During pregnancy, a specialized type of NK cell accumulates in the lining of the uterus (decidua) and interacts with semiallogeneic fetal trophoblast cells. dNK cells are functionally and phenotypically distinct from PB NK and are implicated in regulation of trophoblast transformation of the uterine spiral arteries, which if inadequately performed, can result in pregnancy disorders. Here, we have used uterine artery Doppler RI in the first trimester of pregnancy as a proxy measure of the extent of transformation of the spiral arteries to identify pregnancies with a high RI, indicative of impaired spiral artery remodeling. We have used flow cytometry to examine dNK cells isolated from these pregnancies compared with those from pregnancies with a normal RI. We report a reduction in the proportion of dNK cells from high RI pregnancies expressing KIR2DL/S1,3,5 and LILRB1, receptors for HLA-C and HLA-G on trophoblast. Decreased LILRB1 expression in the decidua was examined by receptor blocking in trophoblast coculture and altered dNK expression of the cytokines CXCL10 and TNF-α, which regulate trophoblast behavior. These results indicate that dNK cells from high RI pregnancies may display altered interactions with trophoblast via decreased expression of HLA-binding cell-surface receptors, impacting on successful transformation of the uterus for pregnancy.

The timing of decidualisation and vascular processes during the implantation period is of paramount importance for the development of a receptive endometrium suitable for implantation. The endometrium transforms during the secretory phase into a well-vascularised receptive tissue characterised by increased vascular permeability, oedema, proliferation and differentiation of stromal cells into decidual cells, invasion of leucocytes, vascular remodelling and angiogenesis. Decidualisation continues in the presence of conception and an influx of immune cells, trophoblasts and vascular adaptation will occur. Vascular changes include spiral artery remodelling, angiogenesis and the induction of angiogenic factors. Disturbances in uterine blood supply are associated with first-trimester miscarriages and third-trimester perinatal morbidity and mortality caused by pre-eclampsia and foetal growth restriction. This article assesses decidual vascular changes during human implantation, and evaluates the involvement of angiogenesis in the pathogenesis of miscarriages, pre-eclampsia and intrauterine growth restriction.

Searchable abstracts of presentations at key conferences on reproductive biology and medicine ISSN 2052-1472 (online)

Decidual natural killer (dNK) cells have been shown to both promote and inhibit trophoblast behavior important for decidual remodeling in pregnancy and have a distinct phenotype compared to peripheral blood NK cells. We investigated whether different levels of oxygen tension, mimicking the physiological conditions of the decidua in early pregnancy, altered cell surface receptor expression and activity of dNK cells and their interactions with trophoblast. dNK cells were isolated from terminated first-trimester pregnancies and cultured in oxygen tensions of 3%, 10%, and 21% for 24 h. Cell surface receptor expression was examined by flow cytometry, and the effects of secreted factors in conditioned medium (CM) on the trophoblast cell line SGHPL-4 were assessed in vitro. SGHPL-4 cells treated with dNK cell CM incubated in oxygen tensions of 10% were significantly more invasive (P < 0.05) and formed endothelial-like networks to a greater extent (P < 0.05) than SGHPL-4 cells treated with dNK cell CM incubated in oxygen tensions of 3% or 21%. After 24 h, a lower percentage of dNK cells expressed CD56 at 21% oxygen (P < 0.05), and an increased percentage of dNK cells expressed NKG2D at 10% oxygen (P < 0.05) compared to other oxygen tensions, with large patient variation. This study demonstrates dNK cell phenotype and secreted factors are modulated by oxygen tension, which induces changes in trophoblast invasion and endovascular-like differentiation. Alterations in dNK cell surface receptor expression and secreted factors at different oxygen tensions may represent regulation of function within the decidua during the first trimester of pregnancy.

Transformation of the uterine spiral arteries (SAs) during pregnancy is critical to support the developing fetus, and is impaired in some pregnancy disorders, including preeclampsia. Decidual natural killer (dNK) cells play a role in SA remodeling, although their interactions with fetal trophoblast remain unclear. A uterine artery Doppler resistance index (RI) in the first trimester of pregnancy can be used as a proxy measure of the extent of SA remodeling; we have used this technique to characterize dNK cells from pregnancies with normal (normal RI) and impaired (high RI) SA remodeling, which display least and highest risk of developing preeclampsia, respectively. We examined the impact of dNK cell secreted factors on trophoblast motility, chemoattraction, and signaling pathways to determine the contribution of dNK cells to SA transformation. We demonstrated that the chemoattraction of the trophoblast by dNK cells is impaired in pregnancies with high RI, as is the ability to induce trophoblast outgrowth from placental villous explants. These processes are dependent on activation of the extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase-Akt signaling pathways, which were altered in trophoblasts incubated with secreted factors from dNK cells from high RI pregnancies. Therefore, by characterizing pregnancies using uterine artery Doppler RI before dNK cell isolation, we have identified that impaired dNK-trophoblast interactions may lead to poor placentation. These findings have implications for pregnancy pathological conditions, such as preeclampsia.

Adequate extravillous trophoblast (EVT) invasion is essential for successful placentation. Although miR-520c-3p plays an important role in CD44-mediated invasion in cancer cells, there is little information on whether miR-520c-3p is involved in the regulatory mechanisms of CD44-mediated EVT invasion.We screened first trimester trophoblast cells and trophoblast cell lines for expression of miR-520c-3p using real-time polymerase chain reaction. The cell invasion assay was performed using EVT cell lines, HTR8/SVneo and HChEpC1b, to investigate the capability of suppressing EVT invasion by miR-520c-3p. Laser microdissection analysis was then used to determine whether miR-520c-3p was present in the first trimester decidua. Finally, the possibility of chorionic villous trophoblast (CVT)-EVT communication via exosomal miR-520c-3p was determined using an in vitro model based on BeWo exosomes and the EVT cell lines as recipient cells.The miR-520c-3p level was significantly downregulated in EVT cell lines and EVTs. Cell invasion was significantly inhibited in miR-520c-3p-overexpressing cell lines, involving a significant reduction of CD44. Laser microdissection analysis showed that miR-520c-3p in the periarterial area of the decidua was significantly higher than that in the non-periarterial area. Using an in vitro model system, BeWo exosomal miR-520c-3p was internalized into the EVT cells with subsequently reduced cell invasion via CD44 repression.EVT invasion is synergistically enhanced by the reciprocal expression of endogenous miR-520c-3p and CD44. The present study supports a novel model involving a placenta-associated miRNA function in cell-cell communication in which CVT exosomal miR-520c-3p regulates cell invasion by targeting CD44 in EVTs.

Objective— During pregnancy, fetal trophoblast disrupt endothelial cell and vascular smooth muscle cell (VSMC) interactions in spiral arteries of the maternal decidua to enable increased nutritional and oxygen delivery to the fetus. Little is known regarding this transformation because of difficulties of studying human pregnancy in vivo. This study investigated how trophoblast-secreted factors affect the interactions of vascular cells and the differentiation status of VSMC during spiral arteries remodeling using 3-dimensional vascular spheroid coculture. Methods and Results— Endothelial cell and VSMC were cocultured in hanging droplets to form spheroids representing an inverted vessel lumen. Control or conditioned media from an extravillous trophoblast (EVT) cell line was incubated with vascular spheroids for 24 hours. Spheroid RNA was then analyzed by Illumina Sentrix BeadChip array. Spheroids incubated with EVT conditioned medium showed significant up/downregulation of 101 genes (>1.5-fold; P <0.05), including an upregulation of C-X-C motif chemokine 10 (IP-10). C-X-C motif chemokine 10 expression was confirmed by qualitative real-time PCR and Western blot analysis of spheroids, and immunohistochemistry of first trimester decidua and ex vivo dissected nonplacental bed spiral arteries. EVT conditioned medium reduced VSMC expression of differentiation markers, and both EVT conditioned medium and C-X-C motif chemokine 10 increased motility of VSMC indicating dedifferentiation of VSMC. Conclusion— EVT-induced C-X-C motif chemokine 10 expression may contribute to spiral arteries remodeling during pregnancy by altering the motility and differentiation status of the VSMC in the vessel.

Impaired trophoblast invasion is associated with pregnancy disorders such as early pregnancy loss and preeclampsia. There is evidence to suggest that the consumption of caffeine during pregnancy may increase the risk of pregnancy loss; however, little is known about the direct effect of caffeine on normal trophoblast biology. Our objectives were to examine the effect of caffeine on trophoblast migration and motility after stimulation with epidermal growth factor (EGF) and to investigate the intracellular signaling pathways involved in this process. Primary first-trimester extravillous trophoblasts (EVT) and the EVT-derived cell line SGHPL-4 were used to study the effect of caffeine on EGF-stimulated cellular motility using time-lapse microscopy. SGHPL-4 cells were further used to study the effect of caffeine and cAMP on EGF-stimulated invasion of fibrin gels. The influence of caffeine and cAMP on EGF-stimulated intracellular signaling pathways leading to the activation of Akt were investigated by Western blot analysis. Caffeine inhibits both EGF-stimulated primary EVT and SGHPL-4 cell motility. EGF stimulation activates phosphatidylinositol 3-kinase, and Akt and caffeine inhibit this activation. Although cAMP inhibits both motility and invasion, it does not inhibit the activation of Akt, indicating that the effects of caffeine seen in this study are independent of cAMP. Further investigation indicated a role for mammalian target of rapamycin complex 2 (mTORC2) as a target for the inhibitory effect of caffeine. In conclusion, we demonstrate that caffeine inhibits EGF-stimulated trophoblast invasion and motility in vitro and so could adversely influence trophoblast biology in vivo.

During pregnancy, maternal uterine spiral arteries (SAs) are remodelled from minimal-flow, high-resistance vessels into larger diameter vessels with low resistance and high flow. Fetal extravillous trophoblasts (EVT) have important roles in this process. Decidual natural killer cells (dNK cells) are the major maternal immune component of the decidua and accumulate around SAs before trophoblast invasion. A role for dNK cells in vessel remodelling is beginning to be elucidated. This review examines the overlapping and dissimilar mechanisms used by EVT and dNK cells in this process and how this may mirror another example of tissue remodelling, namely cancer development.The published literature was searched using Pubmed focusing on EVT, dNK cells and SA remodelling. Additional papers discussing cancer development are also included.Similarities exist between actions carried out by dNK cells and EVT. Both interact with vascular cells lining the SA, as well as with each other, to promote transformation of the SA. EVT differentiation has previously been likened to the epithelialmesenchymal transition in cancer cells, and we discuss how dNKEVT interactions at the maternalfetal interface can also be compared with the roles of immune cells in cancer.The combined role that dNK cells and EVT play in SA remodelling suggests that these interactions could be described as a partnership. The investigation of pregnancy as a multicellular system involving both fetal and maternal components, as well as comparisons to similar examples of tissue remodelling, will further identify the key mechanisms in SA remodelling that are required for a successful pregnancy.

Prostaglandin F2α (PGF2α) is an inflammatory mediator which signals through a G-protein coupled receptor, the F-prostanoid (FP) receptor. We have previously shown elevated FP receptor expression in endometrial adenocarcinoma, a common gynaecological malignancy in Western countries. In this study, the expression of the chemokine CC motif Ligand 20 (CCL20) was determined to be regulated by PGF2α-FP receptor signalling in endometrial adenocarcinoma explants and cell line, and expression of CCL20 and its receptor CCR6 was elevated in endometrial adenocarcinoma compared to non-malignant endometrium. Both CCL20 and CCR6 were localised to neoplastic endometrial epithelial cells. The induction of CCL20 expression by PGF2α-FP signalling in an endometrial adenocarcinoma cell line stably expressing the FP receptor (FPS cells) was found to be dependent on the intracellular signalling of Gq, EGFR, ERK, calcineurin and nuclear factor of activated T-cells (NFAT) proteins. The treatment of FPS cells with recombinant CCL20 caused a significant increase in proliferation. Therefore these data demonstrate a role for the FP receptor in regulation of the chemokine CCL20, which can mediate proliferation of endometrial adenocarcinoma epithelial cells.

Endometrial adenocarcinoma is the most common gynaecological malignancy in western countries. Many of the established risk factors for developing endometrial cancer are associated with excess exposure to oestrogen unopposed by progesterone. These include nulliparity, late onset of the menopause, post-menopausal hormone replacement therapy and obesity. However, a number of risk factors also promote inflammation, another feature proposed to influence cancer development. The human cycling endometrium undergoes regular and cyclical episodes of inflammation. Moreover, hormonal and genetic changes that occur early in the development of endometrial adenocarcinoma can exacerbate the local inflammatory environment. Via alterations in the expression of local mediators and immune cell function, these may contribute to the development of endometrial cancer. This review discusses the contribution of inflammation to the initiation and progression of endometrial adenocarcinoma. Manipulation of inflammatory pathways may therefore represent a therapeutic target in endometrial adenocarcinoma.

In human pregnancy, successful placentation and remodelling of the uterine vasculature require the integration of a number of stages, which are crucial for a healthy pregnancy. As the demands of the developing fetus for nutrients and oxygen increase, the capacity of the maternal blood vessels to supply this must be altered radically, with deficiencies in this process implicated in a number of dangerous pregnancy complications. The complex signalling networks that regulate these tightly co-ordinated events are becoming clearer as more studies of early pregnancy are performed. It is the aim of this review to draw together our knowledge of events that occur to facilitate a successful pregnancy ranging from the preparation for implantation, through the invasion and differentiation of the trophoblast and the regulation of these processes by other cells within the decidual environment, to the active role that the trophoblast and maternal immune cells play in facilitating the remodelling of the uterine spiral arteries. The events involved in a healthy pregnancy will then be compared to aberrant placentation and remodelling, which are characteristics of many pregnancy disorders, and recent advances in detection of abnormal placental development will also be discussed.

Asymmetric dimethylarginine (ADMA) and NG monomethyl-l-arginine (l-NMMA) are endogenous inhibitors of nitric oxide synthases (NOS) and their local concentration is determined by the activity of dimethylarginine dimethylaminohydrolases (DDAHs). The current study in male Wistar rats was designed to immunolocalise DDAH I and II in relation to NOS and to investigate changes in distribution, activity and ADMA content in the acute period following myocardial infarction (MI) resulting from coronary artery ligation. Seven days after the coronary artery ligation, l-Arg and methylated arginine content, as well as DDAH activity were determined in homogenates of left ventricular (LV) infarct and border. The distribution of immunoreactive DDAH I, DDAH II, eNOS and iNOS were determined in sections of LV. In healthy hearts, DDAH I was absent, however, DDAH II was localized to endothelium and endocardium with a similar distribution to that of eNOS. Following MI, LV DDAH activity was increased (to 210±19% of control, P<0.05). Both DDAH I and DDAH II proteins were detected in peri-infarct cardiomyocytes, while DDAH II immunoreactivity was additionally localized to infiltrating inflammatory cells and blood vessels in the healing infarct. Both plasma and LV concentrations of the DDAH substrate, ADMA, were increased post-MI, although the ratio of Arg:ADMA was retained in the LV post-MI relative to sham operated controls. In conclusion, DDAH II has a distribution similar to eNOS in healthy myocardium. The increased levels and activity of DDAH I and DDAH II enzymes following myocardial infarction suggest a potential role for them in local protection of NOS enzymes from inhibition by methylated arginines during infarct healing.