| Background and subject sources:Recurrent spontaneous abortion (RSA) is defined as the loss of three or more consecutive pregnancies before 20th week of gestation, approximately occurred to 1%～3% women during their reproductive years. Several pathogensis mechanisms of RSA have been proposed, including:(1) chromosomal and gene abnormalities such as balanced structural chromosome rearrangement. (2) thrombophilic diseases such as antiphospholipid antibodies syndrome. (3) autoimmue disorders such as various of autoantibodies positive. (4) uterine and anatomical factors such as Asherman syndrome, cervical insufficiency and congenital anomalies. (5) endocrine disorders such as luteal phase insufficiency, polycystic ovarian syndrome, thyroid dysfunction, hyperprolactinemia. (6) infectious diseases. However, the aetiology of 50% RSA still remains unkown. RSA cases due to unknown causes called unexplained recurrent spontaneous abortion (URSA), which has been concerned related to maternal-fetal alloimmune response.Fetus inherited polymorphic gene from father ought to provoke maternal immune response as semi-allogeneic antigen, which subsequently led to fetal rejection. But this immune rejection does not normally occur, which imply that during normal pregnancy, maternal alloimmune response to fetus has been somehow regulated. According to recent research, pregnancy is actually a state of maternal-fetal tolerance, which is actually a state of immune balance achieved by various immune factors’ coordination. If this balance has been broken, fetus will suffer from immunological attack and eventually lead to pregnancy loss. Human leukocyte antigen (HLA) incompatibility, natural killer cell (NK) proportional and functional abnormality, and T cell subsets imbalance has been found in URSA patients.Recently T cell subsets imbalance has become increasingly popular in RSA study. According to their functions, T cells can be mainly devided into T helper cells (Th), cytotoxic T cells (CTL) and regulatory T cells (Tregs). Tregs are very important subset of T cells. Tregs constitutively express CD4 and CD25, with an estimated 5%～10% proportion of peripheral CD4 positive T cells. Foxp3 transcription factor is a specific marker for Tregs. It has been shown that CD4+CD25+Foxp3+Tregs can suppress immune response by cell-to-cell direct contact. CD4+CD25+Foxp3+Tregs also can be indentified as CD4+CD25+CD127lowTregs due to their low expression of CD 127. Therefore suppressive Tregs can be marked by CD4, CD25, CD127 as well. In addition, Foxp3-Tregs are able to play a role in immune regulatory through secretion of cytokines such as interlukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1). IL-10 can suppress macrophages, prevent Thl cells from producing pro-inflammatory cytokines such as IL-2, IFN-γ. TGF-β1 is known as a effective immune suppressing cytokine, which can inhibit self-secretion of T cells, restrain macrophages and NK cells. Furthermore, IL-10 and TGF-β1 can also induce development of peripheral CD4+CD25+Tregs. Tregs play a major role in both preventing autoimmunity and tolerating allogeneic organ grafts, and it seems to also play a key role in maintaining maternal-fetal immune tolerance. Sasaki et al. showed that CD4+CD25+Tregs increased in normal pregnancy, and the proportion of Tregs both in peripheral blood and in decidua have been found significantly decreased in unexplained recurrent spontaneous abortion patients. T helper cells 17 (Th17) is a new lineage of Th cells that selectively produce IL-17, it plays a critical role in induction of inflammation, pathogenesis of autoimmune diseases and immune rejection. Th17 cells are involved in host defense against bacteria, fungi and virus. Several study showed that Th17 cells also take part in pathogenesis of rheumatoid arthritis (RA), psoriasis, multiple sclerosis, and inflammatory bowel disease. Hence, Th 17 cells may play an important role in maternal immune rejection to fetus. Recent study on recurrent spontaneous abortion patients has found negative correlation between the proportion of Thl7 cells and Treg cells both in peripheral blood and decidua. Now is generally accepted that there is Treg/Th17 paradigm imbalance in unexplained recurrent spontaneous abortion.Currently, lymphocyte immunization therapy (LIT) is the most common treatment for unexplained recurrent spontaneous abortion. LIT treatment use lymphocytes from father or third party as immunogen, and inject these lymphocytes intracutaneously to recurrent spontaneous patients so that maternal alloimmune response can be activated and lead to maternal immune tolerance eventually. LIT is a valid treatment which can suppress materal immune response to fetus during pregnancy. However, the mechanism of LIT treatment has not yet been fully elucidated. Hence, it is very necessary to investigate the association between LIT and Treg/Th17 paradigm in URSA patients.In this study,we compared the proportion and function of Treg cells and Th 17 cells and their production in peripheral blood of URSA patients before LIT treatment with the same items after LIT treatment to understand the relationship between Treg/ Thl7 paradigm and lymphocyte immunization therapy in URSA, provide a new entry point in evaluation of LIT treatment efficacy and a promising way to predict pregnancy loss.Objective and significance:This study evaluated the percentage and ratio of Tregs and Th 17 cells and their production in peripheral blood of unexplained recurrent spontaneous abortion patients before and after lymphocyte immunization therapy to gain a better understanding of the aetiology of URSA, and provide theoretical foundation for LIT treatment.Methods:25 Chinese women with unexplained recurrent spontaneous abortion (URSA) were collected from recurrent spontaneous abortion center of Shenzhen Maternity &Child Healthcare Hospital. All subjects should meet following criterion:(1) All subjects have experienced at least three consecutive spontaneous abortion. (2) All subjects are younger than 40 years old. (3) No chromosmal and genetic abnormalities with both parents. (4) No organic diseases, anatomical abnormalities and infecious diesases have been found through vaginal secretion tests, genital chlamydia tests, genital mycoplasms tests, TORCH IgM tests, ultrasonic tests and (or) hysteroscopy. (5) No endocrine disorders have been found through sexual hormone examinations, thyroid fuction tests, blood glucose tests and insulin tests. (6) Thrombophilic tests such as D-Dimer and fibrinogen degradation product are negative. (7) Autoimmune antibodies such as ANA, ACA IgM, ACA IgG, AsAb, EmAb et al. are negative. (8) No abnormities have been found in semen examination. (9) Blocking antibody tests show negative. (10) All subjects have never been treated with lymphocyte immunization therapy.In these 25 unexplained recurrent spontaneous abortion patients, we marked Tregs with CD4, CD25, CD 127, and Th17 with CD8, CD3, IL-17A. We used flow cytometry to investigate the percentage and ratio of Tregs and Th17 cells in peripheral blood and used ELISA kit to measure the concentration of TGF-p 1, IL-10, IL-17 in serum before and after lymphocyte immunization therapy. And then we used t test for statistical analysis of the proportion of Treg cells and Th17 cells and their production in all subjects before and after lymphocyte immunization therapy. Finally we used Pearson’s test to analyse the correlation between results of flow cytometry and ELISA tests. The aim is to determine the relationship between Treg/Th17 paradigm in URSA patients and LIT treatment.Results:1. Results of flow cytometry.(1) The CD4+CD25+Treg cell ratio in peripheral blood of URSA patients before LIT treatment is (2.8+0.5)%, whereas their ratio in peripheral blood after LIT treatment is (4.4+0.8)%, P<0.01.(2) The CD4+CD25+CD127low Treg cell ratio in peripheral blood of URSA patients before LIT treatment is (1.9+0.5)%, whereas their ratio in peripheral blood after LIT treatment is (3.4+0.7)%, P<0.01.(3) The CD8-CD3+IL-17+Th17 cell ratio in peripheral blood of URSA patients before LIT treatment is (3.3±0.5)%, whereas their ratio in peripheral blood after LIT treatment is (1.8±0.5)%, P<0.01.(4) The CD4+CD25+Treg/CD8CD3+IL-17A+Th17 ratio in peripheral blood of URSA patients before LIT treatment is 0.86+0.17, and the CD4+CD25+Treg/ CD8CD3+IL-17A+Thl 7 ratio after LIT treatment is (2.63+0.63)%, P<0.01.(5) The change of CD4+CD25+Tregs proportion in peripheral blood has shown a negative correlation with the change of CD8-CD3+IL-17+Thl7 cells proportion in peripheral blood after LIT therapy (r=-0.704, P< 0.001).(6) The change of CD4+CD25+CD127lowTregs proportion in peripheral blood has shown a negative correlation with the change of CD8-CD3+IL-17+Th17 cells proportion in peripheral blood after LIT therapy (r=-0.623, P<0.001).2. Results of ELISA kit.(1) The concentration of TGF-β1 in serum of URSA patients before LIT treatment is (2582.49 ±732.32)pg/ml, whereas the concentration of TGF-β1 in serum after LIT treatment is (3584.88+665.63)pg/ml, P<0.01.(2) The concentration of IL-10 in serum of URSA patients before LIT treatment is (7.11±2.11l)pg/ml, whereas the concentration of IL-10 in serum after LIT treatment is (9.46±2.19)pg/ml, P<0.01.(3) The concentration of IL-17 in serum of URSA patients before LIT treatment is (3.72±1.11)pg/ml, whereas the concentration of IL-17 in serum after LIT treatment is (2.29±0.79)pg/ml, P<0.01.3. Pearson correlation analysis between results from flow cytometry and ELISA kit(1) The change of IL-17 concentration in serum has shown a positive correlation with the change of CD8-CD3+ IL-17+Th17 cells proportion in peripheral blood after LIT therapy (r=0.687, P<0.001).(2) The change of TGF-β1 concentration in serum has shown a positive correlation with the change of CD4+CD25+Tregs proportion in peripheral blood after LIT therapy (r=0.564, P=0.003).(3) The change of IL-10 concentration in serum has shown a positive correlation with the change of CD4+ CD25+ Tregs proportion in peripheral blood after LIT therapy (r=0.604, P=0.001).Conclusion:1. Lymphocyte immunization therapy can significantly increase peripheral blood CD4+CD25+Tregs ratio and CD4+CD25+ CD127lowTregs ratio of URSA patients, with a significant decrease of CD8-CD3+IL-17+Th17 cell ratio. These imply that LIT might treat URSA patients by affecting their proportion of Tregs and Th17 cells.2. Lymphocyte immunization therapy can significantly increase the concentration of TGF-β1 and IL-10 in serum of URSA patients, and the change of TGF-β1 and IL-10 concentration both positive relate to the increasing CD4+CD25+ Treg cell ratio after LIT treatment. These elucidate that LIT treatment may increase the proportion of Tregs and functionally enhance Tregs, lead to an extensive secretion of TGF-β1 and IL-10 to suppress immune rejection.3. Lymphocyte immunization therapy can decrease the concentration of IL-17 in serum of URSA patients significantly, and the change of 1L-17 concentration positive relate to the decreasing CD8-CD3+IL-17A+Th17 cell ratio after LIT treatment. Accoring to these, LIT may treat URSA patients also by reducing the proportion of Thl7 cells and preventing Thl7 cells from producing IL-17.4. The CD4+CD25+Treg/CD8-CD3+IL-17A+Thl7 ratio in peripheral blood of URSA patients was significantly increased after LIT treatment, and the change of CD4+CD25+Treg cell proportion in peripheral blood of URSA patients after LIT treatment is negatively related to the change of CD8-CD3+IL-17A+Th17 ratio. A negative correlation has also been found between the change of CD4+CD25+CD127lowTregs proportion and CD8CD3+IL-17A+Thl7 cells proportion after LIT therapy. These indicated that LIT therapy might treat URSA patients by targeting and affecting their Treg/Th 17 immunological imbalance. |
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