Obstetrics and Gynaecology - Research Publications
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ItemBlocking Endogenous Leukemia Inhibitory Factor During Placental Development in Mice Leads to Abnormal Placentation and Pregnancy Loss(NATURE PORTFOLIO, 2015-08-14)The placenta forms the interface between the maternal and fetal circulation and is critical for the establishment of a healthy pregnancy. Specialized trophoblast cells derived from the embryonic trophectoderm play a pivotal role in the establishment of the placenta. Leukemia inhibitory factor (LIF) is one of the predominant cytokines present in the placenta during early pregnancy. LIF has been shown to regulate trophoblast adhesion and invasion in vitro, however its precise role in vivo is unknown. We hypothesized that LIF would be required for normal placental development in mice. LIF and LIFRα were immunolocalized to placental trophoblasts and fetal vessels in mouse implantation sites during mid-gestation. Temporally blocking LIF action during specific periods of placental development via intraperitoneal administration of our specific LIFRα antagonist, PEGLA, resulted in abnormal placental trophoblast and vascular morphology and reduced activated STAT3 but not ERK. Numerous genes regulating angiogenesis and oxidative stress were altered in the placenta in response to LIF inhibition. Pregnancy viability was also significantly compromised in PEGLA treated mice. Our data suggest that LIF plays an important role in placentation in vivo and the maintenance of healthy pregnancy.
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ItemLeukemia Inhibitory Factor (LIF) Inhibition during Mid-Gestation Impairs Trophoblast Invasion and Spiral Artery Remodelling during Pregnancy in Mice(PUBLIC LIBRARY SCIENCE, 2015-10-19)The placenta forms the interface between the maternal and fetal circulation and is critical for the establishment of a healthy pregnancy. Trophoblast cell proliferation, migration and invasion into the endometrium are fundamental events in the initiation of placentation. Leukemia inhibitory factor (LIF) has been shown to promote trophoblast invasion in vitro, however its precise role in trophoblast invasion in vivo is unknown. We hypothesized that LIF would be required for normal trophoblast invasion and spiral artery remodeling in mice. Both LIF and its receptor (LIFRα) co-localized with cytokeratin-positive invasive endovascular extravillous trophoblasts (EVT) in mouse implantation sites during mid-gestation. Temporally blocking LIF action during specific periods of placental development via administration of our unique LIFRα antagonist, PEGLA, resulted in abnormal trophoblast invasion and impaired spiral artery remodeling compared to PEG control. PEGLA-treated mouse decidual vessels were characterized by retention of α-smooth muscle actin (αSMA)-positive vascular smooth muscle cells (VSMCs), while PEG control decidual vessels were remodelled by cytokeratin-positive trophoblasts. LIF blockade did not alter F4/80-positive decidual macrophage numbers between treatment groups, but resulted in down-regulation of decidual transcript levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10), which are important immune cell activation factors that promote spiral artery remodeling during pregnancy. Our data suggest that LIF plays an important role in trophoblast invasion in vivo and may facilitate trophoblast-decidual-immune cell cross talk to enable adequate spiral artery remodeling.
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ItemEffects of Estrogens on Adipokines and Glucose Homeostasis in Female Aromatase Knockout Mice(PUBLIC LIBRARY SCIENCE, 2015-08-28)The maintenance of glucose homeostasis within the body is crucial for constant and precise performance of energy balance and is sustained by a number of peripheral organs. Estrogens are known to play a role in the maintenance of glucose homeostasis. Aromatase knockout (ArKO) mice are estrogen-deficient and display symptoms of dysregulated glucose metabolism. We aim to investigate the effects of estrogen ablation and exogenous estrogen administration on glucose homeostasis regulation. Six month-old female wildtype, ArKO, and 17β-estradiol (E2) treated ArKO mice were subjected to whole body tolerance tests, serum examination of estrogen, glucose and insulin, ex-vivo muscle glucose uptake, and insulin signaling pathway analyses. Female ArKO mice display increased body weight, gonadal (omental) adiposity, hyperinsulinemia, and liver triglycerides, which were ameliorated upon estrogen treatment. Tolerance tests revealed that estrogen-deficient ArKO mice were pyruvate intolerant hence reflecting dysregulated hepatic gluconeogenesis. Analyses of skeletal muscle, liver, and adipose tissues supported a hepatic-based glucose dysregulation, with a down-regulation of Akt phosphorylation (a key insulin signaling pathway molecule) in the ArKO liver, which was improved with E2 treatment. Concurrently, estrogen treatment lowered ArKO serum leptin and adiponectin levels and increased inflammatory adipokines such as tumour necrosis factor alpha (TNFα) and interleukin 6 (IL6). Furthermore, estrogen deficiency resulted in the infiltration of CD45 macrophages into gonadal adipose tissues, which cannot be reversed by E2 treatment. This study describes the effects of estrogens on glucose homeostasis in female ArKO mice and highlights a primary phenotype of hepatic glucose dysregulation and a parallel estrogen modified adipokine profile.
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ItemDecidual-Secreted Factors Alter Invasive Trophoblast Membrane and Secreted Proteins Implying a Role for Decidual Cell Regulation of Placentation(PUBLIC LIBRARY SCIENCE, 2012-02-16)Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the 'extravillous trophoblast' (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10(-8) M), medroxyprogesterone acetate (10(-7) M) and cAMP (0.5 mM) for 14 days. Conditioned media (CM) was collected on day 2 (non-decidualized CM) and 14 (decidualized CM) of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN) before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1), dipeptidyl peptidase 1 (DPP1/cathepsin C) and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro-inflammatory condition. Overall, we have demonstrated the potential of a proteomics approach to identify novel proteins expressed by EVT and to uncover the mechanisms leading to disease states.
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ItemTherapeutically blocking Interleukin-11 Receptor-a enhances doxorubicin cytotoxicity in high grade type I endometrioid tumours(IMPACT JOURNALS LLC, 2017-04-04)High grade type I endometrial cancers have poor prognosis. Interleukin (IL)11 is elevated in tumours and uterine lavage with increasing tumour grade in women. IL11 regulates cell cycle, invasion and migration and we recently demonstrated that IL11 receptor (R)α inhibition impaired low and moderate grade endometrial tumourigenesis in vivo. In this report, we hypothesized that micro-RNA(miR)-1 regulates IL11 and that IL11 promotes high grade endometrial tumour growth. We aimed to determine whether combination treatment using an anti-human IL11Rα blocking antibody (Ab) and doxorubicin chemotherapeutic impairs high grade tumour growth. MiR-1 was absent in human endometrial tumours versus human benign endometrium (n = 10/group). Transfection with miR-1 mimic restored miR-1 expression, down-regulated IL11 mRNA and impaired cell viability in grade 3-derived AN3CA human endometrial epithelial cancer cells. AN3CA cell proliferation was reduced in response to Ab and doxorubicin combination treatment versus Ab, IgG control, or doxorubicin alone. Subcutaneous xenograft tumours were established in female Balb/c athymic nude mice using AN3CA cells expressing IL11 and IL11Rα. Administration of recombinant human IL11 to mice (n = 4/group) activated IL11 downstream target, signal transducers and activators of transcription (STAT3) and significantly increased tumour growth (p < 0.05), suggesting that IL11 promotes high grade tumour growth. IL11Rα blocking Ab reduced STAT3 phosphorylation and combination treatment with doxorubicin resulted in a significant reduction in tumour growth (p < 0.05) compared to Ab, doxorubicin, or IgG control. Our data suggest that therapeutically targeting IL11Rα in combination with doxorubicin chemotherapy could inhibit high grade type I endometrioid cancer growth.
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ItemHuman Blastocyst Secreted microRNA Regulate Endometrial Epithelial Cell Adhesion(ELSEVIER SCIENCE BV, 2015-10)Successful embryo implantation requires synchronous development and communication between the blastocyst and the endometrium, however the mechanisms of communication in humans are virtually unknown. Recent studies have revealed that microRNAs (miRs) are present in bodily fluids and secreted by cells in culture. We have identified that human blastocysts differentially secrete miRs in a pattern associated with their implantation outcome. miR-661 was the most highly expressed miR in blastocyst culture media (BCM) from blastocysts that failed to implant (non-implanted) compared to blastocysts that implanted (implanted). Our results indicate a possible role for Argonaute 1 in the transport of miR-661 in non-implanted BCM and taken up by primary human endometrial epithelial cells (HEECs). miR-661 uptake by HEEC reduced trophoblast cell line spheroid attachment to HEEC via PVRL1. Our results suggest that human blastocysts alter the endometrial epithelial adhesion, the initiating event of implantation, via the secretion of miR, abnormalities in which result in implantation failure.
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ItemInterleukin 11 is upregulated in uterine lavage and endometrial cancer cells in women with endometrial carcinoma(BMC, 2010-06-17)BACKGROUND: Interleukin (IL) 11 is produced by human endometrium and endometrial cancer tissue. It has roles in endometrial epithelial cell adhesion and trophoblast cell invasion, two important processes in cancer progression. This study aimed to determine the levels of IL11 in uterine lavage fluid in women with endometrial cancer and postmenopausal women. It further aimed to determine the levels of IL11 protein and its signaling molecules in human endometrial cancer of varying grades, and endometrium from postmenopausal women and IL11 signalling mechanisms in endometrial cancer cell lines. METHODS: IL11 levels in uterine lavage were measured by ELISA. IL11, IL11 receptor(R) alpha, phosphorylated (p) STAT3 and SOCS3 were examined by immunohistochemistry in endometrial carcinomas and in control endometrium from postmenopausal women and normal cycling women. The effect of IL11 on pSTAT3/STAT3 and SOCS3 protein abundance in endometrial cancer cell lines and non-cancer endometrial epithelial cells was determined by Western blot. RESULTS: IL11 was present in uterine flushings and was significantly higher in women with Grade 1 carcinomas compared to postmenopausal women (p < 0.05). IL11 immunostaining was significantly elevated in the endometrial tumour epithelial cells from Grade 1 and 3 compared to endometrial epithelium from postmenopausal and cycling women. IL11R alpha immunostaining intensity was increased in cancer epithelium in the Grades 1 and 2 tumours compared to epithelium from postmenopausal women. Both IL11 and IL11R alpha localized to vascular endothelial and smooth muscle cells while IL11 also localized to subsets of leucocytes in the cancer tissues. pSTAT3 was found in both the tumour epithelial and stromal compartments but was maximal in the tumour epithelial cells, while SOCS3 was predominantly found in the tumour epithelial cells. pSTAT3 staining intensity was significantly higher in Grade 1 and 2 tumour epithelial cells compared to epithelial cells from cycling and postmenopausal women. SOCS3 staining intensity did not differ between between each tumour and postmenopausal endometrial epithelium but SOCS3 in cycling endometrium was significantly higher compared to postmenopausal and Tumour Grades 2 and 3. IL11 increased pSTAT3/STAT3 in all tumour cell lines, while SOCS3 abundance was increased only in one tumour cell line. CONCLUSIONS: The present study suggests that IL11 in uterine washings may be useful as a diagnostic marker for early stage endometrial cancer. It indicates that IL11, along with its specific receptor, IL11R alpha, and downstream signalling molecules, STAT3 and SOCS3, are likely to play a role in the progression of endometrial carcinoma. The precise role of IL11 in endometrial cancer remains to be elucidated.
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ItemLeukemia Inhibitory Factor Enhances Endometrial Stromal Cell Decidualization in Humans and Mice(PUBLIC LIBRARY SCIENCE, 2011-09-23)Adequate differentiation or decidualization of endometrial stromal cells (ESC) is critical for successful pregnancy in humans and rodents. Here, we investigated the role of leukemia inhibitory factor (LIF) in human and murine decidualization. Ex vivo human (H) ESC decidualization was induced by estrogen (E, 10(-8) M) plus medroxyprogesterone acetate (MPA, 10(-7) M). Exogenous LIF (≥50 ng/ml) induced STAT3 phosphorylation in non-decidualized and decidualized HESC and enhanced E+MPA-induced decidualization (measured by PRL secretion, P<0.05). LIF mRNA in HESC was down-regulated by decidualization treatment (E+MPA) whereas LIF receptor (R) mRNA was up-regulated, suggesting that the decidualization stimulus 'primed' HESC for LIF action, but that factors not present in our in vitro model were required to induce LIF expression. Ex vivo first trimester decidual biopsies secreted >100 pg/mg G-CSF, IL6, IL8, and MCP1. Decidualized HESC secreted IL6, IL8, IL15 and MCP1. LIF (50 ng/ml) up-regulated IL6 and IL15 (P<0.05) secretion in decidualized HESC compared to 0.5 ng/ml LIF. In murine endometrium, LIF and LIFR immunolocalized to decidualized stromal cells on day 5 of gestation (day 0 = day of plug detection). Western blotting confirmed that LIF and the LIFR were up-regulated in intra-implantation sites compared to inter-implantation sites on Day 5 of gestation. To determine the role of LIF during in vivo murine decidualization, intra-peritoneal injections of a long-acting LIF antagonist (PEGLA; 900 or 1200 µg) were given just post-attachment, during the initiation of decidualization on day 4. PEGLA treatment reduced implantation site decidual area (P<0.05) and desmin staining immuno-intensity (P<0.05) compared to control on day 6 of gestation. This study demonstrated that LIF was an important regulator of decidualization in humans and mice and data provides insight into the processes underlying decidualization, which are important for understanding implantation and placentation.
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ItemProteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro(BMC, 2011-05-30)BACKGROUND: During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL) 11 regulates human endometrial epithelial cells (hEEC) adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. METHODS: Using a 2D-differential in-gel electrophoresis (DIGE) electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2) and flotillin-1 (FLOT1), were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa) and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle). RESULTS: 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary hEEC and ECC-1 whilst, the lipid-raft protein FLOT1 demonstrated punctate staining in the apical surface of ECC-1 plasma membranes and was upregulated in the epithelium in the receptive phase of the menstrual cycle (p lower or equal 0.05). CONCLUSIONS: This is the first study to use a proteomics approach to identify hEEC plasma membrane proteins that may be useful as infertility markers or pharmacological targets for fertility regulation.
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ItemSubcellular localization of L-selectin ligand in the endometrium implies a novel function for pinopodes in endometrial receptivity(BMC, 2012-06-15)BACKGROUND: Apical surfaces of human endometrial epithelium and endothelium are key elements for the initiation of molecular interactions to capture the blastocyst or leukocyte, respectively. The L-selectin adhesion system has been strongly proposed to play an important role in the initial steps of trophoblast adhesion and promotion of integrin-dependent processes, ultimately culminating in the establishment of the embryo-maternal interface. On the basis of these facts, we hypothesized a novel role for pinopodes as the first embryo-fetal contact sites to contain the highest subcellular expression of L-selectin ligand suggesting its role in early adhesion as predicted. Thus, the objective of this study was therefore to determine the subcellular pattern of distribution of the L-selectin ligand (MECA-79) in human endometrial apical membrane region during the window of implantation. METHODS: Endometrial biopsies of secretory phases from fertile females ranging in age between 25 and 42 years were studied using several approaches, including scanning electron microscopy (SEM), immunostaining for light microscopy and transmission electron microscopy (TEM), and immunoblotting as well as statistical analysis of the area-related numerical densities of immunoreactive MECA-79-bound nanogolds to detect the expression pattern and the subcellular distribution pattern of L-selectin ligand (MECA-79) in human endometrium during the window of implantation. RESULTS: The endometrial biopsies were scored according the dating criteria of Noyes et al. by an experienced histologist. The SEM images of the midluteal phase specimens revealed that fully developed pinopodes were abundant in our samples. HRP-immunostaining and immunofluorescent staining as well as immunoblotting revealed that MECA-79 was expressed in the midluteal phase specimens. The results of immunogold TEM illustrated the expression of MECA-79 in human pinopodes in the midluteal phase and a higher area-relate numerical density in pinopodes compared to that of the uterodome-free areas. CONCLUSIONS: This is the first demonstration of the subcellular localization of MECA-79 in the human pinopodes which may indicate a novel role for pinopodes to be capable of shear-stress-dependent tethering-type adhesion in the initial phases of human embryo implantation.