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The inflammatory process plays a pivotal role during the pathogenesis of human labour, both at term and at preterm. Nicotinamide, a vitamin B(3) derivative, exerts anti-inflammatory and antioxidative properties in several cell types by interaction with various intracellular signalling proteins via modulating the activity of various transcription factors, including activation of the O subfamily of Forkhead/winged helix transcription factors (FoxO) and inhibition of nuclear factor-κB (NF-κB). The aim of this study was to determine the effect of nicotinamide on the expression of pro-labour and mediators in human placenta. The effects of nicotinamide were evaluated, over 24 h, by treating placenta with 0, 25 and 50 mM nicotinamide in the absence or presence of 10 μg/ml lipopolysaccharide (LPS). Nicotinamide treatment resulted in a significant reduction of basal and/or LPS-stimulated release and gene expression of the pro-inflammatory cytokines TNF-α, IL-6 and the chemokine IL-8, and the release of the prostaglandins PGE(2) and PGF(2)α and cyclooxygenase (COX)-2 mRNA expression. Additionally, nicotinamide treatment of human placenta resulted in attenuation of basal and LPS-induced oxidative stress, reducing 8-isoprostane release and increasing gene expression of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT). There was no effect of nicotinamide on NF-κB activation. The anti-inflammatory and antioxidant actions of nicotinamide were abolished by knockdown of FoxO3 using siRNA. In conclusion, nicotinamide exerts anti-inflammatory and antioxidative effects in human placenta, in part, via activation of FoxO3. Further studies should be undertaken to define a possible implication of vitamin B(3) derivatives in the management of preterm labour and delivery. [ABSTRACT FROM AUTHOR]

Abstract: To generate new insights into the process of fetal membrane rupture, we have developed a technique whereby fetal membranes overlying the cervix (i.e. supracervical site, SCS) are tagged in women undergoing term elective Caesarean section. The specific aim is to determine the effect of supracervical apposition on the release of inflammatory mediators and NF-κB signalling in pre-labour fetal membranes. Fetal membranes were collected from both the SCS and from a distal site (DS). The level of NF-κB proteins and its transcriptional co-activator protein CBP and p300 was determined by Western blotting and/or immunohistochemistry (IHC), and cytokine and prostaglandin release was quantified by enzyme immunoassay. Tissues obtained before labour at term possess an area of the fetal membranes (i.e. supracervical site) that exhibit decreased release of IL-1β, IL-6, IL-8, TNF-α and PGE2. IHC revealed that NF-κB signalling proteins, CBP and p300 were significantly increased in SC fetal membranes compared to distal membranes. In summary, data from this study confirm that supracervical fetal membranes display altered structural and biochemical characteristics. [Copyright &y& Elsevier]

Phospholipid-derived mediators, inflammatory cytokines and extracellular matrix remodelling enzymes are all involved in the initiation of human labour and delivery. We have previously demonstrated that natural and synthetic PPAR-gamma ligands regulate LPS-stimulated pro-inflammatory cytokine release from human gestational tissues, however, the effect of these ligands on the basal and/or LPS-induced expression of prostaglandins and proteases is not known. Therefore, the aim of this study was to determine the effects of 15d-PGJ(2) and troglitazone on the expression of basal and LPS-stimulated inflammatory mediators in human gestational tissues. Human placenta, amnion and choriodecidua (n=5) were incubated in the presence or absence of 15 microM 15d-PGJ(2) and 30 microM troglitazone under basal and LPS-stimulated (10 microg/ml) conditions. Treatment of placenta, amnion and choriodecidua with both 15d-PGJ(2) and troglitazone decreased basal and LPS-stimulated IL-1beta, IL-6, IL-10 and TNF-alpha release. Basal type II PLA(2) release from placental tissues was also significantly decreased by 15d-PGJ(2) and troglitazone. There was no effects of 15d-PGJ(2) and troglitazone on cPLA(2) protein expression. Both 15d-PGJ(2) and troglitazone significantly decreased basal and LPS-stimulated PGE(2) and PGF(2alpha) release from placenta and amnion. However, in choriodecidua, although 15d-PGJ(2) decreased basal and/or LPS-stimulated PGE(2) and PGF(2alpha) release, there was an increase in PGE(2) and PGF(2alpha) release in the presence of troglitazone. 15d-PGJ(2) and troglitazone inhibited MMP-9 release from human amnion. NF-kappaB DNA binding activity and NF-kappaB p65 protein expression was inhibited by treatment with 15d-PGJ(2) in human amnion. There was no effect of 15d-PGJ(2) or troglitazone on PPAR-gamma protein, and GW9662 failed to alleviate 15d-PGJ(2) and troglitazone inhibition of IL-6 and TNF-alpha release in placenta, amnion and choriodecidua. The data demonstrated in this study suggest that the 15d-PGJ(2) and troglitazone exhibit anti-inflammatory properties in human gestational tissues via PPAR-gamma independent actions. [ABSTRACT FROM AUTHOR]

Abstract: Phospholipid-derived mediators, inflammatory cytokines and extracellular matrix remodelling enzymes are all involved in the initiation of human labour and delivery. We have previously demonstrated that natural and synthetic PPAR-γ ligands regulate LPS-stimulated pro-inflammatory cytokine release from human gestational tissues, however, the effect of these ligands on the basal and/or LPS-induced expression of prostaglandins and proteases is not known. Therefore, the aim of this study was to determine the effects of 15d-PGJ2 and troglitazone on the expression of basal and LPS-stimulated inflammatory mediators in human gestational tissues. Human placenta, amnion and choriodecidua (n =5) were incubated in the presence or absence of 15μM 15d-PGJ2 and 30μM troglitazone under basal and LPS-stimulated (10μg/ml) conditions. Treatment of placenta, amnion and choriodecidua with both 15d-PGJ2 and troglitazone decreased basal and LPS-stimulated IL-1β, IL-6, IL-10 and TNF-α release. Basal type II PLA2 release from placental tissues was also significantly decreased by 15d-PGJ2 and troglitazone. There was no effects of 15d-PGJ2 and troglitazone on cPLA2 protein expression. Both 15d-PGJ2 and troglitazone significantly decreased basal and LPS-stimulated PGE2 and PGF2α release from placenta and amnion. However, in choriodecidua, although 15d-PGJ2 decreased basal and/or LPS-stimulated PGE2 and PGF2α release, there was an increase in PGE2 and PGF2α release in the presence of troglitazone. 15d-PGJ2 and troglitazone inhibited MMP-9 release from human amnion. NF-κB DNA binding activity and NF-κB p65 protein expression was inhibited by treatment with 15d-PGJ2 in human amnion. There was no effect of 15d-PGJ2 or troglitazone on PPAR-γ protein, and GW9662 failed to alleviate 15d-PGJ2 and troglitazone inhibition of IL-6 and TNF-α release in placenta, amnion and choriodecidua. The data demonstrated in this study suggest that the 15d-PGJ2 and troglitazone exhibit anti-inflammatory properties in human gestational tissues via PPAR-γ independent actions. [Copyright &y& Elsevier]

BackgroundEvidence suggests that women are failing to meet guidelines for nutrition, physical activity, and weight gain during pregnancy. Interventions to promote a healthy lifestyle in pregnancy demonstrate mixed results and many are time and resource intensive. mHealth-delivered interventions offer an opportunity to provide trusted source information in a timely and cost-effective manner. Studies regarding women’s and health professionals’ views of mHealth in antenatal care are limited. ObjectiveThis study aimed to explore women’s and health professionals’ views regarding mHealth information sources and interventions to assist women to eat well, be physically active, and gain healthy amounts of weight in pregnancy. MethodsA descriptive qualitative research approach employed focus groups and in-depth interviews with 15 pregnant or postpartum women and 12 in-depth interviews with health professionals including two from each category: obstetricians, general practitioners, midwives, dietitians, physiotherapists, and community pharmacists. All interviews were transcribed verbatim and thematically analyzed. ResultsWomen uniformly embraced the concept of mHealth information sources and interventions in antenatal care and saw them as central to information acquisition and ideally incorporated into future antenatal care processes. Health professionals exhibited varied views perceiving mHealth as an inevitable, often parallel, service rather than one integrated into the care model. Four key themes emerged: engagement, risk perception, responsibility, and functionality. Women saw their ability to access mHealth elements as a way to self-manage or control information acquisition that was unavailable in traditional care models and information sources. The emergence of technology was perceived by some health professionals to have shifted control of information from trusted sources, such as health professionals and health organizations, to nontrusted sources. Some health professionals were concerned about the medicolegal risks of mHealth (incorrect or harmful information and privacy concerns), while others acknowledged that mHealth was feasible if inherent risks were addressed. Across both groups, there was uncertainty as to who should be responsible for ensuring high-quality mHealth. The absence of a key pregnancy or women’s advocacy group, lack of health funds for technologies, and the perceived inability of maternity hospitals to embrace technology were seen to be key barriers to provision. Women consistently identified the functionality of mHealth as adding value to antenatal care models. For some health professionals, lack of familiarity with and fear of mHealth limited their engagement with and comprehension of the capacity of new technologies to support antenatal care. ConclusionsWomen exhibited positive views regarding mHealth for the promotion of a healthy lifestyle in antenatal care. Conversely, health professionals expressed a much wider variation in attitudes and were more able to identify potential risks and barriers to development and implementation. This study contributes to the understanding of the opportunities and challenges in developing mHealth lifestyle interventions in antenatal care.

Extracellular vesicles (EVs) are nanosized biomolecular packages involved in intercellular communication. EVs are released by all cells, making them broadly applicable as therapeutic, diagnostic, and mechanistic components in (patho)physiology. Sample purity is critical for correctly attributing observed effects to EVs and for maximizing therapeutic and diagnostic performance. Lipoprotein contaminants represent a major challenge for sample purity. Lipoproteins are approximately six orders of magnitude more abundant in the blood circulation and overlap in size, shape, and density with EVs. This study represents the first example of an EV purification method based on the chemically-induced breakdown of lipoproteins. Specifically, a styrene-maleic acid (SMA) copolymer is used to selectively breakdown lipoproteins, enabling subsequent size-based separation of the breakdown products from plasma EVs. The use of the polymer followed by tangential flow filtration or size-exclusion chromatography results in improved EV yield, preservation of EV morphology, increased EV markers, and reduced contaminant markers. SMA-based EV purification enables improved fluorescent labeling, reduces interactions with macrophages, and enhances accuracy, sensitivity, and specificity to detect EV biomarkers, indicating benefits for various downstream applications. In conclusion, SMA is a simple and effective method to improve the purity and yield of plasma-derived EVs, which favorably impacts downstream applications., (© 2023 The Authors. Small published by Wiley‐VCH GmbH.)

Introduction: Previous studies have reported that neonates less than the 25th BWC especially if they were male, were more likely to be associated with birth complications suggesting small neonates often identified as appropriately grown are at risk of adverse outcomes. We have questioned whether smaller neonates not typically categorized as "small for gestational age" may not reach their genetically determined growth due to placental insufficiency., Methods: RNA-Seq was performed on the Illumina NovaSeq 600 using term placentae from neonates that were less than the 10th birthweight centile (BWC) (n = 39), between the 10th and the 30th BWC (n = 15) or greater than the 30th BWC (n = 23). Bioinformatic analyses were conducted and statistical significance was assessed at a level of P < 0.05 for single comparisons or FDR <0.05 unless otherwise noted., Results: Gene set enrichment analysis revealed differences between BWC groups and in relation to the sex of the placenta. Genes associated with hypoxia, inflammatory responses, estrogen responsive genes, and androgen responsive genes were enriched (FDR <0.1) for in placentae of neonates <10th BWC regardless of sex and also in male placentae of neonates between the 10th-30th BWC. Female placenta of neonates between the 10th-30th BWC were comparable to placentae of neonates >30th BWC., Discussion: These findings provide evidence that small male neonates may be at a greater risk of an adverse outcome than females due to changes in gene expression that are associated with placental dysfunction. The current data raises questions of whether placental pathology for smaller appropriately grown neonates should be scientifically and clinically examined in more depth., Competing Interests: Declaration of Competing interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper., (Crown Copyright © 2024. Published by Elsevier Ltd. All rights reserved.)

A birthweight centile (BWC) below the 25th is associated with an elevated risk of adverse perinatal outcomes, particularly among males. This male vulnerability may stem from alterations in placenta-specific androgen signalling, a signalling axis that involves the androgen receptor (AR)-mediated regulation of target genes containing androgen response elements (AREs). In this study, we examined global and ARE-specific transcriptomic signatures in term male placentae (≥37 weeks of gestation) across BWC subcategories (<10th, 10th-30th, >30th) using RNA-seq and gene set enrichment analysis. ARE-containing transcripts in placentae with BWCs below the 10th percentile were upregulated compared to those in the 10th-30th and >30th percentiles, which coincided with the enrichment of gene sets related to hypoxia and the suppression of gene sets associated with mitochondrial function. In the absence of ARE-containing transcripts in silico, <10th and 10th-30th BWC subcategory placentae upregulated gene sets involved in vasculature development, immune function, and cell adhesion when compared to those in the >30th BWC subcategory. Collectively, our in silico findings suggest that changes in the expression of ARE-containing transcripts in male placentae may contribute to impaired placental vasculature and therefore result in reduced fetal growth outcomes.

Placental extracellular vesicles (EVs) can be found in the maternal circulation throughout gestation, and their concentration, content and bioactivity are associated with pregnancy outcomes, including gestational diabetes mellitus (GDM). However, the effect of changes in the maternal microenvironment on the mechanisms associated with the secretion of EVs from placental cells remains to be fully established. Here, we evaluated the effect of high glucose on proteins associated with the trafficking and release of different populations of EVs from placental cells. BeWo and HTR8/SVneo cells were used as placental models and cultured under 5-mM D-glucose (i.e. control) or 25-mM D-glucose (high glucose). Cell-conditioned media (CCM) and cell lysate were collected after 48 h. Different populations of EVs were isolated from CCM by ultracentrifugation (i.e. pellet 2K-g, pellet 10K-g, and pellet 100K-g) and characterised by Nanoparticle Tracking Analysis. Quantitative proteomic analysis (IDA/SWATH) and multiple reaction monitoring protocols at high resolution (MRM HR ) were developed to quantify 37 proteins related to biogenesis, trafficking/release and recognition/uptake of EVs. High glucose increased the secretion of total EVs across the pellets from BeWo cells, an effect driven mainly by changes in the small EVs concentration in the CCM. Interestingly, no effect of high glucose on HTR8/SVneo cells EVs secretion was observed. High glucose induces changes in proteins associated with vesicle trafficking in BeWo cells, including Heat Shock Protein Family A (Hsp70) Member 9 (HSPA9) and Member 8 (HSPA8). For HTR8/SVneo, altered proteins including prostaglandin F2α receptor regulatory protein (FPRP), RAB5A, RAB35, RAB5B, and RB11B, STAM1 and TSG101. These proteins are associated with the secretion and trafficking of EVs, which could explain in part, changes in the levels of circulating EVs in diabetic pregnancies. Further, we identified that proteins RAB11B, PDCD6IP, STAM, HSPA9, HSPA8, SDCBP, RAB5B, RAB5A, RAB7A and ERAP1 regulate EV release in response to high and low glucose when overexpressed in cells. Interestingly, immunohistochemistry analysis of RAB7A revealed distinct changes in placental tissues obtained from women with normal glucose tolerance (NGT, n  = 6) and those with GDM ( n  = 6), influenced by diet or insulin treatment. High glucose regulation of proteins involved in intercellular dynamics and the trafficking of multivesicular bodies to the plasma membrane in placental cells is relevant in the context of GDM pregnancies., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Authors. Journal of Extracellular Biology published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

CD8 + T cells provide robust antiviral immunity, but how epitope-specific T cells evolve across the human lifespan is unclear. Here we defined CD8 + T cell immunity directed at the prominent influenza epitope HLA-A*02:01-M1 58-66 (A2/M1 58 ) across four age groups at phenotypic, transcriptomic, clonal and functional levels. We identify a linear differentiation trajectory from newborns to children then adults, followed by divergence and a clonal reset in older adults. Gene profiles in older adults closely resemble those of newborns and children, despite being clonally distinct. Only child-derived and adult-derived A2/M1 58 + CD8 + T cells had the potential to differentiate into highly cytotoxic epitope-specific CD8 + T cells, which was linked to highly functional public T cell receptor (TCR)αβ signatures. Suboptimal TCRαβ signatures in older adults led to less proliferation, polyfunctionality, avidity and recognition of peptide mutants, although displayed no signs of exhaustion. These data suggest that priming T cells at different stages of life might greatly affect CD8 + T cell responses toward viral infections., (© 2023. The Author(s).)

Pregnancy poses a greater risk for severe COVID-19; however, underlying immunological changes associated with SARS-CoV-2 during pregnancy are poorly understood. We defined immune responses to SARS-CoV-2 in unvaccinated pregnant and nonpregnant women with acute and convalescent COVID-19, quantifying 217 immunological parameters. Humoral responses to SARS-CoV-2 were similar in pregnant and nonpregnant women, although our systems serology approach revealed distinct antibody and FcγR profiles between pregnant and nonpregnant women. Cellular analyses demonstrated marked differences in NK cell and unconventional T cell activation dynamics in pregnant women. Healthy pregnant women displayed preactivated NK cells and γδ T cells when compared with healthy nonpregnant women, which remained unchanged during acute and convalescent COVID-19. Conversely, nonpregnant women had prototypical activation of NK and γδ T cells. Activation of CD4+ and CD8+ T cells and T follicular helper cells was similar in SARS-CoV-2-infected pregnant and nonpregnant women, while antibody-secreting B cells were increased in pregnant women during acute COVID-19. Elevated levels of IL-8, IL-10, and IL-18 were found in pregnant women in their healthy state, and these cytokine levels remained elevated during acute and convalescent COVID-19. Collectively, we demonstrate perturbations in NK cell and γδ T cell activation in unvaccinated pregnant women with COVID-19, which may impact disease progression and severity during pregnancy.

Gestational diabetes mellitus (GDM) and maternal obesity are significant metabolic complications increasingly prevalent in pregnancy. Of major concern, both GDM and maternal obesity can have long-term detrimental impacts on the health of both mother and offspring. Recent research has shown that increased inflammation and oxidative stress are two features central to the pathophysiology of these metabolic conditions. Evidence suggests selenium supplementation may be linked to disease prevention in pregnancy; however, the specific effects of selenium on inflammation and oxidative stress associated with GDM and maternal obesity are unknown. Therefore, this study aimed to investigate the effect of selenium supplementation on an in vitro model of GDM and maternal obesity. Human placental tissue, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) were stimulated with either the bacterial product lipopolysaccharide (LPS) or the pro-inflammatory cytokine TNF-α. Selenium pre-treatment blocked LPS and TNF-α induced mRNA expression and secretion of pro-inflammatory cytokines and chemokines, while increasing anti-inflammatory cytokine and antioxidant mRNA expression in placenta, VAT and SAT. Selenium pre-treatment was also found to inhibit LPS- and TNF-α induced phosphorylation of ERK in placenta, VAT and SAT. These findings indicate that selenium may be able to prevent inflammation and oxidative stress associated with GDM and maternal obesity. Additional in vivo studies are required to identify the efficacy of selenium supplementation in preventing inflammatory pathways activated by GDM and maternal obesity and to elucidate the mechanism involved.

In examining maternal depression, placental 11β-HSD2 mRNA expression and offspring cortisol regulation as a potential fetal programming pathway in relation to later child emotional disorders, it has become clear that sex differences may be important to consider. This study reports on data obtained from 209 participants in the Mercy Pregnancy and Emotional Wellbeing Study (MPEWS) recruited before 20 weeks of pregnancy. Maternal depressive disorders were diagnosed using the SCID-IV and maternal childhood trauma using the Childhood Trauma Questionnaire. Placental 11β-HSD2 mRNA was measured using qRT-PCR. For assessment of stress-induced cortisol reactivity, salivary cortisol samples were taken at 12 months of age. At 4 years of age, measurement of Childhood Emotional Disorders (depression and anxiety) was based on maternal report using the Preschool Age Psychiatric Assessment (PAPA) and internalizing symptoms using the Child Behavior Checklist (CBCL). Maternal depression in pregnancy and postpartum, and infant cortisol reactivity, was associated with internalizing symptoms for females only. For female offspring only, increased 12-month cortisol reactivity was also associated with increased emotional disorders at 4 years of age; however, there was no association with placental 11β-HSD2 mRNA expression. In females only, the combination of lower placental 11β-HSD2 mRNA expression and higher cortisol reactivity at 12 months of age predicted increased internalising problems. These findings suggest there may be sex differences in prenatal predictors and pathways for early childhood depression and anxiety symptoms and disorder., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Introduction: Placental dysfunction and inefficiency, is important in understanding fetal growth restriction and low birth weight. Two recent studies have examined the relationship between antidepressant use in pregnancy and placental weight ratios; one found lower placental weight ratio associated with antidepressant use and the other found a higher ratio., Methods: This study examined 342 women recruited in early pregnancy, including 75 taking antidepressants, 29 with current depression and 238 controls. Antidepressant use was measured through self-report in early and late pregnancy, hospital records at delivery and drug concentrations in umbilical cord and maternal blood obtained at delivery. Maternal depression was measured using the Structured Clinical Interview for the DSM IV (SCID) at recruitment. Placentas were collected at delivery and weighed, and infant birth weight recorded. Placental efficiency was measured using standardised placental weight residuals and included as the outcome in general linear models (ANOVA/ANCOVA) to test hypotheses., Results: While placental weight was higher for those on antidepressants compared to controls (z=.30 c.f. Z=-0.08, p=.012), there were no significant differences between the three groups after adjusting for maternal body mass index at recruitment. When comparing antidepressant groups separately there were small-to-moderate positive associations between (SSRI) concentrations and placental weight (rho's > 0.20, p's > 0.05), which did not reach significance., Conclusion: Antidepressant use in pregnancy was not associated with significant changes in placental efficiency after adjustment for confounding variables. Future research should expand on this to examine other aspects of placental function and include a wide range of potential confounding variables to draw clinically meaningful conclusions., (Crown Copyright © 2022. Published by Elsevier Ltd. All rights reserved.)

Objective: To determine the effect of preexisting maternal obesity and gestational diabetes mellitus (GDM) on the circulating levels of Metrnl in cord and maternal plasma., Design: Metrnl levels were measured on maternal and cord plasma from women with normal glucose tolerance (NGT) (19 non-obese and 20 obese), GDM controlled by diet (18 non-obese and 17 obese) and GDM controlled by insulin (19 non-obese and 18 obese) at the time of term elective cesarean section. Metrnl concentrations were determined by enzyme-linked immunoassay. Correlations of Metrnl levels with anthropometric parameters and laboratory measurements were also assessed., Results: There was no effect of maternal obesity or GDM on maternal plasma Metrnl concentrations. In cord plasma, Metrnl concentrations were significantly lower in NGT obese compared to NGT non-obese women and in non-obese GDM women compared to non-obese NGT women. Significant positive correlations were observed between maternal plasma Metrnl and cord plasma Metrnl. In cord plasma, significant positive correlations were observed between Metrnl levels and GWG and maternal and cord plasma glucose levels at delivery., Conclusions: At the time of term cesarean section, preexisting maternal obesity and GDM are associated with lower Metrnl levels in cord plasma. Alterations in cord plasma Metrnl levels may lead to alterations in fetal growth trajectory and be a determinant for metabolic disorders later in life.

Background: Gestational diabetes mellitus (GDM) is a serious public health issue affecting 9-15% of all pregnancies worldwide. Recently, it has been suggested that extracellular vesicles (EVs) play a role throughout gestation, including mediating a placental response to hyperglycaemia. Here, we investigated the EV-associated miRNA profile across gestation in GDM, assessed their utility in developing accurate, multivariate classification models, and determined the signaling pathways in skeletal muscle proteome associated with the changes in the EV miRNA profile., Methods: Discovery: A retrospective, case-control study design was used to identify EV-associated miRNAs that vary across pregnancy and clinical status (i.e. GDM or Normal Glucose Tolerance, NGT). EVs were isolated from maternal plasma obtained at early, mid and late gestation (n = 29) and small RNA sequencing was performed. Validation: A longitudinal study design was used to quantify expression of selected miRNAs. EV miRNAs were quantified by real-time PCR (cases = 8, control = 14, samples at three times during pregnancy) and their individual and combined classification efficiencies were evaluated. Quantitative, data-independent acquisition mass spectrometry was use to establish the protein profile in skeletal muscle biopsies from normal and GDM., Results: A total of 2822 miRNAs were analyzed using a small RNA library, and a total of 563 miRNAs that significantly changed (p < 0.05) across gestation and 101 miRNAs were significantly changed between NGT and GDM. Analysis of the miRNA changes in NGT and GDM separately identified a total of 256 (NGT-group), and 302 (GDM-group) miRNAs that change across gestation. A multivariate classification model was developed, based on the quantitative expression of EV-associated miRNAs, and the accuracy to correctly assign samples was > 90%. We identified a set of proteins in skeletal muscle biopsies from women with GDM associated with JAK-STAT signaling which could be targeted by the miRNA-92a-3p within circulating EVs. Interestingly, overexpression of miRNA-92a-3p in primary skeletal muscle cells increase insulin-stimulated glucose uptake., Conclusions: During early pregnancy, differently-expressed, EV-associated miRNAs may be of clinical utility in identifying presymptomatic women who will subsequently develop GDM later in gestation. We suggest that miRNA-92a-3p within EVs might be a protected mechanism to increase skeletal muscle insulin sensitivity in GDM., (© 2021. The Author(s).)

Aims/hypothesis: Type 2 diabetes mellitus is a major cause of morbidity and death worldwide. Women with gestational diabetes mellitus (GDM) have greater than a sevenfold higher risk of developing type 2 diabetes in later life. Accurate methods for postpartum type 2 diabetes risk stratification are lacking. Circulating microRNAs (miRNAs) are well recognised as biomarkers/mediators of metabolic disease. We aimed to determine whether postpartum circulating miRNAs can predict the development of type 2 diabetes in women with previous GDM., Methods: In an observational study, plasma samples were collected at 12 weeks postpartum from 103 women following GDM pregnancy. Utilising a discovery approach, we measured 754 miRNAs in plasma from type 2 diabetes non-progressors (n = 11) and type 2 diabetes progressors (n = 10) using TaqMan-based real-time PCR on an OpenArray platform. Machine learning algorithms involving penalised logistic regression followed by bootstrapping were implemented., Results: Fifteen miRNAs were selected based on their importance in discriminating type 2 diabetes progressors from non-progressors in our discovery cohort. The levels of miRNA miR-369-3p remained significantly different (p < 0.05) between progressors and non-progressors in the validation sample set (n = 82; 71 non-progressors, 11 progressors) after adjusting for age and correcting for multiple comparisons. In a clinical model of prediction of type 2 diabetes that included six traditional risk factors (age, BMI, pregnancy fasting glucose, postpartum fasting glucose, cholesterol and triacylglycerols), the addition of the circulating miR-369-3p measured at 12 weeks postpartum improved the prediction of future type 2 diabetes from traditional AUC 0.83 (95% CI 0.68, 0.97) to an AUC 0.92 (95% CI 0.84, 1.00)., Conclusions: This is the first demonstration of miRNA-based type 2 diabetes prediction in women with previous GDM. Improved prediction will facilitate early lifestyle/drug intervention for type 2 diabetes prevention.

Background: Pre-existing diabetes in pregnancy is associated with an increased risk of complications. Likewise, living in rural, regional and remote Victoria, Australia, is also associated with poorer health outcomes. There is a gap in the literature with regard to whether Victorian women with pre-existing diabetes experience a greater risk of adverse pregnancy outcomes compared to their metropolitan counterparts., Aim: Our objective is to compare obstetric and perinatal outcomes for women with pre-existing diabetes delivering in rural vs metropolitan hospitals in Victoria, Australia., Materials and Methods: Retrospective population-based study using routinely collected state-based data of singleton births to women with type 1 and type 2 diabetes who delivered in metropolitan (n = 3233) and rural hospitals (n = 693) in Victoria, Australia, between 2006-2015. Pearson's χ 2 test, Fisher's exact test and MannWhitney U-test were used to compare obstetric and perinatal outcomes between metropolitan and rural locations., Results: Delivery in a rural hospital was associated with higher rates of stillbirth (2.3% vs 1.1%, P = 0.027), macrosomia (25.9% vs 16.9%, P < 0.001), shoulder dystocia (8.4% vs 3.5%, P < 0.001) and admission to the neonatal intensive care unit/special care nursery (73.2% vs 59.3%, P < 0.001). Smoking (18.0% vs 8.9%, P < 0.001), overweight/obesity (P = 0.047) and socioeconomic disadvantage (P < 0.001) were more common in rural women., Conclusions: Women with pre-existing diabetes who deliver in rural hospitals experience a greater risk of adverse perinatal outcomes and present with increased maternal risk factors. These results suggest a need to improve care for women with pre-existing diabetes in rural Victoria., (© 2021 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists.)

Placental 11β-HSD2 has been a focus of research for understanding potential fetal programming associated with maternal emotional disorders. This study examined the pathway from antenatal mental health via placental 11β-HSD2 mRNA to cortisol regulation in the infant offspring. This study reports on data obtained from 236 participants in the Mercy Pregnancy and Emotional Wellbeing Study (MPEWS). At term, placental tissue was collected within 30 min of birth from 52 participants meeting current criteria for a depressive disorder, and 184 control participants. Depressive disorders were diagnosed using the SCID-IV. In addition, antidepressant use, depressive and anxiety symptoms were measured in early and late pregnancy. Placental 11β-HSD2 mRNA expression was measured using qRT-PCR. Infant salivary cortisol samples were taken at 12 months of age. Women on antidepressant medication and with higher trait anxiety had higher placental 11β-HSD2 expression compared to women not taking medication. Furthermore, the offspring of women taking an antidepressant and who also had a current depressive disorder and high trait anxiety had high cortisol reactivity at 12 months of age and this was mediated through 11β-HSD2 mRNA expression. In contrast, offspring of women not taking antidepressant medication with depressive disorder and high anxiety there was low cortisol reactivity observed. Our findings suggest that the relationship between maternal antenatal depression and anxiety and infant cortisol reactivity is mediated through placental 11β-HSD2 mRNA expression. Furthermore, the direction differed for women taking antidepressants, where infant cortisol reactivity was high whereas when compared to those with unmedicated depression and anxiety, where infant cortisol reactivity was low., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Immune effector responses against Plasmodium falciparum include antibody-mediated activation of innate immune cells, which can induce Fc effector functions, including antibody-dependent cellular cytotoxicity, and the secretion of cytokines and chemokines. These effector functions are regulated by the composition of immunoglobulin G (IgG) Fc N-linked glycans. However, a role for antibody-mediated natural killer (NK) cells activation or Fc N-linked glycans in pregnant women with malaria has not yet been established. Herein, we studied the capacity of IgG antibodies from pregnant women, with placental malaria or non-placental malaria, to induce NK cell activation in response to placental malaria-associated antigens DBL2 and DBL3. Antibody-mediated NK cell activation was observed in pregnant women with malaria, but no differences were associated with susceptibility to placental malaria. Elevated anti-inflammatory glycosylation patterns of IgG antibodies were observed in pregnant women with or without malaria infection, which were not seen in healthy non-pregnant controls. This suggests that pregnancy-associated anti-inflammatory Fc N-linked glycans may dampen the antibody-mediated activation of NK cells in pregnant women with malaria infection. Overall, although anti-inflammatory glycans and antibody-dependent NK cell activation were detected in pregnant women with malaria, a definitive role for these antibody features in protecting against placental malaria remains to be proven.

Gestational diabetes mellitus (GDM) is one of the most common endocrine disorders during gestation and affects around 15% of all pregnancies worldwide, paralleling the global increase in obesity and type 2 diabetes. Normal pregnancies are critically dependent on the development of maternal insulin resistance balanced by an increased capacity to secrete insulin, which allows for the allocation of nutrients for adequate foetal growth and development. Several factors including placental hormones, inflammatory mediators and nutrients have been proposed to alter insulin sensitivity and insulin response and underpin the pathological outcomes of GDM. However, other factors may also be involved in the regulation of maternal metabolism and a complete understanding of GDM pathophysiology requires the identification of these factors, and the mechanisms associated with them. Recent studies highlight the potential utility of tissue-specific extracellular vesicles (EVs) in the diagnosis of disease onset and treatment monitoring for several pregnancy-related complications, including GDM. To date, there is a paucity of data defining changes in the release, content, bioactivity and diagnostic utility of circulating EVs in pregnancies complicated by GDM. Placental EVs may engage in paracellular interactions including local cell-to-cell communication between the cell constituents of the placenta and contiguous maternal tissues, and/or distal interactions involving the release of placental EVs into biological fluids and their transport to a remote site of action. Hence, the aim of this review is to discuss the biogenesis, isolation methods and role of EVs in the physiopathology of GDM, including changes in maternal insulin sensitivity during pregnancy., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Naive and memory T cells are maintained in a quiescent state, yet capable of rapid response and differentiation to antigen challenge via molecular mechanisms that are not fully understood. In naive cells, the deletion of Foxo1 following thymic development results in the increased expression of multiple AP-1 family members, rendering T cells less able to respond to antigenic challenge. Similarly, in the absence of FOXO1, post-infection memory T cells exhibit the characteristics of extended activation and senescence. Age-based analysis of human peripheral T cells reveals that levels of FOXO1 and its downstream target, TCF7, are inversely related to host age, whereas the opposite is found for AP-1 factors. These characteristics of aging also correlate with the formation of T cells manifesting features of cellular senescence. Our work illustrates a role for FOXO1 in the active maintenance of stem-like properties in T cells at the timescales of acute infection and organismal life span., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 University of California, San Diego. Published by Elsevier Inc. All rights reserved.)

Gestational diabetes mellitus (GDM) is a global health issue with significant short and long-term complications for both mother and baby. There is a strong need to identify an effective therapeutic that can prevent the development of GDM. A better understanding of the pathophysiology of GDM and the relationship between the adipose tissue, the placenta and fetal growth is required. The placenta regulates fetal growth by modulating nutrient transfer of glucose, amino acids and fatty acids. Various factors secreted by the adipose tissue, such as adipokines, adipocytokines and more recently identified extracellular vesicles, can influence inflammation and interact with placental nutrient transport. In this review, the role of the placental nutrient transporters and the adipose-derived factors that can influence their function will be discussed. A better understanding of these factors and their relationship may make a potential target for therapeutic interventions to prevent the development of GDM and its consequences., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Spontaneous preterm birth is the leading cause of neonatal mortality and morbidity globally. Activation of the maternal immune system leads to a downstream cascade of proinflammatory events that culminate in the activation of spontaneous uterine contractions and the rupture of the foetal membranes. Anti-inflammatory agents may be a novel therapeutic approach to prevent inflammation-induced myometrial contractions and premature rupture of foetal membranes. The polyphenol gallic acid has been previously shown to exert potent anti-inflammatory effects. Thus, this study aimed to determine the effect of gallic acid on proinflammatory and pro-labour mediators in cytokine-stimulated gestational tissues in vitro. In primary human cells isolated from myometrium and foetal membranes (decidua, and amnion mesenchymal and epithelial cells), gallic acid treatment suppressed inflammation-induced expression of proinflammatory cytokines and chemokines and extracellular matrix-degrading and matrix-remodelling enzymes. Gallic acid also significantly inhibited inflammation-induced myometrial activation as evidenced by decreased expression of contraction-associated proteins, the uterotonic PGF2α and collagen cell contractility. Using a global proteomic approach, gallic acid may differentially regulate proteins associated with collagen synthesis, cell contractility and protein synthesis in primary myometrial and decidual cells. In summary, gallic acid inhibited inflammation-induced mediators involved in active labour in primary cells isolated from myometrium and foetal membranes. These in vitro studies suggest that the polyphenol gallic acid may be able to suppress the production of proinflammatory and pro-labour mediators involved in myometrial contractions and rupture of foetal membranes. Future preclinical studies may elucidate the efficacy of gallic acid in preventing inflammation-driven preterm birth.

Introduction: The world is witnessing a steady rise in the prevalence of gestational diabetes mellitus (GDM), correlated with the current obesity epidemic. Both GDM and obesity negatively impact both the health of women but also that of the next generation. GDM and maternal obesity are associated with increased maternal and fetal inflammation and oxidative stress. A safe and effective intervention that can prevent these pathological features, and reduce the intergenerational burden, is required. Phenolic acids, such as punicalagin and curcumin, possess anti-inflammatory and antioxidant properties. Thus, the aim of this study was to examine the effects of punicalagin and curcumin on pro-inflammatory cytokines and chemokines, and antioxidant expression in an in vitro model of inflammation., Methods: Human placenta, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) explants were obtained at term elective Caesarean section and stimulated with TNF alpha (TNF)., Results: We found that punicalagin and curcumin significantly supressed TNF-induced pro-inflammatory cytokine (IL1A, IL1B, and IL6) and chemokine (CCL2-4, CXCL1, CXCL5 and CXCL8) expression in human placenta, VAT and SAT. Anti-inflammatory cytokine IL4 and IL13 mRNA expression was also upregulated by punicalagin and curcumin treatment in placenta, VAT and SAT. Punicalagin and curcumin also altered antioxidant (SOD2 and catalase) mRNA expression in placenta, VAT and SAT, with minimal effect on hydrogen peroxide concentrations in tissue lysates., Conclusion: These findings suggest that the phenolic acids punicalagin and curcumin possess potent anti-inflammatory capabilities in in vitro human models of inflammation. Further studies are warranted to determine their suitability as therapeutic interventions for pro-inflammatory gestational complications, including GDM and maternal obesity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Gestational diabetes mellitus (GDM) affects up to 16% of pregnant women and is associated with significant long-term health detriments for the mother and her offspring. Two central features of GDM are low-grade inflammation and maternal peripheral insulin resistance, therefore therapeutics which target these may be most effective at preventing the development of GDM. Short-chain fatty acids (SCFAs), such as butyrate and propionate, are metabolites produced from the fermentation of dietary fibre by intestinal microbiota. SCFAs possess anti-inflammatory, anti-obesity and anti-diabetic properties. Therefore, this study aimed to investigate the effect of SCFAs on inflammation and insulin signalling defects in an in vitro model of GDM. Human placenta, visceral adipose tissue (VAT) and s.c. adipose tissue (SAT) were stimulated with either the pro-inflammatory cytokine TNF or bacterial product lipopolysaccharide (LPS). The SCFAs butyrate and propionate blocked TNF- and LPS-induced mRNA expression and secretion of pro-inflammatory cytokines and chemokines in placenta, VAT and SAT. Primary human cells isolated from skeletal muscle were stimulated with TNF to assess the effect of SCFAs on inflammation-induced defects in the insulin signalling pathway. Butyrate and propionate were found to reverse TNF-induced increases in IRS-1 serine phosphorylation and decreases in glucose uptake. Butyrate and propionate exerted these effects by preventing ERK activation. Taken together, these results suggest that the SCFAs may be able to improve insulin sensitivity and prevent inflammation induced by sterile or bacterial inflammation. Future in vivo studies are warranted to investigate the efficacy and safety of SCFAs in preventing insulin resistance and inflammation associated with GDM.

Diabetes is an increasingly common diagnosis among pregnant women. Pregestational diabetes is associated with an increase in many adverse pregnancy outcomes, which impact both on the woman and her fetus. The models of pregnancy care for women with diabetes are based largely on observational data or consensus opinion. Strategies for aneuploidy screening and monitoring for fetal well-being should be modified in women with diabetes. There is an increasing understanding of the mechanisms by which congenital anomalies and disorders of fetal growth occur, involving epigenetic modifications, changes in gene expression in critical developmental pathways, and oxidative stress. This knowledge may lead to pathways for improved care for these high-risk pregnancies., (© 2020 John Wiley & Sons, Ltd.)

Spontaneous preterm birth, which can affect up to 20% of all pregnancies, is the greatest contributor to perinatal morbidity and mortality. Infection is the leading pathological cause of spontaneous preterm birth. Infection activates the maternal immune system, resulting in the upregulation of pro-inflammatory and pro-labor mediators that activate myometrial contractions and rupture of fetal membranes. Anti-inflammatory agents therefore have the potential for the prevention of spontaneous preterm birth. Selenium, an essential micronutrient, has been shown to be a potent anti-inflammatory regulator. Notably, clinical and epidemiological studies have suggested a link between selenium and preterm birth. Thus, the aim of this study was to assess the effect of selenite (an inorganic form of selenium) on the expression of pro-inflammatory and pro-labor mediators in human gestational tissues. Human fetal membranes and myometrium were pre-incubated with or without selenite before incubation with the bacterial product lipopolysaccharide (LPS) to stimulate inflammation associated with preterm birth. Selenite blocked LPS-induced expression of pro-inflammatory cytokines and chemokines and enzymes involved in remodelling of myometrium and degradation of fetal membranes. Of note, selenite also suppressed myometrial activation induced by inflammation as evidenced by a decrease in LPS-induced prostaglandin signalling and myometrial cell contractility. These effects of selenite were mediated by the MAPK protein ERK as selenite blunted LPS induced activation of ERK. In conclusion, selenite suppresses key mediators involved in inflammation induced activation of mediators involved in active labor in human fetal membranes and myometrium. These findings support recent clinical studies demonstrating selenium supplementation is associated with decreased incidence of spontaneous preterm birth.

Spontaneous preterm birth is a global health issue affecting up to 20% of pregnancies and leaves a legacy of neurodevelopmental complications. Inflammation has been implicated in a significant proportion of preterm births, where pro-inflammatory insults trigger production of additional pro-inflammatory and pro-labor mediators. Thus, novel therapeutics that can target inflammation may be a novel avenue for preventing preterm birth and improving adverse fetal outcomes. Short-chain fatty acids (SCFAs), such as butyrate and propionate, are dietary metabolites produced by bacterial fermentation of fiber in the gut. SCFAs are known to possess anti-inflammatory properties and have been found to function through G-coupled-receptors and histone deacetylases. Therefore, this study aimed to investigate the effect of SCFAs on pro-inflammatory and pro-labor mediators in an in vitro model of preterm birth. Primary human cells isolated from myometrium and fetal membranes (decidua, amnion mesenchymal and amnion epithelial cells) were stimulated with the pro-inflammatory cytokines tumor necrosis factor alpha (TNF) or interleukin 1B (IL1B). The SCFAs butyrate and propionate suppressed inflammation-induced expression of pro-inflammatory cytokines and chemokines, adhesion molecules, the uterotonic prostaglandin PGF2alpha and enzymes involved in remodeling of myometrium and degradation of the fetal membranes. Notably, propionate and butyrate also suppressed inflammation-induced prostaglandin signaling and myometrial cell contraction. These effects appear to be mediated through suppression of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) activation. These results suggest that the SCFAs may be able to prevent myometrial contractions and rupture of membranes. Further in vivo studies are warranted to identify the efficacy of SCFAs as a novel anti-inflammatory therapeutic to prevent inflammation-induced spontaneous preterm birth., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Understanding fetal programming pathways that underpin the relationship between maternal and offspring mental health necessitates an exploration of potential role of epigenetic variation in early development. Two genes involved in stress response regulation, the glucocorticoid and mineralocorticoid receptors (NR3C1 and NR3C2) have been a focus in understanding stressful exposures and mental health outcomes. Data were obtained from 236 pregnant women from the Mercy Pregnancy Emotional Wellbeing Study (MPEWS), a selected pregnancy cohort, recruited in early pregnancy. Depression was measured using the Structured Clinical Interview for DSM-IV (SCID-IV) and repeated measures of the Edinburgh Postnatal Depression Scale (EPDS). Antidepressant use, stressful events and anxiety symptoms were measured. NR3C1 and NR3C2 DNA methylation was measured in placental and infant buccal samples. Infant cortisol was measured in repeat saliva samples across a task. This study found maternal early pregnancy depressive disorder and symptoms were associated with lower DNA methylation at NR3C2 CpG&#95;24 in placental tissue. There were no significant differences for depression or antidepressant use for DNA methylation of NR3C1. Antenatal depression was associated with lower infant cortisol reactivity at 12 months. DNA methylation in CpG&#95;24 site in NR3C2 in placental samples suppressed the relationship between early maternal depressive symptoms and infant cortisol reactivity. These findings show a relationship between antenatal depression, NR3C2 DNA methylation and infant cortisol response providing support for a specific fetal programming pathway. Further research is required to examine the stability of this epigenetic mark across childhood and long-term mental health outcomes., (Copyright © 2020. Published by Elsevier Ltd.)

Objective: Sleep-disordered breathing (SDB) is characterised by intermittent hypoxemia, sympathetic activation and widespread endothelial dysfunction, sharing pathophysiologic features with the hypertensive disorders of pregnancy. We sought to determine whether coexisting SDB would adversely impact the outcomes of women with gestational hypertension (GH) and preeclampsia (PE), and healthy matched controls., Study Design: Women diagnosed with GH or PE along with BMI- and gestation-matched normotensive controls underwent polysomnography in late pregnancy to establish the presence or absence of SDB (RDI ≥ 5). Clinical outcomes of hypertensive disease severity were compared between groups, and venous blood samples were taken in the third trimester and at delivery to examine for any impact of SDB on the anti-angiogenic markers of PE., Results: Data was available for 17 women with PE, 24 women with GH and 44 controls. SDB was diagnosed in 41% of the PE group, 63% of the GH group and 39% of the control group. Women with PE and co-existing SDB did not have worse outcomes in terms of gestation at diagnosis of PE (SDB = 29.1 (25.9, 32.1) weeks vs. no SDB = 32.0 (29.0, 33.9), p = n.s.) and days between diagnosis of PE and delivery (SDB = 20.0 (4.0, 35.0) days vs. no SDB = 10.5 (9.0, 14.0), p = n.s.). There were also no differences in severity of hypertension, antihypertensive treatment and biochemical, haematological and anti-angiogenic markers of PE between SDB and no SDB groups. Similar results were observed among women with GH. Healthy control women with SDB were no more likely to develop a hypertensive disorder of pregnancy in the later stages of pregnancy (SDB = 5.9% vs. no SDB = 7.4%, p = n.s.). Increasing the threshold for diagnosis of SDB to RDI ≥ 15 did not unmask a worse prognosis., Conclusion: The presence of SDB during pregnancy did not worsen the disease course of GH or PE, and was not associated with high blood pressure or anti-angiogenic markers of hypertensive disease amongst healthy pregnant women. Given the numerous reports of the relationship between SDB and diagnosis of hypertensive disorders of pregnancy, it appears more work is required to distinguish causal, versus confounding, pathways., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Mark Howard receives research support from the Resmed Foundation, Philips Respironics and the Cooperative Research Centre (CRC) for Alertness, Safety and Productivity. Maree Barnes receives research support from AirLiquide Healthcare. This article is not related to either relationship. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Danielle Wilson, Alison Fung, Fergal O’Donoghue, Martha Lappas and Susan Walker declare that no competing interests exist.

Results: GPR91 mRNA expression was significantly higher in myometrium from women during term spontaneous labor compared to no labor. Likewise, in mice, GPR91 mRNA expression was significantly upregulated in myometrium during inflammation-induced preterm labor compared to preterm no labor. In myometrial cells, IL1B and TNF significantly increased GPR91 mRNA expression. Knockdown of GPR91 by siRNA in myometrial cells significantly suppressed the secretion and/or expression of IL1B- and TNF-induced proinflammatory cytokines (GM-CSF, IL1A, IL1B, and IL6) and chemokines (CXCL8 and CCL2), myometrial contractility (expression of the contraction-associated proteins PTGFR and CX43, secretion of the uterotonic PGF 2 α , and in situ collagen gel contraction), and the transcription factor NF- κ B., Conclusion: Our findings demonstrate that GPR91 is involved in the genesis of proinflammatory and prolabor mediators induced by IL1B or TNF and collectively suggest that GPR91 may contribute to augmentation of the labor processes., Competing Interests: The authors have nothing to declare., (Copyright © 2020 Ratana Lim and Martha Lappas.)

The mechanisms underpinning maternal metabolic adaptations to a healthy pregnancy and in gestational diabetes mellitus (GDM) remain poorly understood. We hypothesized that small extracellular vesicles (sEVs) isolated from healthy pregnant women promote islet glucose-stimulated insulin secretion (GSIS) and peripheral insulin resistance in nonpregnant mice and that sEVs from GDM women fail to stimulate insulin secretion and cause exacerbated insulin resistance. Small EVs were isolated from plasma of nonpregnant, healthy pregnant, and GDM women at 24-28 weeks of gestation. We developed a novel approach in nonpregnant mice involving a mini-osmotic pump for continuous 4-day jugular venous infusion of sEVs and determined their effects on glucose tolerance in vivo and islets and skeletal muscle in vitro. Fasting insulin was elevated in mice infused with pregnant sEVs as compared to sEVs from nonpregnant and GDM women. Mice infused with sEVs from GDM women developed glucose intolerance. GSIS was increased in mice infused with healthy pregnancy sEVs compared to mice receiving nonpregnant sEVs. GSIS and muscle basal insulin signaling, and insulin responsiveness were attenuated in mice infused with GDM sEVs. sEVs represent a novel mechanism regulating maternal glucose homeostasis in pregnancy and we speculate that altered sEV content contributes to the development of GDM., (© 2020 Federation of American Societies for Experimental Biology.)

Gestational diabetes mellitus (GDM) is a global health issue, whereby pregnant women are afflicted with carbohydrate intolerance with first onset during pregnancy. GDM is characterized by maternal peripheral insulin resistance, thought to be driven by low-grade maternal inflammation. Nobiletin, a polymethoxylated flavonoid, possesses potent glucose-sensitizing and anti-inflammatory properties; however, its effects in GDM have not been assessed. The present study aimed to determine the effects of nobiletin on glucose metabolism and inflammation associated with GDM in both in vitro human tissues and an in vivo animal model of GDM. In vitro, treatment with nobiletin significantly improved TNF-impaired glucose uptake in human skeletal muscle, and suppressed mRNA expression and protein secretion of pro-inflammatory cytokines and chemokines in human placenta and visceral adipose tissue (VAT). Mechanistically, nobiletin significantly inhibited Akt and Erk activation in placenta, and NF-κB activation in VAT. In vivo, GDM mice treated with 50 mg/kg nobiletin daily via oral gavage from gestational day (gd) 1-17 or via i.p. injections from gd 10-17 significantly improved glucose tolerance. Pregnant GDM mice treated with nobiletin from either gd 1-17 or gd 10-17 exhibited significantly suppressed mRNA expression of pro-inflammatory cytokines and chemokines in placenta, VAT and subcutaneous adipose tissue (SAT). Using a quantitative mass spectrometry approach, we identified differentially abundant proteins associated with the effect of nobiletin in vivo. Together, these studies demonstrate that nobiletin improves glucose metabolism and reduces inflammation associated with GDM and may be a novel therapeutic for the prevention of GDM., (© 2020 The Author(s).)

Mucosal-associated invariant T (MAIT) cells are important for immune responses against microbial infections. Although known to undergo marked numerical changes with age in humans, our understanding of how MAIT cells are altered during different phases across the human life span is largely unknown. Although also abundant in the tissues, our study focuses on MAIT cell analyses in blood. Across the human life span, we show that naive-like MAIT cells in umbilical cord blood switch to a central/effector memory-like profile that is sustained into older age. Whereas low-grade levels of plasma cytokine/chemokine were apparent in older donors (>65 y old), surprisingly, they did not correlate with the ex vivo MAIT hyperinflammatory cytokine profile observed in older adults. Removal of MAIT cells from older individuals and an aged environment resulted in the reversal of the baseline effector molecule profile comparable with MAIT cells from younger adults. An upregulated basal inflammatory profile accounted for reduced Escherichia coli -specific responses in aged MAIT cells compared with their young adult counterparts when fold change in expression levels of GzmB, CD107a, IFN-γ, and TNF was examined. However, the magnitude of antimicrobial MR1-dependent activation remained as potent and polyfunctional as with younger adults. Paired TCRαβ analyses of MAIT cells revealed large clonal expansions in older adults and tissues that rivalled, remarkably, the TCRαβ repertoire diversity of virus-specific CD8 + T cells. These data suggest that MAIT cells in older individuals, although associated with large clonal TCRαβ expansions and increased baseline inflammatory potential, demonstrate plasticity and provide potent antimicrobial immunity., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Objective: To determine whether the presence of co-existing sleep-disordered breathing (SDB) is associated with worse perinatal outcomes among women diagnosed with a hypertensive disorder of pregnancy (HDP), compared with normotensive controls., Study Design: Women diagnosed with HDP (gestational hypertension or preeclampsia) and BMI- and gestation-matched controls underwent polysomnography in late pregnancy to determine if they had coexisting SDB. Fetal heart rate (FHR) monitoring accompanied the sleep study, and third trimester fetal growth velocity was assessed using ultrasound. Cord blood was taken at delivery to measure key regulators of fetal growth., Results: SDB was diagnosed in 52.5% of the HDP group (n = 40) and 38.1% of the control group (n = 42); p = .19. FHR decelerations were commonly observed during sleep, but the presence of SDB did not increase this risk in either the HDP or control group (HDP group-SDB = 35.3% vs. No SDB = 40.0%, p = 1.0; control group-SDB = 41.7% vs. No SDB = 25.0%, p = .44), nor did SDB affect the total number of decelerations overnight (HDP group-SDB = 2.7 ± 1.0 vs. No SDB = 2.8 ± 2.1, p = .94; control group-SDB = 2.0 ± 0.8 vs. No SDB = 2.0 ± 0.7, p = 1.0). Fetal growth restriction was the strongest predictor of fetal heart rate events during sleep (aOR 5.31 (95% CI 1.26-22.26), p = .02). The presence of SDB also did not adversely affect fetal growth; in fact among women with HDP, SDB was associated with significantly larger customised birthweight centiles (43.2% ± 38.3 vs. 16.2% ± 27.0, p = .015) and fewer growth restricted babies at birth (30% vs. 68.4%, p = .026) compared to HDP women without SDB. There was no impact of SDB on measures of fetal growth for the control group. Cord blood measures of fetal growth did not show any adverse effect among women with SDB, either in the HDP or control group., Conclusion: We did not find that the presence of mild SDB worsened fetal acute or longitudinal outcomes, either among women with HDP or BMI-matched normotensive controls. Unexpectedly, we found the presence of SDB conferred a better prognosis in HDP in terms of fetal growth. The fetus has considerable adaptive capacity to withstand in utero hypoxia, which may explain our mostly negative findings. In addition, SDB in this cohort was mostly mild. It may be that fetal sequelae will only be unmasked in the setting of more severe degrees of SDB and/or underlying placental disease., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Mark Howard receives research support from the Resmed Foundation, Philips Respironics and the Cooperative Research Centre (CRC) for Alertness, Safety and Productivity. Maree Barnes receives research support from AirLiquide Healthcare. This article is not related to either relationship. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Danielle Wilson, Alison Fung, Fergal O'Donoghue, Martha Lappas and Susan Walker declare that no competing interests exist.

Preterm birth is a major contributor to neonatal deaths and associated long-term morbidities for the survivors, yet therapies remain elusive, given our incomplete understanding of the mechanisms driving human labor and delivery. Human labor is an inflammatory process, and we investigated whether IRG1 (immunoresponsive gene-1) plays a role in these processes. We demonstrate that IRG1 mRNA and protein expression is significantly increased in myometrium with human term labor, compared to no labor samples, and with preterm (LPS) labor in a mouse model. Pro-labor mediators such as pro-inflammatory cytokines TNF and IL1B, and TLR ligands fsl-1, flagellin, LPS, and poly(I:C) also increased IRG1 mRNA expression in myometrial explants. IRG1 silencing, using siRNA in primary myometrial cells, displayed a decrease in the expression of inflammation-induced pro-inflammatory cytokines (IL1A, IL6), chemokines (CCL2, CXCL1, CXCL8), adhesion molecules (ICAM1, VCAM1), and contractility (PTGFR mRNA expression, prostaglandin F 2α release, and in situ gel contraction assay). Our results suggest that IRG1 is involved when pro-labor mediators activate the inflammatory processes of human labor, warranting further investigation.

Preterm birth is a global healthcare challenge. Spontaneous preterm birth (sPTB) is commonly caused by inflammation, yet there are currently no effective therapies available. The Bromodomain and Extra-Terminal motif (BET) proteins, Bromodomain-containing protein (Brd) 2 (Brd2), Brd3 and Brd4 regulate inflammation in non-gestational tissues. The roles of Brd2-4 in human pregnancy are unknown. Using human and mouse models, the present study has identified the Brd proteins part of the process by which inflammation induces parturition. Using human clinical samples, we demonstrate that labor and infection increase the expression of Brds in the uterus and fetal membranes. In primary human myometrial, amnion and decidual cells, we found that global Brd protein inhibition, as well as selective inhibition of Brds, suppressed inflammation-induced expression of mediators involved in myometrial contractions and rupture of fetal membranes. Importantly, studies in the mouse model demonstrate that the pan-Brd inhibitor JQ1 reduced intrauterine inflammation induced by bacterial endotoxin LPS as well as decreasing the effectiveness of LPS to induce parturition. These results implicate BET proteins as novel therapeutic targets for reducing inflammation associated with spontaneous preterm labor., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: We aimed to investigate the association of breastfeeding on postpartum glucose levels and lipid profiles in women diagnosed with gestational diabetes mellitus (GDM) and women without GDM., Methods: We performed a secondary analysis of a cohort study of 243 women, 159 women with GDM and 84 normally glucose tolerant women between 2012 and 2017. At approximately 6-10 weeks postpartum, we measured fasting blood glucose and plasma lipid levels. Breastfeeding behaviour was self-defined as exclusive breastfeeding or not exclusive breastfeeding., Results: The mean (SD) glucose in the group of women who breastfed exclusively was 4.6 (0.49) mmol/L, compared to 4.9 (0.58) mmol/L (95% CI 0.45, 0.15, p  <  0.001) among women who did not exclusively breastfeed. Among women with GDM, the reduction in fasting glucose in women who were breastfeeding was 0.22 mmol/L (95% CI 0.39, 0.05, p  = 0.004), and in women who were not GDM, the reduction was 0.14 mmol/L (95% CI 0.37, 0.09, p  = 0.24,). After adjustment for GDM status in pregnancy, maternal body mass index (BMI), maternal age and ethnicity, and exclusive breastfeeding was associated with a decreased fasting glucose of 0.19 (95% CI 0.318, 0.061, p  = 0.004). After similar adjustment, there was no significant difference in triglycerides, high density lipoprotein cholesterol or low-density lipoprotein cholesterol between women who were breastfeeding and women who were not breastfeeding., Conclusions: Breastfeeding is associated with a reduction in fasting glucose levels postpartum, but not maternal lipid profile. Breastfeeding may play a role in reducing glucose intolerance in women who have had GDM., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s). 2019.)

Gestational diabetes mellitus (GDM) imposes serious short- and long-term health problems for mother and baby. An effective therapeutic that can reduce the incidence of GDM and improve long-term maternal and fetal outcomes is a major research priority, crucially important for public health. A lack of knowledge about the underlying pathophysiology of GDM has hampered the development of such therapeutics. What we do know, however, is that maternal insulin resistance, low-grade inflammation and endothelial cell dysfunction are three central features of pregnancies complicated by GDM. Indeed, data generated over the past decade have implicated a number of candidate regulators of insulin resistance, inflammation and endothelial cell dysfunction in placenta, maternal adipose tissue and skeletal muscle. These include nuclear factor-κB (NF-κB), peroxisome proliferator-activated receptors (PPARs), sirtuins (SIRTs), 5' AMP-activated protein kinase (AMPK), glycogen synthase kinase 3 (GSK3), PI3K/mTOR, inflammasome and endoplasmic reticulum (ER) stress. In this review, the identification of these as key modulators of GDM will be discussed. The biochemical pathways involved in the formation of these may represent potential sites for intervention that may translate to therapeutic interventions to prevent the development of GDM.

Scope: Gestational diabetes mellitus (GDM), which affects up to 20% of pregnant women, is associated with maternal peripheral insulin resistance, low-grade inflammation, and oxidative stress. The flavonoid naringenin has potent anti-diabetic, anti-inflammatory, and anti-oxidative properties; however, its effects in GDM remain unknown. The study aimed to determine the effects of naringenin on glucose metabolism, inflammation, and oxidative stress associated with GDM both in vitro and in vivo., Methods and Results: In vitro, human tissue samples obtained at term elective Caesarean section are stimulated with tumour necrosis factor alpha (TNF) to develop a GDM-like environment. Naringenin treatment significantly improves TNF-impaired glucose uptake in skeletal muscle. In placenta and visceral adipose tissue (VAT), naringenin significantly reduces expression of pro-inflammatory cytokines and chemokines and increases antioxidant mRNA expression. Mechanistically, naringenin suppresses nuclear factor κB activation. In vivo, pregnant heterozygous db/+ mice are used to model GDM. Daily intraperitoneal injections of GDM mice with naringenin from gestational day 10-17 significantly improve glucose tolerance, reduces IL1A mRNA expression, and increases antioxidant mRNA expression in placenta, VAT, and subcutaneous adipose tissue., Conclusion: Naringenin is shown to improve insulin sensitivity, inflammation, and oxidative stress associated with GDM and shows promise as a novel preventive therapeutic., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Although γδ T cells comprise up to 10% of human peripheral blood T cells, questions remain regarding their role in disease states and T-cell receptor (TCR) clonal expansions. We dissected anti-viral functions of human γδ T cells towards influenza viruses and defined influenza-reactive γδ TCRs in the context of γδ-TCRs across the human lifespan., Methods: We performed 51 Cr-killing assay and single-cell time-lapse live video microscopy to define mechanisms underlying γδ T-cell-mediated killing of influenza-infected targets. We assessed cytotoxic profiles of γδ T cells in influenza-infected patients and IFN-γ production towards influenza-infected lung epithelial cells. Using single-cell RT-PCR, we characterised paired TCRγδ clonotypes for influenza-reactive γδ T cells in comparison with TCRs from healthy neonates, adults, elderly donors and tissues., Results: We provide the first visual evidence of γδ T-cell-mediated killing of influenza-infected targets and show distinct features to those reported for CD8 + T cells. γδ T cells displayed poly-cytotoxic profiles in influenza-infected patients and produced IFN-γ towards influenza-infected cells. These IFN-γ-producing γδ T cells were skewed towards the γ9δ2 TCRs, particularly expressing the public GV9-TCRγ, capable of pairing with numerous TCR-δ chains, suggesting their significant role in γδ T-cell immunity. Neonatal γδ T cells displayed extensive non-overlapping TCRγδ repertoires, while adults had enriched γ9δ2-pairings with diverse CDR3γδ regions. Conversely, the elderly showed distinct γδ-pairings characterised by large clonal expansions, a profile also prominent in adult tissues., Conclusion: Human TCRγδ repertoire is shaped by age, tissue compartmentalisation and the individual's history of infection, suggesting that these somewhat enigmatic γδ T cells indeed respond to antigen challenge., Competing Interests: The authors declare no conflict of interest.

Obesity worsens the age-related tendency towards cardiovascular disease and diabetes. Older adults are vulnerable to medication adverse effects. Intentional weight loss in older adults with obesity has been shown to improve cardiovascular and glycaemic markers. The effect of rapid weight loss induced by very-low-calorie diets (VLCDs) on these markers has not been evaluated in this group. In this 12-week study, participants were randomized to one of healthy eating, hypocaloric diet or VLCD, all combined with three times weekly exercise (Ex/HE, Ex/Diet, Ex/VLCD, respectively). The effects of these interventions on weight, blood pressure, lipids, glucose and HbA 1c , inflammatory markers and cardiovascular and diabetes medication changes were measured. Weight loss was 3.7%, 5.1% and 11.1% in Ex/HE, Ex/Diet and Ex/VLCD, respectively. There were significant improvements in HbA 1c in all groups, but by the greatest degree in Ex/VLCD (0.18 ± 0.07%, 0.18 ± 0.06% and 0.59 ± 0.13%, respectively). Similar patterns were seen in total cholesterol (0.13 ± 0.15, 0.21 ± 0.11 and 0.53 ± 0.13 mmol/L, respectively, P = .047), triglycerides (0.35 ± 0.13, 0.20 ± 0.10 and 0.51 ± 0.09 mmol/L, respectively, P = .011) and systolic blood pressure (9 ± 2, 2 ± 3 and 14 ± 3 mmHg respectively, P = .025). There were no between-group differences in fasting glucose, high-density lipoprotein (HDL) cholesterol, LDL-C and inflammatory markers. Reductions in anti-hypertensive or diabetes medication were made in 4/29, 7/36 and 16/37 participants in Ex/HE, Ex/Diet and Ex/VLCD, respectively (P = .017). Significant weight loss achieved with a VLCD gave rise to improvements in multiple cardiovascular risk markers, despite reduction in medication. Weight loss is an under-utilized method of cardiovascular risk management in this group., (© 2019 World Obesity Federation.)