Higher early pregnancy plasma myo-inositol associates with increased postprandial glycaemia later in pregnancy: Secondary analyses of the NiPPeR randomized controlled trial.

AIM: Myo-inositol supplementation from ~13 weeks' gestation reportedly improves glycaemia regulation in metabolically at-risk women, with speculation that earlier supplementation might bring further improvement. However, the NiPPeR trial of a myo-inositol-containing supplement starting preconception did not lower gestational glycaemia in generally healthy women. We postulated that the earlier timing of supplementation influences the maternal metabolic adaptation for gestational glycaemia regulation. METHODS: In total, 585 women were recruited from Singapore, UK and New Zealand for the NiPPeR study. We examined associations of plasma myo-inositol concentrations at 7 and 28 weeks' gestation with 28 weeks plasma glucose (PG; fasting, and 1 h and 2 h in 75 g oral glucose tolerance test) and insulin indices using linear regression adjusting for covariates. RESULTS: Higher 7-week myo-inositol, but not 28-week myo-inositol, associated with higher 1 h PG [ßadj (95% confidence intervals) 0.05 (0.01, 0.09) loge mmol/L per loge µmol/L, p = .022] and 2 h PG [0.08 (0.03, 0.12), p = .001]; equivalent to 0.39 mmol/L increase in 2 h PG for an average 7-week myo-inositol increase of 23.4 µmol/L with myo-inositol supplementation. Higher 7-week myo-inositol associated with a lower 28-week Stumvoll index (first phase), an approximation of insulin secretion [-0.08 (-0.15, -0.01), p = .020] but not with 28-week Matsuda insulin sensitivity index. However, the clinical significance of a 7-week myo-inositol-related increase in glycaemia was limited as there was no association with gestational diabetes risk, birthweight and cord C-peptide levels. In-silico modelling found higher 28-week myo-inositol was associated with lower gestational glycaemia in White, but not Asian, women after controlling for 7-week myo-inositol effects. CONCLUSION: To our knowledge, our study provides the first evidence that increasing first trimester plasma myo-inositol may slightly exacerbate later pregnancy post-challenge glycaemia, indicating that the optimal timing for starting prenatal myo-inositol supplementation needs further investigation.

Maternal mood, anxiety and mental health functioning after combined myo-inositol, probiotics, micronutrient supplementation from preconception: Findings from the NiPPeR RCT.

Observational studies have reported associations between nutrition during pregnancy and mental wellbeing. As secondary outcomes, the NiPPeR double-blind randomized trial in women planning conception investigated whether a myo-inositol, probiotics and enriched micronutrients formulation (intervention) taken preconception and throughout pregnancy could improve mental wellbeing during pregnancy and post-delivery, compared with a standard micronutrient supplement (control). Mood and anxiety symptoms were ascertained (Edinburgh Postnatal Depression Scale (EPDS), State-Trait Anxiety Inventory (STAI-state)) at preconception (baseline), 7, 28 and 34 weeks gestation, 3-weeks and 6-months post-delivery. EPDS>=13 was categorised as low mood; STAI-state>=45 as high anxiety. Change in mental health functioning was assessed as difference between preconception baseline and 6-month post-delivery 12-item Short-Form Health Survey (SF-12v2) mental component scores. Adjusting for site, ethnicity and baseline scores, there were no robust differences in EPDS and STAI-state scores between intervention and control groups across pregnancy (n = 630) and post-delivery (n = 532). Compared to controls, intervention group women averaged a 1.21 (95 %CI 0.04,2.39) higher change in SF-12v2 mental component score from preconception to 6-months post-delivery. Taking a myo-inositol, micronutrient and probiotic supplement during preconception/pregnancy had no effect on mood and anxiety, but there was evidence of a modest improvement in mental health functioning from preconception to 6-months post-delivery.

Impact of preconception and antenatal supplementation with myo-inositol, probiotics, and micronutrients on offspring BMI and weight gain over the first 2 years.

BACKGROUND: Nutritional intervention preconception and throughout pregnancy has been proposed as an approach to promoting healthy postnatal weight gain in the offspring but few randomised trials have examined this. METHODS: Measurements of weight and length were obtained at multiple time points from birth to 2 years among 576 offspring of women randomised to receive preconception and antenatally either a supplement containing myo-inositol, probiotics, and additional micronutrients (intervention) or a standard micronutrient supplement (control). We examined the influence on age- and sex-standardised BMI at 2 years (WHO standards, adjusting for study site, sex, maternal parity, smoking and pre-pregnancy BMI, and gestational age), together with the change in weight, length, BMI from birth, and weight gain trajectories using latent class growth analysis. RESULTS: At 2 years, there was a trend towards lower mean BMI among intervention offspring (adjusted mean difference [aMD] - 0.14 SD [95% CI 0.30, 0.02], p = 0.09), and fewer had a BMI > 95th percentile (i.e. > 1.65 SD, 9.2% vs 18.0%, adjusted risk ratio [aRR] 0.51 [95% CI 0.31, 0.82], p = 0.006). Longitudinal data revealed that intervention offspring had a 24% reduced risk of experiencing rapid weight gain > 0.67 SD in the first year of life (21.9% vs 31.1%, aRR 0.76 [95% CI 0.58, 1.00], p = 0.047). The risk was likewise decreased for sustained weight gain > 1.34 SD in the first 2 years of life (7.7% vs 17.1%, aRR 0.55 [95% CI 0.34, 0.88], p = 0.014). From five weight gain trajectories identified, there were more intervention offspring in the "normal" weight gain trajectory characterised by stable weight SDS around 0 SD from birth to 2 years (38.8% vs 30.1%, RR 1.29 [95% CI 1.03, 1.62], p = 0.029). CONCLUSIONS: Supplementation with myo-inositol, probiotics, and additional micronutrients preconception and in pregnancy reduced the incidence of rapid weight gain and obesity at 2 years among offspring. Previous reports suggest these effects will likely translate to health benefits, but longer-term follow-up is needed to evaluate this. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02509988 (Universal Trial Number U1111-1171-8056). Registered on 16 July 2015.

Maternal B-vitamin and vitamin D status before, during, and after pregnancy and the influence of supplementation preconception and during pregnancy: Prespecified secondary analysis of the NiPPeR double-blind randomized controlled trial.

BACKGROUND: Maternal vitamin status preconception and during pregnancy has important consequences for pregnancy outcome and offspring development. Changes in vitamin status from preconception through early and late pregnancy and postpartum have been inferred from cross-sectional data, but longitudinal data on vitamin status from preconception throughout pregnancy and postdelivery are sparse. As such, the influence of vitamin supplementation on vitamin status during pregnancy remains uncertain. This study presents one prespecified outcome from the randomized controlled NiPPeR trial, aiming to identify longitudinal patterns of maternal vitamin status from preconception, through early and late pregnancy, to 6 months postdelivery, and determine the influence of vitamin supplementation. METHODS AND FINDINGS: In the NiPPeR trial, 1,729 women (from the United Kingdom, Singapore, and New Zealand) aged 18 to 38 years and planning conception were randomized to receive a standard vitamin supplement (control; n = 859) or an enhanced vitamin supplement (intervention; n = 870) starting in preconception and continued throughout pregnancy, with blinding of participants and research staff. Supplement components common to both treatment groups included folic acid, ß-carotene, iron, calcium, and iodine; components additionally included in the intervention group were riboflavin, vitamins B6, B12, and D (in amounts available in over-the-counter supplements), myo-inositol, probiotics, and zinc. The primary outcome of the study was glucose tolerance at 28 weeks' gestation, measured by oral glucose tolerance test. The secondary outcome reported in this study was the reduction in maternal micronutrient insufficiency in riboflavin, vitamin B6, vitamin B12, and vitamin D, before and during pregnancy. We measured maternal plasma concentrations of B-vitamins, vitamin D, and markers of insufficiency/deficiency (homocysteine, hydroxykynurenine-ratio, methylmalonic acid) at recruitment, 1 month after commencing intervention preconception, in early pregnancy (7 to 11 weeks' gestation) and late pregnancy (around 28 weeks' gestation), and postdelivery (6 months after supplement discontinuation). We derived standard deviation scores (SDS) to characterize longitudinal changes among participants in the control group and measured differences between the 2 groups. At recruitment, the proportion of patients with marginal or low plasma status was 29.2% for folate (<13.6 nmol/L), 7.5% and 82.0% for riboflavin (<5 nmol/L and ≤26.5 nmol/L, respectively), 9.1% for vitamin B12 (<221 pmol/L), and 48.7% for vitamin D (<50 nmol/L); these proportions were balanced between the groups. Over 90% of all participants had low or marginal status for one or more of these vitamins at recruitment. Among participants in the control group, plasma concentrations of riboflavin declined through early and late pregnancy, whereas concentrations of 25-hydroxyvitamin D were unchanged in early pregnancy, and concentrations of vitamin B6 and B12 declined throughout pregnancy, becoming >1 SDS lower than baseline by 28 weeks gestation. In the control group, 54.2% of participants developed low late-pregnancy vitamin B6 concentrations (pyridoxal 5-phosphate <20 nmol/L). After 1 month of supplementation, plasma concentrations of supplement components were substantially higher among participants in the intervention group than those in the control group: riboflavin by 0.77 SDS (95% CI 0.68 to 0.87, p < 0.0001), vitamin B6 by 1.07 SDS (0.99 to 1.14, p < 0.0001), vitamin B12 by 0.55 SDS (0.46 to 0.64, p < 0.0001), and vitamin D by 0.51 SDS (0.43 to 0.60, p < 0.0001), with higher levels in the intervention group maintained during pregnancy. Markers of vitamin insufficiency/deficiency were reduced in the intervention group, and the proportion of participants with vitamin D insufficiency (<50 nmol/L) during late pregnancy was lower in the intervention group (35.1% versus 8.5%; p < 0.0001). Plasma vitamin B12 remained higher in the intervention group than in the control group 6 months postdelivery (by 0.30 SDS (0.14, 0.46), p = 0.0003). The main limitation is that generalizability to the global population is limited by the high-resource settings and the lack of African and Amerindian women in particular. CONCLUSIONS: Over 90% of the trial participants had marginal or low concentrations of one or more of folate, riboflavin, vitamin B12, or vitamin D during preconception, and many developed markers of vitamin B6 deficiency in late pregnancy. Preconception/pregnancy supplementation in amounts available in over-the-counter supplements substantially reduces the prevalence of vitamin deficiency and depletion markers before and during pregnancy, with higher maternal plasma vitamin B12 maintained during the recommended lactational period. TRIAL REGISTRATION: ClinicalTrials.gov NCT02509988; U1111-1171-8056.

The effect of a preconception and antenatal nutritional supplement on children's BMI and weight gain over the first 2 years of life: findings from the NiPPeR randomised controlled trial.

BACKGROUND: Nutritional intervention before and throughout pregnancy might promote healthy infant weight gain; however, clinical evidence is scarce. Therefore, we examined whether preconception and antenatal supplementation would affect the body size and growth of children in the first 2 years of life. METHODS: Women were recruited from the community before conception in the UK, Singapore, and New Zealand, and randomly allocated to either the intervention (myo-inositol, probiotics, and additional micronutrients) or control group (standard micronutrient supplement) with stratification by site and ethnicity. Measurements of weight and length were obtained from 576 children at multiple timepoints in the first 2 years of life. Differences in age and sex standardised BMI at age 2 years (WHO standards) and the change in weight from birth were examined. Written informed consent was obtained from the mothers, and ethics approval was granted by local committees. The NiPPeR trial was registered with ClinicalTrials.gov (NCT02509988) on July 16, 2015 (Universal Trial Number U1111-1171-8056). FINDINGS: 1729 women were recruited between Aug 3, 2015, and May 31, 2017. Of the women randomised, 586 had births at 24 weeks or more of gestation between April, 2016, and January, 2019. At age 2 years, adjusting for study site, infant sex, parity, maternal smoking, maternal prepregnancy BMI, and gestational age, fewer children of mothers who received the intervention had a BMI of more than the 95th percentile (22 [9%] of 239 vs 44 [18%] of 245, adjusted risk ratio 0·51, 95% CI 0·31-0·82, p=0·006). Longitudinal data revealed that the children of mothers who received the intervention had a 24% reduced risk of experiencing rapid weight gain of more than 0·67 SD in the first year of life (58 [21·9%] of 265 vs 80 [31·1%] of 257, adjusted risk ratio 0·76, 95% CI 0·58-1·00, p=0·047). Risk was likewise decreased for sustained weight gain of more than 1·34 SD in the first 2 years (19 [7·7%] of 246 vs 43 [17·1%] of 251, adjusted risk ratio 0·55, 95% CI 0·34-0·88, p=0·014). INTERPRETATION: Rapid weight gain in infancy is associated with future adverse metabolic health. The intervention supplement taken before and throughout pregnancy was associated with lower risk of rapid weight gain and high BMI at age 2 years among children. Long-term follow-up is required to assess the longevity of these benefits. FUNDING: National Institute for Health Research; New Zealand Ministry of Business, Innovation and Employment; Société Des Produits Nestlé; UK Medical Research Council; Singapore National Research Foundation; National University of Singapore and the Agency of Science, Technology and Research; and Gravida.

Time-to-conception and clinical pregnancy rate with a myo-inositol, probiotics, and micronutrient supplement: secondary outcomes of the NiPPeR randomized trial.

OBJECTIVE: To determine whether a combined myo-inositol, probiotics and micronutrient nutritional supplement impacts time-to-natural-conception and clinical pregnancy rates. DESIGN: Secondary outcomes of a double-blind randomized controlled trial. SETTING: Community recruitment. PATIENTS: Women aged 18 to 38 years planning to conceive in the United Kingdom, Singapore, and New Zealand, excluding those with diabetes mellitus or receiving fertility treatment. INTERVENTION: A standard (control) supplement (folic acid, iron, calcium, iodine, ß-carotene), compared with an intervention additionally containing myo-inositol, probiotics, and other micronutrients (vitamins B2, B6, B12, D, zinc). MAIN OUTCOME MEASURES: Number of days between randomization and estimated date of natural conception of a clinical pregnancy, as well as cumulative pregnancy rates at 3, 6, and 12 months. RESULTS: Of 1729 women randomized, 1437 (83%; intervention, n=736; control, n=701) provided data. Kaplan-Meier curves of conception were similar between intervention and control groups; the time at which 20% achieved natural conception was 90.5 days (95% confidence interval: 80.7, 103.5) in the intervention group compared with 92.0 days (76.0, 105.1) in the control group. Cox's proportional hazard ratios (HRs) comparing intervention against control for cumulative achievement of pregnancy (adjusted for site, ethnicity, age, body mass index, and gravidity) were similar at 3, 6, and 12 months. Among both study groups combined, overall time-to-conception lengthened with higher preconception body mass index, and was longer in non-White than in White women. Among women who were overweight the intervention shortened time-to-conception compared with control regardless of ethnicity (12-month HR=1.47 [1.07, 2.02], P=.016; 20% conceived by 84.5 vs. 117.0 days) and improved it to that comparable to nonoverweight/nonobese women (20% conceived by 82.1 days). In contrast, among women with obesity, time-to-conception was lengthened with intervention compared with control (12-month HR=0.69 [0.47, 1.00]; P=.053; 20% conceived by 132.7 vs. 108.5 days); an effect predominantly observed in non-White women with obesity. CONCLUSIONS: Time-to-natural-conception and clinical pregnancy rates within a year were overall similar in women receiving the intervention supplement compared with control. Overweight women had a longer time-to-conception but there was suggestion that the supplement may shorten their time-to-conception to that comparable to the nonoverweight/nonobese women. Further studies are required to confirm this. CLINICAL TRIAL REGISTRATION NUMBER: clinicaltrials.gov (NCT02509988).

Peripartum outcomes after combined myo-inositol, probiotics, and micronutrient supplementation from preconception: the NiPPeR randomized controlled trial.

BACKGROUND: Evidence that nutritional supplementation before and during pregnancy improves peripartum outcomes is sparse. In the Nutritional Intervention Preconception and During Pregnancy to Maintain Healthy Glucose Metabolism and Offspring Health (NiPPeR) trial, we previously reported that a combined myo-inositol, probiotics, and micronutrient supplement started at preconception showed no difference in the primary outcome of gestational glycemia, but did reduce the risk of preterm delivery, preterm prelabor rupture of membranes, and major postpartum hemorrhage. OBJECTIVE: This study aimed to examine the hypothesis that a reduction in major postpartum hemorrhage following a combined nutritional (myo-inositol, probiotics, and micronutrients) intervention is linked with promotion of labor progress and reduced operative delivery. STUDY DESIGN: This double-blind randomized controlled trial recruited 1729 women from the United Kingdom, Singapore, and New Zealand, aged 18 to 38 years, and planning conception between 2015 and 2017. The effects of the nutritional intervention compared with those of a standard micronutrient supplement (control), taken at preconception and throughout pregnancy, were examined for the secondary outcomes of peripartum events using multinomial, Poisson, and linear regression adjusting for site, ethnicity, and important covariates. RESULTS: Of the women who conceived and progressed beyond 24 weeks' gestation with a singleton pregnancy (n=589), 583 (99%) provided peripartum data. Between women in the intervention (n=293) and control (n=290) groups, there were no differences in rates of labor induction, oxytocin augmentation during labor, instrumental delivery, perineal trauma, and intrapartum cesarean delivery. Although duration of the first stage of labor was similar, the second-stage duration was 20% shorter in the intervention than in the control group (adjusted mean difference, -12.0 [95% confidence interval, -22.2 to -1.2] minutes; P=.029), accompanied by a reduction in operative delivery for delayed second-stage progress (adjusted risk ratio, 0.61 [0.48-0.95]; P=.022). Estimated blood loss was 10% lower in the intervention than in the control group (adjusted mean difference, -35.0 [-70.0 to -3.5] mL; P=.047), consistent with previous findings of reduced postpartum hemorrhage. CONCLUSION: Supplementation with a specific combination of myo-inositol, probiotics, and micronutrients started at preconception and continued in pregnancy reduced the duration of the second stage of labor, the risk of operative delivery for delay in the second stage, and blood loss at delivery.

Human non-CpG methylation patterns display both tissue-specific and inter-individual differences suggestive of underlying function.

DNA methylation (DNAm) in mammals is mostly examined within the context of CpG dinucleotides. Non-CpG DNAm is also widespread across the human genome, but the functional relevance, tissue-specific disposition, and inter-individual variability has not been widely studied. Our aim was to examine non-CpG DNAm in the wider methylome across multiple tissues from the same individuals to better understand non-CpG DNAm distribution within different tissues and individuals and in relation to known genomic regulatory features.DNA methylation in umbilical cord and cord blood at birth, and peripheral venous blood at age 12-13 y from 20 individuals from the Southampton Women's Survey cohort was assessed by Agilent SureSelect methyl-seq. Hierarchical cluster analysis (HCA) was performed on CpG and non-CpG sites and stratified by specific cytosine environment. Analysis of tissue and inter-individual variation was then conducted in a second dataset of 12 samples: eight muscle tissues, and four aliquots of cord blood pooled from two individuals.HCA using methylated non-CpG sites showed different clustering patterns specific to the three base-pair triplicate (CNN) sequence. Analysis of CAC sites with non-zero methylation showed that samples clustered first by tissue type, then by individual (as observed for CpG methylation), while analysis using non-zero methylation at CAT sites showed samples grouped predominantly by individual. These clustering patterns were validated in an independent dataset using cord blood and muscle tissue.This research suggests that CAC methylation can have tissue-specific patterns, and that individual effects, either genetic or unmeasured environmental factors, can influence CAT methylation.

Epigenome-wide association study of sarcopenia: findings from the Hertfordshire Sarcopenia Study (HSS).

BACKGROUND: Sarcopenia is the age-related loss of muscle mass, strength, and function. Epigenetic processes such as DNA methylation, which integrate both genetic and environmental exposures, have been suggested to contribute to the development of sarcopenia. This study aimed to determine whether differences in the muscle methylome are associated with sarcopenia and its component measures: grip strength, appendicular lean mass index (ALMi), and gait speed. METHODS: Using the Infinium Human MethylationEPIC BeadChip, we measured DNA methylation in vastus lateralis muscle biopsies of 83 male participants (12 with sarcopenia) with a mean (standard deviation) age of 75.7 (3.6) years from the Hertfordshire Sarcopenia Study (HSS) and Hertfordshire Sarcopenia Study extension (HSSe) and examined associations with sarcopenia and its components. Pathway, histone mark, and transcription factor enrichment of the differentially methylated CpGs (dmCpGs) were determined, and sodium bisulfite pyrosequencing was used to validate the sarcopenia-associated dmCpGs. Human primary myoblasts (n = 6) isolated from vastus lateralis muscle biopsies from male individuals from HSSe were treated with the EZH2 inhibitor GSK343 to assess how perturbations in epigenetic processes may impact myoblast differentiation and fusion, measured by PAX7 and MYHC immunocytochemistry, and mitochondrial bioenergetics determined using the Seahorse XF96. RESULTS: Sarcopenia was associated with differential methylation at 176 dmCpGs (false discovery rate ≤ 0.05) and 141 differentially methylated regions (Stouffer ≤ 0.05). The sarcopenia-associated dmCpGs were enriched in genes associated with myotube fusion (P = 1.40E-03), oxidative phosphorylation (P = 2.78E-02), and voltage-gated calcium channels (P = 1.59E-04). ALMi was associated with 71 dmCpGs, grip strength with 49 dmCpGs, and gait speed with 23 dmCpGs (false discovery rate ≤ 0.05). There was significant overlap between the dmCpGs associated with sarcopenia and ALMi (P = 3.4E-35), sarcopenia and gait speed (P = 4.78E-03), and sarcopenia and grip strength (P = 7.55E-06). There was also an over-representation of the sarcopenia, ALMi, grip strength, and gait speed-associated dmCpGs with sites of H3K27 trimethylation (all P ≤ 0.05) and amongst EZH2 target genes (all P ≤ 0.05). Furthermore, treatment of human primary myoblasts with the EZH2 inhibitor GSK343 inhibitor led to an increase in PAX7 expression (P ≤ 0.05), decreased myotube fusion (P = 0.043), and an increase in ATP production (P = 0.008), with alterations in the DNA methylation of genes involved in oxidative phosphorylation and myogenesis. CONCLUSIONS: These findings show that differences in the muscle methylome are associated with sarcopenia and individual measures of muscle mass, strength, and function in older individuals. This suggests that changes in the epigenetic regulation of genes may contribute to impaired muscle function in later life.

Evaluation of Preconception Dietary Patterns in Women Enrolled in a Multisite Study.

Background: Diet indices are widely used in nutritional research across communities but do not "capture" the full extent of diet variability across multiple countries. Empirically derived dietary patterns can provide additional information because they reflect combinations of foods potentially associated with health outcomes. Limited studies have evaluated preconception dietary patterns in heterogeneous populations. Objectives: In the multisite Nutritional Intervention Preconception and During Pregnancy to Maintain Healthy Glucose Metabolism and Offspring Health (NiPPeR) study, the secondary aims included: 1) derive pooled and site-specific preconception dietary patterns, and 2) evaluate these patterns using anthropometric measures and metabolic biomarkers. Methods: Women planning pregnancy (n = 1720) in the United Kingdom, Singapore, and New Zealand completed interviewer-administered harmonized FFQs and lifestyle questionnaires at recruitment. Across-cohort ("pooled") and site-specific dietary patterns were derived, and associations between dietary pattern scores and BMI, waist-to-hip ratio, plasma lipids, and glycemia assessed using multivariable linear regression, expressing results as SD change in outcome per SD change in dietary pattern score. Results: The pooled analysis identified 3 dietary patterns: "Vegetables/Fruits/Nuts" ("Healthy"), "Fried potatoes/Processed meat/Sweetened beverages" ("Less Healthy"), and "Fish/Poultry/Noodles/Rice" ("Mixed"). The "Healthy" and "Less Healthy" pooled pattern scores were highly correlated with their corresponding site-specific dietary pattern scores ("Healthy": ρ = 0.87-0.93; "Less Healthy": ρ = 0.65-0.88). Women with higher scores for the "Healthy" pooled pattern had a lower waist-to-hip ratio (standardized ß: -0.10; 95% CI: -0.18, -0.01); those with higher scores for the "Less Healthy" pooled pattern had a higher BMI (standardized ß: 0.17; 95% CI: 0.09, 0.24), higher LDL cholesterol (standardized ß: 0.10; 95% CI: 0.01, 0.19), and less optimal glucose profiles. However, we noted higher adherence to the "Healthy" pooled pattern with higher BMI. Conclusions: The "Healthy" and "Less Healthy" pooled patterns were comparable to the corresponding site-specific patterns. Although the associations between these patterns and objective anthropometric/metabolic measures were largely in the expected directions, future studies are required to confirm these findings.This trial is registered at clinicaltrials.gov (NCT02509988).

Myo-Inositol, Probiotics, and Micronutrient Supplementation From Preconception for Glycemia in Pregnancy: NiPPeR International Multicenter Double-Blind Randomized Controlled Trial.

OBJECTIVE: Better preconception metabolic and nutritional health are hypothesized to promote gestational normoglycemia and reduce preterm birth, but evidence supporting improved outcomes with nutritional supplementation starting preconception is limited. RESEARCH DESIGN AND METHODS: This double-blind randomized controlled trial recruited from the community 1,729 U.K., Singapore, and New Zealand women aged 18-38 years planning conception. We investigated whether a nutritional formulation containing myo-inositol, probiotics, and multiple micronutrients (intervention), compared with a standard micronutrient supplement (control), taken preconception and throughout pregnancy could improve pregnancy outcomes. The primary outcome was combined fasting, 1-h, and 2-h postload glycemia (28 weeks gestation oral glucose tolerance test). RESULTS: Between 2015 and 2017, participants were randomized to control (n = 859) or intervention (n = 870); 585 conceived within 1 year and completed the primary outcome (295 intervention, 290 control). In an intention-to-treat analysis adjusting for site, ethnicity, and preconception glycemia with prespecified P < 0.017 for multiplicity, there were no differences in gestational fasting, 1-h, and 2-h glycemia between groups (ß [95% CI] loge mmol/L intervention vs. control -0.004 [-0.018 to 0.011], 0.025 [-0.014 to 0.064], 0.040 [0.004-0.077], respectively). Between the intervention and control groups there were no significant differences in gestational diabetes mellitus (24.8% vs. 22.6%, adjusted risk ratio [aRR] 1.22 [0.92-1.62]), birth weight (adjusted ß = 0.05 kg [-0.03 to 0.13]), or gestational age at birth (mean 39.3 vs. 39.2 weeks, adjusted ß = 0.20 [-0.06 to 0.46]), but there were fewer preterm births (5.8% vs. 9.2%, aRR 0.43 [0.22-0.82]), adjusting for prespecified covariates. CONCLUSIONS: Supplementation with myo-inositol, probiotics, and micronutrients preconception and in pregnancy did not lower gestational glycemia but did reduce preterm birth.

Maternal dysglycaemia, changes in the infant's epigenome modified with a diet and physical activity intervention in pregnancy: Secondary analysis of a randomised control trial.

BACKGROUND: Higher maternal plasma glucose (PG) concentrations, even below gestational diabetes mellitus (GDM) thresholds, are associated with adverse offspring outcomes, with DNA methylation proposed as a mediating mechanism. Here, we examined the relationships between maternal dysglycaemia at 24 to 28 weeks' gestation and DNA methylation in neonates and whether a dietary and physical activity intervention in pregnant women with obesity modified the methylation signatures associated with maternal dysglycaemia. METHODS AND FINDINGS: We investigated 557 women, recruited between 2009 and 2014 from the UK Pregnancies Better Eating and Activity Trial (UPBEAT), a randomised controlled trial (RCT), of a lifestyle intervention (low glycaemic index (GI) diet plus physical activity) in pregnant women with obesity (294 contol, 263 intervention). Between 27 and 28 weeks of pregnancy, participants had an oral glucose (75 g) tolerance test (OGTT), and GDM diagnosis was based on diagnostic criteria recommended by the International Association of Diabetes and Pregnancy Study Groups (IADPSG), with 159 women having a diagnosis of GDM. Cord blood DNA samples from the infants were interrogated for genome-wide DNA methylation levels using the Infinium Human MethylationEPIC BeadChip array. Robust regression was carried out, adjusting for maternal age, smoking, parity, ethnicity, neonate sex, and predicted cell-type composition. Maternal GDM, fasting glucose, 1-h, and 2-h glucose concentrations following an OGTT were associated with 242, 1, 592, and 17 differentially methylated cytosine-phosphate-guanine (dmCpG) sites (false discovery rate (FDR) ≤ 0.05), respectively, in the infant's cord blood DNA. The most significantly GDM-associated CpG was cg03566881 located within the leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) (FDR = 0.0002). Moreover, we show that the GDM and 1-h glucose-associated methylation signatures in the cord blood of the infant appeared to be attenuated by the dietary and physical activity intervention during pregnancy; in the intervention arm, there were no GDM and two 1-h glucose-associated dmCpGs, whereas in the standard care arm, there were 41 GDM and 160 1-h glucose-associated dmCpGs. A total of 87% of the GDM and 77% of the 1-h glucose-associated dmCpGs had smaller effect sizes in the intervention compared to the standard care arm; the adjusted r2 for the association of LGR6 cg03566881 with GDM was 0.317 (95% confidence interval (CI) 0.012, 0.022) in the standard care and 0.240 (95% CI 0.001, 0.015) in the intervention arm. Limitations included measurement of DNA methylation in cord blood, where the functional significance of such changes are unclear, and because of the strong collinearity between treatment modality and severity of hyperglycaemia, we cannot exclude that treatment-related differences are potential confounders. CONCLUSIONS: Maternal dysglycaemia was associated with significant changes in the epigenome of the infants. Moreover, we found that the epigenetic impact of a dysglycaemic prenatal maternal environment appeared to be modified by a lifestyle intervention in pregnancy. Further research will be needed to investigate possible medical implications of the findings. TRIAL REGISTRATION: ISRCTN89971375.

GWAS on longitudinal growth traits reveals different genetic factors influencing infant, child, and adult BMI.

Early childhood growth patterns are associated with adult health, yet the genetic factors and the developmental stages involved are not fully understood. Here, we combine genome-wide association studies with modeling of longitudinal growth traits to study the genetics of infant and child growth, followed by functional, pathway, genetic correlation, risk score, and colocalization analyses to determine how developmental timings, molecular pathways, and genetic determinants of these traits overlap with those of adult health. We found a robust overlap between the genetics of child and adult body mass index (BMI), with variants associated with adult BMI acting as early as 4 to 6 years old. However, we demonstrated a completely distinct genetic makeup for peak BMI during infancy, influenced by variation at the LEPR/LEPROT locus. These findings suggest that different genetic factors control infant and child BMI. In light of the obesity epidemic, these findings are important to inform the timing and targets of prevention strategies.

Mitochondrial oxidative capacity and NAD+ biosynthesis are reduced in human sarcopenia across ethnicities.

The causes of impaired skeletal muscle mass and strength during aging are well-studied in healthy populations. Less is known on pathological age-related muscle wasting and weakness termed sarcopenia, which directly impacts physical autonomy and survival. Here, we compare genome-wide transcriptional changes of sarcopenia versus age-matched controls in muscle biopsies from 119 older men from Singapore, Hertfordshire UK and Jamaica. Individuals with sarcopenia reproducibly demonstrate a prominent transcriptional signature of mitochondrial bioenergetic dysfunction in skeletal muscle, with low PGC-1α/ERRα signalling, and downregulation of oxidative phosphorylation and mitochondrial proteostasis genes. These changes translate functionally into fewer mitochondria, reduced mitochondrial respiratory complex expression and activity, and low NAD+ levels through perturbed NAD+ biosynthesis and salvage in sarcopenic muscle. We provide an integrated molecular profile of human sarcopenia across ethnicities, demonstrating a fundamental role of altered mitochondrial metabolism in the pathological loss of skeletal muscle mass and function in older people.

In Epigenomic Studies, Including Cell-Type Adjustments in Regression Models Can Introduce Multicollinearity, Resulting in Apparent Reversal of Direction of Association.

Background: Association studies of epigenome-wide DNA methylation and disease can inform biological mechanisms. DNA methylation is often measured in peripheral blood, with heterogeneous cell types with different methylation profiles. Influences such as adiposity-associated inflammation can change cell-type proportions, altering measured blood methylation levels. To determine whether associations between loci-specific methylation and outcomes result from cellular heterogeneity, many studies adjust for estimated blood cell proportions, but high correlations between methylation and cell-type proportions could violate the statistical assumption of no multicollinearity. We examined these assumptions in a population-based study. Methods: CDKN2A promoter CpG methylation was measured in peripheral blood from 812 adolescents aged 17 years (Western Australian Pregnancy Cohort Study). Loge adolescent BMI was used as the outcome in a regression analysis with DNA methylation as predictor, adjusting for age/sex. Further regression analyses additionally adjusted for estimated cell-type proportions using the reference-based Houseman method, and simulations modeled the effects of varying levels of correlation between cell proportions and methylation. Correlations between estimated cell proportions and CpG methylation from Illumina 450K were measured. Results: Lower DNA methylation was associated with higher BMI when cell-type adjustment was not included; for CpG4, ß = -0.004 logeBMI/%methylation (95% CI -0.0065, -0.001; p = 0.003). The direction of association reversed when adjustment for six cell types was made; for CpG4, ß = 0.004 logeBMI/%methylation (-0.0002, 0.0089; p = 0.06). Correlations between CpG methylation and cell-type proportions were high, and variance inflation factors (VIFs) were extremely high (25 to 113.7). Granulocyte count was correlated with BMI, and removing granulocytes from the regression model reduced all VIFs to <3.1, with persistence of a positive association between methylation and BMI [CpG4 ß = 0.004 logeBMI/%methylation (-0.0002, 0.0088; p = 0.06)]. Simulations supported major effects of multicollinearity on regression results. Conclusions: Where cell types are highly correlated with other covariates in regression models, the statistical assumption of no multicollinearity may be violated. This can result in reversal of direction of association, particularly when examining associations with phenotypes related to inflammation, as CpG methylation may associate with changes in cell-type proportions. Removing predictors with high correlations from regression models may remove the multicollinearity. However, this might hinder biological interpretability.

Differential SLC6A4 methylation: a predictive epigenetic marker of adiposity from birth to adulthood.

BACKGROUND: The early life environment may influence susceptibility to obesity and metabolic disease in later life through epigenetic processes. SLC6A4 is an important mediator of serotonin bioavailability, and has a key role in energy balance. We tested the hypothesis that methylation of the SLC6A4 gene predicts adiposity across the life course. METHODS: DNA methylation at 5 CpGs within the SLC6A4 gene identified from a previous methyl binding domain array was measured by pyrosequencing. We measured DNA methylation in umbilical cord (UC) from children in the Southampton Women's Survey cohort (n = 680), in peripheral blood from adolescents in the Western Australian Pregnancy Cohort Study (n = 812), and in adipose tissue from lean and obese adults from the UK BIOCLAIMS cohort (n = 81). Real-time PCR was performed to assess whether there were corresponding alterations in gene expression in the adipose tissue. RESULTS: Lower UC methylation of CpG5 was associated with higher total fat mass at 4 years (p = 0.031), total fat mass at 6-7 years (p = 0.0001) and % fat mass at 6-7 years (p = 0.004). Lower UC methylation of CpG5 was also associated with higher triceps skinfold thickness at birth (p = 0.013), 6 months (p = 0.038), 12 months (p = 0.062), 2 years (p = 0.0003), 3 years (p = 0.00004) and 6-7 years (p = 0.013). Higher maternal pregnancy weight gain (p = 0.046) and lower parity (p = 0.029) were both associated with lower SLC6A4 CpG5 methylation. In adolescents, lower methylation of CpG5 in peripheral blood was associated with greater concurrent measures of adiposity including BMI (p ≤ 0.001), waist circumference (p = 0.011), subcutaneous fat (p ≤ 0.001) and subscapular, abdominal and suprailiac skinfold thicknesses (p = 0.002, p = 0.008, p = 0.004, respectively). In adipose tissue, methylation of both SLC6A4 CpG5 (p = 0.019) and expression of SLC6A4 (p = 0.008) was lower in obese compared with lean adults. CONCLUSIONS: These data suggest that altered methylation of CpG loci within SLC6A4 may provide a robust marker of adiposity across the life course.

FUT2 Genetic Variants and Reported Respiratory and Gastrointestinal Illnesses During Infancy.

BACKGROUND: Fucosyltransferase 2 (FUT2) controls the production of digestive and respiratory epithelia of histo-blood group antigens involved in the attachment of pathogens. The aim of our study was to relate FUT2 variants to reported gastrointestinal and respiratory illnesses in infancy. METHODS: In the Southampton Women's Survey, FUT2 genetic variants (single-nucleotide polymorphisms [SNPs] rs601338 and rs602662) were genotyped in 1831 infants and related to infant illnesses, after adjustment for sex, breastfeeding duration, and potential confounders. RESULTS: For FUT2 SNP rs601338, the risk ratios for ≥1 bout of diarrhea during ages 6-12 months and ages 12-24 months per additional risk (G) allele were 1.23 (95% confidence interval [CI], 1.08-1.4; P = .002) and 1.41 (95% CI, 1.24-1.61; P = 1.7 × 10-7), respectively; the risk ratio for ≥1 diagnosis of a lower respiratory illness (ie, pneumonia or bronchiolitis) during ages 12-24 months per additional G allele was 2.66 (95% CI, 1.64-4.3; P = .00007). Similar associations were found between rs602662 and gastrointestinal and respiratory illnesses, owing to the high linkage disequilibrium with rs601338 (R2 = 0.92). Longer breastfeeding duration predicted a lower risk of diarrhea, independent of infant FUT2 genotype. CONCLUSIONS: We confirmed that FUT2 G alleles are associated with a higher risk of infant gastrointestinal illnesses and identified novel associations with respiratory illnesses. FUT2 locus variants need consideration in future studies of gastrointestinal and respiratory illnesses among infants.

Genome-wide association study of offspring birth weight in 86 577 women identifies five novel loci and highlights maternal genetic effects that are independent of fetal genetics.

Genome-wide association studies of birth weight have focused on fetal genetics, whereas relatively little is known about the role of maternal genetic variation. We aimed to identify maternal genetic variants associated with birth weight that could highlight potentially relevant maternal determinants of fetal growth. We meta-analysed data on up to 8.7 million SNPs in up to 86 577 women of European descent from the Early Growth Genetics (EGG) Consortium and the UK Biobank. We used structural equation modelling (SEM) and analyses of mother-child pairs to quantify the separate maternal and fetal genetic effects. Maternal SNPs at 10 loci (MTNR1B, HMGA2, SH2B3, KCNAB1, L3MBTL3, GCK, EBF1, TCF7L2, ACTL9, CYP3A7) were associated with offspring birth weight at P < 5 × 10-8. In SEM analyses, at least 7 of the 10 associations were consistent with effects of the maternal genotype acting via the intrauterine environment, rather than via effects of shared alleles with the fetus. Variants, or correlated proxies, at many of the loci had been previously associated with adult traits, including fasting glucose (MTNR1B, GCK and TCF7L2) and sex hormone levels (CYP3A7), and one (EBF1) with gestational duration. The identified associations indicate that genetic effects on maternal glucose, cytochrome P450 activity and gestational duration, and potentially on maternal blood pressure and immune function, are relevant for fetal growth. Further characterization of these associations in mechanistic and causal analyses will enhance understanding of the potentially modifiable maternal determinants of fetal growth, with the goal of reducing the morbidity and mortality associated with low and high birth weights.

Is cellular heterogeneity merely a confounder to be removed from epigenome-wide association studies?

Excitement about DNA methylation biomarkers has been tempered by a growing appreciation of the complex causal relations with cell fate. Intersample differences in DNA methylation can be partitioned into those that are independent of cellular heterogeneity and those that are caused by differential mixtures of cell types. Generally, the field has assumed that the former are more likely to be causative of disease. The latter has been considered a likely consequence of disease and a confounder to be removed. We argue that the conceptual separation of these signals is artificial and not necessarily informative about causation. DNA methylation is a very sensitive measure of cell fate mix and therefore reveals much about underlying disease etiology including aspects of causation.

Response to Antenatal Cholecalciferol Supplementation Is Associated With Common Vitamin D-Related Genetic Variants.

Context: Single-nucleotide polymorphisms (SNPs) in genes related to vitamin D metabolism have been associated with serum 25-hydroxyvitamin D [25(OH)D] concentration, but these relationships have not been examined following antenatal cholecalciferol supplementation. Objective: To determine whether SNPs in DHCR7, CYP2R1, CYP24A1, and GC are associated with the response to gestational cholecalciferol supplementation. Design: Within-randomization group analysis of the Maternal Vitamin D Osteoporosis Study trial of antenatal cholecalciferol supplementation. Setting: Hospital antenatal clinics. Participants: In total, 682 women of white ethnicity (351 placebo, 331 cholecalciferol) were included. SNPs at rs12785878 (DHCR7), rs10741657 (CYP2R1), rs6013897 (CYP24A1), and rs2282679 (GC) were genotyped. Interventions: 1000 IU/d cholecalciferol from 14 weeks of gestation until delivery. Main Outcome Measure: 25(OH)D at randomization and 34 weeks of gestation were measured in a single batch (Liaison; Diasorin, Dartford, UK). Associations between 25(OH)D and the SNPs were assessed by linear regression using an additive model [ß represents the change in 25(OH)D per additional common allele]. Results: Only rs12785878 (DHCR7) was associated with baseline 25(OH)D [ß = 3.1 nmol/L; 95% confidence interval (CI), 1.0 to 5.2 nmol/L; P < 0.004]. In contrast, rs10741657 (CYP2R1) (ß = -5.2 nmol/L; 95% CI, -8.2 to -2.2 nmol/L; P = 0.001) and rs2282679 (GC) (ß = 4.2 nmol/L; 95% CI, 0.9 to 7.5 nmol/L; P = 0.01) were associated with achieved 25(OH)D status following supplementation, whereas rs12785878 and rs6013897 (CYP24A1) were not. Conclusions: Genetic variation in DHCR7, which encodes 7-dehyrocholesterol reductase in the epidermal vitamin D biosynthesis pathway, appears to modify baseline 25(OH)D. In contrast, the response to antenatal cholecalciferol supplementation was associated with SNPs in CYP2R1, which may alter 25-hydroxylase activity, and GC, which may affect vitamin D binding protein synthesis or metabolite affinity.