Supporting evidences for potential biomarkers of endometriosis detected in peripheral blood

Endometriosis is one of the most common gynecological diseases in women during the reproductive age [2] , [3] . Despite of this, endometriosis is one of the human diseases where a very long time-interval exists between insurgence of the symptoms and final diagnosis, making it one of the most under-diagnosed and under-treated disease [4] , [5] , [6] , [7] . Therefore, the clinical value of a non-invasive diagnostic test for endometriosis would be enormous, because it could allow to immediately identify among women with sub-fertility, those suffering of endometriosis and to rapidly perform laparoscopic surgery, that has been reported to increase fertility [8] , [9] . ELISA tests were performed on a cohort of endometriosis ( n =100) and healthy patients ( n =10) in order to confirm the differential expression of two proteins, complement C3 and human albumin identified through the proteomic approach described in Signorile and Baldi [1] . Interestingly, most of the albumin was depleted before performing the 2D gel analysis. However, albumin was still identified among the differential expressed markers as it is stated in Table 3 of the original paper [1] . ROC analysis of ELISA results confirmed the statistical significance of the differential expression for these proteins in endometriosis patients respect to healthy controls. In Fig. 1 , the ROC curves for these proteins are depicted, together with specificity and sensitivity.

The authors (Pietro G. Signorile and Alfonso Baldi) declare that they have a patent application (WO 2013/171655) related to the themes of the article.

Details about the recruitment of the human subjects can be found in Signorile and Baldi [1] . Briefly, the control group included 10 healthy women in reproductive age, with no clear signs of endometriosis, such as irregular cycling and ovulating, and that had at least one normal pregnancy. The group of endometriosis patients included only subjects where the diagnosis of endometriosis was definitively confirmed by laparoscopy and histology. Exclusion criteria in the group of endometriosis patients were: a) patients using hormonal medication; b) patients operated within 6 months before the time of sample collections and c) patients suffering of degenerative, autoimmune or neoplastic diseases. The clinical characteristics of the patients enrolled are described in Signorile and Baldi [1] . Analysis was done on a subset of the patient enrolled in the previous paper. A specific selection of the patients was not performed, but all the samples still available for the ELISA assay were used. Ten milliliters of peripheral blood were obtained from patients by venipuncture, collected in EDTA tubes and stored at +4 °C. In order to collect sera, samples were centrifuged at 3000 rpm for 10 min at 4 °C, aliquoted in sterilized tubes, labeled and stored at −80°C. Samples that were haemolysed during the procedure, were not included in the study protocol. Elisa tests were performed using the dedicated Abnova kits for human serum albumin (Catalogue #KA0455) and Complement C3 (Catalogue #KA1020), following the manufacturer׳s protocols. Receiver operating characteristics (ROC) curve analyses were performed to determine the best predictor of endometriosis between the tested variables. Statistical analysis was performed with the SPSS package (version 13.05, SPSS, Inc.).