Study On The Expression Of CD4~+T Cells And The Relevant Factors Of ATA~+ Infertile Women And The Effect On The Treatment Outcome Of IVF-ET

ObjectiveTo investigate the expression of the CD4⁺T cells（the Th1 cells,Th2 cells,Th17 cells and Treg cells）and their corresponding cytokines（IFN-γ,IL-4,IL-17A and TGF-β1）and characteristic transcription factors（T-betmRNA,GATA-3mRNA,RORγtmRNA and FoxP3mRNA）in the peripheral blood of patients with normal thyroid function while positive anti-thyroid autoantibodies（ATA⁺）at different time points(on the oocyte retrieval days,the 5^th day of embryo transfer and the 14^th day of embryo transfer),and further to explore the immunological mechanism of adverse outcomes of in vitro fertilization-embryo transfer（IVF-ET）treatment caused by ATA.Methods1.In the first part,a retrospective analysis was conducted on the effects of the ATA⁺patients on pregnancy outcomes of the IVF-ET and the frozen embryo transfer（FET）.2.In the second part,a prospective study was conducted on a total of 80 patients（80cycles）with normal thyroid function under the fresh IVF treatment in the Reproductive Medicine Department,Yantai Yuhuangding Hospital,affiliated to Qingdao University from June 2016 to September 2016,among which 28 patients（28 cycles）with ATA⁺and 32 patients（32 cycles）with negative anti-thyroid autoantibodies（ATA^-）were treated with embryo transfer.3.Peripheral blood samples were collected from the ATA⁺and ATA^-patients with normal thyroid function on the oocyte retrieval days and the 5^th day and the 14^th day of embryo transfer.4.Flow cytometry（FCM）was used to detect the proportion of the CD4⁺T cells,namely the Th1 cells,Th2 cells,Th17 cells and Treg cells in the peripheral blood of the two groups at the oocyte retrieval days and the 5^th day and the 14^th day of embryo transfer.5.Enzyme linked immunosorbent assay（ELISA）was used to detect the concentration of the CD4⁺T cells corresponding cytokines IFN-γ,IL-4,IL-17A and TGF-β1 in the peripheral blood of the two groups at the oocyte retrieval days and the 5^th day and the 14^thh day of embryo transfer.6.Reverse transcription-polymerase chain reaction（RT-PCR）was used to detect the expression of characteristic transcription factors T-betmRNA,GATA-3mRNA,RORγtmRNA and FoxP3mRNA with the CD4⁺T cells in the peripheral blood of the two groups at the oocyte retrieval days and the 5^th day and the 14^th day of embryo transfer.7.A study was done on the proportion of the Th1 cells,Th2 cells,Th17 cells and Treg cells,and the concentration of corresponding cytokines IFN-γ,IL-4,IL-17A and TGF-β1,and the expression of characteristic transcription factors T-betmRNA,GATA-3mRNA,RORγtmRNA and FoxP3mRNA in the peripheral blood of the two groups at the oocyte retrieval days and the 5^th day and the 14^th day of embryo transfer and their effects on the outcome of IVF-ET treatment.8.A study was done on the ratio of the Th1/Th2 and Th17/Treg,the corresponding cytokines the ratio of IFN-γ/IL-4 and IL-17A/TGF-β1,the characteristic transcription factors the ratio of T-betmRNA/GATA-3mRNA and RORγtmRNA/FoxP3mRNA in the peripheral blood of the two groups at the oocyte retrieval days and the 5^th day and the 14^th day of embryo transfer and their effects on the outcome of IVF-ET treatment.9.A study was done on the changes of the CD4⁺T cells,the corresponding cytokines and characteristic transcription factors in the peripheral blood of the ATA⁺group and ATA^-group at different time points and their effects on the outcome of IVF-ET treatment.Results1.In the first part,the retrospective study showed a significant rise in the titers of the TG-Ab and TPO-Ab in the ATA⁺patients in the IVF,while a clear decrease in the IVF excellent embryo rate,the number of the IVF transplanted excellent embryos,the number of the ICSI excellent embryos,and the number of the ICSI transplanted embryos（P<0.05）.In addition,the titers of the TG-Ab and TPO-Ab in the ATA⁺patients were negatively correlated with the IVF fertilization rate.In the FET,the early abortion rate of the ATA⁺patients increased significantly（P<0.05）.2.In the second part,the prospective study showed that the number of the large follicle on HCG day,obtained follicle count and transplanted good quality embryos of the IVF-ET patients in the ATA⁺group were significantly less than those in the ATA^-group（P<0.05）.In addition,the biochemical pregnancy abortion rate of the IVF-ET patients in the ATA⁺group was significantly higher than that in the ATA^-group（P<0.05）.The number of excellent embryos,excellent embryos rate,blastocyst rate as well as the uumber of the transplanted high qualified embryo of the ATA⁺patients in the pregnancy group were significantly more than those in the non-pregnancy group（P<0.05）.3.The result of the study on the proportion of the CD4⁺T cells in the peripheral blood of patients in two groups showed that the proportion of the Th1 cells in the peripheral blood of patients in the ATA⁺group were significantly higher than those in ATA^-group on the oocyte retrieval days;the proportion of the Th2 cells in the peripheral blood of patients in the ATA⁺group were significantly lower than those in the ATA^-group on the 14^th day of embryo transfer;the proportion of the Th17 cells in the peripheral blood of patients at different time points increased significantly while that of the Treg cells decreased clearly in the ATA⁺patients.The proportion of the Th17 cells in the peripheral blood of ATA⁺patients in the pregnancy group at different time points were significantly lower than those in the non-pregnancy group（P<0.05）.4.The result of the study on the balance of the Th1 cells and Th2 cells,Th17 cells and Treg cells in the peripheral blood of patients in two groups showed that the ratio of Th1/Th2,Th17/Treg in the peripheral blood at different time points in the ATA⁺group were significantly higher than those in the ATA^-group（P<0.05）.The balance of the Th1 and Th2cells,Th17 and Treg cells in the peripheral blood of the ATA⁺patients at different time points were broken.The ratio of Th17/Treg in the peripheral blood of ATA⁺patients in the pregnancy group at different time points were significantly lower than those in the non-pregnancy group（P<0.05）.5.The result of the study on the changes of the proportion of the CD⁺T cells in the peripheral blood of patients in the ATA⁺and ATA^-group showed that the proportion of the Th1 cells in the ATA⁺group at the 5^th day of embryo transfer were significantly higher than those at the 14^th day of embryo transfer（P<0.05）;and the proportion of the Th1 cells in the ATA^-group at the 5^th day of embryo transfer were higher than those at the 14^th day of embryo transfer（P>0.05）.6.The result of the study on the concentration of the corresponding cytokines of the CD4⁺T cells showed that the concentration of IFN-γin the peripheral blood of patients in the ATA⁺group were significantly higher than those in the ATA^-group on the oocyte retrieval days;the concentration of IL-4 in the peripheral blood of patients in the ATA⁺group were significantly lower than those in the ATA^-group on the 14^th day of embryo transfer;the concentration of IL-17A in the peripheral blood of patients in the ATA⁺groups at different time points increased significantly while that of TGF-β1 decreased clearly.The concentration of IL-17A in the peripheral blood of ATA⁺patients in the pregnancy group at different time points were significantly lower than those in the non-pregnancy group（P<0.05）.7.The result of the study on the ratio of the corresponding cytokines of the CD4⁺T cells showed that both the ratio of IFN-γ/IL-4 and IL-17A/TGF-β1 in the peripheral blood of patients in the ATA⁺groups at different time points increased significantly.The ratio of IL-17A/TGF-β1 in the peripheral blood of the ATA⁺patients in the pregnancy group at different time points were significantly lower than those in the non-pregnancy group（P<0.05）.8.The result of the study on the concentration changes of the corresponding cytokines of the CD4⁺T cells showed that the concentration of IFN-γin the peripheral blood of patients in the ATA⁺group at the oocyte retrieval days were higher than those at the 5^th day of embryo transfer（P>0.05）,while the concentration of IFN-γin the peripheral blood of patients of the ATA^-group at the oocyte retrieval days were significantly lower than those at the 5^th day of embryo transfer（P<0.05）.The concentration of IL-4 in the peripheral blood of patients in the ATA⁺group at the 14^th day of embryo transfer were higher than those at the 5^thh day of embryo transfer（P>0.05）,while the concentration of IL-4 in the peripheral blood of patients in the ATA^-group at the 14^th day of embryo transfer were significantly higher than those at the 5^th day of embryo transfer（P<0.05）.9.The result of the study on the expression of transcription factors with the CD4⁺T cells corresponding characteristic showed that the expression of T-betmRNA in the peripheral blood of patients in the ATA⁺group were significantly higher than those in the ATA^-group on the oocyte retrieval days;the expression of GATA-3mRNA in the peripheral blood of patients in the ATA⁺group were significantly lower than those in the ATA^-group on the 14^thh day of embryo transfer;the expression of RORγtmRNA in the peripheral blood of patients in the ATA⁺group at different time points increased significantly while that of FoxP3mRNA decreased clearly.The expression of RORγtmRNA in the peripheral blood of ATA⁺patients in the pregnancy group at different time points were significantly lower than those in the non-pregnancy group（P<0.05）.10.The result of the study on the ratio of transcription factors with the CD4⁺T cells corresponding characteristic showed that the ratio of T-betmRNA/GATA-3mRNA and RORγtmRNA/FoxP3mRNA in the peripheral blood drawn at different time points of the ATA⁺group were significantly higher than those in the ATA^-group（P<0.05）.The ratio of RORγtmRNA/FoxP3mRNA in the peripheral blood of the ATA⁺patients in the pregnancy group at different time points were significantly lower than those in the non-pregnancy group（P<0.05）.11.The result of the study on the changes of the expression of the CD4⁺T cells corresponding characteristic transcription factors showed that the expression of T-betmRNA in the peripheral blood of patients in the ATA⁺group at the 14^th day of embryo transfer were significantly higher than those of the oocyte retrieval days（P<0.05）,while the expression of T-betmRNA in the peripheral blood of patients in the ATA^-group at the 14^th day of embryo transfer were lower than those of the oocyte retrieval days（P>0.05）.The expression of RORγtmRNA in the peripheral blood of patients in the ATA⁺group at the 14^th day of embryo transfer were significantly higher than those of the oocyte retrieval days（P<0.05）;while the expression of RORγtmRNA in the peripheral blood of patients in the ATA^-group at the 14^thh day of embryo transfer were higher than those of the oocyte retrieval days（P>0.05）.Conclusion1.The proportion of the Th1 cells and their corresponding cytokines IFN-γand transcription factors T-betmRNA in the peripheral blood of the ATA⁺patients on the oocyte retrieval days were significantly increased,which may be one of the reasons for the low number of oocyte and the number of excellent embryos in the IVF-ET.2.The proportion of the Th2 cells and their corresponding cytokines IL-4 and transcription factors GATA-3mRNA in the peripheral blood of the ATA⁺patients on the 14^thh day of embryo transfer were significantly decreased,which may be one of the reasons for the increase of the biochemical pregnancy abortion rate in the IVF-ET.3.The significant increase of the concentrations of the Th17 cells and their corresponding cytokines IL-17A and transcription factors RORγtmRNA in the peripheral blood of the ATA⁺patients,as well as the significant decrease of the concentrations of the Treg cells and their corresponding cytokines TGF-β1 and transcription factors FoxP3mRNA in the peripheral blood of the ATA⁺patients,might be one of the causes to the poor treatment outcome of the IVF-ET.4.The ratio of the Th1/Th2 and Th17/Treg cells in the peripheral blood of the ATA⁺patients at different time points significantly increased,that is,the balance of Th1 and Th2cells,Th17 and Treg cells in the peripheral blood of the ATA⁺patients was broken,which might be one of the immunological mechanisms of the IVF-ET treatment for adverse pregnancy outcomes in the ATA⁺patients.