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Inhibin/Activin Subunits Are Immunohistochemically Expressed in Complete and Partial Hydatidiform Moles

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Inhibin/Activin Subunits Are Immunohistochemically Expressed in Complete and Partial Hydatidiform Moles ANTICANCER RESEARCH 27: 1995-2000 (2007) Inhibin/Activin Subunits are Immunohistochemically Expressed in Complete and Partial Hydatidiform Moles IOANNIS MYLONAS1, NAIM SHABANI1,2, JULIA VOGL1, JOSEF MAKOVITZKY3, SUSI KUNZE1, CHRISTINA KUHN1, SANDRA SCHULZE1, KLAUS FRIESE1 and UDO JESCHKE1 1Ludwig-Maximilians University Munich, First Department of Obstetrics and Gynaecology, 80337 Munich, Germany; 2Landes-Krankenhaus Schaerding, Abteilung für Frauenheilkunde und Geburtshilfe, A-4780 Schaerding, Austria; 3University of Rostock, Department of Obstetrics and Gynaecology, 18057 Rostock, Germany Abstract. Background and aim: Inhibins are dimeric complete moles, suggesting the utilization of these antibodies glycoproteins, belonging to the transforming growth factor beta as diagnostic differentiation markers between complete and (TGF-‚) family, composed of an ·-subunit (INH-·) and one of partial moles. two possible ‚-subunits (‚A or ‚B). Additionally two further ‚- subunits (‚C and ‚E) have been cloned, although their function Inhibins are dimeric disulphide-linked glycoproteins and are remains still quite unclear. The detection by immuno- members of the transforming growth factor beta (TGF-‚) histochemistry of inhibin/activin subunits has been proposed as a family of cytokines; they were initially isolated from the useful marker of trophoblastic diseases. Interestingly, a complete gonads and identified as modulators of FSH production mole cannot be easily differentiated from a partial mole. from the anterior pituitary gland (1, 2). They are Therefore, the aim of this study was to determine expression heterodimers consisting of one ·-subunit and one of two changes of the five inhibin/activin subunits in partial and possible ‚-subunits (‚A- and ‚B-subunits). The ·-subunit complete moles. Materials and Methods: Histologically diagnosed can dimerize with either ‚A or ‚B to form inhibin-A (·-‚A) complete (n=6) and partial (n=3) hydatidiform moles were or -B (·-‚B), respectively. Activins are homodimers of ‚- immunohistochemical analyzed for INH-·, INH-‚A, INH-‚B, subunits linked by a disulfide bond. Depending on the INH-‚C and INH-‚E subunits. The immunohistochemical combination of the subunits, there are three isoforms of reaction in intermediate trophoblast was analyzed with a semi- activin, namely activin-A (‚A-‚A), activin-B (‚B-‚B) and quantitative score (IRS) and statistical analysis was performed. activin-AB (‚A-‚B) (1, 2). Recently, three additional ‚- Results: Immuno-histochemical reaction with INH-·, INH-‚A, subunits have been identified, determined as ‚C (3), ‚D INH-‚B, INH-‚C and INH-‚E subunits was demonstrated in (Xenopus only) (4) and ‚E (5), although their precise hydatidiform moles. The INH-‚A and INH-‚B expression was function remains unknown. significantly higher in complete compared to partial moles The expression of inhibin/activin subunits have been (p<0.05 each), while INH-·, INH-‚C and INH-‚E did not described in different female tissues, including normal and demonstrate any statistically significant differences. Conclusion: pathological human endometrium (6-9), and in normal and We demonstrated an immunohistochemical expression of all five pathological placenta (10-13), suggesting different important inhibin/activin subunits in partial and complete hydatidiform roles, such as paracrine modulators of reproductive function moles. The expression of INH-‚A and INH-‚B determined (1, 14). Inhibin/activin subunits are also expressed in immunohistochemically was significantly up-regulated in placental decidua, the syncytiotrophoblast (13) and in the trophoblast (10, 12), while recently inhibin/activin subunits were demonstrated in a hydatidiform mole with persistent polymorphic trophoblastic hyperplasia (15). Correspondence to: PD Dr. Udo Jeschke, Ludwig-Maximilians- Hydropic swelling of some or all villi and trophoblastic University Munich, First Department of Obstetrics and proliferation characterize hydatidiform mole, including two Gynaecology, Maistrasse 11, 80337 Munich, Germany. Tel: +49 89 distinct entities, complete and partial hydatidiform moles 5160 4266, Fax: +49 89 5160 4916, e-mail: [email protected] (16). Increased levels of circulating inhibin have previously muenchen.de been reported in molar pregnancy: it has been proposed Key Words: Inhibin/activin subunits, inhibin-·, inhibin-‚A, that serum inhibin concentrations may be of value in the inhibin-‚B, inhibin-‚C, inhibin-‚E, complete hydatidiform mole, management of trophoblastic disease (17) and increased partial hydatidiform mole. serological concentrations of inhibin can be found in 0250-7005/2007 $2.00+.40 1995 ANTICANCER RESEARCH 27: 1995-2000 (2007) Table I. Antibodies used for immunohistochemical characterisation of complete and partial hydatidiform moles by immunohistochemistry. Antibody Clone Isotype Dilution Source Inhibin-· R1 mouse IgG2a 1:50 Serotec, Oxford, UK Inhibin-‚A E4 mouse IgG2b 1:50 Serotec, Oxford, UK Inhibin-‚B C5 mouse IgG2a 1:10 Serotec, Oxford, UK Inhibin-‚C Polyclonal Goat 1:50 R&D Systems, Inhibin-‚E Polyclonal rabbit 1:4000 In house antibody BioGenes, Berlin, Germany hydatidiform mole (18). Recently, it was suggested that complex with the use of the mouse-IgG-Vectastain Elite ABC kit serum inhibin A and activin A measurements might be of (Vector Laboratories, Burlingame, CA, USA) for inhibin-·, -‚A value in diagnosis and short-term follow-up of molar and -‚B as well as goat-IgG-Vectastain Elite ABC kit (Vector pregnancy (19). Inhibin-· and -‚ subunits are consistently Laboratories) for inhibin-‚C and -‚E. The antibodies used are given in Table I. co-expressed immunohistochemically in syncytiotrophoblast Briefly, paraffin-fixed tissue sections were dewaxed using xylol in complete and partial moles, suggesting that these for 15 min, rehydrated in an ascending series of alcohol (70%, 96% glycoproteins might be useful tissue markers in the and 100%) and subjected to antigen retrieval on a high setting for differential diagnosis of trophoblastic lesions (20). 10 min in a pressure cooker in sodium citrate buffer (pH 6.0), Interestingly, complete moles can be reliably distinguished containing citrate acid 0.1M and sodium citrate 0.1M in distilled from non-molar pregnancy, but neither non-molar pregnancy water. After cooling, the slides were washed twice in PBS. nor complete moles are easily differentiated from partial Endogenous peroxidase activity was quenched by immersion in 3% hydrogen peroxide (Merck, Darmstadt, Germany) in methanol for mole (21, 22). However, limited data on histological 20 min. Non-specific binding of the primary antibodies was blocked expression of inhibin/activin subunits expression in these by incubating the sections with diluted normal serum (10 ml PBS two forms of hydatidiform moles exists. Therefore, the containing 150 Ìl horse serum; Vector Laboratories) for 20 min at aims of the present study were to determine expression room temperature. Sections were then incubated at room changes of the five inhibin/activin subunits in partial and temperature for 120 min with the primary antibodies. Inhibin-· was complete moles. diluted in PBS. After washing with PBS, the slides were incubated in diluted biotinylated serum (10 ml PBS containing 50 Ìl horse serum; Vector Laboratories) for another 30 min at room Materials and Methods temperature. After incubation with the avidin-biotin peroxidase complex for another 30 min and repeated washing steps with PBS, Placental tissues diagnosed by a gynecological pathologist as visualisation was performed with substrate and the chromagen 3,3'- complete (n=6) and partial (n=3) hydatidiform moles were diaminobenzidine (DAB; Dako, Glostrup, Denmark) for 8-10 min. obtained from the First Department of Obstetrics and Gynaecology The slides were counterstained further with Mayer’s acidic of the LMU Munich. hematoxylin and washed in a series of alcohol (50-98%). After xylol treatment the slides were covered. Generation of a polyclonal activin-‚E peptide antibody. Anti-activin- Negative controls were performed by replacing the primary ‚E polyclonal antibodies were generated in rabbits against a antibody with normal horse serum (Vector Laboratories). polypeptide of 16 amino acids of activin-‚E (polypeptide-sequence: Positive cells showed a brownish color and the negative control, NH2-CRWGPRRRRQGSRTLL-COOH; amino acid position 144 as well as unstained cells, appeared blue. The standardisation, to 158; accession number: AAH05161). A primary dose of 200 Ìg dilution and optimisation of this protocol for inhibin-·, -‚A and activin-‚E polypeptide was emulsified in Freund’s complete adjuvant -‚B was primarily tested on normal premenopausal ovary tissue, (Sigma-Aldrich, Germany) and administered subcutaneously in while negative controls included postmenopausal ovarian tissue. rabbits. Three doses of the peptide emulsified in Freund’s For inhibin-‚C and inhibin-‚E normal liver tissue was used as incomplete adjuvant were administrated at intervals (6 weeks). After positive control. the third booster injection (14 days), blood was collected from the rabbit and the serum was separated. Antibodies were isolated using Immunohistochemical evaluation and statistical analysis. The column chromatography with a protein A column (Amersham intensity and distribution patterns of specific inhibin/activin subunit Pharmacia Biotech, Freiburg, Germany). immunohistochemical staining reaction was evaluated by two blinded, independent observers, including a gynecological Immunohistochemistry. Immunohistochemistry
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