1. Immune-inflammation gene signatures in endometriosis patients
Soo Hyun Ahn, M.Sc., Kasra Khalaj, M.Sc., Steven L. Young, M.D., Ph.D., Bruce A. Lessey, M.D., Ph.D., Madhuri Koti, D.V.M., Ph.D., Chandrakant Tayade, D.V.M., Ph.D. 
Fertility and Sterility 
Volume 106, Issue 6, Pages e7 (November 2016)
DOI: /j.fertnstert
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Unsupervised hierarchic clustering of 24 samples with the use of GENE-E software for 579 inflammation and immune-related genes. Ectopic endometrium (n = 8), eutopic endometrium (n = 8), and control endometrium samples (n = 8) were subjected to unsupervised hierarchical clustering analysis for (A) all 579 analyzed genes followed by (B) the same analysis using 299 selected genes to assess differential expression of transcriptomics in each group. The dendogram on the right-hand side shows complete segregation of the ectopic endometriotic samples (ectopic 1–8) from the eutopic endometrium (eutopic 1–8) and control endometrium tissue samples (control 1–8). The list of significantly differentially expressed genes for ectopic versus control (396 genes significantly differentially expressed) and ectopic versus eutopic (322 genes significantly differentially expressed) were further compared to identify transcriptomes that were differentially measured in ectopic endometriotic tissues only. This led to the identification of 299 genes that were commonly differentially expressed in transcript counts in the ectopic endometriotic tissue sample (B). The color scale bar denotes maximum counts in red and minimal counts in blue.
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3. Figure 2
Representative immune genes were significantly increased in ectopic endometriotic lesions. The expression of genes encoding for (A–F) inflammatory cytokines and receptors, (G) receptor for FAS, (H–L) TNF superfamily, (M–R) cell surface markers for T-cell activation and antigen-presenting cell activation, (P and Q) chemoattractants for endothelial progenitor cell recruitment, (S and T) cell adhesion molecules, and (U–Y) HLA molecules were significantly increased in the ectopic tissues compared with the control. The graphs represent the scatter plots of all samples. The middle bar denotes the mean of the samples, and the error bars represent the standard deviation. *P≤.05 between ectopic versus eutopic and between ectopic versus control.
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4. Figure 3
Representative immune genes were significantly decreased in ectopic endometriotic lesions compared with matched eutopic endometrium. Genes involved in (A–C) the regulation of natural killer and cytotoxic T-cell activity and (D–F) the cell signaling mechanism for proliferation and apoptosis were significantly decreased in expression in ectopic tissues compared with control. The expression of genes involved in the regulation of T-cell activity and inflammation were significantly decreased in ectopic tissues compared with control (G and H). The graphs represent scatter plots of all samples. ∗P≤.05 between ectopic versus eutopic and between ectopic versus control.
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5. Figure 4
Genes involved in the cell signaling pathway for apoptosis and cell death were significantly decreased in the eutopic endometrium. Genes involved in (A–H) the apoptosis and cell death signaling pathway and (J and K) decidualization were significantly decreased in eutopic endometrium compared with control. (I) BAX, a gene encoding for a protein involved in apoptosis, was significantly increased in eutopic endometrium compared with control. ∗P≤.05.
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6. Supplemental Figure 1
Marker selection analysis between the tissue types ranks 579 genes from most (red) to least (blue) differentially expressed genes. To organize 579 genes into most differentially expressed to least, each comparisons—(A) ectopic versus control, (B) eutopic versus control, and (C) ectopic versus eutopic—were subjected to marker selection analysis with the use of GENE-E software.
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7. Supplemental Figure 2
Flow chart of how 299 “signature genes” for ectopic endometriotic samples were determined. To discern a list of genes that may be differentially regulated only for ectopic endometriotic samples, we compared the list of genes that were statistically significant between ectopic versus control and between ectopic versus eutopic. With the use of a Venn diagram, 299 genes were common in both groups. Unsupervised hierarchic clustering with the use of 299 genes still grouped the ectopic samples away from the samples of eutopic endometrium and control endometrium, assuring that these 299 genes could be used for marker selection analysis. Pathway and network analysis with the use of DAVID bioinformatics resources allowed us to discern KEGG pathways and Gene Ontology terms to better establish the biologic relationship of the genes.
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8. Supplemental Figure 3
Genes involved in angiogenesis, pain, and inflammasome complex were significantly increased in ectopic endometriotic lesions. The expression of genes involved in (A and B) endothelial progenitor cell recruitment and promotion of angiogenesis, (C) prostaglandin activity and pain, and (D and E) inflammasome complex formation were significantly increased in ectopic tissues compared with matched eutopic endometrium and control endometrium. The graphs represent the scatter plots of all samples. *P≤.05 between ectopic versus eutopic and between ectopic versus control.
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9. Supplemental Figure 4
Genes involved in classic and alternate complement protein pathways were aberrantly expressed in ectopic endometriotic tissues. (A–N) Genes involved in complement pathways were significantly increased in ectopic tissues compared with control. (O) C4BPA, a gene encoding for C4b-binding protein, which controls the activation of the classic complement pathway, was the only complement pathway protein that was significantly decreased in expression. *P≤.05.
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10. Supplemental Figure 5
Representative immune genes were significantly increased in the eutopic endometrium. The expression of genes encoding for (A–G) proinflammatory cytokines, chemokines, and receptors for interleukin (IL) 8 and IL-7, (H and I) cell adhesion molecules, and (J–L) HLA molecules were significantly increased in eutopic endometrium compared to the control endometrium. The graphs represent scatter plots of all samples. *P≤.05.
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11. Supplemental Figure 6
Genes encoding for proteins involved in the complement pathway, B and natural killer (NK) cell signaling, cell signaling molecules, and costimulatory molecules were aberrantly expressed in the eutopic endometrium. The expression of genes encoding for (A–C) classic and alternate complement pathway, (E–K) B and NK cell activation and signaling, (L, N, and O) cell signaling molecules, and (P and Q) immune cell costimulatory molecules were significantly increased in eutopic endometrium compared with control. C4A/B (D) and IKBKAP (M) were significantly decreased in eutopic endometrium compared with control. *P≤.05.
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12. Supplemental Figure 7
RORC and IL-17A expression in ectopic endometriotic tissues and eutopic endometrium compared to control endometrium samples. Nanostring mRNA transcript counts of (A and B) RORC and (C and D) IL-17A in (A and C) ectopic versus control (B and D) eutopic versus control shows that RORC transcript counts are lower and IL-17A transcript counts are higher in endometriosis patients compared with control. P values are denoted in each graph.
Fertility and Sterility  , e7DOI: ( /j.fertnstert )
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions