Decoding human fetal liver haematopoiesis.

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, natural killer and innate lymphoid cell precursors in the yolk sac. We demonstrate a shift in the haemopoietic composition of fetal liver during gestation away from being predominantly erythroid, accompanied by a parallel change in differentiation potential of HSC/MPPs, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a reference for harnessing the therapeutic potential of HSC/MPPs.

Anti-Cholestatic Therapy with Obeticholic Acid Improves Short-Term Memory in Bile Duct-Ligated Mice.

Patients with cholestatic liver disease, including those with primary biliary cholangitis, can experience symptoms of impaired cognition or brain fog. This phenomenon remains unexplained and is currently untreatable. Bile duct ligation (BDL) is an established rodent model of cholestasis. In addition to liver changes, BDL animals develop cognitive symptoms early in the disease process (before development of cirrhosis and/or liver failure). The cellular mechanisms underpinning these cognitive symptoms are poorly understood. Herein, the study explored the neurocognitive symptom manifestations, and tested potential therapies, in BDL mice, and used human neuronal cell cultures to explore translatability to humans. BDL animals exhibited short-term memory loss and showed reduced astrocyte coverage of the blood-brain barrier, destabilized hippocampal network activity, and neuronal senescence. Ursodeoxycholic acid (first-line therapy for most human cholestatic diseases) did not reverse symptomatic or mechanistic aspects. In contrast, obeticholic acid (OCA), a farnesoid X receptor agonist and second-line anti-cholestatic agent, normalized memory function, suppressed blood-brain barrier changes, prevented hippocampal network deficits, and reversed neuronal senescence. Co-culture of human neuronal cells with either BDL or human cholestatic patient serum induced cellular senescence and increased mitochondrial respiration, changes that were limited again by OCA. These findings provide new insights into the mechanism of cognitive symptoms in BDL animals, suggesting that OCA therapy or farnesoid X receptor agonism could be used to limit cholestasis-induced neuronal senescence.

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.

Platelet-derived growth factor BB is reduced in endometrial endothelial cells of women with abnormal uterine bleeding-endometrial disorder.

RESEARCH QUESTION: What is the expression pattern of platelet-derived growth factor BB (PDGF-BB), and its receptors, across the menstrual cycle in healthy control women and those with abnormal uterine bleeding-endometrial disorder (AUB-E)? DESIGN: Immunohistochemical staining for PDGF-BB, platelet-derived growth factor receptor alpha (PDGFRα) and platelet-derived growth factor beta (PDGFRß) was performed in control and AUB-E endometrium from the proliferative, early, mid- and late secretory phases of the menstrual cycle (n = 5 each group). Control proliferative phase endometrium was cultured in PDGF-BB (0, 10 ng/ml) and vascular maturation assessed (n = 3). Endothelial cell to vascular smooth muscle cell (VSMC) association was assessed after treatment with PDGF-BB (0, 1, 10 ng/ml). Secretion of angiogenic growth factors by endothelial cells or VSMC was determined. RESULTS: Endothelial cell immunoreactivity for PDGF-BB was reduced in the mid and late secretory phases in AUB-E (P = 0.008). PDGFRα was also reduced in mid secretory phase endothelial cells, proliferative and early secretory phase glandular epithelium in AUB-E (P = 0.008). PDGFRß expression was not altered. Treatment of proliferative phase endometrium with PDGF-BB (10 ng/ml) reduced the percentage of vessels expressing contractile VSMC markers. PDGF-BB had no effect on angiogenic growth factor secretion by endothelial cells or VSMC in vitro and did not affect their association in an in-vitro endothelial cell-VSMC association assay. CONCLUSIONS: Reduced endothelial cell expression of PDGF-BB in the AUB-E endometrium may contribute to the reduced vascular maturation previously observed in these women.

Endometrial vascular development in heavy menstrual bleeding: altered spatio-temporal expression of endothelial cell markers and extracellular matrix components.

STUDY QUESTION: Are there any phenotypic and structural/architectural changes in the vessels of endometrium and superficial myometrium during the normal menstrual cycle in healthy women and those with heavy menstrual bleeding (HMB)? SUMMARY ANSWER: Spatial and temporal differences in protein levels of endothelial cell (EC) markers and components of the extracellular matrix (ECM) were detected across the menstrual cycle in healthy women and these are altered in HMB. WHAT IS KNOWN ALREADY: HMB affects 30% of women of reproductive age with ~50% of cases being idiopathic. We have previously shown that the differentiation status of endometrial vascular smooth muscle cells (VSMCs) is altered in women with HMB, suggesting altered vessel maturation compared to controls. Endometrial arteriogenesis requires the co-ordinated maturation not only of the VSMCs but also the underlying ECs and surrounding ECM. We hypothesized that there are spatial and temporal patterns of protein expression of EC markers and vascular ECM components in the endometrium across the menstrual cycle, which are altered in women with HMB. STUDY DESIGN, SIZE, DURATION: Biopsies containing endometrium and superficial myometrium were taken from hysterectomy specimens from both healthy control women without endometrial pathology and women with subjective HMB in the proliferative (PP), early secretory (ESP), mid secretory (MSP) and late secretory (LSP) phases (N = 5 for each cycle phase and subject group). Samples were fixed in formalin and embedded in paraffin wax. PARTICIPANTS/MATERIALS, SETTING, METHODS: Serial sections (3µm thick) were immunostained for EC markers (factor VIII related antigen (F8RA), CD34, CD31 and ulex europaeus-agglutinin I (UEA-1) lectin), structural ECM markers (osteopontin, laminin, fibronectin and collagen IV) and for Ki67 to assess proliferation. Immunoreactivity of vessels in superficial myometrium, endometrial stratum basalis, stratum functionalis and luminal region was scored using either a modified Quickscore or by counting the number of positive vessels. MAIN RESULTS AND THE ROLE OF CHANCE: In control samples, all four EC markers showed a dynamic expression pattern according to the menstrual cycle phase, in both endometrial and myometrial vessels. EC protein marker expression was altered in women with HMB compared with controls, especially in the secretory phase in the endometrial luminal region and stratum functionalis. For example, in the LSP expression of UEA-1 and CD31 in the luminal region decreased in HMB (mean quickscore: 1 and 5, respectively) compared with controls (3.2 and 7.4, respectively) (both P = 0.008), while expression of F8RA and CD34 increased in HMB (1.4 and 8, respectively) compared with controls (0 and 5.8, respectively) (both P = 0.008). There was also a distinct pattern of expression of the vascular structural ECM protein components osteopontin, laminin, fibronectin and collagen IV in the superficial myometrium, stratum functionalis and stratum basalis during the menstrual cycle, which was altered in HMB. In particular, compared with controls, osteopontin expression in HMB was higher in stratum functionalis in the LSP (7.2 and 11.2, respectively P = 0.008), while collagen IV expression was reduced in stratum basalis in the MSP (4.6 and 2.8, respectively P = 0.002) and in stratum functionalis in the ESP (7 and 3.2, respectively P = 0.008). LIMITATIONS, REASONS FOR CAUTION: The protein expression of vascular EC markers and ECM components was assessed using a semi-quantitative approach in both straight and spiral arterioles. In our hospital, HMB is determined by subjective criteria and levels of blood loss were not assessed. WIDER IMPLICATIONS OF THE FINDINGS: Variation in the protein expression pattern between the four EC markers highlights the importance of choice of EC marker for investigation of endometrial vessels. Differences in expression of the different EC markers may reflect developmental stage dependent expression of EC markers in endometrial vessels, and their altered expression in HMB may reflect dysregulated vascular development. This hypothesis is supported by altered expression of ECM proteins within endometrial vessel walls, as well as our previous data showing a dysregulation in VSMC contractile protein expression in the endometrium of women with HMB. Taken together, these data support the suggestion that HMB symptoms are associated with weaker vascular structures, particularly in the LSP of the menstrual cycle, which may lead to increased and extended blood flow during menstruation. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Wellbeing of Women (RG1342) and Newcastle University. There are no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Expression of melanocortin receptors in human endometrium.

STUDY QUESTION: Are melanocortin receptors (MCR1-5) expressed in the endometrium? SUMMARY ANSWER: MCR1-5 are expressed in endometrium to varying degrees, with MC2R, MC3R and MC5R being the most abundant and the majority of expression being observed in glandular epithelium. WHAT IS KNOWN ALREADY: Women with Addison's disease who were being administered synthetic ACTH reported menstrual complications as a side effect. There is no previous literature on expression of the melanocortin receptors within the endometrium, and therefore whether ACTH may directly affect the endometrial vasculature. STUDY DESIGN, SIZE, DURATION: Endometrial biopsies were taken from hysterectomy specimens in control women without endometrial pathology (n = 4 for each of proliferative and late-secretory phases). Biopsies were formalin fixed and embedded in paraffin wax. Decidual samples (n = 7) were cultured in a range of concentrations of synthetic ACTH for 3 days before being formalin fixed and embedded in paraffin wax. PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometrial paraffin embedded sections were immunostained for MCR1-5 and assessed using a modified quickscore with luminal epithelium, glandular epithelium, stromal cells, endothelial cells and vascular smooth muscle cells all being assessed separately. Cultured decidual biopsy paraffin embedded sections were immunostained for H-caldesmon and the number of layers of vascular smooth muscle cells surrounding the vessel assessed. MAIN RESULTS AND THE ROLE OF CHANCE: All five melanocortin receptors were shown to be immunolocalised to the endometrium, with MC5R, MC2R and MC3R being the most abundant and limited immunostaining being observed for MC1R and MC4R. Treatment of decidual biopsies with synthetic adrenocorticotropin (ACTH) resulted in loss of vascular integrity. LIMITATIONS, REASONS FOR CAUTION: This is an observational study and does not definitively demonstrate a link between synthetic ACTH administration and menstrual complications. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to demonstrate widespread expression of melanocortin receptors within the endometrium. Further study is required to determine the role of this hormone family in endometrial function. STUDY FUNDING/COMPETING INTERESTS: The work was part funded by MRC grant G09000001. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Altered vascular smooth muscle cell differentiation in the endometrial vasculature in menorrhagia.

STUDY QUESTION: How does the smooth muscle content and differentiation stage of vascular smooth muscle cells (VSMCs) in endometrial blood vessels change according to the different phases of the menstrual cycle and is this altered in women with menorrhagia? SUMMARY ANSWER: The smooth muscle content (as a proportion of the vascular cross-sectional area) of endometrial blood vessels remained unchanged during the normal menstrual cycle and in menorrhagia; however, expression of the VSMC differentiation markers, smoothelin and calponin, was dysregulated in endometrial blood vessels in samples from women with menorrhagia compared with controls. WHAT IS KNOWN ALREADY: Menorrhagia affects 30% of women of reproductive age and is the leading indication for hysterectomy. Previous studies have suggested important structural and functional roles for endometrial blood vessels, including impaired vascular contractility. Differentiation of VSMC from a synthetic to contractile state is associated with altered cellular phenotype that contributes to normal blood flow and pressure. This vascular maturation process has been little studied in endometrium both across the normal menstrual cycle and in menorrhagia. STUDY DESIGN, SIZE, DURATION: Endometrial biopsies were taken from hysterectomy specimens or by pipelle biopsy prior to hysterectomy in controls without endometrial pathology and in women with menorrhagia (n = 7 for each of proliferative, early-secretory, mid-secretory and late-secretory phases for both groups). Biopsies were formalin fixed and embedded in paraffin wax. PARTICIPANTS/MATERIALS, SETTING, METHODS: Paraffin-embedded sections were immunostained for α smooth muscle actin (αSMA), myosin heavy chain (MyHC), H-caldesmon, desmin, smoothelin and calponin (h1 or basic). VSMC content was measured in 25 αSMA(+) vascular cross sections per sample and expressed as a ratio of the muscular area:gross vascular cross-sectional area. VSMC differentiation was analysed by the presence/absence of differentiation markers compared with αSMA expression. Smoothelin and calponin expression was also analysed in relation to total number of blood vessels by double immunostaining for endothelial cell markers. MAIN RESULTS AND THE ROLE OF CHANCE: Study of VSMC differentiation markers revealed decreased expression of calponin both in αSMA(+) vessels (P = 0.008) and in relation to total number of vessels (P = 0.001) in late secretory phase endometrium in menorrhagia compared with controls. Smoothelin expression in αSMA(+) vessels was increased (P = 0.03) in menorrhagia, although this was not significant in relation to the total number of vessels. In normal endometrium, the proportion of blood vessels expressing αSMA increased from 63% in proliferative endometrium to 81% in the late secretory phase (P = 0.002). The overall arterial muscle content did not differ between control and menorrhagia at any phase of the menstrual cycle, occupying 78-81% of gross vascular cross-sectional area during the different menstrual cycle phases. LIMITATIONS, REASONS FOR CAUTION: This study included both straight and spiral arterioles and analysed only stratum functionalis. The VSMC differentiation with respect to αSMA expression is an observational study and the data are presented as presence or absence of the differentiation markers in each field of view, corresponding with the vascular cross sections included in the study of vascular muscle content. WIDER IMPLICATIONS OF THE FINDINGS: Smoothelin and calponin have been widely implicated as important regulators of vascular tone, vascular contractility and rate of blood flow. Our results have uncovered a disparate pattern of calponin expression, potentially indicating a dysfunctional contraction mechanism in the endometrial blood vessels in menorrhagia, thus implicating calponin as a potential therapeutic target. STUDY FUNDING/COMPETING INTERESTS: This study was funded by Wellbeing of Women (RG1342) and Newcastle University. There are no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Altered expression of interleukin-6, interleukin-8 and their receptors in decidua of women with sporadic miscarriage.

STUDY QUESTION: Are alterations in decidual expression of interleukin (IL)-6 and IL-8 associated with sporadic miscarriage? SUMMARY ANSWER: IL-6 and IL-8 secretion from decidual uterine natural killer (uNK) cells and macrophages isolated from women with spontaneous miscarriage was reduced compared with normal controls. WHAT IS KNOWN ALREADY: Miscarriage is a common gynaecological problem with huge financial and personal implications. Eleven to twenty per cent of all clinically recognized pregnancies are lost before the 20th week of gestation, with miscarriages often being divided into early (≤ 12 completed weeks from last menstrual period) and late (≥ 13 weeks). Spiral artery remodelling is a key feature of early pregnancy; failure of this process has been implicated in sporadic miscarriage. The molecular triggers that initiate spiral artery remodelling are not clear, although cytokines such as IL-6 and IL-8 may play a role. STUDY DESIGN, SIZE, DURATION: This was a laboratory-based study using decidual and placental bed biopsy samples from women with sporadic miscarriage (n = 30) and termination of pregnancy controls (n = 30). PARTICIPANTS/MATERIALS, SETTING, METHODS: Total adherent decidual cells, CD10(+) stromal cells, CD14(+) macrophages and CD56(+) uNK cells were isolated from decidua from apparently normal pregnancies that were terminated at either 8-10 or 12-14 weeks' gestation. In addition, CD14(+) macrophages and CD56(+) uNK cells were isolated from decidua from sporadic miscarriage at 8-10 weeks' gestation. Secreted IL-8 was measured in all isolated cell populations, while IL-6 was measured in CD14(+) macrophages and CD56(+) uNK cells from both sporadic miscarriage and normal controls. Placental bed biopsies were taken from women after sporadic miscarriage or termination of pregnancy at ≤ 12 completed weeks' or >13 weeks' gestational age, formalin-fixed, paraffin-embedded and immunostained for IL-6, IL-6Rα, GP130, IL-8, CXCR1, CXCR2 and CD13 (aminopeptidase N). Staining intensity for each factor was assessed in extravillous trophoblast cell populations, myometrial and decidual stroma, myometrial and decidual spiral arteries and decidual glandular epithelium. A CPA model was used to assess the potential role of IL-6 and IL-8 in spiral artery remodelling. MAIN RESULTS AND THE ROLE OF CHANCE: IL-8 was secreted by total adherent decidual cells, CD10(+) stromal cells and CD14(+) macrophages at both 8-10 and 12-14 weeks' gestation, with CD14(+) cells secreting the highest levels. Both CD14(+) and CD56(+) cells isolated from decidua of early sporadic miscarriage produced lower IL-6 (P = 0.04, P = 0.01, respectively) and IL-8 levels (P = 0.0007, P = 0.002, respectively) compared with normal cases. In addition, altered expression of IL-6, IL-8 and their receptors was observed in various cell types in placental bed (myometrial stroma, glandular epithelium, interstitial extravillous trophoblast cells, vascular smooth muscle cells and endothelial cells) in sporadic miscarriage, particularly from later gestational ages. IL-6 and IL-8 disrupted vascular smooth muscle morphology and organization in an in vitro model of spiral artery remodelling. LIMITATIONS, REASONS FOR CAUTION: By the nature of sampling at the time of miscarriage, it was not possible to ascertain the cause or effect in the observed alterations of levels of IL-6 and IL-8 in sporadic miscarriage. WIDER IMPLICATIONS OF THE FINDINGS: Alterations in the expression of IL-6, IL-8 and their receptors may be associated with the aetiology of sporadic miscarriage, especially given the potential role of these cytokines in the regulation of trophoblast invasion and spiral artery remodelling. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by funding from Wellbeing of Women (RG1000). The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

The role of vascular smooth muscle cell apoptosis and migration during uterine spiral artery remodeling in normal human pregnancy.

During human uterine spiral artery (SpA) remodeling, vascular smooth muscle cells (VSMCs) are lost and replaced by fibrinoid, incorporating extravillous trophoblast (EVT) cells. The aim of the current study was to determine the relative contributions of apoptosis and migration to VSMC loss during SpA remodeling. Immunohistochemistry (Apoptag, active caspase 3, lamin) of placental bed biopsies (8-20 wk gestation) demonstrated apoptotic cells in all samples; double immunolabeling identified these as trophoblasts, leukocytes, and endothelial cells. In total, 294 SpAs were studied, and only one apoptotic VSMC was identified. H-caldesmon-immunopositive VSMCs were observed surrounding and separate from SpA walls in partially remodeled vessels; the highest level of VSMC migration was observed in vessels with associated EVT cells (number of migrated cells 6.4 ± 1.2; distance migrated 3.5 ± 0.3 pixels) compared with those without (number of migrated cells 3.6 ± 0.5, P<0.001; distance migrated 2.8 ± 0.1 pixels, P<0.0001). VEGF-A, VEGF-C, TGF-ß1, and Ang-2 all stimulated human aorta VSMC invasion in vitro, although EVT cell culture supernatants did not. In summary, apoptosis is unlikely to play a major role in loss of VSMCs from SpAs during remodeling in normal pregnancy, but VSMCs appear to migrate away from the wall of the SpA, an effect enhanced by the presence of EVT cells.

Uterine natural killer cells initiate spiral artery remodeling in human pregnancy.

Uterine spiral artery remodeling is required for successful human pregnancy; impaired remodeling is associated with pregnancy complications, including late miscarriage, preeclampsia, and fetal growth restriction. The molecular triggers of remodeling are not known, but it is now clear that there are "trophoblast-independent" and "trophoblast-dependent" stages. Uterine natural killer (uNK) cells are abundant in decidualized endometrium in early pregnancy; they surround spiral arteries and secrete a range of angiogenic growth factors. We hypothesized that uNK cells mediate the initial stages of spiral artery remodeling. uNK cells and extravillous trophoblast (EVT) cells were isolated from early pregnancy decidua and placenta. Chorionic plate arteries from full-term placentas and spiral arteries from nonpregnant myometrium were cultured with angiogenic growth factors or conditioned medium (CM) from uNK cells or EVT or uNK cell/EVT cocultures. In both vessel models, uNK cell CM induced disruption of vascular smooth muscle cells (VSMCs) and breakdown of extracellular matrix components. Angiopoietin (Ang)-1, Ang-2, interferon-γ, and VEGF-C also disrupted VSMC integrity with an Ang-2 inhibitor abrogating the effect of uNK cell CM. These results provide compelling evidence that uNK cells contribute to the early stages of spiral artery remodeling; failure of this process could contribute to pregnancy pathology.

Effects of interleukin-6 on extravillous trophoblast invasion in early human pregnancy.

Invasion of uterine tissues by extravillous trophoblast cells (EVT) is essential for successful human pregnancy. EVT invasion is tightly regulated by a number of factors, including growth factors and cytokines, but the mechanisms that underlie their regulatory effect remain poorly understood. Interleukin (IL)-6 has been suggested to play a role in controlling EVT invasion. We hypothesized that IL-6 produced by cells in uterine decidua would regulate EVT invasiveness via IL-6Rα and gp130 receptors expressed by trophoblast cells. The effect of IL-6 on EVT signalling and cytokine production was also studied. Supernatants from disaggregated 'total' decidual cells, CD8(+) T cells, CD10(+) decidual stromal cells, CD14 macrophages, CD56(+) uterine natural killer cells, cytotrophoblast and EVT cells contained large quantities of IL-6 protein at both 8-10 and 12-14 weeks gestational age. IL-6Rα and gp130 were immunolocalized to EVT in placental bed biopsies from 8 to 20 weeks gestation and IL-6Rα expression was confirmed by western blotting. IL-6 had no effect on the invasive potential of EVT from chorionic villi or the immortalized EVT cell line HTR-8/SVneo in a Matrigel(®) invasion assay. IL-6 stimulated phosphorylation of several cell signalling proteins in EVT (8-14 weeks' gestation), although significance was lost after correction for multiple comparisons. Incubation with IL-6 decreased secretion of regulated upon activation, normal T-cell expressed and secreted (RANTES) by EVT cells. In conclusion, although IL-6 did not affect trophoblast cell invasion, it stimulated EVT cellular cascades and inhibited secretion of RANTES involved in a number of cellular processes.

Localization of angiogenic growth factors and their receptors in the human endometrium throughout the menstrual cycle and in recurrent miscarriage.

BACKGROUND: Angiogenesis is a key feature of endometrial development. Inappropriate endometrial vascular development has been associated with recurrent miscarriage (RM) with increased amounts of perivascular smooth muscle cells surrounding them. METHODS: In the current study, we have used immunohistochemistry to study temporal and spatial expression of a series of angiogenic growth factors (AGFs) and their receptors; vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-D, VEGF-R1, VEGF-R2, VEGF-R3, platelet-derived growth factor (PDGF)-BB, PDGF-Rα, PDGF-Rß, transforming growth factor (TGF)-ß1, TGF-ßRI, TGF-ßRII, angiopoietin (Ang)-1, Ang-2 and Tie-2, in the proliferative, early secretory and mid-late secretory phase endometrium from control women as well as in the mid-late secretory phase of women with a history of RM. The AGFs and their receptors studied were immunostained and assessed separately in stromal, vascular smooth muscle, endothelial and glandular epithelial cells. Laser capture microdissection and real-time RT-PCR were used to confirm expression patterns observed by immunohistochemistry. RESULTS: Most AGFs investigated showed both temporal and spatial expression patterns in normal cycling endometrium. In addition, immunostaining intensity for several AGFs was altered in women with a history of RM, particularly in vascular smooth muscle cells (VSMCs). VSMC expression of TGF-ß1, VEGF-R1 and VEGF-R2 was increased while expression of PDGF-BB, TGF-ßRI, TGF-ßRII, Ang-2, VEGF-A and VEGF-C was reduced. CONCLUSIONS: This study confirms that the cycling endometrium is a highly angiogenic tissue and that this process is likely to be altered in women with a history of RM and may contribute to the aetiology of this condition.

Prednisolone treatment reduces endometrial spiral artery development in women with recurrent miscarriage.

BACKGROUND: Uterine natural killer (uNK) cells and endometrial blood vessel maturation are increased in the luteal phase of the menstrual cycle in a subset of women with recurrent miscarriage (RM). uNK cell numbers are reduced after treatment with prednisolone (20 mg/day for 3 weeks). HYPOTHESES: Prednisolone treatment reduces endometrial vascular maturation and angiogenic growth factor expression in women with RM with increased uNK cells. METHODS: Endometrial biopsies (n = 18 paired samples) from women with RM at LH + 7 before and during prednisolone treatment (20 mg/day for 3 weeks) were snap frozen. Total RNA and cDNA was prepared and used in a human angiogenesis RT-PCR superarray (84 genes, n = 6 pairs) with results validated using RT-PCR (n = 15 pairs). Immunohistochemistry (n = 15 pairs) was performed for Factor VIII, α-smooth muscle actin (α-SMA) and myosin heavy chain (MyHC) and the total number of vessels and the percentage of vessels completely surrounded by vascular smooth muscle cells (VSMCs) were determined. RESULTS: During prednisolone treatment there was no change in the total number of endometrial blood vessels but the percentage of vessels completely surrounded by VSMCs was decreased (α-SMA P < 0.0001; MyHC P < 0.0001). Endometrial EGF and STAB 1 expression was decreased during prednisolone treatment in samples from woman who went on to have a live birth. CONCLUSIONS: The effect of prednisolone therapy for some women with RM may be due to altered endometrial angiogenic growth factor expression and reduced blood vessel maturation.

Possible roles for folic acid in the regulation of trophoblast invasion and placental development in normal early human pregnancy.

In addition to its role in the prevention of neural tube defects, folic acid has many other physiological functions, including cell proliferation, DNA replication, and antioxidant protection. The aim of this study was to determine the role that folic acid has in regulating placental trophoblast development. Placental explants from placentae at gestational age 7 wk (n = 3) were cultured in folic acid at concentrations of 10(-6) M, 10(-8) M, and 10(-10) M. Extravillous trophoblast (EVT) invasion was assessed following 6-day culture, and explants were used for immunohistochemical evaluation of proliferation (MKI67) and apoptosis (active caspase 3). In addition, an array was performed on cell culture supernatants to examine a range of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). Folic acid increased the invasion of EVT cells in this explant model by between 83% and 19% (P = 0.005), and this was associated with increased MKI67 positivity and decreased active caspase 3 positivity; this effect was concentration dependent and showed a biphasic response. In addition, culture in folic acid increased vascular density, as determined by anti-CD31 immunostaining (P = 0.05). The increase in EVT invasion correlated with increased placental explant secretion of MMP2 (P = 0.01), MMP3 (P = 0.01), and MMP9 (P = 0.02). This study demonstrates that folic acid is potentially important in a number of crucial early stages of placental development, including EVT invasion, angiogenesis, and secretion of MMPs, and highlights the need for further studies to address the benefit of longer-term folic acid supplementation throughout pregnancy to prevent pregnancy disorders associated with deficient placental development, including preeclampsia.

Interaction between uterine natural killer cells and extravillous trophoblast cells: effect on cytokine and angiogenic growth factor production.

BACKGROUND: Uterine natural killer (uNK) cells are a major source of cytokines and angiogenic growth factors (AGFs), with AGF levels decreasing and cytokine levels increasing with gestational age. The factors that regulate AGF and cytokine secretion are unclear but may involve interactions between uNK cells and extravillous trophoblast (EVT) cells. We hypothesize that uNK cell interaction with EVT cells alters their cytokine and AGF secretion. METHODS: Ex vivo co-cultures of uNK cells with either EVT (irradiated or fresh) or villous cytotrophoblast (CTB; control cell type) cells isolated from the same patients at 8-10 or 12-14 weeks gestational age (n = 10 each group) were established. Co-cultures were established with either direct contact between the different cell types or with the cells separated by a 0.4 µm filter. AGFs and cytokines were measured in cell culture supernatants using multiplex analysis (FAST Quant) or ELISA. RESULTS: Secretion of angiopoietin-1 (P < 0.006) and vascular endothelial growth factor-C (P < 0.001) by uNK cells was lower when these cells were co-cultured, either directly or indirectly, with both trophoblast cell types at both gestational ages tested compared with when cultured alone. In contrast, interleukin (IL)-6 (P < 0.0001), IL-8 (P < 0.0001) and transforming growth factor-ß1 (P < 0.002) were decreased only in direct uNK/EVT and uNK/CTB co-culture conditions at 8-10 and 12-14 weeks gestational age. CONCLUSIONS: AGF and cytokine secretion was reduced after co-culture of uNK cells and both EVT and CTB cells. It remains unclear whether uNK cell AGF and cytokine production was reduced after co-culture with trophoblast cells (EVT or CTB) or whether trophoblast cell (EVT or CTB) AGF and cytokine production was reduced after co-culture with uNK cells. Local production of AGFs and cytokines in the placental bed may be lowered when uNK cells come in direct contact with EVT cells.

Transforming Growth Factor (TGF) ß and Endometrial Vascular Maturation.

Appropriate growth and development of the endometrium across the menstrual cycle is key for a woman's quality of life and reproductive well-being. Recurrent pregnancy loss (RPL) and heavy menstrual bleeding (HMB) affect a significant proportion of the female population worldwide. These endometrial pathologies have a significant impact on a woman's quality of life as well as placing a high economic burden on a country's health service. An underlying cause for both conditions is unknown in approximately 50% of cases. Previous research has demonstrated that aberrant endometrial vascular maturation is associated with both RPL and HMB, where it is increased in RPL but reduced in HMB. TGFß1 is one of the key growth factors that regulate vascular maturation, by inducing phenotypic switching of vascular smooth muscle cells (VSMCs) from a synthetic phenotype to a more contractile one. Our previous data demonstrated an increase in TGFß1 in the endometrium of RPL, while others have shown a decrease in women with HMB. However, TGFß1 bioavailability is tightly controlled, and we therefore sought to perform an extensive immunohistochemical analysis of different components in the pathway in the endometrium of normal controls, women with HMB or RPL. In addition, two in vitro models were used to examine the role of TGFß1 in endometrial vascular maturation and endothelial cell (EC):VSMC association. Taken all together, the immunohistochemical data suggest a decrease in bioavailability, receptor binding capacity, and signaling in the endometrium of women with HMB compared with controls. In contrast, there is an increase in the bioavailability of active TGFß1 in the endometrium of women with RPL compared with controls. Endometrial explants cultured in TGFß1 had an increase in the number of vessels associated with contractile VSMC markers, although the total number of vessels did not increase. In addition, TGFß1 increased EC:VSMC association in an in vitro model. In conclusion, TGFß1 is a key regulator of endometrial vascular maturation and could be considered as a therapeutic target for women suffering from HMB and/or RPL.

Regulation of extravillous trophoblast invasion by uterine natural killer cells is dependent on gestational age.

BACKGROUND: Extravillous trophoblast (EVT) cell invasion of uterine decidua and the inner third of myometrium is critical for successful pregnancy. Many decidual factors are likely to play a role in regulating this process. We have previously shown that cytokines, known to be produced by uterine natural killer (uNK) cells, such as TNF-alpha, TGF-beta1 and IFN-gamma inhibit EVT invasion. We therefore hypothesized that supernatants from purified uNK cells would inhibit EVT invasion. METHODS AND RESULTS: Total unfractionated decidual cell supernatants from 8 to 10 weeks gestation increased EVT invasion from placental villous explants, although uNK cell supernatants from 8 to 10 weeks gestation had no effect. In contrast, both total decidual and uNK cell supernatants from 12 to 14 weeks gestation stimulated EVT invasion. MMP-2, uPA, PAI-1 and PAI-2 levels did not differ under any of the conditions tested, whereas MMP-9 levels were increased in the presence of both total decidual and uNK cell supernatants from both gestational age groups. There was a decrease in the level of EVT apoptosis in the presence of uNK cell supernatant from 12 to 14 weeks, but not 8-10 weeks, gestation. CONCLUSIONS: Decidual uNK cell supernatants from 12 to 14 weeks gestational age stimulated EVT invasion, potentially by increasing MMP9 levels and reducing apoptosis. Total decidual cell isolates stimulated EVT invasion at both gestational ages investigated, potentially reflecting the complex nature of these cell culture supernatants.

Transient loss of endothelial cells in human spiral artery remodelling during early pregnancy: Challenging the dogma.

Spiral artery (SpA) remodelling is essential for a successful pregnancy and is best described by its morphological features; vascular smooth muscle cell separation and loss, vessel dilatation, and invasion by extravillous trophoblast cells (EVT). Current opinion holds that EVT fully replace the endothelial cells (EC) of the SpA and take on EC-like characteristics. Placental bed biopsies (6-20 weeks gestation) were immunostained for EC and EVT, showing transient loss of EC. In vessels containing an endovascular EVT plug (n = 28) 77.6 ± 19.3% of the lumen was covered by EC while in vessels without endovascular EVT (n = 100) it was 100 ± 0%.

Uterine natural killer cells and angiogenesis in recurrent reproductive failure.

BACKGROUND: Increased numbers of phenotypically unusual CD56(bright) CD16- uterine natural killer (uNK) cells have been associated with recurrent reproductive failure. uNK cells produce angiogenic growth factors and are potential regulators of decidual angiogenesis in early pregnancy. The final common mechanism for early pregnancy loss is thought to be early onset of the maternal circulation and excessive placental oxidative stress. We tested the hypothesis that increased uNK cells in preimplantation endometrium are associated with altered angiogenesis. METHODS: Women with recurrent reproductive failure (n = 122) were investigated with uterine artery Doppler and endometrial biopsy. Immunohistochemistry was used to identify uNK, endothelial and vascular smooth muscle cells and image analysis was used to assess location, density and differentiation. RESULTS: uNK cell density was positively correlated with the formation of blood (P = 0.005, r = 0.5) and lymphatic vessels (P = 0.0001, r = 0.6), spiral arteriole smooth muscle differentiation (P = 0.01, r = 0.5) and endometrial oedema (P = 0.004). The functional effect of this was a reduced uterine artery resistance to blood flow. CONCLUSIONS: These data suggest that uNK cells may regulate angiogenesis in non-pregnant endometrium. The mechanisms of reproductive failure associated with increased uNK cell density appear to be increased angiogenesis and peri-implantation blood flow, which may lead to early maternal circulation and hence pregnancy failure due to excessive oxidative stress.

Localization of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitors for MMPs (TIMPs) in uterine natural killer cells in early human pregnancy.

BACKGROUND: Invasion by extravillous trophoblast into uterine decidua and myometrium with remodeling of spiral arteries is essential for normal human pregnancy and is tightly regulated. Uterine natural killer (uNK) cells appear to be a major maternal regulator of placentation through the secretion of growth factors, cytokines and proteinases. METHOD: Matrix metalloproteinase (MMP)-2 and MMP-9 activity in placental bed biopsies was studied using in situ gelatin zymography. Expression by uNK cells of MMP-2, MMP-9 and their tissue inhibitors, TIMP-1, TIMP-2 and TIMP-3, was localized in the placental bed by immunohistochemistry. Levels of MMP-2, MMP-9, TIMP-1, TIMP-2 and TIMP-3 secreted into 24 h cell culture supernatants of isolated uNK and unseparated (total) decidual cells (8-10 and 12-14 weeks gestation, n = 10 each group) were determined by gelatin gel zymography or western blot as appropriate. RESULTS: Gelatinase activity in situ appeared greater in decidua than myometrium. uNK cells showed strong immunostaining for MMP-2 and TIMP-2. MMP-9 activity was lower in uNK cells than total decidual supernatants (8-10 weeks: P = 0.0003; 12-14 weeks: P = 0.0012). In contrast, there was no difference in MMP-2 secreted by either uNK cell or total decidual cultures. CONCLUSIONS: uNK cells from early human pregnancy decidua possess innate protease activity, especially MMP-2, providing further evidence for a role for these cells in regulation of trophoblast invasion and spiral artery remodeling in early placentation.