INTERGROWTH-21 Identifies High Prevalence of Low Symphysis-Fundal Height in Indigenous Pregnant Women Experiencing Multiple Infections, Nutrient Deficiencies, and Inflammation: The Maternal Infections, Nutrient Deficiencies, and Inflammation (MINDI) Cohort.

BACKGROUND: In the absence of ultrasound, symphysis-fundal height (SFH) can assess maternal-fetal well-being as it is associated with gestational age, fetal weight, and amniotic fluid volume. However, other modifiers of SFH, including maternal infections, nutrient deficiencies, and inflammation (MINDI), have not been widely explored. OBJECTIVES: Our objectives were 2-fold: 1) to assess prevalence of low SFH in indigenous Panamanian women using both Pan-American Health Organization (PAHO) and INTERGROWTH-21 standards and 2) to explore associations of SFH with maternal health indicators: infections (oral, skin, urogenital, nematode infections), nutrient deficiencies [protein and iron indicators (ferritin, serum iron, serum transferrin receptor, hepcidin), folate, and vitamins A, D, and B-12], and inflammation [leukocytes, C-reactive protein (CRP), cytokines]. METHODS: For this cross-sectional study, low-SFH-for-gestational-age was assessed using PAHO and INTERGROWTH <10th centile in 174 women at ≥16 weeks of gestation. Bootstrapping selected MINDI variables for inclusion in multivariable fractional polynomial (MFP) logistic regressions for low SFH. Associations of MINDI variables with hepcidin were also investigated. RESULTS: Prevalence of low SFH was 8% using PAHO, but using INTERGROWTH, 50.6% had SFH <10th centile, including 37.9% <3rd centile. Both PAHO-SFH <10th centile and INTERGROWTH-SFH <3rd centile were associated with higher hepcidin (OR = 1.12, P = 0.008, and OR = 3.04, P = 0.001, respectively) and with lower TNF-α (OR = 0.73, P = 0.012, and OR = 0.93, P = 0.015, respectively). Wood-smoke exposure increased the odds of PAHO-SFH <10th centile (OR = 1.19, P = 0.009), whereas higher BMI decreased the odds of INTERGROWTH-SFH <3rd centile (OR = 0.87, P = 0.012). Lower pulse pressure (OR = 0.90, P = 0.009) and lower inflammatory responses [lower lymphocytes (OR = 0.21, P = 0.026), IL-17 (OR = 0.89, P = 0.011)] distinguished SFH <3rd centile from SFH ≥3rd to <10th centiles using INTERGROWTH-21 standards. The MFP regression for hepcidin controlling for SFH (adjusted R 2 = 0.40, P = 0.001) revealed associations with indicators of inflammation (CRP, P < 0.0001; IL-17, P = 0.012), acidic urinary pH (P = 0.008), and higher intake of supplements (P = 0.035). CONCLUSIONS: Associations of low SFH with MINDI variables, including hepcidin, highlight its potential for early detection of multicausal in utero growth faltering.

Nodal is required to maintain the uterine environment in an anti-inflammatory state during pregnancy†.

Preterm birth remains the major cause of perinatal mortality and morbidity worldwide, affecting up to 12% of pregnancies and accounting for ~75% of neonatal deaths. However, the mechanisms and causes that underlie it are still largely unknown. One of the major causes of preterm birth is infection or inflammation within the maternal-fetal interface. Our lab has previously shown that a uterine specific deletion of Nodal results in mutant females delivering 2 days prior to term demonstrating an important role for this factor in the maintenance of pregnancy. Here, we have addressed the function of Nodal in the uterus during pregnancy. We demonstrate that Nodal heterozygous mice have an increase in basal levels of pro-inflammatory cytokines IL-1ß, IL-6, IL-12p, TNF-α, and IFN-γ as well as an increase in the number of macrophages in response to the inflammatory agent, lipopolysaccharide (LPS). Using bone marrow-derived macrophages, we demonstrated that pretreatment with recombinant Nodal reduces pro-inflammatory gene expression when these cells are challenged with LPS. Our results demonstrate that Nodal is required to maintain the uterine environment in an anti-inflammatory state by preventing proinflammatory cytokine expression.

Evidence of a gene-environment interaction of NODAL variants and inflammation in preterm birth.

OBJECTIVE: NODAL has been implicated in timing of parturition and immune regulation. We investigated the relationship between NODAL polymorphisms, infection/inflammation, and preterm birth. STUDY DESIGN: For this secondary analysis, 613 women (189 preterm and 424 term) from the Montreal Prematurity Study were genotyped for NODAL polymorphisms and assessed for bacterial vaginosis and placental inflammation. RESULT: NODAL polymorphisms were not associated with preterm birth. However, the rs2231947(C>T) variant allele was associated with increased risk for preterm birth among women with bacterial vaginosis (odds ratio: 2.76, 95% confidence interval: 1.12-6.85). Among women without placental inflammation, the rs1904589(A>G) variant allele was associated with increased risk of preterm birth (odds ratio: 1.31, 95% confidence interval: 1.02-1.70). Among women with placental inflammation, the rs10999338(C>T) variant allele was associated with reduced risk of preterm birth (odds ratio: 0.50, 95% confidence interval: 0.29-0.87). CONCLUSION: The effect of NODAL polymorphisms on preterm birth depends on maternal infection/inflammation status.

Differential expression of genes in fetal brain as a consequence of maternal protein deficiency and nematode infection.

Maternal dietary protein deficiency and gastrointestinal nematode infection during early pregnancy have negative impacts on both maternal placental gene expression and fetal growth in the mouse. Here we used next-generation RNA sequencing to test our hypothesis that maternal protein deficiency and/or nematode infection also alter the expression of genes in the developing fetal brain. Outbred pregnant CD1 mice were used in a 2×2 design with two levels of dietary protein (24% versus 6%) and two levels of infection (repeated sham versus Heligmosomoides bakeri beginning at gestation day 5). Pregnant dams were euthanized on gestation day 18 to harvest the whole fetal brain. Four fetal brains from each treatment group were analyzed using RNA Hi-Seq sequencing and the differential expression of genes was determined by the edgeR package using NetworkAnalyst. In response to maternal H. bakeri infection, 96 genes (88 up-regulated and eight down-regulated) were differentially expressed in the fetal brain. Differentially expressed genes were involved in metabolic processes, developmental processes and the immune system according to the PANTHER classification system. Among the important biological functions identified, several up-regulated genes have known neurological functions including neuro-development (Gdf15, Ing4), neural differentiation (miRNA let-7), synaptic plasticity (via suppression of NF-κß), neuro-inflammation (S100A8, S100A9) and glucose metabolism (Tnnt1, Atf3). However, in response to maternal protein deficiency, brain-specific serine protease (Prss22) was the only up-regulated gene and only one gene (Dynlt1a) responded to the interaction of maternal nematode infection and protein deficiency. In conclusion, maternal exposure to GI nematode infection from day 5 to 18 of pregnancy may influence developmental programming of the fetal brain.

Expression of growth-related genes in the mouse placenta is influenced by interactions between intestinal nematode (Heligmosomoides bakeri) infection and dietary protein deficiency.

Intestinal nematode infection and dietary protein deficiency are common during pregnancy and both have been shown to impair fetal growth in humans, livestock and laboratory animals. The placenta has been linked to fetal growth but its role in mediating the response to maternal infection and protein deficiency is not understood. We used microarrays to test the hypothesis that maternal intestinal nematode infection and protein deficiency alter the expression of placental genes related to fetal growth. Placentas were obtained on day 18 of pregnancy from CD-1 mice fed protein sufficient (24%) or protein deficiency (6%) isoenergetic diets and either uninfected or infected with Heligmosomoides bakeri. Gene expression was analysed using the Affymetrix GeneChip 2.0 ST mouse array (n=3/experimental group). Differentially expressed genes were identified using two-way ANOVA (P<0.02, fold-change >1.25) and pathway analyses were performed using DAVID software. Expression changes for selected genes were confirmed using qPCR. Heligmosomoides bakeri infection down-regulated 109 transcripts, including genes related to oxidative phosphorylation, and up-regulated 214 transcripts, including genes involved in ATP binding and hemopoiesis. Up-regulation of hemopoiesis genes may explain increased placental mass previously reported in H. bakeri-infected mice. Protein deficiency down-regulated 141 annotated transcripts, including genes involved in cell motility and endopeptidase activity, and up-regulated 131 annotated transcripts, including genes related to hemopoiesis. A statistical interaction was detected for 248 transcripts, including several genes with known functions in fetal growth. Notably, expression of the gene Irs1 (insulin receptor substrate) was lower in infected dams but only when they were fed a protein sufficient diet. Also, expression of several genes, including Igf1r (insulin-like growth factor-1 receptor) and Prl (prolactin) was up-regulated by infection in protein deficiency dams and down-regulated by protein deficiency in uninfected dams. Our results highlight that expression of placental genes involved in fetal growth is influenced by the interaction between protein deficiency and H. bakeri infection.

Protein deficiency and intestinal nematode infection in pregnant mice differentially impact fetal growth through specific stress hormones, growth factors, and cytokines.

BACKGROUND: Protein deficiency (PD) and intestinal nematode infections commonly co-occur during pregnancy and impair fetal growth, but the complex network of signals has not been explored. OBJECTIVE: Our objective was to assess those stress hormones, growth factors, and cytokines affected by maternal PD and nematode infection and associated with fetal growth. METHODS: Using a 2 × 2 factorial design, CD-1 mice, fed protein-sufficient (PS; 24%) or protein-deficient (PD; 6%) isoenergetic diets, were either uninfected or infected every 5 d with Heligmosomoides bakeri, beginning on gestational day (GD) 5. Biomarker concentrations were measured on GD 18 in maternal serum (m), fetal serum (f), and amniotic fluid (af) by using Luminex. RESULTS: Maternal PD lowered fetal body mass (PS/uninfected 1.25 ± 0.02 g, PS/infected 1.19 ± 0.02 g vs. PD/uninfected 1.11 ± 0.02 g, PD/infected 0.97 ± 0.02 g; P = 0.02), fetal lung (P = 0.005), and liver (P = 0.003) but not brain mass, whereas maternal infection lowered fetal length (PS/uninfected 2.28 ± 0.02 cm, PD/uninfected 2.27 ± 0.03 cm vs. PS/infected 2.21 ± 0.03 cm, PD/infected 2.11 ± 0.02 cm; P = 0.05) and kidney mass (P = 0.04). PD elevated stress hormones (m-adrenocortiotropic hormone, f-corticosterone, af-corticosterone) and reduced insulin-like growth factor 1 in all compartments (P ≤ 0.01), but these were unassociated with fetal mass or length. Fetal mass was positively associated with f-leptin (R(2) = 0.71, P = 0.0001) and negatively with fetal cytokines [tumor necrosis factor-α: R(2) = 0.62, P = 0.001; interleukin-4 (IL-4): R(2) = 0.63, P = 0.0004]. In contrast, maternal infection lowered f-prolactin (P = 0.02) that was positively associated with fetal length (R(2) = 0.43; P = 0.03); no other biomarker was affected by infection. Regression analyses showed associations between organ growth, cytokines, and growth factors: 1) thymus, spleen, heart, and brain with m-IL-10; 2) brain and kidney with f-vascular endothelial growth factor, af-monocyte chemotactic protein 1, af-interferon-γ, and af-eotaxin; and 3) liver and lung with f-leptin and af-corticosterone (all P ≤ 0.02). CONCLUSIONS: PD and nematode infection impaired fetal mass and linear growth, respectively. Fetal mass, length, and individual organ masses were regulated by different hormones, growth factors, and cytokines.

Protein deficiency alters impact of intestinal nematode infection on intestinal, visceral and lymphoid organ histopathology in lactating mice.

Protein deficiency impairs local and systemic immune responses to Heligmosomoides bakeri infection but little is known about their individual and interactive impacts on tissue architecture of maternal lymphoid (thymus, spleen) and visceral (small intestine, kidney, liver, pancreas) organs during the demanding period of lactation. Using a 2 × 2 factorial design, pregnant CD1 mice were fed a 24% protein sufficient (PS) or a 6% protein deficient (PD) isoenergetic diet beginning on day 14 of pregnancy and were infected with 100 H. bakeri larvae four times or exposed to four sham infections. On day 20 of lactation, maternal organs were examined histologically and serum analytes were assayed as indicators of organ function. The absence of villus atrophy in response to infection was associated with increased crypt depth and infiltration of mast cells and eosinophils but only in lactating dams fed adequate protein. Infection-induced lobular liver inflammation was reduced in PD dams, however, abnormalities in the kidney caused by protein deficiency were absent in infected dams. Bilirubin and creatinine were highest in PD infected mice. Infection-induced splenomegaly was not due to an increase in the lymphoid compartment of the spleen. During lactation, infection and protein deficiency have interactive effects on extra-intestinal pathologies.

Pentachlorophenol induction of the Pseudomonas aeruginosa mexAB-oprM efflux operon: involvement of repressors NalC and MexR and the antirepressor ArmR.

Pentachlorophenol (PCP) induced expression of the NalC repressor-regulated PA3720-armR operon and the MexR repressor-controlled mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa. PCP's induction of PA3720-armR resulted from its direct modulation of NalC, the repressor's binding to PA3720-armR promoter-containing DNA as seen in electromobility shift assays (EMSAs) being obviated in the presence of this agent. The NalC binding site was localized to an inverted repeat (IR) sequence upstream of PA3720-armR and overlapping a promoter region whose transcription start site was mapped. While modulation of MexR by the ArmR anti-repressor explains the upregulation of mexAB-oprM in nalC mutants hyperexpressing PA3720-armR, the induction of mexAB-oprM expression by PCP is not wholly explainable by PCP induction of PA3720-armR and subsequent ArmR modulation of MexR, inasmuch as armR deletion mutants still showed PCP-inducible mexAB-oprM expression. PCP failed, however, to induce mexAB-oprM in a mexR deletion strain, indicating that MexR was required for this, although PCP did not modulate MexR binding to mexAB-oprM promoter-containing DNA in vitro. One possibility is that MexR responds to PCP-generated in vivo effector molecules in controlling mexAB-oprM expression in response to PCP. PCP is an unlikely effector and substrate for NalC and MexAB-OprM--its impact on NalC binding to the PA3720-armR promoter DNA occurred only at high µM levels--suggesting that it mimics an intended phenolic effector/substrate(s). In this regard, plants are an abundant source of phenolic antimicrobial compounds and, so, MexAB-OprM may function to protect P. aeruginosa from plant antimicrobials that it encounters in nature.