Glycodelin A is a prognostic marker to predict poor outcome in advanced stage ovarian cancer patients
- Christoph Scholz†1,
- Sabine Heublein†2,
- Miriam Lenhard3,
- Klaus Friese2, 3,
- Doris Mayr4 and
- Udo Jeschke2Email author
© Scholz et al.; licensee BioMed Central Ltd. 2012
Received: 26 June 2012
Accepted: 22 September 2012
Published: 5 October 2012
Glycodelin is a cell surface glycoprotein offering a unique gender specific carbohydrate configuration. Sialylated carbohydrate structures, which are unusual for mammals, characterize Glycodelin isolated from amniotic fluid (Glycodelin A, GdA). Glycodelin in general exerts multiple, partly opposing functions ranging from immunosuppression to cell differentiation. As these markedly influence tumorigenesis, this study aimed to clarify whether expression of different Glycodelin isoforms is related to clinicopathological characteristics and prognosis of ovarian cancer patients. Further the use of Glycodelin as a serum marker in benign and malignant ovarian diseases was evaluated.
Ovarian cancer specimens (n = 152) were stained for Glycodelin with carbohydrate and peptide specific antibodies. Associations between Glycodelin expression and histological grading, FIGO stage as well as patient’s prognosis were examined. Glycodelin was correlated to expression of gonadotropin receptors and mucin-1, which are discussed as ovarian cancer tissue markers. In addition, Glycodelin serum concentrations were analyzed in patients suffering from benign (n = 73) or malignant (n = 38) ovarian neoplasias.
Glycodelin A was found to be an independent prognostic marker for poor prognosis in advanced ovarian cancer patients. GdA staining correlated with gonadotropin receptor (FSHR and LHCGR) and with hCG expression. Gd expression showed a positive correlation with a tumour-associated epitope of mucin 1 (TA-MUC1). Further, compared to ovarian cancer, serum Gd was increased in patients with benign ovarian tumors.
Glycodelin A might be related to tumor aggressiveness and poor clinical outcome in advanced epithelial ovarian cancer. Glycodelin serum levels found in patients suffering from benign ovarian tumors, might contribute to a more global attenuation during progression of these precursor lesions.
KeywordsOvarian cancer Glycodelin Immunohistochemistry Prognosis
Epithelial ovarian cancer (EOC) represents the most lethal malignancy of the female genital tract. Nowadays ovarian cancer patients’ prognosis mostly relies on completeness of surgical tumor resection [1, 2], clinical staging and histological tumor grading. Thus there is a compelling need to identify and validate tumor specific antigens which are suitable to individualize therapeutic strategies. Interestingly, during EOC evolvement and progression host anti-tumor immune defense seems to be actively blocked by tumor derived mediators. By creating this highly suppressive environment, EOC manages to extensively grow and spread in the peritoneal cavity.
Glycodelin (Gd), a potent immunosuppressive agent of the reproductive tract, is supposed to contribute to this immune tolerant phenotype. Gd is a glycoprotein whose immune-regulatory actions have been highlighted within different biological processes [4–6] and which is abundantly found in the female reproductive tract [7–9]. Structure wise it is part of the lipocalin superfamily and exerts its potent immune-regulatory activity via its unique, heavily sialysiated glycosylation pattern. Apart from its physiologic role as an immunomodulatory agent during implantation of the fetal semiallotransplantant it is also expressed by malignant tissues and contributes to the tumor-micromilieu [10, 11]. Nevertheless, the physiological importance of Gd-expression in malignant diseases remains unknown. Gd is one of very few proteins that show a gender specific glycosylation pattern. Glycodelin, isolated from amniotic fluid (glycodelin A, GdA) is composed of two identical subunits closely connected by non-covalent bonds and a carbohydrate content of 17.5% . A similar glycoprotein, Glycodelin S (GdS), was found in seminal plasma, but with a different glycosylation compared to GdA. While GdA is heavily sialylated, GdS is characterized by fucose-rich carbohydrate structures .
In the current study Gd was detected by antibodies raised against peptide sequences, which are not gender specific or specific for GdA or GdS, and a GdA specific monoclonal antibody [14, 15]. In this work we aimed to clarify whether Gd expression in EOC is of prognostic significance. Further Glycodelin was correlated to expression of gonadotropin receptors and Mucin-1, which are discussed as ovarian cancer tissue markers. Finally we tested whether Glycodelin might be a potentially useful serum biomarker to detect ovarian cancer.
Materials and methods
Patients’ characteristics; Details on patients included in immunohistochemistry (A) and EIA study (B) are shown
Sera of 111 patients, who underwent surgery at the Department of Obstetrics and Gynecology of Ludwig-Maximilians-University Munich between 2002 and 2005, were collected before surgery. Histological diagnoses (Table 1 B, benign ovarian diseases, n = 73 and EOC, n = 38) were made by gynecological pathologists. Written informed consent was obtained from all patients before surgery. Benign ovarian diseases were set up of cystic lesions (n = 21; serous cysts, mucinous cysts, follicle cysts, inclusion cyst, corpus luteum cysts), inflammatory diseases (n = 18; endometriosis cysts, sactosalpinx) and benign tumors (n = 34; serous and mucinous cystadenofibroma, fibroma, teratoma). Sera of EOC patients (n = 38) were analyzed in parallel.
Antibodies used for immunohistochemistry and EIA; GdC15, GdQ13 and GdN20 were purchased from SantaCruz Biotechnologies, Santa Cruz, CA (SCBT)
Reaction system used for detection
Proteinase K (30 min, RT)
UV-Block (45 min)
1:1000 (UV Block)
30 min (RT)
Vectastain elite kit (goat IgG)
citrate buffer (pH 6, 5 min, pressure cooker)
UV-Block (45 min)
1: 300 (UV Block)
Vectastain elite kit (goat IgG)
GdA (Jeschke et al. 2006)
Mixed glycan/protein epitope
citrate buffer (pH 6, 5 min, pressure cooker)
1.5% horse serum (20 min)
1:3000 (DAKO dil.)
Vectastain elite kit (mouse IgG)
Competitive enzyme immuno assay (EIA)
Reaction system used for detection
1% BSA, 0.5% NaN3 in PBS
1 hour (RT)
1% BSA, 0.5% NaN3 in PBS
1 hour (RT)
Analysis of Gd serum levels
Sera were collected prior to surgery and stored at - 80°C. Analysis was performed using a competitive enzyme immune assay (EIA) principle. Briefly, plates were coated with antibodies (Table 2), which were rose against a peptide sequence localized in the C-terminal (GdQ13) or N-terminal (GdN20) end of the Gd protein core, over night at RT and blocked in blocking solution for 30 min. Following washes with PBS plates were incubated with biotinylated Gd standard peptides (GdN20 peptid 6 ng/ml; GdQ13 peptid 1.5 ng/ml) for 1 hour followed by washes in PBS. Serum was diluted 1/2 in PBS and applied onto the plate for 1 hour to perform the competition step. After washes with PBS the plate was incubated with POD streptavidine for 2 min before the reaction was stopped with 1 N sulphuric acid. The readout was performed using an ELISA Reader Dynex MRX (Dynex Technologies, Chantilly, VA). Amniotic fluid or just blocking solution was used as positive and negative controls, respectively.
Data were analysed employing the SPSS (v19, IBM, Armonk, New York) statistic software for MS windows. Statistical significance for pair wise comparisons of unlinked non parametric values was determined by Mann–Whitney-U test whereas differences among three or more groups were tested using Kruskal-Wallis one-way analysis of variance by ranks, respectively. Survival and recurrence free survival was plotted in accordance with Kaplan-Meier survival analysis. Correlations were assessed using Spearman’s rho and gamma correlation coefficient for two independent variables. Statistical significance for all tests was assumed for p < 0.05 and data are presented as mean ± standard error.
Patients’ characteristics and Glycodelin expression
34 patients presented with early disease in stage I (FIGO I). 10 patients had FIGO stage II and 103 patients underwent surgery because of suspected ovarian cancer involving the peritoneal cavity (100 patients FIGO stage III and 3 patients stage IV). All patients who suffered from EOC staged FIGO II - IV received carboplatin and paclitaxel as chemotherapy after surgery. Histological 53 tumors were assigned with WHO grade G3, 50 with G2 and another 37 were classified as G1. Mean follow-up time was 11.03 ± 0.58 years and mean overall survival 7.12 ± 0.63 years. 27 relapses and 102 deaths were documented.
Correlation of Glycodelin with tumor biomarkers, clinicopathological criteria and patients’ prognosis
Correlations between GdC15, GdQ13 and GdA; Correlations between GdC15, GdQ13 and GdA were assessed using Spearman’s rho test for two independent variables
Glycodelin A predicts prognosis in EOC patients
Serum Glycodelin concentrations
This study found that GdA independently predicts unfavorable prognosis in advanced stage ovarian cancer patients. Gd has already been published to correlate with less favorable clinical presentation in breast  and lung cancer , though controversial results were observed in ovarian cancer patients . However a common problem in research on Gd is that most commercially available antisera were raised against peptide epitopes. Since gender specific glycosylation pattern crucially determines Gd-protein function , we employed GdA specific staining [14, 15]. Remarkably, just GdA correlated with patients’ prognosis (Figure 2) and GdA was much more selectively distributed than the peptide epitopes (Figure 1). Thus we hypothesize, that the specific glycosylation pattern of GdA and not necessarily just its presence as determined by the peptide specific antisera, is responsible for its tumor promoting activity. Therefore GdA might be an interesting marker to discriminate between high risk and low risk patients in advanced ovarian cancer (stage III and IV). A former study showed that Gd is also secreted to ascites fluid of ovarian cancer patients and that this Gd has significant differences in its structure of sialyl Lewis-type oligosaccharides compared to GdA. Additionally, ascites Gd inhibits IL-2 stimulated proliferation of peripheral blood leucocytes and inhibits adhesion of SLeX-positive cells to E-selectin. Therefore, Gd could act as an inhibitor of lymphocyte activation and/or adhesion in ovarian cancer .
As several studies proofed the immune suppressive effect of GdA [6, 11], we suppose that GdA interferes with host anti tumor immune defense in EOC as well. Besides, it is widely accepted, that EOC has the ability to escape immune rejection by creating an immune suppressive environment . Thus expression of the immunomodulatory GdA in ovarian carcinomas might contribute to permit immunological destruction of malignant cells and therefore might be related to tumor aggressiveness and poor clinical outcome.
Furthermore GdA staining correlated with gonadotropin receptor (FSHR and LHCGR) and with hCG expression itself (Table 3). There are only few studies on human chorionic gonadotropin and its receptor expression in ovarian cancer tissue [25, 26]. In a recent study we found an opposed prognostic value of LHCGR and FSHR in ovarian cancer  in almost the same panel of patients as described within this study. GdA and hCG stimulate each other in a positive feedback mechanism in the placental decidua/trophoblast cell system as described earlier [27, 28]. Results obtained within this study seem to confirm former results of GdA - hCG interaction. Gd expression showed also a positive correlation with a tumour-associated epitope of MUC1 (TA-MUC1), which was stained by the PankoMabGEX™ antibody. This epitope was found to be steroid hormone receptor dependent in breast cancer . Because it is known that Gd expression is also regulated by steroid hormones , there could be a common regulation pathway for both Gd and TA-MUC1.
The use of Gd as a serum biomarker in ovarian cancer patients has been controversially discussed [31, 32]. As Gd might interfere with tumor development, we decided to examine precursor lesions as well, which turned out to be characterized by elevated serum Gd levels. Interestingly in sera collected from ovarian cancer patients Gd levels were not significantly altered compared to cystic or inflammatory diseases. Benign ovarian tumors are part of a multistep process leading to low grade invasive ovarian cancer . Gd mediated immune attenuation has been described during pregnancy [34–36] as well as in inflammatory processes . Thus, besides of it pathophysiologic role, Gd might be an interesting to detect benign or inflammatory changes in the ovaries, but the detection of GdA with its unique glycosylation may be a more suitable serum marker for ovarian cancer patients.
GdA is related to tumor aggressiveness and poor clinical outcome in advanced ovarian cancer. GdA might be an interesting marker to discriminate between high risk and low risk patients in stage III and IV ovarian cancer.
The authors thank Simone Hofmann, Susanne Kunze and Christina Kuhn for excellent technical support.
- Classe JM, Jaffre I, Frenel JS, Bordes V, Dejode M, Dravet F, Ferron G, Marchal F, Berton Rigaud D, Loussouarn D: Prognostic factors for patients treated for a recurrent FIGO stage III ovarian cancer: a retrospective study of 108 cases. Eur J Surg Oncol. 2011, 37 (11): 971-977. 10.1016/j.ejso.2011.08.138.PubMedView ArticleGoogle Scholar
- Bristow RE, Tomacruz RS, Armstrong DK, Trimble EL, Montz FJ: Survival effect of maximal cytoreductive surgery for advanced ovarian carcinoma during the platinum era: a meta-analysis. J Clin Oncol. 2002, 20 (5): 1248-1259. 10.1200/JCO.20.5.1248.PubMedView ArticleGoogle Scholar
- Lenhard SM, Bufe A, Kumper C, Stieber P, Mayr D, Hertlein L, Kirschenhofer A, Friese K, Burges A: Relapse and survival in early-stage ovarian cancer. Arch Gynecol Obstet. 2009, 280 (1): 71-77. 10.1007/s00404-008-0877-z.PubMedView ArticleGoogle Scholar
- Lee CL, Lam KK, Koistinen H, Seppala M, Kurpisz M, Fernandez N, Pang RT, Yeung WS, Chiu PC: Glycodelin-A as a paracrine regulator in early pregnancy. J Reprod Immunol. 2011, 90 (1): 29-34. 10.1016/j.jri.2011.04.007.PubMedView ArticleGoogle Scholar
- Ren S, Liu S, Howell PM, Zhang G, Pannell L, Samant R, Shevde-Samant L, Tucker JA, Fodstad O, Riker AI: Functional characterization of the progestagen-associated endometrial protein gene in human melanoma. J Cell Mol Med. 2010, 14 (6B): 1432-1442.PubMedPubMed CentralView ArticleGoogle Scholar
- Scholz C, Toth B, Brunnhuber R, Rampf E, Weissenbacher T, Santoso L, Friese K, Jeschke U: Glycodelin A induces a tolerogenic phenotype in monocyte-derived dendritic cells in vitro. Am J Reprod Immunol. 2008, 60 (6): 501-512. 10.1111/j.1600-0897.2008.00647.x.PubMedView ArticleGoogle Scholar
- Jeschke U, Toth B, Scholz C, Friese K, Makrigiannakis A: Glycoprotein and carbohydrate binding protein expression in the placenta in early pregnancy loss. J Reprod Immunol. 2010, 85 (1): 99-105. 10.1016/j.jri.2009.10.012.PubMedView ArticleGoogle Scholar
- Scholl B, Bersinger NA, Kuhn A, Mueller MD: Correlation between symptoms of pain and peritoneal fluid inflammatory cytokine concentrations in endometriosis. Gynecol Endocrinol. 2009, 25 (11): 701-706. 10.3109/09513590903159680.PubMedView ArticleGoogle Scholar
- Bersinger NA, Birkhauser MH, Yared M, Wunder DM: Serum glycodelin pattern during the menstrual cycle in healthy young women. Acta Obstet Gynecol Scand. 2009, 88 (11): 1215-1221. 10.3109/00016340903294264.PubMedView ArticleGoogle Scholar
- Koistinen H, Hautala LC, Seppala M, Stenman UH, Laakkonen P, Koistinen R: The role of glycodelin in cell differentiation and tumor growth. Scand J Clin Lab Invest. 2009, 69 (4): 452-459. 10.1080/00365510903056023.PubMedView ArticleGoogle Scholar
- Scholz C, Rampf E, Toth B, Brunnhuber R, Weissenbacher T, Friese K, Jeschke U: Ovarian cancer-derived glycodelin impairs in vitro dendritic cell maturation. J Immunother. 2009, 32 (5): 492-497. 10.1097/CJI.0b013e3181a59fa9.PubMedView ArticleGoogle Scholar
- Bohn H, Kraus W, Winckler W: New soluble placental tissue proteins: their isolation, characterization, localization and quantification. Placenta Suppl. 1982, 4: 67-81.PubMedGoogle Scholar
- Koistinen H, Koistinen R, Dell A, Morris HR, Easton RL, Patankar MS, Oehninger S, Clark GF, Seppala M: Glycodelin from seminal plasma is a differentially glycosylated form of contraceptive glycodelin-A. Mol Hum Reprod. 1996, 2 (10): 759-765. 10.1093/molehr/2.10.759.PubMedView ArticleGoogle Scholar
- Jeschke U, Bischof A, Speer R, Briese V, Richter DU, Bergemann C, Mylonas I, Shabani N, Friese K, Karsten U: Development of monoclonal and polyclonal antibodies and an ELISA for the determination of glycodelin in human serum, amniotic fluid and cystic fluid of benign and malignant ovarian tumors. Anticancer Res. 2005, 25 (3A): 1581-1589.PubMedGoogle Scholar
- Jeschke U, Kuhn C, Mylonas I, Schulze S, Friese K, Mayr D, Speer R, Briese V, Richter DU, Haase M: Development and characterization of monoclonal antibodies for the immunohistochemical detection of glycodelin A in decidual, endometrial and gynaecological tumour tissues. Histopathology. 2006, 48 (4): 394-406. 10.1111/j.1365-2559.2006.02351.x.PubMedView ArticleGoogle Scholar
- Remmele W, Stegner HE: [Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue]. Pathologe. 1987, 8 (3): 138-140.PubMedGoogle Scholar
- Lenhard M, Lennerova T, Ditsch N, Kahlert S, Friese K, Mayr D, Jeschke U: Opposed roles of follicle-stimulating hormone and luteinizing hormone receptors in ovarian cancer survival. Histopathology. 2011, 58 (6): 990-994. 10.1111/j.1365-2559.2011.03848.x.PubMedView ArticleGoogle Scholar
- Lenhard M, Tsvilina A, Schumacher L, Kupka M, Ditsch N, Mayr D, Friese K, Jeschke U: Human chorionic gonadotropin and its relation to grade, stage and patient survival in ovarian cancer. BMC Cancer. 2012, 12 (1): 2-10.1186/1471-2407-12-2.PubMedPubMed CentralView ArticleGoogle Scholar
- Hautala LC, Greco D, Koistinen R, Heikkinen T, Heikkila P, Aittomaki K, Blomqvist C, Koistinen H, Nevanlinna H: Glycodelin expression associates with differential tumour phenotype and outcome in sporadic and familial non-BRCA1/2 breast cancer patients. Breast Cancer Res Treat. 2011, 128 (1): 85-95. 10.1007/s10549-010-1065-y.PubMedView ArticleGoogle Scholar
- Kunert-Keil C, Steinmuller F, Jeschke U, Gredes T, Gedrange T: Immunolocalization of glycodelin in human adenocarcinoma of the lung, squamous cell carcinoma of the lung and lung metastases of colonic adenocarcinoma. Acta Histochem. 2011, 113 (8): 798-802. 10.1016/j.acthis.2010.11.009.PubMedView ArticleGoogle Scholar
- Mandelin E, Lassus H, Seppala M, Leminen A, Gustafsson JA, Cheng G, Butzow R, Koistinen R: Glycodelin in ovarian serous carcinoma: association with differentiation and survival. Cancer Res. 2003, 63 (19): 6258-6264.PubMedGoogle Scholar
- Dell A, Morris HR, Easton RL, Panico M, Patankar M, Oehniger S, Koistinen R, Koistinen H, Seppala M, Clark GF: Structural analysis of the oligosaccharides derived from glycodelin, a human glycoprotein with potent immunosuppressive and contraceptive activities. J Biol Chem. 1995, 270 (41): 24116-24126. 10.1074/jbc.270.41.24116.PubMedView ArticleGoogle Scholar
- Jeschke U, Mylonas I, Kunert-Keil C, Stahn R, Scholz C, Janni W, Kuhn C, Schroder E, Mayr D, Friese K: Immunohistochemistry, glycosylation and immunosuppression of glycodelin in human ovarian cancer. Histochem Cell Biol. 2009, 131 (2): 283-295. 10.1007/s00418-008-0510-z.PubMedView ArticleGoogle Scholar
- Napoletano C, Bellati F, Landi R, Pauselli S, Marchetti C, Visconti V, Sale P, Liberati M, Rughetti A, Frati L: Ovarian cancer cytoreduction induces changes in T cell population subsets reducing immunosuppression. J Cell Mol Med. 2010, 14 (12): 2748-2759. 10.1111/j.1582-4934.2009.00911.x.PubMedPubMed CentralView ArticleGoogle Scholar
- Zheng W, Lu JJ, Luo F, Zheng Y, Feng Y, Felix JC, Lauchlan SC, Pike MC: Ovarian epithelial tumor growth promotion by follicle-stimulating hormone and inhibition of the effect by luteinizing hormone. Gynecol Oncol. 2000, 76 (1): 80-88. 10.1006/gyno.1999.5628.PubMedView ArticleGoogle Scholar
- Minegishi T, Kameda T, Hirakawa T, Abe K, Tano M, Ibuki Y: Expression of gonadotropin and activin receptor messenger ribonucleic acid in human ovarian epithelial neoplasms. Clin Cancer Res. 2000, 6 (7): 2764-2770.PubMedGoogle Scholar
- Toth B, Roth K, Kunert-Keil C, Scholz C, Schulze S, Mylonas I, Friese K, Jeschke U: Glycodelin protein and mRNA is downregulated in human first trimester abortion and partially upregulated in mole pregnancy. J Histochem Cytochem. 2008, 56 (5): 477-485. 10.1369/jhc.2008.950600.PubMedPubMed CentralView ArticleGoogle Scholar
- Jeschke U, Karsten U, Reimer T, Richter DU, Bergemann C, Briese V, Mylonas I, Friese K: Stimulation of hCG protein and mRNA in first trimester villous cytotrophoblast cells in vitro by glycodelin A. J Perinat Med. 2005, 33 (3): 212-218.PubMedGoogle Scholar
- Dian D, Janni W, Kuhn C, Mayr D, Karsten U, Mylonas I, Friese K, Jeschke U: Evaluation of a novel anti-mucin 1 (MUC1) antibody (PankoMab) as a potential diagnostic tool in human ductal breast cancer; comparison with two established antibodies. Onkologie. 2009, 32 (5): 238-244. 10.1159/000209280.PubMedView ArticleGoogle Scholar
- Jaffe RC, Donnelly KM, Fazleabas AT: The induction of baboon glycodelin expression by progesterone is not through Sp1. Mol Hum Reprod. 2003, 9 (1): 35-40. 10.1093/molehr/gag008.PubMedView ArticleGoogle Scholar
- Horowitz IR, Cho C, Song M, Flowers LC, Santanam N, Parthasarathy S, Ramachandran S: Increased glycodelin levels in gynecological malignancies. Int J Gynecol Cancer. 2001, 11 (3): 173-179. 10.1046/j.1525-1438.2001.01017.x.PubMedView ArticleGoogle Scholar
- Richter C, Baetje M, Bischof A, Makovitzky J, Richter DU, Gerber B, Briese V: Expression of the glycodelin A gene and the detection of its protein in tissues and serum of ovarian carcinoma patients. Anticancer Res. 2007, 27 (4A): 2023-2025.PubMedGoogle Scholar
- Staebler A, Diebold J: [Molecular pathology of epithelial ovarian neoplasias: from the phenotype-genotype correlation to new targets in diagnostics and therapy]. Pathologe. 2007, 28 (3): 180-186. 10.1007/s00292-007-0910-1.PubMedView ArticleGoogle Scholar
- Lang EE, Venkatraman G: Glycodelin gene expression in human peripheral white blood cells. Ir J Med Sci. 2007, 176 (2): 101-104. 10.1007/s11845-007-0019-9.PubMedView ArticleGoogle Scholar
- Tulppala M, Julkunen M, Tiitinen A, Stenman UH, Seppala M: Habitual abortion is accompanied by low serum levels of placental protein 14 in the luteal phase of the fertile cycle. Fertil Steril. 1995, 63 (4): 792-795.PubMedGoogle Scholar
- Dalton CF, Laird SM, Estdale SE, Saravelos HG, Li TC: Endometrial protein PP14 and CA-125 in recurrent miscarriage patients; correlation with pregnancy outcome. Hum Reprod. 1998, 13 (11): 3197-3202. 10.1093/humrep/13.11.3197.PubMedView ArticleGoogle Scholar
- Loukovaara S, Immonen IR, Loukovaara MJ, Koistinen R, Kaaja RJ: Glycodelin: a novel serum anti-inflammatory marker in type 1 diabetic retinopathy during pregnancy. Acta Ophthalmol Scand. 2007, 85 (1): 46-49.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.