Immunological and microbial shifts in the aging rhesus macaque lung during nontuberculous mycobacterial infection.

Nontuberculous mycobacteria (NTM) are environmentally ubiquitous organisms that predominately cause NTM pulmonary disease (NTMPD) in individuals over the age of 65. The incidence of NTMPD has increased in the U.S., exceeding that of Mycobacterium tuberculosis. However, the mechanisms leading to higher susceptibility and severity of NTMPD with aging are poorly defined in part due to the lack of animal models that accurately recapitulate human disease. Here, we compared bacterial load, microbial communities, and host responses longitudinally between three young (two female and one male) and two aged (two female) rhesus macaques inoculated with Mycobacterium avium subsp. hominissuis (MAH) in the right caudal lobe. Unilateral infection resulted in a low bacterial load in both young and aged animals confined to the infected side. Although a robust inflammatory response was only observed in the inoculated lung, immune cell infiltration and antigen-specific T cells were detected in both lungs. Computed tomography, gross pathology, and histopathology revealed increased disease severity and persistence of bacterial DNA in aged animals. Additional analyses showed the translocation of gut and oral-pharyngeal bacterial DNA into the lower respiratory microbiome. Finally, single-cell RNA sequencing revealed a heightened inflammatory response to MAH infection by alveolar macrophages in aged animals. These data are consistent with the model that increased disease severity in the aged is mediated by a dysregulated macrophage response that may be sustained through persistent antigen presence. IMPORTANCE: Nontuberculous mycobacteria (NTM) are emerging as pathogens of high consequence, as cases of NTM pulmonary disease (NTMPD) have exceeded those of Mycobacterium tuberculosis. NTMPD can be debilitating, particularly in patients over 65 years of age, as it causes chronic cough and fatigue requiring prolonged treatments with antibiotics. The underlying mechanisms of this increased disease severity with age are poorly understood, hampering the development of therapeutics and vaccines. Here, we use a rhesus macaque model to investigate the impact of age on host-NTM interactions. This work shows that aging is associated with increased disease severity and bacterial persistence in aged rhesus macaques, thus providing a preclinical model to develop and test novel therapeutics and interventions.

Risk of Adverse Neonatal Outcomes After Combined Prenatal Cannabis and Nicotine Exposure.

Importance: The prevalence of cannabis use in pregnancy is rising and is associated with adverse perinatal outcomes. In parallel, combined prenatal use of cannabis and nicotine is also increasing, but little is known about the combined impact of both substances on pregnancy and offspring outcomes compared with each substance alone. Objective: To assess the perinatal outcomes associated with combined cannabis and nicotine exposure compared with each substance alone during pregnancy. Design, Setting, and Participants: This retrospective population-based cohort study included linked hospital discharge data (obtained from the California Department of Health Care Access and Information) and vital statistics (obtained from the California Department of Public Health) from January 1, 2012, through December 31, 2019. Pregnant individuals with singleton gestations and gestational ages of 23 to 42 weeks were included. Data were analyzed from October 14, 2023, to March 4, 2024. Exposures: Cannabis-related diagnosis and prenatal nicotine product use were captured using codes from International Classification of Diseases, Ninth Revision, Clinical Modification, and International Statistical Classification of Diseases, Tenth Revision, Clinical Modification. Main Outcome and Measures: The main outcomes were infant and neonatal death, infants small for gestational age, and preterm delivery. Results were analyzed by multivariable Poisson regression models. Results: A total of 3 129 259 pregnant individuals were included (mean [SD] maternal age 29.3 [6.0] years), of whom 23 007 (0.7%) had a cannabis-related diagnosis, 56 811 (1.8%) had a nicotine-use diagnosis, and 10 312 (0.3%) had both in pregnancy. Compared with nonusers, those with cannabis or nicotine use diagnoses alone had increased rates of infant (0.7% for both) and neonatal (0.3% for both) death, small for gestational age (14.3% and 13.7%, respectively), and preterm delivery (<37 weeks) (12.2% and 12.0%, respectively). Moreover, risks in those with both cannabis and nicotine use were higher for infant death (1.2%; adjusted risk ratio [ARR], 2.18 [95% CI, 1.82-2.62]), neonatal death (0.6%; ARR, 1.76 [95% CI, 1.36-2.28]), small for gestational age (18.0%; ARR, 1.94 [95% CI, 1.86-2.02]), and preterm delivery (17.5%; ARR, 1.83 [95% CI, 1.75-1.91]). Conclusions and Relevance: These findings suggest that co-occurring maternal use of cannabis and nicotine products in pregnancy is associated with an increased risk of infant and neonatal death and maternal and neonatal morbidity compared with use of either substance alone. Given the increasing prevalence of combined cannabis and nicotine use in pregnancy, these findings can help guide health care practitioners with preconception and prenatal counseling, especially regarding the benefits of cessation.

Vitamin C Supplementation Among Pregnant Smokers and Airway Function Trajectory in Offspring: A Secondary Analysis of a Randomized Clinical Trial.

This secondary analysis of a randomized clinical trial analyzes the association of vitamin C supplementation in women who smoked during pregnancy with airway function trajectory in their offspring at 4 to 6 years of age.

Improvements in lung function following vitamin C supplementation to pregnant smokers are associated with buccal DNA methylation at 5 years of age.

BACKGROUND: We previously reported in the "Vitamin C to Decrease the Effects of Smoking in Pregnancy on Infant Lung Function" randomized clinical trial (RCT) that vitamin C (500 mg/day) supplementation to pregnant smokers is associated with improved respiratory outcomes that persist through 5 years of age. The objective of this study was to assess whether buccal cell DNA methylation (DNAm), as a surrogate for airway epithelium, is associated with vitamin C supplementation, improved lung function, and decreased occurrence of wheeze. METHODS: We conducted epigenome-wide association studies (EWAS) using Infinium MethylationEPIC arrays and buccal DNAm from 158 subjects (80 placebo; 78 vitamin C) with pulmonary function testing (PFT) performed at the 5-year visit. EWAS were performed on (1) vitamin C treatment, (2) forced expiratory flow between 25 and 75% of expired volume (FEF25-75), and (3) offspring wheeze. Models were adjusted for sex, race, study site, gestational age at randomization (≤ OR > 18 weeks), proportion of epithelial cells, and latent covariates in addition to child length at PFT in EWAS for FEF25-75. We considered FDR p < 0.05 as genome-wide significant and nominal p < 0.001 as candidates for downstream analyses. Buccal DNAm measured in a subset of subjects at birth and near 1 year of age was used to determine whether DNAm signatures originated in utero, or emerged with age. RESULTS: Vitamin C treatment was associated with 457 FDR significant (q < 0.05) differentially methylated CpGs (DMCs; 236 hypermethylated; 221 hypomethylated) and 53 differentially methylated regions (DMRs; 26 hyper; 27 hypo) at 5 years of age. FEF25-75 was associated with one FDR significant DMC (cg05814800), 1,468 candidate DMCs (p < 0.001), and 44 DMRs. Current wheeze was associated with 0 FDR-DMCs, 782 candidate DMCs, and 19 DMRs (p < 0.001). In 365/457 vitamin C FDR significant DMCs at 5 years of age, there was no significant interaction between time and treatment. CONCLUSIONS: Vitamin C supplementation to pregnant smokers is associated with buccal DNA methylation in offspring at 5 years of age, and most methylation signatures appear to be persistent from the prenatal period. Buccal methylation at 5 years was also associated with current lung function and occurrence of wheeze, and these functionally associated loci are enriched for vitamin C associated loci. Clinical trial registration ClinicalTrials.gov, NCT01723696 and NCT03203603.

The amniotic fluid proteome changes across gestation in humans and rhesus macaques.

Amniotic fluid is a complex biological medium that offers protection to the fetus and plays a key role in normal fetal nutrition, organogenesis, and potentially fetal programming. Amniotic fluid is also critically involved in longitudinally shaping the in utero milieu during pregnancy. Yet, the molecular mechanism(s) of action by which amniotic fluid regulates fetal development is ill-defined partly due to an incomplete understanding of the evolving composition of the amniotic fluid proteome. Prior research consisting of cross-sectional studies suggests that the amniotic fluid proteome changes as pregnancy advances, yet longitudinal alterations have not been confirmed because repeated sampling is prohibitive in humans. We therefore performed serial amniocenteses at early, mid, and late gestational time-points within the same pregnancies in a rhesus macaque model. Longitudinally-collected rhesus amniotic fluid samples were paired with gestational-age matched cross-sectional human samples. Utilizing LC-MS/MS isobaric labeling quantitative proteomics, we demonstrate considerable cross-species similarity between the amniotic fluid proteomes and large scale gestational-age associated changes in protein content throughout pregnancy. This is the first study to compare human and rhesus amniotic fluid proteomic profiles across gestation and establishes a reference amniotic fluid proteome. The non-human primate model holds promise as a translational platform for amniotic fluid studies.

Prenatal delta-9-tetrahydrocannabinol exposure is associated with changes in rhesus macaque DNA methylation enriched for autism genes.

BACKGROUND: With the growing availability of cannabis and the popularization of additional routes of cannabis use beyond smoking, including edibles, the prevalence of cannabis use in pregnancy is rapidly increasing. However, the potential effects of prenatal cannabis use on fetal developmental programming remain unknown. RESULTS: We designed this study to determine whether the use of edible cannabis during pregnancy is deleterious to the fetal and placental epigenome. Pregnant rhesus macaques consumed a daily edible containing either delta-9-tetrahydrocannabinol (THC) (2.5 mg/7 kg/day) or placebo. DNA methylation was measured in 5 tissues collected at cesarean delivery (placenta, lung, cerebellum, prefrontal cortex, and right ventricle of the heart) using the Illumina MethylationEPIC platform and filtering for probes previously validated in rhesus macaque. In utero exposure to THC was associated with differential methylation at 581 CpGs, with 573 (98%) identified in placenta. Loci differentially methylated with THC were enriched for candidate autism spectrum disorder (ASD) genes from the Simons Foundation Autism Research Initiative (SFARI) database in all tissues. The placenta demonstrated greatest SFARI gene enrichment, including genes differentially methylated in placentas from a prospective ASD study. CONCLUSIONS: Overall, our findings reveal that prenatal THC exposure alters placental and fetal DNA methylation at genes involved in neurobehavioral development that may influence longer-term offspring outcomes. The data from this study add to the limited existing literature to help guide patient counseling and public health polices focused on prenatal cannabis use in the future.

Cessation of chronic delta-9-tetrahydrocannabinol use partially reverses impacts on male fertility and the sperm epigenome in rhesus macaques.

OBJECTIVE: To determine whether discontinuation of delta-9-tetrahydrocannabinol (THC) use mitigates THC-associated changes in male reproductive health using a rhesus macaque model of daily THC edible consumption. DESIGN: Research animal study. SETTING: Research institute environment. PATIENT(S): Adult male rhesus macaques (age, 8-10 years; n = 6). INTERVENTION(S): Chronic daily THC edible administration at medically and recreationally relevant contemporary doses followed by cessation of THC use. MAIN OUTCOME MEASURE(S): Testicular volume, serum male hormones, semen parameters, sperm deoxyribonucleic acid (DNA) fragmentation, seminal fluid proteomics, and whole genome bisulfite sequencing of sperm DNA. RESULT(S): Chronic THC use resulted in significant testicular atrophy, increased gonadotropin levels, decreased serum sex steroid levels, changes in seminal fluid proteome, and increased DNA fragmentation with partial recovery after discontinuation of THC use. For every increase of 1 mg/7 kg/day in THC dosing, there was a significant decrease in the total testicular volume bilaterally by 12.6 cm3 (95% confidence interval [CI], 10.6-14.5), resulting in a 59% decrease in volume. With THC abstinence, the total testicular volume increased to 73% of its original volume. Similarly, with THC exposure, there were significant decreases in the mean total testosterone and estradiol levels and a significant increase in the follicle-stimulating hormone level. With increasing THC dose, there was a significant decrease in the liquid semen ejaculate volume and weight of coagulum; however, no other significant changes in the other semen parameters were noted. After discontinuing THC use, there was a significant increase in the total serum testosterone level by 1.3 ng/mL (95% CI, 0.1-2.4) and estradiol level by 2.9 pg/mL (95% CI, 0.4-5.4), and the follicle-stimulating hormone level significantly decreased by 0.06 ng/mL (95% CI, 0.01-0.11). Seminal fluid proteome analysis revealed differential expression of proteins enriched for processes related to cellular secretion, immune response, and fibrinolysis. Whole genome bisulfite sequencing identified 23,558 CpGs differentially methylated in heavy-THC vs. pre-THC sperm, with partial restoration of methylation after discontinuation of THC use. Genes associated with altered differentially methylated regions were enriched for those involved in the development and function of the nervous system. CONCLUSION(S): This is the first study demonstrating that discontinuation of chronic THC use in rhesus macaques partially restores adverse impacts to male reproductive health, THC-associated sperm differentially methylated regions in genes important for development, and expression of proteins important for male fertility.

A functional genomics pipeline to identify high-value asthma and allergy CpGs in the human methylome.

BACKGROUND: DNA methylation of cytosines at cytosine-phosphate-guanine (CpG) dinucleotides (CpGs) is a widespread epigenetic mark, but genome-wide variation has been relatively unexplored due to the limited representation of variable CpGs on commercial high-throughput arrays. OBJECTIVES: To explore this hidden portion of the epigenome, this study combined whole-genome bisulfite sequencing with in silico evidence of gene regulatory regions to design a custom array of high-value CpGs. This study focused on airway epithelial cells from children with and without allergic asthma because these cells mediate the effects of inhaled microbes, pollution, and allergens on asthma and allergic disease risk. METHODS: This study identified differentially methylated regions from whole-genome bisulfite sequencing in nasal epithelial cell DNA from a total of 39 children with and without allergic asthma of both European and African ancestries. This study selected CpGs from differentially methylated regions, previous allergy or asthma epigenome-wide association studies (EWAS), or genome-wide association study loci, and overlapped them with functional annotations for inclusion on a custom Asthma&Allergy array. This study used both the custom and EPIC arrays to perform EWAS of allergic sensitization (AS) in nasal epithelial cell DNA from children in the URECA (Urban Environment and Childhood Asthma) birth cohort and using the custom array in the INSPIRE [Infant Susceptibility to Pulmonary Infections and Asthma Following RSV Exposure] birth cohort. Each CpG on the arrays was assigned to its nearest gene and its promotor capture Hi-C interacting gene and performed expression quantitative trait methylation (eQTM) studies for both sets of genes. RESULTS: Custom array CpGs were enriched for intermediate methylation levels compared to EPIC CpGs. Intermediate methylation CpGs were further enriched among those associated with AS and for eQTMs on both arrays. CONCLUSIONS: This study revealed signature features of high-value CpGs and evidence for epigenetic regulation of genes at AS EWAS loci that are robust to race/ethnicity, ascertainment, age, and geography.

Analysis of Pregnancy Complications and Epigenetic Gestational Age of Newborns.

Importance: Preeclampsia, gestational hypertension, and gestational diabetes, the most common pregnancy complications, are associated with substantial morbidity and mortality in mothers and children. Little is known about the biological processes that link the occurrence of these pregnancy complications with adverse child outcomes; altered biological aging of the growing fetus up to birth is one molecular pathway of increasing interest. Objective: To evaluate whether exposure to each of these 3 pregnancy complications (gestational diabetes, gestational hypertension, and preeclampsia) is associated with accelerated or decelerated gestational biological age in children at birth. Design, Setting, and Participants: Children included in these analyses were born between 1998 and 2018 and spanned multiple geographic areas of the US. Pregnancy complication information was obtained from maternal self-report and/or medical record data. DNA methylation measures were obtained from blood biospecimens collected from offspring at birth. The study used data from the national Environmental Influences on Child Health Outcomes (ECHO) multisite cohort study collected and recorded as of the August 31, 2021, data lock date. Data analysis was performed from September 2021 to December 2022. Exposures: Three pregnancy conditions were examined: gestational hypertension, preeclampsia, and gestational diabetes. Main Outcomes and Measures: Accelerated or decelerated biological gestational age at birth, estimated using existing epigenetic gestational age clock algorithms. Results: A total of 1801 child participants (880 male [48.9%]; median [range] chronological gestational age at birth, 39 [30-43] weeks) from 12 ECHO cohorts met the analytic inclusion criteria. Reported races included Asian (49 participants [2.7%]), Black (390 participants [21.7%]), White (1026 participants [57.0%]), and other races (92 participants [5.1%]) (ie, American Indian or Alaska Native, Native Hawaiian or other Pacific Islander, multiple races, and other race not specified). In total, 524 participants (29.0%) reported Hispanic ethnicity. Maternal ages ranged from 16 to 45 years of age with a median of 29 in the analytic sample. A range of maternal education levels, from less than high school (260 participants [14.4%]) to Bachelor's degree and above (629 participants [34.9%]), were reported. In adjusted regression models, prenatal exposure to maternal gestational diabetes (ß, -0.423; 95% CI, -0.709 to -0.138) and preeclampsia (ß, -0.513; 95% CI, -0.857 to -0.170), but not gestational hypertension (ß, 0.003; 95% CI, -0.338 to 0.344), were associated with decelerated epigenetic aging among exposed neonates vs those who were unexposed. Modification of these associations, by sex, was observed with exposure to preeclampsia (ß, -0.700; 95% CI, -1.189 to -0.210) and gestational diabetes (ß, -0.636; 95% CI, -1.070 to -0.200), with associations observed among female but not male participants. Conclusions and Relevance: This US cohort study of neonate biological changes related to exposure to maternal pregnancy conditions found evidence that preeclampsia and gestational diabetes delay biological maturity, especially in female offspring.

Effect of Vitamin C Supplementation for Pregnant Smokers on Offspring Airway Function and Wheeze at Age 5 Years: Follow-up of a Randomized Clinical Trial.

Importance: Vitamin C supplementation (500 mg/d) for pregnant smokers has been reported to increase offspring airway function as measured by forced expiratory flow (FEF) through age 12 months; however, its effects on airway function at age 5 years remain to be assessed. Objective: To assess whether vitamin C supplementation in pregnant smokers is associated with increased and/or improved airway function in their offspring at age 5 years and whether vitamin C decreases the occurrence of wheeze. Design, Setting, and Participants: This study followed up the Vitamin C to Decrease the Effects of Smoking in Pregnancy on Infant Lung Function (VCSIP) double-blind, placebo-controlled randomized clinical trial conducted at 3 centers in the US (in Oregon, Washington, and Indiana) between 2012 and 2016. Investigators and participants remain unaware of the treatment assignments. Forced expiratory flow measurements at age 5 years were completed from 2018 to 2021. Interventions: Pregnant smokers were randomized to vitamin C (500 mg/d) or placebo treatment. Main Outcomes and Measures: The primary outcome was the prespecified measurement of FEF between 25% and 75% expired volume (FEF25-75) by spirometry at age 5 years. Secondary outcomes included FEF measurements at 50% and 75% of expiration (FEF50 and FEF75), forced expiratory volume in 1 second (FEV1), and occurrence of wheeze. Results: Of the 251 pregnant smokers included in this study, 125 (49.8%) were randomized to vitamin C and 126 (50.2%) were randomized to placebo. Of 213 children from the VCSIP trial who were reconsented into this follow-up study, 192 (90.1%) had successful FEF measurements at age 5 years; 212 (99.5%) were included in the analysis of wheeze. Analysis of covariance demonstrated that offspring of pregnant smokers allocated to vitamin C compared with placebo had 17.2% significantly higher mean (SE) measurements of FEF25-75 at age 5 years (1.45 [0.04] vs 1.24 [0.04] L/s; adjusted mean difference, 0.21 [95% CI, 0.13-0.30]; P < .001). Mean (SE) measurements were also significantly increased by 14.1% for FEF50 (1.59 [0.04] vs 1.39 [0.04] L/s; adjusted mean difference, 0.20 [95% CI, 0.11-0.30]; P < .001), 25.9% for FEF75 (0.79 [0.02] vs 0.63 [0.02] L/s; 0.16 [95% CI, 0.11-0.22]; P < .001), and 4.4% for FEV1 (1.13 [0.02] vs 1.09 [0.02] L; 0.05 [95% CI, 0.01-0.09]; P = .02). In addition, offspring of pregnant smokers randomized to vitamin C had significantly decreased wheeze (28.3% vs 47.2%; estimated odds ratio, 0.41 [95% CI, 0.23-0.74]; P = .003). Conclusions and Relevance: In this follow-up study of offspring of pregnant smokers randomized to vitamin C vs placebo, vitamin C supplementation during pregnancy resulted in significantly increased airway function of offspring at age 5 years and significantly decreased the occurrence of wheeze. These findings suggest that vitamin C supplementation for pregnant smokers may decrease the effects of smoking in pregnancy on childhood airway function and respiratory health. Trial Registration: ClinicalTrials.gov Identifier: NCT03203603.

Vaping Aerosols from Vitamin E Acetate and Tetrahydrocannabinol Oil: Chemistry and Composition.

The popularity of vaping cannabis products has increased sharply in recent years. In 2019, a sudden onset of electronic cigarette/vaping-associated lung injury (EVALI) was reported, leading to thousands of cases of lung illness and dozens of deaths due to the vaping of tetrahydrocannabinol (THC)-containing e-liquids that were obtained on the black market. A potential cause of EVALI has been hypothesized due to the illicit use of vitamin E acetate (VEA) in cannabis vape cartridges. However, the chemistry that modifies VEA and THC oil, to potentially produce toxic byproducts, is not well understood under different scenarios of use. In this work, we quantified carbonyls, organic acids, cannabinoids, and terpenes in the vaping aerosol of pure VEA, purified THC oil, and an equal volume mixture of VEA and THC oil at various coil temperatures (100-300 °C). It was found under the conditions of our study that degradation of VEA and cannabinoids, including Δ9-THC and cannabigerol (CBG), occurred via radical oxidation and direct thermal decomposition pathways. Evidence of terpene degradation was also observed. The bond cleavage of aliphatic side chains in both VEA and cannabinoids formed a variety of smaller carbonyls. Oxidation at the ring positions of cannabinoids formed various functionalized products. We show that THC oil has a stronger tendency to aerosolize and degrade compared to VEA at a given temperature. The addition of VEA to the e-liquid nonlinearly suppressed the formation of vape aerosol compared to THC oil. At the same time, toxic carbonyls including formaldehyde, 4-methylpentanal, glyoxal, or diacetyl and its isomers were highly enhanced in VEA e-liquid when normalized to particle mass.

Functional, transcriptional, and microbial shifts associated with healthy pulmonary aging in rhesus macaques.

Older individuals are at increased risk of developing severe respiratory infections. However, our understanding of the impact of aging on the respiratory tract remains limited as samples from healthy humans are challenging to obtain and results can be confounded by variables such as smoking and diet. Here, we carry out a comprehensive cross-sectional study (n = 34 adult, n = 49 aged) to define the consequences of aging on the lung using the rhesus macaque model. Pulmonary function testing establishes similar age and sex differences as humans. Additionally, we report increased abundance of alveolar and infiltrating macrophages and a concomitant decrease in T cells were in aged animals. scRNAseq reveals shifts from GRZMB to IFN expressing CD8+ T cells in the lungs. These data provide insight into age-related changes in the lungs' functional, microbial, and immunological landscape that explain increased prevalence and severity of respiratory diseases in the elderly.

Impact of vitamin C supplementation on placental DNA methylation changes related to maternal smoking: association with gene expression and respiratory outcomes.

BACKGROUND: Maternal smoking during pregnancy (MSDP) affects development of multiple organ systems including the placenta, lung, brain, and vasculature. In particular, children exposed to MSDP show lifelong deficits in pulmonary function and increased risk of asthma and wheeze. Our laboratory has previously shown that vitamin C supplementation during pregnancy prevents some of the adverse effects of MSDP on offspring respiratory outcomes. Epigenetic modifications, including DNA methylation (DNAm), are a likely link between in utero exposures and adverse health outcomes, and MSDP has previously been associated with DNAm changes in blood, placenta, and buccal epithelium. Analysis of placental DNAm may reveal critical targets of MSDP and vitamin C relevant to respiratory health outcomes. RESULTS: DNAm was measured in placentas obtained from 72 smokers enrolled in the VCSIP RCT: NCT03203603 (37 supplemented with vitamin C, 35 with placebo) and 24 never-smokers for reference. Methylation at one CpG, cg20790161, reached Bonferroni significance and was hypomethylated in vitamin C supplemented smokers versus placebo. Analysis of spatially related CpGs identified 93 candidate differentially methylated regions (DMRs) between treatment groups, including loci known to be associated with lung function, oxidative stress, fetal development and growth, and angiogenesis. Overlap of nominally significant differentially methylated CpGs (DMCs) in never-smokers versus placebo with nominally significant DMCs in vitamin C versus placebo identified 9059 candidate "restored CpGs" for association with placental transcript expression and respiratory outcomes. Methylation at 274 restored candidate CpG sites was associated with expression of 259 genes (FDR < 0.05). We further identified candidate CpGs associated with infant lung function (34 CpGs) and composite wheeze (1 CpG) at 12 months of age (FDR < 0.05). Increased methylation in the DIP2C, APOH/PRKCA, and additional candidate gene regions was associated with improved lung function and decreased wheeze in offspring of vitamin C-treated smokers. CONCLUSIONS: Vitamin C supplementation to pregnant smokers ameliorates changes associated with maternal smoking in placental DNA methylation and gene expression in pathways potentially linked to improved placental function and offspring respiratory health. Further work is necessary to validate candidate loci and elucidate the causal pathway between placental methylation changes and outcomes of offspring exposed to MSDP. Clinical trial registration ClinicalTrials.gov, NCT01723696. Registered November 6, 2012. https://clinicaltrials.gov/ct2/show/record/NCT01723696 .

Maternal Prenatal Hair Cortisol Is Associated with Child Wheeze among Mothers and Infants with Tobacco Smoke Exposure and Who Face High Socioeconomic Adversity.

The association of co-occurring prenatal stress and tobacco exposures on childhood wheezing and asthma are not well established. In this study, we compared maternal prenatal hair cortisol concentration (HCC) to the maternal report of infant wheezing (y/n) in the first year of life among mother-infant dyads exposed to tobacco smoke and socioeconomic adversity. Data were obtained from the Vitamin C to Decrease Effects of Smoking in Pregnancy on Infant Lung Function study. Maternal adversity was defined by the level of education, household income, and health insurance provider. Hair was collected at delivery, representing average circulating third-trimester cortisol levels. HCC was log transformed and dichotomized into high/low cortisol groups that were placed into a multivariate model predicting wheeze. Subjects (n = 132) were primarily White with ≤high school education and receiving government-provided health insurance. Forty-five percent of infants wheezed. Average HCC was 3.39 pg/mg hair. Women with HCC > 3.55 pg/mg were more than twice as likely to report having a child who wheezed (odds ratio 2.56, 95% confidence interval 1.22-5.40; p = 0.01), adjusting for insurance provider and maternal asthma. Among this sample of dyads with prenatal smoke exposure, elevated maternal HCC was associated with child wheeze that was not diminished after consideration of covariates.

Hair and nail nicotine levels of mothers and their infants as valid biomarkers of exposure to intrauterine tobacco smoke.

INTRODUCTION: Tobacco use remains the single most modifiable cause of adverse pregnancy outcomes. It is crucial to be able to accurately quantify the burden of tobacco exposure on both the mother and fetus to have better measures of efficacy with interventions being studied. METHODS: This is a descriptive and exploratory study conducted within a randomized controlled trial. Pregnant smoking and non-smoking women were followed from ≤22 weeks' gestation through delivery with monthly maternal smoking questionnaires, urine cotinine levels, and collection of maternal and infant hair and nail samples, at delivery. Nicotine was extracted and measured (ng/mg) using high-performance liquid chromatography with electrochemical detection. RESULTS: Forty-six mother-infant dyads (34 pregnant smokers and 12 pregnant non-smokers) had successful completion of maternal and infant hair and nails samples. The median hair nicotine levels of the smoking mothers and their infants was significantly higher than those of the non-smokers (1.015 vs 0.037 ng/ mg, p<0.05 for the mothers; 0.445 vs 0.080 ng/mg, p<0.01 for the infants). Similarly, the median nail nicotine levels for smoking mothers and their infants were significantly higher than the non-smokers (2.130 vs 0.056 ng/mg, p<0.01 for the mothers; 0.594 vs 0.132 ng/mg, p<0.05 for the infants). We found a moderate but significant correlation between maternal hair and nail nicotine (r=0.64, p<0.001), infant hair and nail nicotine (r=0.64; p<0.001), maternal and infant hair nicotine (r=0.61, p<0.001), and maternal and infant nail nicotine levels (r=0.58, p<0.001). CONCLUSIONS: Our study shows that both infant hair and nail nicotine levels are valid biomarkers of intrauterine tobacco smoke exposure, and can be used to identify prenatal smoke exposure, correlating well with the level of maternal nicotine exposure.

Vitamin C to Pregnant Smokers Persistently Improves Infant Airway Function to 12†Months of Age: A Randomised Trial.

BACKGROUND: Vitamin C (500 mg·day-1) supplementation for pregnant smokers has been reported to increase newborn pulmonary function and infant forced expiratory flows (FEFs) at 3 months of age. Its effect on airway function through 12 months of age has not been reported. OBJECTIVE: To assess whether vitamin C supplementation to pregnant smokers is associated with a sustained increased airway function in their infants through 12 months of age. METHODS: This is a prespecified secondary outcome of a randomised, double-blind, placebo-controlled trial that randomised 251 pregnant smokers between 13 and 23 weeks of gestation: 125 to 500 mg·day-1 vitamin C and 126 to placebo. Smoking cessation counselling was provided. FEFs performed at 3 and 12 months of age were analysed by repeated measures analysis of covariance. RESULTS: FEFs were performed in 222 infants at 3 months and 202 infants at 12 months of age. The infants allocated to vitamin C had significantly increased FEFs over the first year of life compared to those allocated to placebo. The overall increased flows were: 40.2 mL·sec-1 for FEF75 (adjusted 95% CI for difference 6.6 to 73.8; p=0.025); 58.3 mL·sec-1 for FEF50 (95% CI 10.9 to 105.8; p=0.0081); and 55.1 mL·sec-1 for FEF25-75 (95% CI, 9.7 to 100.5; p=0.013). CONCLUSIONS: In offspring of pregnant smokers randomised to vitamin C versus placebo, vitamin C during pregnancy was associated with a small but significantly increased airway function at 3 and 12 months of age, suggesting a potential shift to a higher airway function trajectory curve. Continued follow-up is underway.