@2024 Afarand., IRAN

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ISSN: 2251-8215    Sarem Journal of Reproductive Medicine 2019;3(2):59-64

Background
… [1] With the spread of immunology of pregnancy, the importance of maternal immunity to fetus has been increasingly considered, and failure of this tolerance will be associated with pregnancy problems such as recurrent spontaneous abortion (RSA) [2, 3]. ... [4-22]. In recent decades, lymphocyte therapy has been used as an immunological treatment for patients with RSA.
Previous Studies
Improvement in the outcome of pregnancy following lymphocyte immunotherapy in patients with a history of RSA has been reported in many studies [23-26], but conflicting observations suggest the failure of this method of treatment and the extent of its effectiveness is still under discussion [24, 27]. Previous findings suggest that Th2 cells predominate after lymphocyte therapy [28], but, the current study on the changes in the number of Th17 and Regulatory T cells (Treg) after lymphocyte therapy in women with RSA show that the ratio of Th17 to Treg was significantly reduced after treatment, indicating the importance of lymphocyte therapy for the change in the function of Th17 cells toward preserving pregnancy [29].
Aim(s)
The aim of this study was to investigate the effect of lymphocyte therapy on the level of proinflammatory cytokines of Th17 cells in women with RSA.
Research type
The present study is a semi-experimental.
Research society, place and time
The study was performed on patients referring to Sarem Hospital in Tehran from July to November 2013.
Sampling method and number
The sample size (30 patients) was determined according to similar studies, with expert advice in statistics and epidemiology, and using statistical formulas. Patients were evaluated after referral to the RSA clinic and being examined by a specialist physician. Patients with no known cause for RSA were included in this study. Therefore, patients underwent examinations such as karyotype to reject chromosome abnormality, to check thyroid hormone levels, levels of autoantibodies such as antiphospholipid antibodies, anti-cardiolipin antibodies and anticoagulants, coagulation factors such as C and S proteins, prothrombin and homocysteine, anatomical and uterine problems, and in the event of a negative blood test (Cross-match), they were included in the study. Separation of serum: About 5ml of blood were received before and after treatment. In order to separate the 2ml serum, the peripheral blood samples were collected for 10 minutes with a round of 210g centrifuge and serum until complete collection of samples was stored in a -70°C freezer. The 3ml blood sample was transferred to a sub-hood for separation and culture of mononuclear cells into a sterilized heparin tube. Separation of peripheral blood mononuclear cells: With the aid of a ficoll technique, peripheral blood mononuclear cells were separated for cell culture and preparation of supernatant, and after washing twice with Phosphate-buffered saline (PBS) (Betagene; Iran), cell count and cell survival were determined by Trypan Blue (Sigma; United States). Cell culture: After counting mononuclear cells, a cell count of 2.5×106 cell in RPMI 1640 (Sigma; USA) media containing penicillin-streptomycin (100units/ml), 10% fetal bovine serum (FBS) (Sigma; USA), and 30μl actuator PHA (Sigma; USA) at 37°C and 5% carbon dioxide in a 50ml cell culture flask was cultured for 48 hours. The supernatant was, then, separated from the cell culture by centrifugation and stored until complete collection of samples in a -70°C freezer. Lymphocyte therapy: In two consecutive times for 4 weeks, 20ml of heparin blood was obtained from the patient's wife and, maintaining sterile conditions, peripheral blood mononuclear cells (PBMC) were isolated, using ficoll technique. After 3 times washing the cells with the Ringer serum and counting them using Neubauer lam at each turn, 80-100×106 cells per 1ml of Ringer serum was injected with 0.5ml intraperitoneally on the forearm or arm of the patient. The second stage of lymphocyte therapy was performed after the first injection [30]. Measurement of IL-17 and IL-21 in serum and cell culture soup: To measure the level of IL-17 and IL-21 cytokines in serum and supernatant samples of cell culture, the kits were used before and after treatment, using ELISA technique (eBioscience; United States). Measurement of cytokines concentration in patient samples was done in binary form according to the kit's instructions. The sensitivity of the kit for measuring IL-17 and IL-21 cytokines was 0.01pg/ml and 8pg/ml, respectively. SPSS 22 software was used to analyze the data. First, the normal or abnormal distribution of the variables was investigated by the Kolmogorov-Smirnov test. Based on the abnormal distribution of IL-17 and IL-21 variables in serum and cell culture soup, using the non-parametric Wilcoxon test, a significant difference in the level of these cytokines was evaluated before and after the treatment. The results were reported as mid-range and Interquartile range.
Used devices&materials

Findings by Text
The mean age of the patients was 31.37±5.67 years. The concentration of IL-17 and IL-21 cytokines in the serum samples after treatment was significantly reduced compared to the concentration of these cytokines before treatment (p<0.05). The level of IL-17 in cell culture soup was not significantly different after treatment compared to pre-treatment (p=0.13), while IL-21 concentration significantly decreased after treatment (p=0.038; Table 1).
Main comparison to the similar studies
IL-21 is also produced as a proinflammatory cytokine by Th17 cells and, and by examining the polymorphism of the relevant gene, it is partly related to RSA [43]. In contrast, the examining IL-21 gene expression in women with RSA shows that the gene expression level of this cytokine in healthy women is not significantly different from that of patients [44]. In the present study, the level of IL-21 after lymphocyte therapy significantly decreased in serum and supernatant cell culture before treatment, which may indicate a reduction in the inflammatory function of Th17 cells (p=0.004 and p=0.038, respectively). Lymphocyte therapy seems to reduce the level of Th17 associated cytokines that can be effective in reducing the inflammatory response of the mother to the fetus. In the present study, after lymphocyte therapy, Th17 levels of cytokines were significantly reduced in patients’ serum, which was consistent with earlier studies that decreased the ratio of Th17/Treg cells after treatment [29]. Considering the significant reduction of these cytokines after lymphocyte therapy, IL-17 and IL-21 seem to play a significant role in the efficacy of lymphocyte therapy and possibly the success of pregnancy in patients with RSA following this treatment.
Suggestions
The Study of Th17 and Treg cells populations as well as the study of Th17/Treg ratio by Flow cytometry method, And its simultaneous matching with cytokines in RSA patients undergoing lymphocytic therapy are suggested.
Limitations
Limitations of this study included the study of Th17 and Treg cells, as well as the study of Th17/Treg ratio by Flow cytometry method.
Conclusions
Lymphocyte therapy reduces the level of proinflammatory cytokines of Th17 cells in women with RSA.
Acknowledgments
The colleagues of the Department of Recurrent Abortion and the Immunology Department of the Faculty of Medicine of Semnan University of Medical Sciences, as well as the colleagues of the Central Laboratory of Sarem Hospital, are acknowledged.
Conflict of interest
None declared by the authors.
Ethical Permissions
Patients' satisfaction to participate in this study was obtained and the consent form approved by the Ethics Committee of Sarem Hospital was completed by each of the volunteer patients.
Funding Sources
Sarem Cell Research Center and Semnan University of Medical Sciences have funded this research.

TABLES and CHARTS

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