Background North American public health guidelines recommend supplementation with an iron-containing prenatal multivitamin throughout pregnancy to meet the Recommended Daily Allowance (RDA) of 27 mg elemental iron daily. However, whether supplementation with standard prenatal multivitamins is sufficient to prevent maternal iron deficiency is unclear, as needs increase substantially with advancing gestation Objective This study aimed to assess iron status in early and late pregnancy among 60 pregnant women receiving 27 mg/day elemental iron as part of a randomized trial in Vancouver, Canada Methods Study visits were conducted at 8-21 (baseline) and 24-38 (endline) weeks’ gestation. Venous blood specimens were collected for a complete blood count and measurement of iron and inflammatory biomarkers. Supplementation with any additional iron (beyond 27 mg/day) was reported by participants (treatment with additional iron is recommended if ferritin <30 µg/L). Quantile regression was used to explore predictors of endline ferritin concentrations, including ethnicity, education, income, and baseline ferritin Results Overall, n = 60 and n = 54 women participated in baseline and endline visits, respectively. Rates of probable iron deficiency (ferritin <30 µg/L) at baseline and endline were: n = 22 (41%) and n = 44 (81%). Less than half (n = 18; 41%) of participants with probable iron deficiency at endline reported supplementation with additional iron. Ethnicity was the only significant modifier of endline ferritin, with higher concentrations in those of South, East, and Southeast Asian ethnicity compared to European (β = 10.4 µg/L, 95% CI = 0.3-20.5) Conclusion Pregnant individuals may require additional supplemental iron beyond 27 mg to meet requirements in later pregnancy, given the high rates of iron deficiency observed in this clinical trial, despite consumption meeting 100% of the RDA Trial registration: ClinicalTrials.gov (identifier: NCT04022135)

Background: North American health authorities recommend 0.4 mg/day folic acid before conception and throughout pregnancy to reduce the risk of neural tube defects. Folic acid is a synthetic form of folate that must be reduced by dihydrofolate reductase and then further metabolized. Recent evidence suggests that the maximal capacity for this process is limited and unmetabolized folic acid has been detected in the circulation. The biological effects of unmetabolized folic acid are unknown. A natural form of folate, (6S)-5-methyltetrahydrofolic acid (Metafolin®), may be a superior alternative because it does not need to be reduced in the small intestine. Metafolin® is currently used in some prenatal multivitamins; however, it has yet to be evaluated during pregnancy. Methods/design: This double-blind, randomized trial will recruit 60 pregnant women aged 19-42 years. The women will receive either 0.6 mg/day folic acid or an equimolar dose (0.625 mg/day) of (6S)-5-methyltetrahydrofolic acid for 16 weeks. The trial will be initiated at 8-21 weeks' gestation (after neural tube closure) to reduce the risk of harm should (6S)-5methyltetrahydrofolic acid prove less effective. All women will also receive a prenatal multivitamin (not containing folate) to ensure adequacy of other nutrients. Baseline and endline blood samples will be collected to assess primary outcome measures, including serum folate, red blood cell folate and unmetabolized folic acid. The extent to which the change in primary outcomes from baseline to endline differs between treatment groups, controlling for baseline level, will be estimated using linear regression. Participants will have the option to continue supplementing until 1 week postpartum to provide a breastmilk and blood sample. Exploratory analyses will be completed to evaluate breastmilk and postpartum blood folate concentrations.

Objectives The pregnancy-induced alterations in 1-carbon (1C) metabolism, effects of advancing gestation on maternal plasma concentrations of methyl nutrients, and potential implications for maternal dietary intake and infant clinical outcomes are summarized in this narrative review. Background 1C metabolism encompasses a series of pathways where 1C units are transferred among nutrients such as B vitamins, choline, and amino acids (the methyl nutrients). Use of isotopic tracers and measuring methyl nutrients in maternal plasma and infant cord blood has advanced the understanding of 1C flux in pregnancy and kinetics of maternal-placental-fetal transfer. Methyl nutrients are supplied from maternal plasma to the placenta and fetus to support growth and 1C metabolism in these compartments. Methods A literature review was completed in MEDLINE and Google Scholar using search terms related to 1C metabolism, methyl nutrients, and nutrition requirements in pregnancy. English-language articles were reviewed in which 1C metabolism in pregnancy, maternal-placental-fetal transfer of methyl nutrients, and determinants of maternal plasma concentrations of methyl nutrients among healthy pregnant women were assessed. Discussion Adaptations in 1C metabolism occur throughout a healthy pregnancy to support this unique period of accelerated growth. Studies report similar temporal changes in plasma concentrations of many methyl nutrients, including B vitamins, choline, betaine, methionine, and cysteine, among healthy pregnant women from diverse geographic regions. Other key findings discussed in this review include an apparent high degree of B vitamin transfer to the placenta and fetus, influence of choline supplementation on 1C flux and possible benefit of supplementation for infant cognitive development, and that glycine may be conditionally essential in pregnancy. Conclusion Understanding the flux of 1C metabolism in pregnancy and methyl nutrient transfer from maternal plasma is needed to establish appropriate plasma references ranges and, ultimately, dietary recommendations that aim to prevent deficiency and associated adverse health outcomes for mother and baby.

Background: North American health authorities recommend 0.4 mg/day folic acid before conception and throughout pregnancy to reduce the risk of neural tube defects. Folic acid is a synthetic form of folate that must be reduced by dihydrofolate reductase and then further metabolized. Recent evidence suggests that the maximal capacity for this process is limited and unmetabolized folic acid has been detected in circulation. The biological effects of unmetabolized folic acid are unknown. A natural form of folate, (6S)-5-methyltetrahydrofolic acid (Metafolin®), may be a superior alternative because it does not need to be reduced in the small intestine. Metafolin® is currently used in some prenatal multivitamins, however it has yet to be evaluated during pregnancy. Methods: This double-blind randomized trial will recruit 60 pregnant women aged 19-42 years. Women will receive either 0.6 mg/day folic acid or an equimolar dose (0.625 mg/day) of (6S)-5-methyltetrahydrofolic acid for 16-weeks. The trial will be initiated at 8-21 weeks gestation (after neural tube closure) to reduce the risk of harm should (6S)-5-methyltetrahydrofolic acid prove less effective. All women will also receive a prenatal multivitamin (not containing folate) to ensure adequacy of other nutrients. Baseline and endline blood samples will be collected to assess primary outcome measures, including serum folate, red blood cell folate and unmetabolized folic acid. The extent to which the change in primary outcomes from baseline to endline differs between treatment groups, controlling for baseline level, will be estimated using linear regression. Participants will have the option to continue supplementing until 1-week postpartum to provide a breastmilk and blood sample. Exploratory analyses will be completed to evaluate breastmilk and postpartum blood folate concentrations. Discussion: This proof-of-concept trial is needed to obtain estimates of the effect of (6S)-5-methyltetrahydrofolic acid compared to folic acid on circulating biomarkers of folate status during pregnancy. These estimates will inform the design of a definitive trial which will be powered to assess whether (6S)-5-methyltetrahydrofolic acid is as effective as folic acid in raising blood folate concentrations during pregnancy. Ultimately, these findings will inform folate supplementation policies for pregnant women.

Objectives To evaluate the recruitment of pregnant women for a clinical trial in Vancouver, Canada, via social media versus offline methods and to explore optimization of social media campaigns. Methods Facebook was used to run nine social media campaigns (15 weeks total, CA$675). Offline methods were used concurrently over 64 weeks (printing costs: CA$300). The cost, rate of recruitment and conversion rate in each group was calculated. Performance metrics of social media campaigns (reach, impressions, clicks, inquiries, enrolments) were recorded. Linear regression was used to explore the association between metrics and dollars spent per campaign. Results In total, n = 481 inquiries were received: n = 51 (11%) via offline methods and n = 430 (89%) via social media. Enrolees (n = 60 total) included n = 24 (40%) and n = 36 (60%) via offline and social media methods, respectively. Gestational weeks upon inquiry (n = 251; mean ± SD) were not different among groups (offline: 13.3 ± 4.7; social media: 13.2 ± 5.6). Direct cost per enrolee was CA$13 and CA$19 via offline and social media methods, respectively (however, this does not include cost of labour). The rate of recruitment was approximately six times faster via social media. However, the conversion rate was higher via offline methods than social media (47% vs. 8%). The amount spent per campaign was significantly associated with improved clicks and inquiries, but not enrolments. Conclusions Social media was more efficient and effective than offline methods. We gained numerous insights for optimization of social media campaigns (dollars spent, attribution setting, photo testing, automatic optimization) to increase clicks and inquiries, however, this does not necessarily increase enrolments, which was more dependent on study-specific factors (e.g. time of year, study design).

Background Regression to the mean (RTM) is a statistical phenomenon where second measurements are more likely to be closer to the mean. This is particularly observed in those with baseline values further from the mean. Anemic individuals (hemoglobin < 120g/L) are often recruited when evaluating iron supplementation programs, as they are more likely to elicit a greater hemoglobin response; however, they are also at greater risk for RTM as their baseline values are lower than the overall population mean. Objective To calculate and apply RTM to a previously conducted iron supplementation trial of women in Cambodia at increasingly severe baseline anemia cut-offs (hemoglobin < 120g/L,  <115g/L and <110g/L). Methods Women received either 60mg/day iron (n = 191) or placebo (n = 185) for 12 weeks. Hemoglobin was measured at baseline and 12 weeks (endline), and change in hemoglobin was calculated in each group, for each cut-off. RTM was calculated in the placebo group at each cut-off and applied to the change observed at each cut-off in the iron group to obtain the RTM-free effect. Results In the placebo group, mean change in hemoglobin increased as cut-offs became more extreme (0.9g/L to 1.9g/L in those with baseline hemoglobin < 120g/L and <110g/L,  respectively). RTM estimates similarly increased: 1.0g/L (<120g/L),  1.3g/L (<115g/L),  and 1.8g/L (<110g/L). When applying RTM to the iron group, we found that ∼10% of the “treatment effect” could be attributable to RTM at each cut-off. However, iron supplementation was still effective in increasing hemoglobin, with an increased effect in those with lower baseline values, as proven by the RTM-free effect at each cut-off: 8.7g/L (<120g/L),  10.9g/L (<115g/L),  and 13.6g/L (<110g/L). Conclusion RTM may have accounted for ∼10% of the observed change in hemoglobin following iron supplementation; however, appropriate use of a placebo group in the statistical analyses of the trial controls for this potential RTM effect.

Genetic hemoglobinopathies are the most common single-gene disorder worldwide. Some automated hematology analyzers have the capability of flagging individuals who may have hematological disorders based on complete blood count (CBC) biomarkers. We aimed to evaluate the accuracy of a hematology analyzer in identifying genetic hemoglobinopathies in Cambodian women and to determine which hematological biomarkers are the best predictors. A CBC was completed using a Sysmex XN-1000 analyzer and hemoglobinopathies were determined with capillary hemoglobin electrophoresis for 808 nonpregnant Cambodian women. Sysmex XN-1000 Interpretive Program (IP) messages, which flag potential hematological disorders, were produced from CBC results. Then, 2 × 2 tables were used to determine sensitivity and specificity of the IP message “Hemoglobin defect” to detect a genetic hemoglobinopathy. Receiver operating characteristic (ROC) analyses assessed the diagnostic ability of six CBC biomarkers to predict a genetic hemoglobinopathy. In total, 74% of women had a hemoglobinopathy (predominantly Hb E and α-thalassemia). “Hb defect” IP message sensitivity and specificity for genetic hemoglobinopathy detection were 10.4% and 98.6%, respectively. Variable selection strategies yielded a two-variable model including mean corpuscular volume (MCV) and red blood cell (RBC) count (AIC = 99.83, AUCROC = 0.98 (95% CI: 0.97, 0.99)) for the prediction of a homozygous EE disorder. Sensitivity and specificity values do not justify the use of Sysmex XN-1000 IP flag messages for identification of genetic hemoglobinopathies in Cambodian women. Development of an algorithm based on MCV and RBC biomarkers may optimize the screening ability of automated hematology analyzers.

ABSTRACT Background Riboflavin is required for erythropoiesis, which is increased in people with hemoglobinopathies due to increased hemolysis and erythrocyte turnover. Dietary intake and status of riboflavin is poor in Cambodia, where hemoglobinopathies are common. Objective We assessed the association between genetic hemoglobin disorders and riboflavin status in women of reproductive age in Cambodia. Methods Venous blood samples from 515 Cambodian women of reproductive age, 18–45 y, were analyzed for biomarker status of riboflavin [erythrocyte glutathione reductase activation coefficient (EGRac)], genetic hemoglobin (Hb) disorders, and hematological indices. Linear regression analysis was used to estimate the association between EGRac with Hb, ferritin, and Hb genotypes. EGRac was log transformed in the analyses, and the regression coefficients represent the geometric mean differences. Results Genetic Hb disorders were present in 57% of the population, with the homozygous hemoglobin E variant (Hb EE) occurring in ∼10% of women (n = 53). Deficient (EGRac ≥1.40) or marginal riboflavin status (EGRac ≥1.30 and <1.40) was observed in 92% (n = 475) of women. The variant Hb EE genotype was associated with 18% (95% CI: 9%, 28%) higher geometric mean EGRac values than the normal Hb AA genotype (P < 0.001). Conclusions Although riboflavin biomarker deficiency or marginal status is widely prevalent in Cambodian women, lower riboflavin status was observed more frequently in women with the Hb EE genotype than in women with normal Hb AA. The relation between genetic Hb disorders and riboflavin warrants further investigation. This trial was registered at clinicaltrials.gov as NCT01593423 and NCT02481375.