The CD94/NKG2A inhibitory receptor educates uterine NK cells to optimize pregnancy outcomes in humans and mice.

The conserved CD94/NKG2A inhibitory receptor is expressed by nearly all human and ∼50% of mouse uterine natural killer (uNK) cells. Binding human HLA-E and mouse Qa-1, NKG2A drives NK cell education, a process of unknown physiological importance influenced by HLA-B alleles. Here, we show that NKG2A genetic ablation in dams mated with wild-type males caused suboptimal maternal vascular responses in pregnancy, accompanied by perturbed placental gene expression, reduced fetal weight, greater rates of smaller fetuses with asymmetric growth, and abnormal brain development. These are features of the human syndrome pre-eclampsia. In a genome-wide association study of 7,219 pre-eclampsia cases, we found a 7% greater relative risk associated with the maternal HLA-B allele that does not favor NKG2A education. These results show that the maternal HLA-B→HLA-E→NKG2A pathway contributes to healthy pregnancy and may have repercussions on offspring health, thus establishing the physiological relevance for NK cell education. VIDEO ABSTRACT.

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.

Single-cell reconstruction of the early maternal-fetal interface in humans.

During early human pregnancy the uterine mucosa transforms into the decidua, into which the fetal placenta implants and where placental trophoblast cells intermingle and communicate with maternal cells. Trophoblast-decidual interactions underlie common diseases of pregnancy, including pre-eclampsia and stillbirth. Here we profile the transcriptomes of about 70,000 single cells from first-trimester placentas with matched maternal blood and decidual cells. The cellular composition of human decidua reveals subsets of perivascular and stromal cells that are located in distinct decidual layers. There are three major subsets of decidual natural killer cells that have distinctive immunomodulatory and chemokine profiles. We develop a repository of ligand-receptor complexes and a statistical tool to predict the cell-type specificity of cell-cell communication via these molecular interactions. Our data identify many regulatory interactions that prevent harmful innate or adaptive immune responses in this environment. Our single-cell atlas of the maternal-fetal interface reveals the cellular organization of the decidua and placenta, and the interactions that are critical for placentation and reproductive success.

High-Resolution Genetic and Phenotypic Analysis of KIR2DL1 Alleles and Their Association with Pre-Eclampsia.

Killer-cell Ig-like receptor (KIR) genes are inherited as haplotypes. They are expressed by NK cells and linked to outcomes of infectious diseases and pregnancy in humans. Understanding how genotype relates to phenotype is difficult because of the extensive diversity of the KIR family. Indeed, high-resolution KIR genotyping and phenotyping in single NK cells in the context of disease association is lacking. In this article, we describe a new method to separate NK cells expressing allotypes of the KIR2DL1 gene carried by the KIR A haplotype (KIR2DL1A) from those expressing KIR2DL1 alleles carried by the KIR B haplotype (KIR2DL1B). We find that in KIR AB heterozygous individuals, different KIR2DL1 allotypes can be detected in both peripheral blood and uterine NK cells. Using this new method, we demonstrate that both blood and uterine NK cells codominantly express KIR2DL1A and KIR2DL1B allotypes but with a predominance of KIR2DL1A variants, which associate with enhanced NK cell function. In a case-control study of pre-eclampsia, we show that KIR2DL1A, not KIR2DL1B, associates with increased disease risk. This method will facilitate our understanding of how individual KIR2DL1 allelic variants affect NK cell function and contribute to disease risk.

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).

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.

Effect of randomized serum progesterone concentration on secretory endometrial histologic development and gene expression.

STUDY QUESTION: What doses of secretory phase progesterone (P) in women are associated with altered endometrial structure and/or function? SUMMARY ANSWER: Consistently delayed histological maturation was seen at the lowest tested daily P dose (2.5 mg), whereas consistently altered functional response, as reflected by microarray analysis of gene expression was seen at both the 5 and 2.5 mg doses. WHAT IS KNOWN ALREADY: Progesterone is absolutely required for normal embryo implantation and pregnancy survival. Progesterone supplementation is beneficial in ART cycles. STUDY DESIGN, SIZE, DURATION: In this case-control experimental trial, 46 healthy young female volunteers (age 19-34) underwent a single modeled endometrial cycle after GnRH down-regulation or monitored in natural cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: In a university hospital, modeled cycles were obtained by GnRH agonist down-regulation, transdermal estradiol (E2) (0.2 mg/d), and daily injections of P in oil for 10 days: 2.5 mg (n = 6), 5 mg (n = 6), 10 mg (n = 12) or 40 mg (n = 12), after the 10th day of E2. Ten healthy, ovulatory women were used as controls. Endometrial biopsies were obtained on the 10th day of P exposure, or urinary LH surge (in controls). Analysis included histological dating, serum progesterone levels, microarray analysis of the whole genome, RT-PCR, western blot and comparison with the GEO database. MAIN RESULTS AND THE ROLE OF CHANCE: In endometrial biopsies, a morphological delay appears in the 2.5 mg/day of P group. Higher sub-physiological levels of P (≥5 mg/day) resulted in normal histology, but aberrant gene expression. P levels required for consistent histological delay were lower than those in all ovulatory women. Gene expression abnormalities occurred at higher sub-physiological P concentrations, without a change in histology, a functional-morphological disassociation. The expression of some endometrial receptivity-associated genes appeared multiphasic, with peak or nadir of mean or median expression levels between the lowest and highest doses, suggesting sustained supraphysiological doses seen in ART treatment cycles may not be optimal. LARGE SCALE DATA: GEO DataSets ID: 200056980; GSE 56980. LIMITATIONS, REASONS FOR CAUTION: These results were obtained in fertile women, who may respond differently from infertile subjects. WIDER IMPLICATIONS OF THE FINDINGS: The dose of P required for normal endometrial structure (5 mg/day) corresponds to a P concentration well below that seen in ovulatory women, suggesting that persistently delayed mid-secretory histology cannot be solely due to inadequate P concentrations in an ovulatory cycle. Endometrial gene expression is differentially regulated by different doses of progesterone. The apparent multiphasic response of some genes to P dose suggests the possibility that P concentration kinetics may play a role in normal endometrial preparation for receptivity. These findings strongly confirm that histologic development is not a reliable measure of endometrial P action. STUDY FUNDING/COMPETING INTEREST(S): Supported by The Eunice Kennedy Shriver National Institute for Child Health and Disease, National Institute of Health, USA (NICHD/NIH) (R01HD067721 and U54HD30476; SLY and BAL) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 240239/2012-1 (RFS). All authors have no competing interests.

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.

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.

DNA methylation mediated up-regulation of TERRA non-coding RNA is coincident with elongated telomeres in the human placenta.

STUDY QUESTION: What factors regulate elongated telomere length in the human placenta? SUMMARY ANSWER: Hypomethylation of TERRA promoters in the human placenta is associated with high TERRA expression, however, no clear mechanistic link between these phenomena and elongated telomere length in the human placenta was found. WHAT IS KNOWN ALREADY: Human placenta tissue and trophoblasts show longer telomere lengths compared to gestational age-matched somatic cells. However, telomerase (hTERT) expression and activity in the placenta is low, suggesting a role for an alternative lengthening of telomeres (ALT). While ALT is observed in 10-15% of human cancers and in some mouse stem cells, ALT has never been reported in non-cancerous human tissues. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Human term placental tissue and matched cord blood mononuclear cells (CBMCs) were collected as part of the Peri/Postnatal Epigenetic Twins study (PETS). In addition, first trimester placental villi, purified cytotrophoblasts, choriocarcinoma cell lines and a panel of ALT-positive cancer cell lines were tested. Telomere length was determined using the Terminal Restriction Fragment (TRF) assay and a relative quantitative PCR method. DNA methylation levels at several CpG rich subtelomeric TERRA promoters were determined using bisulfite conversion and the SEQUENOM EpiTYPER platform. Expression of TERRA and hTERT was determined using quantitative RT-PCR. ALT was assessed using the C-circle assay (CCA). MAIN RESULTS AND THE ROLE OF CHANCE: The human placenta tissue and purified first trimester trophoblasts showed low subtelomeric (TERRA) DNA methylation compared to matched CBMCs and other somatic cells. Interestingly placental TERRA methylation was lower than ALT-cancer cell lines, previously reported to be hypomethylated at these loci. Low TERRA methylation was associated with higher expression of TERRA RNA in placenta compared to matched CBMCs. Detectable levels of C-circles were observed in first trimester placental villi, but not term placenta, suggesting that the ALT mechanism may be active in specific placental cells in early gestation. C-circle analysis of purified first trimester trophoblasts and ALT-associated PML bodies (APB) staining of first trimester villi cross-sections failed to identify this specific cell type population. LIMITATIONS, REASONS FOR CAUTION: While first trimester villi showed detectable levels of C-circles, these levels were very low compared with those observed in ALT-positive tumours and cell lines. This is consistent with a small sub-population of ALT-positive cells but this requires further investigation. Finally, no mechanistic link was established between TERRA DNA methylation, the presence of C-circles and longer telomere length. WIDER IMPLICATIONS OF THE FINDINGS: Given the previously described role of TERRA ncRNA as a negative regulator of telomerase, the finding of elevated TERRA and long telomeres is counterintutive. ALT as a mechanism for telomere length maintenance has only been reported in certain human cancers, and recently in mouse embryonic stem cells and embryos. As with many aspects of cancer, it appears that ALT activity in tumours may be the inappropriate activation of a pathway found in very specific cell types in human development. Our data are the first supportive evidence for ALT in a non-cancerous human tissue, a result that requires further investigation and replication. The level of TERRA methylation in the human placenta is significantly lower than found in ALT cancer cell lines and somatic cells, raising the possibility of a novel mechanism in maintaining low methylation at subtelomeric regions. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by NHMRC early career fellowship (B.N.), NHMRC Senior Research Fellowship (R.S.) and the Victoria Government Infrastructure Grant. R.R. holds a patent for the C-circle assay. No other conflicts declared.

Kir6.1, a component of an ATP-sensitive potassium channel, regulates natural killer cell development.

Involved in immunity and reproduction, natural killer (NK) cells offer opportunities to develop new immunotherapies to treat infections and cancer or to alleviate pregnancy complications. Most current strategies use cytokines or antibodies to enhance NK-cell function, but none use ion channel modulators, which are widely used in clinical practice to treat hypertension, diabetes, epilepsy, and other conditions. Little is known about ion channels in NK cells. We show that Kcnj8, which codes for the Kir6.1 subunit of a certain type of ATP-sensitive potassium (K ATP ) channel, is highly expressed in murine splenic and uterine NK cells compared to other K + channels previously identified in NK cells. Kcnj8 expression is highest in the most mature subset of splenic NK cells (CD27 - CD11b + ) and in NKG2A + or Ly49C/I + educated uterine NK cells. Using patch clamping, we show that a subset of NK cells expresses a current sensitive to the Kir6.1 blocker PNU-37883A. Kcnj8 does not participate in NK cell degranulation in response to tumor cells in vitro or rejection of tumor cells in vivo . Transcriptomics show that genes previously implicated in NK cell development are amongst those differentially expressed in CD27 - CD11b + NK cells deficient of Kcnj8 . Indeed, we found that mice with NK-cell specific Kcnj8 gene ablation have fewer CD11b + CD27 - and KLRG-1 + NK cells in the bone barrow and spleen. These results show that the K ATP subunit Kir6.1 has a key role in NK-cell development.

Human uterine natural killer cells regulate differentiation of extravillous trophoblast early in pregnancy.

In humans, balanced invasion of trophoblast cells into the uterine mucosa, the decidua, is critical for successful pregnancy. Evidence suggests that this process is regulated by uterine natural killer (uNK) cells, but how they influence reproductive outcomes is unclear. Here, we used our trophoblast organoids and primary tissue samples to determine how uNK cells affect placentation. By locating potential interaction axes between trophoblast and uNK cells using single-cell transcriptomics and in vitro modeling of these interactions in organoids, we identify a uNK cell-derived cytokine signal that promotes trophoblast differentiation at the late stage of the invasive pathway. Moreover, it affects transcriptional programs involved in regulating blood flow, nutrients, and inflammatory and adaptive immune responses, as well as gene signatures associated with disorders of pregnancy such as pre-eclampsia. Our findings suggest mechanisms on how optimal immunological interactions between uNK cells and trophoblast enhance reproductive success.

The science of implantation emerges blinking into the light.

Although embryo implantation is essential for human survival, it remains an enigmatic biological phenomenon. Following fertilization, the resulting blastocyst must signal its presence to the mother, attach to the luminal epithelium of the endometrium and embed into the decidualising stroma. Failure to do so results in infertility, which affects around 9% of women. Subsequent placental development requires remodelling of maternal blood vessels by trophoblast cells from the placenta, that invade deep into the decidua. Failure in these very early stages can compromise fetal development, resulting in diseases of pregnancy such as intrauterine growth restriction or pre-eclampsia which can also impact on health in adulthood. Abnormal implantation therefore constitutes a significant disease burden in humans. Although we have known for many years that successful implantation requires an embryo that is competent to implant and an endometrium that is receptive, the molecular basis of these processes remains poorly understood. Our inability to identify implantation-competent embryos or to diagnose/treat the non-receptive endometrium therefore limits our ability to intervene through assisted reproduction techniques. This Implantation Symposium aims to review recent exciting developments in our understanding of the biology of early implantation and to highlight the rapid progress being made to translate these into improved diagnosis and treatment.

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.

The effect of pregnancy on the uterine NK cell KIR repertoire.

The major leukocyte population in the decidua during the first trimester of pregnancy consists of NK cells that express receptors capable of recognizing MHC class I molecules expressed by placental trophoblast. These include members of the killer immunoglobulin-like receptor (KIR) family, the two-domain KIR (KIR2D), which recognize HLA-C. Interactions between decidual NK (dNK) cell KIR2D and placental HLA-C contribute to the success of pregnancy and dNK cells express KIR2D at higher frequency than peripheral NK (pNK) cells. Thus, they are biased toward recognizing HLA-C. In order to investigate when this unusual KIR repertoire appears, we compared the phenotype of NK cells isolated from non-pregnant (endometrium) and pregnant (decidua) human uterine mucosa. Endometrial NK (eNK) cells did not express KIR2D at a higher level than matched pNK cells, so the bias toward HLA-C recognition occurs as a response to pregnancy. Furthermore, HLA-C expression was upregulated on uterine stromal cells as the mucosa transformed from endometrium to decidua at the onset of pregnancy. As uterine NK (uNK) cells can mature from NK precursors and acquire KIR expression in utero, the pregnancy-specific bias of uNK cells toward HLA-C recognition could arise as developing uNK cells interact with uterine stromal cells, which express higher levels of HLA-C during pregnancy.

Chemokine scavenger D6 is expressed by trophoblasts and aids the survival of mouse embryos transferred into allogeneic recipients.

Proinflammatory CC chemokines are thought to drive recruitment of maternal leukocytes into gestational tissues and regulate extravillous trophoblast migration. The atypical chemokine receptor D6 binds many of these chemokines and is highly expressed by the human placenta. D6 is thought to act as a chemokine scavenger because, when ectopically expressed in cell lines in vitro, it efficiently internalizes proinflammatory CC chemokines and targets them for destruction in the absence of detectable chemokine-induced signaling. Moreover, D6 suppresses inflammation in many mouse tissues, and notably, D6-deficient fetuses in D6-deficient female mice show increased susceptibility to inflammation-driven resorption. In this paper, we report strong anti-D6 immunoreactivity, with specific intracellular distribution patterns, in trophoblast-derived cells in human placenta, decidua, and gestational membranes throughout pregnancy and in trophoblast disease states of hydatidiform mole and choriocarcinoma. We show, for the first time, that endogenous D6 in a human choriocarcinoma-derived cell line can mediate progressive chemokine scavenging and that the D6 ligand CCL2 can specifically associate with human syncytiotrophoblasts in term placenta in situ. Moreover, despite strong chemokine production by gestational tissues, levels of D6-binding chemokines in maternal plasma decrease during pregnancy, even in women with pre-eclampsia, a disease associated with increased maternal inflammation. In mice, D6 is not required for syngeneic or semiallogeneic fetal survival in unchallenged mice, but interestingly, it does suppress fetal resorption after embryo transfer into fully allogeneic recipients. These data support the view that trophoblast D6 scavenges maternal chemokines at the fetomaternal interface and that, in some circumstances, this can help to ensure fetal survival.

Beyond Maternal Tolerance: Education of Uterine Natural Killer Cells by Maternal MHC Drives Fetal Growth.

Reproductive immunology has moved on from the classical Medawar question of 60 years ago "why doesn't the mother reject the fetus?". Looking beyond fetal-maternal tolerance, modern reproductive immunology focuses on how the maternal immune system supports fetal growth. Maternal uterine natural killer (uNK) cells, in partnership with fetal trophoblast cells, regulate physiological vascular changes in the uterus of pregnant women and mice. These vascular changes are necessary to build the placenta and sustain fetal growth. NK cell functions in the uterus and elsewhere, including anti-viral and anti-tumour immunity mediated mostly by blood NK cells, are modulated by NK cell education, a quantifiable process that determines cellular activation thresholds. This process relies largely on interactions between self-MHC class I molecules and inhibitory NK cell receptors. By getting to know self, the maternal immune system sets up uNK cells to participate to tissue homeostasis in the womb. Placentation can be viewed as a form of natural transplantation unique in vertebrates and this raises the question of how uNK cell education or missing-self recognition affect their function and, ultimately fetal growth. Here, using combinations of MHC-sufficient and -deficient mice, we show that uNK cell education is linked to maternal and not fetal MHC, so that MHC-deficient dams produce more growth-restricted fetuses, even when the fetuses themselves express self-MHC. We also show that, while peripheral NK cells reject bone marrow cells according to the established rules of missing-self recognition, uNK cells educated by maternal MHC do not reject fetuses that miss self-MHC and these fetuses grow to their full potential. While these results are not directly applicable to clinical research, they show that NK education by maternal MHC-I is required for optimal fetal growth.

Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors.

BACKGROUND: The human placenta facilitates the exchange of nutrients, gas and waste between the fetal and maternal circulations. It also protects the fetus from the maternal immune response. Due to its role at the feto-maternal interface, the placenta is subject to many environmental exposures that can potentially alter its epigenetic profile. Previous studies have reported gene expression differences in placenta over gestation, as well as inter-individual variation in expression of some genes. However, the factors contributing to this variation in gene expression remain poorly understood. RESULTS: In this study, we performed a genome-wide DNA methylation analysis of gene promoters in placenta tissue from three pregnancy trimesters. We identified large-scale differences in DNA methylation levels between first, second and third trimesters, with an overall progressive increase in average methylation from first to third trimester. The most differentially methylated genes included many immune regulators, reflecting the change in placental immuno-modulation as pregnancy progresses. We also detected increased inter-individual variation in the third trimester relative to first and second, supporting an accumulation of environmentally induced (or stochastic) changes in DNA methylation pattern. These highly variable genes were enriched for those involved in amino acid and other metabolic pathways, potentially reflecting the adaptation of the human placenta to different environments. CONCLUSIONS: The identification of cellular pathways subject to drift in response to environmental influences provide a basis for future studies examining the role of specific environmental factors on DNA methylation pattern and placenta-associated adverse pregnancy outcomes.

Ex vivo functional responses to HLA-G differ between blood and decidual NK cells.

Restricted expression of human leucocyte antigen-G (HLA-G) to fetal extravillous trophoblast cells, which invade the decidua during implantation, suggests a role for HLA-G in placentation. In this study, we have investigated several aspects of HLA-G expression and function. Surface levels of HLA-G expression were measured in 70 normal pregnancies. We show the dimeric conformation that is unique to HLA-G forms after passage through the Golgi apparatus. Differences were found in the receptor repertoire of decidual natural killer (dNK) cells that express the leucocyte immunoglobulin-like receptor B1 (LILRB1), which binds dimeric HLA-G strongly. We then measured functional responses of dNK cells with LILRB1, when stimulated by HLA-G in both monomeric and dimeric conformations. Degranulation, interferon-γ and interleukin-8 production by dNK cells freshly isolated from the first trimester implantation site were either undetected or not affected by HLA-G. These findings should be considered when inferring the activity of tissue NK cells from results obtained with cell lines, peripheral NK or cultured dNK cells.