1. Differential effects of the CpG-toll-like receptor 9 axis on pregnancy outcome in nonobese diabetic mice and wild-type controls 
Yun Sun, M.D., Xiaoli Qin, M.D., Bin Shan, M.D., Wenjing Wang, M.S., Qinling Zhu, M.S., Surendra Sharma, M.D., Ph.D., Ji Wu, Ph.D., Yi Lin, M.D. 
Fertility and Sterility 
Volume 99, Issue 6, Pages e4 (May 2013)
DOI: /j.fertnstert
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
CpG ODN treatment increased serum and decidual mKC production in NOD mice. Serum mKC was measured using ELISA at E9.5 (A) and E15.5 (B) in NOD and WT mice. Data represent means ± SD from 10 different serum samples in each group. Decidual mKC was assayed by flow cytometry at E9.5 (C) and E15.5 (D) in NOD and WT mice using single-cell suspensions of UMGCs. Data shown are representative of experiments that were performed independently 4 times per condition. A significant increase in the proportion of mKC+ cell subpopulation was observed in CpG ODN-treated NOD mice above control ODN-treated NOD mice (P<.05) (C).
Fertility and Sterility  , e4DOI: ( /j.fertnstert )
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3. Figure 2
TNF-α is the key cytotoxic factor leading to fetal resorption or preterm birth upon CpG ODN stimulation in NOD mice. Serum TNF-α was measured using ELISA in NOD and WT mice at E9.5 (A) or E15.5 (B). Data represent means ± SD from 10 different serum samples. UMGCs were collected at E9.5 (C) or E15.5 (D) from NOD mice treated with control ODN or CpG ODN to determine the proportion of TNF-α+ cell population. Histograms are gated on CD45+ cells and are representative of data from four independently conducted experiments. (E) Neutralizing anti-TNF-α mAb was given IP to inhibit TNF-α activity induced by CpG ODN. The percentage of fetal resorption was analyzed at E9.5 (n = 10 in each group).
Fertility and Sterility  , e4DOI: ( /j.fertnstert )
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4. Figure 3
Anti-Gr-1 mAb successfully depleted Gr-1, but Gr-1 depletion does not rescue CpG ODN-induced fetal resorption increase in NOD mice. (A) Anti-Gr-1 was used to deplete Gr-1+ uterine cells. The proportion of Gr-1+ cells in the CD11b+ subpopulation (left panel) and the corresponding proportion of TNF-α+ cells in the CD45+ subpopulation (right panel) were analyzed by flow cytometry. Histograms are representative of independently conducted experiments, 4 times per condition. The percentage of fetal resorption (B) and preterm birth (C) was shown. Ten animals were used under each condition in both fetal resorption and preterm birth models.
Fertility and Sterility  , e4DOI: ( /j.fertnstert )
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5. Figure 4
Anti-F4/80 Ab depletes F4/80+ cells, diminishes TNF-α activity, and abrogates CpG-induced fetal resorption and preterm birth. (A) Anti-TNF-α Ab was used to deplete F4/80+ uterine cells. The proportion of F4/80+ cells in the CD11b+ subpopulation (left panel) and the corresponding proportion of TNF-α+ cells in the CD45+ subpopulation (right panel) was analyzed by flow cytometry. Histograms are representative of independently conducted experiments, 4 times per condition. The percentage of fetal resorption (B) and preterm birth (C) was shown. Ten animals were used under each condition in both fetal resorption and preterm birth models.
Fertility and Sterility  , e4DOI: ( /j.fertnstert )
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6. Supplemental Figure 1
CpG-mediated induction of fetal resorption and preterm birth in WT and NOD mice. CpG or control ODN was injected IP as indicated. (A) Mice were injected with CpG or control ODN at E6.5, and fetal resorption was assessed at E9.5. (B) Injection was performed at E14.5, and the preterm birth rate was assessed. Pup delivery before E18.5 was defined as preterm birth. *Maximum preterm birth (100%) was observed at the dose of CpG used in the experiments. Ten female mice were used in each group.
Fertility and Sterility  , e4DOI: ( /j.fertnstert )
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7. Supplemental Figure 2
CpG ODN-mediated fetal resorption in NOD mice is TLR9 dependent. (A) Mice were injected with CpG ODN 1826 alone at 25 μg/dam, or CpG ODN 2088 alone at 100 μg/dam, or CpG 1826 (25 μg) plus CpG 2088 (100 μg) at E6.5. Fetal resorption was assessed at E9.5. (B) Injection was performed at E14.5, and preterm birth rate was assessed. Pup delivery before E18.5 was defined as preterm birth; n = 10 for each group.
Fertility and Sterility  , e4DOI: ( /j.fertnstert )
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8. Supplemental Figure 3
The proportion of uNK cell populations does not change upon CpG ODN stimulation in NOD mice. (A) Mice were injected with CpG 1826 at 25 μg/dam at E6.5, and the proportion of uNK cells (DBA-lectin+) in CD45+ mononuclear and granular cells was analyzed at E9.5 using flow cytometry. (B) Injection was performed at E14.5, and the proportion of uNK cells (DBA-lectin+) in CD45+ mononuclear and granular cells was analyzed at E15.5 using flow cytometry. (C) In CpG-treated mice, the effect of anti-asialo GM1 on fetal resorption was assessed at E9.5. (D) In CpG-treated mice, the effect of anti-asialo GM1 on preterm birth was assessed. Pup delivery before E18.5 was defined as preterm birth; n = 10 for each group.
Fertility and Sterility  , e4DOI: ( /j.fertnstert )
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9. Supplemental Figure 4
CD11b+F4/80+ and CD11b+Gr-1+ uterine cell populations amplify in response to CpG ODN in NOD but not in WT mice. Single-cell suspensions of UMGCs were obtained from NOD (A and C) or WT (B and D) mice at E9.5 or E15.5 after being treated with control or CpG ODN. Cells were gated for CD11b+F4/80+ (A and B) and CD11b+Gr-1+ in the CD45+ subpopulation (C and D). A representative dot plot of the isotype controls was shown (E). The data shown are representative of experiments performed independently 4 times per condition.
Fertility and Sterility  , e4DOI: ( /j.fertnstert )
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions