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Edurne Novella-Maestre, Ph. D. , Sonia Herraiz, Ph. D

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1 Effect of antiangiogenic treatment on peritoneal endometriosis-associated nerve fibers 
Edurne Novella-Maestre, Ph.D., Sonia Herraiz, Ph.D., José María Vila-Vives, M.D., Carmen Carda, Ph.D., M.D., Amparo Ruiz-Sauri, Ph.D., M.D., Antonio Pellicer, Ph.D., M.D.  Fertility and Sterility  Volume 98, Issue 5, Pages (November 2012) DOI: /j.fertnstert Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

2 Figure 1 Macroscopic evaluation of human endometrial implants on the peritoneal wall of mice. (A) Experimental lesions show a well-developed vascular net and adhesions that resemble human endometriotic lesions. (B) Histologic study shows implants present a cellular stroma surrounding the glandular areas attached to the peritoneal murine tissue (stain: hematoxylin and eosin; magnification: ×100). (C–D) Control mice lesions present a cellular stroma with rich areas of blood vessels and full areas of (C) hemosiderin-macrophage pigments (stain: hematoxylin and eosin; magnification: ×200) and (D) mast cells (stain: blue toluidine; magnification: ×200). (E–F) Deprived areas of (E) macrophages (stain: hematoxylin and eosin; magnification: ×200) and (F) mast cells (stain: blue toluidine; magnification: ×200) are observed in the lesion from Cb2-treated mice when compared with control mice. (G–H) Morphometric study of the samples reveals a statistically significant decrease (P<.05) of (G) macrophages and (H) total inactivated and activated mast cells in the lesions from Cb2-treated mice when compared with the control group. Fertility and Sterility  , DOI: ( /j.fertnstert ) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

3 Figure 2 Vascularization analysis in controls and the Cb2-treated group. (A) Lesions from the control group presented a higher prevalence of newly formed blood vessels areas (red: +vWF(red)/−αSMA) with respect to mature blood vessels areas (green: +vWF/+αSMA), indicating an active angiogenic process in untreated mice lesions. (B) In the lesions from Cb2-treated mice, the reversal of this process was observed with predominant mature blood vessel areas over the newly formed ones, indicating an inhibition of the angiogenic process. (C) When morphometric analysis was performed, a decreasing trend was observed for microvascular density (MVD) in the Cb2-treated groups, and immature blood vessels (IBV) were statistically significantly decreased (*P<.001) in Cb2-treated lesions when compared with controls. Fertility and Sterility  , DOI: ( /j.fertnstert ) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

4 Figure 3 Nerve fibers analysis with controls and the Cb2-treated group. (A) Protein gene product (PGP 9.5) staining was absent in human endometrial samples before implantation in the mouse peritoneum (magnification: ×200). (B–E) PGP 9.5 was fully expressed in all the lesions (B, D: magnification ×100; and C, E: magnification ×200) and healthy mouse peritoneum (F: magnification ×200). However, Cb2-treated lesions show poor PGP9.5 stained areas (D, E: magnification ×100 and ×200, respectively). The nerve fibers density (NFD) study showed a statistically significantly lower (*P<.05) NFD in Cb2-treated lesions when compared with controls (G). Fertility and Sterility  , DOI: ( /j.fertnstert ) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

5 Figure 4 Dopamine neurotransmitter binds to dopamine receptor-2, located on the endothelial cell surface, and promotes the VEGFR-2 endocytosis, preventing VEGF-VEGFR-2 from binding. The receptor phosphorylation is blocked, and the angiogenesis process is inhibited. Consequently, there is blood vessel regression and a neuroendocrine and immune disequilibrium in the endometriotic tissue. Fertility and Sterility  , DOI: ( /j.fertnstert ) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions


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