Lenhard et al. BMC Cancer 2012, 12:2 http://www.biomedcentral.com/1471-2407/12/2 Background Due to missing early clinical symptoms, ovarian cancer is often diagnosed at an advanced stage [1]. Primary treatment includes operative cytoreduction and subse- quent combined platinum-based chemotherapy. Though reported primary response rates range around 80%, ovarian cancer is the most lethal gynecological malig- nancy since 60-70% of patients relapse or die within 5 years after primary diagnosis [2-4]. The molecular mechanism of ovarian cancer develop- ment is still discussed controversially [5]. As ovaries are the target organs of gonadotropins, a relation to the development or growth of ovarian cancer has been pos- tulated [6]. An increased risk for the development of ovarian cancer was assumed in women treated for infer- tility who had therefore been stimulated with gonado- tropins [7-9]. Human gonadotropin (hCG) is expressed in placental trophoblasts, but also in a large number of tumors. HCG and the gonadotropin luteal hormone (LH) bind to the same receptor (LH-R) and have similar biological functions, although hCG is more potent because of its higher receptor binding affinity and its longer circula- tory half life. Human chorionic gonadotropin is a glyco- protein produced by the fetal trophoblast during pregnancy and is secreted into the maternal circulation [10]. The commitment of cytotrophoblasts to syncytio- trophoblasts is associated with activation of a- and b- hCG subunit genes [11]. These intermediates are transi- ent, they differentiate to syncytiotrophoblasts and the expression of b-hCG RNA declines [12]. Also in chorion carcinoma cells consisting of clusters of cytotrophoblast- like and large multinucleated cells, a- and b-hCG RNA is expressed [13]. In these cells, hCG has been used as a tumor marker for a long time [14]. There are only few studies with small patient numbers on human chorionic gonadotropin and its receptor expression in ovarian cancer tissue [15,16]. In a previous study we found a prognostic value of LH-R and FSH-R in ovarian cancer patients [17]. The present study was designed to further analyze hCG expression in a large cohort of ovarian cancer patients and its relation to his- tological subtype, grade, stage, gonadotropin receptor expression and patient survival. In addition, we deter- mined hCG serum concentrations in patients with ovar- ian cancer and compared the results to patients with benign ovarian tumors. Methods Sera Sera of patients diagnosed with an ovarian tumor between 2003 and 2006 were obtained before surgery and stored at -80°C. After surgery, histological diagnos- tic evaluation including staging and grading of tumor Page 2 of 8 tissue were performed by an experienced gynecologic pathologist (D.M.) according to the criteria of the Inter- national Federation of Gynaecologists and Obstetricians (FIGO) and the World Health Organization (WHO). Tissue samples All tissue samples were gained at surgery in patients who had been treated for primary ovarian cancer at our institution between 1990 and 2002. Staging and grading were performed by an experienced gynecologic patholo- gist according to the criteria of the International Federa- tion of Gynaecologists and Obstetricians (FIGO) and the World Health Organization (WHO). Patients with ovar- ian borderline tumor were excluded from the study. Clinical data of the patients’ disease were available from patient charts, aftercare files and tumor registry database information. The main outcomes assessed were disease recurrence and patient survival. Ethics approval The study has been approved by the local ethics com- mittee of the Ludwig-Maximilians University Munich (approval with the reference number 138/03) and has been carried out in compliance with the guidelines of the Helsinki Declaration of 1975. The study participants gave their written informed consent and samples and clinical information were used anonymously. hCG-ELISA Concentration of hCG was obtained by an ELISA and using the Immulite 2000 automated diagnostic system (Siemens, Munich, Germany). Standard deviation for precision at 6.5 m IU/ml is 0.43 with a variation coeffi- cient (CV) of 6.6%. Precision analysis showed no cross reactivity with human FSH (26.8 ng/ml analyzed), LH (16.5 ng/ml analyzed) or TSH (860 ng/ml analyzed). Immunohistochemistry was performed as previously described elsewhere, using a combination of pressure cooker heating and the standard streptavidin-biotin-per- oxidase complex with the use of the rabbit-IgG-Vectas- tain Elite ABC kit (Vector Laboratories, Burlingame, CA) [18,19]. Antibodies used for staining were the anti- hCG (17.75 μg/ml, rabbit IgG, polyclonal, dilution 1:400, Dako, Glostrup, Denmark) and anti-LH (LH/hCG-R, 1 mg/ml, rabbit IgG, polyclonal, dilution 1:25, Dianova, Hamburg, Germany). In short, paraffin-fixed tissue sections were dewaxed with xylol for 15 min and then dehydrated in ascending concentrations of alcohol (70%, 96%, and 100%). After- wards, they were exposed for epitope retrieval for 10 min in a pressure cooker using sodium citrate buffer (pH 6.0) containing 0.1 M citric acid and 0.1 M sodium citrate in distilled water. After cooling, slides were washed in PBS twice. Endogenous peroxidase activity