Anti-platelet therapy is efficacious in treating endometriosis induced in mouse  Sun-Wei Guo, Ding Ding, Xishi Liu  Reproductive BioMedicine Online  Volume 33, Issue 4, Pages 484-499 (October 2016) DOI: 10.1016/j.rbmo.2016.07.007 Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 (A) Boxplot of lesion area among different treatment groups. (B) Changes in mean (±SD) hotplate latency in different treatment groups. U or UT (n = 11): untreated group; L or LO (n = 10), low-dose Ozagrel; H or HO (n = 10), high-dose Ozagrel. * P < 0.05, NS = not statistically significant. For time 0 week versus 2 weeks P < 0.001. Reproductive BioMedicine Online 2016 33, 484-499DOI: (10.1016/j.rbmo.2016.07.007) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Boxplots of different marker staining levels in different treatment groups of C57BL/6 mice in the ectopic lesions. (A) CD41 labelled platelets; (B) VEGF (vascular endothelial growth factor); (C) MVD (microvessel density, CD31 labelled endothelial cells); (D) PCNA (proliferating cell nuclear antigen); (E) F4/80 labelled macrophages; (F) CD163 labelled M2 macrophages. (G) p-p65; (H) COX-2; (I) FGFR2; (J) fibronectin; (K) α-SMA (smooth muscle actin); (L) collagen I. Staining level based on the average number of pixels counted. The dashed line represents the regression line of the data. Symbols of ‘*’, ‘**’, ‘***’ denote statistical significance levels of P < 0.05, P < 0.01, P < 0.001, respectively, as compared with the untreated (UT) group. UT (n = 11): untreated group; LO (n = 10), low-dose Ozagrel; HO (n = 10): high-dose Ozagrel. Reproductive BioMedicine Online 2016 33, 484-499DOI: (10.1016/j.rbmo.2016.07.007) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Hierarchical clustering heatmap of hotplate latency (HPL), lesion area and immunoreactivity measurements in mice. The heatmap was organized by clustering mice by rows (U: untreated [brown]; L: low-dose Ozagrel group [blue]; H: high-dose Ozagrel group [dark blue]) and HPL, lesion area and immunoreactive marker measurements by columns (Phenotype). Red represents the minimal values and white the maximal values. All phenotypes formed 5 clusters: cluster 1 consists of hotplate latency alone; cluster 2, α-SMA, lesion area and FGFR2; cluster 3, fibronectin and collagen I; cluster 4, the extent of platelet aggregation (CD41), p-p65 (NF-κB activation), and PCNA (proliferation marker); and cluster 5, VEGF, COX-2, number of M2 macrophages (CD163), number of macrophages (F4/80), and MVD (angiogenesis). All mice could be grouped roughly into 2 clusters. Cluster 1 (I), consists exclusively of untreated mice, exhibiting a larger lesion area, and elevated immunoreactivity to VEGF, PCNA, COX-2, p-p65, collagen I, α-SMA, fibronectin and FGFR2, more platelet aggregation, more (all and M2) macrophage infiltration, higher MVD, and shorter HPL in the ectopic endometrium. In contrast cluster 2 (II) exhibits smaller lesion area, reduced immunoreactivity to VEGF, PCNA, COX-2, p-p65, collagen I, α-SMA, fibronectin and FGFR2, less platelet aggregation and macrophage infiltration, lower MVD, and longer HPL. This cluster includes exclusively all mice that received Ozagrel treatment. FGFR2 = fibroblast growth factor receptor 2; MVD = microvessel density; PCNA = proliferating cell nuclear antigen; SMA = smooth muscle actin; VEGF = vascular endothelial growth factor. Reproductive BioMedicine Online 2016 33, 484-499DOI: (10.1016/j.rbmo.2016.07.007) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Results of multidimensional scaling analysis to classify all mice using data on fibronectin and α-SMA staining levels, the extent of platelet aggregation, the number of infiltrated M2-type macrophages, and MVD. All mice are represented by their group labels, which are the same as used in Figure 1B. MVD = microvessel density; SMA = smooth muscle actin. Reproductive BioMedicine Online 2016 33, 484-499DOI: (10.1016/j.rbmo.2016.07.007) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 (A) Boxplot of lesion weight (in grams) among different treatment groups. (B) Changes in mean (±SD) hotplate latency in different treatment groups. For day −2 versus day 12 in all groups P < 0.001. (C) Boxplot of the percentage of F4/80 and CD11b dual positive macrophages in peritoneal fluid from the 4 groups of mice using FACS. (D) Boxplot of the percentage of CD206+ macrophages found in the peritoneal fluid from the 4 groups of mice using FACS. ND, no depletion; ED, early platelet depletion; LD, later platelet depletion; DD, early platelet depletion followed by later macrophage depletion. Each group had 8 mice. *: P < 0.05; **: P < 0.01; ***: P < 0.001 (Wilcoxon test) for the comparison between the treatment group and the group that received non-immune IgG antibody (ND). FACS = fluorescence-activated cell sorting; IgG = immunoglobulin G; NS = not statistically significant. Reproductive BioMedicine Online 2016 33, 484-499DOI: (10.1016/j.rbmo.2016.07.007) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 Boxplot of the number of F4/80 + macrophages infiltrating the ectopic endometrium (A) in experiment 2; the extent of CD41+ platelet aggregation, based on the number of CD41+ platelets in a unit area (B), staining levels of VEGF (C), microvessel density (CD31) (D), staining levels of TGF-β1 (E), of α-SMA (F), and of collagen I (G), and the percentage of epithelial cells in ectopic endometrium among different treatment groups. The P-value of the difference among the 4 groups is shown in each figure. ND (n = 8), no depletion; ED (n = 8), early platelet depletion; LD (n = 8), later platelet depletion; DD (n = 8), early platelet depletion followed by later macrophage depletion. SMA = smooth muscle actin; TGF = transforming growth factor; VEGF = vascular endothelial growth factor. Reproductive BioMedicine Online 2016 33, 484-499DOI: (10.1016/j.rbmo.2016.07.007) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 7 Hierarchical clustering heatmap of hotplate latency (HPL), lesion weight (WT), percentage of epithelial cells in endometria (EPI%) and immunoreactivity measurements in mice. The heatmap was organized by clustering both mice by rows (ED: early platelet depletion group [green]; ND: No depletion [Indian red]; LD: later platelet depletion group [dark blue]; DD: early platelet depletion plus later macrophage depletion group [purple]) and HPL, WT, EPI% and immunoreactive marker measurements by columns. Red represents the minimal values and white the maximal values. Cluster I includes almost all mice in group ND. Cluster II includes all the rest mice. CD41: extent of CD41-labelled platelet aggregation; aSMA: α-smooth muscle actin (α-SMA); COL_I: Collagen I; F4/80+MP: number of F4/80-labelled macrophage infiltrated into the ectopic endometrium; MVD: CD-31-labelled microvessel density; TGFbeta1: transforming growth factor β1 or TGF-β1; VEGF: vascular endothelial growth factor. See text for more details. Reproductive BioMedicine Online 2016 33, 484-499DOI: (10.1016/j.rbmo.2016.07.007) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions