Evaluation of changes in exhausted T lymphocytes and miRNAs expression in the different trimesters of pregnancy in pregnant women.

BACKGROUND: Throughout the three trimesters of a typical pregnancy, we looked at changes in the expression of miRNAs and exhausted T lymphocytes for this study. METHODS AND RESULTS: Fifty healthy subjects were included in this study. The frequency of exhausted T lymphocytes was measured in isolated PBMCs using flow cytometry. PD-1, TIM-3, and related miRNAs gene expression were assessed using qRT-PCR. The analyses revealed a significant decline in PD-1 and Tim-3 expression in PBMCs from RPL women (p = 0.0003 and p = 0.001, respectively). In addition, PD-1 and TIM-3 expression increased significantly in the 2nd trimester compared with the 1st trimester of healthy pregnant women (p < 0.0001 and p = 0.0002, respectively). PD-1 and TIM-3 expression was down-regulated in the 3rd trimester compared with the 1st and 2nd trimesters. In the present study, we demonstrated that TIM-3+/CD4+, TIM-3+/CD8+, PD-1+/CD4+, and PD-1+/CD8 + exhausted T lymphocytes increased in the circulation of women in the 2nd trimester compared to the 1st and 3rd trimester. In the 3rd trimester, the expression of miR-16-5p increased significantly (p < 0.0001). miR-125a-3p expression was down and upregulated in 2nd (p < 0.0001) and 3rd (p = 0.0007) trimesters compared to 1st trimester, respectively. This study showed a significant elevation of miR-15a-5p in 3rd trimester compared to 1st trimester of pregnant women (p = 0.0002). CONCLUSIONS: Expression pattern of PD-1 and TIM3 in exhausted T lymphocytes is different not only between normal pregnant and RPL women but also in different trimesters of pregnancy. So, our results showed the role of these markers in the modulation lymphocytes activity in different stages of pregnancy.

Dominant immune cells in pregnancy and pregnancy complications: T helper cells (TH1/TH2, TH17/Treg cells), NK cells, MDSCs, and the immune checkpoints.

Pregnancy problems including recurrent pregnancy loss, repeated implantation failure and pre-eclampsia  are common problems in the reproductive ages. Different reasons such as genetic, immunological, and environmental agents and also infections could develop these complications. In those cases in which the cause of the abortion is diagnosed, the chance of a successful pregnancy is increased by eliminating defective factors. However, in patients with unknown causes, there may be an imbalance in immune cells pattern. As a matter of fact, an inappropriate immune response is often associated with a failed pregnancy. Hence, the focus of treatment is to increase tolerance, not to suppress maternal immune system. These findings are linked to an elevated number of Treg cells and immune checkpoints through normal pregnancy. The present review discusses the balance of myeloid-derived suppressor cells, natural killer cells, T cells, and immune checkpoints, and also targeting them to maintain pregnancy and prevent associated complications.

Sirolimus- and cyclosporine-loaded nanostructured lipid carriers: Development, characterization, and in vitro evaluation in T-cell profiles of patients with a history of recurrent pregnancy loss.

Purpose: The authors developed nanostructured lipid carriers (NLCs) loaded with sirolimus (SRL) and cyclosporine (CsA) to improve their therapeutic efficacy in recurrent pregnancy loss (RPL) patients. Methods: Mono-delivery and co-delivery of SRL and CsA by NLCs (S-NLCs, C-NLCs, and S-C-NLCs) were developed. The MTT assay was used to study the optimum dose of formulations. PCR, Western blotting, and ELISA were also conducted. Results: Well-designed nanodrugs with a suitable size, zeta potential, desirable encapsulation efficiency drug loading, and cellular uptake confirmed optimum formulations. Based on cell viability, the amounts of SRL and CsA could be reduced greatly due to the co-delivery by NLCs. Following S-NLCs and C-NLCs interventions in T cells of patients with RPL and immune abnormality, a significant difference was observed in transcription factors and cytokine levels of Th1, Th17, and Tregs compared with healthy samples. Thus, a higher level of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-17, and IL-21) and their regulators (T-bet and RORγt), as well as a lower level of an anti-inflammatory cytokine (IL-10) and its regulatory (Foxp3), were observed. However, no significant difference was found following the S-C-NLCs intervention. Conclusions: S-C-NLCs effectively balance the immune responses in peripheral T cells in RPL patients to induce maternal immune tolerance.

Role of miRNAs interference on ovarian functions and premature ovarian failure.

Premature ovarian failure is a to some extent unknown and intricate problem with diverse causes and clinical manifestations. The lack of ovarian sex hormones presumably is effective in the occurrence of ovarian failure. Our progress in this field has been very little despite undertaken scientific research endeavors; scholars still are trying to understand the explanation of this dilemmatic medical condition. In contrast, the practice of clinical medicine has made meaningful strides in providing assurance to the women with premature ovarian insufficiency that their quality of life as well as long-term health can be optimized through timely intervention. Very recently Scientists have investigated the regulating effects of small RNA molecules on steroidogenesis apoptosis, ovulation, gonadal, and corpus luteum development of ovaries. In this literature review, we tried to talk over the mechanisms of miRNAs in regulating gene expression after transcription in the ovary. Video abstract.

TIGIT and CD155 as Immune-Modulator Receptor and Ligand on CD4+ T cells in Preeclampsia Patients.

One of the main characteristics of preeclampsia (PE) is systemic inflammation. CD4+ FoxP3+ cells play a critical role in both fetomaternal tolerance and successful pregnancy. T-cell immunoglobulin, as well as immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT)/CD155 pathway, possesses critical parts in the development of normal pregnancy by promoting regulatory T (Treg) cells. However, in PE, the relationship between TIGIT/CD155 and Treg differentiation has not been entirely clarified. In the current report, we aimed to assess the frequency of TIGIT and CD155 expressing TCD4+ cells in both PE and healthy pregnant women, as well as evaluating the amount of inflammatory and inhibitory cytokines at both mRNA and protein levels before and after blocking TIGIT and CD155. In the present report, 59 healthy, and 52 PE patients were designated to obtain their venous blood. The isolation of peripheral blood mononuclear cells (PBMCs) was performed from the blood samples, and PBMCs were then cultured in the RPMI1640 medium. The percentage of CD155+ and TIGIT+ CD4+ cells was assessed by flow cytometry in PBMCs. Cell culture supernatants were utilized to evaluate the secretory levels of transforming growth factor beta (TGF-ß), interleukin (IL)-10, IL-17, tumor necrosis factor alpha (TNF-α), and IL-1 ß, using enzyme-linked immunosorbent assay technique in pregnant women with or without PE both before and after blocking TIGIT and CD155. The mRNA expression of Foxp3, TIGIT, CD155, SHP-1, TGF-ß, IL-10, IL-17, TNF-α, and IL-1ß was also assessed by qRT-PCR in PBMCs before and after blocking TIGIT and CD155 in both populations. The data showed a significant decrease in the frequency of TIGIT+ CD4+ and CD155+ CD4+ T cells in PE women, compared to the control group. Our results showed decreased protein and mRNA levels of TIGIT, CD155, IL-10, FOXP3, and SHP-1 in PE patients. In addition, significant improvements in the levels of IL-17, TNF-α, and IL-1ß were observed in PE patients, as compared with the controls. However, blocking TIGIT and CD155 could increase these inflammatory cytokines and decrease anti-inflammatory cytokines. The data obtained in this report illustrated that there existed an imbalance between inflammatory and anti-inflammatory profiles, with an inflammatory status polarization, in PE patients. Additionally, TIGIT/CD155 showed a positive effect on immune regulation by activating ITIM, demonstrating the potential therapeutic value of the TIGIT/CD155 pathway in PE treatment. Also, using some proteins or materials that increased TIGIT/CD155 pathways activity and can be a therapeutic approach in PE.

Evaluation of CD39, CD73, HIF-1α, and their related miRNAs expression in decidua of preeclampsia cases compared to healthy pregnant women.

BACKGROUND: The Preeclampsia (PE) molecular mechanisms are not fully revealed and different biological processes are involved in the pathogenesis of PE. We aimed to evaluate adenosine and hypoxia-related signaling molecules in PE patients in the current study. METHODS: Decidua tissue and peripheral blood samples were taken from 25 healthy pregnant and 25 PE women at delivery time. CD39, CD73, and Hypoxia-inducible factor-alpha (HIF-α) were evaluated in mRNA and protein level using real-time PCR and western blotting techniques, respectively. Also, miR-30a, miR-206, and miR-18a expression were evaluated by real-time PCR. At last, secretion levels of IGF and TGF-ß in the taken serum of blood samples were measured by ELISA. RESULTS: Our results revealed that Expression of CD39 is decreased in PE cases versus healthy controls at mRNA and protein levels (p = 0.0003 for both). CD73 and HIF-α showed an increased level of expression in PE patients at RNA and protein status (p = 0.0157 and p < 0.0001 for protein evaluation of CD73 and HIF-α, respectively). The miRNA-30a (p = 0.0037) and miR-206 (p = 0.0113) showed elevated expression in the decidua of the PE group. The concentration of secreted IGF-1 (p = 0.0002) and TGF-ß (p = 0.0101) in serum samples of PE cases compared to the healthy group were decreased. CONCLUSION: In conclusion, our results showed that aberrant expression of molecules that are involved in ATP catabolism and the hypoxic conditions is observed in PE cases and involved in their hypertension and inflammation could be served as PE prognosis by more confirming in comprehensive future studies. miR-206 and miR-30a play a role by regulating CD39 and CD73 as molecules that are involved in ATP catabolism as well as regulating the production of IGF-1 in the process of hypertension, which is the main feature in patients with preeclampsia. On the other hand, decreased level of miR-18a lead to upregulation of HIF-1a, and the consequence condition of hypoxia increases hypertension and inflammation in these patients.

Down-Regulation of circulating miR-23a-3p, miR-101-3p, and miR-let-7c in Women with Idiopathic Recurrent Pregnancy Loss.

BACKGROUND: Recently, circulating microRNAs have attracted much attention because they can serve as reliable, non-invasive diagnostic biomarkers for human diseases. This study aimed to quantify miR-23a-3p, miR-101-3p, and miR-let-7c expression levels in plasma samples of patients with idiopathic recurrent pregnancy loss (iRPL) and healthy subjects and to evaluate their potential diagnostic value in diagnosis of iRPL. METHODS: A total of 120 plasma samples were obtained from sixty women with a history of iRPL and sixty healthy fertile women to evaluate the expression levels of the circulating miR-23a-3p, miR-101-3p, and miR-let-7c by quantitative real-time polymerase chain reaction (qPCR) technique. The correlation between miR-23a-3p, miR-101-3p, and miR-let-7c and clinicopathological parameters was also assessed. Receiver operating characteristic (ROC) curve was plotted to determine the diagnostic accuracy of studied miRNAs in iRPL. RESULTS: Our results showed that the miR-23a-3p expression level in plasma of iRPL patients was lower than those in healthy controls but without a statistically significant difference (p = 0.113). The expression levels of miR-101-3p and miR-let-7c were significantly downregulated in iRPL patients compared with healthy subjects (p < 0.05). The plasma levels of miR-23a-3p and miR-let-7c were negatively correlated with number of abortions in iRPL patients. We observed statistically significant positive correlation between miR-23a-3p and miR-101-3p (r = 0.478, p = 0.001), miR-23a-3p and miR-let-7c (r = 0.561, p = 0.0001), and miR-101-3p and miR-let-7c (r = 0.533, p = 0.0001) in patients with iRPL. CONCLUSIONS: The current study provides evidence indicating that downregulation of miR-23a-3p, miR-101-3p, and miR-let-7c may be associated with iRPL.

Immunological and oxidative stress biomarkers in Ankylosing Spondylitis patients with or without metabolic syndrome.

Ankylosing Spondylitis (AS) is a chronic inflammatory disorder of the spine and sacroiliac joints with unidentified etiology closely associated with metabolic syndrome (MetS). Recent studies have reported that immunological and oxidative stress factors are implicated in AS pathogenesis. The aim of this study was to investigate the oxidative and immunological factors in AS patients with or without MetS compare to control group. Real-Time PCR measured expression level of cytokines, transcription factors and related miRNAs. In addition, Th17 and Treg frequencies and cytokines secretion were evaluated by flowcytometry and ELISA methods, respectively. The oxidative stress biomarkers were also assessed with biochemical methods. In AS patients with MetS, higher Th17 and lower Treg frequency were observed. Increased levels of NF-kB and AP-1 mRNA expression were seen in AS patients with MetS (p = 0.0263 and p = 0.0104, respectively). MiR-146a and miR-223 were significantly decreased (p = 0.0005, p = 0.0161, respectively) and increase in miR-21 (p = 0.0002) was observed in AS patients with MetS compared to AS patients without MetS. Additionally, the secretion of TNF-α (p = 0.0167), IL-1ß (p = 0.303), CCL2 (p = 0.0254), CCL3 (p = 0.0119), CXCL8 (p = 0.0364), adiponectin (p = 0.0183) and the levels of SOD (p = 0.0421), NO (p = 0.0451) and CAT (p = 0.0128) were increased in AS patients with MetS. We were not observed significant differences in TOS and GPX levels between studied groups. The higher levels of oxidative stress and immunological inflammatory markers in AS patients with MetS provide further evidences on the oxidative stress and immunological relationship in these patients.

Cyclosporine A improves pregnancy outcomes in women with recurrent pregnancy loss and elevated Th1/Th2 ratio.

Recurrent pregnancy loss (RPL) is a one of the most common obstetrical complications. Since, the successful pregnancy occurs in T helper 2 (Th2)-dominant situation and since, Th1 type immunity is related to pregnancy failure, we investigated the effects of cyclosporine on Th1 and Th2 cells in RPL women. Totally, 76 RPL patients (38 women as treated group and 38 as control group) were included in this study. Flow cytometry was utilized to analyze the frequency of Th1 and Th2 in blood samples. Also, real-time polymerase chain reaction was carried out to assess the messenger RNA (mRNA) expression of transcription factors and enzyme-linked immunosorbent assay was used to evaluate Th1 and Th2 related cytokines. Significant decrease in Th1 frequency (p = 0.0004), Th1/Th2 ratio (p < 0.0001), T-bet mRNA expression (p < 0.0001), interferon-γ (p = 0.0007), and tumor necrosis factor α (p = 0.0002) secretion level were observed in cyclosporine group. Moreover, significant increase in Th2 frequency (p < 0.0001), mRNA expression of GATA binding protein 3 (p = 0.0001), and interleukin 10 secretion level (p = 0.0027) was also evident in treated group. At the end of the investigation, 31 (81.5%) patients in cyclosporine-treated group had successful childbirth when compared with 16 (42.1%) women in control group (p = 0.0001). Given this, cyclosporine treatment for RPL patients with elevated Th1/Th2 ratio can result in improved pregnancy outcome.

Evaluation of ovarian cancer risk in granulosa cells treated with steroid-depleted endometriosis serum: Role of NF-κB/RelA and AKT.

BACKGROUND: Despite at the beginning known as a benign disease, endometriosis is defined as a risk factor for developing ovarian carcinoma. The effect of endometriosis on ovarian malignancy is known but its role in granulosa cell tumor is still unclear. METHODS AND MATERIALS: In this study, serum samples were collected from patients with endometriosis and divided into whole and steroid-depleted groups. Desertification was performed according to the charcoal-dextran protocol and sera were added to the culture media of granulosa cells retrieved from tubal or male factor infertile women. Quantitative real-time polymerase chain reaction and flow cytometry were performed to determine the expression level of inflammatory and apoptotic genes and apoptosis level of granulosa cells. The total concentration of lipid was measured using gas chromatography method in the granulosa cells. RESULTS: Results revealed that the expression of AKT and NF-κB/RelA gene was significantly higher in the granulosa cells treated with steroid-depleted serum obtained from patients with distrificated endometriosis (DE) compared with the control group (9.39- and 7.9-folds, respectively; p < 0.0001). In the DE group, the declined pattern of expression was observed for the genes related to apoptosis. The synthesis of saturated fatty acids was significantly decreased; however, unsaturated fatty acids showed increased levels in the DE group. CONCLUSION: The effect of steroids on endometriosis is contradictory. The level of cortisol and sex hormones could be affected by endometriosis, causing alterations of the disease progression. Reduced level of steroid hormones in patients with endometriosis may be considered as a critical risk factor for granulosa cell tumor.

NK cell frequency and cytotoxicity in correlation to pregnancy outcome and response to IVIG therapy among women with recurrent pregnancy loss.

BACKGROUND: Recurrent miscarriage (RM) has a multifactorial etiology mainly due to chromosomal abnormalities and immunological factors. Treating RM has remained to be a challenging issue and the role of intravenous immunoglobulin (IVIG) in treating RM is still controversial. MATERIALS AND METHODS: This study aimed to evaluate the changes in natural killer (NK) cells' frequency and cytotoxicity in patients with RM who received the IVIG therapy. A total of 78 women with a history of three or more recurrent miscarriages were included and their peripheral blood was drawn in case of positive pregnancy test. On the same date, 400 mg/kg of IVIG was administrated intravenously in 38 women and it continued every four weeks through weeks 30-32 of gestation. The remaining 40 patients with RM were included to be the untreated control group. Then, the effects of IVIG on NK cell frequency, cytotoxic activity, and the expression of inhibitory and activating receptors in the patients with RM, pre and posttreatment were assessed. RESULTS: NK cells percentage and cytotoxicity were significantly reduced in the IVIG-treated patients after 32 weeks of gestation (p < 0.0001). Expression levels of inhibitory receptors was increased, however, the expression levels of activating receptors were significantly decreased after the IVIG therapy. Pregnancy outcome after the treatment was significantly higher (86.8%) in the IVIG-treated patients than controls (45%; p = 0.0006). CONCLUSION: Our results suggested that women with RM may benefit from IVIG as a therapeutic approach and the frequency and functional status of peripheral NK cells may serve as a valuable predictive factor of therapy response.

The role of neutrophils and neutrophil extracellular traps (NETs) in stages, outcomes and pregnancy complications.

Neutrophils are the main components of innate immunity to eliminate infectious pathogens. Neutrophils play a role in several stages of the reproductive cycle, and their presence in the female reproductive system is highly regulated, so their function may change during pregnancy. Emerging evidence suggests that neutrophils are important at all stages of pregnancy, from implantation, placentation, and connective tissue regeneration to birth, as well as birth itself. Neutrophil extracellular traps (NETs) are defined as extracellular strands of unfolded DNA together with histone complexes and neutrophil granule proteins. NET formation is a new mechanism of these cells for their defense function. These strands containing DNA and antimicrobial peptides were initially recognized as one of the defense mechanisms of neutrophils, but later it was explained that they are involved in a variety of non-infectious diseases. Since the source of inflammation and tissue damage is the irregular activity of neutrophils, it is not surprising that NETosis are associated with a number of inflammatory conditions and diseases. The overexpression of NET components or non-principled NET clearance is associated with the risk of production and activation of autoantibodies, which results in participation in autoinflammatory and autoimmune disorders (SLE, RA), fibrosis, sepsis and other disorders such as vascular diseases, for example, thrombosis and atherosclerosis. Recent published articles have shown the role of neutrophils and extracellular traps (NETs) in pregnancy, childbirth and pregnancy-related diseases. The aim of this study was to identify and investigate the role of neutrophils and neutrophil extracellular traps (NETs) in the stages of pregnancy, as well as the complications caused by these cells.

Analysis of Th17 cell population and expression of microRNA and factors related to Th17 in patients with premature ovarian failure.

Folliculogenesis is the process where follicles in the ovaries develop and eventually lead to ovulation. Any disruption to this process can cause premature ovarian failure. miR-326 is one of the microRNAs whose expression leads to Th17 production. Th17 activates the immune system to respond more vigorously, and by producing interlukins and cytokines causes inflammation and autoimmune disorders. Th17-induced inflammation and Th17/Treg imbalance can result in POF. This investigation took samples from 30 POF patients and 30 healthy people. The study utilized PCR to assess the expression levels of cytokines, specific transcription factor (ROR-γt), and miR-326. Additionally, ELISA was employed to analyze serum levels of IL-17, IL-21, IL-23. Furthermore, flow cytometry was utilized to determine the frequency of Th17. Compared to the control group, our results demonstrated a rise in the transcription factor RORɣt and a considerable rise in the frequency of Th17 cells in patients with POF. The level of inflammatory cytokines IL-17, IL-21, and IL-23 secreted in serum samples of patients with POF increased significantly compared to the control group. Results of investigating microRNA associated with Th17 cells also showed increased expression of miR-326 in females suffering from POF. The elevation of pro-inflammatory markers in women with POF contrary to the control group underscores the significant involvement of the immune system in pregnancy disorders pathogenesis. Consequently, immunological factors may serve as promising biomarkers for predicting POF likelihood in high-risk women in the future.

The association of Treg and Th17 cells development factors and anti-TPO autoantibodies in patients with recurrent pregnancy loss.

OBJECTIVES: Thyroid autoimmunity is considered as the most prevalent autoimmune condition in women in fertility age. There are different clinical evidences indicating the association between thyroid autoimmunity and increased risk of RPL. This study aimed to analyze the association of Tregs and Th17 cells development factors and anti-thyroid peroxidase (anti-TPO) antibodies in RPL patients. Healthy controls (n = 36), TPO + controls (n = 25) and TPO + RPL (n = 32) participated in this study. After blood sampling, the frequency of Th17 and Tregs was evaluated using flow cytometry. Real-time PCR and ELISA was used to assess the status of Tregs and Th17 related transcription factors and cytokines in mRNA and protein level, respectively. RESULTS: TPO + RPL group showed a higher Th17 frequency compared to healthy controls and TPO + controls groups (p = 0.0002 and p = 0.04, respectively). Additionally, mRNA expression levels of RORγT and IL-17 were significantly higher in TPO + RPL compared to healthy controls and TPO + controls groups. In contrast, Foxp3 and TGFß expression was lower in TPO + RPL. ELISA findings also indicated a significantly higher IL-17 and lower TGFß secretion in TPO + RPL compared to healthy controls and TPO + controls. Thyroid autoimmunity should intensely be controlled specially in patients with RPL history.

The evaluation of CD39, CD73, and HIF-1 α expression besides their related miRNAs in PBMCs of women with recurrent pregnancy loss.

The molecular mechanisms involved in the pathogenesis of recurrent pregnancy loss (RPL) are not completely recognized. The present study aimed to assess the molecules associated with ATP catabolism and hypoxia besides their related miRNAs in patients with RPL. The frequency of Th17 and Treg cells in PBMCs of RPL women and healthy pregnant women were evaluated with Flow cytometry. The expression levels of CD39, CD73, and Hypoxia-inducible factor-alpha (HIF-1α), miR-18a, miR-30a, and miR-206 in PBMCs of two groups were measured with real-time PCR and western blotting. Then, serum levels of IGF-1, TGF-ß, and HIF-1α were measured by ELISA. Our results indicated a higher (p = 0.0002) and lower (p < 0.0001) frequency of Th17 and Treg lymphocytes in RPL women, respectively. The expression level of CD39 decreased in PBMCs of RPL women whereas the level of CD73 and HIF-α increased (p = 0.0010, 0.0023, 0.0006 respectively). The results of CD39 and CD37 were also confirmed by protein analysis (p = 0.0047, 0.0364 respectively). Almost, the same results for CD39 and CD73 expression at mRNA and protein levels were observed in isolated Treg cells. Moreover, we found the higher expression of miR-206 and miRNA-30a (p = 0.0038, 0.0123), but the lower expression of miRNA-18a (p = 0.0101) in RPL. The concentration level of IGF-1, and TGF-ß reduced (p = 0.0017, 0.0065 respectively) while the level of HIF-α elevated (p = 0.0235) in serum samples of RPL. In conclusion, we observed the dysregulation of molecules that are involved in ATP catabolism and hypoxia, including CD39, CD73, and HIF-1a which is related to miR-18a, miR-30a, and miR-206 change in RPL women. It may be potentially used for RPL prognosis by more comprehensive future studies.

Preeclampsia-Derived Exosomes Imbalance the Activity of Th17 and Treg in PBMCs from Healthy Pregnant Women.

The disturbance of maternofetal immune tolerance is identified as one of the important issues in the pathology of preeclampsia (PE). PE exosomes are believed to possess significant roles in immune abnormalities. In this study, to assess the possible effects of PE exosomes in the pathophysiology of preeclampsia patients, exosomes were isolated from the serum of PE patients and incubated with peripheral blood mononuclear cells (PBMCs) of healthy pregnant women. Also, exosomes from healthy pregnant women were utilized as the control. Th17/Treg ratio in PE and healthy pregnant women and the effects of PE exosomes on expression level of Th17 and Treg transcription factors, as well as their related cytokines in PBMCs of healthy pregnant women, were evaluated. A significant decrease in Treg cell number and increase in Th17 cells and Th17/Treg ratio were observed in PE patients. Following PE-exosome intervention, a significant increase in mRNA expression level of RORγt, IL-17, IL-23, IL-1ß, and IL-6, and significant decrease in IL-10 and TGFß were evident. On the other hand, no significant difference in FoxP3 level was detected. Additionally, increased IL-6, IL-17, IL-23, and IL-1ß levels and decreased IL-10 level in the supernatant of cultured PBMCs from healthy pregnant women following PE-exosome intervention were exhibited. However, TGF-ß level did not change significantly. Based on our findings, PE exosomes are able to alter the activity of Th17 and Treg cells as well as their related gene expression and cytokine profiles. These findings support the probable role of PE exosomes in PE pathogenesis.

The effect of lymphocyte immunotherapy (LIT) in modulating immune responses in patients with recurrent pregnancy loss (RPL).

In order to prevent miscarriage in RPL patients, the goal of this study was to determine how well lymphocyte immunotherapy (LIT) works in modifying immunological responses produced by cells, cytokines, transcription factors, and microRNAs. 200 RPL patients and 200 healthy controls were included in the study. Using flow cytometry, it was possible to compare the frequency of cells before and after lymphocyte treatment. Real-time PCR was used to assess the gene expression levels of transcription factors, cytokines, and microRNAs. ELISA method was used to evaluate the level of secretion of cytokines in the serum. Primary evaluation of the immune profile between healthy controls and RPL cases showed a higher frequency of Th17, NK, B cells and a lower frequency of Treg cells in RPL cases. Also, pro-inflammatory cytokines showed increased expression at mRNA and protein levels in the RPL group in comparison with the control group. Whereas, anti-inflammatory cytokines showed decreased expression in RPL patients. Decreased and increased frequency of Th17 and Treg lymphocytes observed in RPL cases following LIT, respectively. The same results obtained for RORγt and FoxP3 mRNA expression as transcription factor of Th17 and Treg cells, respectively. NK cell cytotoxicity decreased after LIT in RPL patients. miR-326a and miR-155 expression after LIT reduced, but miR-146a and miR-10a expression increased in RPL instances. LIT in RPL cases causes to elevation and modulation of anti-inflammatory and pro-inflammatory cytokines. Our data showed that lymphocyte therapy can be proposed as an effective therapeutic agent in RPL patients with immunological background by a modulating inflammatory condition.

Understanding main pregnancy complications through animal models.

Since human pregnancy is an inefficient process, achieving desired and pleasant outcome of pregnancy - the birth of a healthy and fit baby - is the main goal in any pregnancy. Spontaneous pregnancy failure is actually the most common complication of pregnancy and Most of these pregnancy losses are not known. Animal models have been utilized widely to investigate the system of natural biological adaptation to pregnancy along with increasing our comprehension of the most important hereditary and non-hereditary factors that contribute to pregnancy disorders. We use model organisms because their complexity better reproduces the human condition. A useful animal model for the disease should be pathologically similar to the disease conditions in humans. Animal models deserve a place in research because of the ethical limitations that apply to pregnant women's experiments. The present review provides insights into the overall risk factors involved in recurrent miscarriage, recurrent implant failure and preeclampsia and animal models developed to help researchers identify the source of miscarriage and the best research and treatment strategy for women with Repeated miscarriage and implant failure.

Expression level of immune checkpoint inhibitory factors in preeclampsia.

Preeclampsia (PE) is a severe complication in pregnancy, and its symptoms (proteinuria and hypertension) manifest after 20 weeks of gestation, affecting up to 8 % of pregnancies. The pregnant women's immune system uses different tolerance mechanisms to deal with a semi-allogeneic fetus. The T-cell subsets including CD8+, CD4+, and Treg play a critical role in maintaining pregnancies. The expression of immune checkpoint molecules in T-cells can ensure pregnancy at the feto-maternal interface by controlling immune responses. This research aims to evaluate the expression level of immune checkpoint factors, including PD-1, LAG-3, CTLA-4, and TIM-3 in normal pregnant women and PE patients. Decidual tissue was collected from 50 participants (25 PE and 25 control). For evaluating the genes expression, real-time PCR was employed. The western blot was used to assess the proteins level. The results of real-time PCR indicated significantly decreased expression level of these immune checkpoints in PE patients. In parallel to gene expression results, the protein level of PD-1, LAG-3, CTLA-4, and TIM-3 in the PE group was also reduced. We revealed that the profile of proteins and genes expression of immune checkpoints in the decidua of PE mothers are different from normal pregnancy and these results indicate aberrant expression of immune checkpoints such as PD-1, LAG-3, CTLA-4, and TIM-3 may cause maladaptation immune response which results in PE manifestation.

A comparison of 4- and 24-hour urine samples for the diagnosis of proteinuria in pregnancy.

BACKGROUND: Preeclampsia is a serious complication of pregnancy, and it is vital to diagnosis the condition as early as possible. Proteinuria is an important symptom of preeclampsia, and repeated urine analysis to screen for the condition is part of the standard antenatal care. The purpose of this study was to determine the correlation between 4- and 24-hour urine total protein values to examine whether the 4-hour urine samples could be used for the diagnosis of proteinuria in hypertensive disorders of pregnancy. METHODS: A cross-sectional study was performed on 110 pregnant (after gestational week 20 of pregnancy) patients who were hypertensive (blood pressure ≥140/90 mmHg) and had proteinuria as defined by positive urinary protein of at least 1+ in dipstick. Patients' urine samples were collected over 24 hours; the first 4 hours were collected separately from the next 20-hours. Patients, who did not collect the 24-hour urine, were excluded from the study. One hundred patients met the criteria, and were included in the study. The urine volume, total protein and creatinine levels of 4- and 24-hours samples were measured. The correlation between 4-hour and 24-hour samples was examined using Pearson correlation test. RESULTS: Of the 100 patients, 42 had no proteinuria, 44 had mild proteinuria, and 14 had severe proteinuria. The urine protein values of 4-hour samples correlated with those of the 24-hours samples for patients with mild and severe forms of the disease (P<0.001, r=0.86). CONCLUSION: This study showed there was a correlation between 4-hour and 24-hour urine proteins. The finding indicates that a random 4-hour sample might be used for the initial assessment of proteinuria.