Two-dimensional gel electrophoresis in peritoneal fluid samples identifies differential protein regulation in patients suffering from peritoneal or ovarian endometriosis Endometriosis is determined by local and systemic proinflammatory dysregulation and therefore differential protein expression in peritoneal fluid (PF). Of highest interest is lesion formation and the establishment and persistence of endometriosis. In this study we analyzed well-characterized PF samples of patients with ovarian or peritoneal endometriosis and compared them to control samples. We found 11 proteins differentially regulated, of which some might play a key role in the pathogenesis of endometriosis. (Fertil Steril 2011;95:2764–8. 2011 by American Society for Reproductive Medicine.) Key Words: Endometriosis, peritoneal fluid, 2-dimensional gel electrophoresis, tandem mass spectrometry, protein identification
Endometriosis is characterized by the presence of endometrial glands and stroma outside of the uterine cavity. It affects millions of women of reproductive age (1). The mechanism whereby shed endometrial fragments avoid immune eradication, implant on extrauterine sites, and form persistent endometriotic lesions, are poorly understood (2). Ovarian (OE) and peritoneal endometriosis (PE) appear to be implantations of regurgitated endometrial cells, whereas deep infiltrating endometriosis of the rectovaginal septum Monika M. W€olfler, M.D.a,b Ivo M. Meinhold-Heerlein, M.D.a Linda S€ohngen, M.D.a Werner Rath, M.D.a Ruth Kn€uchel, M.D.b Joseph Neulen, M.D.c Nicolai Maass, M.D.a Corinna Henkel, Ph.D.b a Department of Obstetrics and Gynecology, Medical Faculty of the RWTH Aachen University, Aachen, Germany b Institute of Pathology, Medical Faculty of the RWTH Aachen University, Aachen, Germany c Department of Gynecological Endocrinology and Reproductive Medicine, Medical Faculty of the RWTH Aachen University, Aachen, Germany Received December 22, 2010; revised March 6, 2011; accepted March 21, 2011; published online April 16, 2011. M.M.W. has nothing to disclose. I.M.M.-H. has nothing to disclose. L.S. has nothing to disclose. W.R. has nothing to disclose. R.K. has nothing to disclose. J.N. has nothing to disclose. N.M. has nothing to disclose. C.H. has nothing to disclose. Supported by a research grant from TAKEDA Pharma GmbH, Aachen, Germany. € lfler, M.D., Department of Obstetrics Reprint requests: Monika M. Wo and Gynecology, Medical Faculty of the RWTH Aachen University, 52074 Aachen, Germany (E-mail:
[email protected]).
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relate more to adenomyotic nodules. However, ovarian, peritoneal, and rectovaginal endometriosis might be different entities (3, 4). This study was conducted to explore differential protein expression in peritoneal fluid (PF) of patients with OE or PE compared with controls to understand the local dysregulation potentially involved in the pathogenesis of endometriosis. Peritoneal fluid samples from patients in the midproliferative phase (days 8–11) of the natural menstrual cycle were collected prospectively from symptomatic women aged 18–48 years presenting to this university hospital for diagnosis and/or treatment of dysmenorrhea, dyspareunia, chronic pelvic pain, or subfertility. Before enrollment, all participating patients signed an informed consent. This study was approved by the Institutional Review Board (project EK 137/04). In the studied population, estrogen (E)-dependent diseases other than endometriosis were excluded by gynecologic examination and transvaginal ultrasound. None of the patients received hormonal therapy such as oral contraceptives (OC), progestins, or GnRH analogues at least 3 months before inclusion in the study. Endometriosis was diagnosed by laparoscopic visualization followed by histopathologic assessment of putative lesions. The staging of endometriosis was performed according to the revised American Society for Reproductive Medicine classification standards (5). Peritoneal fluid samples were collected by aspiration from the cul-de-sac during laparoscopy and immediately processed to protect protein integrity. For two-dimensional gel electrophoresis (2DE) samples were solubilized in a buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, and 30 mM Tris. A dilution of sample and buffer containing 400 mg of protein was then filled to 450 mL using rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.3% DTT, 2% ampholyte IPG buffer pH 3-11; GE Healthcare Life Sciences, Piscataway, NJ) and was loaded onto 24-cm IPG strips (Immobiline DryStrip, pH 3–11; GE Healthcare) for rehydration overnight
Fertility and Sterility Vol. 95, No. 8, June 30, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
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and isoelectric focusing. Isoelectric focusing was carried out by the IPGphor III system (GE Healthcare Bio-Sciences) according to G€ org et al. (6). The second dimension was carried out with 9%–18% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Gel images were poststained with Sypro Ruby (Invitrogen, Carlsbad, CA), scanned using the Typhoon 9410 imager (GE Healthcare) at the appropriate wavelength, and were cropped with ImageQuant software (GE Healthcare). The experimental design consisted of 24 well-characterized samples (8 OE, 8 PE, 8 controls) and only gels from three consecutive runs that showed little variability. The two-dimensional images of the gels were analyzed using Delta 2D 4.0 software (Decodon, Greifswald, Germany). To examine the protein spots for significant individual differences between groups a nonparametric test (Kruskal-Wallis) was applied and a fold-change of 1.4 or higher was taken into consideration to allow for proper protein detection in validating Western blot analyses. The significance level was set to P65 were considered (8). For data validation we performed
Western blotting and PF samples were then depleted of highly abundant proteins like albumin and IgG using the Aurum Protein Mini Kit (BioRad, Munich, Germany). In the present study we compared PF samples of eight patients with OE, eight patients with PE, and eight control subjects. The samples evaluated derived from well-characterized patients and demographic data between groups were comparable (Table 1A). In the 2DE 840 paired protein spots were visualized and analyzed for differential expression between groups by the calculation of the spot volumes using Delta 2D software (Decodon). Eleven proteins were differentially expressed at the significance level of P65 is considered to be statistically significant. a Marked proteins were confirmed by Western blotting. W€olfler. Correspondence. Fertil Steril 2011.
ITIH4 has been discussed as a serum biomarker for malignancies (9), chronic obstructive pulmonary disease (10), and ITIH4 levels have been found to be decreased in urine samples of patients suffering from ovarian cancer compared with controls (11). Differential regulation in OE has been found for afamin and apolipoprotein A IV. Afamin was 2.7-fold up-regulated in OE, whereas for PE no significant regulation was detected. Afamin is a vitamin E transporting protein that plays a key role in tissue protection against oxidative damage and was shown to be differentially regulated in benign ovarian conditions and ovarian cancer (12). In an extensive analysis of PF, afamin was shown to make up a higher percentage of the protein content of PF in patients with endometriosis, which was more pronounced in PE (stages I
and II) than in advanced stages of endometriosis (13). Ovarian endometriosis as a separate entity, however, was not evaluated in this study. Apolipoprotein A IV was slightly down-regulated in OE (1.4-fold) and not regulated in PE. Interestingly, a recent study reported downregulation of apolipoprotein A IV in serum of patients suffering from ovarian cancer and benign ovarian conditions (12). Haptoglobin was up-regulated in OE (3.4-fold) and PE (2.9-fold) at the highest levels ascertained in this study, indicating a significant relevance for endometriosis development. Haptoglobin, an acute phase protein, is crucial for the transport and binding of hemoglobin to the reticuloendothelial system and for the excretion of iron to
TABLE 1C Regulation of protein spots identified by tandem mass spectrometry.
1 2 3 4 5 6 7 8 9 10 11
Protein
CO
Mean vol%
SD
OE
Mean vol%
SD
PE
Mean vol%
SD
Complement component 4A Anti-RhD monoclonal T125 gamma1 heavy chain inter-alpha globulin inhibitor ITIH4a Hemopexin Vitronectina Hemoglobin subunit beta Afamina Isoform 1 of alpha-1 antitrypsin Isoform 1 of serum albumin Apolipoprotein A IV Haptoglobina
1 1
0.087 0.08
0.076 0.032
3.4 1.5
0.025 0.048
0.032 0.035
1.4 1.1
0.062 0.086
0.05 0.081
1 1 1 1 1 1 1 1 1
0.063 0.123 0.111 0.055 0.017 0.094 0.059 1.302 0.335
0.022 0.111 0.109 0.022 0.009 0.041 0.029 0.405 0.436
1.2 4.8 1.9 1.1 2.7 1.7 1 1.4 3.4
0.073 0.026 0.214 0.059 0.044 0.164 0.062 0.994 1.122
0.028 0.013 0.051 0.059 0.042 0.067 0.042 0.129 0.356
1.5 5.8 1.7 1.7 1.2 1.6 1.5 1.1 2.9
0.095 0.021 0.184 0.032 0.014 0.153 0.088 1.388 1.116
0.068 0.016 0.139 0.03 0.009 0.1 0.038 0.139 0.156
Note: Differential protein expression was measured by the calculation of the spot volume using Delta 2D 4.0 software (Decodon). According to the algorithms of this software, the significantly differentially expressed protein spots displayed are characterized by the percentage of the mean spot volume (mean vol %) and the SD. The regulation (ratio) of the protein in ovarian (OE) and peritoneal (PE) endometriosis compared with controls (CO) were calculated. To facilitate the comparison between groups by means of a ratio and to indicate the regulation of the protein in endometriosis compared with CO, the mean spot volume in CO was set to 1. All displayed differences were significant at a level of P< .05. a Marked proteins were confirmed by Western blotting. W€olfler. Correspondence. Fertil Steril 2011.
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prevent oxidative damage and toxic effects on the kidneys. SharpeTimms et al. (14) postulated that endometriotic lesions synthesize and secrete haptoglobin. In addition, this study showed that haptoglobin adheres to peritoneal macrophages, decreases adhesion, which may influence phagocytic function, and up-regulates interleukin-6 production, which might increase endometriotic haptoglobin production by a feed-forward regulation and thereby promote the establishment of endometriosis. Another study showed that retrogradely shed endometrial tissues recovered from PF robustly express haptoglobin, which supports a mechanism whereby these purported precursors of endometriotic lesions escape immune destruction (15). In addition, there is evidence for haptoglobin up-regulation in PF in stages I and II endometriosis (16). This evidence thus confirms our findings and suggests an up-regulation of PF haptoglobin expression due to endometriotic lesions within the peritoneal cavity. Another protein involved in the excretion of iron is hemopexin, which was down-regulated (OE 4.8-fold; PE 5.8-fold) in the present study. Hemopexin is the plasma protein with the highest binding affinity to heme, which is potentially highly toxic because of its ability to intercalate into the lipid membrane and to produce hydroxyl radicals. Being the major vehicle for the transportation of heme, hemopexin prevents heme-mediated oxidative stress and heme-bound iron loss (17). In addition, hemopexin has been shown to down-regulate lipopolysaccharides-induced proinflammatory cytokines from macrophages (18). The role of hemopexin in patients suffering from endometriosis has not yet been evaluated and therefore it is impossible to determine whether differential hemopexin regulation is the cause or the consequence of disease. Vitronectin, an abundant glycoprotein found in the extracellular matrix that promotes cell migration, cell adhesion, and invasion, was found up-regulated in PE (1.7-fold) and OE (1.9-fold). Vitronectin is a secreted protein that binds and stabilizes plasminogen activator inhibitor-1, binds to urokinase and its receptor in a dimeric structure, and is the main ligand for the integrin receptor. Involvement of vitronectin in hemostasis and cancer metastasis has been shown previously (19–21). Involvement in endometriosis lesion formation, however, has not yet been investigated. The involvement of the immune system in the pathogenesis of endometriosis is pivotal. In the present study we detected a downregulation of the complement component 4A in OE (3.4-fold) and PE (1.4-fold). Down-regulation of complement components in PF
has previously been demonstrated in infertile women suffering from endometriosis (22). The interrelation of inflammation and endometriosis is well documented. Increases of SERPINA1 (isoform 1 of alpha-1-antitrypsin), the main blood-borne serine, occur in diseases (e.g., rheumatoid arthritis, vasculitis), infections, and other diseases associated with an inflammatory component (23). In the present evaluation of PF samples we found SERPINA1 up-regulated in OE (1.8-fold) and PE (1.6-fold). SERPINA1 has been evaluated and discussed as a biomarker for preeclampsia (24). Whether SERPINA1 is differentially expressed in larger series of patients with endometriosis and whether it might be a feasible biomarker for endometriosis has not yet been evaluated. In summary, by means of 2DE and protein identification we found a series of differentially regulated proteins in PF that might have an impact on the development and establishment of endometriosis lesions or are differentially regulated due to endometriosis. In the present study, acute phase proteins, like haptoglobin and SERPINA1, were found to be up-regulated in endometriosis PF samples, which sustains the theory of a proinflammatory dysregulation as a key feature in endometriosis (25, 26). In addition, oxidative stress in the peritoneal cavity possibly due to iron overload after retrograde menstruation, as postulated by Defrere et al. (27) might also be a contributing factor in the development of endometriosis. The differential regulation of haptoglobin and hemopexin in endometriosis PF samples found this study therefore requests further investigation. Vitronectin as a glycoprotein with potential involvement in tumor metastasis will draw attention on further studies for its possible role in endometriosis lesion formation. Down-regulation of apolipoprotein A IV and afamin in OE confirms recent findings for benign ovarian disease and ovarian cancer (12), albeit more substantial data will be requested to evaluate the involvement of these proteins in ovarian diseases (i.e., OE). Afamin has been reported to be altered in the PF of patients with endometriosis and its role needs to be further elucidated. Acknowledgments: The authors thank Nadine Reulen and Sonja Gostek for their skillful technical assistance and Christin Lebing from Decodon for the professional backup regarding statistical analysis of the 2DE data.
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