A molecular staging model for accurately dating the endometrial biopsy.

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.

Using a Quantitative High-Throughput Screening Platform to Identify Molecular Targets and Compounds as Repurposing Candidates for Endometriosis.

Endometriosis, defined as the growth of hormonally responsive endometrial-like tissue outside of the uterine cavity, is an estrogen-dependent, chronic, pro-inflammatory disease that affects up to 11.4% of women of reproductive age and gender-diverse people with a uterus. At present, there is no long-term cure, and the identification of new therapies that provide a high level of efficacy and favourable long-term safety profiles with rapid clinical access are a priority. In this study, quantitative high-throughput compound screens of 3517 clinically approved compounds were performed on patient-derived immortalized human endometrial stromal cell lines. Following assay optimization and compound criteria selection, a high-throughput screening protocol was developed to enable the identification of compounds that interfered with estrogen-stimulated cell growth. From these screens, 23 novel compounds were identified, in addition to their molecular targets and in silico cell-signalling pathways, which included the neuroactive ligand-receptor interaction pathway, metabolic pathways, and cancer-associated pathways. This study demonstrates for the first time the feasibility of performing large compound screens for the identification of new translatable therapeutics and the improved characterization of endometriosis molecular pathophysiology. Further investigation of the molecular targets identified herein will help uncover new mechanisms involved in the establishment, symptomology, and progression of endometriosis.

Endometrial uNK cell counts do not predict successful implantation in an IVF population.

STUDY QUESTION: Are uterine natural killer (uNK) cell numbers and their distribution relative to endometrial arterioles altered in women with recurrent implantation failure (RIF) compared to women with embryo implantation success (IS)? SUMMARY ANSWER: uNK cell numbers and their distribution relative to endometrial arterioles are not significantly different in women with RIF compared to women in whom embryo implantation occurs successfully following IVF. WHAT IS ALREADY KNOWN: uNK cells are regulators of decidual angiogenesis and spiral arteriole remodelling during early pregnancy. Although some studies have shown that uNK cell numbers may be altered in women with RIF, the methods used to measure uNK cell numbers have proven inconsistent, making reproduction of these results difficult. It is unclear, therefore, whether the results reported so far are reproducible. Moreover, it is not known how uNK cell numbers may impact IVF outcomes. Despite the lack of conclusive evidence, uNK cell numbers are often evaluated as a prognostic criterion in women undergoing assisted reproductive procedures. STUDY DESIGN, SIZE, DURATION: Endometrial pipelle biopsies were collected 6-8 days post-LH surge in natural cycles from women with RIF (n = 14), women with IS (n = 11) and women with potential RIF at the time of the study (PRIF; n = 9) from 2013 to 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: uNK cells (i.e. CD56+ and/or CD16+ phenotypes) and their distribution relative to endometrial arterioles were investigated by standard immunohistochemistry protocols and quantified using Aperio ScanScopeXT images digitized by ImageJ and deconvoluted into binary images for single cell quantification using a Gaussian Blur and Yen algorithm. MAIN RESULTS AND THE ROLE OF CHANCE: There was no significant difference in the cell density of CD56+ or CD16+ uNK cells in women with RIF compared to women with IS or PRIF. There was a higher proportion of uNK cells in the distal regions compared to the regions closest to the arterioles in all patient groups. Further, we identified a significant reduction in uNK cell density in women who had a previous pregnancy compared to those who had not, regardless of their current implantation status. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Spiral arterioles could not always be accurately identified by digital image analysis; therefore, all endometrial arterioles were selected and analysed. Patient numbers for the study were low. However, as the clinical phenotypes of each patient were well defined, and endometrial dating was accurately determined by three independent pathologists, differences between patient groups with respect to the uNK numbers and distribution should have been measurable if uNK cell counts were to be useful as a prognostic marker of RIF. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that CD56+ and CD16+ uNK cell numbers are not significantly different in women with RIF in a typical cohort of women undergoing IVF. Further, prior pregnancy was associated with a significantly reduced number of uNK cells in both the RIF and IS patient groups, suggestive of a long-term pregnancy induced suppression of uNK cells. Combined, these findings do not support the clinical value of using uNK cell numbers as a prognostic indicator of implantation success with IVF treatment. STUDY FUNDING/COMPETING INTEREST(S): Funding for this work was provided by Royal Women's Hospital Foundation. P.P. was supported by an NHMRC Early Career Fellowship [TF 11/14] and W.T.T. was supported by an NHMRC Postgraduate Scholarship [1055814]. The authors do not have any competing interests with this study.

Generation of immortalized human endometrial stromal cell lines with different endometriosis risk genotypes.

Endometriotic lesions are composed in part of endometrial-like stromal cells, however, there is a shortage of immortalized human endometrial stromal cultures available for research. As genetic factors play a role in endometriosis risk, it is important that genotype is also incorporated into analysis of pathological mechanisms. Human telomerase reverse transcriptase (hTERT) immortalization (using Lenti-hTERT-green fluorescent protein virus) took place following genotype selection; 13 patients homozygous for either the risk or non-risk 'other' allele for one or more important endometriosis risk single nucleotide polymorphism on chromosome 1p36.12 (rs3820282, rs56318008, rs55938609, rs12037376, rs7521902 or rs12061255). Short tandem repeat DNA profiling validated that donor tissue matched that of the immortalized cell lines and confirmed that cultures were genetically novel. Expression of morphological markers (vimentin and cytokeratin) and key genes of interest (telomerase, estrogen and progesterone receptors and LINC00339) were examined and functional assays for cell proliferation, steroid hormone and inflammatory responses were performed for 7/13 cultures. All endometrial stromal cell lines maintained their fibroblast-like morphology (vimentin-positive) and homozygous endometriosis-risk genotype following introduction of hTERT. Furthermore, the new stromal cultures demonstrated positive and diverse responses to hormones (proliferation and decidualisation changes) and inflammation (dose-dependent response), while maintaining hormone receptor expression. In conclusion, we successfully developed a range of human endometrial stromal cell lines that carry important endometriosis-risk alleles. The wider implications of this approach go beyond advancing endometriosis research; these cell lines will be valuable tools for multiple endometrial pathologies offering a level of genetic and phenotypic diversity not previously available.

Characterization of Diffuse Intrinsic Pontine Glioma Radiosensitivity using Synchrotron Microbeam Radiotherapy and Conventional Radiation Therapy In Vitro.

Synchrotron microbeam radiation therapy is a promising preclinical radiotherapy modality that has been proposed as an alternative to conventional radiation therapy for diseases such as diffuse intrinsic pontine glioma (DIPG), a devastating pediatric tumor of the brainstem. The primary goal of this study was to characterize and compare the radiosensitivity of two DIPG cell lines (SF7761 and JHH-DIPG-1) to microbeam and conventional radiation. We hypothesized that these DIPG cell lines would exhibit differential responses to each radiation modality. Single cell suspensions were exposed to microbeam (112, 250, 560, 1,180 Gy peak dose) or conventional (2, 4, 6 and 8 Gy) radiation to produce clonogenic cell-survival curves. Apoptosis induction and the cell cycle were also analyzed five days postirradiation using flow cytometry. JHH-DIPG-1 cells displayed greater radioresistance than SF7761 to both microbeam and conventional radiation, with higher colony formation and increased accumulation of G2/M-phase cells. Apoptosis was significantly increased in SF7761 cells compared to JHH-DIPG-1 after microbeam irradiation, demonstrating cell-line specific differential radiosensitivity to microbeam radiation. Additionally, biologically equivalent doses to microbeam and conventional radiation were calculated based on clonogenic survival, furthering our understanding of the response of cancer cells to these two radiotherapy modalities.

Characterization of Diffuse Intrinsic Pontine Glioma Radiosensitivity using Synchrotron Microbeam Radiotherapy and Conventional Radiation Therapy In Vitro.

Synchrotron microbeam radiation therapy is a promising preclinical radiotherapy modality that has been proposed as an alternative to conventional radiation therapy for diseases such as diffuse intrinsic pontine glioma (DIPG), a devastating pediatric tumor of the brainstem. The primary goal of this study was to characterize and compare the radiosensitivity of two DIPG cell lines (SF7761 and JHH-DIPG-1) to microbeam and conventional radiation. We hypothesized that these DIPG cell lines would exhibit differential responses to each radiation modality. Single cell suspensions were exposed to microbeam (112, 250, 560, 1,180 Gy peak dose) or conventional (2, 4, 6 and 8 Gy) radiation to produce clonogenic cell-survival curves. Apoptosis induction and the cell cycle were also analyzed five days postirradiation using flow cytometry. JHH-DIPG-1 cells displayed greater radioresistance than SF7761 to both microbeam and conventional radiation, with higher colony formation and increased accumulation of G2/M-phase cells. Apoptosis was significantly increased in SF7761 cells compared to JHH-DIPG-1 after microbeam irradiation, demonstrating cell-line specific differential radiosensitivity to microbeam radiation. Additionally, biologically equivalent doses to microbeam and conventional radiation were calculated based on clonogenic survival, furthering our understanding of the response of cancer cells to these two radiotherapy modalities.

EGFRvIII-mediated transactivation of receptor tyrosine kinases in glioma: mechanism and therapeutic implications.

A truncation mutant of the epidermal growth factor receptor, EGFRvIII, is commonly expressed in glioma, an incurable brain cancer. EGFRvIII is tumorigenic, in part, through its transactivation of other receptor tyrosine kinases (RTKs). Preventing the effects of this transactivation could form part of an effective therapy for glioma; however, the mechanism by which the transactivation occurs is unknown. Focusing on the RTK MET, we show that MET transactivation in U87MG human glioma cells in vitro is proportional to EGFRvIII activity and involves MET heterodimerization associated with a focal adhesion kinase (FAK) scaffold. The transactivation of certain other RTKs was, however, independent of FAK. Simultaneously targeting EGFRvIII (with panitumumab) and the transactivated RTKs themselves (with motesanib) in an intracranial mouse model of glioma resulted in significantly greater survival than with either agent alone, indicating that cotargeting these RTKs has potent antitumor efficacy and providing a strategy for treating EGFRvIII-expressing gliomas, which are usually refractory to treatment.

Glioma-specific Domain IV EGFR cysteine mutations promote ligand-induced covalent receptor dimerization and display enhanced sensitivity to dacomitinib in vivo.

A feature of many gliomas is the amplification of the epidermal growth factor receptor (EGFR), resulting in its overexpression. Missense mutations or deletions within the extracellular domain are associated with this amplification and can lead to constitutive activation of the receptor, with the Domain I/II deletion, EGFRvIII, being the most common. These changes have also been associated with increased sensitivity to EGFR inhibition using small molecule inhibitors. We have expressed, in human glioma cells, EGFR containing four glioma-specific EGFR missense mutations within Domain IV (C620Y, C624F, C628Y and C636Y) to analyze their biological properties and sensitivity to EGFR inhibition. One of these mutants, C620Y, exhibited an enhanced basal phosphorylation, which was partially dependent on an EGFR-ligand autocrine loop. All Domain IV mutants responded equally as well as wildtype EGFR (wtEGFR) to ligand stimulation. Biochemical analysis revealed that a pre-formed, disulfide-bonded dimer associated with these mutations was underglycosylated, inactive and cytoplasmically retained. Ligand stimulation resulted in the formation of a tyrosine-phosphorylated, disulfide-bonded dimer for all Domain IV mutants but not for wtEGFR. Following treatment with the next-generation, irreversible pan-ErbB inhibitor dacomitinib, the C620Y, C624F and EGFRvIII mutants were inactivated, covalently dimerized and were retained in the cytoplasm, resulting in cell-surface receptor loss and, for C620Y and C624F, decreased binding of EGF. Dacomitinib treatment significantly reduced the in vivo growth of human glioma xenografts bearing C620Y, but not wtEGFR. Collectively, these data indicate that the unique biochemical traits of Domain IV EGFR cysteine mutants can be exploited for enhanced sensitivity to EGFR small molecule inhibitors, with potential clinical applications.

Endometrial lymphangiogensis.

This article briefly summarises some of the more important recent advances in understanding of lymphangiogenesis, and then reviews current knowledge of the lymphatics and lymphangiogenesis in the endometrium. The recent identification of vascular endothelial growth factor-C (VEGF-C) and VEGF-D, as well as specific lymphatic endothelial cell (LEC) markers such as vascular endothelial growth factor receptor-3 (VEGF-R3), lymphatic endothelial hyaluronan receptor-1 (LYVE-1), podoplanin, and prospero-related homeobox-1 (PROX1), has provided the tools to characterize and investigate lymphatic development and function in a wide range of tissues. There are conflicting reports on the distribution of endometrial lymphatics, with some studies reporting lymphatics in the functional zone of human endometrium, others only in the endometrial basalis, and some reporting none at all. Using immunohistochemical methods we have shown that lymphatic vessels of the functionalis were small and sparsely distributed whereas the basalis lymphatics are larger, more frequent and often closely associated with spiral arterioles. Based on comparisons of serial sections, the majority of lymphatic vessels are positive for CD31 but not FVIII or CD34. By comparing CD31 with D2-40 (labels lymphatic endothelial cells) vessel immunostaining, it was estimated that 13% of the vessel profiles in the functionalis, 43% in the basalis and 28% in the myometrium were lymphatics. The lymphangiogenic growth factor VEGF-C is immunolocalized most prominently in the glandular cells, vascular endothelium and some stromal cells in normal cycling endometrium. There is no difference in staining intensity observed between the basalis and functionalis. VEGF-D is immunolocalized throughout the endometrial and myometrial tissues, with no difference in intensity between endometrial glands and stroma or between the basalis and functionalis across the normal cycle. In conclusion, despite an apparently similar distribution of VEGF-C, VEGF-D and VEGF-R3 in endometrial functionalis and basalis, the lymphatic vascular density is 4-5 times higher in the basalis compared to the functionalis. There is also a close association between some lymphatics in the basalis and the spiral arterioles, thus identifying a potential mechanism for a vascular control feedback loop.

Effects of nitrovasodilators on the human fetal-placental circulation in vitro.

This study examines the vasorelaxation of isolated human placental chorionic plate arteries and the perfused fetal-placental vasculature, in vitro, to a variety of nitrovasodilator compounds including glyceryl trinitrate (GTN) sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), S-nitroso-N-glutathione (SNG) and NaNO(2). The effects of these compounds were also examined under conditions of high (>450 mmHg) and low oxygen (<50 mmHg) tension. In a separate series of experiments the effects of GTN and NaNO(2)were further investigated with addition of the antioxidants cysteine (100 microm), glutathione (100 microm) or superoxide dismutase (SOD) (30 I.U./ml). The order of nitrovasodilator potency, when added directly to isolated fetal vessels was GTN=SNP>SNAP=SNG>NaNO(2). The order under low oxygen tension was similar, GTN=SNP>SNG= SNAP>or=NaNO(2). SNG ( approximately fourfold) and NaNO(2)( approximately 50-fold) were significantly more potent under low oxygen conditions. Cysteine, glutathione and SOD were without effect on GTN induced vasodilatation. However, all three agents significantly enhanced (six- to ninefold) the effects of NaNO(2)under similar conditions. When infused directly into the fetal-placental circulation during in vitro perfusion experiments the order of potency was GTN>SNP>or=SNG>or=SNAP>or=NaNO(2). When the nitrovasodilators were infused indirectly via the maternal intervillous space the order of potency was GTN>or=SNP>or=NaNO(2)>or=SNAP=SNG. Our observations suggest that there are important differences in the action of different classes of nitrovasodilator compounds on the fetal-placental circulation. The changes observed with SNG and NaNO(2)may be influenced by levels of tissue oxygenation.

Fetal placental vascular responses to corticotropin-releasing hormone in vitro. Effects of variation in oxygen tension.

In this study, using the human placenta perfused in vitro with Krebs' bicarbonate solution, we have examined the effects of changes in oxygen tension on the vasoreactivity of fetal placental blood vessels to corticotropin releasing hormone (CRH). Vasodilatory responses to human synthetic CRH were measured during sub-maximal vasoconstriction of the fetal placental circulation with prostaglandin F(2alpha)(PGF(2alpha)) (1-100 micrometer). Decreases in fetal placental arterial perfusion pressure (FAP) were obtained with CRH under conditions of high oxygen or low oxygen tension, >/=450 mmHg and