High throughput, cell type-specific analysis of key proteins in human endometrial biopsies of women from fertile and infertile couples.

BACKGROUND: Although histological dating of endometrial biopsies provides little help for prediction or diagnosis of infertility, analysis of individual endometrial proteins, proteomic profiling and transcriptome analysis have suggested several biomarkers with altered expression arising from intrinsic abnormalities, inadequate stimulation by or in response to gonadal steroids or altered function due to systemic disorders. The objective of this study was to delineate the developmental dynamics of potentially important proteins in the secretory phase of the menstrual cycle, utilizing a collection of endometrial biopsies from women of fertile (n = 89) and infertile (n = 89) couples. METHODS AND RESULTS: Progesterone receptor-B (PGR-B), leukemia inhibitory factor, glycodelin/progestagen-associated endometrial protein (PAEP), homeobox A10, heparin-binding EGF-like growth factor, calcitonin and chemokine ligand 14 (CXCL14) were measured using a high-throughput, quantitative immunohistochemical method. Significant cyclic and tissue-specific regulation was documented for each protein, as well as their dysregulation in women of infertile couples. Infertile patients demonstrated a delay early in the secretory phase in the decline of PGR-B (P < 0.05) and premature mid-secretory increases in PAEP (P < 0.05) and CXCL14 (P < 0.05), suggesting that the implantation interval could be closing early. Correlation analysis identified potential interactions among certain proteins that were disrupted by infertility. CONCLUSIONS: This approach overcomes the limitations of a small sample number. Protein expression and localization provided important insights into the potential roles of these proteins in normal and pathological development of the endometrium that is not attainable from transcriptome analysis, establishing a basis for biomarker, diagnostic and targeted drug development for women with infertility.

Function-specific intracellular signaling pathways downstream of heparin-binding EGF-like growth factor utilized by human trophoblasts.

Heparin-binding EGF-like growth factor (HBEGF) is expressed by trophoblast cells throughout gestation. First-trimester cytotrophoblast cells are protected from hypoxia-induced apoptosis because of the accumulation of HBEGF through a posttranscriptional autocrine mechanism. Exogenous application of HBEGF is cytoprotective in a hypoxia/reoxygenation (H/R) injury model and initiates trophoblast extravillous differentiation to an invasive phenotype. The downstream signaling pathways induced by HBEGF that mediate these various cellular activities were identified using two human first-trimester cytotrophoblast cell lines, HTR-8/SVneo and SW.71, with similar results. Recombinant HBEGF (1 nM) induced transient phosphorylation of MAPK3/1 (ERK), MAPK14 (p38), and AKT within 15 min and JNK after 1-2 h. To determine which downstream pathways regulate the various functions of HBEGF, cells were treated with specific inhibitors of the ERK upstream regulator MEK (U0126), the AKT upstream regulator phosphoinositide-3 (PI3)-kinase (LY294002), MAPK14 (SB203580), and JNK (SP600125), as well as with inactive structural analogues. Only SB203580 specifically prevented HBEGF-mediated rescue during H/R, while each inhibitor attenuated HBEGF-stimulated cell migration. Accumulation of HBEGF at reduced oxygen was blocked only by a combination of U0126, SB203580, and SP600125. We conclude that HBEGF advances trophoblast extravillous differentiation through coordinate activation of PI3 kinase, ERK, MAPK14, and JNK, while only MAPK14 is required for its antiapoptotic activity. Additionally, hypoxia induces an autocrine increase in HBEGF protein levels through MAPK14, JNK or ERK. These experiments reveal a complexity of the intracellular signaling circuitry that regulates trophoblast functions critical for implantation and placentation.

Diverse functions of HBEGF during pregnancy.

The establishment of pregnancy requires an intimate physical interaction and a molecular dialogue between the conceptus and the maternal reproductive tract that commences at implantation and continues until the placenta is formed and fully functional. Failure of the regulatory processes that ensure the fidelity of this relationship can precipitate a catastrophic pregnancy loss. One of the earliest identified molecular mediators of blastocyst implantation is heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF), which signals between the endometrium and implanting trophoblast cells to synchronize their corresponding developmental programs. HBEGF expression by trophoblast cells of the developing placenta appears to regulate extravillous differentiation and provide cytoprotection in a sometimes-hostile environment. This versatile member of the EGF signaling system will be examined in light of its associations with key events during early pregnancy.

Epidemiology and treatment patterns of epithelial ovarian cancer.

INTRODUCTION: While ovarian cancer (OC) is relatively rare, it remains one of the most fatal cancers. Lack of robust screening methods for eOC lead to detection of most cases at advanced stages, and most patients relapse following initial treatment. Areas covered: This review summarizes epidemiology and treatment patterns of epithelial ovarian cancer (eOC). MEDLINE, EMBASE, conference proceedings, and the Cochrane Library were searched using key terms and Medical Subject Headings for ovarian cancer, treatment patterns, and epidemiology to identify articles published from 2005-2015. Expert commentary: To improve early detection, future studies should focus on the identification of biomarkers that can detect asymptomatic disease. Following diagnosis and eventual relapse, response to first-line platinum appears to guide physicians' choice of subsequent therapies, but we do not understand what patients ultimately receive or its relationship to categories of response to first-line platinum. Improved understanding of later-line treatment patterns, by initial response to platinum, could correlate with overall outcomes among relapsed patients and promote development of more effective treatment guidelines. Novel treatment approaches, such as immunotherapies, would fulfill a need for an effective strategy against advanced stages of OC that results in fewer toxic side effects.

Trophoblast survival signaling during human placentation requires HSP70 activation of MMP2-mediated HBEGF shedding.

Survival of trophoblast cells in the low oxygen environment of human placentation requires metalloproteinase-mediated shedding of HBEGF and downstream signaling. A matrix metalloproteinase (MMP) antibody array and quantitative RT-PCR revealed upregulation of MMP2 post-transcriptionally in human first trimester HTR-8/SVneo trophoblast cells and placental villous explants exposed to 2% O2. Specific MMP inhibitors established the requirement for MMP2 in HBEGF shedding and upregulation. Because α-amanitin inhibited the upregulation of HBEGF, differentially expressed genes were identified by next-generation sequencing of RNA from trophoblast cells cultured at 2% O2 for 0, 1, 2 and 4 h. Nine genes, all containing HIF-response elements, were upregulated at 1 h, but only HSPA6 (HSP70B') remained elevated at 2-4 h. The HSP70 chaperone inhibitor VER 155008 blocked upregulation of both MMP2 and HBEGF at 2% O2, and increased apoptosis. However, both HBEGF upregulation and apoptosis were rescued by exogenous MMP2. Proximity ligation assays demonstrated interactions between HSP70 and MMP2, and between MMP2 and HBEGF, supporting the concept that MMP2-mediated shedding of HBEGF, initiated by HSP70, contributes to trophoblast survival at the low O2 concentrations encountered during the first trimester, and is essential for successful pregnancy outcomes. Trophoblast survival during human placentation, when oxygenation is minimal, required HSP70 activity, which mediated MMP2 accumulation and the transactivation of anti-apoptotic ERBB signaling by HBEGF shedding.

Regulation of HBEGF by Micro-RNA for Survival of Developing Human Trophoblast Cells.

INTRODUCTION: The growth factor HBEGF is upregulated post-transcriptionally in the low O2 environment of the human placenta during the first 10 weeks of pregnancy. We have examined the possible roles of HBEGF turnover and micro-RNA (miRNA) in its regulation by O2 in human first trimester trophoblast. METHODS: HTR-8/SVneo trophoblast cells were cultured at 2% or 20% O2. The cells were transfected with a dual luciferase reporter construct (psiCHECK-2) containing no insert (control), the HBEGF 3' untranslated region (3'UTR), or sub-regions of the 3'UTR, as well as with siRNA for DGCR8. RNA was extracted from trophoblast cells cultured at 2% O2 for 0-4 h for next-generation sequencing. HBEGF was quantified by ELISA. HBEGF, DGCR8, and ß-actin were examined by western blotting. RESULTS: Protein turnover studies, using 10 µg/ml cyclohexamide, 1 µg/ml lactocystin, or 100 µg/ml MG132, demonstrated faster HBEGF degradation at 20% O2 than 2% O2, mediated by the proteasome. However, proteasome inhibition failed to initiate HBEGF accumulation at 20% O2. Reporter assays, comparing to empty vector, demonstrated that the intact HBEGF 3' UTR inhibited expression (0.26), while fragments containing only its flanking regions increased reporter activity (3.15; 3.43). No differential expression of miRNAs was found in trophoblast cells cultured at 2% and 20% O2. Nevertheless, HBEGF upregulation at 2% O2 was blocked when the miRNA-processing protein DGCR8 was silenced, suggesting a role for miRNA. CONCLUSION: Our findings suggest involvement of flanking regions of the 3'UTR in activating HBEGF protein synthesis in response to 2% O2, possibly through a miRNA-mediated mechanism.