AbstractObjectiveTo evaluate whether human epididymis protein 4 (HE4) andCA125 correlate with known high‐risk prognostic factors for endometrial cancer.DesignProspective multicenter study.SettingThree Danish tertiary gynecological oncology centers.PopulationA total of 352 patients with endometrial cancer and atypical endometrial hyperplasia consecutively referred between 1 September 2009 and 1 January 2012.MethodsPreoperative blood samples were obtained from all patients. Biomarker levels were correlated with pathological characteristics of hysterectomy specimens.Main outcome measuresFIGOstage, depth of myometrial invasion, cervical involvement, lymph node metastases, and histological type and grade of tumor.ResultsWe found that bothHE4 andCA125 were significantly positively correlated with histological grade (HE4:p = 0.002 andCA125:p = 0.027), lymph node metastases (HE4:p = 0.013 andCA125:p < 0.0001), myometrial invasion (p < 0.0001) and cervical involvement (p < 0.0001). Furthermore, a significant increase was found with increasingFIGOstage for both markers (p < 0.0001). In a combined index including age, the diagnostic value increases. Area under the receiver operating characteristics curves were higher for the index compared with the markers individually for all our endpoints. The calculated plots for the combined index may assist gynecologists in predicting the risk of deep myometrial invasion, cervical involvement and lymph node metastases. The analyses emphasize that the combined markers should be used in the prediction of prognostic factors.ConclusionThis study confirmed that the markers are significantly elevated in patients with prognostic high‐risk factors and may, therefore, be used as an additional tool in combination with imaging and clinical information when planning the treatment of endometrial cancer patients.