A Preliminary Investigation Of T Lymphocyte Subpopulation Imbalance And Epigenetic Mechanism In IVF Offspring Mice

Background:In vitro fertilization(IVF)is the most important treatment for infertility.At present,more than 10 million children have been born by IVF worldwide,and it is still growing rapidly.Because IVF technology involves many unnatural conception processes,such as controlled ovarian hyperstimulation,in vitro fertilization,embryo culture and transfer,the long-term health of this group has increasingly become the focus of public concern.Previous studies have found that IVF is associated with a high incidence of immune-related diseases,including metabolic disorders,asthma,and allergic diseases,suggesting that IVF technology may also have a potential impact on the immune system of the offspring.However,there are few studies on whether the immune system changes in IVF offspring,and its mechanism is still not reported.The "developmental origins of health and disease"(DOHaD)theory proposes that epigenetic remodeling is one of the possible mechanisms by which environmental factors exposed from gametogenesis to embryonic and fetal development affect offspring phenotypic changes.A large number of animal studies have provided favorable evidence that genomic DNA methylation modification and other epigenetic modifications in embryos and appendages of assisted reproductive technology,especially in vitro fertilization,significantly alter the expression of different genes compared with natural pregnancy.Although there are significant ethical limitations to performing such epigenetic studies in human embryos,alterations in epigenetic modifications have also been found in human IVF embryo appendage(placenta,cord blood,umbilical cord).So,as a technology that can greatly affect the environment from gametogenesis to early embryonic stage,does IVF cause changes in the immune system of the offspring,and if so,does epigenetic regulation participate?Methods:In order to explore whether the immune system of IVF offspring changes,the proportion and number of T lymphocytes in immune organs or tissues were detected by flow cytometry and other methods by constructing IVF mouse model.In order to further explore the role of epigenetics in the development and differentiation of T lymphocytes in IVF offspring,we performed DNA methylation sequencing and mRNA sequencing of spleen and thymus,and used bioinformatics combined analysis to preliminarily explore the epigenetic remodeling characteristics of thymus and spleen genome and the regulatory mechanism of T lymphocyte development and differentiation in IVF offspring.Result:1.By constructing an IVF mouse model,flow cytometry was used to detect and analyze T lymphocyte subsets in central and peripheral immune organs.It was found that the proportion of double negative T cells in thymus of IVF offspring mice decreased.Spleen T lymphocyte subsets were unbalanced,the number of T lymphocytes increased;,and the proportion and number of Tcl cells decreased.HE staining revealed chronic inflammation in the spleen.Decreased levels of IFN-y and IL-6 in serum were detected by ELISA.2.DNA methylation sequencing and mRNA sequencing were performed on thymus and spleen,and the differential genes were found to be enriched in the differentiation pathway of T lymphocyte subsets and the TNF signaling pathway related to inflammation.According to the correlation analysis,the potential target genes causing the imbalance of T lymphocyte subsets were found.Conclusion:Immune alterations are present in VF offspring mice and imbalance in T lymphocyte subsets in IVF offspring mice,as evidenced by a decrease in the proportion of thymic double-negative T cells,an increase in the number of splenic T lymphocytes,an increase in the proportion of Treg cells,and a decrease in the proportion and number of Tcl cells.The epigenetic mechanism may be due to DNA methylation reprogramming of genes associated with cell developmental differentiation and inflammation in the thymus.