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Immune regulation during pregnancy is complex, and thus an optimal therapy for pregnancy complications is always a big challenge to reproductive medicine. Cyclosporin A (CsA), a potent immunosuppressant, prevents rejection of allografts by hosts, but little is known about the modulating effect of CsA on the materno-fetal relationship. Here, pregnant CBA/J females mated with DBA/2 males as an abortion-prone model were administered with CsA on day 4.5 of gestation, and the pregnant CBA/J females mated with BALB/c males were established as successful pregnancy control. It was demonstrated that administration of CsA at the window of implantation significantly up-regulated the expression of CTLA-4, while down-regulating the levels of CD80, CD86, and CD28 at the materno-fetal interface in the CBA/J×DBA/2 abortion-prone matings, and the embryo resorption rate of the abortion-prone matings reduced significantly after CsA treatment, implying that modulation of costimulatory molecule expression by CsA might contribute to preventing the fetus from maternal immune attack. In addition, treatment with CsA induced enhanced growth and reduced cell apoptosis of the murine trophoblast cells. Together, these findings indicate that CsA has a beneficial effect on the materno-fetal interface in abortion-prone matings, leading to a pregnancy outcome improvement, which might provide new therapeutics for spontaneous pregnancy wastage.

Decidualization is the functional transformation process of endometrium in response to ovarian steroids dedicated to support embryo development. Defective decidualization is closely associated with various pregnancy complications such as recurrent miscarriage (RM). Dual specificity MAPK phosphatases (MKPs) are a family of phosphatases specifically regulating mitogen-activated protein kinase (MAPK) signaling with dual specificity for threonine and tyrosine. Here, using RNA-seq,we found that dual specificity phosphatase 1 (DUSP1) expression was prominently elevated among the MKP family members in db-cAMP treated primary human endometrial stromal cells (ESCs). We verified that its induction by db-cAMP in ESCs was in a dose- and time-dependent manner and that primary human decidual stromal cells (DSCs) present higher expression of DUSP1 than ESCs. A protein kinase A (PKA) inhibitor H-89 abolished its induction in ESCs, but not ESI-09, an EPAC1/2 inhibitor. Knock-down of TORC2/3 but not CREB by siRNA in ESCs diminished its induction by db-cAMP. Furthermore, knock-down of DUSP1, as well as TORC2/3 by siRNA caused abnormal activation of JNK during db-cAMP induction in ESCs, accompanied by decreased IGFBP1 expression, an ESC decidualization indicator, which could be fully rescued by a JNK inhibitor SP600125. In addition, Western blot showed that DUSP1 expression was reduced in the DSCs of patients with RM, along with JNK overactivation and decreased IGFBP1 expression. In conclusion, our results demonstrated that TORC2/3-mediated DUSP1 upregulation in response to the cAMP/PKA signaling safeguards IGFBP1 expression via restraining JNK activity, indicating its involvement in ESC decidualization, and that aberrant expression of DUSP1 in DSCs might engage in the pathogenesis of RM.

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.

Deficient decidualization of endometrial stromal cells (ESCs) can cause adverse pregnancy outcomes including miscarriage, intrauterine growth restriction, and pre-eclampsia. Decidualization is regulated by multiple factors such as hormones and circadian genes. Melatonin, a circadian-controlled hormone, is reported to be important for various reproductive processes, including oocyte maturation and placenta development. Its receptor, MT1, is considered to be related to intrauterine growth restriction and pre-eclampsia. However, the role of melatonin-MT1 signal in decidualization remains unknown. Here, we reported that decidual stromal cells from miscarriages displayed deficient decidualization with decreased MT1 expression. The expression level of MT1 is gradually increased with the process of decidualization induction in vitro. MT1 knockdown suppressed the decidualization level, while the overexpression of MT1 promoted the decidualization process. Moreover, changing MT1 level could regulate the expression of decidualization-related transcription factor FOXO1. Melatonin promoted decidualization and reversed the decidualization deficiency due to MT1 knockdown. Using in vitro and in vivo experiments, we further identified that lipopolysaccharide (LPS) could induce inflammation and decidualization resistance with downregulated MT1 expression, and melatonin could reverse the inflammation and decidualization resistance induced by LPS. These results suggested that the melatonin-MT1 signal might be essential for decidualization and might provide a novel therapeutic target for decidualization deficiency-associated pregnancy complications.

Decidual stromal cells (DSCs) modulate the function of trophoblasts through various factors. Wnt signaling pathway is active at the maternal–fetal interface. Here, we isolated endometrial stromal cells (ESCs) from women of reproductive ages and DSCs from normal pregnancy during the first trimester (6–10 weeks). Real-time quantitative PCR and western blotting were used to screen out the most variable WNT ligands between ESCs and DSCs, which turned out to be WNT16. Both culture mediums from DSCs and recombinant protein of human WNT16 enhanced the survival and invasion of HTR8/SVneo trophoblastic cells. Furthermore, the regulation of DSCs on trophoblast was partly blockaded after we knocked down WNT16 in DSCs. Treating HTR8/SVneo trophoblastic cells with small molecular inhibitors and small interfering RNA (siRNA), we found that the activity of AKT/beta-catenin (CTNNB1) correlated with the effect of WNT16. The crosstalk of WNT16/AKT/beta-catenin between DSCs and trophoblasts was determined to be downregulated in unexplained recurrent spontaneous abortion. This study suggests that WNT16 from DSCs promotes HTR8/SVneo trophoblastic cells invasion and survival via AKT/beta-catenin pathway at the maternal–fetal interface in human early pregnancy. The disturbance of this crosstalk between DSCs and trophoblasts might cause pregnancy failure.

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.

There is delicate crosstalk between fetus-derived trophoblasts (Tros) and maternal cells during normal pregnancy. Dysfunctions in interaction are highly linked to some pregnancy complications, such as recurrent spontaneous abortion (RSA), pre-eclampsia and fetal growth restriction. Hyaluronan (HA), the most abundant component of extracellular matrix, has been reported to act as both a pro- and an anti-inflammatory molecule. Previously, we reported that HA promotes the invasion and proliferation of Tros by activating PI3K/Akt and MAPK/ERK1/2 signaling pathways. While lower HA secretion by Tros was observed during miscarriages than that during normal pregnancies, in the present study, we further confirmed that higher secretion of HA by Tros could induce M2 polarization of macrophages at the maternal–fetal interface by interacting with CD44 and activating the downstream PI3K/Akt-STAT-3/STAT-6 signaling pathways. Furthermore, HA could restore the production of IL-10 and other normal pregnancy markers by decidual macrophages (dMφs) from RSA. These findings underline the important roles of HA in regulating the function of dMφs and maintaining a normal pregnancy.