1. Interleukin-17A is present in neutrophils in endometrioma and stimulates the secretion of growth-regulated oncogene–α (Gro-α) from endometrioma stromal cells 
Masashi Takamura, M.D., Ph.D., Yutaka Osuga, M.D., Ph.D., Gentaro Izumi, M.D., Osamu Yoshino, M.D., Ph.D., Kaori Koga, M.D., Ph.D., Ako Saito, M.D., Ph.D., Tetsuya Hirata, M.D., Ph.D., Yasushi Hirota, M.D., Ph.D., Miyuki Harada, M.D., Ph.D., Akiko Hasegawa, M.D., Ph.D., Yuji Taketani, M.D., Ph.D. 
Fertility and Sterility 
Volume 98, Issue 5, Pages e2 (November 2012)
DOI: /j.fertnstert
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Effects of IL-17A on Gro-α secretion from EoSCs. Endometrial stromal cells (5 × 104 cells/well) were treated with IL-17A (0.1–100 ng/mL) or vehicle (control) for 24 hours. The conditioned medium was collected and assayed for Gro-α concentrations using a specific ELISA. Values are expressed as the means ± SEM of pentaplicate cultures. The data shown are representative of 10 repeated experiments using different samples from 10 women. *P<.001 vs. control.
Fertility and Sterility  , e2DOI: ( /j.fertnstert )
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Neutrophil chemotaxis was increased by secretions from IL-17A-stimulated EoSCs. The neutrophil-chemotactic activity of conditioned medium of EoSCs (4 × 105 cells/well) stimulated with 100 ng/mL IL-17A or vehicle (control) was evaluated using Chemotaxicell chamber. The conditioned medium of EoSCs stimulated with IL-17A attracted more neutrophils than that stimulated with vehicle. This effect was suppressed, though partly, by adding 2 μg/L anti-Gro-α-neutralizing antibody to the conditioned medium 15 minutes before the migration assay. Values are the means ± SEM of the quadruplicate cultures. The data shown are representative of 10 different experiments using different samples from 10 women. Different letters denote significant differences between groups (P<.05).
Fertility and Sterility  , e2DOI: ( /j.fertnstert )
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Immunostaining of IL-17A, Gro-α, and neutrophil elastase in endometrioma tissue. The sections observed in low-power field (×100) and in high-power field (×400) are demonstrated. The data shown are representative of 10 examined tissues. Black bars show 200 μm (A, D, G) and 100 μm (B, C, E, F, H, I).
Fertility and Sterility  , e2DOI: ( /j.fertnstert )
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Interleukin-17A was present in neutrophils in endometrioma tissues. The upper panels show the sections subjected to dual-color immunofluorescence staining for MPO (green) and IL-17A (red), with DAPI counterstaining. The lower panels show the merged images. The left panels show the figures with low-power field (×100) and the right panels show the figures with high-power field (×400). The area in the white broken line in the left under panel corresponds to the whole area of the right under panel. Blue broken lines indicate the epithelium. White solid line shows 200 μm (left) and 100 μm (right). A large portion of IL-17A-positive cells were MPO positive in the stromal area beneath the epithelium. In contrast, IL-17A-positive cells located in the inner area, shown only in figures with low-power field, were negative for MPO. Arrow heads indicate double-positive cells with segmented nuclei. The data shown are representative of the three examined tissues.
Fertility and Sterility  , e2DOI: ( /j.fertnstert )
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

6. Supplemental Figure 1
Effects of IL-17A on Gro-α secretion from EoSCs. Endometria stromal cells (5 × 104 cells/well) were treated with IL-17A (0.1–100 ng/mL) or vehicle (control) for 24 hours. The conditioned medium was collected and assayed for Gro-α concentrations using a specific ELISA. Values are expressed as the means ± SEM of pentaplicate cultures. The data shown are representative—other than Figure 1—of 10 repeated experiments using different samples from 10 women. *P<.001 vs. control.
Fertility and Sterility  , e2DOI: ( /j.fertnstert )
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

7. Supplemental Figure 2
Neutrophil chemotaxis was increased by secretions from IL-17A stimulated EoSCs. The neutrophil-chemotactic activity of conditioned medium of EoSCs (4 × 105 cells/well) stimulated with 100 ng/mL IL-17A or vehicle (control) was evaluated using Chemotaxicell chamber. The conditioned medium of EoSCs stimulated with IL-17A attracted more neutrophils than that stimulated with vehicle. This effect was suppressed, though partly, by adding 2 μg/mL anti-Gro-α neutralizing antibody to the conditioned medium 15 minutes before the migration assay. Values are the means ± SEM of the quadruplicate cultures. The data shown are representative—other than Figure 2—of 10 different experiments using different samples from 10 women. Different letters denote significant differences between groups (P<.05).
Fertility and Sterility  , e2DOI: ( /j.fertnstert )
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions