Obstetrics and Gynaecology - Research Publications

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    A molecular staging model for accurately dating the endometrial biopsy
    Teh, WT ; Chung, J ; Holdsworth-Carson, SJ ; Donoghue, JF ; Healey, M ; Rees, HC ; Bittinger, S ; Obers, V ; Sloggett, C ; Kendarsari, R ; Fung, JN ; Mortlock, S ; Montgomery, GW ; Girling, JE ; Rogers, PAW (NATURE PORTFOLIO, 2023-10-06)
    Natural variability in menstrual cycle length, coupled with rapid changes in endometrial gene expression, makes it difficult to accurately define and compare different stages of the endometrial cycle. Here we develop and validate a method for precisely determining endometrial cycle stage based on global gene expression. Our 'molecular staging model' reveals significant and remarkably synchronised daily changes in expression for over 3400 endometrial genes throughout the cycle, with the most dramatic changes occurring during the secretory phase. Our study significantly extends existing data on the endometrial transcriptome, and for the first time enables identification of differentially expressed endometrial genes with increasing age and different ethnicities. It also allows reinterpretation of all endometrial RNA-seq and array data that has been published to date. Our molecular staging model will significantly advance understanding of endometrial-related disorders that affect nearly all women at some stage of their lives, such as heavy menstrual bleeding, endometriosis, adenomyosis, and recurrent implantation failure.
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    The genetic basis of endometriosis and comorbidity with other pain and inflammatory conditions
    Rahmioglu, N ; Mortlock, S ; Ghiasi, M ; Moller, PL ; Stefansdottir, L ; Galarneau, G ; Turman, C ; Danning, R ; Law, MH ; Sapkota, Y ; Christofidou, P ; Skarp, S ; Giri, A ; Banasik, K ; Krassowski, M ; Lepamets, M ; Marciniak, B ; Noukas, M ; Perro, D ; Sliz, E ; Sobalska-Kwapis, M ; Thorleifsson, G ; Topbas-Selcuki, NF ; Vitonis, A ; Westergaard, D ; Arnadottir, R ; Burgdorf, KS ; Campbell, A ; Cheuk, CSK ; Clementi, C ; Cook, J ; De Vivo, I ; DiVasta, A ; Dorien, O ; Donoghue, JF ; Edwards, T ; Fontanillas, P ; Fung, JN ; Geirsson, RT ; Girling, JE ; Harkki, P ; Harris, HR ; Healey, M ; Heikinheimo, O ; Holdsworth-Carson, S ; Hostettler, IC ; Houlden, H ; Houshdaran, S ; Irwin, JC ; Jarvelin, M-R ; Kamatani, Y ; Kennedy, SH ; Kepka, E ; Kettunen, J ; Kubo, M ; Kulig, B ; Kurra, V ; Laivuori, H ; Laufer, MR ; Lindgren, CM ; MacGregor, S ; Mangino, M ; Martin, NG ; Matalliotaki, C ; Matalliotakis, M ; Murray, AD ; Ndungu, A ; Nezhat, C ; Olsen, CM ; Opoku-Anane, J ; Padmanabhan, S ; Paranjpe, M ; Peters, M ; Polak, G ; Porteous, DJ ; Rabban, J ; Rexrode, KM ; Romanowicz, H ; Saare, M ; Saavalainen, L ; Schork, AJ ; Sen, S ; Shafrir, AL ; Siewierska-Gorska, A ; Slomka, M ; Smith, BH ; Smolarz, B ; Szaflik, T ; Szyllo, K ; Takahashi, A ; Terry, KL ; Tomassetti, C ; Treloar, SA ; Vanhie, A ; Vincent, K ; Vo, KC ; Werring, DJ ; Zeggini, E ; Zervou, M ; Stefansson, K ; Nyegaard, M ; Uimari, O ; Yurttas-Beim, P ; Tung, JY ; Adachi, S ; Buring, JE ; Ridker, PM ; D'Hooghe, T ; Goulielmos, GN ; Hapangama, DK ; Hayward, C ; Horne, AW ; Low, S-K ; Martikainen, H ; Chasman, D ; Rogers, PAW ; Saunders, PT ; Sirota, M ; Spector, T ; Strapagiel, D ; Whiteman, DC ; Giudice, LC ; Velez-Edwards, DR ; Kraft, P ; Salumets, A ; Nyholt, DR ; Magi, R ; Becker, CM ; Steinthorsdottir, V ; Missmer, SA ; Montgomery, GW ; Morris, AP ; Zondervan, KT (NATURE PORTFOLIO, 2023-03)
    Endometriosis is a common condition associated with debilitating pelvic pain and infertility. A genome-wide association study meta-analysis, including 60,674 cases and 701,926 controls of European and East Asian descent, identified 42 genome-wide significant loci comprising 49 distinct association signals. Effect sizes were largest for stage 3/4 disease, driven by ovarian endometriosis. Identified signals explained up to 5.01% of disease variance and regulated expression or methylation of genes in endometrium and blood, many of which were associated with pain perception/maintenance (SRP14/BMF, GDAP1, MLLT10, BSN and NGF). We observed significant genetic correlations between endometriosis and 11 pain conditions, including migraine, back and multisite chronic pain (MCP), as well as inflammatory conditions, including asthma and osteoarthritis. Multitrait genetic analyses identified substantial sharing of variants associated with endometriosis and MCP/migraine. Targeted investigations of genetically regulated mechanisms shared between endometriosis and other pain conditions are needed to aid the development of new treatments and facilitate early symptomatic intervention.
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    Gene expression of the endocannabinoid system in endometrium through menstrual cycle
    Tanaka, K ; Amoako, AA ; Mortlock, S ; Rogers, PAW ; Holdsworth-Carson, SJ ; Donoghue, JF ; Teh, WT ; Montgomery, GW ; McKinnon, B (NATURE PORTFOLIO, 2022-06-07)
    Endocannabinoids mediate cellular functions and their activity is controlled by a complex system of enzymes, membrane receptors and transport molecules. Endocannabinoids are present in endometrium, a cyclical regenerative tissue requiring tightly regulated cellular mechanisms for maturation. The objective of this study was to investigate the gene expression of key elements involved in the endocannabinoid system across the menstrual cycle. RNA was isolated from endometrial tissue and genome-wide gene expression datasets were generated using RNA-sequencing. An a priori set of 70 genes associated with endocannabinoid system were selected from published literature. Gene expression across the menstrual cycle was analyzed using a moderated t test, corrected for multiple testing with Bonferroni's method. A total of 40 of the 70 genes were present in > 90% of the samples, and significant differential gene expression identified for 29 genes. We identified 4 distinct regulation patterns for synthesizing enzymes, as well as a distinct regulation pattern for degradations and transporting enzymes. This study charts the expression of endometrial endocannabinoid system genes across the menstrual cycle. Altered expression of genes that control endocannabinoid may allow fine control over endocannabinoid concentrations and their influence on cellular function, maturation and differentiation as the endometrium matures through the menstrual cycle.
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    Dilated Thin-Walled Blood and Lymphatic Vessels in Human Endometrium: A Potential Role for VEGF-D in Progestin-Induced Break-Through Bleeding (vol 7, e30916, 2012)
    Donoghue, JF ; McGavigan, CJ ; Lederman, FL ; Cann, LM ; Fu, L ; Dimitriadis, E ; Girling, JE ; Rogers, PAW (PUBLIC LIBRARY SCIENCE, 2021-10-26)
    [This corrects the article DOI: 10.1371/journal.pone.0030916.].
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    Peripheral, Central, and Cross Sensitization in Endometriosis-Associated Pain and Comorbid Pain Syndromes.
    McNamara, HC ; Frawley, HC ; Donoghue, JF ; Readman, E ; Healey, M ; Ellett, L ; Reddington, C ; Hicks, LJ ; Harlow, K ; Rogers, PAW ; Cheng, C (Frontiers Media S.A., 2021)
    Endometriosis-associated pain and the mechanisms responsible for its initiation and persistence are complex and difficult to treat. Endometriosis-associated pain is experienced as dysmenorrhea, cyclical pain related to organ function including dysuria, dyschezia and dyspareunia, and persistent pelvic pain. Pain symptomatology correlates poorly with the extent of macroscopic disease. In addition to the local effects of disease, endometriosis-associated pain develops as a product of peripheral sensitization, central sensitization and cross sensitization. Endometriosis-associated pain is further contributed to by comorbid pain conditions, such as bladder pain syndrome, irritable bowel syndrome, abdomino-pelvic myalgia and vulvodynia. This article will review endometriosis-associated pain, its mechanisms, and its comorbid pain syndromes with a view to aiding the clinician in navigating the literature and terminology of pain and pain syndromes. Limitations of our current understanding of endometriosis-associated pain will be acknowledged. Where possible, commonalities in pain mechanisms between endometriosis-associated pain and comorbid pain syndromes will be highlighted.
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    Vascular endothelial growth factor-D over-expressing tumor cells induce differential effects on uterine vasculature in a mouse model of endometrial cancer
    Girling, JE ; Donoghue, JF ; Lederman, FL ; Cann, LM ; Achen, MG ; Stacker, SA ; Rogers, PAW (BIOMED CENTRAL LTD, 2010-07-08)
    BACKGROUND: It has been hypothesised that increased VEGF-D expression may be an independent prognostic factor for endometrial cancer progression and lymph node metastasis; however, the mechanism by which VEGF-D may promote disease progression in women with endometrial cancer has not been investigated. Our aim was to describe the distribution of lymphatic vessels in mouse uterus and to examine the effect of VEGF-D over-expression on these vessels in a model of endometrial cancer. We hypothesised that VEGF-D over-expression would stimulate growth of new lymphatic vessels into the endometrium, thereby contributing to cancer progression. METHODS: We initially described the distribution of lymphatic vessels (Lyve-1, podoplanin, VEGFR-3) and VEGF-D expression in the mouse uterus during the estrous cycle, early pregnancy and in response to estradiol-17beta and progesterone using immunohistochemistry. We also examined the effects of VEGF-D over-expression on uterine vasculature by inoculating uterine horns in NOD SCID mice with control or VEGF-D-expressing 293EBNA tumor cells. RESULTS: Lymphatic vessels positive for the lymphatic endothelial cell markers Lyve-1, podoplanin and VEGFR-3 profiles were largely restricted to the connective tissue between the myometrial circular and longitudinal muscle layers; very few lymphatic vessel profiles were observed in the endometrium. VEGF-D immunostaining was present in all uterine compartments (epithelium, stroma, myometrium), although expression was generally low. VEGF-D immunoexpression was slightly but significantly higher in estrus relative to diestrus; and in estradiol-17beta treated mice relative to vehicle or progesterone treated mice. The presence of VEGF-D over-expressing tumor cells did not induce endometrial lymphangiogenesis, although changes were observed in existing vessel profiles. For myometrial lymphatic and endometrial blood vessels, the percentage of profiles containing proliferating endothelial cells, and the cross sectional area of vessel profiles were significantly increased in response to VEGF-D in comparison to control tumor cells. In contrast, no significant changes were noted in myometrial blood vessels. In addition, examples of invading cells or tumor emboli were observed in mice receiving VEGF-D expressing 293EBNA cells. CONCLUSIONS: These results illustrate that VEGF-D over-expression has differential effects on the uterine vasculature. These effects may facilitate VEGF-D's ability to promote endometrial cancer metastasis and disease progression.
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    The regulation of mitochondrial DNA copy number in glioblastoma cells
    Dickinson, A ; Yeung, KY ; Donoghue, J ; Baker, MJ ; Kelly, RDW ; McKenzie, M ; Johns, TG ; St John, JC (NATURE PUBLISHING GROUP, 2013-12)
    As stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions. It is not understood whether tumor-initiating cells regulate their mtDNA in a similar manner or whether mtDNA is essential for tumorigenesis. We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity. Differentiating multipotent glioblastoma cells failed to match the expansion in mtDNA copy number, patterns of gene expression and increased respiratory capacity observed in hNSCs. Partial depletion of glioblastoma cell mtDNA rescued mtDNA replication events and enhanced cell differentiation. However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH). The transfer of glioblastoma cells depleted to varying degrees of their mtDNA content into immunocompromised mice resulted in tumors requiring significantly longer to form compared with non-depleted cells. The number of tumors formed and the time to tumor formation was relative to the degree of mtDNA depletion. The tumors derived from mtDNA depleted glioblastoma cells recovered their mtDNA copy number as part of the tumor formation process. These outcomes demonstrate the importance of mtDNA to the initiation and maintenance of tumorigenesis in glioblastoma multiforme.
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    Non-Agonistic Bivalent Antibodies That Promote c-MET Degradation and Inhibit Tumor Growth and Others Specific for Tumor Related c-MET
    Greenall, SA ; Gherardi, E ; Liu, Z ; Donoghue, JF ; Vitali, AA ; Li, Q ; Murphy, R ; Iamele, L ; Scott, AM ; Johns, TG ; Li, J (PUBLIC LIBRARY SCIENCE, 2012-04-12)
    The c-MET receptor has a function in many human cancers and is a proven therapeutic target. Generating antagonistic or therapeutic monoclonal antibodies (mAbs) targeting c-MET has been difficult because bivalent, intact anti-Met antibodies frequently display agonistic activity, necessitating the use of monovalent antibody fragments for therapy. By using a novel strategy that included immunizing with cells expressing c-MET, we obtained a range of mAbs. These c-MET mAbs were tested for binding specificity and anti-tumor activity using a range of cell-based techniques and in silico modeling. The LMH 80 antibody bound an epitope, contained in the small cysteine-rich domain of c-MET (amino acids 519-561), that was preferentially exposed on the c-MET precursor. Since the c-MET precursor is only expressed on the surface of cancer cells and not normal cells, this antibody is potentially tumor specific. An interesting subset of our antibodies displayed profound activities on c-MET internalization and degradation. LMH 87, an antibody binding the loop connecting strands 3d and 4a of the 7-bladed β-propeller domain of c-MET, displayed no intrinsic agonistic activity but promoted receptor internalization and degradation. LMH 87 inhibited HGF/SF-induced migration of SK-OV-3 ovarian carcinoma cells, the proliferation of A549 lung cancer cells and the growth of human U87MG glioma cells in a mouse xenograft model. These results indicate that c-MET antibodies targeting epitopes controlling receptor internalization and degradation provide new ways of controlling c-MET expression and activity and may enable the therapeutic targeting of c-MET by intact, bivalent antibodies.
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    Mitochondrial DNA copy number is regulated by DNA methylation and demethylation of POLGA in stem and cancer cells and their differentiated progeny
    Lee, W ; Johnson, J ; Gough, DJ ; Donoghue, J ; Cagnone, GLM ; Vaghjiani, V ; Brown, KA ; Johns, TG ; St John, JC (NATURE PUBLISHING GROUP, 2015-02)
    Mitochondrial DNA (mtDNA) copy number is strictly regulated during differentiation so that cells with a high requirement for ATP generated through oxidative phosphorylation have high mtDNA copy number, whereas those with a low requirement have few copies. Using immunoprecipitation of DNA methylation on 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), which distinguish between de novo DNA methylation and demethylation, respectively, we set out to determine whether DNA methylation at exon 2 of the human mtDNA-specific polymerase (DNA polymerase gamma A (POLGA)) regulates cell-specific mtDNA copy number in highly proliferative and terminally differentiated cells. Highly proliferative cancer and pluripotent and multipotent cells possessed low mtDNA copy number and were highly methylated at exon 2 of POLGA in contrast to post-mitotic cells. Unlike neural stem cells, cancer cells were unable to differentiate and remained extensively DNA methylated at exon 2 of POLGA. However, mtDNA depletion of cancer cells reduced DNA methylation at exon 2 of POLGA as they replenished mtDNA to form tumours in mice. Glioblastoma cells treated with the DNA demethylation agent 5-azacytidine over 28 days of astrocyte-induced differentiation demethylated exon 2 of POLGA leading to increased mtDNA copy number and expression of the astrocyte endpoint marker glial fibrillary acidic protein (GFAP). However, the demethylation agent vitamin C (VitC) was unable to sustain increased mtDNA copy number and differentiation, as was the case when VitC was withdrawn after short-term treatment. These data demonstrate that DNA demethylation of POLGA is an essential regulator of mtDNA copy number and cellular fate and that cancer cells are only able to modulate DNA methylation of POLGA and mtDNA copy number in the presence of a DNA demethylation agent that inhibits de novo methyltransferase 1 activity.
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    Glioma Specific Extracellular Missense Mutations in the First Cysteine Rich Region of Epidermal Growth Factor Receptor (EGFR) Initiate Ligand Independent Activation.
    Ymer, SI ; Greenall, SA ; Cvrljevic, A ; Cao, DX ; Donoghue, JF ; Epa, VC ; Scott, AM ; Adams, TE ; Johns, TG (MDPI AG, 2011-04-18)
    The epidermal growth factor receptor (EGFR) is overexpressed or mutated in glioma. Recently, a series of missense mutations in the extracellular domain (ECD) of EGFR were reported in glioma patients. Some of these mutations clustered within a cysteine-rich region of the EGFR targeted by the therapeutic antibody mAb806. This region is only exposed when EGFR activates and appears to locally misfold during activation. We expressed two of these mutations (R324L and E330K) in NR6 mouse fibroblasts, as they do not express any EGFR-related receptors. Both mutants were autophosphorylated in the absence of ligand and enhanced cell survival and anchorage-independent and xenograft growth. The ECD truncation that produces the de2-7EGFR (or EGFRvIII), the most common EGFR mutation in glioma, generates a free cysteine in this same region. Using a technique optimized for detecting disulfide-bonded dimers, we definitively demonstrated that the de2-7EGFR is robustly dimerized and that ablation of the free cysteine prevents dimerization and activation. Modeling of the R324L mutation suggests it may cause transient breaking of disulfide bonds, leading to similar disulfide-bonded dimers as seen for the de2-7EGFR. These ECD mutations confirm that the cysteine-rich region of EGFR around the mAb806 epitope has a significant role in receptor activation.