Ferritin light chain deficiency-induced ferroptosis is involved in preeclampsia pathophysiology by disturbing uterine spiral artery remodelling.

The proteomic analysis from samples of patients with preeclampsia (PE) displayed a low level of ferritin light chains (FTL), but we do not know what the significance of reduced FTL in PE pathophysiology is. To address this question, we first demonstrated that FTL was expressed in first- and third-trimester cytotrophoblasts, including extravillous trophoblasts (EVTs), of the human placenta. Furthermore, a pregnant rat model of FTL knockdown was successfully established by intravenously injecting adenoviruses expressing shRNA targeting FTL. In pregnant rats with downregulated FTL, we observed PE-like phenotypes and impaired spiral arterial remodelling, implying a causal relationship between FTL downregulation and PE. Blocking ferroptosis with ferrostatin-1 (Fer-1) significantly rescued the above PE-like phenotypes in pregnant rats with FTL knockdown. Furthermore, using trophoblast cell line and chorionic villous explant culture assays, we showed that FTL downregulation induced cell death, especially ferroptosis, resulting in defective uterine spiral artery remodelling. Eventually, this conclusion from the animal model was verified in PE patients' placental tissues. Taken together, this study revealed for the first time that FTL reduction during pregnancy triggered ferroptosis and then caused defective uterine spiral artery remodelling, thereby leading to PE.

Killer Timing: The Temporal Uterine Natural Killer Cell Differentiation Pathway and Implications for Female Reproductive Health.

Natural killer (NK) cells are the predominant maternal uterine immune cell component, and they densely populate uterine mucosa to promote key changes in the post-ovulatory endometrium and in early pregnancy. It is broadly accepted that (a) immature, inactive endometrial NK (eNK) cells in the pre-ovulatory endometrium become activated and transition into decidual NK (dNK) cells in the secretory stage, peri-implantation endometrium, and continue to mature into early pregnancy; and (b) that secretory-stage and early pregnancy dNK cells promote uterine vascular growth and mediate trophoblast invasion, but do not exert their killing function. However, this may be an overly simplistic view. Evidence of specific dNK functional killer roles, as well as opposing effects of dNK cells on the uterine vasculature before and after conception, indicates the presence of a transitory secretory-stage dNK cell (s-dNK) phenotype with a unique angiodevelopmental profile during the peri-implantation period, that is that is functionally distinct from the angiomodulatory dNK cells that promote vessel destabilisation and vascular cell apoptosis to facilitate uterine vascular changes in early pregnancy. It is possible that abnormal activation and differentiation into the proposed transitory s-dNK phenotype may have implications in uterine pathologies ranging from infertility to cancer, as well as downstream effects on dNK cell differentiation in early pregnancy. Further, dysregulated transition into the angiomodulatory dNK phenotype in early pregnancy will likely have potential repercussions for adverse pregnancy outcomes, since impaired dNK function is associated with several obstetric complications. A comprehensive understanding of the uterine NK cell temporal differentiation pathway may therefore have important translational potential due to likely NK phenotypic functional implications in a range of reproductive, obstetric, and gynaecological pathologies.

From stem cells to spiral arteries: A journey through early placental development.

Early placental development lays the foundation of a healthy pregnancy, and numerous tightly regulated processes must occur for the placenta to meet the increasing nutrient and oxygen exchange requirements of the growing fetus later in gestation. Inadequacies in early placental development can result in disorders such as fetal growth restriction that do not present clinically until the second half of gestation. Indeed, growth restricted placentae exhibit impaired placental development and function, including reduced overall placental size, decreased branching of villi and the blood vessels within them, altered trophoblast function, and impaired uterine vascular remodelling, which together combine to reduce placental exchange capacity. This review explores the importance of early placental development across multiple anatomical aspects of placentation, from the stem cells and lineage hierarchies from which villous core cells and trophoblasts arise, through extravillous trophoblast invasion and spiral artery remodelling, and finally remodelling of the larger uterine vessels.

Effect of lncRNA-ATB/miR-651-3p/Yin Yang 1 pathway on trophoblast-endothelial cell interaction networks.

Our previous studies have confirmed that lncRNA-ATB may be involved in the pathogenesis of preeclampsia, however, it is uncertain whether lncRNA-ATB influence the interaction between trophoblast and endothelial cells, which is crucial to the uterine spiral artery remodelling. Scratch wound healing and transwell invasion assay were conducted to test the migration and invasion of trophoblast cells. Co-culture model was used to simulate the physiological environment in vivo. The expression levels of lncRNA-ATB were analyzed in placenta tissues from healthy pregnant women and preeclampsia patients. Subsequently, the binding site of lncRNA-ATB and miR-651-3p was verified using dual-luciferase reporter assay, and the rescue experiment was used to study the effects of these two on the biological function. The direct effects of miR-651-3p and Yin Yang 1 (YY1) were verified using similar methods. LncRNA-ATB was found to be down-regulated in the placenta of preeclampsia patients. LncRNA-ATB knockdown decreased trophoblast migration, invasion and colocalisation with human umbilical vein endothelial cells. MiR-651-3p was a direct target of lncRNA-ATB and they had opposite effects. Moreover, the expression of lncRNA-ATB and miR-651-3p in placental tissues was negatively correlated. MiR-651-3p has been confirmed to directly target the 3' untranslated region of YY1. The inhibitory effects of YY1 low expression on biological function was rescued by miR-651-3p depletion. Western blot analysis showed that lncRNA-ATB could regulate YY1 expression by sponging miR-651-3p. LncRNA-ATB functioned as a competitive endogenous RNA of miR-651-3p to regulate YY1 on progress of spiral artery remodelling.

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%.

Molecular regulation of vascular smooth muscle cell phenotype switching by trophoblast cells at the maternal-fetal interface.

INTRODUCTION: Establishment of hemochorial placenta is associated with development and remodelling of uterine vasculature at the maternal fetal interface. This results in calibration of high resistance uterine arteries to flaccid low resistance vessels resulting in increased blood flow to the placenta and fetus in humans and rodents. Mechanisms underlying these remodelling events are poorly understood. In this report, we examine regulation of vascular remodelling using vascular smooth muscle cell (VSMC) phenotype switching as a primary parameter. METHODS: Cellular dynamics was assessed by Immunofluorescence, qRT-PCR, western blotting in timed pregnant rat tissue. In vitro co-culture of trophoblast cells with vascular smooth muscle cells was used to understand regulation mechanism. RESULTS: Analysis of cellular dynamics on days 13.5, 16.5 and 19.5 of gestation in the rat metrial gland, the entry point of uterine arteries, revealed that invasion of trophoblast cells preceded disappearance of VSMC α-SMA, a contractile state marker. Co-culture of VSMCs with trophoblast cells in vitro recapitulated trophoblast-induced de-differentiation of VSMCs in vivo. Interestingly, co-culturing with trophoblast cells activated PDGFRß signalling in VSMCs, an effect mediated by secreted PDGF-BB from trophoblast cells. Trophoblast cells failed to elicit its effect on VSMC de-differentiation upon inhibition of PDGFRß signalling using a selective inhibitor. Moreover, co-culturing with trophoblast cells also led to substantial increase in Akt activation and a modest increase in Erk phosphorylation in VSMCs and this effect was abolished by PDGFRß inhibition. DISCUSSION: Our results highlight that trophoblast cells direct VSMC phenotype switching and trophoblast derived PDGF-BB is one of the modulator.

Deficiency of DICER reduces the invasion ability of trophoblasts and impairs the pro-angiogenic effect of trophoblast-derived microvesicles.

DICER is a key rate-limiting enzyme in the canonical miRNAs biogenesis pathway, and DICER and DICER-dependent miRNAs have been proved to play essential roles in many physiological and pathological processes. However, whether DICER is involved in placentation has not been studied. Successful spiral artery remodelling is one of the key milestones during placentation, which depends mostly on the invasion of trophoblasts and the crosstalk between trophoblasts and endothelial cells. In the present study, we show that DICER knockdown impairs the invasion ability of both primary extravillous trophoblasts (EVT) and HTR8/SVneo (HTR8) cell lines. The decreased invasion of HTR8 cells upon DICER knockdown (sh-Dicer) was partly due to the up-regulation of miR-16-2-3p, which led to a reduced expression level of the collagen type 1 alpha 2 chain (COL1A2) protein. Moreover, microvesicles (MVs) can be secreted by HTR8 cells and promote the tube formation ability of human umbilical cord vein endothelial cells (HUVECs). However, conditioned medium and MVs derived from sh-Dicer HTR8 cells have an anti-angiogenic effect, due to reduced angiogenic factors and increased anti-angiogenic miRNAs (including let-7d, miR-1-6-2 and miR-15b), respectively. In addition, reduced protein expression of DICER is found in PE placenta by immunoblotting and immunohistochemistry. In summary, our study uncovered a novel DICER-miR-16-2-COL1A2 mediated pathway involved in the invasion ability of EVT, and DICER-containing MVs mediate the pro-angiogenic effect of trophoblast-derived conditioned medium on angiogenesis, implying the involvement of DICER in the pathogenesis of PE.

Resveratrol promotes trophoblast invasion in pre-eclampsia by inducing epithelial-mesenchymal transition.

Impairment spiral arteries remodelling was considered to be the underlying cause of pathogenesis of pre-eclampsia (PE). Resveratrol (RE) was reported that it could modulate cellar phenotype to ameliorate diverse human diseases. However, the biological function of RE in PE remains poorly understood. In this report, we investigated the effect of RE on trophoblast phenotype both in vivo and in vitro. We conducted MTT and transwell assays to explore cell proliferation and invasion events in HTR-8/SVneo. In mice model, the clinical characteristics of PE were established through the injection of NG-nitro-l-arginine methyl ester (L-NAME). Furthermore, related experiments were performed to detect cellar phenotype-associated signalling pathway, including epithelial-mesenchymal transition (EMT) and Wnt/ß-catenin. Cell assays indicated that RE could increase trophoblasts migration and invasion. In addition, hypertension and proteinuria were markedly ameliorated by RE compared with the controls in PE mice model. Moreover, treatment by RE in trophoblasts or in PE model, we found that RE activated EMT progress through the regulation of E-cadherin, ß-catenin, N-cadherin, vimentin expression, and further altered the WNT-related gene expression, including WNT1, WNT3 and WNT5B. Our findings demonstrated that RE might stimulate the invasive capability of human trophoblasts by promoting EMT and mediating the Wnt/ß-catenin pathway in PE.

Aberrant maternal inflammation as a cause of pregnancy complications: A potential therapeutic target?

Pre-eclampsia (PE), fetal growth restriction (FGR), pre-term labour and fetal death are common complications of pregnancy often associated with abnormal maternal inflammation. Though the precise causes of these complications remain obscure, altered maternal blood flow to the placenta is an underlying hallmark, especially with respect to the pathogenesis of PE, FGR and fetal demise. Furthermore, deficient trophoblast-mediated spiral artery remodelling is often cited as the primary cause of impaired utero-placental perfusion. Considerably less attention has been directed towards investigating other factors, including maternal vasoconstriction or hemostatic alterations, as contributors to poor utero-placental perfusion. This review provides a rationale for investigating the role of abnormal maternal inflammation in the pathophysiology of pregnancy complications including PE, FGR and fetal demise. In particular, the association between aberrant maternal inflammation and inadequate utero-placental perfusion is considered in the context of inflammation-associated alterations in maternal hemostasis and vasoconstriction. Finally, the role of aberrant maternal inflammation as a cause of local oxidative/nitrosative stress is examined and the possibility of targeting deficient nitric oxide signalling as a therapeutic intervention for the treatment of inflammation-associated pregnancy complications is discussed.

Decidual cytokines and pregnancy complications: focus on spontaneous miscarriage.

The establishment of pregnancy requires the co-ordinated implantation of the embryo into the receptive decidua, placentation, trophoblast invasion of the maternal decidua and myometrium in addition to remodelling of the uterine spiral arteries. Failure of any of these steps can lead to a range of pregnancy complications, including miscarriage, pre-eclampsia, fetal growth restriction, placenta accreta and pre-term birth. Cytokines are small multifunctional proteins often derived from leucocytes and have primarily been described through their immunomodulatory actions. The maternal-fetal interface is considered to be immunosuppressed to allow development of the semi-allogeneic placental fetal unit. However, cytokine profiles of the decidua and different decidual cell types suggest that the in vivo situation might be more complex. Data suggest that decidual-derived cytokines not only play roles in immunosuppression, but also in other aspects of the establishment of pregnancy, including the regulation of trophoblast invasion and spiral artery remodelling. This review focuses on the potential role of decidua-derived cytokines in the aetiology of unexplained spontaneous miscarriage.

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.