http://informahealthcare.com/gye ISSN: 0951-3590 (print), 1473-0766 (electronic) Gynecol Endocrinol, 2014; 30(7): 520–524 ! 2014 Informa UK Ltd. DOI: 10.3109/09513590.2014.898055
ENDOMETRIOSIS
Increased levels of biglycan in endometriomas and peritoneal fluid samples from ovarian endometriosis patients Vida Kocbek1*, Neli Hevir-Kene1*, Nick A. Bersinger2, Michael D. Mueller2, and Tea Lanisˇnik Rizˇner1 1
Faculty of Medicine, Institute of Biochemistry, University of Ljubljana, Ljubljana, Slovenia and 2Department of Obstetrics and Gynaecology, Endometriosis Centre, Inselspital, University of Berne, Berne, Switzerland Abstract
Keywords
In our previous low-density-array gene-expression analysis we found an increased expression of biglycan gene in ovarian endometriosis patients. In the present study we evaluated biglycan expression at the protein level in tissue, serum and peritoneal fluid (PF) from ovarian endometriosis patients, patients with benign ovarian cysts and healthy women. Twenty samples of endometriomas and 27 of control tissues (benign ovarian cysts and eutopic endometrium of healthy women) were obtained laparoscopically or by curettage. Serum and PF samples were collected from 56 ovarian endometriosis patients and 40 controls (patients with benign cysts and healthy women). Tissue biglycan levels and serum and PF biglycan concentrations were determined by Western blotting and ELISA, respectively. Biglycan was detected in endometriomas and in benign cysts tissues but differed in glycosylation levels. The PF biglycan concentrations were significantly increased in ovarian endometriosis patients (mean ± SD ¼ 220.3 ± 190.5 pg/mg protein) compared to the whole control group (101.9 ± 94.7 pg/mg protein, p50.001), while serum concentrations did not differ significantly. Biglycan appears to be involved in ovarian pathologies and probably has different roles in benign cysts as compared to ovarian endometriomas.
Benign ovarian cysts, biomarker, ELISA, Western blotting
Introduction Endometriosis is a frequent benign gynaecological disease that is characterised by endometrial explants in the peritoneal cavity (peritoneal endometriosis) and/or in the wall of the pelvic organs (deep infiltrating endometriosis), or in the form of ovarian endometrioma (ovarian endometriosis). It affects up to 10% of premenopausal women [1,2] and can result in severe abdominal pain, and reduced fertility [3,4]. The pathogenesis of endometriosis is complex and it is most often explained by retrograde menstruation, a disturbed immune system, and/or changes in other physiological pathways [5]. As a result, endometrial cells can survive and proliferate at ectopic sites [6,7]. To date, there are no reliable biomarkers for ovarian endometriosis and laparoscopic surgery is still required for definitive diagnosis. Although more than 100 potential biomarkers have been investigated, none of these have been validated for clinical application [8]. In our previous low-density-array gene-expression analysis [9], we searched for potential biomarkers among genes encoding secretory proteins and found an eight-fold increased expression of the biglycan (BGN) gene in tissue samples from ovarian endometriosis patients. Biglycan is a proteoglycan composed of a 42 kDa core protein and glycosaminoglycan side chains of chondroitin sulphate and/or dermatan sulphate [10–12]. It can also exist in monoglycanated *These authors contributed equally to this work. Address for correspondence: Dr. Tea Lanisˇnik Rizˇner, Faculty of Medicine, Institute of Biochemistry, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia. Tel: +386-1-5437657. Fax: +386-15437641. E-mail: [email protected]
History Received 8 January 2014 Accepted 21 February 2014 Published online 18 March 2014
and non-glycanated forms [13–15]. Biglycan is a component of the extracellular matrix that not only has a role in cell attachment, proliferation and migration, growth factor and cytokine interaction, apoptosis, but also in direct regulation of inflammatory responses [16,17]. During tissue injury or after secretion from activated macrophages, biglycan can be released as a soluble molecule and can interact with Toll-like receptors (TLR2 and TLR4) on the surface of activated macrophages, and can activate inflammasome [18]. In these manners biglycan can be involved in the pathogenesis of endometriosis. The aim of the present study was to evaluate BGN expression at the protein level in tissue, serum and PF samples of ovarian endometriosis patients, patients with benign ovarian cysts, and healthy controls.
Materials and methods Samples Between 2008 and 2011, PF and serum of patients with ovarian endometriosis, benign ovarian cysts, and healthy women were collected at the University Clinical Centre, Ljubljana (Slovenia). Ovarian endometriomas, benign ovarian cysts and eutopic endometrial tissue were also collected from respective patients. The serum progesterone concentration was measured using a radioimmunoassay (Coat-a-countÕ DPC, Allschwil, Switzerland). The PF and serum samples were collected from 96 women: 56 women were characterised by the presence of endometriosis based on laparoscopy and histology and 40 by the absence (S1). The case group comprised ovarian endometriosis patients, while the control group included women who were diagnosed of benign
Biglycan in endometriosis
DOI: 10.3109/09513590.2014.898055
Table 1. Presence of biglycan in ovarian endometriomas, benign ovarian cysts and eutopic endometrium. Eutopic endometrium of healthy women
Ovarian endometriomas Sample E1 E2 E3 E4 E5 E6 E16 E18 E19 E21 E22 E23 E24 E25 E26 E29 E45 E54 E68 E120
SI
Type of Endom
++ OE, PE ++ OE, PE, DIE +++ OE, PE ++ OE, DIE ++ OE, PE +++ OE, PE +++ OE +++ OE + OE ++ OE ++ OE + OE ++ OE + OE ++ OE – OE ++ OE +++ OE +++ OE ++ OE
RAFS (Stages) III III III IV III III IV III II III III III III IV III IV IV I IV III
Sample
SI
NE41 NE42 NE45 NE46 NE47 NE48 NE49 NE51 NE55 NE56 NE60 NE66 NE69 NE81 NE82 NE84 NE89 NE92 NE95
– – – – – – – + – – – – – – – – – – –
Benign ovarian cysts*
521
The sensitivity of the assay was 10 pg/ml with a linear detection range from 10 pg/ml to 100 ng/ml. The results for serum and PF are shown in ng/ml and pg/mg total protein, respectively. Biglycan cleavage with chondroitinase ABC
C145 +++
Aliquots of 150 mg protein from tissue samples were precipitated, dried, dissolved in 150 ml reaction buffer (50 mM Tris, pH 8.0, 60 mM sodium acetate, 0.02% bovine serum albumin) and incubated overnight with chondroitinase ABC (0.01 U/ 20 mg protein; Sigma, C2905) in a waterbath at 37 C. Aliquots of 30 mg chondroitinase-treated and control untreated protein were used for further analysis by SDS-PAGE and Western blotting.
C146 +++
Statistical analysis
C149
+
C153
+
C173
+
C179
++
The data were analysed using GraphPad Prism, version 5.00 (GraphPad Software, La Jolla, CA, USA). Square-root transformation of the data was applied, and the differences in biglycan levels between the cases and the two control groups were analysed using one-way analysis of variance (one-way ANOVA), followed by Bonferroni’s multiple comparison post-hoc tests. Two-way ANOVA was performed for the two-factorial comparisons of biglycan concentration, depending on the sample (ovarian endometriosis or control) and on the endometrial phase, age or use of oral contraception. Values of p50.05 were considered statistically significant.
Sample
SI
C104 +++ C106
–
SI: Signal Intensity; E: ovarian endometrioma; NE: normal endometrium; C: benign cysts; no signal; + weak signal; ++ moderate signal; +++ strong signal; * serous, mucinous, dermatoid, paraovarian cysts.
ovarian cysts (n ¼ 10) or underwent tubal sterilisation (n ¼ 30). A total of 47 tissue biopsies were also collected: 20 samples of endometriomas and eight samples of benign ovarian cysts were removed by laparoscopy, while 19 eutopic endometrium samples were collected by curettage from women undergoing sterilisation (Table 1). Based on laparoscopic examination, the women in control group were free of endometriosis. The National Medical Ethics Committee of the Republic of Slovenia approved the study, and written informed consent was obtained from the women enrolled. Western blotting The protein fractions were isolated from tissue samples, separated by SDS PAGE and transferred to polyvinylidene difluoride membranes. The membranes were incubated with the primary rabbit anti-biglycan polyclonal antibodies (overnight; dilution, 1:1000; Sigma-Aldrich, St. Louis, MO, USA) and with the secondary peroxidase conjugated goat anti-rabbit antibodies (1hour incubation; dilution, 1:6000; 111-035-045, Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). The RapidStep ECL reagent (Calbiochem, San Diego, CA, USA) was used for detection and quantification was carried out with the Multi-Gauge software using a Fujifilm LAS400 image reader (Fujifilm, Tokyo, Japan). GAPDH (dilution, 1:3000; Sigma G8795) was used as the normalisation control. Enzyme-linked immunosorbent assays We established sandwich ELISA for measuring biglycan concentrations using rabbit (dilution, 1:1000; Sigma-Aldrich HPA003157) and goat (dilution, 1:400; R&D Systems, Minneapolis, MN, USA) anti-biglycan polyclonal antibodies and the recombinant biglycan protein (R&D Systems 2667-ICM-050). A horseradish-peroxidase-conjugated mouse anti-goat secondary antibody was used as a conjugate, and 3,30 ,5,50 -tetramethylbenzidine as a substrate. The absorbance was measured at 460 nm.
Results We evaluated the biglycan levels in tissue, serum and PF samples from patients with ovarian endometriosis and from the control group that comprised patients with benign ovarian cysts and healthy women (Table 1, Figure 1). Biglycan was detected in ovarian endometriomas and benign ovarian cysts, but not in eutopic endometrium of healthy women We detected biglycan with an approximate molecular mass of 42 kDa in 19 samples of ovarian endometriomas out of 20. In contrast, there was no signal for biglycan in 18 out of 19 samples of eutopic endometrium from healthy women (Table 1, Figure 1a). In most of the endometrioma samples, proteins with higher molecular masses were observed (between 100 and 250 kDa), which represent biglycan forms with additional glycosaminoglycan chains, as shown by the treatment of these samples with chondroitinase ABC (Figure 1b). The biglycan levels were also examined in eight samples of benign cyst tissues where we found bands that corresponded to the biglycan core (42 kDa) in all but one of these eight samples but no bands with higher molecular masses (Table 1, Figure 1c). Biglycan concentrations were increased in PF samples from ovarian endometriosis patients We established an ELISA (see Methods) to determine the biglycan concentrations in the PF and serum samples. The PF biglycan levels were significantly higher in ovarian endometriosis patients (mean ± SD: 220.3 ± 190.5 pg/mg protein) as compared to healthy women (101.0 ± 87.9 pg/mg protein; p50.01) (Figure 2a) and the whole control group (101.9 ± 94.7 pg/mg protein; p50.001) (data not shown). On the other hand, the serum biglycan concentrations did not differ significantly between ovarian endometriosis patients, patients with benign ovarian cysts, and healthy women (13.8 ± 7.0 ng/ml versus 14.5 ± 11.8 ng/ml and 12.9 ± 9.1 ng/ml, respectively; p ¼ 0.7487) (Figure 2b).
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Figure 1. Detection of biglycan in tissue samples. Representative ECL detection of biglycan in the protein samples of (a) ovarian endometriomas (E) and samples of normal endometrium (NE); (b) ovarian endometriomas (E) and samples treated with chondroitinase ABC (Ch); (c) benign cysts (C). The ovarian endometrioma (E) tissue, recombinant biglycan protein (RP) and placenta tissue (P) were used as positive controls. GAPDH was used as a reference protein, and its ECL detection is shown below each image. M, molecular weight standards.
Our case and control groups differed in age, BMI, menstrual phase, and use of oral contraceptives, thus further statistical analysis was performed, which revealed that age, BMI and oral contraception do not affect the PF biglycan concentrations in ovarian endometriosis patients compared to controls (S2). However, stratification of the samples based on the menstrual phase of the patients revealed 1.3-fold and 1.8-fold higher PF biglycan levels in the proliferative phase (119.6 ± 90.3 and 266.5 ± 214.2 pg/mg protein) than in the secretory phase (90.2 ± 95.7 and 139.5 ± 102.5 pg/mg protein) in controls and endometriosis patients, respectively (Figure 2c).
Discussion Our recent gene expression analysis showed overexpression of BGN at the mRNA level in endometrioma tissues [9]. In the
same tissues, biglycan was also detected at the cellular level [9], although to date biglycan has not been measured at the protein level in tissue, serum or PF of endometriosis patients. The present study is thus the first that has evaluated biglycan concentrations in both of these physiological fluids, and has also detected biglycan in endometriomas and benign cysts tissue. In contrast to previous studies which used different antibodies [19] we detected biglycan by Western blot only in 1 out of 19 normal eutopic endometrium tissues, which suggests its low expression. On the other hand, we detected biglycan in all but one sample of endometriomas and all of the samples of benign cysts. This data suggest that biglycan is not specifically associated only with ovarian endometriosis, but also with other ovarian pathologies, thus supporting published data on increased BGN expression in polycystic ovaries versus normal ovarian tissue [20]. As biglycan may be secreted from ovarian endometriomas or
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DOI: 10.3109/09513590.2014.898055
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cysts, possibly due to the different glycosylation and localisation of this proteoglycan. Babelova et al. [22] reported that only glycated biglycan interacts with macrophages and initiates inflammatory responses, while neither the biglycan core nor the glycosaminoglycan chains themselves showed any activity. Based on these data, we speculate that inflammation does not have a key role in pathogenesis of benign ovarian cyst, as biglycan in cysts is present in the non-glycated form. In contrast, in ovarian endometriosis, biglycan is glycated and might thus be involved in inflammation. Although further studies are needed to explain why biglycan secretion differs between ovarian endometriosis and benign ovarian cysts, biglycan may have important role in the pathogenesis of both diseases. In combination with other marker molecules, biglycan may also have a potential as a semi-invasive biomarker of ovarian endometriosis.
Acknowledgements We thank our study participants, the personnel from the Department of Obstetrics and Gynaecology, University Clinical Centre, Ljubljana, Slovenia, and especially Dr. Martina Ribicˇ-Pucelj, M.D. and Mrs. Marika Andonova, and Mrs. Anne Vaucher from the University of Berne.
Declaration of interest Figure 2. Biglycan concentrations in peritoneal fluid and serum. Biglycan concentrations in (a) peritoneal fluid and (b) serum from healthy women (HW), patients with benign ovarian cysts (BC) and patients with ovarian endometriosis (OE). (c) Influence of menstrual phase on peritoneal fluid biglycan levels. The two-way ANOVA assessed the influence of variations that derive from differences between controls and OE (Sample (Controls, OE) p value), variations from differences between menstrual phases (Menstrual phase p value) and the interaction between both factors (Interaction p value).
benign cysts tissue, we determined its concentrations in PF and serum, and found significantly increased concentrations in PF from ovarian endometriosis patients as compared to healthy women or the whole control group. The increased concentrations of biglycan in PF but not in serum support the concept that endometriosis is a local pelvic inflammatory disease, and consequently, the PF more closely reflects the pathological processes. Age, BMI and oral contraception did not affect biglycan concentrations in PF, while menstrual phase had an effect which was more evident in the endometriosis group where higher PF biglycan concentrations were seen in the proliferative phase. This is in contrast to studies in human endometrial microvascular endothelial cells that associated increased biglycan levels with the secretory phase [19]. However, our Western blot data suggest that PF biglycan from our case and control groups originates primarily from pathological ovarian tissue, and potentially other sources. Furthermore, studies in mice have shown that regulation of BGN expression may differ between endometrial and ovarian tissue as in ovaries significantly higher BGN levels were observed in the pro-estrous and estrous phases, which are characterized by estradiol production [21]. The detection of biglycan in endometriomas and benign cysts shows that biglycan might have a more general role in different ovarian pathologies. These data are only partially supported by our ELISA, where there were significantly increased PF biglycan concentrations in ovarian endometriosis patients, but not in patients with benign ovarian cysts versus the healthy women. This discrepancy might be explained by differences in biglycan secretion from endometriotic cysts as compared to benign ovarian
The authors report no declarations of interest. This study was supported by a Slovene Human Resource Scholarship 2011 to V.K., and a J3-4135 grant to T.L.R. from the Slovenian Research Agency.
References 1. Signorello LB, Harlow BL, Cramer DW, et al. Epidemiologic determinants of endometriosis: a hospital-based case-control study. Ann Epidemiol 1997;7:267–741. 2. Goldman MB, Cramer DW. The epidemiology of endometriosis. Prog Clin Biol Res 1990;323:15–31. 3. Sinaii N, Plumb K, Cotton L, et al. Differences in characteristics among 1,000 women with endometriosis based on extent of disease. Fertil Steril 2008;89:538–45. 4. Falcone T, Lebovic DI. Clinical management of endometriosis. Obstet Gynecol 2011;118:691–705. 5. Halme J, Hammond MG, Hulka JF, et al. Retrograde menstruation in healthy women and in patients with endometriosis. Obstet Gynecol 1984;64:151–4. 6. Witz CA, Thomas MR, Montoya-Rodriguez IA, et al. Shortterm culture of peritoneum explants confirms attachment of endometrium to intact peritoneal mesothelium. Fertil Steril 2001; 75:385–90. 7. Winterhager E, Fazleabas A, Hillier S. Endometriosis: science and sense. Mol Hum Reprod 2009;15:575. 8. May KE, Conduit-Hulbert SA, Villar J, et al. Peripheral biomarkers of endometriosis: a systematic review. Hum Reprod Update 2010;16: 651–74. 9. Vouk K, Smuc T, Guggenberger C, et al. Novel estrogen-related genes and potential biomarkers of ovarian endometriosis identified by differential expression analysis. J Steroid Biochem Mol Biol 2011;125:231–42. 10. Kresse H, Hausser H, Schonherr E. Small proteoglycans. Experientia 1993;49:403–16. 11. Fisher LW, Termine JD, Young MF. Deduced protein sequence of bone small proteoglycan I (biglycan) shows homology with proteoglycan II (decorin) and several nonconnective tissue proteins in a variety of species. J Biol Chem 1989;264:4571–6. 12. Schaefer L, Iozzo RV. Biological functions of the small leucine-rich proteoglycans: from genetics to signal transduction. J Biol Chem 2008;283:21305–9. 13. Neame PJ, Choi HU, Rosenberg LC. The isolation and primary structure of a 22-kDa extracellular matrix protein from bovine skin. J Biol Chem 1989;264:5474–9.
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14. Roughley PJ, White RJ, Magny MC, et al. Non-proteoglycan forms of biglycan increase with age in human articular cartilage. Biochem J 1993;295:421–6. 15. Schonherr E, Beavan LA, Hausser H, et al. Differences in decorin expression by papillary and reticular fibroblasts in vivo and in vitro. Biochem J 1993;290:893–9. 16. Flannery CR. Usurped SLRPs novel arthritis biomarkers exposed by catabolism of small leucine-rich proteoglycans? Arthritis Res Therapy 2006;8:106. 17. Iozzo RV, Schaefer L. Proteoglycans in health and disease: novel regulatory signaling mechanisms evoked by the small leucine-rich proteoglycans. FEBS J 2010;277:3864–75. 18. Schaefer L, Babelova A, et al. The matrix component biglycan is proinflammatory and signals through Toll-like receptors 4 and 2 in macrophages. J Clin Invest 2005;115:2223–33.
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19. Kitaya K, Yasuo T. Dermatan sulfate proteoglycan biglycan as a potential selectin L/CD44 ligand involved in selective recruitment of peripheral blood CD16(-) natural killer cells into human endometrium. J Leukocyte Biol 2009;85: 391–400. 20. Jansen E, Laven JS, Dommerholt HB, et al. Abnormal gene expression profiles in human ovaries from polycystic ovary syndrome patients. Mol Endocrinol 2004;18:3050–63. 21. Oksjoki S, Sallinen S, Vuorio E, Anttila L. Cyclic expression of mRNA transcripts for connective tissue components in the mouse ovary. Mol Hum Reprod 1999;5:803–8. 22. Babelova A, Moreth K, Tsalastra-Greul W, et al. Biglycan, a danger signal that activates the NLRP3 inflammasome via toll-like and P2X receptors. J Biol Chem 2009;284: 24035–48.
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ENDOMETRIOSIS
Increased levels of biglycan in endometriomas and peritoneal fluid samples from ovarian endometriosis patients Vida Kocbek1*, Neli Hevir-Kene1*, Nick A. Bersinger2, Michael D. Mueller2, and Tea Lanisˇnik Rizˇner1 1
Faculty of Medicine, Institute of Biochemistry, University of Ljubljana, Ljubljana, Slovenia and 2Department of Obstetrics and Gynaecology, Endometriosis Centre, Inselspital, University of Berne, Berne, Switzerland Abstract
Keywords
In our previous low-density-array gene-expression analysis we found an increased expression of biglycan gene in ovarian endometriosis patients. In the present study we evaluated biglycan expression at the protein level in tissue, serum and peritoneal fluid (PF) from ovarian endometriosis patients, patients with benign ovarian cysts and healthy women. Twenty samples of endometriomas and 27 of control tissues (benign ovarian cysts and eutopic endometrium of healthy women) were obtained laparoscopically or by curettage. Serum and PF samples were collected from 56 ovarian endometriosis patients and 40 controls (patients with benign cysts and healthy women). Tissue biglycan levels and serum and PF biglycan concentrations were determined by Western blotting and ELISA, respectively. Biglycan was detected in endometriomas and in benign cysts tissues but differed in glycosylation levels. The PF biglycan concentrations were significantly increased in ovarian endometriosis patients (mean ± SD ¼ 220.3 ± 190.5 pg/mg protein) compared to the whole control group (101.9 ± 94.7 pg/mg protein, p50.001), while serum concentrations did not differ significantly. Biglycan appears to be involved in ovarian pathologies and probably has different roles in benign cysts as compared to ovarian endometriomas.
Benign ovarian cysts, biomarker, ELISA, Western blotting
Introduction Endometriosis is a frequent benign gynaecological disease that is characterised by endometrial explants in the peritoneal cavity (peritoneal endometriosis) and/or in the wall of the pelvic organs (deep infiltrating endometriosis), or in the form of ovarian endometrioma (ovarian endometriosis). It affects up to 10% of premenopausal women [1,2] and can result in severe abdominal pain, and reduced fertility [3,4]. The pathogenesis of endometriosis is complex and it is most often explained by retrograde menstruation, a disturbed immune system, and/or changes in other physiological pathways [5]. As a result, endometrial cells can survive and proliferate at ectopic sites [6,7]. To date, there are no reliable biomarkers for ovarian endometriosis and laparoscopic surgery is still required for definitive diagnosis. Although more than 100 potential biomarkers have been investigated, none of these have been validated for clinical application [8]. In our previous low-density-array gene-expression analysis [9], we searched for potential biomarkers among genes encoding secretory proteins and found an eight-fold increased expression of the biglycan (BGN) gene in tissue samples from ovarian endometriosis patients. Biglycan is a proteoglycan composed of a 42 kDa core protein and glycosaminoglycan side chains of chondroitin sulphate and/or dermatan sulphate [10–12]. It can also exist in monoglycanated *These authors contributed equally to this work. Address for correspondence: Dr. Tea Lanisˇnik Rizˇner, Faculty of Medicine, Institute of Biochemistry, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia. Tel: +386-1-5437657. Fax: +386-15437641. E-mail: [email protected]
History Received 8 January 2014 Accepted 21 February 2014 Published online 18 March 2014
and non-glycanated forms [13–15]. Biglycan is a component of the extracellular matrix that not only has a role in cell attachment, proliferation and migration, growth factor and cytokine interaction, apoptosis, but also in direct regulation of inflammatory responses [16,17]. During tissue injury or after secretion from activated macrophages, biglycan can be released as a soluble molecule and can interact with Toll-like receptors (TLR2 and TLR4) on the surface of activated macrophages, and can activate inflammasome [18]. In these manners biglycan can be involved in the pathogenesis of endometriosis. The aim of the present study was to evaluate BGN expression at the protein level in tissue, serum and PF samples of ovarian endometriosis patients, patients with benign ovarian cysts, and healthy controls.
Materials and methods Samples Between 2008 and 2011, PF and serum of patients with ovarian endometriosis, benign ovarian cysts, and healthy women were collected at the University Clinical Centre, Ljubljana (Slovenia). Ovarian endometriomas, benign ovarian cysts and eutopic endometrial tissue were also collected from respective patients. The serum progesterone concentration was measured using a radioimmunoassay (Coat-a-countÕ DPC, Allschwil, Switzerland). The PF and serum samples were collected from 96 women: 56 women were characterised by the presence of endometriosis based on laparoscopy and histology and 40 by the absence (S1). The case group comprised ovarian endometriosis patients, while the control group included women who were diagnosed of benign
Biglycan in endometriosis
DOI: 10.3109/09513590.2014.898055
Table 1. Presence of biglycan in ovarian endometriomas, benign ovarian cysts and eutopic endometrium. Eutopic endometrium of healthy women
Ovarian endometriomas Sample E1 E2 E3 E4 E5 E6 E16 E18 E19 E21 E22 E23 E24 E25 E26 E29 E45 E54 E68 E120
SI
Type of Endom
++ OE, PE ++ OE, PE, DIE +++ OE, PE ++ OE, DIE ++ OE, PE +++ OE, PE +++ OE +++ OE + OE ++ OE ++ OE + OE ++ OE + OE ++ OE – OE ++ OE +++ OE +++ OE ++ OE
RAFS (Stages) III III III IV III III IV III II III III III III IV III IV IV I IV III
Sample
SI
NE41 NE42 NE45 NE46 NE47 NE48 NE49 NE51 NE55 NE56 NE60 NE66 NE69 NE81 NE82 NE84 NE89 NE92 NE95
– – – – – – – + – – – – – – – – – – –
Benign ovarian cysts*
521
The sensitivity of the assay was 10 pg/ml with a linear detection range from 10 pg/ml to 100 ng/ml. The results for serum and PF are shown in ng/ml and pg/mg total protein, respectively. Biglycan cleavage with chondroitinase ABC
C145 +++
Aliquots of 150 mg protein from tissue samples were precipitated, dried, dissolved in 150 ml reaction buffer (50 mM Tris, pH 8.0, 60 mM sodium acetate, 0.02% bovine serum albumin) and incubated overnight with chondroitinase ABC (0.01 U/ 20 mg protein; Sigma, C2905) in a waterbath at 37 C. Aliquots of 30 mg chondroitinase-treated and control untreated protein were used for further analysis by SDS-PAGE and Western blotting.
C146 +++
Statistical analysis
C149
+
C153
+
C173
+
C179
++
The data were analysed using GraphPad Prism, version 5.00 (GraphPad Software, La Jolla, CA, USA). Square-root transformation of the data was applied, and the differences in biglycan levels between the cases and the two control groups were analysed using one-way analysis of variance (one-way ANOVA), followed by Bonferroni’s multiple comparison post-hoc tests. Two-way ANOVA was performed for the two-factorial comparisons of biglycan concentration, depending on the sample (ovarian endometriosis or control) and on the endometrial phase, age or use of oral contraception. Values of p50.05 were considered statistically significant.
Sample
SI
C104 +++ C106
–
SI: Signal Intensity; E: ovarian endometrioma; NE: normal endometrium; C: benign cysts; no signal; + weak signal; ++ moderate signal; +++ strong signal; * serous, mucinous, dermatoid, paraovarian cysts.
ovarian cysts (n ¼ 10) or underwent tubal sterilisation (n ¼ 30). A total of 47 tissue biopsies were also collected: 20 samples of endometriomas and eight samples of benign ovarian cysts were removed by laparoscopy, while 19 eutopic endometrium samples were collected by curettage from women undergoing sterilisation (Table 1). Based on laparoscopic examination, the women in control group were free of endometriosis. The National Medical Ethics Committee of the Republic of Slovenia approved the study, and written informed consent was obtained from the women enrolled. Western blotting The protein fractions were isolated from tissue samples, separated by SDS PAGE and transferred to polyvinylidene difluoride membranes. The membranes were incubated with the primary rabbit anti-biglycan polyclonal antibodies (overnight; dilution, 1:1000; Sigma-Aldrich, St. Louis, MO, USA) and with the secondary peroxidase conjugated goat anti-rabbit antibodies (1hour incubation; dilution, 1:6000; 111-035-045, Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). The RapidStep ECL reagent (Calbiochem, San Diego, CA, USA) was used for detection and quantification was carried out with the Multi-Gauge software using a Fujifilm LAS400 image reader (Fujifilm, Tokyo, Japan). GAPDH (dilution, 1:3000; Sigma G8795) was used as the normalisation control. Enzyme-linked immunosorbent assays We established sandwich ELISA for measuring biglycan concentrations using rabbit (dilution, 1:1000; Sigma-Aldrich HPA003157) and goat (dilution, 1:400; R&D Systems, Minneapolis, MN, USA) anti-biglycan polyclonal antibodies and the recombinant biglycan protein (R&D Systems 2667-ICM-050). A horseradish-peroxidase-conjugated mouse anti-goat secondary antibody was used as a conjugate, and 3,30 ,5,50 -tetramethylbenzidine as a substrate. The absorbance was measured at 460 nm.
Results We evaluated the biglycan levels in tissue, serum and PF samples from patients with ovarian endometriosis and from the control group that comprised patients with benign ovarian cysts and healthy women (Table 1, Figure 1). Biglycan was detected in ovarian endometriomas and benign ovarian cysts, but not in eutopic endometrium of healthy women We detected biglycan with an approximate molecular mass of 42 kDa in 19 samples of ovarian endometriomas out of 20. In contrast, there was no signal for biglycan in 18 out of 19 samples of eutopic endometrium from healthy women (Table 1, Figure 1a). In most of the endometrioma samples, proteins with higher molecular masses were observed (between 100 and 250 kDa), which represent biglycan forms with additional glycosaminoglycan chains, as shown by the treatment of these samples with chondroitinase ABC (Figure 1b). The biglycan levels were also examined in eight samples of benign cyst tissues where we found bands that corresponded to the biglycan core (42 kDa) in all but one of these eight samples but no bands with higher molecular masses (Table 1, Figure 1c). Biglycan concentrations were increased in PF samples from ovarian endometriosis patients We established an ELISA (see Methods) to determine the biglycan concentrations in the PF and serum samples. The PF biglycan levels were significantly higher in ovarian endometriosis patients (mean ± SD: 220.3 ± 190.5 pg/mg protein) as compared to healthy women (101.0 ± 87.9 pg/mg protein; p50.01) (Figure 2a) and the whole control group (101.9 ± 94.7 pg/mg protein; p50.001) (data not shown). On the other hand, the serum biglycan concentrations did not differ significantly between ovarian endometriosis patients, patients with benign ovarian cysts, and healthy women (13.8 ± 7.0 ng/ml versus 14.5 ± 11.8 ng/ml and 12.9 ± 9.1 ng/ml, respectively; p ¼ 0.7487) (Figure 2b).
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Figure 1. Detection of biglycan in tissue samples. Representative ECL detection of biglycan in the protein samples of (a) ovarian endometriomas (E) and samples of normal endometrium (NE); (b) ovarian endometriomas (E) and samples treated with chondroitinase ABC (Ch); (c) benign cysts (C). The ovarian endometrioma (E) tissue, recombinant biglycan protein (RP) and placenta tissue (P) were used as positive controls. GAPDH was used as a reference protein, and its ECL detection is shown below each image. M, molecular weight standards.
Our case and control groups differed in age, BMI, menstrual phase, and use of oral contraceptives, thus further statistical analysis was performed, which revealed that age, BMI and oral contraception do not affect the PF biglycan concentrations in ovarian endometriosis patients compared to controls (S2). However, stratification of the samples based on the menstrual phase of the patients revealed 1.3-fold and 1.8-fold higher PF biglycan levels in the proliferative phase (119.6 ± 90.3 and 266.5 ± 214.2 pg/mg protein) than in the secretory phase (90.2 ± 95.7 and 139.5 ± 102.5 pg/mg protein) in controls and endometriosis patients, respectively (Figure 2c).
Discussion Our recent gene expression analysis showed overexpression of BGN at the mRNA level in endometrioma tissues [9]. In the
same tissues, biglycan was also detected at the cellular level [9], although to date biglycan has not been measured at the protein level in tissue, serum or PF of endometriosis patients. The present study is thus the first that has evaluated biglycan concentrations in both of these physiological fluids, and has also detected biglycan in endometriomas and benign cysts tissue. In contrast to previous studies which used different antibodies [19] we detected biglycan by Western blot only in 1 out of 19 normal eutopic endometrium tissues, which suggests its low expression. On the other hand, we detected biglycan in all but one sample of endometriomas and all of the samples of benign cysts. This data suggest that biglycan is not specifically associated only with ovarian endometriosis, but also with other ovarian pathologies, thus supporting published data on increased BGN expression in polycystic ovaries versus normal ovarian tissue [20]. As biglycan may be secreted from ovarian endometriomas or
Biglycan in endometriosis
DOI: 10.3109/09513590.2014.898055
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cysts, possibly due to the different glycosylation and localisation of this proteoglycan. Babelova et al. [22] reported that only glycated biglycan interacts with macrophages and initiates inflammatory responses, while neither the biglycan core nor the glycosaminoglycan chains themselves showed any activity. Based on these data, we speculate that inflammation does not have a key role in pathogenesis of benign ovarian cyst, as biglycan in cysts is present in the non-glycated form. In contrast, in ovarian endometriosis, biglycan is glycated and might thus be involved in inflammation. Although further studies are needed to explain why biglycan secretion differs between ovarian endometriosis and benign ovarian cysts, biglycan may have important role in the pathogenesis of both diseases. In combination with other marker molecules, biglycan may also have a potential as a semi-invasive biomarker of ovarian endometriosis.
Acknowledgements We thank our study participants, the personnel from the Department of Obstetrics and Gynaecology, University Clinical Centre, Ljubljana, Slovenia, and especially Dr. Martina Ribicˇ-Pucelj, M.D. and Mrs. Marika Andonova, and Mrs. Anne Vaucher from the University of Berne.
Declaration of interest Figure 2. Biglycan concentrations in peritoneal fluid and serum. Biglycan concentrations in (a) peritoneal fluid and (b) serum from healthy women (HW), patients with benign ovarian cysts (BC) and patients with ovarian endometriosis (OE). (c) Influence of menstrual phase on peritoneal fluid biglycan levels. The two-way ANOVA assessed the influence of variations that derive from differences between controls and OE (Sample (Controls, OE) p value), variations from differences between menstrual phases (Menstrual phase p value) and the interaction between both factors (Interaction p value).
benign cysts tissue, we determined its concentrations in PF and serum, and found significantly increased concentrations in PF from ovarian endometriosis patients as compared to healthy women or the whole control group. The increased concentrations of biglycan in PF but not in serum support the concept that endometriosis is a local pelvic inflammatory disease, and consequently, the PF more closely reflects the pathological processes. Age, BMI and oral contraception did not affect biglycan concentrations in PF, while menstrual phase had an effect which was more evident in the endometriosis group where higher PF biglycan concentrations were seen in the proliferative phase. This is in contrast to studies in human endometrial microvascular endothelial cells that associated increased biglycan levels with the secretory phase [19]. However, our Western blot data suggest that PF biglycan from our case and control groups originates primarily from pathological ovarian tissue, and potentially other sources. Furthermore, studies in mice have shown that regulation of BGN expression may differ between endometrial and ovarian tissue as in ovaries significantly higher BGN levels were observed in the pro-estrous and estrous phases, which are characterized by estradiol production [21]. The detection of biglycan in endometriomas and benign cysts shows that biglycan might have a more general role in different ovarian pathologies. These data are only partially supported by our ELISA, where there were significantly increased PF biglycan concentrations in ovarian endometriosis patients, but not in patients with benign ovarian cysts versus the healthy women. This discrepancy might be explained by differences in biglycan secretion from endometriotic cysts as compared to benign ovarian
The authors report no declarations of interest. This study was supported by a Slovene Human Resource Scholarship 2011 to V.K., and a J3-4135 grant to T.L.R. from the Slovenian Research Agency.
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