Cholesterol deficiency as a mechanism for autism: A valproic acid model.

Dysregulated cholesterol metabolism represents an increasingly recognized feature of autism spectrum disorder (ASD). Children with fetal valproate syndrome caused by prenatal exposure to valproic acid (VPA), an anti-epileptic and mood-stabilizing drug, have a higher incidence of developing ASD. However, the role of VPA in cholesterol homeostasis in neurons and microglial cells remains unclear. Therefore, we examined the effect of VPA exposure on regulation of cholesterol homeostasis in the human microglial clone 3 (HMC3) cell line and the human neuroblastoma cell line SH-SY5Y. HMC3 and SH-SY5Y cells were each incubated in increasing concentrations of VPA, followed by quantification of mRNA and protein expression of cholesterol transporters and cholesterol metabolizing enzymes. Cholesterol efflux was evaluated using colorimetric assays. We found that VPA treatment in HMC3 cells significantly reduced ABCA1 mRNA, but increased ABCG1 and CD36 mRNA levels in a dose-dependent manner. However, ABCA1 and ABCG1 protein levels were reduced by VPA in HMC3. Furthermore, similar experiments in SH-SY5Y cells showed increased mRNA levels for ABCA1, ABCG1, CD36, and 27-hydroxylase with VPA treatment. VPA exposure significantly reduced protein levels of ABCA1 in a dose-dependent manner, but increased the ABCG1 protein level at the highest dose in SH-SY5Y cells. In addition, VPA treatment significantly increased cholesterol efflux in SH-SY5Y, but had no impact on efflux in HMC3. VPA differentially controls the expression of ABCA1 and ABCG1, but regulation at the transcriptional and translational levels are not consistent and changes in the expression of these genes do not correlate with cholesterol efflux in vitro.

Ex vivo toxicity of E-cigarette constituents on human placental tissues.

Globally, ∼50 % of women smoke during pregnancy and the prevalence of vaping is increasing among women of reproductive age. However, the health effects of vaping during pregnancy are largely unknown. This study examined the effects of e-cig constituents alone and in combination (propylene glycol [PG], vegetable glycerin [VG], and nicotine) on human placental tissue viability (MTT assay) and immunoassayed levels of placenta-derived biomarkers, i.e., 8-isoprostane (8-IsoP), heme oxygenase-1 (HO-1), interleukin-6 (IL-6), ß-estradiol (E2), progesterone (P4), allopregnanolone (AP), and brain-derived neurotrophic factor (BDNF). Placental explant cultures were exposed ex vivo for 24 h to media-containing either nicotine (0-5000 nM), PG/VG (0-8 % v/v at 50/50 ratio), or a combination of both. No effects on tissue viability were observed at PG/VG concentrations < 8 % (v/v), while viability significantly reduced at PG/VG concentrations ≥ 10 % (v/v); biomarker studies employed only non-cytotoxic doses. Exposure to PG/VG decreased levels of 8-IsoP, IL-6, and E2, and treatment with 2 % or 8 % PG/VG significantly reduced HO-1 levels, compared to non-treated controls. Exposure to nicotine alone at 2,500 nM and 5,000 nM reduced MTT activity by 20 % (P = 0.04) and 70 % (P < 0.001), respectively, and significantly increased (P < 0.001) levels of HO-1 and BDNF, compared to controls. Treatment with nicotine alone and in combination with PG/VG reduced IL-6 and E2 levels. Interestingly, nicotine-induced toxicity was attenuated by PG/VG addition to nicotine-treated groups. These studies demonstrate that e-cig constituents negatively impact the human placenta and alters production of critical placental biomarkers, suggesting that vaping is an unsafe alternative for pregnant women or their unborn fetus.

Effect of 2,6-xylidine (DMA) on secretion of biomarkers for inflammation and neurodevelopment by the placenta.

Cigarette smoke enhances placental inflammation and interferes with steroidogenesis. However, the chemicals in the smoke responsible for these biological activities are unclear. 2,6 xylidine (also called 2,6 Dimethylaniline, DMA) is a component of cigarette smoke that has carcinogenic properties but its effects on the placenta are unknown. Therefore, we hypothesized that DMA may interfere with placental steroidogenesis or enhance placental inflammation. Placental explant cultures were treated with 0-50,000 nM DMA and concentrations of progesterone (P4), estradiol (E2), testosterone (T), IL-1ß, TNF-α, IL-6, sgp130, HO-1, IL-10, 8-Isoprostane (8-IsoP), and BDNF in the conditioned medium were quantified. Since many environmental toxins enhance the proinflammatory host response to infection, we also performed experiments on placental cultures co-stimulated with 107 heat-killed E. coli. DMA alone significantly reduced P4 and T secretion but enhanced E2 secretion. The toxin also reduced placental secretion of IL-6, sgp130, and BDNF. For bacteria-stimulated cultures, DMA increased secretion of P4 and T, and proinflammatory cytokines (IL-1ß, TNF-α) but had mixed effects on anti-inflammatory markers, increasing some (sgp130, IL-10) and reducing others (HO-1). However, DMA enhanced 8-IsoP levels by bacteria-stimulated placental cultures, suggesting that it increases oxidative stress by the tissues. These studies suggest that DMA affects secretion of biomarkers by the placenta and may promote inflammation. Further studies are needed to determine if these observed changes occur in vivo and the extent to which DMA exposure increases the risk of adverse pregnancy outcomes associated with smoking in pregnancy.

Perinatal outcomes after bariatric surgery.

BACKGROUND: Bariatric surgery is a widely used treatment option for obesity that often provides long-term weight control and health benefits. Although a growing number of women are becoming pregnant after bariatric surgery, only a few population-based studies have assessed the impact thereof on perinatal outcomes. OBJECTIVE: This study aimed to examine the association between bariatric surgery and adverse perinatal outcomes in pregnant women and to examine whether the risk for adverse perinatal outcomes is modified by the postsurgery weight, gestational weight gain, type of bariatric surgery, timing of pregnancy after bariatric surgery, and maternal comorbidities. STUDY DESIGN: A retrospective cohort study was performed with the use of the Bariatric Surgery Registry and hospital inpatient and outpatient physician encounter records. The International Classification of Diseases, Ninth and Tenth Revision codes from hospitalizations during pregnancy and infant birth records were used to ascertain the outcomes of interest. Women eligible for BS who delivered at ≥20 weeks of gestation (n=20,213) at Kaiser Permanente Southern California hospitals (January 1, 2007 to December 31, 2018) were included in the study. Adjusted odds ratios were derived from logistic regression models with inverse probability of treatment weighting to adjust for confounding using propensity scores. RESULTS: Bariatric surgery was associated with a reduction in the risks for gestational diabetes (adjusted odds ratio, 0.60; 95% confidence interval, 0.53-0.69; P<.001), preeclampsia (adjusted odds ratio, 0.53; 95% confidence interval, 0.46-0.61; P<.001), chorioamnionitis (adjusted odds ratio, 0.45; 95% confidence interval, 0.32-0.63; P<.001), cesarean delivery (adjusted odds ratio, 0.65; 95% confidence interval, 0.59-0.72; P<.001), large for gestational age neonate (adjusted odds ratio, 0.23; 95% confidence interval, 0.19-0.29; P<.001), macrosomia (adjusted odds ratio, 0.24; 95% confidence interval, 0.19-0.30; P<.001), and neonatal intensive care unit admission (adjusted odds ratio, 0.70; 95% confidence interval, 0.61-0.81; P<.001). However, bariatric surgery was also associated with a significantly increased risk for small for gestational age neonates (adjusted odds ratio, 2.46; 95% confidence interval, 2.16-2.79; P<.001). The risk for the adverse outcomes is independent of the time interval between the surgery and subsequent pregnancy. CONCLUSION: These data suggest that there are many pregnancy outcome benefits for women with severe obesity who undergo bariatric surgery; however, women who have undergone bariatric surgery before pregnancy should be monitored closely to reduce the risk for small for gestational age neonates and postpartum hemorrhage.

Exposure to polybrominated diphenyl ether-47 increases the risk of post-partum depression.

INTRODUCTION: Post-partum depression (PPD) affects up to 19.1% of pregnancies and is associated with increased levels of proinflammatory cytokines, inflammation, and reductions in brain-derived neurotrophic factor (BDNF). Previous work by our team suggests that environmental toxins such as polybrominated diphenyl ethers (PBDEs) enhance placental inflammation and reduce BDNF production. Nearly, 100% of studied women in California have some level of exposure to these compounds due to extensive use of the flame retardants. High levels of exposure to PBDEs has been linked to increased risk of adverse pregnancy complications associated with placental inflammation such as preterm birth and gestational diabetes but their effects on risk of PPD is unclear. OBJECTIVE: To determine if PPD is associated with higher levels of PBDE-47, the most common PBDE congener in maternal plasma. METHODS: PBDE-47 was quantified in first trimester plasma samples collected from a cohort of 367 asymptomatic pregnant women that were routinely screened for depressive symptoms for 1 year post-partum. Data were analyzed using general linear models and multivariable logistic regression to determine if higher levels of PBDE-47 in the first trimester are associated with development of PPD. RESULTS: Women who developed PPD (n = 22) had significantly higher PBDE-47 levels in their plasma (p=.031) relative to those in which PPD was not diagnosed. Logistic regression analysis suggested that each two-fold increase in PBDE-47 concentrations increased the risk of PPD by 22% (OR = 1.22, 95% CI: 1.03, 1.47). Groups were similar regarding PTB rate, race-ethnicity, parity, child's sex, maternal pre-pregnancy obesity status, maternal age, family income, and study center. Results remained significant after adjustment for these possible confounding factors. CONCLUSIONS: These results suggest that PBDE-47 exposure in the first trimester is associated with increased risk of PPD.

Enhancement of placental inflammation by Dibutyl Phthalate.

Recent studies suggest that women with high exposures to dibutyl phthalate (DBP) are at increased risk for preterm birth, a condition associated with aberrant inflammation in the placenta often caused by subclinical infections. Placental inflammation is also a risk factor for neurodevelopmental disorders whose risk may also be enhanced by DBP. It is unclear, however, if DBP enhances placental inflammation. Therefore, we studied the effects of DBP on the production of biomarkers of placental inflammation and neurodevelopment under basal conditions and a setting of mild infection. Placental explant cultures established from women undergoing elective caesarean delivery were treated with DBP with and without co-stimulation by 107 CFU/mL heat-killed E. coli for 24 h at 37 °C. Conditioned medium was harvested and concentrations of IL-1ß, TNF-α, IL-10, HO-1 and BDNF, a biomarker for neurodevelopment, were quantified. DBP significantly enhanced IL-6 production in basal cultures but had no significant on the other biomarkers quantified. Both TNF-α and IL-1ß production was enhanced by DBP for cultures co-stimulated with E. coli. Although marginal enhancement of IL-6, and IL-10 were observed for bacteria co-treated cultures, results were either non-monotonic or only approached statistical significance. HO-1 production tended to be reduced at the highest concentration of DBP tested and BDNF production was reduced by DBP in a dose-dependent manner for bacteria-stimulated cultures. These results suggest that DBP enhances basal IL-6 production but has little or no effect on other biomarkers studied. However, DBP enhances IL-1ß and TNF-α production but reduces BDNF production by bacteria-stimulated cultures.

Novel Insights into the Regulatory Role of Nuclear Factor (Erythroid-Derived 2)-Like 2 in Oxidative Stress and Inflammation of Human Fetal Membranes.

Fetal membrane dysfunction in response to oxidative stress (OS) is associated with adverse pregnancy outcomes. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is one of the regulators of innate OS response. This study evaluated changes in Nrf2 expression and its downstream targets heme oxygenase (HO-1) and peroxisome proliferator-activated receptor gamma (PPARγ) in fetal membranes during OS and infection in vitro. Furthermore, we tested the roles of sulforaphane (SFN; an extract from cruciferous vegetables) and trigonelline (TRN; an aromatic compound in coffee) in regulating Nrf2 and its targets. Fetal membranes (n = 6) collected at term were placed in an organ explant system were treated with water-soluble cigarette smoke extract (CSE), an OS inducer (1:10), and lipopolysaccharide (LPS; 100 ng/mL). Nrf2 expression, expression, its enhancement by sulforaphane (SFN, 10 µM/mL) and down regulation by TRN (10uM/mL) was determined by western blots. Expression of Nrf2 response elements PPARγ (western) heme oxygenase (HO-1), and IL-6 were quantified by ELISA. CSE and LPS treatment of fetal membranes increased nrf2, but reduced HO-1 and PPARγ and increased IL-6. Co-treatment of SFN, but not with TRN, with CSE and LPS increased Nrf2 substantially, as well as increased HO-1 and PPARγ and reduced IL-6 expression. Risk factor-induced Nrf2 increase is insufficient to generate an antioxidant response in fetal membranes. Sulforaphane may enhance innate antioxidant and anti-inflammatory capacity by increasing NRF-2 expression.

Effect of bisphenol-A (BPA) on placental biomarkers for inflammation, neurodevelopment and oxidative stress.

Background Bisphenol-A (BPA) is a widespread pollutant whose effects on pregnant women are poorly understood. Therefore, we investigated the effects of BPA on basal and bacteria-stimulated production of proinflammatory cytokines [interleukin (IL)-1ß, tumor necrosis factor-α (TNF-α) and IL-6], anti-inflammatory mediators [soluble glycoprotein 130 (sgp) 130, heme oxidase-1 (HO-1) and IL-10] and biomarkers for neurodevelopment [brain-derived neurotrophic factor (BDNF)], and oxidative stress [8-isoprostane (8-IsoP)] by the placenta. Methods Placental explant cultures were treated with BPA (0-10,000 nM) in the presence or absence of 107 colony-forming unit (CFU)/mL heat-killed Escherichia coli for 24 h. Biomarker concentrations in conditioned medium were quantified by the enzyme-linked immunosorbent assay (ELISA). Results Under basal conditions, IL-1ß and IL-6 production was enhanced by BPA in a dose-dependent manner. Sgp130, a soluble receptor that reduces IL-6 bioactivity, was suppressed by BPA at 1000-10,000 nM. BPA also enhanced BDNF production at 1000 and 10,000 nM, and 8-IsoP expression at 10 and 100 nM. For bacteria-treated cultures, BPA increased IL-6 production at 100 nM and reduced sgp130 at 1000 nM but had no effect on IL-1ß, TNF-α, BDNF, HO-1, 8-IsoP or IL-10 production. Conclusion BPA may increase placental inflammation by promoting IL-1ß and IL-6 but inhibiting sgp130. It may also disrupt oxidative balance and neurodevelopment by increasing 8-IsoP and BDNF production.

Effect of Tetrabromobisphenol A on expression of biomarkers for inflammation and neurodevelopment by the placenta.

OBJECTIVE: Polybrominated diphenyl ethers (PBDEs) enhance basal and bacteria-stimulated production of proinflammatory cytokines by the placenta and may also increase the risk of preterm birth and neurodevelopmental disorders. Whether or not other brominated flame retardants such as Tetrabromobisphenol A (TBBPA) have similar properties is unclear. Therefore, we evaluated the effects of TBBPA on the production of steroids, as well as biomarkers for inflammation, oxidative stress, and neurodevelopment by the placenta. METHODS: Placental explant cultures were established from women undergoing elective Cesarean sections at term and treated with 5-50,000 nM TBBPA in the presence and absence of 107 CFU/ml heat-killed E. coli. Concentrations of P4, E2, testosterone (T), IL-1ß, TNF-α, IL-10, HO-1, IL-6, 8-IsoP and BDNF were quantified in the conditioned medium. RESULTS: In the absence of bacteria, TBBPA tended to increase P4 and T as well as IL-6 and 8-IsoP. For bacteria-treated cultures, TBBPA generally inhibited P4, IL-1ß, and HO-1 production but enhanced TNF-α and IL-6 production. CONCLUSIONS: TBBPA alters placental steroidogenesis to favor T production and increases oxidative stress and placental inflammation as suggested by its promotion of 8-IsoP and IL-6 production. TBBPA may also function as a risk modifier where it enhances bacteria-stimulated production TNF-α and IL-6 but reduces HO-1 production, however, this was balanced by reductions in IL-1ß. Further studies are warranted to determine if TBBPA increases the risk of adverse pregnancy outcomes such as preterm birth, pPROM and neurodevelopmental disorders such as autism that have been associated with increased production of proinflammatory cytokines, oxidative stress and/or T.

Polybacterial stimulation suggests discrete IL-6/IL-6R signaling in human fetal membranes: Potential implications on IL-6 bioactivity.

The polybacterial invasion of the amniotic cavity and risk of preterm birth is often due to cervicovaginal bacteria such as genital mycoplasmas (Mycoplasma hominis and Ureaplasma urealyticum) and Gardnerella vaginalis. The most studied biomarker associated with preterm birth is interleukin-6 (IL-6), a pleiotropic cytokine that performs different functions based on classical or trans-signaling mechanisms. This study evaluated the changes in IL-6 and IL-6 function associated accessory molecules by human fetal membranes to determine the functional availability of IL-6 assessment in an in vitro model of polybacterial infection. Fetal membranes were treated with LPS or heat-inactivated genital mycoplasmas and G. vaginalis alone or in combination. IL-6 and its soluble receptors (sgp130, sIL-6R) were assessed in conditioned medium by immunoassays and membrane-bound receptors were evaluated in the tissue using immunohistochemistry and RT-PCR. Data from protein and gene expression were evaluated using linear mixed effects models. Data from immunohistochemistry were evaluated using one-way analysis of variance followed by the Tukey test. Genital mycoplasmas alone, or in combination, inhibited IL-6 trans-signaling with increased sgp130 production. G. vaginalis activated the classical IL-6 signaling pathway, as did LPS. Polybacterial treatment resulted in a balanced response with neither pathway being favored. The increase in IL-6 production by fetal membranes in response to infection is likely a non-specific innate response and not an indicator of a functional mediator of any labor-inducing pathways. This suggests that correlating the risk of adverse pregnancy outcomes and designing interventions based on IL-6 levels without considering soluble receptors may be an ineffective strategy.

Effects of tributyltin on placental cytokine production.

Objective Tributyltin (TBT) is a persistent pollutant but its effects on placental function are poorly understood as are its possible interactions with infection. We hypothesized that TBT alters the production of sex hormones and biomarkers for inflammation and neurodevelopment in an infection-dependent manner. Methods Placental explant cultures were treated with 0-5000 nM TBT in the presence and absence of Escherichia coli. A conditioned medium was harvested and concentrations of steroids (progesterone, P4; testosterone, T and estradiol, E2) as well as biomarkers of inflammation [interleukin (IL)-1ß (IL-1ß), tumor necrosis factor (TNF-α), IL-10, IL-6, soluble glycoprotein 130 (sgp-130) and heme oxygenase-1 (HO-1)], oxidative stress [8-iso-prostaglandin (8-IsoP)] and neurodevelopment [brain-derived neurotrophic factor (BDNF)] were quantified. Results TBT increased P4 slightly but had little or no effect on T or E2 production. IL-1ß, IL-6, sgp-130, IL-10 and 8-IsoP production was enhanced by TBT. P4 and IL-6 production was also enhanced by TBT for bacteria-stimulated cultures but TBT significantly inhibited bacteria-induced IL-1ß and sgp-130 production. High doses of TBT also inhibited BDNF production. Conclusions TBT increases P4 but has minimal effect on downstream steroids. It enhances the production of inflammatory biomarkers such as IL-1ß, TNF-α, IL-10 and IL-6. Inhibition of sgp-130 by TBT suggests that TBT may increase bioactive IL-6 production which has been associated with adverse neurodevelopmental outcomes. Reduced expression of BDNF also supports this possibility.

Effect of polybrominated diphenyl ether congeners on placental cytokine production.

Polybrominated diphenyl ethers (PBDEs) are pollutants that may increase the risk of preterm birth. In previous studies, we found that a mixture of PBDEs altered the expression of biomarkers for preterm birth by the placenta. However, there are 209 different PBDE congeners with different tissue distributions. How these different congeners may alter the production of immunomodulators by the placenta that help to maintain the survival of the fetal allograft is unclear. Therefore, we compared the effects 5 common congeners on basal and bacteria-stimulated cytokine production by the placenta. Placental explant cultures were incubated with 20 µM of PBDE congeners 47, 99, 100, 153, 209 or vehicle in the presence and absence of Escherichia coli for 20 h. Conditioned medium was harvested and concentrations of IL-1ß, TNF-α, IL-6, sgp130, HO-1, IL-10, BDNF, and 8-IsoP quantified. For unstimulated cultures, all congeners, except for PBDE-47, reduced the production of IL-1ß and IL-6 production was enhanced by PBDE-153. BDNF concentrations tended to be reduced by most PBDE congeners and IL-10 production was enhanced by PBDE-99, -153, and -209. 8-IsoP production was enhanced by PBDE-153, but not the other congeners. For bacteria-stimulated cultures, PBDE-47 increased IL-1ß production and PBDE-47, -153, and -209 tended to reduce TNF-α production. IL-6 production was enhanced by all PBDEs except 153. IL-10 production was enhanced by all congeners except for PBDE-47. All congeners significantly enhanced BDNF and 8-IsoP. These results suggest that PBDEs can alter the expression of placental biomarkers in a congener and infection-dependent manner.

Linking Inflammation and Parkinson Disease: Hypochlorous Acid Generates Parkinsonian Poisons.

Inflammation is a common feature of Parkinson Disease and other neurodegenerative disorders. Hypochlorous acid (HOCl) is a reactive oxygen species formed by neutrophils and other myeloperoxidase-containing cells during inflammation. HOCl chlorinates the amine and catechol moieties of dopamine to produce chlorinated derivatives collectively termed chlorodopamine. Here, we report that chlorodopamine is toxic to dopaminergic neurons both in vivo and in vitro Intrastriatal administration of 90 nmol chlorodopamine to mice resulted in loss of dopaminergic neurons from the substantia nigra and decreased ambulation-results that were comparable to those produced by the same dose of the parkinsonian poison, 1-methyl-4-phenylpyridinium (MPP+). Chlorodopamine was also more toxic to differentiated SH SY5Y cells than HOCl. The basis of this selective toxicity is likely mediated by chlorodopamine uptake through the dopamine transporter, as expression of this transporter in COS-7 cells conferred sensitivity to chlorodopamine toxicity. Pharmacological blockade of the dopamine transporter also mitigated the deleterious effects of chlorodopamine in vivo The cellular actions of chlorodopamine included inactivation of the α-ketoglutarate dehydrogenase complex, as well as inhibition of mitochondrial respiration. The latter effect is consistent with inhibition of cytochrome c oxidase. Illumination at 670 nm, which stimulates cytochrome c oxidase, reversed the effects of chlorodopamine. The observed changes in mitochondrial biochemistry were also accompanied by the swelling of these organelles. Overall, our findings suggest that chlorination of dopamine by HOCl generates toxins that selectively kill dopaminergic neurons in the substantia nigra in a manner comparable to MPP+.

Efficacy of midtrimester short cervix interventions is conditional on intraamniotic inflammation.

BACKGROUND: Midtrimester ultrasound is a valuable method for identifying asymptomatic women at risk for spontaneous preterm delivery (PTD). However, response to various treatments (cerclage, progestogen) has been variable in the clinical setting. It remains unclear how other biomarkers may be used to guide intervention strategies. OBJECTIVE: We applied an amniotic fluid inflammatory scoring system to determine if the degree of inflammation is associated with intervention efficacy in patients with midtrimester short cervix. STUDY DESIGN: Women carrying a singleton fetus between 16-24 weeks' gestation with a short cervix (≤25 mm) on transvaginal ultrasound underwent amniocentesis and were assigned to McDonald cerclage, no cerclage, or weekly 17-alpha hydroxyprogesterone caproate (17OHP-C). Our previously described inflammatory risk score (comprised of 14 inflammatory markers) was used to classify patients as high (score ≥8) or low (score <8) risk for inflammation. Gestational age at delivery was compared for each intervention and risk score status. Risk of delivering as a function of the remaining gestation was evaluated using modified Cox proportional hazards models with incorporation of methods to account for both left and right truncation bias. RESULTS: Ninety patients were included: 24 were in the nonintervention control group, 51 received cerclage, and 15 received 17OHP-C. Inflammation status at time of sampling influenced the efficacy of the treatment (P < .001). Compared to the nonintervention control group, in patients with low inflammation (score < 8), both cerclage (adjusted hazard ratio [HR], 2.86; 95% confidence interval [CI], 1.28-6.37) and 17OHP-C (HR, 3.11; 95% CI, 1.04-9.30) were associated with increased hazard of PTD. In contrast, in patients with high inflammation (score ≥8) both cerclage (HR, 0.22; 95% CI, 0.08-0.65) and 17OHP-C (HR, 0.20; 95% CI, 0.05-0.81) were associated with lower hazard of delivering preterm. CONCLUSION: Cerclage placement or administration of 17OHP-C therapy for midtrimester short cervix for PTD prevention appears beneficial only in the subset of patients with high inflammation. Knowledge of the amniotic fluid inflammatory status may aid in guiding the appropriate therapy for women presenting with midtrimester short cervix who are at increased risk of PTD.

Environmental Pollutant Polybrominated Diphenyl Ether, a Flame Retardant, Induces Primary Amnion Cell Senescence.

OBJECTIVE: Polybrominated diphenyl ethers (PBDEs) are documented to increase the risk for spontaneous preterm birth (PTB). We hypothesize that PBDEs cause oxidative stress (OS) that leads to fetal cell senescence and inflammation associated with PTB. METHODS: Primary amnion epithelial cells (n = 5) isolated from term, not in labor pregnancies, were exposed to PBDE congeners 47 and 99 (each 5 µM). ROS kinetics was monitored. Morphologic changes, phospho-p38 MAPK (P-p38) activation, development of senescence, and induction of uterotonins (COX-2 expression) were quantified using light microscopy, Western blot, senescence-associated ß-galactosidase (SA ß-gal) staining, and qRT-PCR, respectively, after 48 and 72 hr of exposure. RESULTS: Both PBDE congeners induced ROS within 2 min compared to controls (P < 0.05). P-p38 activation was significant after PBDE-99 treatment than controls (P < 0.05). After 72 hr of treatment, both PBDE-treated cells showed cell death-associated morphologic changes with significantly higher SA ß-gal-stained cells than control. COX-2 expression was higher after 72 hr of treatment with PBDE-99. Overall, the PBDE-99 response was more pronounced than PBDE-47. CONCLUSIONS: Congener-dependent OS response, p38 MAPK activation, senescence, and COX-2 expression were seen in human amnion cells by PBDEs. These findings demonstrate environment pollutant-induced senescence activation and inflammation can lead to pathways resulting in PTB.

Does carbon monoxide inhibit proinflammatory cytokine production by fetal membranes?

AIM: Infection-induced inflammation is a common cause of preterm birth. Pharmacologic inhibition of proinflammatory cytokines improves pregnancy outcome in animal models but there are no universally effective therapies for preterm birth in women. Carbon monoxide (CO) has anti-inflammatory properties at low concentrations but its effects on reproductive tissues is unclear. Therefore, we studied the effect of supplemental CO on the production of cytokines associated with preterm birth by fetal membranes. METHODS: Cross-sections of whole fetal membranes, isolated choriodecidua, and isolated amnion were prepared using tissues collected from women who had normal vaginal deliveries at term. Tissues were placed in an organ explant culture system and stimulated with up to 10(8) CFU/mL Escherichia coli. Cultures were incubated under room air or room air+250 ppm CO for 18 h and cytokine concentrations in conditioned medium were quantified by ELISA. RESULTS: CO inhibited IL-1ß and TNF-α (P≤0.001) production by cultures stimulated with 10(7) CFU/mL bacteria but had no detectable effect on IL-10 by full-thickness membranes. Although CO also tended to reduce TNF-α production (P=0.053), no effect of CO was detected for IL-10 or IL-1ß for membranes stimulated with 10(8) CFU/mL E. coli. TNF-α, but not IL-1ß or IL-10 production, was inhibited by CO for choriodecidual cultures stimulated with 10(7) or 10(8) CFU/mL E. coli (P<0.001). IL-1ß production was significantly inhibited by CO for amnion cultures stimulated with 10(7) (P=0.002) and 10(8) (P=0.017) CFU/mL E. coli. Exposure to bacteria had no effect on TNF-α or IL-10 production but CO tended to increase IL-10 production by amnion cultures stimulated with 10(8) CFU/mL E. coli (P=0.037). CONCLUSIONS: These results suggest that CO may help promote an anti-inflammatory environment during intrauterine infections by inhibiting TNF-α and IL-1ß production.

Effect of oxygen tension on bacteria-stimulated cytokine production by fetal membranes.

AIM: Tissue culture studies indicate that bacterial products stimulate the production of proinflammatory cytokines by reproductive tissues. However, most of these studies have been performed under room air conditions, supplemented with 5% CO2. In this study, we tested whether O2 tension affects bacteria-stimulated cytokine production by extra-placental fetal membranes. METHODS: Cultures of full-thickness membranes, isolated choriodecidua, and isolated amnion were exposed to bacteria and incubated under 21% (room air) or 5% O2 for 18 h. Cytokine concentrations in conditioned medium was quantified by immunoassay. RESULTS: Culture under 5% O2 increased production of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, but reduced IL-10 and IL-6 production by full membranes. Isolated choriodecidua responded to 5% O2 with increased IL-1ß production and reduced IL-6 production, but had no effect on TNF-α and IL-10 production was not detected. No effect of O2 tension on IL-1ß or IL-6 production by isolated amnion was detected, however, Escherichia coli-stimulated IL-10, TNF-α and IL-8 production was enhanced by culture under 5% O2. CONCLUSIONS: Increased oxygen tension reduces the pro-inflammatory responsiveness of cell cultures to E. coli and promotes an anti-inflammatory cytokine profile. Differential effects of O2 tension on choriodecidua and amnion suggests a network of paracrine factors that regulate cytokine levels in response to changes in O2 tension.

Can carbon monoxide prevent infection-mediated preterm birth in a mouse model?

PROBLEM: Preterm birth is frequently caused by intrauterine infection and inflammation. Recent studies have demonstrated that carbon monoxide (CO), which is produced endogenously, has potent anti-inflammatory properties. Whether or not CO can prevent infection-mediated preterm birth is unknown. METHODS: Mice were assigned to one of four groups: sham infection, sham infection + CO, infection, or infection + CO. Infections were established by intra-uterine injection of Escherichia coli on day 14 of pregnancy. Animals received daily i.p. injections of 1 mL CO-saturated lactated ringers solution (LRS) or LRS alone beginning on the morning of surgery. Gestational age at delivery and litter characteristics was noted. In second experiment, animals were sacrificed 24 hrs post-surgery and tissues were harvested for cytokine analyses. RESULTS: Escherichia coli intrauterine infection increased the number of animals delivering preterm. This effect was significantly ameliorated by CO-LRS. CO-treatment also increased litter size and weights of the surviving offspring. Cytokines in the amniotic fluid and the placenta were increased by E. coli exposure, but CO had no detectible effect on E. coli-stimulated cytokine production. No effects of CO were detected in sham-infected animals. CONCLUSION: Supplemental CO improves pregnancy outcome after intrauterine infection and may function at a point downstream of, or through pathways independent of, induction of proinflammatory cytokines.

Fetal membrane biomarker network diversity and disease functions induced by intra-amniotic pathogens.

PROBLEM: Intra-amniotic pathogens and by-products activate innate immune responses encompassing multitudes of signaling molecules and pathways that can result in spontaneous preterm birth (PTB). This study investigates fetal membrane response to bacterial stimulation using a bioinformatics approach. METHOD OF STUDY: Dysregulated biomarker (IL1-ß, IL-2, IL-8, IL-10, and TNF-α) data from fetal membranes at term stimulated with Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis, E. coli, Group B Streptococci, Polyporhans gingivalis, or Gardnerella vaginalis with 50% (v/v) amniotic fluid (AF) were analyzed by Ingenuity Pathway Analysis. RESULTS: In racially stratified analysis, networks representing late-stage immune inflammation were seen in African-Americans in AF absence. Inflammation was dominant in AF presence as well. In Caucasians, late-stage immune response was dominant with AF, but not in its absence. CONCLUSIONS: Fetal membrane biofunctions in response to bacteria reflect early- and late-stage innate immune defenses that vary based on the presence of AF and subject race.

Amniotic fluid and maternal race influence responsiveness of fetal membranes to bacteria.

Spontaneous preterm birth (PTB) and preterm prelabor rupture of membranes (pPROM) occur more frequently in African-American women than in other racial groups. This may be due to an enhanced inflammatory response to pathogens associated with the condition. It is also possible that amniotic fluid (AF) has different immunomodulatory properties in African-American women that increase their risk of PTB and pPROM. To test this, we cultured fetal membranes from European-American and African-American women with sterile medium (control), Escherichia coli, Gardnerella vaginalis, Group B streptococci (GBS), Polyporphorans gingivalis, Mycoplasma hominis, Ureaplasma urealyticum or Ureaplasma parvum in the presence and absence of 50% autologous AF. Cytokine concentrations were quantified in the conditioned medium. All bacterial species increased IL-8 production. IL-1ß and TNF-α production were stimulated by LPS, E. coli, and G. vaginalis compared with control, but responses to Group B streptococci and P. gingivalis were limited to IL-1ß and TNF-α respectively. Genital mycoplasmas stimulated TNF-α and IL-10 but had no effect on IL-1ß production. African-Americans had twice the IL-1ß response to E. coli as European-Americans (P=0.031). Conversely, European-Americans produced more IL-8 in response to LPS than African-Americans (P=0.026). AF had both pro- and anti-inflammatory properties that varied between races and pathogens. These results suggest that the host response to fetal membrane infections is complex and not generalizable. Interventions to prevent PTB and pPROM may need to be customized based on a patient's race, type of bacterial infection and factors in her AF.