Local injury of the endometrium induces an inflammatory response that promotes successful implantation Yulia Gnainsky, Ph.D., Irit Granot, Ph.D., Paulomi.

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Local injury of the endometrium induces an inflammatory response that promotes successful implantation Yulia Gnainsky, Ph.D., Irit Granot, Ph.D., Paulomi B. Aldo, M.S., Amihai Barash, M.D., Yuval Or, M.D., Edna Schechtman, Ph.D., Gil Mor, Ph.D., Nava Dekel, Ph.D. Fertility and Sterility Volume 94, Issue 6, Pages 2030-2036 (November 2010) DOI: 10.1016/j.fertnstert.2010.02.022 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Evaluation of the cytokine/chemokine profile in the endometrial samples. Protein levels of (A) growth-regulated oncogene-α (GRO-α), (B) interleukin-15 (IL-15), (C) macrophage inflammatory protein 1B (MIP-1B) were determined by multiple cytokine Luminex analysis. (D, E) Relative MIP-1B and tumor necrosis factor-α (TNF-α) messenger RNA (mRNA) levels were tested by quantitative real-time polymerase chain reaction. The box plot horizontal lines represent the median and the 25th to 75th percentile. Comparison between days 8 to 12 and 21 within the experimental group was performed by Wilcoxon's signed rank test. Comparison between day-21 samples of the experimental and control groups was performed using Mann-Whitney test (∗∗P<.01, ∗P<.05). The effect of TNF-α on the expression of MIP-1B, MUC1, Gro-α, and IL-15 was tested in (F) freshly isolated endometrial stromal cells (ESC) from day-21 control samples and in (G) epithelial cell line ECC-1. Results of in vitro experiments are mean ± standard error of the mean from three independent experiments. Endometrial stromal cells were prepared from three different tissues. ∗, #, and † are significantly different from their respective control (P<.05; student t-test). Fertility and Sterility 2010 94, 2030-2036DOI: (10.1016/j.fertnstert.2010.02.022) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Characterization of macrophages/dendritic cells (DCs) in the endometrial samples by flow cytometry. Leukocytes region was defined using (A) characteristic size (FSC) and granularity (SSC) parameters (R1) as well as by positive staining with (B) anti-CD45 (R2). (C) The HLA-DR+CD56– cells (R3) within leukocyte region (R1 and R2) that exhibited a positive staining for (D) CD11c were defined as macrophages/DCs. (E) HLA-DR+CD11c+ cells (R4) were separated to (F) macrophages and DCs according to CD14 staining. (G) A representative analysis of HLA-DR+CD11c+cells in endometrial samples from days 8 to 12 and day 21 of the experimental group and the day-21 sample of the control group. (H) The abundance of HLA-DR+CD11c+ cells was calculated of total leukocytes (R1 and R2). The box plot horizontal lines represent the median and the 25th to 75th percentile. Comparison between days 8 to 12 and 21 within the experimental group was performed by Wilcoxon's test signed rank test. Comparison between the day-21 samples of the experimental and control groups was performed using Mann-Whitney test. ∗∗∗P<.001. Fertility and Sterility 2010 94, 2030-2036DOI: (10.1016/j.fertnstert.2010.02.022) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Expression of osteopontin (OPN) and vascular endothelial growth factor (VEGF) in the endometrial samples. The box plot horizontal lines represent the median and the 25th to 75th percentile of (A) VEGF messenger RNA (mRNA) levels, (B) OPN mRNA, and (C) protein levels. ∗P<.05, ∗∗P<.01 (Mann-Whitney test). Fertility and Sterility 2010 94, 2030-2036DOI: (10.1016/j.fertnstert.2010.02.022) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Comparison of the abundance of (A) macrophages/dendritic cells (HLA-DR+CD11c+ cells), (B) messenger RNA (mRNA) levels of macrophage inflammatory protein 1B (MIP-1B), (C) tumor necrosis factor-α (TNF-α), and (D) osteopontin (OPN) between pregnant (+) and not pregnant (–) IVF patients. The box plot horizontal lines represent the median and the 25th to 75th percentile. ∗P<.05 (Mann-Whitney test). Fertility and Sterility 2010 94, 2030-2036DOI: (10.1016/j.fertnstert.2010.02.022) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions