“Spot”-ting differences between the ectopic and eutopic endometrium of endometriosis patients  Nina Chehna-Patel, M.Sc., Geetanjali Sachdeva, Ph.D., Rahul Gajbhiye, M.B.B.S., Neeta Warty, M.D., Vrinda Khole, Ph.D.  Fertility and Sterility  Volume 94, Issue 6, Pages 1964-1971.e1 (November 2010) DOI: 10.1016/j.fertnstert.2010.01.048 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) A representative silver-stained 2D gel of ectopic endometrium (ECE), indicating the positions of the 17 ectopic-exclusive spots (N1 to N17). (B) Detection of haptoglobin, Rho-GDIα, and SM-22α in three control endometrium (CE) and three paired eutopic endometrium (EUE) and ECE samples via immunoblotting. Densitometry analysis of immunoreactive bands appearing at the expected size was carried out to measure relative quantity of the protein of interest. The density of the band for the protein of interest was divided by that of GAPDH (an internal protein-loading control) for each sample. (C) Relative levels of haptoglobin and Rho-GDIα in ECE (n = 3) compared with its EUE counterpart from the same patient (n=3) and CE from control women (n = 3). Fold change (FC) in the protein expression was calculated by dividing the mean of the normalized density of the immunoreactive band of haptoglobin/Rho-GDIα in ECE with that of CE or EUE. ∗Statistically significant values (P<.05) calculated by one-way analysis of variance with Tukey multiple-comparison test. Fertility and Sterility 2010 94, 1964-1971.e1DOI: (10.1016/j.fertnstert.2010.01.048) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Immunolocalization of haptoglobin (B, E, H, and K) and α-SMA (C, F, I, and L) in proliferative control (A, B, and C), secretory control (D, E, and F), eutopic (G, H, and I), and ectopic (J, K, and L) endometrial sections. Immunoreactive haptoglobin in ECE (K) was localized in apical region of the glands, indicative of its secretory nature, with weak staining in the stromal compartment. In contrast, in EUE (H) of the same patient immunostaining was maximal in the stroma. In CE (B and E) the localization pattern of haptoglobin was similar to that of the EUE but the intensity of staining significantly less. An increase in the number of blood vessels was seen, as indicated by α-SMA–stained endothelial cells in the EUE (i) compared with the CE (C and F), although in the ECE the immunostaining for α-SMA was intense in the cytoplasm in a majority of the stromal cells (l). Negative control samples (A, D, G, and J) with primary antibodies omitted are also shown. Magnification: ×40 for A, B, D, E, G, H, J, and K; ×10 for C, F, I, and L; insets ×40. Fertility and Sterility 2010 94, 1964-1971.e1DOI: (10.1016/j.fertnstert.2010.01.048) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Immunolocalization of Rho-GDIα (A, D, G, and J), SM-22α (B, E, H, and K), and Rab37 (C, F, I, and L) in proliferative CE (A, B, and C), secretory CE (D, E, and F), EUE (G, H, and I), and ECE (J, K, and L) sections. Immunoreactive Rho-GDIα in ECE (J) was intensely localized in the cytoplasm of a subset of cells, present both in the stroma and in the glands. In contrast, in EUE (G) of the same patient and CE (A and D), a decrease in the staining intensity was seen. SM-22α demonstrated cytoplasmic immunostaining in ECE (K). In contrast, in EUE (H) of the same patient as well as CE (B and E), negligible immunostaining was seen. Rab37 was immunolocalized to cytoplasmic cells in the ECE (L), and in EUE (I) of the same patient as well as CE (C and F) negligible immunostaining was seen. CD10 immunostaining in ECE (K1 and L1) confirmed that the cells are of endometrial origin. Negative control samples with primary antibodies omitted are also shown as insets (A1, D1, G1, and J1). All images are at ×40. Fertility and Sterility 2010 94, 1964-1971.e1DOI: (10.1016/j.fertnstert.2010.01.048) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions