the blood,follicular fluid,granulosa cells,and exosomes of PCOS patients.The level of miR-320 in human follicular fluid can be used as an indicator for evaluating embryo quality.Interfering with miR-320 expression in mouse oocytes affects early embryonic development.ObjectiveTo study the expression of miR-320-3p in granulosa cells of PCOS patients,granulosa cells and oocytes of PCOS mice.Detect the expression of target genes and pluripotency genes in early embryos,and perform transcriptome sequencing of early embryos.Preliminary exploration of the molecular mechanism of the effect of abnormal expression of PCOS miR-320-3p on embryonic developmental potential.Methods1.Expression of miR-320-3p in human granulosa cells detected by q RT-PCR.2.Subcutaneous injection of dehydroepiandrosterone to establish PCOS model.3.Expression of miR-320-3p in mouse granulosa cells and MⅡ oocytes detected by q RT-PCR.4.Intracytoplasmic injection of miR-320-3p mimics or NCs into mouse oocytes and grouping: Control + NC group,PCOS + NC group,PCOS + mimic group（miR-320-3p mimics）.In vitro fertilization and culture after injection,Record the number of4-cell stage embryos and blastocysts.5.The blastocysts were stained with trophectoderm molecular marker CDX2 and detected by TUNEL cell apoptosis.The total number of cells,trophectoderm（TE）cells,inner cell mass（ICM）cells and apoptotic cells were counted in each group.6.q RT-PCR method to detect the expression changes of miR-320-3p target genes and embryonic development-related pluripotency genes in 4-cell embryos.7.The differential expression of m RNAs in mouse 4-cell embryos was analyzed by transcriptome sequencing and bioinformatics,and the differential genes were verified by q RT-PCR method.8.Statistical analysis of the data using SPSS 25.0 software.Result1.Compared with the control group,the expression of miR-320-3p in the follicular fluid granulosa cells of PCOS patients was down-regulated（P<0.05）.2.Compared with the mice in the control group,the estrous cycle of the mice in the PCOS group was disordered,and most of them were in the estrus and diestrus;the ovarian tissue sections of the mice in the PCOS group showed that the number of follicles increased significantly,some follicles had cystic expansion,and the granulosa cells were loosely arranged and undisturbed.Irregular,significantly reduced corpus luteum.3.The expression of miR-320-3p was down-regulated in granulosa cells and MⅡstage oocytes in PCOS group（P<0.05）4.Compared with the control+NC group,the blastocyst rate in the PCOS+NC group was significantly decreased,and compared with the PCOS+NC group,the blastocyst rate in the PCOS+mimics group was significantly increased（P<0.05）.5.The total number of cells,TE cells,and ICM cells in the control+NC group and PCOS+mimics group were significantly more than those in the PCOS+NC group,and the number of apoptotic cells was significantly less than that in the PCOS+NC group（P<0.05）.6.Compared with the control+NC group and the PCOS+mimics group,the expressions of Ulk1,Pbx3,Myc,and Dppa3 were significantly down-regulated in the4-cell embryos of the PCOS+NC group,and the expressions of Kdm5 b and Stab2 were significantly up-regulated（P<0.05）.Compared with control+NC group,Wdr5 expression was up-regulated in both PCOS+NC group and PCOS+mimics group.（P<0.05）.7.Transcriptome sequencing of 4-cell embryos: There were 242 differential genes in 4-cell embryos in control+NC group and PCOS+NC group,and 94 differential genes in 4-cell embryos in PCOS+NC group and PCOS+mimics group.Among them,25 differentially expressed genes were abnormally expressed in the PCOS+NC group,and their expression levels were restored in the PCOS+mimics group,tending to the expression levels of the control+NC group.Compared with the control+NC group and the PCOS+mimics group,the expressions of Vrk2,Ptpn3,Kit1 and Reep3 were upregulated in the 4-cell embryos of the PCOS+NC group,the expressions of Meterf2,Ampd1 and Il1 rap were down-regulated（P<0.05）.ConclusionDown-regulated expression of miR-320-3p in granulosa cells of PCOS patients and granulosa cells and oocytes of PCOS mice.Low expression of miR-320-3p in PCOS mouse oocytes affects preimplantation development and reflects the quality of oocytes and embryos.Compensating miR-320-3p in PCOS oocytes can improve embryonic developmental potential and improve embryo quality.