Decidual Leukocytes Research Articles

Decidual leukocytes respond to African lineage Zika virus infection with mild anti-inflammatory changes during acute infection in rhesus macaques.

Zika virus (ZIKV) can be vertically transmitted during pregnancy resulting in a range of adverse pregnancy outcomes. The decidua is commonly found to be infected by ZIKV, yet the acute immune response to infection remains understudied in vivo. We hypothesized that in vivo African-lineage ZIKV infection induces a pro-inflammatory response in the decidua. To test this hypothesis, we evaluated the decidua in pregnant rhesus macaques within the first two weeks following infection with an African-lineage ZIKV and compared our findings to gestationally aged-matched controls. Decidual leukocytes were phenotypically evaluated using spectral flow cytometry, and cytokines and chemokines were measured in tissue homogenates from the decidua, placenta, and fetal membranes. The results of this study did not support our hypothesis. Although ZIKV RNA was detected in the decidual tissue samples from all ZIKV infected dams, phenotypic changes in decidual leukocytes and differences in cytokine profiles suggest that the decidua undergoes mild anti-inflammatory changes in response to that infection. Our findings emphasize the immunological state of the gravid uterus as a relatively immune privileged site that prioritizes tolerance of the fetus over mounting a pro-inflammatory response to clear infection.

Deciphering decidual leukocyte traffic with serial intravascular staining.

The decidual immunome is dynamic, dramatically changing its composition across gestation. Early pregnancy is dominated by decidual NK cells, with a shift towards T cells later in pregnancy. However, the degree, timing, and subset-specific nature of leukocyte traffic between the decidua and systemic circulation during gestation remains poorly understood. Herein, we employed intravascular staining in pregnant C57BL/6J mice and cynomolgus macaques (Macaca fascicularis) to examine leukocyte traffic into the decidual basalis during pregnancy. Timed-mated or virgin mice were tail-vein injected with labelled αCD45 antibodies 24 hours and 5 minutes before sacrifice. Pregnant cynomolgus macaques (GD155) were infused with labelled αCD45 at 2 hours or 5 mins before necropsy. Decidual cells were isolated and resulting suspensions analyzed by flow cytometry. We found that the proportion of intravascular (IVAs)-negative leukocytes (cells labeled by the 24h infusion of αCD45 or unlabeled) decreased across murine gestation while recent immigrants (24h label only) increased in mid- to late-gestation. In the cynomolgus model our data confirmed differential labeling of decidual leukocytes by the infused antibody, with the 5 min infused animal having a higher proportion of IVAs+ cells compared to the 2hr infused animal. Decidual tissue sections from both macaques showed the presence of intravascularly labeled cells, either in proximity to blood vessels (5min infused animal) or deeper into decidual stroma (2hr infused animal). These results demonstrate the value of serial intravascular staining as a sensitive tool for defining decidual leukocyte traffic during pregnancy.

Emerging role of C5aR2: novel insights into the regulation of uterine immune cells during pregnancy.

Pregnancy is a fascinating immunological phenomenon because it allows allogeneic fetal and placental tissues to survive inside the mother. As a component of innate immunity with high inflammatory potential, the complement system must be tightly regulated during pregnancy. Dysregulation of the complement system plays a role in pregnancy complications including pre-eclampsia and intrauterine growth restriction. Complement components are also used as biomarkers for pregnancy complications. However, the mechanisms of detrimental role of complement in pregnancy is poorly understood. C5a is the most potent anaphylatoxin and generates multiple immune reactions via two transmembrane receptors, C5aR1 and C5aR2. C5aR1 is pro-inflammatory, but the role of C5aR2 remains largely elusive. Interestingly, murine NK cells have been shown to express C5aR2 without the usual co-expression of C5aR1. Furthermore, C5aR2 appears to regulate IFN-γ production by NK cells in vitro. As IFN-γ produced by uterine NK cells is one of the major factors for the successful development of a vital pregnancy, we investigated the role anaphylatoxin C5a and its receptors in the establishment of pregnancy and the regulation of uterine NK cells by examinations of murine C5ar2-/- pregnancies and human placental samples. C5ar2-/- mice have significantly reduced numbers of implantation sites and a maternal C5aR2 deficiency results in increased IL-12, IL-18 and IFN-γ mRNA expression as well as reduced uNK cell infiltration at the maternal-fetal interface. Human decidual leukocytes have similar C5a receptor expression patterns showing clinical relevance. In conclusion, this study identifies C5aR2 as a key contributor to dNK infiltration and pregnancy success.

Dendritic Cells and the Establishment of Fetomaternal Tolerance for Successful Human Pregnancy.

Pregnancy is a remarkable event where the semi-allogeneic fetus develops in the mother's uterus, despite genetic and immunological differences. The antigen handling and processing at the maternal-fetal interface during pregnancy appear to be crucial for the adaptation of the maternal immune system and for tolerance to the developing fetus and placenta. Maternal antigen-presenting cells (APCs), such as macrophages (Mφs) and dendritic cells (DCs), are present at the maternal-fetal interface throughout pregnancy and are believed to play a crucial role in this process. Despite numerous studies focusing on the significance of Mφs, there is limited knowledge regarding the contribution of DCs in fetomaternal tolerance during pregnancy, making it a relatively new and growing field of research. This review focuses on how the behavior of DCs at the maternal-fetal interface adapts to pregnancy's unique demands. Moreover, it discusses how DCs interact with other cells in the decidual leukocyte network to regulate uterine and placental homeostasis and the local maternal immune responses to the fetus. The review particularly examines the different cell lineages of DCs with specific surface markers, which have not been critically reviewed in previous publications. Additionally, it emphasizes the impact that even minor disruptions in DC functions can have on pregnancy-related complications and proposes further research into the potential therapeutic benefits of targeting DCs to manage these complications.

Methods for Co-culture of Primary Human Extravillous Trophoblast Cells and Uterine Natural Killer Cells.

During early pregnancy, fetal-derived extravillous trophoblast cells (EVT) from the placenta invade the maternal decidua and inner third of the uterus where they establish fetal tolerance and remodel the uterine spiralarteries, which ensures establishment of a successful pregnancy. Aberrant EVT invasion and spiral artery remodeling is associated with a number of pregnancy complications including miscarriage, preeclampsia, fetal growth restriction, and placenta accrete. During invasion of the maternal tissues, the EVT interact with a number of different cell types including the decidual leukocytes. EVT express HLA-C, HLA-G, HLA-E, and HLA-F and interact with uterine natural killer (uNK) cells through a series of different receptors. Epidemiological evidence suggests that different combinations of HLA-C and killer cell Ig-like receptor (KIR) haplotypes impact pregnancy success. Therefore, there is much interest in the functional consequence of interactions between EVT and uNK cells, and several different methodologies have been used to isolate these different cell types and their co-culture.

The impact of maternal SARS-CoV-2 vaccination and first trimester infection on feto-maternal immune responses.

ProblemCOVID‐19 infection during pregnancy increases maternal and fetal morbidity and mortality. Infection in the second or third trimester leads to changes in the decidual leukocyte populations. However, it is not known whether COVID‐19 infection in the first trimester or COVID‐19 vaccination during pregnancy alters the decidual immune environment.Method of studyWe examined decidual biopsies obtained at delivery from women who had COVID‐19 in the first trimester (n = 8), were fully vaccinated against COVID‐19 during pregnancy (n = 17), or were neither infected nor vaccinated during pregnancy (n = 9). Decidual macrophages, NK cells, and T cells were quantified by immunofluorescence. Decidual IL‐6, IL‐10, and IP‐10 were quantified by ELISA.ResultsThere were no differences in decidual macrophages, NK cells, T cells, or cytokines between the first trimester COVID‐19 group and the control group. The vaccinated cohort had lower levels of macrophages and NK cells compared to the control group. There were no differences in cytokines between the vaccinated and control groups.ConclusionsCOVID‐19 infection in the first trimester did not cause significant decidual leukocyte or cytokine changes at the maternal‐fetal interface. Additionally, vaccination was not associated with decidual inflammation, supporting the safety of SARS‐CoV‐2 vaccination during pregnancy.

From decidualization to pregnancy progression: An overview of immune and metabolic effects of VIP.

A tight immune and metabolic regulation underlies the early maternal-placental interaction to assist the energetic dynamic demands of the fetus throughout pregnancy. The vasoactive intestinal peptide (VIP) holds biochemical, metabolic and immune properties consistent with a regulatory role during pregnancy. Here we overview critical aspects of embryo implantation and placental development with special focus on the immune and metabolic effects of VIP expressed by decidual and trophoblast cells. During decidualization, endometrial stromal cells undergo reticular stress and trigger unfolded protein response (UPR) that enable expansion of their endoplasmic reticulum and immunomodulatory factor synthesis. These processes appear differentially affected in recurrent abortion and in vitro fertilization failure suggesting their relevance in reproductive pathologies. Similarly, defective placentation associates with altered immune, vascular and trophoblast interaction resulting in complicated pregnancies that threaten maternal and neonatal health and underlie metabolic programming of adult life. We discuss the most recent research on decidual, trophoblast and immune cell interaction on the light of VIP regulation. Its role in decidualization and UPR associated with a sterile inflammatory response and angiogenesis is discussed. Evidence on VIP modulation of cytotrophoblast cell function, metabolism and immune profile is revised as well as the shaping of decidual leukocyte phenotype and function from decidualization to term. The broad spectrum of effects of VIP from implantation to term in normal and pathological conditions summarized here might contribute to the identification of novel biomarkers for diagnosis and pharmacological targeting.

Single-cell RNA sequencing reveals immunological rewiring at the maternal-fetal interface following asymptomatic/mild SARS-CoV-2 infection.

While severe coronavirus 2019 (COVID-19) is associated with immune activation at the maternal-fetal interface, responses to asymptomatic/mild severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during pregnancy remain unknown. Here, we assess immunological adaptations in blood and term decidua in response to asymptomatic/mild disease in pregnant women. We report attenuated antigen presentation and type I interferon (IFN) signaling pathways, loss of tissue-resident decidual macrophages, and upregulated cytokine/chemokine signaling in monocyte-derived decidual macrophages. Furthermore, we describe increased frequencies of activated tissue-resident T cells and decreased abundance of regulatory T cells with infection while frequencies of cytotoxic CD4/CD8 T cells are increased in the blood. In contrast to decidual macrophages, type I IFN signaling is higher in decidual T cells. Finally, infection leads to a narrowing of T cell receptor diversity in both blood and decidua. Collectively, these observations indicate that asymptomatic/mild COVID-19 during pregnancy results in remodeling of the immunological landscape of the maternal-fetal interface, with a potential for long-term adverse outcomes for the offspring.

Decidual immune response following COVID-19 during pregnancy varies by timing of maternal SARS-CoV-2 infection

While COVID-19 infection during pregnancy is common, fetal transmission is rare, suggesting that intrauterine mechanisms form an effective blockade against SARS-CoV-2. Key among these is the decidual immune environment of the placenta. We hypothesize that decidual leukocytes are altered by maternal SARS-CoV-2 infection in pregnancy and that this decidual immune response is shaped by the timing of infection during gestation. To address this hypothesis, we collected decidua basalis tissues at delivery from women with symptomatic COVID-19 during second (2nd Tri COVID, n = 8) or third trimester (3rd Tri COVID, n = 8) and SARS-CoV-2-negative controls (Control, n = 8). Decidual natural killer (NK) cells, macrophages and T cells were evaluated using quantitative microscopy, and pro- and anti-inflammatory cytokine mRNA expression was evaluated using quantitative reverse transcriptase PCR (qRT-PCR). When compared with the Control group, decidual tissues from 3rd Tri COVID exhibited significantly increased macrophages, NK cells and T cells, whereas 2nd Tri COVID only had significantly increased T cells. In evaluating decidual cytokine expression, we noted that IL-6, IL-8, IL-10 and TNF-α were significantly correlated with macrophage cell abundance. However, in 2nd Tri COVID tissues, there was significant downregulation of IL-6, IL-8, IL-10, and TNF-α. Taken together, these results suggest innate and adaptive immune responses are present at the maternal-fetal interface in maternal SARS-CoV-2 infections late in pregnancy, and that infections earlier in pregnancy show evidence of a resolving immune response. Further studies are warranted to characterize the full scope of intrauterine immune responses in pregnancies affected by maternal COVID-19.

Trophectoderm differentiation to invasive syncytiotrophoblast is promoted by endometrial epithelial cells during human embryo implantation.

STUDY QUESTIONHow does the human embryo breach the endometrial epithelium at implantation?SUMMARY ANSWEREmbryo attachment to the endometrial epithelium promotes the formation of multinuclear syncytiotrophoblast from trophectoderm, which goes on to breach the epithelial layer.WHAT IS KNOWN ALREADYA significant proportion of natural conceptions and assisted reproduction treatments fail due to unsuccessful implantation. The trophectoderm lineage of the embryo attaches to the endometrial epithelium before breaching this barrier to implant into the endometrium. Trophectoderm-derived syncytiotrophoblast has been observed in recent in vitro cultures of peri-implantation embryos, and historical histology has shown invasive syncytiotrophoblast in embryos that have invaded beyond the epithelium, but the cell type mediating invasion of the epithelial layer at implantation is unknown.STUDY DESIGN, SIZE, DURATIONFresh and frozen human blastocyst-stage embryos (n = 46) or human trophoblast stem cell (TSC) spheroids were co-cultured with confluent monolayers of the Ishikawa endometrial epithelial cell line to model the epithelial phase of implantation in vitro. Systems biology approaches with published transcriptomic datasets were used to model the epithelial phase of implantation in silico.PARTICIPANTS/MATERIALS, SETTING, METHODSHuman embryos surplus to treatment requirements were consented for research. Day 6 blastocysts were co-cultured with Ishikawa cell layers until Day 8, and human TSC spheroids modelling blastocyst trophectoderm were co-cultured with Ishikawa cell layers for 48 h. Embryo and TSC morphology was assessed by immunofluorescence microscopy, and TSC differentiation by real-time quantitative PCR (RT-qPCR) and ELISA. Single-cell human blastocyst transcriptomes, and bulk transcriptomes of TSC and primary human endometrial epithelium were used to model the trophectoderm–epithelium interaction in silico. Hypernetworks, pathway analysis, random forest machine learning and RNA velocity were employed to identify gene networks associated with implantation.MAIN RESULTS AND THE ROLE OF CHANCEThe majority of embryos co-cultured with Ishikawa cell layers from Day 6 to 8 breached the epithelial layer (37/46), and syncytiotrophoblast was seen in all of these. Syncytiotrophoblast was observed at the embryo-epithelium interface before breaching, and syncytiotrophoblast mediated all pioneering breaching events observed (7/7 events). Multiple independent syncytiotrophoblast regions were seen in 26/46 embryos, suggesting derivation from different regions of trophectoderm. Human TSC spheroids co-cultured with Ishikawa layers also exhibited syncytiotrophoblast formation upon invasion into the epithelium. RT-qPCR comparison of TSC spheroids in isolated culture and co-culture demonstrated epithelium-induced upregulation of syncytiotrophoblast genes CGB (P = 0.03) and SDC1 (P = 0.008), and ELISA revealed the induction of hCGβ secretion (P = 0.03). Secretory-phase primary endometrial epithelium surface transcriptomes were used to identify trophectoderm surface binding partners to model the embryo-epithelium interface. Hypernetwork analysis established a group of 25 epithelium-interacting trophectoderm genes that were highly connected to the rest of the trophectoderm transcriptome, and epithelium-coupled gene networks in cells of the polar region of the trophectoderm exhibited greater connectivity (P < 0.001) and more organized connections (P < 0.0001) than those in the mural region. Pathway analysis revealed a striking similarity with syncytiotrophoblast differentiation, as 4/6 most highly activated pathways upon TSC-syncytiotrophoblast differentiation (false discovery rate (FDR < 0.026)) were represented in the most enriched pathways of epithelium-coupled gene networks in both polar and mural trophectoderm (FDR < 0.001). Random forest machine learning also showed that 80% of the endometrial epithelium-interacting trophectoderm genes identified in the hypernetwork could be quantified as classifiers of TSC-syncytiotrophoblast differentiation. This multi-model approach suggests that invasive syncytiotrophoblast formation from both polar and mural trophectoderm is promoted by attachment to the endometrial epithelium to enable embryonic invasion.LARGE SCALE DATANo omics datasets were generated in this study, and those used from previously published studies are cited.LIMITATIONS, REASONS FOR CAUTION In vitro and in silico models may not recapitulate the dynamic embryo-endometrial interactions that occur in vivo. The influence of other cellular compartments in the endometrium, including decidual stromal cells and leukocytes, was not represented in these models.WIDER IMPLICATIONS OF THE FINDINGSUnderstanding the mechanism of human embryo breaching of the epithelium and the gene networks involved is crucial to improve implantation success rates after assisted reproduction. Moreover, early trophoblast lineages arising at the epithelial phase of implantation form the blueprint for the placenta and thus underpin foetal growth trajectories, pregnancy health and offspring health.STUDY FUNDING/COMPETING INTEREST(S)This work was funded by grants from Wellbeing of Women, Diabetes UK, the NIHR Local Comprehensive Research Network and Manchester Clinical Research Facility, and the Department of Health Scientist Practitioner Training Scheme. None of the authors has any conflict of interest to declare.

Purification of Primary Decidual Natural Killer Cells for Functional Analysis.

Decidual NK cells (dNK) are a unique type of NK cells found at the maternal-fetal interface during pregnancy. dNK play a key role in placental development, trophoblast invasion, and immunity to viral and bacterial infection of the placenta. dNK are the predominant leukocyte population in first trimester placental tissues and comprise around 70% of the total CD45+ leukocytes. dNK remain present throughout pregnancy but their proportion decreases to 20-40% of term placenta decidual tissue leukocytes. Investigation of dNK function throughout pregnancy is of high clinical relevance for understanding the development of placental inflammatory disorders as well as maternal-to-fetal transmission of pathogens. In this chapter, we describe in detail the methods we developed to purify dNK from first trimester and term pregnancy placental tissues. These methods are suitable to assess their protein and gene expression profiles as well as their function.

Uterine Natural Killer Cells and Implantation

Decidual leukocytes make up approximately 30% of all decidual stromal cells in early pregnancy, of which 70% are uterine natural killer (uNK) cells. uNK cells are phenotypically distinct from peripheral blood NK cells, being CD56[Formula: see text]CD16[Formula: see text]. A recent single-cell sequencing project of the decidua identified three subsets of uNK cells, but we are yet to determine how they differ functionally. Several roles for uNK cells in implantation are starting to emerge including biosensing of poor-quality embryos, killing of bacteria infected trophoblast, spiral artery remodeling, and regulation of trophoblast invasion. Altered numbers of uNK cells have been identified in several pathological conditions, but whether this is causative of the condition is currently unclear.

Regulation of the innate immune cells during pregnancy: An immune checkpoint perspective.

The foetus can be regarded as a half‐allograft implanted into the maternal body. In a successful pregnancy, the mother does not reject the foetus because of the immune tolerance mechanism at the maternal‐foetal interface. The innate immune cells are a large part of the decidual leukocytes contributing significantly to a successful pregnancy. Although the contributions have been recognized, their role in human pregnancy has not been completely elucidated. Additionally, the accumulated evidence demonstrates that the immune checkpoint molecules expressed on the immune cells are co‐inhibitory receptors regulating their activation and biological function. Therefore, it is critical to understand the immune microenvironment and explore the function of the innate immune cells during pregnancy. This review summarizes the classic immune checkpoints such as PD‐1, CTLA‐4 and some novel molecules recently identified, including TIM‐3, CD200, TIGIT and the Siglecs family on the decidual and peripheral innate immune cells during pregnancy. Furthermore, it emphasizes the role of the immune checkpoint molecules in pregnancy‐associated complications and reproductive immunotherapy.

BCG-trained innate immunity leads to fetal growth restriction by altering immune cell profile in the mouse developing placenta.

Trained immunity is a new concept illustrating that innate immune cells are able to undergo a long-term metabolic and epigenetic reprogramming after infection or vaccination, thus displaying either a pro- or an anti-inflammatory phenotype during a sequential unrelated challenge. Innate immune cells such as natural killer (NK) cells and macrophages constitute a large part of the decidual leukocyte population at the maternal-fetal interface, playing an important role in placental development and as such in fetal growth and development. In this study, we hypothesized that training the innate immune cells before pregnancy could have an impact on pregnancy. To test this hypothesis, we used CBA/J x DBA/2 mouse model to investigate pregnancy outcomes and leukocyte population at the maternal-fetal interface. Although we were not able to show a beneficial effect of LPS-tolerogenic training on fetal resorption, Bacillus Calmette-Guérin (BCG) training, known to prime innate immune cells to be proinflammatory, led to fetal growth restriction, without aggravating the fetal resorption rate. We also found that BCG training led to less NK cells and macrophages at the maternal-fetal interface at the early stage of placentation (E9.5), associated with a down-regulation of Ccr3 and Lif mRNA expression. This induced altered leucocyte population profile can be an explanation for the subsequent fetal growth restriction. These data suggest that preconceptional infections-induced trained immunity could influence pregnancy outcomes.

Immune Profile of the Normal Maternal-Fetal Interface in Rhesus Macaques and Its Alteration Following Zika Virus Infection.

The maternal decidua is an immunologically complex environment that balances maintenance of immune tolerance to fetal paternal antigens with protection of the fetus against vertical transmission of maternal pathogens. To better understand host immune determinants of congenital infection at the maternal-fetal tissue interface, we performed a comparative analysis of innate and adaptive immune cell subsets in the peripheral blood and decidua of healthy rhesus macaque pregnancies across all trimesters of gestation and determined changes after Zika virus (ZIKV) infection. Using one 28-color and one 18-color polychromatic flow cytometry panel we simultaneously analyzed the frequency, phenotype, activation status and trafficking properties of αβ T, γδ T, iNKT, regulatory T (Treg), NK cells, B lymphocytes, monocytes, macrophages, and dendritic cells (DC). Decidual leukocytes showed a striking enrichment of activated effector memory and tissue-resident memory CD4+ and CD8+ T lymphocytes, CD4+ Tregs, CD56+ NK cells, CD14+CD16+ monocytes, CD206+ tissue-resident macrophages, and a paucity of B lymphocytes when compared to peripheral blood. t-distributed stochastic neighbor embedding (tSNE) revealed unique populations of decidual NK, T, DC and monocyte/macrophage subsets. Principal component analysis showed distinct spatial localization of decidual and circulating leukocytes contributed by NK and CD8+ T lymphocytes, and separation of decidua based on gestational age contributed by memory CD4+ and CD8+ T lymphocytes. Decidua from 10 ZIKV-infected dams obtained 16-56 days post infection at third (n=9) or second (n=1) trimester showed a significant reduction in frequency of activated, CXCR3+, and/or Granzyme B+ memory CD4+ and CD8+ T lymphocytes and γδ T compared to normal decidua. These data suggest that ZIKV induces local immunosuppression with reduced immune recruitment and impaired cytotoxicity. Our study adds to the immune characterization of the maternal-fetal interface in a translational nonhuman primate model of congenital infection and provides novel insight in to putative mechanisms of vertical transmission.

Single-Cell RNA-Seq of Mouse Decidual Leukocytes Reveals Intriguing Gestational Changes in the Immune Cell Landscape and Effects of SRC-1/-2 Double-Deficiency

Single-cell RNA-seq of Mouse Decidual Leukocytes Reveals Intriguing Gestational Changes in the Immune Cell Landscape and Effects of SRC-1/-2 Double-DeficiencyOur previous findings suggest that the fetus signals the mother when it is ready to be born through secretion of surfactant components/immune modulators, surfactant protein A (SP-A) and platelet-activating factor (PAF) by the fetal lung into amniotic fluid (AF). This occurs with increased proinflammatory cytokine expression by fetal AF macrophages (Mϕ), increased myometrial NF-κB activation and contractile (CAP) gene expression. Steroid receptor coactivator (Src)-1 and Src-2 are critical for developmental induction SP-A and PAF by the fetal lung. The finding that pregnant wild-type (WT) mice carrying Src-1 and Src-2 double-deficient fetuses (Src-1/-2d/d) manifested a marked delay (~38h) in parturition, further suggests that signals for parturition arise from the fetus and that Src-1 and Src-2 serve a critical role. Infiltrating leukocytes at the maternal-fetal interface (MFI)/decidua are known to play a central role in pregnancy maintenance and parturition timing. However, there is limited knowledge regarding gestational changes in immune cell composition toward term. To analyze gestational changes in the composition of immune cells within decidua and effects of Src-1/-2d/d, we conducted single-cell RNA-seq of ~17,000 decidual leukocytes (CD45+) from pregnant mice at 15.5 and 18.5 dpc carrying WT or Src-1/-2d/d fetuses. Unsupervised clustering identified 22 distinct decidual immune cell clusters, comprised of Mϕ, B cells, natural killer cells, neutrophils, dendritic cells and monocytes. Significant differences in cell type composition and transcriptional profiles were found across study groups (WT @ 15.5 dpc vs. 18.5 dpc; Src-1/-2d/dvs. WT @15.5 dpc and 18.5 dpc). Interestingly, in deciduas of pregnant mice carrying WT fetuses, Mϕ and B cell clusters markedly increased between 15.5 and 18.5 dpc, whereas, neutrophils declined; however, these gestational changes did not occur in pregnant mice carrying Src-1/-2d/d fetuses. Differential gene expression and gene ontology enrichment analyses revealed specific gene expression patterns distinguishing these immune cell subtypes to uncover their putative functions. These findings highlight the complexity and dynamics of the decidual immune cell landscape during the transition from myometrial quiescence to contractility, and the fetal-maternal interactions leading to parturition. Support: NIH grants R01-HL050022 (CRM) and P01-HD087150 (CRM), Burroughs Wellcome Preterm Birth Grant #1019823 (CRM).

Decidual-derived RANKL facilitates macrophages accumulation and residence at the maternal-fetal interface in human early pregnancy.

During the first trimester, the accumulation of macrophages, which is the second largest decidual leukocyte population (~20%) at the maternal-fetal interface, is quite vital for a successful pregnancy, including embryo implantation, trophoblast invasion, and vascular remodeling. The mechanism of the enrichment and redistribution of macrophages in the uterine decidua of early pregnancy is largely unclear. A total of 37 women with normal early pregnancies were included. Primary decidual macrophages (dMφs) (n=37) and primary decidual stromal cells (DSCs) (n=37) were isolated, and the adhesion molecules were analyzed by flow cytometry (FCM). Adhesive experiment was carried out to evaluate the adhesion capacity by counting cell numbers of dMφs adhered to DSCs in a co-culture system. We found that RANK+ dMφs was the dominating subtype at the maternal-fetal interface. The expression of adhesion molecules (eg, CD29, CD31, CD54, and CD62L) on the surface of RANK+ dMφs was higher than that of RANK- dMφs. After co-culture with DSCs, the expression of adhesion molecules on dMφs was up-regulated in a RANKL-dependent manner. Meanwhile, dMφs promoted the releasing of RANKL on DSCs after co-culture. Consistently, dMφs exhibited the lessoned capacity of adhesion to DSCs when blocking the crosstalk of RANKL-RANK between the DSCs and dMφs in vitro. These results suggest that the interaction of RANKL-RANK up-regulates the expression of adhesion molecules on the surface of dMφs, contributing to the accumulation and residence of dMφs in human early pregnancy.

Role of the HLA-G immune checkpoint molecule in pregnancy

The non-classical HLA class I molecule HLA-G is expressed in trophoblasts where it contributes to maternal-fetal tolerance. HLA-G has been implicated in the control of trophoblast invasion, uterine vascular remodeling, and maintenance of a local immunosuppressive state. Understanding HLA-G biology at the maternal-fetal interface is therefore a critical issue in reproduction. In this regard, we review here: (i) the effects of HLA-G on decidual leucocytes and stromal cells, (ii) the contribution of trogocytosis in HLA-G expression on decidual cells, (iii) its interaction with the ILT2, ILT4 and KIR2DL4 receptors, (iv) the link between HLA-G polymorphism and pregnancy disorders, and (v) the expression of newly-described HLA-G isoforms at the maternal-fetal interface.

Single-cell Immune Landscape of Human Recurrent Miscarriage

Successful pregnancy in placental mammals substantially depends on the establishment of maternal immune tolerance to the semi-allogenic fetus. Disorders in this process are tightly associated with adverse pregnancy outcomes including recurrent miscarriage (RM). However, an in-depth understanding of the systematic and decidual immune environment in RM remains largely lacking. In this study, we utilized single-cell RNA-sequencing (scRNA-seq) to comparably analyze the cellular and molecular signatures of decidual and peripheral leukocytes in normal and unexplained RM pregnancies at the early stage of gestation. Integrative analysis identifies 22 distinct cell clusters in total, and a dramatic difference in leukocyte subsets and molecular properties in RM cases is revealed. Specifically, the cytotoxic properties of CD8+ effector T cells, nature killer (NK), and mucosal-associated invariant T (MAIT) cells in peripheral blood indicates apparently enhanced pro-inflammatory status, and the population proportions and ligand–receptor interactions of the decidual leukocyte subsets demonstrate preferential immune activation in RM patients. The molecular features, spatial distribution, and the developmental trajectories of five decidual NK (dNK) subsets have been elaborately illustrated. In RM patients, a dNK subset that supports embryonic growth is diminished in proportion, while the ratio of another dNK subset with cytotoxic and immune-active signature is significantly increased. Notably, a unique pro-inflammatory CD56+CD16+ dNK subset substantially accumulates in RM decidua. These findings reveal a comprehensive cellular and molecular atlas of decidual and peripheral leukocytes in human early pregnancy and provide an in-depth insight into the immune pathogenesis for early pregnancy loss.

Single-cell RNA Sequencing Deciphers Immune Landscape of Human Recurrent Miscarriage

Single-cell RNA Sequencing Deciphers Immune Landscape of Human Recurrent Miscarriage