Pharmacological blockade of the interleukin-1 receptor suppressed Escherichia coli lipopolysaccharide-induced neuroinflammation in preterm fetal sheep.

BACKGROUND: Intraamniotic inflammation is associated with preterm birth, especially in cases occurring before 32 weeks' gestation, and is causally linked with an increased risk for neonatal mortality and morbidity. Targeted anti-inflammatory interventions may assist in improving the outcomes for pregnancies impacted by intrauterine inflammation. Interleukin-1 is a central upstream mediator of inflammation. Accordingly, interleukin-1 is a promising candidate target for intervention therapies and has been targeted previously using the interleukin-1 receptor antagonist, anakinra. Recent studies have shown that the novel, noncompetitive, allosteric interleukin-1 receptor inhibitor, rytvela, partially resolved inflammation associated with preterm birth and fetal injury. In this study, we used a preterm sheep model of chorioamnionitis to investigate the anti-inflammatory efficacy of rytvela and anakinra, administered in the amniotic fluid in the setting of intraamniotic Escherichia coli lipopolysaccharide exposure. OBJECTIVE: We hypothesized that both rytvela and anakinra would reduce lipopolysaccharide-induced intrauterine inflammation and protect the fetal brain. STUDY DESIGN: Ewes with a singleton fetus at 105 days of gestation (term is ∼150 days) were randomized to one of the following groups: (1) intraamniotic injections of 2 mL saline at time=0 and time=24 hours as a negative control group (saline group, n=12); (2) intraamniotic injection of 10 mg Escherichia coli lipopolysaccharide in 2 mL saline and intraamniotic injections of 2 mL saline at time=0 hours and time=24 hours as an inflammation positive control group (lipopolysaccharide group, n=11); (3) intraamniotic injection of Escherichia coli lipopolysaccharide in 2 mL saline and intraamniotic injections of 2.5 mg rytvela at time=0 hours and time=24 hours to test the anti-inflammatory efficacy of rytvela (lipopolysaccharide + rytvela group, n=10); or (4) intraamniotic injection of Escherichia coli lipopolysaccharide in 2 mL saline and intraamniotic injections of 100 mg anakinra at time=0 hours and time=24 hours to test the anti-inflammatory efficacy of anakinra (lipopolysaccharide + anakinra group, n=12). Amniotic fluid was sampled at time 0, 24, and 48 hours (ie, at each intervention and at delivery). Fetal umbilical cord blood was collected at delivery for differential blood counts and chemical studies. Inflammation was characterized by the analysis of fetal tissue cytokine and chemokine levels using quantitative polymerase chain reaction, enzyme-linked inmmunosorbent assay, and histology. The primary study outcome of interest was the assessment of anakinra and rytvela brain-protective effects in the setting of Escherichia coli lipopolysaccharide-induced intrauterine inflammation. Secondary outcomes of interest were to assess protection from fetal and intrauterine (ie, amniotic fluid, chorioamnion) inflammation. RESULTS: Intraamniotic administration of lipopolysaccharide caused inflammation of the fetal lung, brain, and chorioamnionitis in preterm fetal sheep. Relative to treatment with saline only in the setting of lipopolysaccharide exposure, intraamniotic administration of both rytvela and anakinra both significantly prevented periventricular white matter injury, microglial activation, and histologic chorioamnionitis. Anakinra showed additional efficacy in inhibiting fetal lung myeloperoxidase activity, but its use was associated with metabolic acidaemia and reduced fetal plasma insulin-like growth factor-1 levels at delivery. CONCLUSION: Intraamniotic administration of rytvela or anakinra significantly inhibited fetal brain inflammation and chorioamnionitis in preterm fetal sheep exposed to intraamniotic lipopolysaccharide. In addition, anakinra treatment was associated with potential negative impacts on the developing fetus.

Characterisation of Mid-Gestation Amniotic Fluid Cytokine and Bacterial DNA Profiles in Relation to Pregnancy Outcome in a Small Australian Cohort.

A well-established association exists between intrauterine bacteria and preterm birth. This study aimed to explore this further through documenting bacterial and cytokine profiles in Australian mid-gestation amniotic fluid samples from preterm and term births. Samples were collected during amniocenteses. DNA was extracted and the full-length 16S rRNA gene was amplified and sequenced. Levels of the cytokines IL-1ß, IL-6, IL-10, TNF-α and MCP-1 were determined using the Milliplex MAGPIX system. Bacterial DNA profiles were low in diversity and richness, with no significant differences observed between term and preterm samples. No differences in the relative abundance of individual OTUs between samples were identified. IL-1ß and TNF-α levels were significantly higher in samples containing reads mapping to Sphingomonas sp.; however, this result should be interpreted with caution as similar reads were also identified in extraction controls. IL-6 levels were significantly increased in samples with reads that mapped to Pelomonas sp., whilst TNF-α levels were elevated in fluid samples from pregnancies that subsequently delivered preterm. Bacterial DNA unlikely to have originated from extraction controls was identified in 20/31 (64.5%) mid-gestation amniotic fluid samples. Bacterial DNA profiles, however, were not predictive of preterm birth, and although cytokine levels were elevated in the presence of certain genera, the biological relevance of this remains unknown.

Longitudinal effects of prenatal exposure to plastic-derived chemicals and their metabolites on asthma and lung function from childhood into adulthood.

BACKGROUND AND OBJECTIVE: Environmental exposure to phthalates and bisphenol A (BPA), chemicals used in the production of plastics, may increase risk for asthma and allergies. However, little is known about the long-term effects of early life exposure to these compounds. We investigated if prenatal exposure to these compounds was associated with asthma, allergy and lung function outcomes from early childhood into adulthood in a cohort study. METHODS: Maternal serum samples collected from 846 pregnant women in the Raine Study were assayed for BPA and phthalate metabolites. The children of these women were followed up at 5, 13 and 22 years where spirometry and respiratory questionnaires were conducted to determine asthma and allergy status. Lung function trajectories were derived from longitudinal spirometry measurements. Multinomial logistic regression and weighted quantile sum regression was used to test associations of individual and chemical mixtures with asthma phenotypes and lung function trajectories. RESULTS: Effects of prenatal BPA and phthalates on asthma phenotypes were seen in male offspring, where BPA was associated with increased risk for persistent asthma, while mono-iso-butyl phthalate and mono-iso-decyl phthalate was associated with increased risk for adult asthma. Prenatal BPA had no effect on lung function trajectories, but prenatal phthalate exposure was associated with improved lung function. CONCLUSION: Prenatal BPA exposure was associated with increased likelihood of persistent asthma in males, while prenatal phthalate exposure was associated with increased likelihood of adult asthma in males. Results suggest that prenatal exposure to prenatal BPA and phthalates affect asthma risk, particularly in males, however lung function was not adversely affected.

Associations between Prenatal Exposure to Phthalates and Features of the Metabolic Syndrome in Males from Childhood into Adulthood.

Phthalate metabolites are detectable within the majority of the population. Evidence suggests that a prenatal exposure to phthalates may be associated with the subsequent risks of obesity and elevated blood pressure. We hypothesised that a prenatal exposure to phthalates would lead to an increase in adverse cardiometabolic parameters through childhood and adulthood. The maternal serum phthalate measurements from the stored samples taken from Gen1 mothers at 18 and 34 weeks gestation were examined in relation to the cardiometabolic measures in 387 male offspring from the Raine Study. Data from the Gen2 follow-ups between 3 and 27 years were used. The primary outcomes were analysed longitudinally using linear mixed models for the repeated measures. Non-alcoholic fatty liver disease (NAFLD) was assessed at 17 years using logistic regression. A consistent positive relationship was observed between a prenatal exposure to mono-carboxy-iso-octyl phthalate (MCiOP) through adolescence into adulthood with systolic blood pressure. There were no other consistent cardiovascular associations. Mid-levels of prenatal exposures to Mono-n-butyl phthalate (MnBP) were associated with a greater incidence of NAFLD. Detectable Mono-3-carboxypropyl phthalate (MCPP) was associated with a lower serum HDL-C through late childhood into adulthood, while a higher prenatal exposure to mono-iso-butyl phthalate (MiBP), was associated with a higher LDL-C at 22 years of age. A mid-level prenatal exposure to mono-2-ethylhexyl phthalate (MEHP) metabolites was associated with higher insulin in adulthood, while a higher prenatal exposure to the sum of the Di-(2-ethyl-hexyl) phthalate (DEHP) and Di-iso-nonyl phthalate (DiNP) metabolites was associated with higher fasting serum glucose in adulthood. In conclusion, our study demonstrated that higher prenatal phthalate exposures to some phthalate metabolites was associated with some adverse metabolic profiles through adolescence into adulthood, although the consistent themes were limited to a few metabolites and the outcomes of systolic blood pressure, fasting insulin and glucose.

Asthma and allergies in a cohort of adolescents conceived with ART.

RESEARCH QUESTION: Are asthma and allergies more common in adolescents conceived with assisted reproductive technologies (ART) compared with adolescents conceived without? DESIGN: The Growing Up Healthy Study (GUHS) is a prospective cohort study including ART-conceived offspring born between 1991 and 2001 in Perth, Australia. Their long-term health outcomes, including asthma and allergy parameters, were compared with those of their counterparts conceived without ART from the Raine Study Generation 2 (Gen2), born in 1989-1991. At age 14, 152 GUHS and 1845 Gen2 participants completed the following assessments: the International Studies of Asthma and Allergies in Childhood (ISAAC) questionnaire, spirometry, methacholine challenge testing and skin prick testing (SPT). RESULTS: No differences were detected in the prevalence of current asthma (7.7% versus 10.8%, adjusted odds ratio [aOR] 0.82 (95% CI 0.44-1.52), P = 0.530). Spirometry-measured lung volumes were larger in the ART adolescents. Bronchial hyperresponsiveness was less prevalent in the ART cohort (8.8 versus 18.6%, P = 0.006). Current allergic rhinoconjunctivitis (ARC) rates were significantly higher in the ART cohort (32.4% versus 25.2%, aOR 1.52 [95% CI 1.03-2.26], P = 0.036), with no cohort differences in atopic dermatitis. Food allergies were more prevalent in the ART cohort (20.7 versus 10.9%, aOR 1.89 [95% CI 1.17-3.06], P = 0.010) with more adolescents having a positive SPT (68.0% versus 45.4%, aOR 3.03 [95% 1.99-4.63], P < 0.001). CONCLUSIONS: This study reports no differences in asthma prevalence, slightly altered lung function, an increase in ARC, food allergies and positive SPT in the ART-conceived adolescents. These findings are important to families and healthcare providers and may open up possibilities for targeted screening and treatment. Further studies are required to confirm these findings.

Randomised controlled trial of nebulised gentamicin in children with bronchiectasis.

AIM: Following trials of inhaled antibiotics in adults, this study investigates the efficacy of nebulised gentamicin to improve respiratory function in children with bronchiectasis. METHODS: This is a randomised, double-blind, placebo-controlled, crossover trial of 12-week nebulised placebo/gentamicin, 6-week washout, 12-week gentamicin/placebo. Participants were children (5-15 years) with bronchiectasis, chronic infection (any pathogen), and able to perform spirometry from a hospital bronchiectasis clinic. Primary outcomes were change in forced expiratory volume in 1 s (FEV1 ) and hospitalisation days. Secondary outcomes included sputum bacterial density, sputum inflammatory markers, additional antibiotics and symptom severity. Analyses were on an intention-to-treat basis. RESULTS: Fifteen children (mean 11.7-years-old) completed the study. There was no significant change in mean FEV1 (56%/55%, P = 0.38) or annual rate of hospital admissions (1.1/0, P = 0.12) between gentamicin and placebo, respectively. However, Haemophilus influenzae sputum growth (27% vs. 80%, P = 0.002) and bacterial density (2.4 log10 cfu/mL lower P < 0.001) improved with gentamicin. Sputum inflammatory markers interleukin-1ß (P < 0.001), interleukin-8 (P < 0.001) and tumour necrosis factor-α (P = 0.003) were lower with gentamicin. Poor recruitment limited study power and treatment adherence was challenging for this cohort. CONCLUSIONS: In this crossover study of nebulised gentamicin in children with bronchiectasis, there was a reduction in sputum bacterial density and inflammation. However, there were no major improvements in clinical outcomes and adherence was a challenge.

Asthma and allergies in offspring conceived by ART: a systematic review and meta-analysis.

BACKGROUND: Currently, 1 in 25 children born in Australia are conceived through ARTs such as IVF and ICSI. Worldwide over 8 million children have been born after ART. There is evidence that these children are at an increased risk of congenital malformations, preterm birth, low birth weight and neonatal morbidity. However, studies on long-term health outcomes of offspring conceived after ART are lacking. Atopic disorders, such as asthma, atopic dermatitis and various allergies are increasingly common within society, and concerns have been raised that ART increases the risk of atopy amongst offspring. OBJECTIVE AND RATIONALE: The aim of this study was to systematically summarise and quantify the risk of atopic disorders in offspring conceived with ART compared to those conceived without ART. SEARCH METHODS: A systematic review was conducted according to the PRISMA guidelines. Several systematic searches were performed in the following international databases: Medline, Embase, Cinahl, PsychINFO, AMED, Global Health and ISI Web of Science. Search terms utilised were all terms pertaining to ART, IVF, ICSI, asthma, atopic dermatitis and allergies. The search period was 1978-2021. Included observational studies stated a primary outcome of asthma or allergies in offspring conceived after ART, with a comparison group conceived without ART. Individual studies were scored on quality and risk of bias, using the Newcastle-Ottawa scale (NOS). OUTCOMES: There were 26 studies which met the inclusion criteria; of these, 24 studies investigated asthma in offspring conceived after ART. While 10 studies, including the two largest population-based studies, reported a significantly increased risk of asthma in offspring conceived after ART (adjusted odds ratio (aOR) range: 1.20-2.38), 14 smaller cohort studies found no difference (aOR range 0.70-1.27). In the meta-analysis of the 14 highest-quality studies (NOS ≥ 7), a modest yet significantly increased risk of asthma was demonstrated in offspring conceived after ART [risk ratio (RR) 1.28 (1.08-1.51)]. Although heterogeneity in these 14 studies was high (I2 = 85%), the removal of outliers and high weight studies significantly reduced heterogeneity (I2 = 0% and I2 = 34% respectively) while still demonstrating a significantly increased risk [RR 1.19 (1.10-1.28) and RR 1.31 (1.03-1.65), respectively]. The increased asthma risk was also observed in most subgroup and sensitivity analyses. The allergy rates were not increased in offspring conceived after ART in 9 of 12 studies (aOR range 0.60-1.30). In summary, the findings of this systematic review and meta-analysis suggest a trend towards a significantly increased risk of asthma, but not allergies, in offspring conceived after ART. There was no evidence of publication bias in the asthma studies and minimal evidence of publication bias in the allergy studies (both P > 0.05). WIDER IMPLICATIONS: Asthma brings considerable burden to the quality of life of individuals and to society. Hence, it is of great importance to untangle potential causal pathways. Although ART use is common, knowledge about its long-term health effects is required to provide evidence-based advice to couples considering ART, and to be vigilant for any potential adverse health effects on offspring conceived after ART.

Associations between Prenatal Exposure to Phthalates and Timing of Menarche and Growth and Adiposity into Adulthood: A Twenty-Years Birth Cohort Study.

Phthalates are ubiquitous environmental chemicals with endocrine disrupting properties and potentially obesogenic effects. We hypothesised that antenatal phthalate exposure may influence growth and adiposity patterns in girls through childhood into adolescence. Among 1342 Raine Study singleton females, 462 had maternal serum and at least one outcome available up to 20 years of age. Individuals' maternal serum collected at 18 and 34 weeks gestation was pooled and analyzed for concentrations of 32 metabolites of 15 phthalate diesters. Cox regression and linear models were used to determine associations between maternal phthalate levels and age at menarche, change in height and weight z-scores between birth and two years, height from birth to 20 years, BMI from two to 20 years, deviation from mid-parental height at age 20 and DEXA scan measures at age 20. Weak negative associations were detected with some phthalate metabolites and change in height and weight z-score during infancy. Weak positive associations between some of the high molecular weight phthalate metabolites and height z-score were detected during childhood. While still within the normal range, age at menarche was slightly delayed in girls with higher prenatal exposure to the higher molecular weight phthalate metabolites. We derived some associations between prenatal phthalate exposure with early growth patterns and age at menarche.

Prenatal Testosterone Associates With Blood Pressure in Young Adults: A Prospective Cohort Study.

[Figure: see text].

The relationship between oxytocin blood concentrations and antidepressants over pregnancy and the postpartum.

Antidepressant treatment of perinatal depression is increasingly common and accepted in clinical guidelines. It has been suggested that serotonergic antidepressants may effect changes in the oxytocinergic system, including oxytocin levels, and that this may be one of the beneficial mechanisms of action for these drugs. Furthermore, oxytocin has been associated with the quality of the parent-child relationship, which may be important in treatment of perinatal depression. This study will explore if there is a relationship between antidepressant use over the perinatal period and oxytocin levels. Data from a pregnancy cohort study are used from 279 women across three groups: women taking antidepressants in pregnancy (n = 48), women with untreated depression (n = 31) and healthy control women (n = 200). Data included antidepressant use, maternal depression and oxytocin plasma concentrations in pregnancy and up to 12 months postpartum. We found that concurrent oxytocin blood concentrations were not associated with perinatal antidepressant use. However, oxytocin blood concentrations increased more steeply in those on antidepressants across the perinatal period compared to control women. A steeper increase for Selective Serotonergic Reuptake Inhibitors was observed, however, this effect was on the boarder of statistical significance. In conclusion, although antidepressant use and oxytocin was not associated at any time point, women taking antidepressants during pregnancy had larger increases in oxytocin over the perinatal period. Future research could examine specific agents and class of antidepressant and the relationship to parenting.

A specific bacterial DNA signature in the vagina of Australian women in midpregnancy predicts high risk of spontaneous preterm birth (the Predict1000 study).

BACKGROUND: Intrauterine infection accounts for a quarter of the cases of spontaneous preterm birth; however, at present, it is not possible to efficiently identify pregnant women at risk to deliver preventative treatments. OBJECTIVE: This study aimed to establish a vaginal microbial DNA test for Australian women in midpregnancy that will identify those at increased risk of spontaneous preterm birth. STUDY DESIGN: A total of 1000 women with singleton pregnancies were recruited in Perth, Australia. Midvaginal swabs were collected between 12 and 23 weeks' gestation. DNA was extracted for the detection of 23 risk-related microbial DNA targets by quantitative polymerase chain reaction. Obstetrical history, pregnancy outcome, and demographics were recorded. RESULTS: After excluding 64 women owing to losses to follow-up and insufficient sample for microbial analyses, the final cohort consisted of 936 women of predominantly white race (74.3%). The overall preterm birth rate was 12.6% (118 births); the spontaneous preterm birth rate at <37 weeks' gestation was 6.2% (2.9% at ≤34 weeks' gestation), whereas the preterm premature rupture of the membranes rate was 4.2%. No single individual microbial target predicted increased spontaneous preterm birth risk. Conversely, women who subsequently delivered at term had higher amounts of Lactobacillus crispatus, Lactobacillus gasseri, or Lactobacillus jensenii DNA in their vaginal swabs (13.8% spontaneous preterm birth vs 31.2% term; P=.005). In the remaining women, a specific microbial DNA signature was identified that was strongly predictive of spontaneous preterm birth risk, consisting of DNA from Gardnerella vaginalis (clade 4), Lactobacillus iners, and Ureaplasma parvum (serovars 3 and 6). Risk prediction was improved if Fusobacterium nucleatum detection was included in the test algorithm. The final algorithm, which we called the Gardnerella Lactobacillus Ureaplasma (GLU) test, was able to detect women at risk of spontaneous preterm birth at <37 and ≤34 weeks' gestation, with sensitivities of 37.9% and 44.4%, respectively, and likelihood ratios (plus or minus) of 2.22 per 0.75 and 2.52 per 0.67, respectively. Preterm premature rupture of the membranes was more than twice as common in GLU-positive women. Adjusting for maternal demographics, ethnicity, and clinical history did not improve prediction. Only a history of spontaneous preterm birth was more effective at predicting spontaneous preterm birth than a GLU-positive result (odds ratio, 3.6). CONCLUSION: We have identified a vaginal bacterial DNA signature that identifies women with a singleton pregnancy who are at increased risk of spontaneous preterm birth and may benefit from targeted antimicrobial therapy.

The influence of prenatal exposure to phthalates on subsequent male growth and body composition in adolescence.

Phthalates are ubiquitous environmental chemicals with predominantly anti-androgenic, and potentially obesogenic effects. We hypothesised that antenatal phthalate exposure may influence subsequent boy's growth and body composition through childhood and adolescence. Among 1399 singleton males from the Raine Study, 410 had maternal serum and at least one height, BMI or DEXA outcome available after birth and up to 20 years of age. Maternal serum collected at 18 and 34 weeks' gestation was pooled, and analyzed for concentrations of 32 metabolites of 15 phthalate diesters. Their serum concentrations were categorized into undetectable/detectable levels or tertiles. Linear mixed models were used to determine associations between maternal serum phthalate levels and longitudinal height and body mass index (BMI) z-scores in boys from birth to 20 years of age (n = 250 and n = 295 respectively). Linear regression was used to determine associations between maternal phthalate levels and deviation from mid-parental height (n = 177) and DEXA scan outcomes (n = 191) at the 20 year follow-up. Weak positive associations of participants height z-score increase were detected with exposure to some phthalate metabolites in particular to the lower molecular weight phthalate metabolites. Less consistent findings, by mixed model analyses, were detected for BMI and body composition, by dual energy X-ray absorptiometry (DEXA), with some positive associations of phthalate metabolites with BMI and some negative associations with DEXA fat tissue measures, although no consistent findings were evident. In conclusion, we derived some associations of childhood growth with prenatal phthalate exposure, particularly with respect to the lower molecular weight phthalate metabolites.

Targeting Toll-like receptor-4 to tackle preterm birth and fetal inflammatory injury.

Every year, 15 million pregnancies end prematurely, resulting in more than 1 million infant deaths and long-term health consequences for many children. The physiological processes of labour and birth involve essential roles for immune cells and pro-inflammatory cytokines in gestational tissues. There is compelling evidence that the mechanisms underlying spontaneous preterm birth are initiated when a premature and excessive inflammatory response is triggered by infection or other causes. Exposure to pro-inflammatory mediators is emerging as a major factor in the 'fetal inflammatory response syndrome' that often accompanies preterm birth, where unscheduled effects in fetal tissues interfere with normal development and predispose to neonatal morbidity. Toll-like receptors (TLRs) are critical upstream gatekeepers of inflammatory activation. TLR4 is prominently involved through its ability to sense and integrate signals from a range of microbial and endogenous triggers to provoke and perpetuate inflammation. Preclinical studies have identified TLR4 as an attractive pharmacological target to promote uterine quiescence and protect the fetus from inflammatory injury. Novel small-molecule inhibitors of TLR4 signalling, specifically the non-opioid receptor antagonists (+)-naloxone and (+)-naltrexone, are proving highly effective in animal models for preventing preterm birth induced by bacterial mimetic LPS, heat-killed Escherichia coli, or the TLR4-dependent pro-inflammatory lipid, platelet-activating factor (PAF). Here, we summarise the rationale for targeting TLR4 as a master regulator of inflammation in fetal and gestational tissues, and the potential utility of TLR4 antagonists as candidates for preventative and therapeutic application in preterm delivery and fetal inflammatory injury.

Use of stored serum in the study of time trends and geographical differences in exposure of pregnant women to phthalates.

BACKGROUND: Exposure to some phthalate diesters has been associated with adverse reproductive health outcomes in both rodents and humans indicative of anti-androgenic effects. Exposure during sensitive periods of development, such as prenatally, is of particular concern. OBJECTIVES: We wished to investigate whether phthalate metabolites measured in maternal serum samples from historical birth cohorts can be used to assess prenatal exposure. Further, we aimed to study temporal and geographical trends in phthalate exposure across three different birth cohorts. METHODS: We compared phthalate metabolite levels in maternal serum samples from an Australian (1989-91) and a Danish (1997-2001) birth cohort with levels in serum and urine samples from a recent Danish birth cohort (2012-14). Samples were analysed for 32 phthalate metabolites from 15 phthalate diesters by isotope-diluted liquid chromatography-tandem mass spectrometry (LC-MS/MS). Correlations between metabolites were tested by Spearman rank correlation test, and differences between the cohorts were tested by Mann-Whitney U test. RESULTS: Overall, we observed large variations in serum phthalate metabolite levels between individuals. Secondary metabolites of di-(2-ethyl-hexyl) phthalate (DEHP) and di-iso-nonyl phthalate (DiNP) in serum were weakly to moderately and positively correlated to the levels measured in urine, and secondary metabolites of DEHP were also moderately to strongly and significantly correlated in serum. Correlations with mono-(2-ethyl-hexyl) phthalate (MEHP) and mono-iso-nonyl phthalate (MiNP), the two primary metabolites of DEHP and DiNP, were inconsistent, and we found indications of sample contamination. We observed some significant differences in phthalate metabolite levels between the three cohorts with generally higher levels in the older birth cohorts. CONCLUSION: Based on comparison across two older birth cohorts and a recent cohort, our results support the concept that historical biobanked serum samples may be used for assessment of prenatal exposure to phthalates when using serum levels of the monoesters of the low-molecular weight (LMW) phthalates and the secondary metabolites of the high-molecular weight (HMW) phthalates. Serum phthalate measurements are, however, not suitable for human biomonitoring and should only be used to exploit historical samples from cohorts, where urine samples were not collected. Our findings suggest that phthalate exposure may have decreased over time from the early 1990s to the 2010s.

Placental and intra-amniotic inflammation are associated with altered fetal immune responses at birth.

INTRODUCTION: High-grade placental inflammation is associated with preterm birth and poor neonatal outcomes. Recent reports suggest that low-grade placental inflammation is common in uncomplicated pregnancies. The relationship between placental inflammation and innate immune anti-microbial responses is unknown. In this study we sought to identify any association between placental inflammation and fetal immune responses. METHODS: Cord blood samples collected from late preterm and full-term Caesarean section deliveries (n = 44) were exposed to various immune challenges (resiquimod, LPS, PGN, poly (I:C), cGAMP, and 5'ppp-dsRNA) and production of inflammatory mediators (G-CSF, IFN-γ, IL-1ß, IL-6, IL-8, IL-10, and TNF-α) was measured by multiplex assay. Hospital histology reports were used to assess the extent of inflammation in the placenta. RESULTS: Almost half (47.7%) of placentae examined here showed histological evidence of inflammation. Resiquimod, LPS, and PGN elicited strong inflammatory responses in neonatal cord blood, while poly (I:C), cGAMP, and 5'ppp-dsRNA elicited weaker responses. Fetuses with evidence of chorioamnionitis and fetal inflammatory reaction in their placentae had significantly increased immune responses to cGAMP and 5'ppp-dsRNA (ligands for STING and RIG-I, respectively) and significantly decreased immune responses to poly (I:C) (a TLR3 agonist). Interestingly, STING, RIG-I, and TLR3 are all involved in viral response pathways, suggesting that fetuses exposed to chorioamnionitis or fetal inflammatory reaction might respond differently to viruses postnatally. CONCLUSION: Our data suggest that low-level placental inflammation is associated with altered innate cytokine responses at birth.

The Not-so-Sterile Womb: Evidence That the Human Fetus Is Exposed to Bacteria Prior to Birth.

The human microbiome includes trillions of bacteria, many of which play a vital role in host physiology. Numerous studies have now detected bacterial DNA in first-pass meconium and amniotic fluid samples, suggesting that the human microbiome may commence in utero. However, these data have remained contentious due to underlying contamination issues. Here, we have used a previously described method for reducing contamination in microbiome workflows to determine if there is a fetal bacterial microbiome beyond the level of background contamination. We recruited 50 women undergoing non-emergency cesarean section deliveries with no evidence of intra-uterine infection and collected first-pass meconium and amniotic fluid samples. Full-length 16S rRNA gene sequencing was performed using PacBio SMRT cell technology, to allow high resolution profiling of the fetal gut and amniotic fluid bacterial microbiomes. Levels of inflammatory cytokines were measured in amniotic fluid, and levels of immunomodulatory short chain fatty acids (SCFAs) were quantified in meconium. All meconium samples and most amniotic fluid samples (36/43) contained bacterial DNA. The meconium microbiome was dominated by reads that mapped to Pelomonas puraquae. Aside from this species, the meconium microbiome was remarkably heterogeneous between patients. The amniotic fluid microbiome was more diverse and contained mainly reads that mapped to typical skin commensals, including Propionibacterium acnes and Staphylococcus spp. All meconium samples contained acetate and propionate, at ratios similar to those previously reported in infants. P. puraquae reads were inversely correlated with meconium propionate levels. Amniotic fluid cytokine levels were associated with the amniotic fluid microbiome. Our results demonstrate that bacterial DNA and SCFAs are present in utero, and have the potential to influence the developing fetal immune system.

Murine exposure to gold nanoparticles during early pregnancy promotes abortion by inhibiting ectodermal differentiation.

BACKGROUND: Gold nanoparticles (AuNPs) have been widely studied for biomedical applications, although their safety and potential toxicity in pregnancy remains unknown. The aim of this study is to explore the effect of AuNPs maternal exposure at different gestational ages on fetal survival and development, as well as the potential mechanism of AuNPs affecting embryos and fetuses. METHODS: Thirty nm polyethylene glycol (PEG)-coated AuNPs (A30) were administered to pregnant mice via intravenous injection (5 µg Au/g body weight) over three days at either early or late pregnancy. Fetal abortion rate and morphological development in E16.5 were then detected in detail. The pregnant mice physiological states with A30 exposure were examined by biochemical, histological or imaging methods; and materno-fetal distribution of gold elements was assayed by electron microscopy and mass spectrometry. Murine embryonic stem cells derived embryoid-bodies or neuroectodermal cells were treated with A30 (0.0025 to 0.25 µg Au/mL) to examine A30 effects on expression levels of the germ differentiation marker genes. Tukey's method was used for statistical analysis. RESULTS: Exposure to A30 during early (A30E) but not late (A30L) pregnancy caused a high abortion rate (53.5%), lower fetal survival rate and abnormal decidualization compared with non-exposed counterparts. The developmental damage caused by A30 followed an "all-or-nothing" pattern, as the non-aborted fetuses developed normally and pregnancies maintained normal endocrine values. A30 caused minor impairment of liver and kidney function of A30E but not A30L mice. TEM imaging of fetal tissue sections confirmed the transfer of A30 into fetal brain and live as aggregates. qPCR assays showed A30 suppressed the expression of ectodermal, but not mesodermal and endodermal differentiation markers. CONCLUSIONS: These results illustrate that maternal A30 exposure in early pregnant results in A30 transfer into embryonic tissues, inhibiting ectodermal differentiation of embryonic stem cells, leading to abnormal embryonic development and abortion. While exposure to A30 during late pregnancy had little or no impact on dams and fetuses. These findings suggest the safety of biomedical applications employing AuNPs during pregnancy is strongly influenced by fetal maturity and gestational age at exposure and provide the clues for AuNPs safe application period in pregnancy.

Intracellular speciation of gold nanorods alters the conformational dynamics of genomic DNA.

Gold nanorods are one of the most widely explored inorganic materials in nanomedicine for diagnostics, therapeutics and sensing1. It has been shown that gold nanorods are not cytotoxic and localize within cytoplasmic vesicles following endocytosis, with no nuclear localization2,3, but other studies have reported alterations in gene expression profiles in cells following exposure to gold nanorods, via unknown mechanisms4. In this work we describe a pathway that can contribute to this phenomenon. By mapping the intracellular chemical speciation process of gold nanorods, we show that the commonly used Au-thiol conjugation, which is important for maintaining the noble (inert) properties of gold nanostructures, is altered following endocytosis, resulting in the formation of Au(I)-thiolates that localize in the nucleus5. Furthermore, we show that nuclear localization of the gold species perturbs the dynamic microenvironment within the nucleus and triggers alteration of gene expression in human cells. We demonstrate this using quantitative visualization of ubiquitous DNA G-quadruplex structures, which are sensitive to ionic imbalances, as an indicator of the formation of structural alterations in genomic DNA.

Relationship between umbilical cord sex hormone binding globulin, sex steroids, and age at menarche: a prospective cohort study.

OBJECTIVE: To study the role of the prenatal environment in regulating reproductive development by measuring the prospective association between umbilical cord concentrations of sex hormone binding globulin (SHBG; principal regulator of sex steroid activity), bioavailable sex steroids, and age at menarche. DESIGN: Prospective population-based cohort. SETTING: Not applicable. PATIENT(S): In 286 female members of the Western Australian Pregnancy (Raine) cohort, concentrations of SHBG and steroids (estrogens: estrone, estradiol, estriol and estetrol [E4]; androgens: total testosterone, Δ4-androstenedione, androstenedione and dehydroepiandrosterone) were measured by liquid chromatography-tandem mass spectrometry from archived umbilical cord blood samples collected at birth. Bioavailable concentrations of testosterone and estradiol were calculated along with total composite measures of androgen and estrogen bioactivity. SHBG was measured by ELISA. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Age of menarche was calculated from date of menarche, collected prospectively by questionnaire sent home with participants at the year 10 follow-up. RESULT(S): Higher maternal education, higher body mass index, and the presence of antepartum hemorrhage were all significantly associated with earlier age at menarche. The bioavailable sex steroid measures accounted for 8.3% of the variance in age at menarche. Further, both SHBG and E4 concentrations accounted for a significant proportion of unique variance in age at menarche. CONCLUSION(S): Lower SHBG and higher E4 concentrations in umbilical cord blood were associated with earlier age at menarche. These results suggest that the prenatal sex steroid environment contributes toward pubertal development and age at menarche.