The presence of endometrial cells in the peritoneal cavity enhances monocyte recruitment and induces inflammatory cytokines in mice: Implications for endometriosis  Xuan Cao, M.D., DongZi Yang, M.D., Ph.D., MingQing Song, M.D., Ph.D., Ana Murphy, M.D., Sampath Parthasarathy, Ph.D.  Fertility and Sterility  Volume 82, Pages 999-1007 (October 2004) DOI: 10.1016/j.fertnstert.2004.04.040 Copyright © 2004 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Identification of cells as differentiated macrophages by their abilities to internalize fluorescent acetyl low-density lipoprotein (LDL). Vertical lines indicate the different groups. A, a, 1 is the control group; B, b, 2 is the experimental group injected with epithelial cells; and C, c, 3 is the experimental group injected with stromal cells. Upper panels (A, B, C) show the peritoneal macrophages that were cultured overnight before adding in acetyl LDL. Middle panels (a, b, c) show the peritoneal macrophages that were cultured overnight and then were cultured an additional 4 hours with acetyl LDL. Bottom panels (1, 2, 3) show acetyl LDL fluorescence images of peritoneal macrophages. The same amounts and dilutions of isolated macrophage from the peritoneal lavage in different groups were used. Fertility and Sterility 2004 82, 999-1007DOI: (10.1016/j.fertnstert.2004.04.040) Copyright © 2004 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Increased protein levels of MCP-1/JE in peritoneal lavage. Yellow: control group. Blue: epithelial group. Red: stromal group. The increased protein levels of MCP-1/JE could be seen at as early as 4 hours, and subsequently decreased at 24 hours and at 72 hours. (**P<.01). At the 4-hour point, the line shows the mean MCP-1/JE protein level in the control group to be 645.6 pg/mL. The mean MCP-1/JE protein level in the group of injected epithelial cells is 1885.4 pg/mL (compare with control group; P<.01). The mean protein level in the group of injected stromal cells is 1471.7 pg/mL (compare with control group; P<.01). The protein levels of MCP-1/JE are radically reduced by the time; the range is 40–87 pg/mL, and no statistically significant differences were found in the experimental group or control group at the 24-hour and 72-hour points. Fertility and Sterility 2004 82, 999-1007DOI: (10.1016/j.fertnstert.2004.04.040) Copyright © 2004 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 The real-time PCR analysis of MCP-1/JE gene from the mesothelium. The products in the 32nd cycle were fractionated by agarose gel electrophoresis, stained with ethidium bromide as in the bottom figures. The maximum relative gene expression of MCP-1/JE in the mesothelium was observed at 4 hours after injection of the epithelial and stromal cells, after which the relative gene expression ratio with the control group was reduced. At 4 hours, the gene expression ofMCP-1/JE in the injected epithelial (blue) group is 3.5, in stromal cells (red) group is 2.5, and the control group is 1 (yellow). There are statistically significant increased gene expressions in the experimental groups and the control group (P<.05, n = 7). Fertility and Sterility 2004 82, 999-1007DOI: (10.1016/j.fertnstert.2004.04.040) Copyright © 2004 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 The increased protein levels of IL-1α and IL-6 in peritoneal lavage. Yellow: control group. Blue: epithelial group. Red: stromal group. (Left) The protein level of IL-1α in peritoneal lavage was increased at 4 hours, and then decreased at 24 hours. At 4 hours, the mean protein level in the control group was 137.9 pg/mL. The mean protein level of the group injected with epithelial cells was 314.4 pg/mL, which is statistically significantly different from that of the control group (P<.01). The average protein level of lL-1α in the group injected with stromal cells was 263.8 pg/mL. The range of protein levels of IL-1α at 24 hours was 57.8 – 65.7 pg/mL. No statistically significant difference was found between the experiment groups and the control group. (Right) The protein level of IL-6 in the peritoneal lavage was increased at 4 hours, and then decreased at 24 hours. The protein levels of lL-6 in the control group and the group injected with epithelial cells and stromal cells were 318.7, 3411.7, and 1640.8 pg/mL, respectively, at 4 hours. This was a statistically significant increase in the groups of injected epithelial cells (P<.05) and stromal cells (P<.05) compared with the findings in the control group. The range of IL-6 protein levels at 24 hours ranged from 14.3 to 27.7 pg/mL. No statistically significant difference was found between the experiment groups and the control group. Fertility and Sterility 2004 82, 999-1007DOI: (10.1016/j.fertnstert.2004.04.040) Copyright © 2004 American Society for Reproductive Medicine Terms and Conditions