Maternal prebiotic supplementation during pregnancy and lactation modifies the microbiome and short chain fatty acid profile of both mother and infant.

BACKGROUND & AIMS: Improving maternal gut health in pregnancy and lactation is a potential strategy to improve immune and metabolic health in offspring and curtail the rising rates of inflammatory diseases linked to alterations in gut microbiota. Here, we investigate the effects of a maternal prebiotic supplement (galacto-oligosaccharides and fructo-oligosaccharides), ingested daily from <21 weeks' gestation to six months' post-partum, in a double-blinded, randomised placebo-controlled trial. METHODS: Stool samples were collected at multiple timepoints from 74 mother-infant pairs as part of a larger, double-blinded, randomised controlled allergy intervention trial. The participants were randomised to one of two groups; with one group receiving 14.2 g per day of prebiotic powder (galacto-oligosaccharides GOS and fructo-oligosaccharides FOS in ratio 9:1), and the other receiving a placebo powder consisting of 8.7 g per day of maltodextrin. The faecal microbiota of both mother and infants were assessed based on the analysis of bacterial 16S rRNA gene (V4 region) sequences, and short chain fatty acid (SCFA) concentrations in stool. RESULTS: Significant differences in the maternal microbiota profiles between baseline and either 28-weeks' or 36-weeks' gestation were found in the prebiotic supplemented women. Infant microbial beta-diversity also significantly differed between prebiotic and placebo groups at 12-months of age. Supplementation was associated with increased abundance of commensal Bifidobacteria in the maternal microbiota, and a reduction in the abundance of Negativicutes in both maternal and infant microbiota. There were also changes in SCFA concentrations with maternal prebiotics supplementation, including significant differences in acetic acid concentration between intervention and control groups from 20 to 28-weeks' gestation. CONCLUSION: Maternal prebiotic supplementation of 14.2 g per day GOS/FOS was found to favourably modify both the maternal and the developing infant gut microbiome. These results build on our understanding of the importance of maternal diet during pregnancy, and indicate that it is possible to intervene and modify the development of the infant microbiome by dietary modulation of the maternal gut microbiome.

Study Protocol for a Randomised Controlled Trial Investigating the Effects of Maternal Prebiotic Fibre Dietary Supplementation from Mid-Pregnancy to Six Months' Post-Partum on Child Allergic Disease Outcomes.

Infant allergy is the most common early manifestation of an increasing propensity for inflammation and immune dysregulation in modern environments. Refined low-fibre diets are a major risk for inflammatory diseases through adverse effects on the composition and function of gut microbiota. This has focused attention on the potential of prebiotic dietary fibres to favourably change gut microbiota, for local and systemic anti-inflammatory effects. In pregnancy, the immunomodulatory effects of prebiotics may also have benefits for the developing fetal immune system, and provide a potential dietary strategy to reduce the risk of allergic disease. Here, we present the study protocol for a double-blinded, randomised controlled trial investigating the effects of maternal prebiotics supplementation on child allergic disease outcomes. Eligible pregnant women have infants with a first-degree relative with a history of medically diagnosed allergic disease. Consented women are randomised to consume either prebiotics (galacto-oligosaccharides and fructo-oligosaccharides) or placebo (maltodextrin) powder daily from 18-20 weeks' gestation to six months' post-partum. The target sample size is 652 women. The primary outcome is infant medically diagnosed eczema; secondary outcomes include allergen sensitisation, food allergies and recurrent wheeze. Breast milk, stool and blood samples are collected at multiple timepoints for further analysis.

Effects of maternal n-3 fatty acid supplementation on placental cytokines, pro-resolving lipid mediators and their precursors.

The aim of this study was to determine whether supplementation with fish oil-derived n-3 polyunsaturated fatty acids (n-3 PUFA) during pregnancy modifies placental PUFA composition, the accumulation of specialised pro-resolving lipid mediators (SPMs, specifically resolvins (Rv), protectins (PD) and upstream precursors) and inflammatory gene expression. Placentas were collected from women (n=51) enrolled in a randomised, placebo controlled trial of n-3 PUFA supplementation from 20-week gestation. Lipids were extracted for fatty acid analysis and SPMs were quantitated by mass spectrometry. Gene expression was determined by qRT-PCR. Using multiple regression analysis, data were correlated for placental n-3 PUFA and SPM levels with PUFA levels in maternal and cord blood erythrocytes. Supplementation with n-3 PUFAs increased placental docosahexaenoic acid (DHA) levels, but not eicosapentaenoic acid (EPA) levels (P<0.05), and increased the levels of the SPM precursors 18-hydroxyeicosapentaenoic acid and 17-hydroxydocosahexaenoic acid (17-HDHA) by two- to threefold (P<0.0005). RvD1, 17R-RvD1, RvD2 and PD1 were detectable in all placentas, but concentrations were not significantly increased by n-3 PUFA supplementation. Placental DHA levels were positively associated with maternal and cord DHA levels (P<0.005), and with placental 17-HDHA concentrations (P<0.0001). Placental mRNA expression of PTGS2, IL1ß, IL6 and IL10 was unaffected by n-3 PUFA supplementation, but TNFα expression was increased by 14-fold (P<0.05). We conclude that n-3 PUFA supplementation in pregnancy i) enhances placental accumulation of DHA and SPM precursors, ii) does not alter placental EPA levels, and iii) has no stimulatory effects on inflammatory gene expression. Further studies are required to ascertain the biological significance of SPMs in the placenta and the potential immunomodulatory effects of elevating placental SPM levels.

Maternal dietary omega-3 fatty acid intake increases resolvin and protectin levels in the rat placenta.

Placental inflammation is associated with several pregnancy disorders. Inflammation is limited by anti-inflammatory and proresolving mechanisms, the latter partly mediated by resolvins and protectins derived from omega-3 polyunsaturated fatty acids (n-3PUFA). We examined effects of dietary n-3PUFAs on levels of resolvins, protectins, and lipoxygenase (ALOX) enzymes in the rat placenta. Rats consumed standard (Std) or high n-3PUFA (Hn3) diets from day 1 of pregnancy; tissues were collected on day 17 or 22 (term = day 23). Maternal Hn3 diet increased resolvin and protectin precursors, 18R/S-HEPE (P < 0.001), and 17R/S-HDHA (P < 0.01) at both days. Resolvins (17R-RvD1 and RvD1) increased at day 22 (P < 0.001) after Hn3 consumption, coincident with higher Alox15b and Alox5 mRNA expression, while RvD2 increased at both days (P < 0.05). Protectins, PD1, and 10S,17S-DiHDHA increased over late gestation (P < 0.001), coincident with higher Alox15 mRNA expression (P < 0.001) and further increased with Hn3 diet (P < 0.05). Maternal systemic and placental proinflammatory mediators were not suppressed by Hn3 diet; systemic IL1ß, placental Il1ß, and Il6 mRNA expression increased marginally with Hn3 at day 22 (P < 0.001), while Ptgs1 (Cox1) expression increased both days (P < 0.05). Our data indicate that maternal n-3PUFA supplementation enhances expression of enzymes in the n-3PUFA metabolic pathway and increases placental levels of resolvins and protectins.

Maternal dietary omega-3 fatty acid supplementation reduces placental oxidative stress and increases fetal and placental growth in the rat.

Placental oxidative stress plays a key role in the pathophysiology of several placenta-related disorders including intrauterine growth restriction. Oxidative stress occurs when accumulation of reactive oxygen species damages DNA, proteins, and lipids, an outcome normally limited by antioxidant defenses. Dietary supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFAs) may limit oxidative stress by increasing antioxidant capacity, but n-3 PUFAs are also highly susceptible to lipid peroxidation; so n-3 PUFA supplementation is potentially harmful. Here we examined the effect of n-3 PUFAs on placental oxidative stress and on placental and fetal growth in the rat. We also investigated whether diet-induced changes in maternal plasma fatty acid profiles are associated with comparable changes in placental and fetal tissues. Rats were fed either standard or high n-3 PUFA diets from Day 1 of pregnancy, and tissues were collected on Day 17 or 22 (term = Day 23). Dietary supplementation with n-3 PUFAs increased fetal (6%) and placental (12%) weights at Day 22, the latter attributable primarily to growth of the labyrinth zone (LZ). Increased LZ weight was accompanied by reduced LZ F(2)-isoprostanes (by 31% and 11% at Days 17 and 22, respectively), a marker of oxidative damage. Maternal plasma PUFA profiles were altered by dietary fatty acid intake and were strongly predictive of corresponding profiles in placental and fetal tissues. Our data indicate that n-3 PUFA supplementation reduces placental oxidative stress and enhances placental and fetal growth. Moreover, fatty acid profiles in the mother, placenta, and fetus are highly dependent on dietary fatty acid intake.

Development of a model for functional studies of ABCG2 (breast cancer resistance protein) efflux employing a standard BeWo clone (B24).

Human choriocarcinoma-derived BeWo cells express high levels of breast cancer resistance protein (BCRP/ABCG2) with no functional P-glycoprotein (P-gp) (ABCB1) activity, making them a potential model to study bidirectional ABCG2-mediated drug transport. However, the original BeWo clone (B24) available to researchers does not form confluent monolayers with tight junctions required by the model. Our aim was to adapt culture conditions to attempt to generate confluent BeWo monolayers for drug transport studies using the standard B24 clone. BeWo cells (B24; American Type Culture collection [ATCC]) were cultured in six-well plates or polycarbonate millicell inserts in a number of media formulations, growth supplements, and basement membrane substitutes. Cells were examined for confluence by microscopy, and transepithelial electrical resistance (TEER) was measured daily; monolayer permeability was assessed when TEER had stabilized. Optimal growth rates were achieved in culture conditions consisting of Medium 199 (M199) supplemented with epidermal growth factor (EGF; 20 ng/mL), vitamin supplements, and 10% fetal calf serum (FCS) with collagen coating. A TEER of 170 Ω in 0.6 cm(2) inserts was achieved 2 weeks after seeding under optimal conditions. The cell-impermeable diffusion marker 5(6) carboxy-2,7dichlorodihydrofluorescein (C-DCDHF) had a permeability coefficient of 3.5×10(-6) cm/s, indicative of minimal paracellular permeability. ABCG2 expression, as determined by immunoblotting, remained unaffected by confluency. In conclusion, we describe culture conditions for the B24 BeWo clone that facilitate the formation of monolayers with tighter junctions and reduced paracellular transport compared to previously published models. These growth conditions provide a good model of ABCG2-mediated drug transport in a human placental cell line.