OBJECTIVE: The decidualization process conditions monocytes to the immunosuppressive and tolerogenic dendritic cell (DC)-10 profile, a DC subset with high IL-10 production. Since the implantation process implies an embryo-endometrium-immune crosstalk, here we focused on the ability of embryonic soluble factors to modify decidual DC conditioning accordingly with its quality.
METHODS: Human endometrial stromal cell line (HESC) decidualized with medroxyprogesterone and dibutyryl-cAMP (Dec) was stimulated with human embryo-conditioned media (ECM), classified as normal (ND) or impaired developed (ID) for 48 h (n = 18/group). Monocytes isolated from six healthy women were differentiated to DCs with rhGM-CSF+rhIL-4 in the presence/absence of conditioned media (CM) from decidualized cells stimulated with ECM or nontreated.
RESULTS: We found that decidualized cells stimulated with ECM sustain a myeloid regulatory cell profile on monocyte-derived culture with increased frequency of CD1a-CD14+ and CD83+CD86low cells. ND-Dec sustained the higher expression of the DC-10 markers, HLA-G and IL-10 whereas ID-Dec diminished IL-10 production (ID-Dec: 135 ± 37.4 vs. Dec: 223.3 ± 49.9 pg/mL, p < 0.05). The treatment with ECM-Dec sustained a higher IL-10 production and prevented the increase of CD83/CD86 after LPS challenge regardless of embryo quality. Notably, TNF-α production increased in ID-Dec cultures (ID-Dec: 475.1 ± 134.7 vs. Dec: 347.5 ± 98 pg/mL, p < 0.05).
CONCLUSIONS: Although remaining in a tolerogenic profile compatible with DC-10, DCs can differentially respond to decidual secreted factors based on embryo quality, changing their secretome. These results suggest that in the presence of arrested embryo, DCs could differentially shape the immunological microenvironment, contributing to arrested embryo clearance during the menstrual phase.

Endocytosis represents a category of regulated active transport mechanisms. These encompass clathrin-dependent and -independent mechanisms, as well as fluid phase micropinocytosis and macropinocytosis, each demonstrating varying degrees of specificity and capacity. Collectively, these mechanisms facilitate the internalization of cargo into cellular vesicles. Pregnancy is one such physiological state during which endocytosis may play critical roles. A successful pregnancy necessitates ongoing communication between maternal and fetal cells at the maternal-fetal interface to ensure immunologic tolerance for the semi-allogenic fetus whilst providing adequate protection against infection from pathogens, such as viruses and bacteria. It also requires transport of nutrients across the maternal-fetal interface, but restriction of potentially harmful chemicals and drugs to allow fetal development. In this context, trogocytosis, a specific form of endocytosis, plays a crucial role in immunological tolerance and infection prevention. Endocytosis is also thought to play a significant role in nutrient and toxin handling at the maternal-fetal interface, though its mechanisms remain less understood. A comprehensive understanding of endocytosis and its mechanisms not only enhances our knowledge of maternal-fetal interactions but is also essential for identifying the pathogenesis of pregnancy pathologies and providing new avenues for therapeutic intervention.

UNASSIGNED: Kisspeptin, a protein encoded by the KISS1 gene, functions as an essential factor in suppressing tumor growth. The intricate orchestration of cellular processes such as proliferation and differentiation is governed by the Notch1/Akt/Foxo1 signaling pathway, which assumes a central role in maintaining cellular homeostasis. In the specific context of this investigation, the focal point lies in a meticulous exploration of the intricate mechanisms underlying the regulatory effect of kisspeptin on the process of endometrial decidualization. This investigation delves into the interplay between kisspeptin and the Notch1/Akt/Foxo1 signaling pathway, aiming to elucidate its significance in the pathophysiology of recurrent spontaneous abortion (RSA).
UNASSIGNED: We enrolled a cohort comprising 45 individuals diagnosed with RSA, who were admitted to the outpatient clinic of the Reproductive Center at the Second Affiliated Hospital of Soochow University between June 2020 and December 2020. On the other hand, an additional group of 50 women undergoing elective abortion at the outpatient clinic of the Family Planning Department during the same timeframe was also included. To comprehensively assess the molecular landscape, Western blot and RT-qPCR were performed to analyze the expression levels of kisspeptin (and its gene KISS1), IGFBP1 (an established marker of decidualization), Notch1, Akt, and Foxo1 within the decidua. Human endometrial stromal cells (hESC) were given targeted interventions, including treatment with siRNA to disrupt KISS1 or exposure to kisspeptin10 (the bioactive fragment of kisspeptin), and were subsequently designated as the siKP group or the KP10 group, respectively. A control group comprised hESC was transfected with blank siRNA, and cell proliferation was meticulously evaluated with CCK8 assay. Following in vitro induction for decidualization across the three experimental groups, immunofluorescence assay was performed to identify differences in Notch1 expression and decidualization morphology between the siKP and the KP10 groups. Furthermore, RT-qPCR and Western blot were performed to gauge the expression levels of IGFBP1, Notch1, Akt, and Foxo1 across the three cell groups. Subsequently, decidualization was induced in hESC by adding inhibitors targeting Notch1, Akt, and Foxo1. The expression profiles of the aforementioned proteins and genes in the four groups were then examined, with hESC induced for decidualization without adding inhibitors serving as the normal control group. To establish murine models of normal pregnancy (NP) and RSA, CBA/J×BALB/c and CBA/J×DBA/2 mice were used. The mice were respectively labeled as the NP model and RSA model. The experimental groups received intraperitoneal injections of kisspeptin10 and kisspeptin234 (acting as a blocker) and were designated as RSA-KP10 and NP-KP234 groups. On the other hand, the control groups received intraperitoneal injections of normal saline (NS) and were referred to as RSA-NS and NP-NS groups. Each group comprised 6 mice, and uterine tissues from embryos at 9.5 days of gestation were meticulously collected for observation of embryo absorption and examination of the expression of the aforementioned proteins and genes.
UNASSIGNED: The analysis revealed that the expression levels of kisspeptin, IGFBP1, Notch1, Akt, and Foxo1 were significantly lower in patients diagnosed with RSA compared to those in women with NP (P<0.01 for kisspeptin and P<0.05 for IGFBP1, Notch1, Akt, and Foxo1). After the introduction of kisspeptin10 to hESC, there was an observed enhancement in decidualization capability. Subsequently, the expression levels of Notch1, Akt, and Foxo1 showed an increase, but they decreased after interference with KISS1. Through immunofluorescence analysis, it was observed that proliferative hESC displayed a slender morphology, but they transitioned to a rounder and larger morphology post-decidualization. Concurrently, the expression of Notch1 increased, suggesting enhanced decidualization upon the administration of kisspeptin10, but the expression decreased after interference with KISS1. Further experimentation involved treating hESC with inhibitors specific to Notch1, Akt, and Foxo1 separately, revealing a regulatory sequence of Notch1/Akt/Foxo1 (P<0.05). In comparison to the NS group, NP mice administered with kisspeptin234 exhibited increased fetal absorption rates (P<0.001) and decreased expression of IGFBP1, Notch1, Akt, and Foxo1 (P<0.05). Conversely, RSA mice administered with kisspeptin10 demonstrated decreased fetal absorption rates (P<0.001) and increased expression levels of the aforementioned molecules (P<0.05).
UNASSIGNED: It is suggested that kisspeptin might exert its regulatory influence on the process of decidualization through the modulation of the Notch1/Akt/Foxo1 signaling cascade. A down-regulation of the expression levels of kisspeptin could result in suboptimal decidualization, which in turn might contribute to the development or progression of RSA.

The expression and function of podoplanin (PDPN) in the normal human placenta has been debated in placental evaluation. This study emphasizes the importance of a multimodal approach of PDPN expression in normal human placentas. A complete examination is performed using immunohistochemistry, RNAscope and automated Digital Image examination (DIA) interpretation. QuPath DIA-based analysis automatically generated the stromal and histological scores of PDPN expression for immunohistochemistry and RNAscope stains. The umbilical cord\'s isolated fibroblasts and luminal structures expressed PDPN protein and PDPN_mRNA. RNAscope detected PDPN_mRNA upregulation in syncytial placental knots trophoblastic cells, but immunohistochemistry did not certify this at the protein level. The study found a significant correlation between the IHC and RNAscope H-Score (p = 0.033) and Allred Score (p = 0.05). A successful multimodal strategy for PDPN assessment in human placentas confirmed PDPN expression heterogeneity in the full-term human normal placenta and umbilical cord at the protein and mRNA level. In placental syncytial knots trophoblastic cells, PDPN showed mRNA overexpression, suggesting a potential role in placenta maturation.

Pregnancy is a highly regulated biological phenomenon that involves the development of a semi-allogeneic fetus inside the uterus of the mother. The maternal-fetal interface is a critical junction where communication takes place between the fetal and maternal immune systems, which determine the outcome of the pregnancy. The interface is composed of the decidua and placenta. The main cells present at the maternal-fetal interface include invading trophoblasts, maternal immune cells, and decidual stromal cells. Although maternal tolerance is crucial for maintaining a successful pregnancy, the role of the placenta in pregnancy is also important. Dysregulation of the placenta leads to various placenta-mediated complications, such as preeclampsia, intrauterine growth restriction, and placental abruption. Although the exact mechanism involving these complications is unclear, research has elucidated various factors involved in these pregnancy disorders. This review aimed to provide a summary of the maternal-fetal interface and immune mechanisms involved in placenta-mediated complications.

BACKGROUND: Factors contributing to recurrent pregnancy loss (RPL) in more than half of the cases are still unknown. The incidence and societal impact of this condition requires urgent elucidation of the mechanisms behind it, which could aid in significant improvement of clinical management.
METHODS: Using a highly efficient in-solution digestion method and label-free data-independent LC-MS/MS acquisition with ion mobility, we performed comparative proteomics analysis of the decidua tissues from 19 RPL patients and 10 controls. Differentially abundant proteins (DAPs) were compared and correlated with 3 publicly available transcriptomic datasets and the expression of selected biomarkers was tested by qPCR in decidua and chorionic villi from an extended cohort.
RESULTS: From 1952 proteins identified based on ≥2 peptides, the statistically significant difference in abundance (Anova p ≤ 0.05) and fold change ≥1.2 showed 85 proteins. Pathway analysis using Reactome, KEGG and Wiki pathways identified enrichment of \"Signaling by ROBO receptors\", \"RNA degradation\" and \"Cytoplasmic Ribosomal Proteins\". The correlation between protein and gene expression in decidua revealed that the down-regulated ribosomal proteins in our dataset (RPS15, RPS17, RPL27A, RPL35A and RPL18) showed the same regulation trend at the mRNA level, which was later confirmed for transcripts of RPS15 and RPL18 in our cohort.
CONCLUSIONS: Our data suggests that the potential causes of RPL from the maternal side could be associated with impaired RNA processing machinery. Furthermore, the list of DAPs in RPL opens future investigations in terms of screening novel gene variants predisposing to pregnancy failure and developing biomarkers for RPL risk.

The process of human parturition involves inflammation at the interface where fetal chorion trophoblast cells interact with maternal decidual stromal (DS) cells and maternal immune cells in the decidua (endometrium of pregnancy). This study tested the hypothesis that inflammation at the chorion-decidua interface (CDI) induces labor by negating the capacity for progesterone (P4) to block labor and that this is mediated by inactivation of P4 in DS cells by aldo-keto reductase family 1 member C1 (AKR1C1). In human, Rhesus macaque, and mouse CDI, AKR1C1 expression increased in association with term and preterm labor. In a human DS cell line and in explant cultures of term human fetal membranes containing the CDI, the prolabor inflammatory cytokine, interleukin-1ß (IL-1ß), and media conditioned by LPS-stimulated macrophages increased AKR1C1 expression and coordinately reduced nuclear P4 levels and P4 responsiveness. Loss of P4 responsiveness was overcome by inhibition of AKR1C1 activity, inhibition of AKR1C1 expression, and bypassing AKR1C1 activity with a P4 analog that is not metabolized by AKR1C1. Increased P4 activity in response to AKR1C1 inhibition was prevented by the P4 receptor antagonist RU486. Pharmacologic inhibition of AKR1C1 activity prevented parturition in a mouse model of inflammation-induced preterm parturition. The data suggest that inflammatory stimuli at the CDI drive labor by inducing AKR1C1-mediated P4 inactivation in DS cells and that inhibiting and/or bypassing of AKR1C1-mediated P4 inactivation is a plausible therapeutic strategy to mitigate the risk of inflammation-associated preterm birth.

Myeloid-derived suppressor cells (MDSCs) play a crucial role in maintaining maternal-fetal tolerance by expressing some immune-suppressive molecules, such as indoleamine 2,3-dioxygenase (IDO). Toxoplasma gondii (T. gondii) infection can break the immune microenvironment of maternal-fetal interface, resulting in adverse pregnancy outcomes. However, whether T. gondii affects IDO expression in dMDSCs and the molecular mechanism of its effect are still unclear. Here we show, the mRNA level of IDO is increased but the protein level decreased in infected dMDSCs. Mechanistically, the upregulation of transcriptional levels of IDO in dMDSCs is regulated through STAT3/p52-RelB pathway and the decrease of IDO expression is due to its degradation caused by increased SOCS3 after T. gondii infection. In vivo, the adverse pregnancy outcomes of IDO-/- infected mice are more severe than those of wide-type infected mice and obviously improved after exogenous kynurenine treatment. Also, the reduction of IDO in dMDSCs induced by T. gondii infection results in the downregulation of TGF-β and IL-10 expression in dNK cells regulated through Kyn/AhR/SP1 signal pathway, eventually leading to the dysfunction of dNK cells and contributing the occurrence of adverse pregnancy outcomes. This study reveals a novel molecular mechanism in adverse pregnancy outcome induced by T. gondii infection.

The pathogenesis of preeclampsia (PE) has not been elucidated, but immune imbalance is known to be one of the main pathogeneses. Dysfunction of decidual macrophages can lead to PE, and the PD-1/PD-L1 signaling pathway is associated with macrophage polarization. However, the relationship between the influence of the PD-1/PD-L1 signaling pathway on macrophage polarization and the onset of PE has not been fully elucidated. In this study, we analyzed the expression of CD68, iNOS, CD206, PD-1 and PD-L1 and the coexpression of CD68+PD-1+ and CD68+PD-L1+ in the decidual tissue of PE patients (n= 18) and healthy pregnant women (n=20). We found that CD68 and iNOS expression was increased in the decidua of PE patients (P < 0.001) and that CD206, PD-1 and PD-L1 expression and CD68+PD-1+ and CD68+PD-L1+ coexpression were decreased (P < 0.001). To assess the influence of the PD-1/PD-L1 signaling pathway on macrophage polarization, we added an anti-PD-1 mAb (pembrolizumab) or an anti-PD-L1 mAb (durvalumab) during THP-1 differentiation into M1 macrophages. Then, we detected the polarization of CD68+CD80+ macrophages and the expression of iNOS. To examine the effect of macrophage polarization on the invasion ability of trophoblast cells, macrophages were cocultured with HTR8/SVneo cells, and the invasion ability of HTR8/SVneo cells was detected via transwell assays. We found that CD68+CD80+ macrophage polarization was enhanced (P<0.05) and that iNOS expression was greater (P<0.01) in the pembrolizumab group. In the durvalumab group, CD68+CD80+ macrophage polarization and iNOS expression were also increased (P<0.05 and P<0.001). Compared with that in the untreated group, the aggressiveness of HTR8/SVneo cells was decreased in both the pembrolizumab group (P < 0.01) and the durvalumab group (P < 0.001). These findings indicate that the PD-1/PD-L1 signaling pathway may play an important role in the pathogenesis of PE by influencing macrophage polarization and reducing the invasion ability of trophoblasts.

BACKGROUND: Studies have shown that EMT (epithelial-mesenchymal transition) and energy metabolism influence each other, and it is unclear whether the trophoblast energy metabolism phenotype is dominated by glycolysis or mitochondrial respiration, and the relationship between trophoblast energy metabolism and EMT is still unclear.
METHODS: Exosomes were isolated from the DSC of URSA patients and their miRNA profile was characterized by miRNA sequencing. Wound healing assays and transwell assays were used to assess the invasion and migration ability of trophoblasts. Mitochondrial stress and glycolysis stress test were used to evaluate energy metabolism phenotype of trophoblast. Luciferase reporter assays, qRT-PCR and WB were conducted to uncover the underlying mechanism. Finally, animal experiments were employed to explore the effect of DSC-exos on embryo absorption in mice.
RESULTS: Our results showed that URSA-DSC-exos suppressed trophoblast EMT to reduce their migration and invasion, miR-22-5p_R-1 was the most upregulated miRNAs. URSA-DSC-exos can suppress trophoblast MGS (metabolic switch from mitochondrial respiration to glycolysis) and inhibit trophoblast migration and invasion by transferring miR-22-5p_R-1. Mechanistically, miR-22-5p_R-1 suppress trophoblast MGS and inhibit trophoblast EMT by directly suppressing PDK4 expression at the post-transcriptional level. Furthermore, in vivo experiment suggested that URSA-DSC-exos aggravated embryo absorption in mice. Clinically, PDK4 and EMT molecule were aberrant in villous of URSA patients, and negative correlations were found between miR-22-5p_R-1 and PDK4.
CONCLUSIONS: Our findings indicated that URSA-DSC-exos induced MGS obstacle playing an important role in intercellular communication between trophoblast and DSC, illuminating a novel mechanism in DSC regulation of trophoblasts and their role in URSA.