
==== Front
Mol Metab
Mol Metab
Molecular Metabolism
2212-8778
Elsevier

S2212-8778(24)00135-2
10.1016/j.molmet.2024.102004
102004
Original Article
Maternal gut Bifidobacterium breve modifies fetal brain metabolism in germ-free mice
Lopez-Tello Jorge jorge.lopeztello@uam.es
jl898@cam.ac.uk
12⁎
Kiu Raymond 367
Schofield Zoe 3
Zhang Cindy X.W. 1
van Sinderen Douwe 4
Le Gall Gwénaëlle 5
Hall Lindsay J. 3671
Sferruzzi-Perri Amanda N. 11
1 Department of Physiology, Development, and Neuroscience, Centre for Trophoblast Research, University of Cambridge, Cambridge, UK
2 Department of Physiology, Faculty of Medicine. Autonomous University of Madrid, Spain
3 Food, Microbiome & Health, Quadram Institute Bioscience, Norwich Research Park, Norwich, UK
4 APC Microbiome Institute, University College Cork, Cork, Ireland
5 Norwich Medical School, University of East Anglia, Bob Champion Research and Education Building, James Watson Road, Norwich Research Park, Norwich NR4 7UQ, UK
6 Institute of Microbiology & Infection, University of Birmingham, Birmingham, UK
7 Department of Microbes, Infection & Microbiomes, School of Infection, Inflammation & Immunology, University of Birmingham, Birmingham, UK
⁎ Corresponding author. Department of Physiology, Development, and Neuroscience, Centre for Trophoblast Research, University of Cambridge, Cambridge, UK. jorge.lopeztello@uam.esjl898@cam.ac.uk
1 Contributed equally.

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© 2024 The Authors
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https://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Background

Recent advances have significantly expanded our understanding of the gut microbiome's influence on host physiology and metabolism. However, the specific role of certain microorganisms in gestational health and fetal development remains underexplored.

Objective

This study investigates the impact of Bifidobacterium breve UCC2003 on fetal brain metabolism when colonized in the maternal gut during pregnancy.

Methods

Germ-free pregnant mice were colonized with or without B. breve UCC2003 during pregnancy. The metabolic profiles of fetal brains were analyzed, focusing on the presence of key metabolites and the expression of critical metabolic and cellular pathways.

Results

Maternal colonization with B. breve resulted in significant metabolic changes in the fetal brain. Specifically, ten metabolites, including citrate, 3-hydroxyisobutyrate, and carnitine, were reduced in the fetal brain. These alterations were accompanied by increased abundance of transporters involved in glucose and branched-chain amino acid uptake. Furthermore, supplementation with this bacterium was associated with elevated expression of critical metabolic pathways such as PI3K-AKT, AMPK, STAT5, and Wnt-β-catenin signaling, including its receptor Frizzled-7. Additionally, there was stabilization of HIF-2 protein and modifications in genes and proteins related to cellular growth, axogenesis, and mitochondrial function.

Conclusions

The presence of maternal B. breve during pregnancy plays a crucial role in modulating fetal brain metabolism and growth. These findings suggest that Bifidobacterium could modify fetal brain development, potentially offering new avenues for enhancing gestational health and fetal development through microbiota-targeted interventions.

Highlights

• B. breve in maternal gut alters fetal brain cell cycle and axogenesis genes.

• B. breve in maternal gut alters fetal brain metabolism, reducing 10 key metabolites.

• B. breve in maternal gut modifies nutrient transporters in fetal brain.

• B. breve impacts mitochondrial TCA cycle, HIF and Wnt-β-catenin in fetal brain.

Keywords

Pregnancy
Microbiota
Fetus
Brain
Metabolism
==== Body
pmc1 Introduction

Fetal growth restriction (FGR) is a severe condition defined as the failure of the fetus to reach its growth potential due to pathological compromise. A main cause of FGR is placental insufficiency during gestation [1]. Human epidemiological studies and experimental animal models have found that FGR and placental insufficiency can affect fetal organ development, including changes in key organs like the heart, kidneys and brain [[2], [3], [4], [5], [6], [7], [8], [9]]. Studies have also shown that disruption of fetal brain development due to suboptimal intrauterine environments can lead to neurodevelopmental disorders postnatally, including motor and cognitive dysfunctions, learning impairments and cerebral palsy [[10], [11], [12], [13], [14], [15]]. Managing placental insufficiency and FGR in a clinical setting can present substantial challenges. Pharmacological interventions like aspirin, heparin and sildenafil citrate, which target inflammatory, coagulation and blood flow pathways are controversial due to variability in their effects on feto-placental and pregnancy outcomes [16,17]. Hence, there is an imminent need to devise effective treatments that can prevent and/or mitigate the adverse consequences associated with FGR.

In recent years, there has been an explosion of studies describing the importance of the gut microbiota in regulating developmental processes, from neurogenesis [18] to ageing [19,20]. Moreover, a perturbed gut microbiota has been linked to neurological conditions, like Parkinson's disease [21] and schizophrenia [22], and metabolic-related disorders, including type-2 diabetes [23]. In the context of pregnancy, previous studies have demonstrated dramatic changes in the composition of the maternal gut microbiota during pregnancy [24] and in women who developed a hypertensive disorder of pregnancy, preeclampsia or had abnormal placental growth [25,26]. Of note, the genus Bifidobacterium increases in abundance in the maternal gut during pregnancy in both women and mice [27] and has been shown to possess multiple benefits. For example, Bifidobacterium protects against infectious diseases and is involved in modulating host immune responses [[28], [29], [30], [31]]. Moreover, we have previously shown that administering three consecutive doses of Bifidobacterium (specifically, Bifidobacterium breve UCC2003) on gestational days 10, 12, and 14 to pregnant germ-free (GF) mice (referred to as the BIF group throughout the text; see Fig. S1), and non-pregnant specific-pathogen-free mice results in stable gut colonization [29,32]. This timing and dosing were chosen based on the observation that levels of Bifidobacterium increase throughout pregnancy [27] and to reduce the need for repeated handling of the mice, which can lead to stress and spontaneous abortions. Additionally, our approach could potentially align with supplementation studies in pregnant women, as we started the experiments after pregnancy was confirmed.

Herein we tested the hypothesis that maternal gut B. breve abundance would associate with changes in the development and metabolism of the fetal brain. Working with germ-free mice that lack a maternal microbiota (GF) and GF mice that received B. breve UCC2003 treatment during pregnancy, our results demonstrate the significance of maternal gut B. breve in fetal brain development and metabolism.

2 Results

2.1 Maternal gut B. breve UCC2003 modifies expression of genes involved in cell cycle and axogenesis

Using this approach, we have previously shown that administration of B. breve UCC2003 to pregnant GF mice improves fetal growth together with beneficial structural and functional alterations in the placenta and changes in metabolic genes in the fetal liver [32]. Moreover, supplementation with B. breve UCC2003 (BIF) improved liver size compared to untreated GF mice (GF). However, brain weight did not differ between untreated GF and BIF fetuses, suggesting that although untreated GF fetuses were growth restricted, they exhibited preserved brain development [32]. To understand the molecular basis of this, we used biobanked samples from our previous study and quantified the mRNA levels of key growth, cell cycle, microglia, and neurogenesis genes in the brain of fetuses from GF mice following maternal B. breve UCC2003 administration. This revealed that there was no difference in the expression of vascular gene Vegf or apoptotic genes Tp53, Casp3 and Bax in the brain between fetuses of the GF and BIF groups (Figure 1A). However, mRNA levels of the transcriptional activator Foxm1 and the mitotic cycling Cdk1 gene were significantly reduced in the fetal brain of the BIF compared to the untreated GF group (Figure 1B). No changes were observed in other assessed cell cycle genes (Cdk2, Cdk4, Cdc42, Ccne1, Gli1 and Axin2) or in genes involved in microglia activation (e.g. Ier3, Klf2 or Egr1) between GF and BIF groups (Figure 1B–C). Quantification of expression levels of key axonogenesis genes revealed that Plxna3 was significantly down-regulated in the fetal brain of BIF compared to untreated GF mice (Figure 1D). Also, Slit1 showed a tendency to be reduced in the BIF treated group (p = 0.05). The expression levels of the other axonogenesis genes assessed (Sema3f, Ntn1, Nrcam, Sv2b, Gabra1, Gabrg1 and Gabrg2) were shown to be similar between the two experimental groups (Figure 1D). Collectively, these data demonstrate that maternal gut B. breve UCC2003 modulates the expression of specific genes involved in cell cycle control and axonogenesis in the fetal brain.Figure 1 Maternal gut B. breve supplementation modifies genes involved in fetal brain cell growth and axon development. Relative mRNA levels of genes involved in cell cycle (A-B), microglia activation (C), and axonogenesis (D). Gene expression is relative to two housekeeping genes (Gapdh and Actb). Data analysed by ANOVA with the group as fixed effect and means comparisons was made by Fisher test (further details in the methods section). Data are means ± SEM with individual data points shown. ∗P < 0.05; ∗∗P < 0.01.

Figure 1

2.2 Maternal gut B. breve UCC2003 modifies fetal brain metabolism

To investigate if maternal B. breve UCC2003 supplementation affects fetal brain metabolism, we performed metabolomic profiling of fetal brain lysates from untreated and BIF treated GF mice. We quantified a total of 78 metabolites, of which 10, primarily amino acids and citrate were significantly reduced in the BIF compared to untreated GF group (Table 1 and Table S1). Considering that the levels of certain amino acids such as leucine and valine were altered in the BIF group, we quantified the mRNA levels of transporters involved in branched-chain amino acid uptake and metabolism in the fetal brain. This analysis identified that expression of the large neutral amino acid transporter 1 (LAT1), encoded by the Slc7a5 gene, but not large neutral amino acid transporter 2 (LAT2; encoded by Slc7a8) was significantly elevated in the BIF treated group compared to untreated GF mice (Figure 2A). No differences were found in the expression levels of genes encoding other amino acid transporters, namely the system A amino acid transporters (Slc38a1,2,4 - SNATs) in the fetal brain between BIF versus untreated GF mice (Figure 2B).Table 1 Maternal gut B. breve supplementation induce changes in fetal brain metabolites. Data analysed by one-way ANOVA, with the group as fixed effect and means comparisons made by Fisher test (general linear model-GLM model). Litter size added as a covariate. Data displayed as mean ± SEM. Values were considered statistically significant with P < 0.05. Further data about the metabolites analysed can be found in Table S1.

Table 1Metabolite (mmol/Kg)	GF group (n = 5)	BIF group (n = 5)	P value	
3-Hydroxyisobutyrate	0.063 ± 0.004	0.041 ± 0.007	0.048	
Alanine	3.523 ± 0.578	1.776 ± 0.163	0.020	
Arginine	0.192 ± 0.030	0.107 ± 0.015	0.045	
Aspartate	1.937 ± 0.228	1.119 ± 0.150	0.026	
Carnitine	0.370 ± 0.047	0.210 ± 0.023	0.022	
Dimethylamine	0.481 ± 0.192	0.072 ± 0.053	0.002	
Leucine	0.261 ± 0.019	0.177 ± 0.017	0.027	
Threonine	2.209 ± 0.292	1.170 ± 0.249	0.020	
Valine	0.378 ± 0.048	0.262 ± 0.027	0.035	
Citrate	0.219 ± 0.025	0.154 ± 0.023	0.046	

Figure 2 Maternal gut B. breve supplementation modifies the expression of nutrient transporters in the fetal brain. (A–E) Relative mRNA levels of genes encoding nutrient transporters measured in fetal brain samples, expression is relative to two housekeeping genes (Gapdh and Actb). Statistical analysis performed by ANOVA with the group as fixed effect (each fetus as a repeated measure) and means comparisons was made by Fisher test (linear mixed model). Data are means ± SEM with individual datapoints shown. Data obtained from a total of 5 GF and 6 BIF pregnant dams/litters (9 fetuses per group analysed). ∗P < 0.05; ∗∗P < 0.01.

Figure 2

In previous work we reported that circulating levels of glucose were increased in response to B. breve UCC2003 administration of GF pregnant mice [32]. Glucose is the predominant source of energy used by the fetal brain to support its development [33]. Hence, we quantified the mRNA levels of the main glucose transporter-encoding genes Slc2a1 and Slc2a3 (GLUT1 and GLUT3, respectively) in fetal brain tissue. This revealed that expression of both glucose transporters was significantly elevated in the fetal brain of BIF treated mice compared to untreated GF group (Figure 2C). Moreover, the mRNA level of Slc37a4 (encoding G6PT), an important gene that controls glucose homeostasis by regulating the transport of glucose-6-phosphate from the cytoplasm to the lumen of the endoplasmic reticulum [34], was also elevated in the BIF group (Figure 2C). In addition, the expression of solute carrier family 16 members 1,2,4,8 - MCTs which are involved in the movement of monocarboxylates, including lactate and pyruvate, and ketone bodies like d-β-hydroxybutyrate [35], were all shown to be significantly elevated in the fetal brain of BIF treated pregnant mice compared to the untreated GF group (Figure 2D). There was no difference in the expression of genes involved in lipid uptake (solute carrier family 27 members - FATP) in brain lysates of BIF treated pregnant GF mice compared to untreated GF mice (Figure 2E). These findings indicate that the presence of maternal gut B. breve UCC2003 impacts on the levels of specific metabolites and expression of key nutrient transporters in the fetal brain.

2.3 Maternal gut B. breve UCC2003 modifies cellular and metabolic pathways in the fetal brain

Driven by the changes detected in metabolites and nutrient transporters, we proceeded to analyze cellular and metabolic pathways in the fetal brain. Therefore, we analysed a variety of signalling pathways that have been previously implicated in the execution of different processes in the brain, including: cellular growth, proliferation and survival (e.g. PI3K-AKT [36,37]), nutrient uptake (e.g. AMPK [38]), dendritic organization (e.g. ERK [39]), neuronal development/differentiation and astrogliogenesis (e.g. AMPK [40], STAT3-5 [41]), and cell fate transition (e.g. Wnt/β-catenin [42]).

Between the BIF and untreated GF groups, at gene level, we observed elevated levels of Mapk1 (encoding mitogen-activated protein kinase 1), reduced Stat5b (signal transducer and activator of transcription 5B), and unchanged expression of Pkb and Prkaa1 (encoding AKT serine/threonine kinase 1 and protein kinase AMP-activated catalytic subunit alpha 1, respectively) in the fetal brain (Figure 3A). Immunoblotting analysis of PI3K-AKT, MAPK, AMPK and STAT5 signalling pathways (Figure 3B) however revealed increased levels of PI3K-p110β, AMPK and STAT5 proteins in fetal brain lysates of BIF treated compared to untreated pregnant GF mice (Figure 3C). Informed by phosphorylation levels, we also observed enhanced activation of the AKT signalling pathway, indicated by increased threonine 308 residue phosphorylation levels. No differences were observed in the other signalling pathways (Figure 3D). Collectively, these data show that the presence of maternal gut B. breve UCC2003 is associated with the activation of key cellular and metabolic pathways in the fetal brain.Figure 3 Maternal gut B. breve supplementation modifies genes and proteins involved in fetal brain cell metabolism and growth. (A) Relative mRNA levels of genes encoding cell signalling pathways measured in fetal brain samples, expression is relative to two housekeeping genes (Gapdh and Actb). (B–F) Immunoblots and relative protein expression values of proteins involved in cellular metabolism and growth. Protein levels were normalized to total protein (in the case of phosphorylated proteins), actin or tubulin abundance. Data analysed by ANOVA with the group as fixed effect and means comparisons was made by Fisher test (further details in the methods section). Data are means ± SEM with individual datapoints shown. ∗P < 0.05; ∗∗P < 0.01.

Figure 3

2.4 Maternal gut B. breve UCC2003 induces changes in HIF affecting mitochondrial TCA cycle and Wnt-β-catenin signalling in the fetal brain

Molecular pathways like the PI3K-AKT can regulate the stability of hypoxia inducible factors (HIFs) [43]. Moreover HIFs regulate brain development and function even in normoxic conditions [44]. We therefore analysed the mRNA and protein levels of HIF-1α and HIF-2α. Whilst we found no difference in the mRNA levels of Hif1α or Hif2α (Figure 4A), HIF-2α protein abundance was significantly up-regulated in the fetal brain of BIF treated pregnant mice compared to the untreated GF group (no change HIF-1α; Figure 4B). HIF can regulate the tricarboxylic acid (TCA) cycle by repressing mitochondrial function and activating expression of the gene encoding pyruvate dehydrogenase kinase 1 (PDHK1) [45]. We therefore measured the abundance of PDHK1 in fetal brain lysates and found that total protein content was significantly elevated in the BIF treated group compared to the GF (Figure 4C). Moreover, we quantified mitochondrial ATP production capacity by assessing oxidative phosphorylation (OXPHOS) via analysis of OXPHOS complexes. We were able to detect 4 out of the 5 respiratory chain complexes and found that complex-II was significantly elevated in the fetal brain of BIF treated compared to the untreated GF mice. No changes were observed in the levels of other OXPHOS complexes detected (Figure 4D).Figure 4 Maternal gut B. breve supplementation results in stabilization of HIF2α and changes in mitochondrial function and Wnt-β-catenin signalling in the fetal brain. (A) Relative mRNA levels of Hif1α and Hif2α. (B-C) Immunoblots and relative protein expression values of HIF1α, HIF2α and PDHK1 normalized to actin or HSP90 levels. (D) Immunoblot and relative protein abundance of mitochondrial complexes (C) normalized to Ponceau Staining. (E) Immunoblots and relative protein abundance of Frizzled-7 and β-catenin normalized to actin or tubulin levels. (F) Relative mRNA levels of Ascl1. (G) Immunoblot and relative protein abundance of HIRA. All gene expression is relative to two housekeeping genes (Gapdh and Actb). Western blotting performed with 5 fetuses/group from 5 dams per group (different litters). Data analysed by ANOVA with the group as fixed effect and means comparisons was made by Fisher test (further details in the methods section). Data are means ± SEM with individual datapoints shown. ∗P < 0.05; ∗∗P < 0.01.

Figure 4

HIF is also known to activate multiple genes and signalling cascades, including the Wnt and β-catenin pathway [46,47]. We therefore quantified the levels of proteins in these pathways and observed that the abundance of Frizzled-7, a cognate Wnt receptor [48] and total levels of β-catenin protein were significantly elevated in the fetal brain of BIF treated GF mice (Figure 4E). However, the phosphorylated levels of β-catenin at the serine 675 residue, which promotes β-catenin stability and increases β-catenin transcriptional activity [[49], [50], [51], [52]], were unaltered when normalized to total β-catenin (Figure 4E). Moreover, the abundance of Ascl1, a pro-neural transcription factor that also induces the Wnt signalling pathway [53,54] was significantly increased in the brain of fetuses from BIF treated GF mice compared to the untreated GF group (Figure 4F). Finally, the abundance of histone cell cycle regulator (HIRA), a histone chaperone that regulates neurogenesis by enhancing β-catenin expression [55], was enhanced in the fetal brain of GF mice treated with BIF versus untreated GF mice (Figure 4G). Taken together, these results suggest that the presence of B. breve in the maternal gut stabilizes HIF2-α, enhances mitochondrial oxidative phosphorylation capacity and promotes accumulation of Wnt and β-catenin levels in the fetal brain.

3 Discussion

Previous studies have established that the gut microbiome can influence brain function and behaviour [56,57], including a recent murine model study revealing that the maternal gut microbiota can modulate fetal axonogenesis via microbially regulated metabolites [18]. These data suggest that targeting the maternal gut microbiota, such as through probiotic supplementation, may lead to defined and specific changes in brain development across the perinatal period. Numerous species and strains of Bifidobacterium are commonly found in healthy breast-fed infants, as well as being used as probiotics, where they exert a range of beneficial health effects in the host, including protection against inflammatory insults and promoting barrier function and differentiation of intestinal epithelial cells [28,29]. In terms of brain function, prior work has demonstrated that strains of Bifidobacterium, like Bifidobacterium longum 1714™, can modify brain activity by improving stress responses and cognitive function in human and animal models [[58], [59], [60], [61]]. In the current study, we report that oral administration of B. breve UCC2003 to pregnant mice can induce changes in the developing fetal brain, namely through modifying fetal brain metabolism, and genes and signalling pathways involved in cell growth and axonogenesis. Our results have potential translational implications, as around 14% of pregnant women use probiotics during pregnancy in European countries like the Netherlands [62]. However, the utilization of probiotics in clinical and nutritional contexts, particularly during pregnancy, remains a topic of continual debate. Thus, to comprehensively understand their influence on the developing offspring, it is essential to define the mechanisms supporting their beneficial effects.

In this present investigation, our data suggest that B. breve UCC2003 strongly influences fetal brain metabolism. Notably, the fetal brain of the BIF group exhibited a significant reduction in ten specific metabolites. These findings are particularly intriguing, as our previous study did not reveal any changes in the concentrations of these metabolites in the fetal liver or in the placental labyrinth zone, thus highlighting that Bifidobacterium supplementation induces distinct responses in different fetal tissues [32]. Among the metabolites, multiple amino acids were reduced, suggesting that these amino acids, including alanine, leucine and valine may have been used by the fetus. Moreover, other amino acids like arginine or carnitine have previously been reported to be impacted by the maternal microbiota, as shown in studies comparing fetal brains of GF versus specific pathogen free mice [63]. Leucine, which is transported by the Slc7a5 (elevated at gene level in the BIF group) and regulated by MTORC1 [64], has been shown to be regulated by fetal glycaemia, as glucose concentration decreases leucine oxidation independent of insulin [65]. Our previous work showed that administration of B. breve UCC2003 resulted in upregulation of the Slc2a1 transporter in the placental labyrinth zone and in the fetal liver. Moreover, fetal glycaemia was improved in the BIF group and compared to the specific-pathogen-free fetus [32]. Although in this study we did not observe changes in the glucose concentrations in the fetal brain, we found that the abundance of key glucose transporters, specifically, Slc2a1, Slc2a3 and Slc37a1, were increased in the BIF group. Interestingly, the mRNA levels of Slc2a3 and Slc37a1 in the fetal liver were unaltered in the BIF group compared to GF mice [32], highlighting once again the different metabolic responses of the fetus based on tissue type. Future work should explore the role of fetal glycaemia and its impact on the metabolism of amino acids like leucine in the different organs of the fetus.

Another metabolite that was reduced in the BIF group was 3-hydroxybutyrate. This was in line with the lower levels of valine, as 3-hydroxybutyrate serves as a crucial intermediate in the metabolism of branched-chain amino acids like valine [66]. Previous studies indicate that 3-hydroxybutyrate, which can be used as an alternative energy source for the brain and other tissues when glucose becomes scarce [67], is highly involved in mitochondrial function as it can modify mitochondrial membrane permeability transition [68]. Moreover, it is known that neurons treated with 3-hydroxybutyrate exhibit an increase in mitochondrial respiration [68]. We also found two additional metabolites that were reduced in the BIF group, namely carnitine and citrate, which are involved in the tricarboxylic acid (TCA) cycle. Carnitine plays a crucial role in the transport of long-chain fatty acids into the mitochondria [69], whilst citrate is an intermediate of the TCA cycle involved in nicotinamide adenine dinucleotide metabolism [70]. Analysis of mitochondrial electron transfer system (ETS) components revealed that the fetal brains of BIF treated mothers had increased levels of the mitochondrial complex-II. Both citrate and 3-hydroxybutyrate are well known inhibitors of succinate dehydrogenase (mitochondrial complex-II) [71], suggesting a potential link between these results. Moreover, we found that the mRNA levels of the transcription factor Foxm1 and cyclin Cdk1 were reduced in the BIF group. Prior work has shown that Foxm1 and Cdk1 can also control mitochondrial oxygen consumption rates and mitochondrial abundance [72]. Future experiments could be undertaken to understand the potential interplay between these metabolites and the elevated mitochondrial ETS complex II activity, such as using fetal brain explants or cerebral organoids cultured with and without these metabolites, and using high-resolution mitochondrial respirometry analysis. Moreover, aside from in vitro studies to improve our understanding of the mechanisms of action, animal studies using additional control experimental groups are needed [32].

In our study, we did not observe changes in the mRNA levels of well-known genes involved in angiogenesis or apoptosis, such as Vegf, Tp53, Casp3 or Bax. However, we observed reduced mRNA levels of Plxna3. This gene, controlled by the maternal microbiota [18], encodes a class 3 semaphorin receptor that regulates multiple neurodevelopmental processes including axonal growth and guidance [73,74], and neuronal death [75]. Semaphorins have been shown to interact with critical pathways involved in cell proliferation, growth, and apoptosis including the PI3K/AKT pathway [76]. Brains from the BIF group showed activation of the PI3K pathway as evidenced by increased protein levels of PI3K-p110β and elevated phosphorylation levels of AKT. Activation of this signalling pathway has been linked to enhanced dendritic branching, cellular proliferation, neuronal hypertrophy [37]. Moreover, the PI3K/AKT has been implicated in the regulation of MCTs [35,77], which were found to be elevated in the BIF group. In our study it is unclear if the administration of this bacterium induced structural changes in the fetal brain and if so, whether the PI3K/AKT pathway could be driving such changes. Therefore, further experiments using structural/histological analysis are needed in order to determine changes in cell division, cell death, and axon development.

Prior research in mice has demonstrated the crucial role of the HIF-2α in facilitating proper brain formation, neural network development and the migration of neural stem cells [44,78]. Additionally, HIF-2α has been identified as having protective functions for neural stem cells, promoting neurogenesis, and regulating angiogenic and apoptotic processes [79]. In our study, we observed that B. breve UCC2003 administration led to the stabilization of HIF-2α in the fetal brain without inducing changes in HIF-1α protein stabilization. The unaltered state of HIF-1α in this model suggests the absence of intrauterine hypoxic conditions in the BIF group. In this regard, previous research has indicated that HIF-2α, but not HIF-1α, is present in the brains of adult mice under normoxic conditions, and that HIF-1α protein stabilization occurs only under hypoxic conditions [44]. However, other authors suggest that the stability of HIF-2α protein is similar to that of HIF-1α and relies on oxygen-dependent degradation [80]. Following this line of research, in vitro work using neonatal rat neuronal cultures has demonstrated that hypoxia causes up-regulation of AMPK. This metabolic signalling pathway aside from acting as a metabolic sensor regulating glycolysis, has neuroprotective and pro-apoptotic effects [38]. In our work we found that total levels, but not phosphorylated AMPK, were increased in the BIF group. Moreover, additional pathways that have been associated with HIFs and found dysregulated in this model are the Wnt/β-catenin and STAT5 signalling pathways. Both total protein levels of Wnt/β-catenin and STAT5 were increased and in the case of STAT5, the mRNA levels were decreased, suggesting a potential change in the stability, translational efficiency, and/or protein degradation rates in response to B. breve administration [81]. The activity of Wnt/β-catenin pathway is closely related with low oxygen levels [82] and is critical for the development of brain regions, including the proper formation of the midbrain and anterior hindbrain. Inhibition of this pathway results in brain abnormalities due to changes in cell proliferation and cell death [83,84]. STAT5 is a target gene of HIF-2α in haematopoietic stem cells [85] and is particularly important in the forebrain region development and axon guidance [86]. Moreover, we found that PDHK1 was significantly elevated in the BIF group. It is known that HIF can upregulate the expression of PDHK1, which indeed was found elevated in the fetal brain of the BIF group. This adaptive mechanism is used to sustain energy production by inhibiting the conversion of pyruvate to acetyl-CoA, and promoting glycolysis over oxidative phosphorylation [87]. Other studies have found that HIF-2α protein is inhibited or knocked down, the expression of Ascl1 (which was increased in the BIF group) is enhanced; resulting in the induction of sympathetic nervous system differentiation marker genes [88]. Taken together, our data suggest that oxygen levels may be altered in fetuses exposed to B. breve UCC2003 when compared to GF untreated mice. Although we could not measure oxygen levels at the tissue level in this model to verify whether the fetuses were hypoxic or not, we can also speculate another potential mechanism linked to the HIF-2α protein stabilization that may relate to changes in the transport region of the placenta [32]. In this regard, we found that B. breve UCC2003 oral administration resulted in a thinner placental barrier thickness, and this reduction would be expected to aid in the diffusion of oxygen from the mother to the fetus [89]. Therefore, it is less likely that the fetuses of BIF pregnant mice are hypoxic, but further work is certainly required.

While the exact mechanisms linking B. breve in the maternal gut and the fetal brain are complex and likely multifaceted, we propose several potential mechanisms. Changes may be initiated by short-chain fatty acids, such as acetate, produced by B. breve, as these can affect the vagus nerve, are transported across the placenta and are known to alter blood-brain barrier permeability in both the mother and fetus [[90], [91], [92], [93], [94]]. Another possible mechanism is through the release of bacterial extracellular vesicles (BEVs), which have been linked to changes in host immunity [95]. However, the direct contribution of B. breve liberated short-chain fatty acids and BEVs to placental and fetal growth, let alone the developing fetal brain, has yet to be described. As exposed previously [32], our findings suggest that changes in fetal development could be related to alterations in the structure and function of transport labyrinth region of the placenta in B. breve supplemented GF mice. However, we have not explored whether B. breve could also alter the endocrine region of the placenta, which is a critical determinant of fetal development [96,97]. In conclusion, through the use of germ-free mice as a proof of concept, our previous and current studies have underscored the pivotal role of gut microbes, specifically, B. breve UCC2003, in the control of metabolic and cellular pathways in the placenta and in the fetal liver and fetal brain (summarized in Figure 5). While the precise mechanisms governing the alterations induced by B. breve UCC2003 necessitate further exploration, our study provides clear evidence that maternal oral intake of probiotics can influence fetal organogenesis.Figure 5 Summary Figure illustrating changes induced by Maternal gut B. breve supplementation. The illustration summarizes the most relevant results obtained from our previous study [32] and the new study comparing GF versus GF treated with B. breve during development. C-II refers to the mitochondrial complex-II OXPHOS. Abbreviations: p (phosphorylated protein levels), t (total protein levels).

Figure 5

4 Limitations of the study

While we have identified changes in gene and protein expression, how these relate to the structural organization of the fetal brain following maternal administration of B. breve UCC2003 remains unclear. Additionally, to fully understand the molecular changes induced in the fetal brain by maternal supplementation of B. breve, unbiased analyses like RNA-sequencing and/or phospho-proteomics are needed. As the brain is very heterogenous in nature, with functionally specialised regions under discrete developmental control, future research should employ single cell and spatial techniques to elucidate how distinct areas and cell types in the fetal brain respond to maternal B. breve supplementation [98]. It is noteworthy that, although samples analysed represented both fetal sexes (see Table S4), our study is under-powered to determine whether males and females varied in their response to maternal BIF treatment. This is an important area for future research, given that previous work has identified genes can be sexually-dimorphically expressed in the developing brain even prior to gonadal hormone production [99]. Additionally, the inclusion of specific-pathogen-free mice in the study, as we did previously [32], would have provided valuable information to comprehend the direction of the metabolic changes observed in the BIF group. This is important to highlight as the exposure to a microbial challenge in the GF mouse could have also resulted in elevated immune responses. In this regard, it is plausible that the metabolite levels in the BIF group could be within normal ranges for the gestational day and for fetal brain development of the specific-pathogen-free mice (rather than the metabolites being used up more in the BIF treated compared to untreated GF mice). Conversely, untreated GF fetuses might increase the abundance of these metabolites as a mechanism to preserve fetal brain development and maturation in response to growth restriction. For instance, in growth-restricted piglets, there is an increased activity of aromatic amino acid decarboxylase [100]. Additionally, the amino acid alanine, which our study found to be altered, is elevated in rats experiencing fetal growth restriction [101]. A more sensitive mass spectrometry method may also allow neurotransmitter homologues like GABA to be assessed. This is relevant as there are some data that indicate Bifidobacterium can produce these and would be relevant for interpreting our findings [102]. Furthermore, the lack of postnatal investigations, including behavioural studies, has constrained our ability to ascertain potential implications of B. breve UCC2003 on both short- and long-term neurocognitive outcomes in offspring. In this regard, previous studies have shown that the microbiota can affect host behaviour [57]. Interestingly, another Bifidobacterium strain, B. longum NCC3001, has been shown to normalize anxiety-like behaviour in a mouse model of chronic colitis by activating vagus-nerve pathways at the level of the enteric nervous system, reinforcing the concept of a gut–brain communication axis [103]. Considering the changes that we have found in utero, future work should explore the potential fetal programming effects on the autonomic nervous system (enteric nervous system and vagus nerve), the neuroendocrine system and the hypothalamic–pituitary–adrenal (HPA) axis. Studies are also needed to monitor the growth and behaviour of offspring into adult-life.

5 Materials and methods

5.1 Bacterial strain, growing conditions and lyophilization

B. breve UCC2003 generation/growing conditions were previously described [32]. Briefly, B. breve was grown in De Man, Rogosa and Sharpe agar (MRS) under anaerobic conditions overnight. Bacterial cell pellet was resuspended in 10% milk powder and lyophilised in 200 ml volume. Lyophilised B. breve was reconstituted with 500 μl PBS. Concentration of B. breve was 1010 CFU/ml and all batches were tested for contamination upon reconstitution on Luria-Bertani (LB) and Brain-Heart Infusion (BHI) plates under anaerobic and aerobic conditions at 37 °C.

5.2 Animal work ethics statement

All animal work was conducted at the University of East Anglia (UK) under the UK Regulation of Animals (Scientific Procedures) Act of 1986 and approved by the UK Home Office and the UEA Ethical Review Committee (project license PDADA1B0C).

5.3 Animal model and experimental design

The animal model employed for this study was previously described [32] and the experimental design is depicted in Figure S1. Briefly, GF mice were housed under a 12:12 h light/dark with free access to food and water. GF-C57BL/6J mice were time mated and on gestational day (GD) GD9.5, pregnant GF mice were transferred to individually ventilated cages. Treatment with the probiotic started on GD10 (+2 extra doses on GD12 and GD14) by providing 100 μL of reconstituted lyophilised B. breve UCC2003 or 100 μL vehicle control (PBS, 4 % skimmed milk powder) by oral gavage. The group that received the three doses of B. breve UCC2003 received the name of BIF group (n = 6 pregnant mice), and the other cohort of mice (treated with vehicle solution) was named as GF group (n = 5 pregnant mice). As previously described, our probiotic was formulated in a similar fashion to commercial probiotics preparations. Moreover, the time frame was selected to reflect a potential frame in which women would take probiotics once their pregnancy is confirmed. The colonisation levels of B. breve in pregnant mice can be found in our previous publication [32].

5.4 RNA extraction

Fetal brain RNA was extracted from 1 to 3 fetuses randomly selected per litter with RNeasy plus Mini Kits (Qiagen) as previously described [96]. Reverse transcription was performed using the cDNA reverse transcription kit (Applied Biosystems) following manufacturer's instructions. Samples were analysed with a StepOne real-time PCR machine (ThermoFisher) in duplicates using SYBR Green qPCR master mix (Applied Biosystems, ThermoFisher). Gene expression was normalized to the geometric mean expression of two reference genes, Actb and Gapdh. Analysis was performed using the 2-ΔΔCt method [104] and the primer sequences used can be found in Table S2.

5.5 Protein extraction and western blotting assays

Brain protein extraction and Western blotting were performed as previously described [96]. Briefly, brain samples (1 fetus randomly selected per litter) were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (R0278-50M, Sigma Aldrich) supplemented with protease inhibitor cocktail mix (11836170001, Roche), 1 mM β-glycerophosphate (G-9891, Sigma Aldrich) and 1 mM sodium orthovanadate (S65089891, Sigma Aldrich). After protein quantification with BCA protein assay kit (23,225, ThermoFisher), samples were mixed with SDS gel loading buffer (L-4390, Sigma Aldrich) and protein denaturalization performed at 90 °C for 5 min. After electrophoresis, membranes were blocked with 5% fetal bovine serum (A2153-100G, Sigma Aldrich) or semi-skimmed milk (Marvel) and incubated overnight with primary antibodies described in Table S3. The day after, membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) (1:10,000 NA934 or NA931, Amersham) and exposed to ECL substrate (SuperSignal West Femto, ThermoFisher) for chemioluminiscence detection. Images were taken with the iBright instrument (ThermoFisher) Pixel intensity of protein bands was analysed with ImageJ software.

5.6 Metabolite extraction and nuclear magnetic resonance (NMR) spectroscopy

Fetal brain metabolites were extracted as described elsewhere [32]. Briefly, frozen tissue (∼14 mg) from 1 fetus randomly selected per litter was mixed with 200 μL of ice-cold methanol (Fisher Scientific) and 42.5 μL of ultra-pure cold water. Samples were vortexed and tissue was disrupted with a a tissue lyser (Qiagen) and ∼ 15–20 glass beads (Merck) for for 2 × 2 min. Subsequently, 100 μL of ice-cold chloroform (Merck) and 100 μL of ultra-pure cold water were added to the mixture. Samples were incubated on ice for 15 min and then transferred into sterile microcentrifuge tubes and centrifuged for 3 min at 17,000×g. The aqueous phase was transferred into new tubes and speed-vacuumed for 30 min at 50 °C and 30 min without heating prior to reconstitution with phosphate buffer solution at 600 μL. Samples were subjected to NMR spectroscopy. The 1H NMR spectra were recorded at 600 MHz on a Bruker AVANCE spectrometer (Bruker BioSpin GmbH, Germany) running Topspin 2.0 software. Fetal brain metabolites were quantified with the software Chenomx® NMR Suite 7.0™.

5.7 Fetal sex

Fetal sex, determined by the detection of the Sry gene, was performed retrospectively on fetal tissue incubated with lysis buffer (composed with KCl, 1M Tris-HCl, 1M MgCl2, Gelatin, Tween-20, Nonidet P-40 and Proteinase K). Then, samples were mixed with Taq Ready PCR system (Sigma), specific primers (Sry: FPrimer: 5′-GTGGGTTCCTGTCCCACTGC-3′, RPrimer: 5′-GGCCATGTCAAGCGCCCCAT-3′ and PCR autosomal gene control: FPrimer: 5′-TGGTTGGCATTTTATCCCTAGAAC-3′, RPrimer: 5′-GCAACATGGCAACTGGAAACA-3′). Samples were run in agarose gel electrophoresis.

5.8 Statistical analysis

All statistical analyses and sample sizes are shown in each figure legend. Only samples from viable fetuses were analysed. Statistical analysis was performed with GraphPad Prism software (GraphPad v9, San Diego, CA), SAS/STAT 9.0 (Statistical System Institute Inc. Cary, NC, USA) and Microsoft Excel (v2010). Identification and removal of outliers was performed with the ROUT method [105]. For parameters involving only one fetus per litter (Western blot data and metabolomics), a one-way ANOVA was employed with group as a fixed effect and means comparisons were made using the Fisher test (general linear model-GLM model). Conversely, parameters involving more than one fetus per litter (qPCR) were analysed using a one-way ANOVA with the group as fixed effect and each fetus treated as a repeated measure, employing the Fisher test for means comparisons (linear mixed model - MIXED model). In the MIXED model, fetuses coming from the same litter were nested. In both statistical analyses, litter size served as a covariate. The significance threshold for all statistical tests used in this study was set at p < 0.05. Figures in the manuscript show mean ± SEM alongside individual data points (raw data). However, the reported mean ± SEM bars have been adjusted for repeated measures and/or litter size. Data were graphed in GraphPad and figure panels were merged with Adobe Illustrator to display corrected mean ± SEM and individual dots.

Funding

This work was supported by (JL-T) Sir Henry 10.13039/100010269 Wellcome Postdoctoral Fellowship (220456/Z/20/Z ), 10.13039/501100000288 Newton International Fellowship from the Royal Society (NF170988/RG90199 ) and Attraction of Talent Grant from the Community of Madrid (grant No. 2023-T1/SAL-GL-28960 , CESAR NOMBELA fellowship). L.J.H. is supported by Wellcome Trust Investigator Award 220876/Z/20/Z ; the 10.13039/501100000268 Biotechnology and Biological Sciences Research Council (BBSRC) , Institute Strategic Programme Gut Microbes and Health BB/R012490/1 , and its constituent projects BBS/E/F/000PR10353 and BBS/E/F/000PR10356 , and the BBSRC Institute Strategic Programme Food Microbiome and Health BB/X011054/1 and its constituent project BBS/E/F/000PR13631 . ANS-P is supported by a 10.13039/501100001255 Lister Institute of Preventative Medicine Research Prize (RG93692 ). DvS is a member of the 10.13039/501100014745 APC Microbiome Ireland research centre funded by 10.13039/501100001602 Science Foundation Ireland (SFI) through the Irish Government’s National Development Plan (Grant numbers SFI/12/RC/2273a and SFI/12/RC/2273b ).

CRediT authorship contribution statement

Jorge Lopez-Tello: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Raymond Kiu: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. Zoe Schofield: Writing – review & editing, Methodology, Investigation, Data curation. Cindy X.W. Zhang: Writing – review & editing, Investigation. Douwe van Sinderen: Writing – review & editing, Resources. Gwénaëlle Le Gall: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. Lindsay J. Hall: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization. Amanda N. Sferruzzi-Perri: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

Appendix A Supplementary data

The following are the Supplementary data to this article:figs1 figs1

Table S1

List of metabolites analysed in fetal brains. Data analysed by one-way ANOVA, with the group as fixed effect and means comparisons made by Fisher test (general linear model-GLM model). Litter size added as a covariate. Data displayed as mean ± SEM. Values were considered statistically significant with P < 0.05. Additional metabolites can be found in Table 1.

Table S1

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Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.molmet.2024.102004.
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References

1 Malhotra A. Allison B.J. Castillo-Melendez M. Jenkin G. Polglase G.R. Miller S.L. Neonatal morbidities of fetal growth restriction: pathophysiology and impact Front Endocrinol 10 2019
2 Luna R.L. Kay V.R. Rätsep M.T. Khalaj K. Bidarimath M. Peterson N. Placental growth factor deficiency is associated with impaired cerebral vascular development in mice Mol Hum Reprod 22 2 2016 130 142 10.1093/molehr/gav069 26646502
3 López-Tello J. Arias-Alvarez M. Jimenez-Martinez M.A. Garcia-Garcia R.M. Rodriguez M. Lorenzo Gonzalez P.L. Competition for materno-fetal resource partitioning in a rabbit model of undernourished pregnancy PLoS One 12 1 2017 e0169194 10.1371/journal.pone.0169194
4 López-Tello J. Arias-Álvarez M. Jiménez-Martínez M.-Á. Barbero-Fernández A. García-García R.M. Rodríguez M. The effects of sildenafil citrate on feto-placental development and haemodynamics in a rabbit model of intrauterine growth restriction Reprod Fertil Dev 29 6 2017 1239 1248 10.1071/RD15330 27209378
5 Brown L.D. Hay W.W. Impact of placental insufficiency on fetal skeletal muscle growth Mol Cell Endocrinol 435 2016 69 77 10.1016/j.mce.2016.03.017 26994511
6 Camm E.J. Botting K.J. Sferruzzi-Perri A.N. Near to one's heart: the intimate relationship between the placenta and fetal heart Front Physiol 9 2018 629 10.3389/fphys.2018.00629 29997513
7 Hinchliffe S.A. Sargent P.H. Howard C.V. Chan Y.F. van Velzen D. Human intrauterine renal growth expressed in absolute number of glomeruli assessed by the disector method and Cavalieri principle Laboratory Investigation; a Journal of Technical Methods and Pathology 64 6 1991 777 784 2046329
8 Saha K. Shahida S.M. Chowdhury N.I. Mostafa G. Saha S.K. Jahan S. Relationship between estimated foetal weight and renal volume in intra uterine growth retarded foetus in Bangladeshi women Mymensingh Med J: Md Med J 23 4 2014 752 757
9 Gilchrist C.P. Cumberland A.L. Kondos-Devcic D. Hill R.A. Khore M. Quezada S. Hippocampal neurogenesis and memory in adolescence following intrauterine growth restriction Hippocampus 31 3 2021 321 334 10.1002/hipo.23291 33320965
10 Miller S.L. Huppi P.S. Mallard C. The consequences of fetal growth restriction on brain structure and neurodevelopmental outcome J Physiol 594 4 2016 807 823 10.1113/JP271402 26607046
11 Benítez-Marín M.J. Marín-Clavijo J. Blanco-Elena J.A. Jiménez-López J. González-Mesa E. Brain sparing effect on neurodevelopment in children with intrauterine growth restriction: a systematic review Children 8 9 2021 745 10.3390/children8090745 34572177
12 Mikaelsson M.A. Constância M. Dent C.L. Wilkinson L.S. Humby T. Placental programming of anxiety in adulthood revealed by Igf2-null models Nat Commun 4 2013 2311 10.1038/ncomms3311 23921428
13 Vossbeck S. de Camargo O.K. Grab D. Bode H. Pohlandt F. Neonatal and neurodevelopmental outcome in infants born before 30 weeks of gestation with absent or reversed end-diastolic flow velocities in the umbilical artery Eur J Pediatr 160 2 2001 128 134 10.1007/s004310000680 11271385
14 Morsing E. Asard M. Ley D. Stjernqvist K. Marsál K. Cognitive function after intrauterine growth restriction and very preterm birth Pediatrics 127 4 2011 e874 e882 10.1542/peds.2010-1821 21382944
15 Guellec I. Lapillonne A. Renolleau S. Charlaluk M.-L. Roze J.-C. Marret S. Neurologic outcomes at school age in very preterm infants born with severe or mild growth restriction Pediatrics 127 4 2011 e883 e891 10.1542/peds.2010-2442 21382951
16 Bettiol A. Lombardi N. Crescioli G. Avagliano L. Mugelli A. Ravaldi C. Pharmacological interventions for the prevention of fetal growth restriction: protocol for a systematic review and network meta-analysis BMJ Open 9 7 2019 e029467 10.1136/bmjopen-2019-029467
17 Pels A. Derks J. Elvan-Taspinar A. van Drongelen J. de Boer M. Duvekot H. Maternal sildenafil vs placebo in pregnant women with severe early-onset fetal growth restriction: a randomized clinical trial JAMA Netw Open 3 6 2020 e205323 10.1001/jamanetworkopen.2020.5323
18 He V. Gn P. Dw W. Ejl C. El S. A Q. The maternal microbiome modulates fetal neurodevelopment in mice Nature 586 7828 2020 10.1038/s41586-020-2745-3
19 O'Toole P.W. Jeffery I.B. Gut microbiota and aging Science (New York, N.Y.) 350 6265 2015 1214 1215 10.1126/science.aac8469 26785481
20 Bárcena C. Valdés-Mas R. Mayoral P. Garabaya C. Durand S. Rodríguez F. Healthspan and lifespan extension by fecal microbiota transplantation into progeroid mice Nat Med 25 8 2019 1234 1242 10.1038/s41591-019-0504-5 31332389
21 Sampson T.R. Debelius J.W. Thron T. Janssen S. Shastri G.G. Ilhan Z.E. Gut microbiota regulate motor deficits and neuroinflammation in a model of Parkinson's disease Cell 167 6 2016 1469 1480.e12 10.1016/j.cell.2016.11.018 27912057
22 Zhu F. Guo R. Wang W. Ju Y. Wang Q. Ma Q. Transplantation of microbiota from drug-free patients with schizophrenia causes schizophrenia-like abnormal behaviors and dysregulated kynurenine metabolism in mice Mol Psychiatr 25 11 2020 2905 2918 10.1038/s41380-019-0475-4
23 Gurung M. Li Z. You H. Rodrigues R. Jump D.B. Morgun A. Role of gut microbiota in type 2 diabetes pathophysiology EBioMedicine 51 2020 102590 10.1016/j.ebiom.2019.11.051
24 Koren O. Goodrich J.K. Cullender T.C. Spor A. Laitinen K. Bäckhed H.K. Host remodeling of the gut microbiome and metabolic changes during pregnancy Cell 150 3 2012 470 480 10.1016/j.cell.2012.07.008 22863002
25 Huang L. Cai M. Li L. Zhang X. Xu Y. Xiao J. Gut microbiota changes in preeclampsia, abnormal placental growth and healthy pregnant women BMC Microbiol 21 2021 265 10.1186/s12866-021-02327-7 34607559
26 Miao T. Yu Y. Sun J. Ma A. Yu J. Cui M. Decrease in abundance of bacteria of the genus Bifidobacterium in gut microbiota may be related to pre-eclampsia progression in women from East China Food Nutr Res 2021 10.29219/fnr.v65.5781
27 Nuriel-Ohayon M. Neuman H. Ziv O. Belogolovski A. Barsheshet Y. Bloch N. Progesterone increases Bifidobacterium relative abundance during late pregnancy Cell Rep 27 3 2019 730 736.e3 10.1016/j.celrep.2019.03.075 30995472
28 Kiu R. Treveil A. Harnisch L.C. Caim S. Leclaire C. van Sinderen D. Bifidobacterium breve UCC2003 induces a distinct global transcriptomic program in neonatal murine intestinal epithelial cells iScience 23 7 2020 101336 10.1016/j.isci.2020.101336
29 Hughes K.R. Harnisch L.C. Alcon-Giner C. Mitra S. Wright C.J. Ketskemety J. Bifidobacterium breve reduces apoptotic epithelial cell shedding in an exopolysaccharide and MyD88-dependent manner Open Biology 7 1 2017 160155 10.1098/rsob.160155
30 Hidalgo-Cantabrana C. Delgado S. Ruiz L. Ruas-Madiedo P. Sánchez B. Margolles A. Bifidobacteria and their health-promoting effects Microbiol Spectr 5 3 2017 10.1128/microbiolspec.BAD-0010-2016
31 O'Callaghan A. van Sinderen D. Bifidobacteria and their role as members of the human gut microbiota Front Microbiol 7 2016 925 10.3389/fmicb.2016.00925 27379055
32 Lopez-Tello J. Schofield Z. Kiu R. Dalby M.J. van Sinderen D. Le Gall G. Maternal gut microbiota Bifidobacterium promotes placental morphogenesis, nutrient transport and fetal growth in mice Cell Mol Life Sci: CM 79 7 2022 386 10.1007/s00018-022-04379-y
33 Vannucci R.C. Vannucci S.J. Glucose metabolism in the developing brain Semin Perinatol 24 2 2000 107 115 10.1053/sp.2000.6361 10805166
34 Pan C.-J. Chen S.-Y. Lee S. Chou J.Y. Structure-function study of the glucose-6-phosphate transporter, an eukaryotic antiporter deficient in glycogen storage disease type Ib Mol Genet Metabol 96 1 2009 32 37 10.1016/j.ymgme.2008.10.005
35 Pérez-Escuredo J. Van Hée V.F. Sboarina M. Falces J. Payen V.L. Pellerin L. Monocarboxylate transporters in the brain and in cancer Biochim Biophys Acta 1863 10 2016 2481 2497 10.1016/j.bbamcr.2016.03.013 26993058
36 Chong Z.Z. Li F. Maiese K. Activating Akt and the brain's resources to drive cellular survival and prevent inflammatory injury Histol Histopathol 20 1 2005 299 315 10.14670/HH-20.299 15578447
37 Wang L. Zhou K. Fu Z. Yu D. Huang H. Zang X. Brain development and akt signaling: the crossroads of signaling pathway and neurodevelopmental diseases J Mol Neurosci 61 3 2017 379 384 10.1007/s12031-016-0872-y 28025777
38 Muraleedharan R. Dasgupta B. AMPK in the brain: its roles in glucose and neural metabolism FEBS J 289 8 2022 2247 2262 10.1111/febs.16151 34355526
39 Kim I.-J. Drahushuk K.M. Kim W.-Y. Gonsiorek E.A. Lein P. Andres D.A. Extracellular signal-regulated kinases regulate dendritic growth in rat sympathetic neurons J Neurosci 24 13 2004 3304 3312 10.1523/JNEUROSCI.3286-03.2004 15056710
40 Ramamurthy S. Chang E. Cao Y. Zhu J. Ronnett G.V. AMPK activation regulates neuronal structure in developing hippocampal neurons Neuroscience 259 2014 13 24 10.1016/j.neuroscience.2013.11.048 24295634
41 Wang T. Yuan W. Liu Y. Zhang Y. Wang Z. Zhou X. The role of the JAK-STAT pathway in neural stem cells, neural progenitor cells and reactive astrocytes after spinal cord injury Biomedical Reports 3 2 2015 141 146 10.3892/br.2014.401 25798237
42 Eze U.C. Bhaduri A. Haeussler M. Nowakowski T.J. Kriegstein A.R. Single-cell atlas of early human brain development highlights heterogeneity of human neuroepithelial cells and early radial glia Nat Neurosci 24 4 2021 584 594 10.1038/s41593-020-00794-1 33723434
43 Joshi S. Singh A.R. Zulcic M. Durden D.L. A macrophage-dominant PI3K isoform controls hypoxia-induced HIF1α and HIF2α stability and tumor growth, angiogenesis, and metastasis Mol Cancer Res: MCR 12 10 2014 1520 1531 10.1158/1541-7786.MCR-13-0682 25103499
44 Kleszka K. Leu T. Quinting T. Jastrow H. Pechlivanis S. Fandrey J. Hypoxia-inducible factor-2α is crucial for proper brain development Sci Rep 10 2020 19146 10.1038/s41598-020-75838-4
45 Kim J. Tchernyshyov I. Semenza G.L. Dang C.V. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia Cell Metabol 3 3 2006 177 185 10.1016/j.cmet.2006.02.002
46 Dunwoodie S.L. The role of hypoxia in development of the Mammalian embryo Dev Cell 17 6 2009 755 773 10.1016/j.devcel.2009.11.008 20059947
47 Shen X. Li M. Wang C. Liu Z. Wu K. Wang A. Hypoxia is fine-tuned by Hif-1α and regulates mesendoderm differentiation through the Wnt/β-Catenin pathway BMC Biol 20 1 2022 219 10.1186/s12915-022-01423-y 36199093
48 Phesse T. Flanagan D. Vincan E. Frizzled7: a promising achilles' heel for targeting the Wnt receptor complex to treat cancer Cancers 8 5 2016 50 10.3390/cancers8050050 27196929
49 Taurin S. Sandbo N. Qin Y. Browning D. Dulin N.O. Phosphorylation of β-catenin by cyclic AMP-dependent protein kinase J Biol Chem 281 15 2006 9971 9976 10.1074/jbc.M508778200 16476742
50 Hino S. Tanji C. Nakayama K.I. Kikuchi A. Phosphorylation of beta-catenin by cyclic AMP-dependent protein kinase stabilizes beta-catenin through inhibition of its ubiquitination Mol Cell Biol 25 20 2005 9063 9072 10.1128/MCB.25.20.9063-9072.2005 16199882
51 W,van V. Nh L. W H. L B. M T. Er B. β-catenin tyrosine 654 phosphorylation increases Wnt signalling and intestinal tumorigenesis Gut 60 9 2011 10.1136/gut.2010.233460
52 Taurin S. Sandbo N. Yau D.M. Sethakorn N. Dulin N.O. Phosphorylation of beta-catenin by PKA promotes ATP-induced proliferation of vascular smooth muscle cells Am J Physiol Cell Physiol 294 5 2008 C1169 C1174 10.1152/ajpcell.00096.2008 18353896
53 Woods L.M. Ali F.R. Gomez R. Chernukhin I. Marcos D. Parkinson L.M. Elevated ASCL1 activity creates de novo regulatory elements associated with neuronal differentiation BMC Genom 23 1 2022 255 10.1186/s12864-022-08495-8
54 Tenjin Y. Kudoh S. Kubota S. Yamada T. Matsuo A. Sato Y. Ascl1-induced Wnt11 regulates neuroendocrine differentiation, cell proliferation, and E-cadherin expression in small-cell lung cancer and Wnt11 regulates small-cell lung cancer biology Laboratory Investigation; a Journal of Technical Methods and Pathology 99 11 2019 1622 1635 10.1038/s41374-019-0277-y 31231131
55 Li Y. Jiao J. Histone chaperone HIRA regulates neural progenitor cell proliferation and neurogenesis via β-catenin J Cell Biol 216 7 2017 1975 1992 10.1083/jcb.201610014 28515277
56 Sherwin E. Bordenstein S.R. Quinn J.L. Dinan T.G. Cryan J.F. Microbiota and the social brain Science (New York, N.Y.) 366 6465 2019 eaar2016 10.1126/science.aar2016
57 Collins S.M. Surette M. Bercik P. The interplay between the intestinal microbiota and the brain Nat Rev Microbiol 10 11 2012 735 742 10.1038/nrmicro2876 23000955
58 Savignac H.M. Kiely B. Dinan T.G. Cryan J.F. Bifidobacteria exert strain-specific effects on stress-related behavior and physiology in BALB/c mice Neuro Gastroenterol Motil 26 11 2014 1615 1627 10.1111/nmo.12427
59 Savignac H.M. Tramullas M. Kiely B. Dinan T.G. Cryan J.F. Bifidobacteria modulate cognitive processes in an anxious mouse strain Behav Brain Res 287 2015 59 72 10.1016/j.bbr.2015.02.044 25794930
60 Allen A.P. Hutch W. Borre Y.E. Kennedy P.J. Temko A. Boylan G. Bifidobacterium longum 1714 as a translational psychobiotic: modulation of stress, electrophysiology and neurocognition in healthy volunteers Transl Psychiatry 6 11 2016 e939 10.1038/tp.2016.191 27801892
61 Wang H. Braun C. Murphy E.F. Enck P. Bifidobacterium longum 1714TM strain modulates brain activity of healthy volunteers during social stress Am J Gastroenterol 114 7 2019 1152 1162 10.14309/ajg.0000000000000203 30998517
62 Rutten N. Van der Gugten A. Uiterwaal C. Vlieger A. Rijkers G. Van der Ent K. Maternal use of probiotics during pregnancy and effects on their offspring's health in an unselected population Eur J Pediatr 175 2 2016 229 235 10.1007/s00431-015-2618-1 26319129
63 Pessa-Morikawa T. Husso A. Kärkkäinen O. Koistinen V. Hanhineva K. Iivanainen A. Maternal microbiota-derived metabolic profile in fetal murine intestine, brain and placenta BMC Microbiol 22 1 2022 46 10.1186/s12866-022-02457-6 35130835
64 Torigoe M. Maeshima K. Ozaki T. Omura Y. Gotoh K. Tanaka Y. l-Leucine influx through Slc7a5 regulates inflammatory responses of human B cells via mammalian target of rapamycin complex 1 signaling Mod Rheumatol 29 5 2019 885 891 10.1080/14397595.2018.1510822 30092695
65 Liechty E.A. Boyle D.W. Moorehead H. Liu Y.M. Denne S.C. Increased fetal glucose concentration decreases ovine fetal leucine oxidation independent of insulin Am J Physiol 265 4 Pt 1 1993 E617 E623 10.1152/ajpendo.1993.265.4.E617 7901997
66 Xu M. Che L. Niu L. Wang L. Li M. Jiang D. Molecular mechanism of valine and its metabolite in improving triglyceride synthesis of porcine intestinal epithelial cells Sci Rep 13 1 2023 2933 10.1038/s41598-023-30036-w 36806358
67 Newman J.C. Verdin E. β-Hydroxybutyrate Annu Rev Nutr 37 2017 51 76 10.1146/annurev-nutr-071816-064916 28826372
68 Kim D.Y. Simeone K.A. Simeone T.A. Pandya J.D. Wilke J.C. Ahn Y. Ketone bodies mediate antiseizure effects through mitochondrial permeability transition Ann Neurol 78 1 2015 77 87 10.1002/ana.24424 25899847
69 Ferreira G.C. McKenna M.C. l-Carnitine and acetyl-l-carnitine roles and neuroprotection in developing brain Neurochem Res 42 6 2017 1661 1675 10.1007/s11064-017-2288-7 28508995
70 Icard P. Coquerel A. Wu Z. Gligorov J. Fuks D. Fournel L. Understanding the central role of citrate in the metabolism of cancer cells and tumors: an update Int J Mol Sci 22 12 2021 6587 10.3390/ijms22126587 34205414
71 Hillar M. Lott V. Lennox B. Correlation of the effects of citric acid cycle metabolites on succinate oxidation by rat liver mitochondria and submitochondrial particles J Bioenerg 7 1 1975 1 16 10.1007/BF01558459 1176438
72 Kobiita A. Silva P.N. Schmid M.W. Stoffel M. FoxM1 coordinates cell division, protein synthesis, and mitochondrial activity in a subset of β cells during acute metabolic stress Cell Rep 42 8 2023 112986 10.1016/j.celrep.2023.112986
73 Steele J.L. Morrow M.M. Sarnat H.B. Alkhunaizi E. Brandt T. Chitayat D.A. Semaphorin-plexin signaling: from axonal guidance to a new X-linked intellectual disability syndrome Pediatr Neurol 126 2022 65 73 10.1016/j.pediatrneurol.2021.10.008 34740135
74 Sakurai A. Doci C. Gutkind J.S. Semaphorin signaling in angiogenesis, lymphangiogenesis and cancer Cell Res 22 1 2012 23 32 10.1038/cr.2011.198 22157652
75 Ben-Zvi A. Manor O. Schachner M. Yaron A. Tessier-Lavigne M. Behar O. The Semaphorin receptor PlexinA3 mediates neuronal apoptosis during dorsal root ganglia development J Neurosci: The Official Journal of the Society for Neuroscience 28 47 2008 12427 12432 10.1523/JNEUROSCI.3573-08.2008
76 Castro-Rivera E. Ran S. Brekken R.A. Minna J.D. Semaphorin 3B inhibits the phosphatidylinositol 3-kinase/akt pathway through neuropilin-1 in lung and breast cancer cells Cancer Res 68 20 2008 8295 8303 10.1158/0008-5472.CAN-07-6601 18922901
77 Zhang P. Ma J. Gao J. Liu F. Sun X. Fang F. Downregulation of monocarboxylate transporter 1 inhibits the invasion and migration through suppression of the PI3K/Akt signaling pathway in human nasopharyngeal carcinoma cells J Bioenerg Biomembr 50 4 2018 271 281 10.1007/s10863-018-9763-y 29882205
78 Leu T. Fandrey J. Schreiber T. (H)IF applicable: promotion of neurogenesis by induced HIF-2 signalling after ischaemia Pflueg Arch Eur J Physiol 473 8 2021 1287 1299 10.1007/s00424-021-02600-8
79 Lopez-Barneo J. Pardal R. Ortega-Sáenz P. Cellular mechanism of oxygen sensing Annu Rev Physiol 63 2001 259 287 10.1146/annurev.physiol.63.1.259 11181957
80 Loboda A. Jozkowicz A. Dulak J. HIF-1 and HIF-2 transcription factors--similar but not identical Mol Cell 29 5 2010 435 442 10.1007/s10059-010-0067-2
81 Koussounadis A. Langdon S.P. Um I.H. Harrison D.J. Smith V.A. Relationship between differentially expressed mRNA and mRNA-protein correlations in a xenograft model system Sci Rep 5 2015 10775 10.1038/srep10775
82 Mazumdar J. O'Brien W.T. Johnson R.S. LaManna J.C. Chavez J.C. Klein P.S. O2 regulates stem cells through Wnt/β-catenin signalling Nat Cell Biol 12 10 2010 1007 1013 10.1038/ncb2102 20852629
83 Brafman D. Willert K. Wnt/β-catenin signaling during early vertebrate neural development Developmental Neurobiology 77 11 2017 1239 1259 10.1002/dneu.22517 28799266
84 Serbedzija G.N. Dickinson M. McMahon A.P. Cell death in the CNS of the Wnt-1 mutant mouse J Neurobiol 31 3 1996 275 282 10.1002/(SICI)1097-4695(199611)31:3<275::AID-NEU1>3.0.CO;2-7 8910786
85 Fatrai S. Wierenga A.T.J. Daenen S.M.G.J. Vellenga E. Schuringa J.J. Identification of HIF2α as an important STAT5 target gene in human hematopoietic stem cells Blood 117 12 2011 3320 3330 10.1182/blood-2010-08-303669 21263150
86 Markham K. Schuurmans C. Weiss S. STAT5A/B activity is required in the developing forebrain and spinal cord Mol Cell Neurosci 35 2 2007 272 282 10.1016/j.mcn.2007.03.001 17462911
87 Erdem A. Marin S. Pereira-Martins D.A. Cortés R. Cunningham A. Pruis M.G. The Glycolytic Gatekeeper PDK1 defines different metabolic states between genetically distinct subtypes of human acute myeloid leukemia Nat Commun 13 2022 1105 10.1038/s41467-022-28737-3 35232995
88 Pietras A. Hansford L.M. Johnsson A.S. Bridges E. Sjölund J. Gisselsson D. HIF-2α maintains an undifferentiated state in neural crest-like human neuroblastoma tumor-initiating cells Proc Natl Acad Sci USA 106 39 2009 16805 16810 10.1073/pnas.0904606106 19805377
89 Woods L. Perez-Garcia V. Hemberger M. Regulation of placental development and its impact on fetal growth—new insights from mouse models Front Endocrinol 9 2018 10.3389/fendo.2018.00570
90 Braniste V. Al-Asmakh M. Kowal C. Anuar F. Abbaspour A. Tóth M. The gut microbiota influences blood-brain barrier permeability in mice Sci Transl Med 6 263 2014 263ra158 10.1126/scitranslmed.3009759
91 O'Riordan K.J. Collins M.K. Moloney G.M. Knox E.G. Aburto M.R. Fülling C. Short chain fatty acids: microbial metabolites for gut-brain axis signalling Mol Cell Endocrinol 546 2022 111572 10.1016/j.mce.2022.111572
92 Duttaroy A.K. Transport of fatty acids across the human placenta: a review Prog Lipid Res 48 1 2009 52 61 10.1016/j.plipres.2008.11.001 19041341
93 Ziętek M. Celewicz Z. Szczuko M. Short-chain fatty acids, maternal microbiota and metabolism in pregnancy Nutrients 13 4 2021 1244 10.3390/nu13041244 33918804
94 Usta-Gorgun B. Yilmaz-Ersan L. Short-chain fatty acids production by Bifidobacterium species in the presence of salep Electron J Biotechnol 47 2020 29 35 10.1016/j.ejbt.2020.06.004
95 Chronopoulos A. Kalluri R. Emerging role of bacterial extracellular vesicles in cancer Oncogene 39 46 2020 6951 6960 10.1038/s41388-020-01509-3 33060855
96 Lopez-Tello J. Sferruzzi-Perri A.N. Characterization of placental endocrine function and fetal brain development in a mouse model of small for gestational age Front Endocrinol 14 2023
97 Lopez-Tello J. Yong H.E.J. Sandovici I. Dowsett G.K.C. Christoforou E.R. Salazar-Petres E. Fetal manipulation of maternal metabolism is a critical function of the imprinted Igf2 gene Cell Metabol 35 7 2023 1195 1208.e6 10.1016/j.cmet.2023.06.007
98 La Manno G. Siletti K. Furlan A. Gyllborg D. Vinsland E. Mossi Albiach A. Molecular architecture of the developing mouse brain Nature 596 7870 2021 92 96 10.1038/s41586-021-03775-x 34321664
99 Dewing P. Shi T. Horvath S. Vilain E. Sexually dimorphic gene expression in mouse brain precedes gonadal differentiation Brain Research. Molecular Brain Research 118 1–2 2003 82 90 10.1016/s0169-328x(03)00339-5 14559357
100 Bauer R. Walter B. Vorwieger G. Bergmann R. Füchtner F. Brust P. Intrauterine growth restriction induces up-regulation of cerebral aromatic amino acid decarboxylase activity in newborn piglets: [18F]fluorodopa positron emission tomographic study Pediatr Res 49 4 2001 474 480 10.1203/00006450-200104000-00007 11264429
101 Chanez C. Rabin O. Heroux M. Giguere J.F. Cerebral amino acid changes in an animal model of intrauterine growth retardation Metab Brain Dis 8 1 1993 61 72 10.1007/BF01000530 8492785
102 Duranti S. Ruiz L. Lugli G.A. Tames H. Milani C. Mancabelli L. Bifidobacterium adolescentis as a key member of the human gut microbiota in the production of GABA Sci Rep 10 1 2020 14112 10.1038/s41598-020-70986-z
103 Morais L.H. Schreiber H.L. Mazmanian S.K. The gut microbiota–brain axis in behaviour and brain disorders Nat Rev Microbiol 19 4 2021 241 255 10.1038/s41579-020-00460-0 33093662
104 Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method Methods 25 4 2001 402 408 10.1006/meth.2001.1262 11846609
105 Motulsky H.J. Brown R.E. Detecting outliers when fitting data with nonlinear regression – a new method based on robust nonlinear regression and the false discovery rate BMC Bioinf 7 1 2006 123 10.1186/1471-2105-7-123
