Effects Of Gonadotropin Releasing Hormone Agonist On Regulatory T Cells

Embryo implantation is the most critical step in the reproductive process.Low embryo implantation rate is one of the unsolved problems all along in the field of reproductive medicine,and also limits the development of assisted reproductive technology.Existing data shows that 2/3 embryo implantation failure is associated with endometrial receptivity.However,the clinical measures to improve endometrial receptivity are very limited.Endometrial receptivity is affected by multiple factors.The immune microenvironment of maternal-fetal interface is one of the important factors that affect endometrial receptivity,which determines embryo implant rate.It may reveal the relationship of hormone immune function and embryo implantation by researching effects of hormone on immune cells.Gonadotropin releasing hormone(GnRH)is an important hormone in the reproductive process,which has multiple immune regulatory functions.Its agonist(GnRHa)has been used in vitro fertility(IVF)for more than 30 years,which is one of the most commonly used drugs for assisted reproduction.Regulatory T cells(Tregs)has function of immunosuppression,and it occurs dynamic change in menstrual cycle and pregnant period,which is an important immune factor to successfully embryo implantation or sustained pregnancy.However,it is still unclear about GnRHa’s effect on endometrial immune status.There still has no clear report if there has effects on Tregs after GnRHa treatment at present.This study intends to observe the changes of quantity and quality on Tregs after GnRHa treatment,to assess the state of immune tolerance of endometrium,further reveal the relationship of hormone immune function and embryo implantation and provide the basis for optimization and improvement of IVF treatment.The study is divided into the following two sections.1.Effects of GnRHa on Tregs in infertile patientsCollected peripheral blood of infertile patients and divided into two groups based on if they were treated with GnRHa: GnRHa group and non-GnRHa group.Tested the ratio of CD3~+ T lymphocyte,CD3~+ CD4 ~+ and CD3 ~+ CD8 ~+ T cells and CD4 ~+ CD25 ~+ Foxp3 ~+ Tregs by flow cytometry.Collected secretory endometrium of infertile patients and divided into three groups: GnRHa group,non-GnRHa and Control group.Tested the level of Foxp3 m RNA and CTLA-4 mRNA by q RT-PCR and the expression of Foxp3 positive cells by immunohistochemistry.The results showed that it had no obvious change of CD4~+CD25~+Foxp3~+/CD4~+ proportion in peripheral blood mononuclear cells after GnRHa treatment.The level of Foxp3 m RNA and CTLA-4 mRNA in secretory endometrium in GnRHa group had upward tendency compared with non-GnRHa group,and no obvious difference compared with Control group.The level of Foxp3 m RNA and CTLA-4 m RNA in non-GnRHa group were significantly lower than that of Control group.The rate of Foxp3 positive cells expression in secretory endometrium had no significant difference among three groups.The results revealed that in infertile patients,the percentage of Tregs in peripheral blood mononuclear cells has no change and the level of Foxp3 m RNA and CTLA-4 mRNA in secretory endometrium have increased with GnRHa treatment.2.Effects of GnRHa on Tregs in adult female miceKunming adult female mice with normal estrous cycle were chosen for studies,including two groups: trial(GnRHa/COH)group and control(NS/COH)group.Detected the percentages of CD3~+ T lymphocytes and CD3~+CD4~+ and CD3~+CD8~+ T cells and CD4 ~+CD25~+Foxp3~+ Tregs in peripheral blood,spleen,para-aortic lymph node,inguinal lymph node by flow cytometry.Tested the level of Foxp3 m RNA in endomentrium by q RT-PCR.The serum concentration of TGF-β1 was detected by ELISA.After GnRHa downregulation,the percentage of CD4~+ CD25~+ Foxp3~+/CD4~+ in peripheral blood,spleen,para-aortic lymph node,inguinal lymph node and the serum concentration of TGF-β1 have no obvious change,and the level of Foxp3 mRNA in endomentrium has significantly increased.With GnRHa/COH treatment,the percentage of CD4~+CD25~+Foxp3~+/CD4~+ in blood and spleen has significantly increased and has no obvious change in para-aortic lymph node and inguinal lymph node,and the level of Foxp3 mRNA in endomentrium has significantly increased,and the serum concentration of TGF-β1 have no obvious change.The results revealed that in adult female mice,the level of Foxp3 m RNA in endometrium has increased with GnRHa downregulation,and the percentage of Tregs in peripheral blood,spleen and the level of Foxp3 m RNA in endometrium has increased with GnRHa/COH treatment.In conclusion,the number of Tregs in peripheral blood and secondary lymphoid organs has no change,but the level of Foxp3 mRNA and CTLA-4 mRNA in endometrium have increased after GnRHa treatment.The number of Tregs in peripheral blood and spleen and the level of Foxp3 mRNA in endometrium have increased with GnRHa/COH treatment.It could be speculated that endometrial immune tolerance may be improved with GnRHa treatment,and it may be improved of the immune tolerance status of maternal system and endometrial local condition with GnRHa/COH treatment,which provide advantages for embryo implantation.The serum concentration of TGF-β1 has no change with GnRHa treatment,so it could be speculated that the secretion of TGF-β1 of Tregs is not influenced GnRHa.