Study On The Mechanism Of 1,25（OH）₂D₃ Regulating NK Cell Function In Recurrent Spontaneous Abortion

Objective:Recurrent spontaneous abortion(RSA)is defined as two or more consecutive fetal losses before 28 weeks of gestation with the same sexual partner.With the postponement of women’s childbearing age and the introduction of the "universal twochild" policy and the "three-child" policy,the incidence of RSA has increased year by year,which is an important health problem nationwide and even worldwide.The etiology of RSA is complex and diverse,and the clinical manifestations are lack of specificity.When diagnosing and treating RSA,systematic screening is often required to clarify the etiology.At present,the known etiology of RSA mainly includes genetic factors,anatomical factors,endocrine factors,coagulation factors,immune factors,infection factors and psychological factors,among which the immune factors is a hotspot and difficulty in the field of reproductive immunology.Compared with the mother,the embryo can be regarded as a semi-allograft.A successful pregnancy requires the maternal immune system to develop immune tolerance to the embryo carrying paternal antigens.Immune imbalance will lead to adverse pregnancy outcomes.In recent years,studies have shown that more than half of RSA is related to maternal immune dysfunction,and some studies believe that it is closely related to the abnormal number and activity of natural killer(NK)cells.As an important part of innate immunity of organism,the function of natural killer cells has been gradually recognized,which can not only participate in cellular immunity,but also participate in the occurrence and development of RSA through humoral immunity.Therefore,NK cells have become the focus of research on the immune factors of RSA in recent years.Vitamin D(VD)is an essential nutrient for maintaining normal physiological activities of human body.Recent studies have found that it can not only play a "classic" role in affecting bone metabolism,but also act on target genes through vitamin D receptor(VDR),playing a variety of "non-classical" biological functions,including regulating cell proliferation,differentiation,apoptosis and immune regulation.Vitamin D receptor is expressed in placenta,decidua and ovary,and participates in hormone synthesis and secretion and affects pregnancy outcome;VDR is also expressed in endometrial stromal cells,regulating the process of embryo implantation.The expression of vitamin D in the decidua of the uterus changes dramatically during pregnancy.Vitamin D metabolism-related enzymes are abundantly expressed in the decidua of early pregnancy,is highly expressed in the decidua of the uterus.And the local concentration of its active form,1,25-dihydroxy-vitamin D3(1,25-dihydroxy-vitamin D3,1,25-dihydroxy-vitamin D3)in placenta increased 3-4 times than that before pregnancy,suggesting that vitamin D may play an important physiological role in maternal-fetal immune interaction in early pregnancy.The increase of local vitamin D in placenta/decidua during early pregnancy is related to the increase of catalytic formation and the decrease of decomposition and inactivation.The expression of 1-αhydroxylase(CYP27B1)increased;The expression of 24 hydroxylase(CYP24A1)involved in vitamin D decomposition was reduced.Low vitamin D levels are common in patients with recurrent spontaneous abortion.The miscarriage rate of women with vitamin D insufficiency or deficiency is significantly higher than that of women with normal vitamin D levels,suggesting that low vitamin D levels may be associated with an increased risk of RSA.It has been found that compared with normal pregnant women,RSA patients have abnormal vitamin D metabolic pathways at decidual level,such as decreased expression of VDR and CYP27B1.More and more clinical evidences suggest that VD deficiency or insufficiency is associated with an increased risk of obstetric complications,such as repeated embryo implantation failure,spontaneous abortion,recurrent miscarriage,gestational diabetes mellitus and hypertensive disorders of pregnancy.Studies have found that vitamin D can down regulate the cytotoxicity and perforin secretion of peripheral blood NK cells in RSA patients,suggesting that vitamin D also has an immunomodulatory effect on peripheral blood NK cells.Vitamin D may be a key regulator of immune function at the maternal-fetal interface during pregnancy and participate in the immune regulation of pregnancy.However,the specific mechanism by which it regulates the body’s immune function is still unclear.In our previous study,we found that the peripheral blood vitamin D level of RSA abortion patients was significantly lower than that of healthy pregnant women during the same period.Therefore,we hypothesized that 1,25(OH)2D3 could play an important role in early pregnancy by regulating the number and function of NK cells in the peripheral blood and decidua.This study will be divided into four parts to study the pathogenesis of 1,25(OH)2D3 participating in recurrent spontaneous abortion by regulating NK cells:Part I:The relationship between serum vitamin D level in early pregnancy and the percentage of peripheral blood and decidual NK cell subsets,as well as the expression levels of VDR,CYP27B1 and CYP24A1 proteins in decidua of RSA patients were studied.Part Ⅱ:The changes in the percentage of peripheral blood NK cell subsets and serum cytokines in RSA patients after vitamin D3 supplementation were studied.Part Ⅲ:The role and mechanism of 1,25(OH)2D3 in regulating NK cell function and cytokine secretion.Part Ⅳ:The pregnancy outcomes of vitamin D deficient recurrent spontaneous abortion(RSA)mouse model were studied.Methods:1.Part Ⅰ:From October 2020 to January 2021,select 15 RSA patients who went to the Family Planning Clinic of Hospital for artificial abortion due to the termination of embryo development as RSA abortion group,17 healthy pregnant women who terminated their pregnancy due to personal wishes were selected as the control group,and 26 RSA patients with normal pregnancy were selected as the RSA pregnancy group.The fasting elbow venous blood of the three groups in the first trimester of pregnancy was collected.The serum 25-hydroxyvitamin D3 level was detected by Enzyme linked immunosorbent assay(ELISA),and the percentage of NK cell subsets in peripheral blood was detected by flow cytometry;The decidual tissues of RSA abortion group and control group in the first trimester of pregnancy during artificial abortion were collected,decidual immune cells were extracted by density gradient centrifugation,and the percentage of decidual NK cell subsets was detected by flow cytometry;The expressions of VDR,CYP27B1 and CYP24A1 proteins in decidual tissues were detected by Western blot.2.Part II:A total of 134 patients with immune RSA who visited the Department of Gynecology and Obstetrics clinic of Hospital for the first time from June 2020 to April 2021,and had serum 25(OH)D deficiency(serum 25(OH)D<20ng/ml)on the day of the first visit were selected as the research subjects.The same vitamin D3 preparation was given at 1725 IU/d for 8 weeks.The fasting elbow vein blood of the study subjects was drawn before and after the treatment,the serum 25(OH)D level was detected by electrochemiluminescence method,the percentage of NK cell subsets in peripheral blood was detected by flow cytometry,and the changes of serum cytokines were detected by immunological analysis and flow cytometer analysis.3.Part Ⅲ:Stable passaged human peripheral blood NK-92 (?) MI cells were selected for experiments.After 48 hours of incubation with 1,25(OH)2D3 at 0,10,and 100 nM,the changes of VDR mRNA expression were detected by quantitative realtime polymerase chain reaction(RT-qPCR),and the expression of VDR protein was detected by Western blot.The optimal treatment concentration was determined to be 100 nM.NK 92(?) MI cells were cultured with 100nM of 1,25(OH)2D3 and and NF-κB inhibitor BAY 11-7082 for 48 hours.The mRNA expression of IL-2,IFN-γ,TNF-α,perforin and granzyme B were detected by PCR.The protein expression of perforin,granzyme B and NF-κB was detected by WB assay,and the expression of IFN-γ and TNFα was detected by ELISA.Part Ⅳ:a Vitamin D deficient CBA/J mouse model was constructed by Vitamin D deficient diet.The mice were divided into vitamin D normal(VDN)and vitamin D deficiency(VDD)groups.The CBA/J female mice,DBA/2 and BALB/c male mice were used to establish recurrent spontaneous abortion models or normal pregnancy models respectively.The serum levels of 25(OH)D3 were detected by ELISA.The pregnant mice in each group were killed on the day 13.5 of pregnancy.The embryo absorption rate of each group was calculated to explore the effect of vitamin D deficiency on the pregnancy outcome of recurrent spontaneous abortion mouse model.Results:Part Ⅰ:1.The general clinical data of the subjects were comparable.2.The serum vitamin D level in RSA abortion group was significantly lower than that in control group and RSA pregnancy group(P<0.05).3.Comparison of the percentage of different NK cell subsets in peripheral blood:The percentage of total NK cells in RSA abortion group was significantly higher than that in RSA pregnancy group and control group,P<0.05;The percentage of CD56dimCD16+NK cell subsets in peripheral blood of RSA abortion group was higher than that of RSA pregnancy group and control group(P<0.05);The percentage of CD56bright CD 16-NK cells in RSA abortion patients was lower than that in RSA pregnancy group and normal early pregnancy group(P<0.05).4.Comparison of the percentage of decidual NK cell subsets:the percentage of CD56+decidual NK cells in RSA group was significantly lower than that in control group(P<0.05).5.Compared with the control group,the expression levels of VDR and CYP27B1 protein in decidua of RSA abortion group decreased,and the expression level of CYP24A1 protein increased(P<0.05).Part Ⅱ:1.After vitamin D3 supplementation,the serum 25(OH)D3 level in RSA patients increased significantly,P<0.05.2.The percentage of peripheral blood CD56brightCD16-NK cell subsets increased,and the levels of Th1 type cytokine IL-2 and IFN-γ decreased(P<0.05).Part Ⅲ:After NK 92(?) MI cells were cultured with 0,10 and 100nM/ml 1,25(OH)2D3 medium for 48h,VDR mRNA and protein expression increased in a concentration-dependent manner.After NK-92(?) MI cells were cultured with 100nM 1,25(OH)2D3 medium and BAY11-7082 for 48h,the expression levels of IFN-γ and TNFα were down-regulated by inhibiting the expression of NF-κB p65 protein.100nM 1,25(OH)2D3 could down-regulate the expression of perforin and granzyme B proteins.PartⅣ:In the vitamin D deficient recurrent spontaneous abortion mouse model,the serum vitamin D level was significantly reduced,and the embryo absorption rate was significantly increased.Vitamin D supplementation with food or vitamin D3 preparation before or after pregnancy can increase the serum 25(OH)D3 level to a certain extent,reduce the embryo absorption rate and improve the pregnancy outcomes.Conclusion:Part Ⅰ:The serum vitamin D level in RSA patients was significantly lower than that in normal pregnant women and RSA patients.The percentages of total NK cells and CD56dimCD16+NK cell subsets in peripheral blood increased,while the percentages of CD56brightCD16-NK cell subsets and decidual NK cells decreased.Vitamin D metabolism disorder at the decidual level.It is suggested that low level of vitamin D,imbalance of peripheral blood and decidual NK cell subsets,and disorder of decidual vitamin D metabolism are risk factors for pregnancy failure.Part Ⅱ:Exogenous vitamin D supplementation can increase the serum vitamin D level and the percentage of CD56brightCD16-NK cells in peripheral blood of RSA patients,reduce the production of Th1 type cytokines IL-2 and IFN-γ,regulate the body’s Th1/Th2 type immune shift to the direction of Th2,and promote the formation of immune tolerance.Part Ⅲ:1,25(OH)2D3 can down regulate the expression of perforin and granzyme in NK-92(?) MI cells through VDR signal pathway,thereby reducing their cytotoxicity;By inhibiting NF-κB p65 protein synthesis reduces expression of proinflammatory factor IFN-γ and TNF-α.PartⅣ:Vitamin D deficiency increases the embryo absorption rate of recurrent spontaneous abortion mice model,which is one of the risk factors for early spontaneous abortion.Timely supplementation of vitamin D to normal level can reduce the occurrence of spontaneous abortion in the first trimester of pregnancy.