Expression of microtubule associated protein 2 and synaptophysin in endometrium: high levels in deep infiltrating endometriosis lesions  Martina Gori, B.Sc., Alice Luddi, Ph.D., Giuseppe Belmonte, B.Sc., Paola Piomboni, Ph.D., Claudia Tosti, M.D., Lucia Funghi, Ph.D., Errico Zupi, M.D., Lucia Lazzeri, M.D., Ph.D., Felice Petraglia, M.D.  Fertility and Sterility  Volume 105, Issue 2, Pages 435-443 (February 2016) DOI: 10.1016/j.fertnstert.2015.10.024 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 NGF, SYP, and MAP-2 mRNA expression in DIE or OMA lesions, in eutopic endometrium, and in healthy control (proliferative phase). Fold change (y-axis) represents NGF, SYP, or MAP-2 expression normalized to HPRT1, relative to healthy endometrium considered to be equal to 1. The expression of NGF was significantly stronger in the lesions than in the eutopic endometrium and healthy endometrium. SYP mRNA expression levels for DIE were the highest. MAP-2 mRNA expression levels for both OMA and DIE were the highest. Data represent value obtained by ΔΔCt method. ***P<.001. Fertility and Sterility 2016 105, 435-443DOI: (10.1016/j.fertnstert.2015.10.024) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Double-staining immunofluorence of the NGF- (green), MAP-2- (red), and SYP-positive cells (green) in healthy endometrium. Double-staining immunofluorence of the NGF- (green), MAP-2- (red), and SYP-positive cells (green) in OMA and DIE. Nerve growth factor showed an intense signal in glandular epithelium and in stromal tissue surrounding glands of both OMA and DIE lesions. Microtubule-associated protein 2 became intense and diffuse in OMA and DIE. Synaptophysin staining was very weak in OMA samples, whereas the labeling was clearly detectable in DE stroma. Scale bar = 100 μm. Fertility and Sterility 2016 105, 435-443DOI: (10.1016/j.fertnstert.2015.10.024) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Top: Colocalization of NGF and MAP-2 is negative in endometrium but is evident in glandular epithelium of OMA and in DE tissue. Colocalization of NGF and SYP was negative in endometrium and OMA but was clearly present in DIE stroma. Scale bar = 100 μm. Bottom: The intensity of NGF (left histogram), MAP-2 (middle histogram) and SYP (right histogram) is shown in endometrium, OMA, and DIE, in comparison with the endometrium (considered to be equal to 1). Data are represented as mean ± SD. Significant differences between groups are analyzed by one-way analysis of variance. *P<.05. Fertility and Sterility 2016 105, 435-443DOI: (10.1016/j.fertnstert.2015.10.024) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 4 NGF (A) and SYP (B) mRNA expression in endometrial stromal cells prepared from healthy endometrium. Fold change (y-axis) represents NGF or SYP expression normalized to HPRT1, relative to untreated HESCs, considered to be equal to 1. NGF and SYP mRNA expression levels increased after TNF-α treatment at 0.5 and 5 ng/mL. Data represent value obtained by ΔΔCt method. **P<.01; ***P<.001. Fertility and Sterility 2016 105, 435-443DOI: (10.1016/j.fertnstert.2015.10.024) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions