The Role And Mechanism Of Vitamin D Regulating Treg/Th17 Balance In Recurrent Pregnancy Loss

Partâ… Peripheral Vitamin D Levels,Treg and Th17 cells Percentages and Their Correlation in Women with Recurrent Pregnancy Loss Objective:To investigate the vitamin D levels,regulatory T cells（Treg）,helper 17 T（Th17）cells and intermediate Foxp3⁺IL-17⁺T cells percentages in peripheral blood of normal pregnancy（NP）and recurrent pregnancy loss（RPL）women.To analysis the correlation of vitamin D levels and Treg cells,Th17 cells,Foxp3⁺IL-17⁺T cells percentages.Methods:Peripheral blood serum of NP group（n=77）and RPL group（n=215）were collected,andvitaminDlevels（25（OH）D₃）weredetectedby electrochemiluminescence.The percentages of Treg（marked by CD4⁺Foxp3⁺）,Th17（marked by CD4⁺IL-17A⁺）and Foxp3⁺IL-17⁺T（marked by CD4⁺Foxp3⁺IL-17⁺）intermediate cells in peripheral blood mononuclear cells（PBMC）from NP group（n=48）and RPL group（n=107）were detected by flow cytometry（FCM）technology.In addition,vitamin D levels and the percentages of Treg,Th17 and Foxp3⁺IL-17⁺T intermediate cells of RPL group were compared by Spearman analysis.Results:（1）Compared with NP group（24.68 ng/ml±8.91 ng/ml）,vitamin D levels in peripheral blood of RPL group（21.34 ng/ml±7.81 ng/ml）were significantly lower（P<0.01）;Compared with NP group（5.72%±2.69%）,the percentage of Treg cells in the RPL group（4.80%±1.95%）decreased（P<0.05）;Compared with the NP group（1.26%±0.81%）,the percentage of Th17 cells in the RPL group（1.61%±0.97%）increased（P<0.05）;Compared with NP group（0.20%±0.17%）,the percentage of Foxp3⁺IL-17⁺T cells（0.27%±0.23%）in the RPL group increased（P<0.05）;Compared with the NP group（4.75±2.60）,the Treg/Th17 ratio in the RPL group（3.60±1.98）decreased（P<0.01）.（2）In RPL group,the vitamin D levels were positively correlated with the percentage of Treg cells（r=0.511,P<0.001）;Vitamin D levels were negatively correlated with the percentage of Th17 cells（r=-0.336,P<0.001）;Vitamin D levels were negatively correlated with Foxp3⁺IL-17⁺T cells percentage（r=-0.268,P<0.01）;Vitamin D levels were positively correlated with Treg/Th17 ratio（r=0.587,P<0.001）.Conclusion:There were lower vitamin D levels and Treg/Th17 imbalance in peripheral blood of patients with RPL.Vitamin D levels and Treg,Th17 percentages were relevant.Part II Effect of Vitamin D Supplementation on Regulation of Treg/Th17 Imbalance in Women with RPL and Its Impact on Clinical OutcomeObjectives: To compare vitamin D levels and Treg,Th17,Foxp3+IL-17+T cells in the peripheral blood of study group（SG）（supplement with vitamin D）and control group（CG）（without vitamin D supplementation）.The pregnancy rate and miscarriage rate were compared between the SG group and the CG group after 8 months.Methods: RPL patients with vitamin D levels below 30 ng/m L were randomly divided into SG groups（n=50）and CG group（n=50）.The SG group received oral vitamin D capsules for 2 months,2000 IU per day;While the CG group did not receive vitamin D.Two months later,the levels of vitamin D（25（OH）D3）in the peripheral blood of the two groups were measured again by electrochemiluminescence.At the same time,the percentages of Treg,Th17 and Foxp3+IL-17+T cells in the peripheral blood of the two groups were detected by FCM.At the 8th month after the start of the trial,clinical outcomes were tracked.Results: Compared with the CG group（18.29 ng/ml ± 6.02 ng/ml）,vitamin D levels in the peripheral blood of the SG group（23.89 ng/ml ± 7.27 ng/ml）were significantly increased after two months of vitamin D supplementation（P < 0.001）.Compared with the CG group（4.63% ± 1.81%）,the percentage of Treg cells in the SG group（5.53% ± 2.28%）increased（P < 0.05）;Compared with the CG group（1.48% ± 1.05%）,the percentage of Th17 cells in the SG group（1.19% ± 0.93%）had no significant difference（P > 0.05）;Compared with CG group（0.30% ± 0.17%）,the percentage of Foxp3+IL-17+T cells in SG group（0.21% ± 0.17%）was decreased（P < 0.01）;Compared with the CG group（2.99 ± 1.53）,the Treg/Th17 ratio in the SG group（4.77 ± 2.61）was increased（P < 0.001）.The clinical outcomes of 21 cases in the SG group and 17 cases in the CG group were followed up.There were no significant difference in the clinical pregnancy rate and miscarriage rate between the two groups statistically（P > 0.05）.Conclusion: After supplementation of vitamin D,the vitamin D levels and Treg cells percentage increased,and Th17 cells percentage and Foxp3+IL-17+ intermediate cells percentage decreased in RPL patients.Vitamin D supplementation had no effect on the outcomes of RPL.Part â…¢ The Mechanism of Vitamin D in Regulating Treg/Th17 BalanceObjectives: To investigate the mechanism of vitamin D in Treg/Th17 balance in peripheral blood.Methods: FCM was used to detect the percentages of Treg,Th17,and Foxp3+IL-17+T intermediate cells the PBMC in non-pregnant women treated with different vitamin D3 concentrations（100 n M/L,10 n M/L,1 n M/L）after cultured for 4.5 days（n=8）.RT-PCR was used to detect Foxp3,ROR-γT,VDR,CYP27B1 and CYP24A1 m RNA（n=13）.Results:（1）The percentages of Treg cells in the 1 n M,10 n M,and 100 n M groups（10.86% ± 2.54%,10.92% ± 2.42%,14.04% ± 2.77%）were significantly higher than the control group（6.31% ± 1.60%）（P < 0.01）;Compared with the control group（2.32% ± 1.31%）,the percentage of Th17 cells in the 100 n M group（1.08% ± 0.48%）was decreased（P < 0.05）;Compared with the control group（3.86 ± 3.25）,the Treg/Th17 ratio in 100 n M group（13.92 ± 8.85）was increased（P < 0.01）;The percentage of Foxp3+IL-17+T cells in each group were not significantly different（P > 0.05）.（2）Compared with the control group,Foxp3 m RNA expression was increased in 100 n M group（P < 0.01）;ROR-γT m RNA expression were increased in 10 n M and 100 n M groups（P < 0.001）;VDR m RNA expression were increased in 10 n M and 100 n M group（P < 0.01）;CYP24A1 m RNA expression in 10 n M and 100 n M groups were significantly increased（P < 0.001）;There were no significant difference in CYP27B1 m RNA expression between the groups（P > 0.05）.Conclusion: Vitamin D can regulate Treg/Th17 balance,probably by increasing vitamin D receptor and metabolic enzymes activity in PBMC and promoting the transformation from Treg to Th17 cells.