Elevated Maternal Folate Status and Changes in Maternal Prolactin, Placental Lactogen and Placental Growth Hormone Following Folic Acid Food Fortification: Evidence from Two Prospective Pregnancy Cohorts.

Folic acid (FA) food fortification in Australia has resulted in a higher-than-expected intake of FA during pregnancy. High FA intake is associated with increased insulin resistance and gestational diabetes. We aimed to establish whether maternal one-carbon metabolism and hormones that regulate glucose homeostasis change in healthy pregnancies post-FA food fortification. Circulating folate, B12, homocysteine, prolactin (PRL), human placental lactogen (hPL) and placental growth hormone (GH2) were measured in early pregnancy maternal blood in women with uncomplicated pregnancies prior to (SCOPE: N = 604) and post (STOP: N = 711)-FA food fortification. FA food fortification resulted in 63% higher maternal folate. STOP women had lower hPL (33%) and GH2 (43%) after 10 weeks of gestation, but they had higher PRL (29%) and hPL (28%) after 16 weeks. FA supplementation during pregnancy increased maternal folate and reduced homocysteine but only in the SCOPE group, and it was associated with 54% higher PRL in SCOPE but 28% lower PRL in STOP. FA food fortification increased maternal folate status, but supplements no longer had an effect, thereby calling into question their utility. An altered secretion of hormones that regulate glucose homeostasis in pregnancy could place women post-fortification at an increased risk of insulin resistance and gestational diabetes, particularly for older women and those with obesity.

Low-dose thiamine supplementation of lactating Cambodian mothers improves human milk thiamine concentrations: a randomized controlled trial.

BACKGROUND: Infantile beriberi-related mortality is still common in South and Southeast Asia. Interventions to increase maternal thiamine intakes, and thus human milk thiamine, are warranted; however, the required dose remains unknown. OBJECTIVES: We sought to estimate the dose at which additional maternal intake of oral thiamine no longer meaningfully increased milk thiamine concentrations in infants at 24 wk postpartum, and to investigate the impact of 4 thiamine supplementation doses on milk and blood thiamine status biomarkers. METHODS: In this double-blind, 4-parallel arm randomized controlled dose-response trial, healthy mothers were recruited in Kampong Thom, Cambodia. At 2 wk postpartum, women were randomly assigned to consume 1 capsule, containing 0, 1.2 (estimated average requirement), 2.4, or 10 mg of thiamine daily from 2 through 24 weeks postpartum. Human milk total thiamine concentrations were measured using HPLC. An Emax curve was plotted, which was estimated using a nonlinear least squares model in an intention-to-treat analysis. Linear mixed-effects models were used to test for differences between treatment groups. Maternal and infant blood thiamine biomarkers were also assessed. RESULTS: In total, each of 335 women was randomly assigned to1 of the following thiamine-dose groups: placebo (n = 83), 1.2 mg (n = 86), 2.4 mg (n = 81), and 10 mg (n = 85). The estimated dose required to reach 90% of the maximum average total thiamine concentration in human milk (191 µg/L) is 2.35 (95% CI: 0.58, 7.01) mg/d. The mean ± SD milk thiamine concentrations were significantly higher in all intervention groups (183 ± 91, 190 ± 105, and 206 ± 89 µg/L for 1.2, 2.4, and 10 mg, respectively) compared with the placebo group (153 ± 85 µg/L; P < 0.0001) and did not significantly differ from each other. CONCLUSIONS: A supplemental thiamine dose of 2.35 mg/d was required to achieve a milk total thiamine concentration of 191 µg/L. However, 1.2 mg/d for 22 wk was sufficient to increase milk thiamine concentrations to similar levels achieved by higher supplementation doses (2.4 and 10 mg/d), and comparable to those of healthy mothers in regions without beriberi. This trial was registered at clinicaltrials.gov as NCT03616288.

Effect of Selenium and Iodine on Oxidative Stress in the First Trimester Human Placenta Explants.

Imbalanced maternal micronutrient status, poor placentation, and oxidative stress are associated with greater risk of pregnancy complications, which impact mother and offspring health. As selenium, iodine, and copper are essential micronutrients with key roles in antioxidant systems, this study investigated their potential protective effects on placenta against oxidative stress. First trimester human placenta explants were treated with different concentrations of selenium (sodium selenite), iodine (potassium iodide), their combination or copper (copper (II) sulfate). The concentrations represented deficient, physiological, or super physiological levels. Oxidative stress was induced by menadione or antimycin. Placenta explants were collected, fixed, processed, and embedded for laser ablation inductively coupled plasma-mass spectrometry (LA ICP-MS) element imaging or immunohistochemical labelling. LA ICP-MS showed that placenta could uptake selenium and copper from the media. Sodium selenite and potassium iodide reduced DNA damage and apoptosis (p < 0.05). Following oxidative stress induction, a higher concentration of sodium selenite (1.6 µM) was needed to reduce DNA damage and apoptosis while both concentrations of potassium iodide (0.5 and 1 µM) were protective (p < 0.05). A high concentration of copper (40 µM) increased apoptosis and DNA damage but this effect was no longer significant after induction of oxidative stress. Micronutrients supplementation can increase their content within the placenta and an optimal maternal micronutrient level is essential for placenta health.

Assessment of salt intake to consider salt as a fortification vehicle for thiamine in Cambodia.

Thiamine deficiency is a public health issue in Cambodia. Thiamine fortification of salt has been proposed; however, the salt intake of lactating women, the target population, is currently unknown. We estimated salt intakes among lactating women (<6 months postpartum) using three methods: repeat observed-weighed intake records and 24-h urinary sodium excretions (n = 104), and household salt disappearance (n = 331). Usual salt intake was estimated by adjusting for intraindividual intakes using the National Cancer Institute method, and a thiamine salt fortification scenario was modeled using a modified estimated average requirement (EAR) cut-point method. Unadjusted salt intake from observed intakes was 9.3 (8.3-10.3) g/day, which was not different from estimated salt intake from urinary sodium excretions, 9.0 (8.4-9.7) g/day (P = 0.3). Estimated salt use from household salt disappearance was 11.3 (10.7-11.9) g/person/day. Usual (adjusted) salt intake from all sources was 7.7 (7.4-8.0) g/day. Assuming no stability losses, a modeled fortification dose of 275 mg thiamine/kg salt could increase thiamine intakes from fortified salt to 2.1 (2.0-2.2) mg/day, with even low salt consumers reaching the EAR of 1.2 mg/day from fortified salt alone. These findings, in conjunction with future sensory and stability research, can inform a potential salt fortification program in Cambodia.

Maternal folate, one-carbon metabolism and pregnancy outcomes.

Single nucleotide polymorphisms and pre- and peri-conception folic acid (FA) supplementation and dietary data were used to identify one-carbon metabolic factors associated with pregnancy outcomes in 3196 nulliparous women. In 325 participants, we also measured circulating folate, vitamin B12 and homocysteine. Pregnancy outcomes included preeclampsia (PE), gestational hypertension (GHT), small for gestational age (SGA), spontaneous preterm birth (sPTB) and gestational diabetes mellitus (GDM). Study findings show that maternal genotype MTHFR A1298C(CC) was associated with increased risk for PE, whereas TCN2 C766G(GG) had a reduced risk for sPTB. Paternal MTHFR A1298C(CC) and MTHFD1 G1958A(AA) genotypes were associated with reduced risk for sPTB, whereas MTHFR C677T(CT) genotype had an increased risk for GHT. FA supplementation was associated with higher serum folate and vitamin B12 concentrations, reduced uterine artery resistance index and increased birth weight. Women who supplemented with <800 µg daily FA at 15-week gestation had a higher incidence of PE (10.3%) compared with women who did not supplement (6.1%) or who supplemented with ≥800 µg (5.4%) (P < .0001). Higher serum folate levels were found in women who later developed GDM compared with women with uncomplicated pregnancies (Mean ± SD: 37.6 ± 8 nmol L-1 vs. 31.9 ± 11.2, P = .007). Fast food consumption was associated with increased risk for developing GDM, whereas low consumption of green leafy vegetables and fruit were independent risk factors for SGA and GDM and sPTB and SGA, respectively. In conclusion, maternal and paternal genotypes, together with maternal circulating folate and homocysteine concentrations, and pre- and early-pregnancy dietary factors, are independent risk factors for pregnancy complications.

Effect of parenteral lipid emulsion on preterm infant PUFAs and their downstream metabolites.

OBJECTIVE: Oxylipins synthesized by oxidation of long-chain polyunsaturated fatty acids (LCPUFAs) are bioactive downstream lipid mediators. The aim of this study was to describe oxylipin levels in preterm infants born 30 to 33 weeks' gestation who were enrolled in a randomized controlled trial in which peripheral parenteral nutrition (P-PN), including lipid emulsion (containing soy, medium chain triglyceride, olive and fish oil), was compared with 10% glucose on growth during the transition to enteral feeds. METHODS: Of the 92 infants randomized to the P-PN study, the first 72 (P-PN n = 34, control n = 38) had blood taken for fatty acid analyses. P-PN infants received parenteral nutrition including 3% protein, 8% glucose and 17% SMOFlipid® lipid (containing soy, medium chain triglyceride, olive and fish oil), and control infants 10% glucose. Both groups commenced enteral feeds when clinically stable. 32 oxylipins and 5 free fatty acids were screened (using ultra-high-performance liquid chromatography-tandem mass spectrometry), and 5 total LCPUFA were measured (using gas chromatography), on study days 1 (baseline), 2, 4, 7, 14 and 21. RESULTS: Both total and free LA, ALA and EPA were significantly higher in the P-PN group compared with control over the first week of life. Whereas total AA was significantly lower and free DHA significantly higher over the same time period. All LA, ALA, EPA and four DHA derived oxylipins detected were significantly higher in the P-PN group compared with the control group during the first week of life, with three AA derived oxylipins significantly lower and one significantly higher. CONCLUSIONS: Parenteral lipid emulsion resulted in a change in total and free fatty acids and related oxylipins with the profiles suggesting increased omega-6 driven inflammation. Further studies to investigate the association between the oxylipin levels and nutrition and to determine whether the oxylipin profiles influence the clinical outcome in preterm infants are warranted.

Effect of Iodine and Selenium on Proliferation, Viability, and Oxidative Stress in HTR-8/SVneo Placental Cells.

Adequate maternal micronutrition is vital for placental formation, fetal growth, and development. Oxidative stress adversely affects placental development and function and an association between deficient placental development, oxidative stress, and micronutrient deficiency has been observed. Selenium and iodine are two essential micronutrients with antioxidant properties. Epidemiological studies have shown that poor micronutrient status in pregnant women is associated with a higher incidence of pregnancy complications. The aim of this study was to determine how selenium, iodine, and their combination impact oxidative stress in placental trophoblast cells. HTR8/SVneo extravillous trophoblasts were supplemented with a concentration range of organic and inorganic selenium, potassium iodide, or their combination for 24 h. Oxidative stress was then induced by treating cells with menadione or H2O2 for 24 h. Cell viability and lipid peroxidation as the biomarker of oxidative stress were assessed at 48 h. Both menadione and H2O2 reduced cell viability and increased lipid peroxidation (P < 0.05). Greater cell viability was found in selenium-supplemented cells when compared with vehicle treated cells (P < 0.05). Selenium and iodine supplementation separately or together were associated with lower lipid peroxidation compared with vehicle control (P < 0.05). Supplementation with the combination of selenium and iodine resulted in a greater reduction in lipid peroxidation compared with selenium or iodine alone (P < 0.05). Oxidative stress negatively impacts trophoblast cell survival and cellular integrity. Selenium and iodine protect placental trophoblasts against oxidative stress. Further research is warranted to investigate the molecular mechanisms by which selenium and iodine act in the human placenta.

Weekly iron-folic acid supplements containing 2.8 mg folic acid are associated with a lower risk of neural tube defects than the current practice of 0.4 mg: a randomised controlled trial in Malaysia.

INTRODUCTION: Weekly iron-folic acid (IFA) supplements are recommended for all menstruating women in countries where anaemia prevalence is >20%. Anaemia caused by folate deficiency is low worldwide, and the need to include folic acid is in question. Including folic acid might reduce the risk of a neural tube defect (NTD) should a woman become pregnant. Most weekly supplements contain 0.4 mg folic acid; however, WHO recommends 2.8 mg because it is seven times the daily dose effective in reducing NTDs. There is a reluctance to switch to supplements containing 2.8 mg of folic acid because of a lack of evidence that this dose would prevent NTDs. Our aim was to investigate the effect of two doses of folic acid, compared with placebo, on red blood cell (RBC) folate, a biomarker of NTD risk. METHODS: We conducted a three-arm double-blind efficacy trial in Malaysia. Non-pregnant women (n=331) were randomised to receive 60 mg iron and either 0, 0.4, or 2.8 mg folic acid once weekly for 16 weeks. RESULTS: At 16 weeks, women receiving 0.4 mg and 2.8 mg folic acid per week had a higher mean RBC folate than those receiving 0 mg (mean difference (95% CI) 84 (54 to 113) and 355 (316 to 394) nmol/L, respectively). Women receiving 2.8 mg folic acid had a 271 (234 to 309) nmol/L greater mean RBC folate than those receiving 0.4 mg. Moreover, women in the 2.8 mg group were seven times (RR 7.3, 95% CI 3.9 to 13.7; p<0.0001) more likely to achieve an RBC folate >748 nmol/L, a concentration associated with a low risk of NTD, compared with the 0.4 mg group. CONCLUSION: Weekly IFA supplements containing 2.8 mg folic acid increases RBC folate more than those containing 0.4 mg. Increased availability and access to the 2.8 mg formulation is needed. TRAIL REGISTRATION NUMBER: This trial is registered with the Australian New Zealand Clinical Trial Registry (ACTRN12619000818134).

Effect of once weekly folic acid supplementation on erythrocyte folate concentrations in women to determine potential to prevent neural tube defects: a randomised controlled dose-finding trial in Malaysia.

INTRODUCTION: Folic acid (0.4 mg) taken prior to and during early pregnancy reduces the risk of neural tube defects (NTDs). Because these birth defects occur early in pregnancy, before women may know they are pregnant, many countries have mandated the addition of folic acid to food staples. In countries where fortification is not possible, and weekly iron folic acid programmes exist to reduce anaemia, the WHO recommends that 2.8 mg (7×0.4 mg) folic acid be given instead of the current weekly practice of 0.4 mg. Currently, there is a lack of evidence to support if the 2.8 mg folic acid per week dose is sufficient to raise erythrocyte folate concentrations to a level associated with a reduced risk of a NTD-affected pregnancy. We aim to conduct a three-arm randomised controlled trial to determine the effect of weekly folic acid with iron on erythrocyte folate, a biomarker of NTD risk. METHODS AND ANALYSIS: We will recruit non-pregnant women (n=300; 18-45 years) from Selangor, Malaysia. Women will be randomised to receive either 2.8, 0.4 or 0.0 (placebo) mg folic acid with 60 mg iron weekly for 16 weeks, followed by a 4-week washout period. The primary outcome will be erythrocyte folate concentration at 16 weeks and the mean concentration will be compared between randomised treatment groups (intention-to-treat) using a linear regression model adjusting for the baseline measure. ETHICS AND DISSEMINATION: Ethical approval was obtained from the University of British Columbia (H18-00768) and Universiti Putra Malaysia (JKEUPM-2018-255). The results of this trial will be presented at scientific conferences and published in peer-reviewed journals. TRIAL REGISTRATION NUMBERS: ACTRN12619000818134 and NMRR-19-119-45736.

Intravenous fat induces changes in PUFA and their bioactive metabolites: Comparison between Japanese and Australian preterm infants.

OBJECTIVE: Oxylipins are biologically active signaling molecules that initiate and resolve inflammation; they are synthesized by oxidation of polyunsaturated fatty acids (PUFAs) and reflect PUFA intake and status. The PUFA intake in preterm infants differs between countries because of the type of lipid emulsions used and the PUFA content of breast milk. We compared total blood PUFA, free PUFA and their oxylipin levels in dried whole blood samples from preterm infants born in Australia and Japan. METHODS: We enrolled 30 and 14 preterm infants born less than 31 weeks' gestation, from Adelaide and Japan respectively. Blood samples were obtained from cord blood, and on postnatal days 4, 7, 14 and 28. Total PUFAs were measured using gas chromatography, while free fatty acids and oxylipins were screened using ultra high-performance liquid chromatography mass spectroscopy. RESULTS: Differences in the levels of blood PUFA between the centres were found which were in line with the timing and type of lipid emulsion administration. Significant differences in longitudinal levels were seen more often in free PUFA and their oxylipins than in total blood PUFA. This was particularly true for AA and DHA. In contrast, differences in the levels could be seen in total blood EPA, as well as in free EPA and its oxylipins. Further, levels of many free PUFA and their oxylipins were higher in Japanese infants than in Australian infants. CONCLUSION: Differences in total and free fatty acids and unesterified oxylipins, were observed during the first weeks of life and between preterm infants born in Australia and Japan, which were likely a reflection of the type of lipid emulsion and timing of administration. The clinical significance of these changes remains to be explored.

Thiamine dose response in human milk with supplementation among lactating women in Cambodia: study protocol for a double-blind, four-parallel arm randomised controlled trial.

INTRODUCTION: Thiamine (vitamin B1) deficiency remains a concern in Cambodia where women with low thiamine intake produce thiamine-poor milk, putting their breastfed infants at risk of impaired cognitive development and potentially fatal infantile beriberi. Thiamine fortification of salt is a potentially low-cost, passive means of combating thiamine deficiency; however, both the dose of thiamine required to optimise milk thiamine concentrations as well as usual salt intake of lactating women are unknown. METHODS AND ANALYSIS: In this community-based randomised controlled trial, 320 lactating women from Kampong Thom, Cambodia will be randomised to one of four groups to consume one capsule daily containing 0, 1.2, 2.4 or 10 mg thiamine as thiamine hydrochloride, between 2 and 24 weeks postnatal. The primary objective is to estimate the dose where additional maternal intake of thiamine no longer meaningfully increases infant thiamine diphosphate concentrations 24 weeks postnatally. At 2, 12 and 24 weeks, we will collect sociodemographic, nutrition and health information, a battery of cognitive assessments, maternal (2 and 24 weeks) and infant (24 weeks only) venous blood samples (biomarkers: ThDP and transketolase activity) and human milk samples (also at 4 weeks; biomarker: milk thiamine concentrations). All participants and their families will consume study-provided salt ad libitum throughout the trial, and we will measure salt disappearance each fortnight. Repeat weighed salt intakes and urinary sodium concentrations will be measured among a subset of 100 participants. Parameters of Emax dose-response curves will be estimated using non-linear least squares models with both 'intention to treat' and a secondary 'per-protocol' (capsule compliance ≥80%) analyses. ETHICS AND DISSEMINATION: Ethical approval was obtained in Cambodia (National Ethics Committee for Health Research 112/250NECHR), Canada (Mount Saint Vincent University Research Ethics Board 2017-141) and the USA (University of Oregon Institutional Review Board 07052018.008). Results will be shared with participants' communities, as well as relevant government and scientific stakeholders via presentations, academic manuscripts and consultations. TRIAL REGISTRATION NUMBER: NCT03616288.

Maternal Selenium, Copper and Zinc Concentrations in Early Pregnancy, and the Association with Fertility.

Trace elements such as zinc, copper, and selenium are essential for reproductive health, but there is limited work examining how circulating trace elements may associate with fertility in humans. The aim of this study was to determine the association between maternal plasma concentrations of zinc, copper, and selenium, and time to pregnancy and subfertility. Australian women (n = 1060) who participated in the multi-centre prospective Screening for Pregnancy Endpoints study were included. Maternal plasma concentrations of copper, zinc and selenium were assessed at 15 ± 1 weeks' gestation. Estimates of retrospectively reported time to pregnancy were documented as number of months to conceive; subfertility was defined as taking more than 12 months to conceive. A range of maternal and paternal adjustments were included. Women who had lower zinc (time ratio, 1.20 (0.99-1.44)) or who had lower selenium concentrations (1.19 (1.01-1.40)) had a longer time to pregnancy, equivalent to a median difference in time to pregnancy of around 0.6 months. Women with low selenium concentrations were also at a 1.46 (1.06-2.03) greater relative risk for subfertility compared to women with higher selenium concentrations. There were no associations between copper and time to pregnancy or subfertility. Lower selenium and zinc trace element concentrations, which likely reflect lower dietary intakes, associate with a longer time to pregnancy. Further research supporting our work is required, which may inform recommendations to increase maternal trace element intake in women planning a pregnancy.