Surgical removal of endometriotic lesions alters local and systemic proinflammatory cytokines in endometriosis patients  Stephany P. Monsanto, M.Sc., Andrew K. Edwards, Ph.D., Juhua Zhou, Ph.D., Prakash Nagarkatti, Ph.D., Mitzi Nagarkatti, Ph.D., Steven L. Young, M.D., Ph.D., Bruce A. Lessey, M.D., Ph.D., Chandrakant Tayade, D.V.M., Ph.D.  Fertility and Sterility  Volume 105, Issue 4, Pages 968-977.e5 (April 2016) DOI: 10.1016/j.fertnstert.2015.11.047 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) Plasma levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL) 2, IL-8, and IL-10 decreased significantly after removal of lesions. However, post hoc analysis revealed that this difference was significant only in pre- vs. post-op samples, not in pre- vs. 3-month samples. No significant differences were observed for other cytokines. One-way Friedman analysis of variance (ANOVA) and Tukey post hoc test; n = 19. (B) When stratified by disease stage, plasma levels of GM-CSF were found to decrease significantly after surgery in advanced endometriosis patients (stages III–IV), but not in early endometriosis patients (stages I–II). However, post hoc analysis revealed that these differences were not significant. One-way ANOVA and Tukey post hoc test; n = 14 (early stage); n = 5 (advanced stage). (C) When stratified by disease stage and time point, early-stage patients had lower plasma levels of Regulated on activation, normal T expressed and secreted (RANTES) than advanced-stage patients. No differences were seen in early- vs. advanced-stage patients at later time points. No differences were observed for other cytokines. Bars represent the mean, and the whiskers represent the 95% confidence intervals. t test; n = 14 (early stage); n = 5 (advanced stage). *P<.05. Fertility and Sterility 2016 105, 968-977.e5DOI: (10.1016/j.fertnstert.2015.11.047) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Differential expression of nine cytokines was detected in eutopic tissue compared with ectopic tissue. Levels of GM-CSF and IL-15 were lower in eutopic tissue. In contrast, levels of basic fibroblast growth factor (FGF-basic), interferon-inducible protein (IP) 10, IL-1 receptor antagonist (IL-1ra), granulocyte colony–stimulating factor (G-CSF), macrophage inflammatory protein (MIP) 1β, IL-7, and IL-5 were higher in eutopic than in ectopic tissue. Other abbreviations as in Figure 1. Bars represent the mean, and the whiskers represent the 95% confidence intervals. *P<.05; paired t test or Wilcoxon signed ranked test for nonparametric data (n = 18). Fertility and Sterility 2016 105, 968-977.e5DOI: (10.1016/j.fertnstert.2015.11.047) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 When samples were stratified by stage of disease, differential expression of IL-1ra, IL-5, IL-7, IL-9, IL-15, FGF-basic, G-CSF, GM-CSF, IP-10, MIP-1β, and tumor necrosis factor (TNF) α was observed between eutopic and ectopic tissues of early-stage patients, but not in advanced-stage patients. Only IL-2 was found to be differentially expressed in eutopic vs. eutopic tissue from advanced-stage patients. Abbreviations as in Figures 1 and 2. Bars represent the mean, and the whiskers represent the 95% confidence intervals. *P<.05; n = 13; paired t test or Wilcoxon signed ranked test. Fertility and Sterility 2016 105, 968-977.e5DOI: (10.1016/j.fertnstert.2015.11.047) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 When tissue samples were stratified by disease stage and tissue type, levels of platelet-derived growth factor (PDGF) bb, interleukin (IL) 4, interferon (IFN) γ, IL-9, basic fibroblast growth factor (FGF-basic), and tumor necrosis factor (TNF) α were found to be higher in early- vs. advanced-stage eutopic samples. In early- vs. advanced-stage ectopic samples, granulocyte-macrophage colony-stimulating factor (GM-CSF) levels were higher in early-stage compared to advanced-stage, whereas granulocyte colony–stimulating factor (G-CSF) levels where lower in early-stage compared with advanced-stage ectopic samples. No differences were observed for other cytokines. Bars represent the mean, and the whiskers represent the 95% confidence intervals. *P<.05; n = 13 (early stage); n = 5 (advanced stage); paired t test or Wilcoxon signed ranked test. Fertility and Sterility 2016 105, 968-977.e5DOI: (10.1016/j.fertnstert.2015.11.047) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 (A) Peritoneal fluid levels of IL-5 changed significantly according to endometriosis stage (P=.042), but post hoc tests revealed that the differences were not significant. Differences in peritoneal fluid levels of IL-12 were significant only when comparing stage 3 vs. stage 1. *P<.05; n = 36; one-way analysis of variance (ANOVA) and Tukey test (IL-5); Kruskal-Wallis ANOVA and Dunn multiple comparison (IL-12). (B) Mean cytokine concentrations in the peritoneal fluid of patients. IP = interferon-inducible protein; MCP = monocyte chemotactic protein; MIP = macrophage inflammatory protein; RANTES = Regulated on activation, normal T expressed and secreted; VEGF = vascular endothelial growth factor; other abbreviations as in Supplemental Figure 1. Bars represent the mean, and the whiskers represent the 95% confidence intervals. Fertility and Sterility 2016 105, 968-977.e5DOI: (10.1016/j.fertnstert.2015.11.047) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions