Paternal microbiome perturbations impact offspring fitness.

The gut microbiota operates at the interface of host-environment interactions to influence human homoeostasis and metabolic networks1-4. Environmental factors that unbalance gut microbial ecosystems can therefore shape physiological and disease-associated responses across somatic tissues5-9. However, the systemic impact of the gut microbiome on the germline-and consequently on the F1 offspring it gives rise to-is unexplored10. Here we show that the gut microbiota act as a key interface between paternal preconception environment and intergenerational health in mice. Perturbations to the gut microbiota of prospective fathers increase the probability of their offspring presenting with low birth weight, severe growth restriction and premature mortality. Transmission of disease risk occurs via the germline and is provoked by pervasive gut microbiome perturbations, including non-absorbable antibiotics or osmotic laxatives, but is rescued by restoring the paternal microbiota before conception. This effect is linked with a dynamic response to induced dysbiosis in the male reproductive system, including impaired leptin signalling, altered testicular metabolite profiles and remapped small RNA payloads in sperm. As a result, dysbiotic fathers trigger an elevated risk of in utero placental insufficiency, revealing a placental origin of mammalian intergenerational effects. Our study defines a regulatory 'gut-germline axis' in males, which is sensitive to environmental exposures and programmes offspring fitness through impacting placenta function.

Effects of quercetin on Aroclor 1254-induced expression of CYP450 and cytokines in pregnant rats.

The present study aimed to investigate the protective effect of quercetin on polychlorinated biphenyls (PCB)-induced liver and embryo damage in pregnant Sprague-Dawley rats. Pregnant rats were divided into five groups, and then were orally gavaged daily with peanut oil (vehicle) or a commercial PCB mixture (Aroclor 1254) - with or without co-treatment with 75, 150, or 300 mg/kg quercetin - on gestation days (GD) 4-7. At GD 9, all rats were euthanized, and their blood, liver, and uterus were collected. Expressions of CYP450 mRNA and protein in liver, cytokines (IFNγ, IL-2, IL-4, and IL-6) and IFNγ/IL-4 ratios in liver and sera, liver morphology, and the status of implanted embryos were analyzed. The results showed Aroclor 1254 treatment alone caused hepatic cord damage (i.e. cell disorganization, swelling, decreased cytoplasm, vacuolization), and that quercetin co-treatment appeared to mitigate this damage. Similarly, levels of CYP1A1 and CYP2B1 mRNA in livers of Aroclor 1254-only-treated rats were significantly higher than those in rats co-treated with quercetin. Hepatic and sera levels of IFNγ, IL-2, IL-6, and IFNγ/IL-4 ratios, and the ratio of delayed-development embryos, all increased in Aroclor 1254-treated rats, but were relatively decreased as a result of quercetin co-treatments. IL-4 levels were decreased by Aroclor 1254 and tended to increase back to normal when quercetin was used. The results indicated that quercetin imparted a protective effect against Aroclor 1254-induced toxicity in pregnant rats, in part, by modulating levels of important pro-inflammatory cytokines and reducing induced CYP1A1 and CYP2B1 expression.

Exosomes derived from umbilical cord mesenchymal stem cells reduce microglia-mediated neuroinflammation in perinatal brain injury.

BACKGROUND: Preterm newborns are at high risk of developing neurodevelopmental deficits caused by neuroinflammation leading to perinatal brain injury. Human Wharton's jelly mesenchymal stem cells (hWJ-MSC) derived from the umbilical cord have been suggested to reduce neuroinflammation, in part through the release of extracellular vesicle-like exosomes. Here, we studied whether exosomes derived from hWJ-MSC have anti-inflammatory effects on microglia-mediated neuroinflammation in perinatal brain injury. METHODS: Using ultracentrifugation, we isolated exosomes from hWJ-MSC culture supernatants. In an in vitro model of neuroinflammation, we stimulated immortalized BV-2 microglia and primary mixed glial cells with lipopolysaccharide (LPS) in the presence or absence of exosomes. In vivo, we introduced brain damage in 3-day-old rat pups and treated them intranasally with hWJ-MSC-derived exosomes. RESULTS: hWJ-MSC-derived exosomes dampened the LPS-induced expression of inflammation-related genes by BV-2 microglia and primary mixed glial cells. The secretion of pro-inflammatory cytokines by LPS-stimulated primary mixed glial was inhibited by exosomes as well. Exosomes interfered within the Toll-like receptor 4 signaling of BV-2 microglia, as they prevented the degradation of the NFκB inhibitor IκBα and the phosphorylation of molecules of the mitogen-activated protein kinase family in response to LPS stimulation. Finally, intranasally administered exosomes reached the brain and reduced microglia-mediated neuroinflammation in rats with perinatal brain injury. CONCLUSIONS: Our data suggest that the administration of hWJ-MSC-derived exosomes represents a promising therapy to prevent and treat perinatal brain injury.

Prenatal exposure to pyrrolizidine alkaloids induced hepatotoxicity and pulmonary injury in fetal rats.

Hepatic and pulmonary toxicity in fetal rats induced by pyrrolizidine alkaloids (PAs) was investigated. Retrorsine (RTS) or monocrotaline (MCT) was intragastrically administered during pregnancy. The reduction of body and tail lengths was consistent with body weight loss in PA-exposed fetuses, and pathological lesions in liver and lung were observed only in fetuses. Both PAs reduced fetal serum transaminase activities. The GSH/GSSG ratio, GSH peroxidase and superoxide dismutase activities also decreased but glutathione S-transferase activity increased in fetal lung, especially for MCT. The pyrrole-protein adducts in fetal liver and lung could be detected, and those adducts in RTS fetal lungs were about 65% of those in MCT group. In conclusion, prenatal PAs exposure induced fetal hepatic and pulmonary toxicities through the generation of pyrrole metabolites and oxidative injury. The difference on fetal pulmonary redox homeostasis between two PAs groups might be associated with the content of PAs migrated to fetal lungs.

Role for Cystathionine γ Lyase (CSE) in an Ethanol (E)-Induced Lesion in Fetal Brain GSH Homeostasis.

Earlier, we reported that gestational ethanol (E) can dysregulate neuron glutathione (GSH) homeostasis partially via impairing the EAAC1-mediated inward transport of Cysteine (Cys) and this can affect fetal brain development. In this study, we investigated if there is a role for the transulfuration pathway (TSP), a critical bio-synthetic point to supply Cys in E-induced dysregulation of GSH homeostasis. These studies utilized an in utero E binge model where the pregnant Sprague⁻Dawley (SD) rat dams received five doses of E at 3.5 g/kg by gastric intubation beginning embryonic day (ED) 17 until ED19 separated by 12 h. The postnatal day 7 (PN7) alcohol model employed an oral dosing of 4 g/kg body weight split into 2 feedings at 2 h interval and an iso-caloric and iso-volumic equivalent maltose-dextrin milk solution served as controls. The in vitro model consisted of cerebral cortical neuron cultures from embryonic day (ED) 16⁻17 fetus from SD rats and differentiated neurons from ED18 rat cerebral cortical neuroblasts. E concentrations were 4 mg/mL. E induced an accumulation of cystathionine in primary cortical neurons (PCNs), 2nd trimester equivalent in utero binge, and 3rd trimester equivalent PN7 model suggesting that breakdown of cystathionine, a required process for Cys supply is impaired. This was associated with a significant reduction in cystathionine γ-lyase (CSE) protein expression in PCN (p < 0.05) and in fetal cerebral cortex in utero (53%, p < 0.05) without a change in the expression of cystathionine ß-synthase (CBS). Concomitantly, E decreased Cse mRNA expression in PCNs (by 32% within 6 h of exposure, p < 0.05) and in fetal brain (33%, p < 0.05). In parallel, knock down of CSE in differentiated rat cortical neuroblasts exaggerated the E-induced ROS, GSH loss with a pronounced caspase-3 activation and cell death. These studies illustrate the importance of TSP in CSE-related maintenance of GSH and the downstream events via Cys synthesis in neurons and fetal brain.

Role of oxidative injury in affecting the foetal & placental weights on exposure to tobacco smoke.

OBJECTIVE: To investigate the role of oxidative injury in affecting foetal and placental weights in mice by exposing them to tobacco smoke with or without supplementation with antioxidants. METHODS: The randomized control trial of pregnant mice at day one of gestation was conducted at Anatomy Department CPSP Regional Center Islamabad, from March 2005 to October 2005. The mice were divided into three groups: Group C had controls, while the two other groups, groups S and SV were exposed to secondary tobacco smoke in a whole body exposure chamber with and without supplementation with vitamins respectively. At term, the animals were sacrificed and the placentae and foetuses were weighed. The average values were calculated. The means for each group were analysed and the foetal placental ratio was calculated. SPSS 17 was used for statistical analysis. RESULTS: There were 44 mice; 15(34%) each in S and SV groups, while Group C had 14(32%) mice who acted as the controls. The mean foetal weight in Group S was 0.65±0.52g which was significantly less (p<0.0001) than1.48±0.19g in Group C. The mean foetal weight in Group SV was 0.97±0.65g which was not significantly different from S (p=0.124). The mean placental weight in Group S was 0.16±0.02g which was significantly less than 0.21±0.05gin Group C (p=0.014). In Group SV it was significantly more than Group S (p<0.0001). The ratio of mean foetal and mean placental weights in the groups C, S and SV were 7.05, 3.92 and4.41 respectively. CONCLUSIONS: Prenatal exposure to tobacco smoke decreased the mean foetal and placental weights and the foetal-placental ratio. This may partly be attributed to oxidative injury induced by free radicals in the tobacco smoke as it is prevented to some extent by simultaneous administration of antioxidants.

Microglia toxicity in preterm brain injury.

Microglia are the resident phagocytic cells of the central nervous system. During brain development they are also imperative for apoptosis of excessive neurons, synaptic pruning, phagocytosis of debris and maintaining brain homeostasis. Brain damage results in a fast and dynamic microglia reaction, which can influence the extent and distribution of subsequent neuronal dysfunction. As a consequence, microglia responses can promote tissue protection and repair following brain injury, or become detrimental for the tissue integrity and functionality. In this review, we will describe microglia responses in the human developing brain in association with injury, with particular focus on the preterm infant. We also explore microglia responses and mechanisms of microglia toxicity in animal models of preterm white matter injury and in vitro primary microglia cell culture experiments.

Prenatal vitamin C deficiency results in differential levels of oxidative stress during late gestation in foetal guinea pig brains.

Antioxidant defences are comparatively low during foetal development making the brain particularly susceptible to oxidative stress during antioxidant deficiencies. The brain is one of the organs containing the highest concentration of vitamin C (VitC) and VitC deficiency during foetal development may place the brain at risk of redox status imbalance. In the present study, we investigated the developmental pattern and effect of VitC deficiency on antioxidants, vitamin E and superoxide dismutase (SOD), assessed oxidative damage by measuring malondialdehyde (MDA), hydroxynonenal (HNE) and nitrotyrosine (NT) and analysed gene and protein expression of apoptosis marker caspase-3 in the guinea pig foetal brain at two gestational (GD) time points, GD 45/pre-term and GD 56/near term following either a VitC sufficient (CTRL) or deficient (DEF) maternal dietary regime. We show that except for SOD, antioxidants and oxidative damage markers are differentially expressed between the two GDs, with high VitC (p<0.0001), NT modified proteins (p<0.0001) and active caspase-3 levels (p<0.05) at pre-term and high vitamin E levels (p<0.0001), HNE (p<0.0001) and MDA (p<0.0001) at near term. VitC deficiency significantly increased SOD activity (p<0.0001) compared to CTRLs at both GDs indicating a compensatory response, however, low levels of VitC significantly elevated MDA levels (p<0.05) in DEF at near term. Our results show a differential regulation of the investigated markers during late gestation and suggest that immature brains are susceptible to oxidative stress due to prenatal vitC deficiency in spite of an induction of protective adaptation mechanisms.

Sevoflurane anesthesia in pregnant mice induces neurotoxicity in fetal and offspring mice.

BACKGROUND: Each year, over 75,000 pregnant women in the United States undergo anesthesia care. The authors set out to assess the effects of the anesthetic sevoflurane on neurotoxicity in pregnant mice and on learning and memory in fetal and offspring mice. METHODS: Pregnant mice (gestational day 14) and mouse primary neurons were treated with 2.5% sevoflurane for 2 h and 4.1% sevoflurane for 6 h, respectively. Brain tissues of both fetal and offspring mice (P31) and the primary neurons were harvested and subjected to Western blot and immunohistochemistry to assess interleukin-6, the synaptic markers postsynaptic density-95 and synaptophysin, and caspase-3 levels. Separately, learning and memory function in the offspring mice was determined in the Morris water maze. RESULTS: Sevoflurane anesthesia in pregnant mice induced caspase-3 activation, increased interleukin-6 levels (256 ± 50.98% [mean ± SD] vs. 100 ± 54.12%, P = 0.026), and reduced postsynaptic density-95 (61 ± 13.53% vs. 100 ± 10.08%, P = 0.036) and synaptophysin levels in fetal and offspring mice. The sevoflurane anesthesia impaired learning and memory in offspring mice at P31. Moreover, interleukin-6 antibody mitigated the sevoflurane-induced reduction in postsynaptic density-95 levels in the neurons. Finally, environmental enrichment attenuated the sevoflurane-induced increases in interleukin-6 levels, reductions of synapse markers, and learning and memory impairment. CONCLUSIONS: These results suggest that sevoflurane may induce detrimental effects in fetal and offspring mice, which can be mitigated by environmental enrichment. These findings should promote more studies to determine the neurotoxicity of anesthesia in the developing brain.

Decreased expression of Flightless I, a gelsolin family member and developmental regulator, in early-gestation fetal wounds improves healing.

Up until late in the third trimester of gestation and through to adulthood, the healing response acts more to regenerate than to repair a wound. The mechanisms underlying this "scar-free" healing remain unknown although the actin cytoskeleton has a major role. Flightless I (Flii), an actin-remodelling protein and essential developmental regulator, negatively affects wound repair but its effect on scar-free fetal healing is unknown. Using fetal skin explants from E17 (regenerate) and E19 (repair) rats, the function of Flii in fetal wound repair was determined. Expression of Flii increased between E17 and E19 days of gestation and wounding transiently increased Flii expression in E17 but not E19 wounds. However, both confocal and immunofluorescent analysis showed E17 keratinocytes immediately adjacent to the wounds downregulated Flii. As a nuclear coactivator and inhibitor of proliferation and migration, the absence of Flii in cells at the edge of the wound could be instrumental in allowing these cells to proliferate and migrate into the wound deficit. In contrast, Flii was strongly expressed within the cytoplasm and nucleus of keratinocytes within epidermal cells at the leading edge of E19 wounded fetal skin explants. This increase in Flii expression in E19 wounds could affect the way these cells migrate into the wound space and contribute to impaired wound healing. Neutralising Flii protein improved healing of early- but not late-gestation wounds. Flii did not colocalise with actin cables formed around E17 wounds suggesting an independent mechanism of action distinct from its actin-binding function in scar-free wound repair.

[Molecular mechanisms of reparation in prenatality].

Analysis of the specific features of an inflammatory reaction, wound contraction, the synthesis of cell mediators, cytokines, growth factors, and extracellular matrix modulators in response to a prenatal injury has allowed the discussion of the factors contributing to scar-free healing and the possible ways of minimizing fibrosis.

Regenerative healing in fetal skin: a review of the literature.

In mature skin, wound repair typically begins with hemostasis and inflammation. This is followed by a proliferative phase with reepithelialization, angiogenesis, and collagen production, and ends with the generation of a permanent scar. However, animal studies and clinical observations have shown that a different type of healing occurs in fetal skin in the first two trimesters of development. In early fetal skin, wounds exhibit a unique pattern of wound healing leading to regeneration. Notably, repair in the fetus takes place with little or no inflammation, faster reepithelialization, and no scarring. Although research in scarless fetal healing began several decades ago, the exact mechanisms of how this regenerative process takes place remain unknown. Knowing how the fetus will respond to potential injury from invasive diagnostic procedures or surgery is essential, especially given the development of less invasive fetal surgical techniques which could increase the number of fetal surgeries. In addition, insights into regenerative healing may provide information about how to accelerate postnatal wound healing as well as how to improve healing from a cosmetic standpoint. Future research directions include identification of the molecular controls responsible for scarless healing, with the intention that this new information will lead to improved therapeutic strategies for wound healing.

Tissue factor: a link between C5a and neutrophil activation in antiphospholipid antibody induced fetal injury.

Fetal loss in patients with antiphospholipid (aPL) antibodies has been ascribed to thrombosis of placental vessels. However, we have shown that inflammation, specifically activation of complement with generation of the anaphylotoxin C5a, is an essential trigger of fetal injury. In this study, we analyzed the role of the procoagulant molecule tissue factor (TF) in a mouse model of aPL antibody-induced pregnancy loss. We found that either blockade of TF with a monoclonal antibody in wild-type mice or a genetic reduction of TF prevented aPL antibody-induced inflammation and pregnancy loss. In response to aPL antibody-generated C5a, neutrophils express TF potentiating inflammation in the deciduas and leading to miscarriages. Importantly, we showed that TF in myeloid cells but not fetal-derived cells (trophoblasts) was associated with fetal injury, suggesting that the site for pathologic TF expression is neutrophils. We found that TF expression in neutrophils contributes to respiratory burst and subsequent trophoblast injury and pregnancy loss induced by aPL antibodies. The identification of TF as an important mediator of C5a-induced oxidative burst in neutrophils in aPL-induced fetal injury provides a new target for therapy to prevent pregnancy loss in the antiphospholipid syndrome.

Stress in pregnancy activates neurosteroid production in the fetal brain.

Neurosteroids such as allopregnanolone are potent agonists at the GABA(A) receptor and suppress the fetal CNS activity. These steroids are synthesized in the fetal brain either from cholesterol or from circulating precursors derived from the placenta. The concentrations of allopregnanolone are remarkably high in the fetal brain and rise further in response to acute hypoxic stress, induced by constriction of the umbilical cord. This response may result from the increased 5alpha-reductase and cytochrome P-450(SCC) expression in the brain. These observations suggest that the rise in neurosteroid concentrations in response to acute hypoxia may represent an endogenous protective mechanism that reduces excitotoxicity following hypoxic stress in the developing brain. In contrast to acute stress, chronic hypoxemia induces neurosteroidogenic enzyme expression without an increase in neurosteroid concentrations and, therefore, may pose a greater risk to the fetus. At birth, the allopregnanolone concentrations in the brain fall markedly, probably due to the loss of placental precursors; however, stressors, including hypoxia and endotoxin-induced inflammation, raise allopregnanolone concentrations in the newborn brain. This may protect the newborn brain from hypoxia-induced damage. However, the rise in allopregnanolone concentrations was also associated with increased sleep. This rise in sedative steroid levels may depress arousal and contribute to the risk of sudden infant death syndrome. Our recent findings indicate that acute hypoxic stress in pregnancy initiates a neurosteroid response that may protect the fetal brain from hypoxia-induced cell death, whereas the decline in allopregnanolone levels after birth may result in greater vulnerability to brain injury in neonates.

Morphometric analysis of brain lesions in rat fetuses prenatally exposed to low-level lead acetate: correlation with lipid peroxidation.

The effect of prenatal lead acetate exposure was studied microscopically together with the concentration of lead and lipid fluorescent products (LFP) in the brain of rat fetuses. Wistar rats were intoxicated with a lead solution containing either 160 or 320 ppm of lead acetate solution during 21 days through drinking water. The control group (ten rats) received deionized water for the same period. The rats were killed on gestation day 21 and fetuses were obtained; the placenta, umbilical cord and parietal cortex (Cx), striatum (St), thalamus (Th) and cerebellum (Ce) were collected for measuring tissue lead concentration, LFP as an index of lipid peroxidation and histopathologic examination. Lead contents were increased in placenta, umbilical cord, St, Th and Cx in both lead-exposed groups. Lead exposure increased (LFP) in placenta and umbilical cord, St, Th and Ce as compared to the control group. Histopathological examination showed severe vascular congestion in placenta, the Cx, St, Th and Ce with hyperchromatic and shrunken cells. Interstitial oedema was found in all regions studied of both lead exposed groups. The morphometric evaluation of the studied brain regions showed an absolute decrease in total cell number and increased number of damaged cells and interstitial oedema. Our results show that morphological changes in rat brain are correlated with increased lipid peroxidation, and the lead levels of the umbilical cord, however it is not clear whether oxidative stress is the cause or the consequence of these neurotoxic effects of lead.