Placental pathology after mild maternal immune challenge at E12.5. A–C: Placentas were harvested from pregnant mice 24 hours after E12.5 saline or LPS treatment. Placentas show typical dark areas of hemorrhage in LPS-challenged pregnancies. Scale bar = 1 cm. D: Dark-field image of a saline mouse placental cross section showing reflective red blood cells (yellow) within the umbilical cord (UC) and labyrinth layer where the maternal-fetal blood exchange occurs. The spongiotrophoblast (Sp) layer appears darker. Scale bar = 100 μm. E–J: Uteri and placentas were harvested from pregnant mice at 6 hours or 24 hours after E12.5 treatment with saline or LPS (60 or 100 μg/kg). Vascular engorgement is visible in placentas stained with H&E at 6 hours (F and G) or 24 hours (E and H–J) after LPS treatment (60 μg/kg) within the labyrinth (Lab) layer. Tracts of eosinophilic necrotic tissue are also seen within the spongiotrophoblast (Sp) layer 24 hours after LPS challenge, indicated by arrows (E) and a boxed area (I), which is shown also at higher magnification (J). Yellow dotted lines mark the border between the spongiotrophoblast and labyrinth layers. Scale bar = 100 μM (E). Original magnification: ×10 (E); ×100 (F–I); ×400 (J).

Cytokine levels in the placenta and maternal serum. Pregnant mice were injected with saline or LPS (60 μg/kg) at E12.5. Maternal serum and placentas were collected at 2, 6, or 24 hours after treatment and were analyzed for cytokine abundance using a Luminex bead array. Data are plotted as pg cytokine/mg total protein for placenta (A) or pg/mL of maternal serum (B), in order by time (2, 6, and 24 hours). A: Statistically significant effects of treatment in the placenta were examined by two-way analysis of variance as follows: IL-1β (P = 0.0167), IL-6 (P = 0.0702), TNF-α (P = 0.0037), IL-12 p40 (P = 0.0259), CXCL1 (P = 0.0076), CCL2 (P < 0.0001), and CCL5 (P = 0.0002). B: All seven molecules also showed statistically significant treatment effects in the maternal serum by two-way analysis of variance: IL-1β (P < 0.0001), IL-6 (P = 0.0238), TNF-α (P = 0.0146), IL-12 p40 (P < 0.0001), CXCL1 (P = 0.0043), CCL2 (P < 0.0001), and CCL5 P = 0.0008) (n = 4 to 6 serum, n = 6 to 8 placentas, from at least three pregnancies per time point for each group). Differences in individual time points were assessed by Bonferroni post hoc tests. *P < 0.05; **P < 0.01; ***P < 0.001. Data are reported as means ± SEM.

Blockade of TNF-α signaling prevents fetal loss and placental pathology. TNFR2-IgG (5 mg/kg) was administered intraperitoneally to pregnant mice at E12.5, 1 hour before treatment with saline or LPS. A: Fetal loss was assessed at 24 hours after treatment. TNFR2-IgG-treated mice were protected from LPS-induced fetal loss (P = 0.015 treatment effect by two-way analysis of variance; n = 4 to 6 pregnancies). **P < 0.01 versus WT by Bonferroni post hoc test. B: Placentas were harvested at 24 hours after saline or LPS (60 μg/kg) treatment at E12.5 and stained with H&E. The percent area of the spongiotrophoblast layer that had a necrotic, hypereosinophilic appearance was measured. WT LPS-treated mice showed an increase in necrotic tissue that was reduced by treatment with TNFR2-IgG (P = 0.001 by one-way analysis of variance; n = 6 to 10 placentas per group). **P < 0.01; ***P < 0.001 by Bonferroni post hoc test. Data are reported as means ± SEM.

Dose-dependent risk of fetal loss but lack of growth restriction or premature birth after maternal LPS administration in a mouse model. A: The number of surviving fetuses was counted 24 hours after treatment with LPS at different embryonic ages and doses (n = 3 to 40 pregnancies per group). The effect of treatment on survival was significant at E10.5 (P = 0.0022), E12.5 (P < 0.0001), and E14.5 (P < 0.0001) by one-way analysis of variance. Analysis of variance could not be performed for E8.5 treatments because of lack of variance within groups. **P < 0.01; ***P < 0.001 by Dunnett's multiple comparison post hoc test. B: Pregnant mice were treated with saline (n = 13) or LPS (60 μg/kg) (n = 17) on E12.5. Maternal weights were monitored daily and were expressed as a percentage of starting weight at the time of treatment. LPS-treated maternal weights are slightly but significantly lower than for saline-injected control animals (P < 0.0001 by two-way analysis of variance). *P < 0.05; ***P < 0.001 by Bonferroni post hoc test). C: Pregnant mice were treated with saline (n = 18) or LPS (60 μg/kg) (n = 21) on E12.5 and the time of birth was recorded. The length of gestation in LPS-injected animals was increased by approximately 0.5 days. *P = 0.014 by two-tailed t-test. D and E: Pregnant mice were treated with saline (n = 36) or LPS (60 μg/kg) (n = 31) on E12.5. Fetuses were collected and weighed at E17.5 or at birth. Fetal growth was unaffected in LPS-treated pregnancies (E17.5: P = 0.30 by two-tailed t-test; birth: P = 0.27 by two-tailed t-test). Data are reported as means ± SEM.

Hypoxia and impaired proliferation in the fetal brain. Pregnant mice were treated with pimonidazole 15 minutes before saline or LPS (60 μg/kg) at E12.5. Two hours after saline or LPS treatment, fetuses were harvested and brains were processed and stained with an antibody against pimonidazole adducts or phosphohistone H3 (pHH3) and DAPI to mark all nuclei. A: A coronal section of the fetal brain stained with DAPI (blue) to mark nuclei; the boxed area indicates the approximate region magnified in the accompanying images (B–D). B and C: Representative images of saline- and LPS-treated fetal brain. Brains were stained with an antibody against pimonidazole adducts (red) and DAPI to mark nuclei (blue). Scale bars = 100 μm. D: An example of a saline-treated control brain stained with an antibody against pHH3 (red) and DAPI to mark nuclei (blue). Scale bar = 100 μm. E: Pimonidazole (pimo) staining was quantified as the proportion of pimo-positive pixels relative to DAPI-positive pixels. The amount tissue area in the cortex positively staining for pimonidazole increased dramatically after LPS administration (n = 9 saline and 12 LPS brains from 3 to 4 pregnancies per group). ***P < 0.0001 by two-tailed Student's t-test. F: The number of pHH3⁺ metaphase cells was counted per unit tissue area using DAPI. The density of pHH3⁺ metaphase cells was reduced in fetal brain tissues from pregnancies challenged with LPS (n = 9 saline and 12 LPS brains from 3 to 4 pregnancies per group). *P = 0.0199 by two-tailed Student's t-test. Data are reported as mean ± SEM.

TLR4 and TNF-α signaling through TNFR1 is necessary for fetal loss and placental pathology. A: WT (C57BL/10J) females were bred with WT males to produce WT fetuses or TLR4^−/− females were bred with WT males to produce TLR4^+/− fetuses in at TLR4^−/− maternal environment. Pregnant mice were injected with saline or LPS (60 μg/kg) at E12.5. Placentas were harvested at 24 hours after treatment and stained with H&E. The percent area of the spongiotrophoblast layer that had a necrotic, hypereosinophilic appearance was measured. WT mice showed an increase in necrosis in the spongiotrophoblast layer with LPS, but fetuses in a TLR4^−/− environment did not (n = 6 to 9 placentas from at 2 to 3 pregnancies per group). P = 0.0002 by one-way analysis of variance. **P < 0.01; ***P < 0.001 by Bonferroni post hoc test. B: Pregnant mice were injected with saline or LPS at different doses at E12.5. Fetal loss in WT mice (C57BL/6) or mice deficient in TNF-α, TNFR1, or TNFR2 was assessed at 24 hours after treatment. TNF-α^−/− and TNFR1^−/−, but not TNFR2^−/−, mice were protected from LPS-induced fetal loss (n = 4 to 7 saline, 6 to 11 LPS for all strains). There was a significant effect of strain (P < 0.0001), dose (P = 0.0002), and interaction (P = 0.0239) by two-way analysis of variance. **P < 0.01 versus WT by Bonferroni post hoc test. C: WT females were bred with WT males to produce WT fetuses, TNFR1^−/− females were bred with TNFR1^−/− males to produce TNFR1^−/− fetuses, and TNFR1^−/− females were bred with WT males to produce TNFR1^+/− fetuses in a TNFR1^−/− maternal environment. Pregnant mice were injected with saline or LPS (μg/kg) at E12.5. Placentas were harvested and analyzed as described for panel A. LPS-treated mice showed significant levels of necrotic tissue within the placenta in all genotypes: WT (**P = 0.0067; n = 14 saline, 18 LPS), TNFR1^−/− (*P = 0.0279; n = 14 saline, 10 LPS), and TNFR1^+/− (*P = 0.0183; n = 6 saline, 9 LPS) (two-tailed t-tests). A two-way analysis of variance revealed significant effects of strain (P = 0.0473), and treatment (P = 0.0033). TNFR1^−/− LPS placentas were highly protected from pathology, compared with WT LPS placentas, but TNFR1^+/− LPS placentas did not differ from WT LPS placentas (**P < 0.01, Bonferroni post hoc test; n = at least 6 placentas per group). D: Pregnant mice were treated with pimonidazole 15 minutes before saline or LPS (60 μg/kg) treatment. Two hours after saline or LPS, fetuses were harvested and pimonidazole staining was quantified as the proportion of pimo-positive pixels relative to total tissue area. Pimonidazole staining increased after LPS administration in WT but not in TNFR1^−/− mice (P = 0.0067 by one-way analysis of variance; n = 6 to 9 brains from 2 to 3 pregnancies per group). *P < 0.05 versus WT saline by Bonferroni post hoc test. E: The number of pHH3⁺ metaphase cells was also scored and compared with saline controls for each strain. The density of pHH3⁺ metaphase cells was reduced in fetal brain tissues from pregnancies challenged with LPS in WT mice (*P = 0.028, two-tailed t-test; n = 12 saline, 18 LPS) but not in TNFR1^−/− mice (P = 0.41; n = 9 saline, 9 LPS). Data are reported as means ± SEM.