Evaluation of erythrocyte band 3 phosphotyrosine level, glutathione content, CA-125, and human epididymal secretory protein E4 as combined parameters in endometriosis  Luciana Bordin, Ph.D., Cristina Fiore, M.Sc., Gabriella Donà, M.Sc., Alessandra Andrisani, M.D., Guido Ambrosini, M.D., Diego Faggian, Ph.D., Mario Plebani, M.D., Giulio Clari, M.Sc., Decio Armanini, M.D.  Fertility and Sterility  Volume 94, Issue 5, Pages 1616-1621 (October 2010) DOI: 10.1016/j.fertnstert.2009.10.038 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Decrease in total glutathione content after diamide treatment (ΔGSH, expressed as 1 − GSH(D)/GSH(B)) and increase in Tyr-P, CA-125, and HE4 in three groups. Data are expressed as means ± SEM. +P<.05; +++P<.001, EG versus PS-EG. ∗P<.05; ∗∗P<.01; ∗∗∗P<.0005, EG or PS-EG versus CG. Fertility and Sterility 2010 94, 1616-1621DOI: (10.1016/j.fertnstert.2009.10.038) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Values of CA-125, HE4, diamide-induced band 3 Tyr-P level, total GSH content in both basal and diamide treated RBC and combination of both, in CG (control group), EG (endometriosis group), Non-EG (non-endometriosis group) and PS-EG (post surgery-endometriosis group). Values are expressed as mean ± SD of serum concentrations of CA-125 and HE4, and of erythrocyte parameters, band 3 Tyr-P level, GSH content in basal(B), or following diamide treatment(D), or combination of both (ΔGSH), in the different groups of patients. Arrows indicate statistical comparisons, determined by ANOVA followed by paired (EG vs PS-EG) or unpaired (EG or PS-EG vs CG) Student's t test (two tailed). GSH is expressed as μmol/ml of packed red blood cells. GSH(B) = total content of glutathione in untreated erythrocytes; GSH(D) = total content of glutathione in diamide-treated erythrocytes; ΔGSH = total decrease of glutathione content after diamide treatment expressed as 1-GSH(D)/GSH(B), n.s. = not significant, ∗ P<0.05, ∗∗ P<0.01, ∗∗∗ P<0.001. Fertility and Sterility 2010 94, 1616-1621DOI: (10.1016/j.fertnstert.2009.10.038) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Diamide-induced P-Tyr levels in CG, EG, and PS-EG. Fresh blood was collected from all patients and controls, and erythrocytes, isolated as described in the Methods section, were incubated with and without 1.5 mM diamide. Membranes (10 μg), obtained as described in the Methods section, underwent Western blotting and were immunostained with anti-P-Tyr antibody or anti-actin antibody as a loading control. This figure is representative of more than 30 separate experiments. Fertility and Sterility 2010 94, 1616-1621DOI: (10.1016/j.fertnstert.2009.10.038) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 ROC curve for ΔGSH (panel A) and Tyr-P (panel B) in erythrocytes as a test to discriminate between patients before and after surgery. For ΔGSH, AUC was 0.87, sensitivity was 82.76%, specificity was 78.57%, and the cutoff was 0.31 ΔGSH. For Tyr-P, AUC was 0.75, sensitivity was 48.28%, specificity was 92.86%, and the cutoff was 193% Tyr-P. Fertility and Sterility 2010 94, 1616-1621DOI: (10.1016/j.fertnstert.2009.10.038) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions