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Iron is necessary for various critical biological processes, but iron overload is also dangerous since labile iron is redox-active and toxic. We found that low serum iron and decidual local iron deposition existed simultaneously in recurrent pregnancy loss (RPL) patients. Mice fed with a low-iron diet (LID) also showed iron deposition in the decidua and adverse pregnancy outcomes. Decreased ferroportin (cellular iron exporter) expression that inhibited the iron export from decidual stromal cells (DSCs) might be the reason for local iron deposition in DSCs from low-serum-iron RPL patients and LID-fed mice. Iron supplementation reduced iron deposition in the decidua of spontaneous abortion models and improved pregnancy outcomes. Local iron overload caused ferroptosis of DSCs by downregulating glutathione (GSH) and glutathione peroxidase 4 levels. Both GSH and cystine (for the synthesis of GSH) supplementation reduced iron-induced lipid reactive oxygen species (ROS) and cell death in DSCs. Ferroptosis inhibitor, cysteine, and GSH supplementation all effectively attenuated DSC ferroptosis and reversed embryo loss in the spontaneous abortion model and LPS-induced abortion model, making ferroptosis mitigation a potential therapeutic target for RPL patients. Further study that improves our understanding of low-serum-iron-induced DSC ferroptosis is needed to inform further clinical evaluations of the safety and efficacy of iron supplementation in women during pregnancy.

The T-box transcription factor protein eomesodermin (Eomes) is known for both homeostasis and function of effector and memory CD8+T cells. However, much less is known about the functional regulation of Eomes on CD8+ T cells during pregnancy. In the present study, we concluded the higher Eomes expression dCD8+T cells during normal early pregnancy. The number of Eomes+dCD8+T cells decreased in miscarriage. This Eomes+dCD8+T cell subset also expressed less growth-promoting factors, shifted toward pro-inflammatory phenotype in miscarriage. Primary Trophoblasts and HTR8/SVneo cell line could increase Eomes expression of dCD8+T cells from both normal early pregnancy and miscarriage, which might provide a new strategy for therapy to promote maternal–fetal tolerance and prevent pregnancy loss. These findings indicated that Eomes might be promising early warming targets of miscarriage. In addition, this study suggested that the reproductive safety must be a criterion considered in modulating the dose and function of Eomes in CD8+T cells to reverse T cell exhaustion.

To obtain a successful pregnancy, trophoblasts must provide a physical barrier, suppress maternal reactivity, produce immunosuppressive hormones locally, and enhance the production of blocking factors that are able to bind to several antigenic sites. Inadequate placental perfusion has been closely associated with several pregnancy-associated diseases. Galectin-9 (Gal-9) has a wide variety of regulatory functions in innate and adaptive immunity during infection, tumor growth, and organ transplantation. We utilized immortalized human first-trimester extravillous trophoblast cells (HTR8/SVneo) for our functional study and examined the effects of Gal-9 on apoptosis, cytokine production and angiogenesis of HTR8/SVneo cells. Gal-9 inhibited the apoptosis and IFN-γ and IL-17A production, promoted IL-4 production, and coordinated the crosstalk between HTR8/SVneo cells and human umbilical vein endothelial cells via its interaction with Tim-3. Blockade of JNK signaling inhibited Gal-9 activities in HTR8/SVneo cells. In addition, we detected a correlation between low levels of Gal-9 and spontaneous abortion. So Gal-9 could inhibit the apoptosis and proinflammatory cytokine expression, and promote the angiogenesis and IL-4 production in HTR8/SVneo cells via Tim-3 in a JNK dependent manner to help the maintenance of normal pregnancy. These findings possibly identify Gal-9 as a key regulator of trophoblast cells and suggest its potential as a biomarker and target for the treatment of recurrent pregnancy loss.

T-cell immunoglobulin mucin-3 (Tim-3) plays roles in the functional regulation of both adaptive and innate immune cells and is greatly involved in many diseases. However, the precise roles of Tim-3 on macrophages (Mφs) in pregnancy remain unstated. In the current study, we found the higher frequency of Tim-3

Maternal decidual CD8+ T (dCD8+ T) cells must integrate the antithetical demands of maternal–fetal tolerance and anti-viral immunity to establish a successful pregnancy. T-cell immunoglobulin mucin-3 (Tim-3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are two important co-inhibitory molecules that regulating CD8+ T cells responses during infection and tumor. In the present study, we examined the co-expression of Tim-3 and CTLA-4 on CD8+ T cells during pregnancy and found the higher frequency of Tim-3+CTLA-4+dCD8+ T cells in response to trophoblasts. This Tim-3+CTLA-4+dCD8+ T cells subset showed an active status and produced more anti-inflammatory cytokines. Furthermore, the decreased number and altered function of Tim-3+CTLA-4+dCD8+ T cells correlated to miscarriage. Combined blocking Tim-3 and CTLA-4 pathways were highly effective in inhibiting the production of anti-inflammatory cytokines and were detrimental to the maintenance of pregnancy. Together, these findings supported that Tim-3 and CTLA-4 pathways might play positive roles in the establishment and/or maintenance of maternal–fetal tolerance so to promote the maintenance of normal pregnancy. So the reproductive safety must be considered, especially when anti-Tim-3/CTLA-4 antibody (and other immune checkpoint inhibitors) are used in pregnancy.

Normal pregnancy is associated with several immune adaptations in both systemic and local maternal–fetal interface to allow the growth of semi-allogeneic conceptus. A failure in maternal immune tolerance to the fetus may result in abnormal pregnancies, such as recurrent spontaneous abortion. The regulation of T-cell homeostasis during pregnancy has important implications for maternal tolerance and immunity. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) and T-cell immunoglobulin mucin-3 (Tim-3) are important negative immune regulatory molecules involved in viral persistence and tumor metastasis. Here we described the lower frequency of splenic T cells co-expressing CTLA-4 and Tim-3 accompanied by higher levels of proinflammatory but lower anti-inflammatory cytokines production in abortion-prone mouse model. Blockade of CTLA-4 and Tim-3 pathways leaded to the dysfunction of splenic T cells. By the higher expression during normal pregnancy, CTLA-4 and Tim-3 co-expression on splenic T cells linked to immunosuppressive phenotype. As the spleen is an important site for peripheral immune activation, our data suggest potential noninvasive biomarkers and therapeutic targets for miscarriage.

Decidual CD4

A successful pregnancy requires the maternal immune system to accept a fetus expressing allogeneic paternal antigens and provide competent responses to infections. Accordingly, maternal-fetal immune abnormalities may have an important role in the development of recurrent spontaneous abortion (RSA). Ever since the establishment of the association between immunologic abnormalities and RSA, various types of immune therapy to restore normal immune homeostasis have been increasingly developed. Although previous studies have focused on the maternal-fetal interface, non-invasive examination is of great importance in clinical practice. The present study investigated the balance between type-17 T-helper (Th17) and T-regulatory (Treg) cells in the peripheral blood to improve the current understanding of the pathogenesis of RSA. Imbalances in Th17/Treg cells and associated molecular profiles were observed in patients with RSA. Furthermore, it was determined that the immunosuppressant cyclosporine A reduced the proportion of Th17 cells and promoted Treg-cell dominance by upregulating the expression of co-inhibitory molecules in pregnant females with a history of RSA. Progesterone, the traditional maternal-care drug, also had a certain immunomodulatory role through restoring the levels of several co-inhibitory molecules (including T-cell immunoglobulin mucin family member-3, programmed cell death-1 and cytotoxic T-lymphocyte associated protein-4) in the treatment of RSA. Changes in these immune molecules within the maternal peripheral blood may be indicators for monitoring pregnancy and prediction of RSA.

The single and/or combination use of immune checkpoint blockade therapies in human infectious diseases and cancer are rapidly expanding. Despite early efforts, substantial uncertainty remains about the safety and efficacy of immune checkpoint blockade in some populations. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and T-cell immunoglobulin mucin-3 (Tim-3) are the major targetable co-inhibitory receptors on T cells. Here we showed that in animal studies, treatment with either CTLA-4- or Tim-3-blocking antibody caused greater susceptibility to fetal loss with altered cytokine profiles by decidual CD4+T (dCD4+T) cells. CTLA-4 and Tim-3 pathways appeared to play key roles in maintaining maternal-fetal tolerance by regulating the function of dCD4+T cells. In addition, the abnormality in number and functionality of dCTLA-4+Tim-3+CD4+T cells was associated with miscarriage. These findings underscored the important roles of the CTLA-4 and Tim-3 pathways in regulating dCD4+T cells function and maintaining normal pregnancy. Our study also emphasized the importance of careful consideration of reproductive safety when choosing immune checkpoint blockade therapies in real world clinical care.

The aim of the current study was to determine the pattern of immune cells and related functional molecules in peripheral blood and at the maternal-fetal interface in women with unexplained recurrent spontaneous abortion (URSA). In part I, 155 women were included and divided into four groups: non-pregnant controls with no history of URSA (NPCs), pregnant controls with no history of URSA (PCs), non-pregnant women with a history of URSA (NPUs), and pregnant women with a history of URSA (PUs). Venous blood samples were collected and analyzed. In part II, 35 subjects with URSA and 40 subjects in the early stage of normal pregnancy who chose to undergo an abortion were recruited. Samples of the decidua were collected, and the proportion of immune cells and the expression of related molecules were evaluated. Peripheral regulatory T cells (Treg cells) increased in PCs compared to NPCs, but in women with URSA the flux of Treg cells disappeared when pregnancy occurred. Levels of interleukin-10 (IL-10), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and IL-17 and the ratio of Th17/Treg cells in peripheral blood remained stable among the four groups. At the maternal-fetal interface, the percentage of Treg cells, the level of CTLA-4 of CD4+CD25+CD127lo cells and CD4+Foxp3+ cells were significantly lower in women with URSA compared to controls, respectively. Levels of transforming growth factor-β1 (TGF-β1) mRNA and protein in the decidua significantly decreased in URSA while levels of IL-6 and tumor necrosis factor-ɑ (TNF-ɑ) and the Th17/Treg ratio significantly increased. In conclusion, peripheral Treg cells did not increase in pregnant women with URSA. The decrease in Treg cells and levels of CTLA-4 and TGF-β1 and as well as the increase in levels of IL-6 and TNF-ɑ, and the Th17/Treg ratio at the maternal-fetal interface might contribute to inappropriate maternal-fetal immune tolerance in URSA.

A novel microporous metal-organic framework (MOF) based on Zn(II) ion as node with the chemical formula of {[Zn(H2O)(HL)]·(DMF)2(H2O)2}n (1, H3L ​= ​2-(4-carboxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid, DMF ​= ​N,N-dimethylformamide), which has rich water-coordinated metal centers and high density of free N-donor groups has been successfully prepared via H3L ligand and Zn(NO3)2·6H2O reaction within H2O, EtOH along with DMF mixed solvent. The presence of open metal sites along with the basic N-donor sites in the resulting activated 1a induces a moderate high sorption capacity of CO2. In addition, because of basic N-donor groups along with Lewis acidic Zn2+ ions, 1a has become an efficient heterogeneous catalyst, which can chemically fix CO2 in the carbonate of cyclic annular at mild room temperature. Furthermore, the influence of compound on the activation of PI3K/AKT signaling pathway was studied by Western blot. The ELISA was carried out the evaluate the IL-18 and IL-6 content in synovial cells after compound treatment.

Problem During normal pregnancy, delicate crosstalk is established between fetus-derived trophoblasts and maternal immune cells to ensure maternal-fetal tolerance and successful placentation. Dysfunction in these interactions has been highly linked to certain pregnancy complications. Method of study Naive CD4+ T cells were cultivated with or without 1st trimester derived trophoblast cell line HTR8/SVneo cells in the absence or presence of T helper 17 (Th17) or regulatory (Treg)cell-inducing differentiation conditions. After 5 days of co-culture, HTR8/SVneo cells and CD4+ T cells were harvested and analyzed using flow cytometry. Results CD4+ T cells exposed to HTR8/SVneo cells showed enhanced induction of CD4+ Foxp3+ Treg cells with strong expression of TGF-β1 and inhibitory molecules (cytotoxic T lymphocyte-associated protein-4 [CTLA-4], T-cell immunoglobulin mucin-3 [Tim-3], and programmed cell death-1 [PD-1]). Though not effecting Th17 differentiation, exposure to HTR8/SVneo cells promoted increased expression of proliferative and apoptotic markers on Th17 cells. Co-culture with Th0 cells, or differentiated Th17 or Treg cells, down-regulated Caspase-3 and MMP-9 (but not MMP-2) expression in HTR8/SVneo cells, while promoting Ki67 expression. Conclusions HTR8/SVneo cells regulated maternal CD4+ T-cell differentiation, resulting in the expansion of immunosuppressive Treg cells, while CD4+ T cells might promote the growth, and control the invasiveness of HTR8/SVneo cells. Thus, a bidirectional regulatory loop might exist between trophoblasts and maternal immune cell subsets, thereby promoting harmonious maternal-fetal crosstalk.

The peroxisome proliferator-activated receptor-γ (PPARG) is a member of the nuclear hormone receptor superfamily that has attracted considerable attention as a candidate gene for gestational diabetes mellitus (GDM) based on its function as a key factor involved in the regulation of adipocyte differentiation as well as lipid and glucose metabolism and insulin sensitivity. Many studies have examined the association between P12A polymorphism (rs1801282) in the PPARG gene and risk of GDM, but the results have been inconsistent. To derive a more precise estimation of the relationship, a meta-analysis of 2,858 GDM patients and 6,890 controls from nine published case-control studies was performed. An overall random effects odds ratio of 0.89 (95 % confidence interval [CI]: 0.77-1.04, P = 0.15) was found for 12A allele. In the subgroup analysis by ethnicity, significantly decreased risks were found in East Asians, while no significant associations were detected among Caucasian and Middle Eastern populations. Similar results were also observed using dominant genetic model. This meta-analysis suggested an overall weak association between the P12A polymorphism and GDM risk among East Asians. However, additional very large-scale studies are warranted to provide conclusive evidence on the effects of the PPARG gene on risk of GDM.

To assess the efficacy and safety of mifepristone combined with oral or vaginal misoprostol for termination of pregnancy between 8 and 16 weeks of gestation.This was a randomized, multi-center, open clinical trial. A total of 625 women at 8-16 weeks of gestation were randomized to receive 200 mg oral mifepristone followed by either oral misoprostol 400 µg every 3 hours or vaginal misoprostol 400 µg every 6 hours for a maximum of 4 doses 36-48 hours later. There were 417 women in oral group with 198 at 8-9 weeks and 219 at 10-16 weeks, while 208 women in vaginal group with 99 at 8-9 weeks and 109 at 10-16 weeks. The outcome measures were the success abortion rate, induction to abortion interval, the amount of bleeding, reoccurrence of menstruation and adverse events.Abortion rate was significantly higher in vaginal group [98.1% (202/206)] than that in oral group [94.0% (390/415), P = 0.023]; concerning termination of pregnancy at 8-9 weeks and 10-16 weeks respectively, there were no significant differences between oral and vaginal groups (P = 0.156, P = 0.073). The induction to abortion interval was no significant difference in oral and vaginal group in different gestational weeks (P = 0.238, P = 0.273). The average induction to abortion interval was (4.1 ± 6.6) hours and (6.0 ± 4.5) hours respectively in terminating 8-9 weeks and 10-16 weeks of gestation. Concerning the amount of bleeding within 2 hours of placenta expulsion, there was significant difference between oral group [(63 ± 46) ml] and vaginal group [(55 ± 45) ml] in terminating 8-9 weeks of gestation (P = 0.047), while there was no significant difference between groups in terminating 10-16 weeks of gestation [oral group (76 ± 52) ml versus vaginal group (76 ± 61) ml, P = 0.507]. The reoccurrence of menstruation was about 37 days in both oral and vaginal groups. Two cases of incomplete abortion were serious adverse events (SAE) relating to treatment. The common adverse events (AE) of nausea and vomiting were significantly higher in oral group [57.2% (239/417), 36.3% (151/417)] than those in vaginal group [45.4% (94/208), 26.1% (54/208); P = 0.005, 0.011].Oral or vaginal misoprostol combined with mifepristone, is effective and safe for termination of pregnancy between 8 and 16 weeks of gestation.

Trophoblasts play a crucial role in embryo implantation and maintenance of normal pregnancy. Recently, oxidative stress has been considered as one important factor in the pathogenesis of spontaneous abortion and preeclampsia. Many studies have reported that the plasma levels of hydrogen peroxide (H2O2) are significantly increased in women with preeclampsia, but the mechanisms involved in H2O2-induced cell cytotoxicity in trophoblasts are still not completely explained. Our present study was undertaken to provide a united understanding of the role of oxidative stress generated by H2O2 on human trophoblasts and the underlying intracellular signaling pathways. Exposure to H2O2 resulted in a concentration-dependent growth decrease and apoptosis in human trophoblast-like JEG-3 cells. H2O2 treatment also caused intracellular reactive oxygen species (ROS) production and concomitant dissipation of the mitochondrial membrane potential. The three MAPK subfamilies, ERK1/2, JNK and p38 kinase, were all activated under H2O2-induced oxidative stress. Blocking the activation of JNK and p38 kinase increased cell viability and decreased apoptosis induced by H2O2 with their respective inhibitors, SP600125 and SB203580. However, preventing ERK1/2 activation further increased H2O2-induced cell death with U0126, an inhibitor of ERK upstream kinase MEK1/2. Taken together, these findings suggest that the mitochondria-dependent pathways and JNK-p38 kinase pathways are involved in H2O2-induced oxidative damage of human trophoblast-like JEG-3 cells, while ERK1/2 pathway may play an active role in cell survival following oxidant injury.