Primitive haematopoiesis in the human placenta gives rise to macrophages with epigenetically silenced HLA-DR.

The earliest macrophages are generated during embryonic development from erythro-myeloid progenitors (EMPs) via primitive haematopoiesis. Although this process is thought to be spatially restricted to the yolk sac in the mouse, in humans, it remains poorly understood. Human foetal placental macrophages, or Hofbauer cells (HBC), arise during the primitive haematopoietic wave ~18 days post conception and lack expression of human leukocyte antigen (HLA) class II. Here, we identify a population of placental erythro-myeloid progenitors (PEMPs) in the early human placenta that have conserved features of primitive yolk sac EMPs, including the lack of HLF expression. Using in vitro culture experiments we demonstrate that PEMP generate HBC-like cells lacking HLA-DR expression. We find the absence of HLA-DR in primitive macrophages is mediated via epigenetic silencing of class II transactivator, CIITA, the master regulator of HLA class II gene expression. These findings establish the human placenta as an additional site of primitive haematopoiesis.

Single-cell roadmap of human gonadal development.

Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.

Mapping the temporal and spatial dynamics of the human endometrium in vivo and in vitro.

The endometrium, the mucosal lining of the uterus, undergoes dynamic changes throughout the menstrual cycle in response to ovarian hormones. We have generated dense single-cell and spatial reference maps of the human uterus and three-dimensional endometrial organoid cultures. We dissect the signaling pathways that determine cell fate of the epithelial lineages in the lumenal and glandular microenvironments. Our benchmark of the endometrial organoids reveals the pathways and cell states regulating differentiation of the secretory and ciliated lineages both in vivo and in vitro. In vitro downregulation of WNT or NOTCH pathways increases the differentiation efficiency along the secretory and ciliated lineages, respectively. We utilize our cellular maps to deconvolute bulk data from endometrial cancers and endometriotic lesions, illuminating the cell types dominating in each of these disorders. These mechanistic insights provide a platform for future development of treatments for common conditions including endometriosis and endometrial carcinoma.

How Do Uterine Natural Killer and Innate Lymphoid Cells Contribute to Successful Pregnancy?

Innate lymphoid cells (ILCs) are the most abundant immune cells in the uterine mucosa both before and during pregnancy. Circumstantial evidence suggests they play important roles in regulating placental development but exactly how they contribute to the successful outcome of pregnancy is still unclear. Uterine ILCs (uILCs) include subsets of tissue-resident natural killer (NK) cells and ILCs, and until recently the phenotype and functions of uILCs were poorly defined. Determining the specific roles of each subset is intrinsically challenging because of the rapidly changing nature of the tissue both during the menstrual cycle and pregnancy. Single-cell RNA sequencing (scRNAseq) and high dimensional flow and mass cytometry approaches have recently been used to analyse uILC populations in the uterus in both humans and mice. This detailed characterisation has significantly changed our understanding of the heterogeneity within the uILC compartment. It will also enable key clinical questions to be addressed including whether specific uILC subsets are altered in infertility, miscarriage and pregnancy disorders such as foetal growth restriction and pre-eclampsia. Here, we summarise recent advances in our understanding of the phenotypic and functional diversity of uILCs in non-pregnant endometrium and first trimester decidua, and review how these cells may contribute to successful placental development.

BAP1/ASXL complex modulation regulates epithelial-mesenchymal transition during trophoblast differentiation and invasion.

Normal function of the placenta depends on the earliest developmental stages when trophoblast cells differentiate and invade into the endometrium to establish the definitive maternal-fetal interface. Previously, we identified the ubiquitously expressed tumour suppressor BRCA1-associated protein 1 (BAP1) as a central factor of a novel molecular node controlling early mouse placentation. However, functional insights into how BAP1 regulates trophoblast biology are still missing. Using CRISPR/Cas9 knockout and overexpression technology in mouse trophoblast stem cells, here we demonstrate that the downregulation of BAP1 protein is essential to trigger epithelial-mesenchymal transition (EMT) during trophoblast differentiation associated with a gain of invasiveness. Moreover, we show that the function of BAP1 in suppressing EMT progression is dependent on the binding of BAP1 to additional sex comb-like (ASXL1/2) proteins to form the polycomb repressive deubiquitinase (PR-DUB) complex. Finally, both endogenous expression patterns and BAP1 overexpression experiments in human trophoblast stem cells suggest that the molecular function of BAP1 in regulating trophoblast differentiation and EMT progression is conserved in mice and humans. Our results reveal that the physiological modulation of BAP1 determines the invasive properties of the trophoblast, delineating a new role of the BAP1 PR-DUB complex in regulating early placentation.

Menstrual flow as a non-invasive source of endometrial organoids.

Assessment of the endometrium often necessitates a biopsy, which currently involves an invasive, transcervical procedure. Here, we present an alternative technique based on deriving organoids from menstrual flow. We demonstrate that organoids can be derived from gland fragments recovered from menstrual flow. To confirm they faithfully reflect the in vivo state we compared organoids derived from paired scratch biopsies and ensuing menstrual flow from patients undergoing in vitro fertilisation (IVF). We demonstrate that the two sets of organoids share the same transcriptome signature, derivation efficiency and proliferation rate. Furthermore, they respond similarly to sex steroids and early-pregnancy hormones, with changes in morphology, receptor expression, and production of 'uterine milk' proteins that mimic those during the late-secretory phase and early pregnancy. This technique has wide-ranging impact for non-invasive investigation and personalised approaches to treatment of common gynaecological conditions, such as endometriosis, and reproductive disorders, including failed implantation after IVF and recurrent miscarriage.

Phenotypic and functional characterization of first-trimester human placental macrophages, Hofbauer cells.

Hofbauer cells (HBCs) are a population of macrophages found in high abundance within the stroma of the first-trimester human placenta. HBCs are the only fetal immune cell population within the stroma of healthy placenta. However, the functional properties of these cells are poorly described. Aligning with their predicted origin via primitive hematopoiesis, we find that HBCs are transcriptionally similar to yolk sac macrophages. Phenotypically, HBCs can be identified as HLA-DR-FOLR2+ macrophages. We identify a number of factors that HBCs secrete (including OPN and MMP-9) that could affect placental angiogenesis and remodeling. We determine that HBCs have the capacity to play a defensive role, where they are responsive to Toll-like receptor stimulation and are microbicidal. Finally, we also identify a population of placenta-associated maternal macrophages (PAMM1a) that adhere to the placental surface and express factors, such as fibronectin, that may aid in repair.

Genetic predisposition to hypertension is associated with preeclampsia in European and Central Asian women.

Preeclampsia is a serious complication of pregnancy, affecting both maternal and fetal health. In genome-wide association meta-analysis of European and Central Asian mothers, we identify sequence variants that associate with preeclampsia in the maternal genome at ZNF831/20q13 and FTO/16q12. These are previously established variants for blood pressure (BP) and the FTO variant has also been associated with body mass index (BMI). Further analysis of BP variants establishes that variants at MECOM/3q26, FGF5/4q21 and SH2B3/12q24 also associate with preeclampsia through the maternal genome. We further show that a polygenic risk score for hypertension associates with preeclampsia. However, comparison with gestational hypertension indicates that additional factors modify the risk of preeclampsia.

Investigation of human trophoblast invasion in vitro.

BACKGROUND: In humans, inadequate trophoblast invasion into the decidua is associated with the 'great obstetrical syndromes' which include pre-eclampsia, foetal growth restriction (FGR) and stillbirth. The mechanisms regulating invasion remain poorly understood, although interactions with the uterine environment are clearly of central importance. Extravillous trophoblast (EVT) cells invade the uterus and transform the spiral arteries. Progress in understanding how they invade has been limited due to the lack of good in vitro models. Firstly, there are no non-malignant cell lines that have an EVT phenotype. Secondly, the invasion assays used are of limited use for the small numbers of primary EVT available from first-trimester placentas. We discuss recent progress in this field with the generation of new EVT lines and invasion assays using microfluidic technology. OBJECTIVE AND RATIONALE: Our aim is to describe the established models used to study human trophoblast invasion in vivo and in vitro. The difficulties of obtaining primary cells and cell lines that recapitulate the phenotype of EVT are discussed together with the advantages and pitfalls of the different invasion assays. We compare these traditional end point assays to microfluidic assays where the dynamics of migration can be measured. SEARCH METHODS: Relevant studies were identified by PubMed search, last updated on February 2020. A search was conducted to determine the number of journal articles published using the cell lines JEG-3, BeWo, JAR, HTR-8/Svneo, Swan-71 and primary human extravillous trophoblast in the last 5 years. OUTCOMES: Deep trophoblast invasion into the maternal decidua is a particular feature of human pregnancy. This invasion needs to be finely regulated to allocate resources between mother and baby. A reliable source of EVT is needed to study in vitro how the uterine environment regulates this process. First, we critically discuss the issues with the trophoblast cell lines currently used; for example, most of them lack expression of the defining marker of EVT, HLA-G. Recently, advances in human stem cell and organoid technology have been applied to extraembryonic tissues to develop trophoblast cell lines that can grow in two (2D) and three dimensions (3D) and differentiate to EVT. This means that the 'trophoblast' cell lines currently in use should rapidly become obsolete. Second, we critically discuss the problems with assays to study trophoblast invasion. These lack physiological relevance and have simplified migration dynamics. Microfluidic assays are a powerful tool to study cell invasion because they require only a few cells, which are embedded in 3D in an extracellular matrix. Their major advantage is real-time monitoring of cell movement, enabling detailed analysis of the dynamics of trophoblast migration. WIDER IMPLICATIONS: Trophoblast invasion in the first trimester of pregnancy remains poorly understood despite the importance of this process in the pathogenesis of pre-eclampsia, FGR, stillbirth and recurrent miscarriage. The new technologies described here will allow investigation into this critical process.

In vitro fertilization add-ons for the endometrium: it doesn't add-up.

The probability of live birth from an in vitro fertilization (IVF) cycle is modest. Many additional treatments (add-ons) are available which promise to improve the success of IVF. This review summarizes the current evidence for common IVF add-ons which are suggested to improve endometrial receptivity. Systematic reviews of randomized controlled trials and individual trials were included. Five add-ons were included: immune therapies, endometrial scratching, endometrial receptivity array, uterine artery vasodilation, and human chorionic gonadotropin instillation. The results suggest there is no robust evidence that these add-ons are effective or safe. Many IVF add-ons are costly, consuming precious resources which may be better spent on evidence-based treatments or further IVF. Large randomized controlled trials and appropriate safety assessment should be mandatory before the introduction of IVF add-ons into routine practice.

Development of the human placenta.

The placenta is essential for normal in utero development in mammals. In humans, defective placental formation underpins common pregnancy disorders such as pre-eclampsia and fetal growth restriction. The great variation in placental types across mammals means that animal models have been of limited use in understanding human placental development. However, new tools for studying human placental development, including 3D organoids, stem cell culture systems and single cell RNA sequencing, have brought new insights into this field. Here, we review the morphological, molecular and functional aspects of human placental formation, with a focus on the defining cell of the placenta - the trophoblast.

Tissue stiffness at the human maternal-fetal interface.

STUDY QUESTION: What is the stiffness (elastic modulus) of human nonpregnant secretory phase endometrium, first trimester decidua, and placenta? SUMMARY ANSWER: The stiffness of decidua basalis, the site of placental invasion, was an order of magnitude higher at 103 Pa compared to 102 Pa for decidua parietalis, nonpregnant endometrium and placenta. WHAT IS KNOWN ALREADY: Mechanical forces have profound effects on cell behavior, regulating both cell differentiation and migration. Despite their importance, very little is known about their effects on blastocyst implantation and trophoblast migration during placental development because of the lack of mechanical characterization at the human maternal-fetal interface. STUDY DESIGN, SIZE, DURATION: An observational study was conducted to measure the stiffness of ex vivo samples of human nonpregnant secretory endometrium (N = 5) and first trimester decidua basalis (N = 6), decidua parietalis (N = 5), and placenta (N = 5). The stiffness of the artificial extracellular matrix (ECM), Matrigel®, commonly used to study migration of extravillous trophoblast (EVT) in three dimensions and to culture endometrial and placental organoids, was also determined (N = 5). PARTICIPANTS/MATERIALS, SETTING, METHODS: Atomic force microscopy was used to perform ex vivo direct measurements to determine the stiffness of fresh tissue samples. Decidua was stained by immunohistochemistry (IHC) for HLA-G+ EVT to confirm whether samples were decidua basalis or decidua parietalis. Endometrium was stained with hematoxylin and eosin to confirm the presence of luminal epithelium. Single-cell RNA sequencing data were analyzed to determine expression of ECM transcripts by decidual and placental cells. Fibrillin 1, a protein identified by these data, was stained by IHC in decidua basalis. MAIN RESULTS AND THE ROLE OF CHANCE: We observed that decidua basalis was significantly stiffer than decidua parietalis, at 1250 and 171 Pa, respectively (P < 0.05). The stiffness of decidua parietalis was similar to nonpregnant endometrium and placental tissue (250 and 232 Pa, respectively). These findings suggest that it is the presence of invading EVT that is driving the increase in stiffness in decidua basalis. The stiffness of Matrigel® was found to be 331 Pa, significantly lower than decidua basalis (P < 0.05). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Tissue stiffness was derived by ex vivo measurements on blocks of fresh tissue in the absence of blood flow. The nonpregnant endometrium samples were obtained from women undergoing treatment for infertility. These may not reflect the stiffness of endometrium from normal fertile women. WIDER IMPLICATIONS OF THE FINDINGS: These results provide direct measurements of tissue stiffness during the window of implantation and first trimester of human pregnancy. They serve as a basis of future studies exploring the impact of mechanics on embryo implantation and development of the placenta. The findings provide important baseline data to inform matrix stiffness requirements when developing in vitro models of trophoblast stem cell development and migration that more closely resemble the decidua in vivo. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Centre for Trophoblast Research, the Wellcome Trust (090108/Z/09/Z, 085992/Z/08/Z), the Medical Research Council (MR/P001092/1), the European Research Council (772426), an Engineering and Physical Sciences Research Council Doctoral Training Award (1354760), a UK Medical Research Council and Sackler Foundation Doctoral Training Grant (RG70550) and a Wellcome Trust Doctoral Studentship (215226/Z/19/Z).

Trophoblast organoids as a model for maternal-fetal interactions during human placentation.

The placenta is the extraembryonic organ that supports the fetus during intrauterine life. Although placental dysfunction results in major disorders of pregnancy with immediate and lifelong consequences for the mother and child, our knowledge of the human placenta is limited owing to a lack of functional experimental models1. After implantation, the trophectoderm of the blastocyst rapidly proliferates and generates the trophoblast, the unique cell type of the placenta. In vivo, proliferative villous cytotrophoblast cells differentiate into two main sub-populations: syncytiotrophoblast, the multinucleated epithelium of the villi responsible for nutrient exchange and hormone production, and extravillous trophoblast cells, which anchor the placenta to the maternal decidua and transform the maternal spiral arteries2. Here we describe the generation of long-term, genetically stable organoid cultures of trophoblast that can differentiate into both syncytiotrophoblast and extravillous trophoblast. We used human leukocyte antigen (HLA) typing to confirm that the organoids were derived from the fetus, and verified their identities against four trophoblast-specific criteria3. The cultures organize into villous-like structures, and we detected the secretion of placental-specific peptides and hormones, including human chorionic gonadotropin (hCG), growth differentiation factor 15 (GDF15) and pregnancy-specific glycoprotein (PSG) by mass spectrometry. The organoids also differentiate into HLA-G+ extravillous trophoblast cells, which vigorously invade in three-dimensional cultures. Analysis of the methylome reveals that the organoids closely resemble normal first trimester placentas. This organoid model will be transformative for studying human placental development and for investigating trophoblast interactions with the local and systemic maternal environment.

Variants in the fetal genome near FLT1 are associated with risk of preeclampsia.

Preeclampsia, which affects approximately 5% of pregnancies, is a leading cause of maternal and perinatal death. The causes of preeclampsia remain unclear, but there is evidence for inherited susceptibility. Genome-wide association studies (GWAS) have not identified maternal sequence variants of genome-wide significance that replicate in independent data sets. We report the first GWAS of offspring from preeclamptic pregnancies and discovery of the first genome-wide significant susceptibility locus (rs4769613; P = 5.4 × 10-11) in 4,380 cases and 310,238 controls. This locus is near the FLT1 gene encoding Fms-like tyrosine kinase 1, providing biological support, as a placental isoform of this protein (sFlt-1) is implicated in the pathology of preeclampsia. The association was strongest in offspring from pregnancies in which preeclampsia developed during late gestation and offspring birth weights exceeded the tenth centile. An additional nearby variant, rs12050029, associated with preeclampsia independently of rs4769613. The newly discovered locus may enhance understanding of the pathophysiology of preeclampsia and its subtypes.

A microfluidics assay to study invasion of human placental trophoblast cells.

Pre-eclampsia, fetal growth restriction and stillbirth are major pregnancy disorders throughout the world. The underlying pathogenesis of these diseases is defective placentation characterized by inadequate invasion of extravillous placental trophoblast cells into the uterine arteries. How trophoblast invasion is controlled remains an unanswered question but is influenced by maternal uterine immune cells called decidual natural killer cells. Here, we describe an in vitro microfluidic invasion assay to study the migration of primary human trophoblast cells. Each experiment can be performed with a small number of cells making it possible to conduct research on human samples despite the challenges of isolating primary trophoblast cells. Cells are exposed to a chemical gradient and tracked in a three-dimensional microenvironment using real-time high-resolution imaging, so that dynamic readouts on cell migration such as directionality, motility and velocity are obtained. The microfluidic system was validated using isolated trophoblast and a gradient of granulocyte-macrophage colony-stimulating factor, a cytokine produced by activated decidual natural killer cells. This microfluidic model provides detailed analysis of the dynamics of trophoblast migration compared to previous assays and can be modified in future to study in vitro how human trophoblast behaves during placentation.

Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium.

In humans, the endometrium, the uterine mucosal lining, undergoes dynamic changes throughout the menstrual cycle and pregnancy. Despite the importance of the endometrium as the site of implantation and nutritional support for the conceptus, there are no long-term culture systems that recapitulate endometrial function in vitro. We adapted conditions used to establish human adult stem-cell-derived organoid cultures to generate three-dimensional cultures of normal and decidualized human endometrium. These organoids expand long-term, are genetically stable and differentiate following treatment with reproductive hormones. Single cells from both endometrium and decidua can generate a fully functional organoid. Transcript analysis confirmed great similarity between organoids and the primary tissue of origin. On exposure to pregnancy signals, endometrial organoids develop characteristics of early pregnancy. We also derived organoids from malignant endometrium, and so provide a foundation to study common diseases, such as endometriosis and endometrial cancer, as well as the physiology of early gestation.

Activating KIR2DS4 Is Expressed by Uterine NK Cells and Contributes to Successful Pregnancy.

Tissue-specific NK cells are abundant in the pregnant uterus and interact with invading placental trophoblast cells that transform the maternal arteries to increase the fetoplacental blood supply. Genetic case-control studies have implicated killer cell Ig-like receptor (KIR) genes and their HLA ligands in pregnancy disorders characterized by failure of trophoblast arterial transformation. Activating KIR2DS1 or KIR2DS5 (when located in the centromeric region as in Africans) lower the risk of disorders when there is a fetal HLA-C allele carrying a C2 epitope. In this study, we investigated another activating KIR, KIR2DS4, and provide genetic evidence for a similar effect when carried with KIR2DS1 KIR2DS4 is expressed by ∼45% of uterine NK (uNK) cells. Similarly to KIR2DS1, triggering of KIR2DS4 on uNK cells led to secretion of GM-CSF and other chemokines, known to promote placental trophoblast invasion. Additionally, XCL1 and CCL1, identified in a screen of 120 different cytokines, were consistently secreted upon activation of KIR2DS4 on uNK cells. Inhibitory KIR2DL5A, carried in linkage disequilibrium with KIR2DS1, is expressed by peripheral blood NK cells but not by uNK cells, highlighting the unique phenotype of uNK cells compared with peripheral blood NK cells. That KIR2DS4, KIR2DS1, and some alleles of KIR2DS5 contribute to successful pregnancy suggests that activation of uNK cells by KIR binding to HLA-C is a generic mechanism promoting trophoblast invasion into the decidua.

What Is Trophoblast? A Combination of Criteria Define Human First-Trimester Trophoblast.

Controversy surrounds reports describing the derivation of human trophoblast cells from placentas and embryonic stem cells (ESC), partly due to the difficulty in identifying markers that define cells as belonging to the trophoblast lineage. We have selected criteria that are characteristic of primary first-trimester trophoblast: a set of protein markers, HLA class I profile, methylation of ELF5, and expression of microRNAs (miRNAs) from the chromosome 19 miRNA cluster (C19MC). We tested these criteria on cells previously reported to show some phenotypic characteristics of trophoblast: bone morphogenetic protein (BMP)-treated human ESC and 2102Ep, an embryonal carcinoma cell line. Both cell types only show some, but not all, of the four trophoblast criteria. Thus, BMP-treated human ESC have not fully differentiated to trophoblast. Our study identifies a robust panel, including both protein and non-protein-coding markers that, in combination, can be used to reliably define cells as characteristic of early trophoblast.

Composition, Development, and Function of Uterine Innate Lymphoid Cells.

Innate lymphoid cells (ILCs), including NK cells, contribute to barrier immunity and tissue homeostasis. In addition to the role of uterine NK cells in placentation and fetal growth, other uterine ILCs (uILCs) are likely to play roles in uterine physiology and pathology. In this article, we report on the composition of uILCs in the endometrium during the luteal phase and in the decidua during early pregnancy. Whereas nonkiller uILC1s and uILC2s are barely detectable in mouse and not detected in humans, a sizeable population of uILC3s is found in human endometrium and decidua, which are mostly NCR(+) and partially overlap with previously described IL-22-producing uterine NK cells. Development of mouse uILC3 is Nfil3 independent, suggesting unique features of uILCs. Indeed, although the cytokine production profile of mouse uILCs recapitulates that described in other tissues, IL-5, IL-17, and IL-22 are constitutively produced by uILC2s and uILC3s. This study lays the foundation to understand how ILCs function in the specialized uterine mucosa, both in tissue homeostasis and barrier immunity and during pregnancy.

Tissue-Specific Education of Decidual NK Cells.

During human pregnancy, fetal trophoblast cells invade the decidua and remodel maternal spiral arteries to establish adequate nutrition during gestation. Tissue NK cells in the decidua (dNK) express inhibitory NK receptors (iNKR) that recognize allogeneic HLA-C molecules on trophoblast. Where this results in excessive dNK inhibition, the risk of pre-eclampsia or growth restriction is increased. However, the role of maternal, self-HLA-C in regulating dNK responsiveness is unknown. We investigated how the expression and function of five iNKR in dNK is influenced by maternal HLA-C. In dNK isolated from women who have HLA-C alleles that carry a C2 epitope, there is decreased expression frequency of the cognate receptor, KIR2DL1. In contrast, women with HLA-C alleles bearing a C1 epitope have increased frequency of the corresponding receptor, KIR2DL3. Maternal HLA-C had no significant effect on KIR2DL1 or KIR2DL3 in peripheral blood NK cells (pbNK). This resulted in a very different KIR repertoire for dNK capable of binding C1 or C2 epitopes compared with pbNK. We also show that, although maternal KIR2DL1 binding to C2 epitope educates dNK cells to acquire functional competence, the effects of other iNKR on dNK responsiveness are quite different from those in pbNK. This provides a basis for understanding how dNK responses to allogeneic trophoblast affect the outcome of pregnancy. Our findings suggest that the mechanisms that determine the repertoire of iNKR and the effect of self-MHC on NK education may differ in tissue NK cells compared with pbNK.