Study Design
In this two-arm, double-blind randomized trial, we will recruit 60 pregnant women aged 19–42 years living in Vancouver, BC, Canada. Participants will be randomized to supplement daily for 16-weeks with either 0.6 mg/day folic acid or an equimolar dose (0.625 mg/day) of (6S)–5-methyltetrahydrofolic acid (Fig 2). The 16-week intervention was based on the estimated half-life of RBC folate and to enable measurement of long-term folate status in RBCs (past 12–16 weeks).37 All participants will also receive a prenatal multivitamin not containing any folate, to ensure adequacy of other nutrients (e.g. iron) during pregnancy. Fasting venous blood samples will be collected at baseline and endline to measure primary outcome measures, including serum folate, RBC folate and UMFA; exploratory measures will include plasma S-adenosyl-methionine, S-adenosyl-homocysteine, total homocysteine, total cysteine, methionine, vitamin B–12, pyridoxal–5-phosphate, free choline and betaine. Genotyping of the MTHFR (677 C>T, rs1801133, and 1298 A>C, rs1801131) and DHFR (rs1643649 and rs70991108) variants and a complete blood count will also be determined. The study protocol has been developed in accordance with the 2013 SPIRIT guidelines (Fig 3)..38 See Additional file 1 (SPIRIT checklist) for further details.
The primary objective of this pilot study is to gain estimates for the change in serum folate, RBC folate and UMFA following supplementation with (6S)–5-methyltetrahydrofolic acid and folic acid for 16-weeks of pregnancy to inform the design of an adequately powered definitive trial. The secondary objective is to obtain recruitment and participation rates to assist in the design and feasibility of a definitive trial.
Figure 2 Participant flow diagram
Figure 3 Standard Protocol Items: Recommendation for Interventional Trials (SPIRIT) schedule of enrollment, interventions and assessments
Participants, Recruitment & Informed Consent
Inclusion criteria:i) pregnant woman (singleton pregnancy); ii) living in the greater Vancouver area; iii) ≤21 weeks gestation at time of consent; iv) 19–42 years of age; v) able to provide informed consent.
Exclusion criteria: i) pre-existing medical conditions known to impact maternal folate status (malabsorptive and inflammatory bowel diseases, active celiac disease, gastric bypass surgery, atrophic gastritis, epilepsy, advanced liver disease, kidney dialysis, type 1 or 2 diabetes mellitus, sickle cell trait/anemia);7 ii) lifestyle factors known to impact maternal folate status (current smoking, alcohol consumption, recreational drug use);7 iii) medium to high risk for development of an NTD-affected pregnancy (applies to women or their male partner: personal or family history [parents or siblings] of other folate sensitive congenital anomalies, personal NTD history or a previous NTD-affected pregnancy);7 iv) medications known to interfere with B-vitamin metabolism (Chloramphenicol, Methotrexate, Metformin, Sulfasalazine, Phenobarbital, Phenytoin, Primidone, Triamterene, Barbiturates);7 v) pre-pregnancy body mass index (BMI) ≥30 kg/m2; or vi) allergy to any study supplement ingredients.
Sample Size: We will require 50 women (25 in each group) to reliably estimate the distributions of serum and RBC folate concentrations.39 Thus, to account for dropouts or loss to follow up, we will recruit a total of 60 women (30 in each group).
Recruitment: Posters will be displayed at the BC Women’s Hospital, in medical and prenatal clinics and in retail establishments for pregnant women (e.g. prenatal classes, fitness studios, maternity clothing stores) throughout Vancouver, BC, Canada. Study details will be shared with staff and health care professionals, including physicians, midwives, nurses and others as applicable. Advertising on social media (Facebook and Instagram) will be used and targeted towards pregnant women aged 19–42 years in the greater Vancouver, BC area.
Obtaining Informed Consent & Confirming Eligibility: Women interested in participating will contact the research coordinator (KMC) directly. At this time, study details, eligibility and informed consent will be described. Eligible participants who would like to be enrolled will be given a study ID and their baseline meeting will be scheduled. Participants will be instructed to continue any current prenatal multivitamin/folate supplementation until their baseline visit.
Study Visits
Study visits will be facilitated at the University of British Columbia, Food, Nutrition and Health Building (Vancouver, BC, Canada). Baseline8–21 weeks gestation): Both the participant and the research coordinator (KMC) will sign the informed consent form; a copy will be scanned and e-mailed to the participant following the study visit. Participants will complete a baseline questionnaire to capture medical and nutrition history and demographic data. A validated food frequency questionnaire will be used to calculate dietary folate equivalents (DFEs). Weight and height will be taken using an electronic scale and stadiometer and recorded to 0.1 kg and 0.1 m respectively. A 12 mL 3 hr-fasting venous blood sample will be collected. Participants will receive their study supplements (either folic acid or (6S)–5-methyltetrahydrofolic acid) and be provided with a supplement diary to record daily intake. Endline24–37 weeks gestation):A 12 mL 3-hr fasting venous blood sample will be collected, weight will be taken as described above and participants will complete an endline questionnaire to determine any changes in health status or medication use since baseline. Participants will return all supplement bottles (including any remaining capsules) and their supplement diary.
Study Supplements
The folic acid and (6S)–5-methyltetrahydrofolic acid will be provided separately from the prenatal multivitamin. This decision was made because some women report intolerance when consuming prenatal multivitamins; this is typically due to the high iron content and resolves after the first trimester.40 Should temporary intolerance arise, participants may skip or take a half dose of their multivitamin, while continuing to supplement with the full dose of folate.
Bulk ingredients for the folic acid and prenatal multivitamins have been provided by Natural Factors (Coquitlam, Canada). Bulk (6S)–5-methyltetrahydrofolic acid (Metafolin®) has been provided by Merck & Cie (Schaffhausen, Switzerland). All bulk ingredients have been compounded into vegetable gel-capsules at Natural Factors (Coquitlam, Canada). Folic acid and (6S)–5-methyltetrahydrofolic acid capsules are identical in appearance to accommodate double-blinding. The prenatal multivitamin contains the same micronutrient formulation as WN Pharmaceuticals Ltd Prenatal (NPN 80025456), except the folic acid has been removed. A notice of authorization for the clinical trials application (Submission No. 244456) was provided by the natural and non-prescription health products directorate of Health Canada on July 26th, 2019.
Double blinding & randomization
An independent research assistant from Natural Factors (Coquitlam, Canada) will assign blinding codes (A or B) to the folic acid and (6S)–5-methyltetrahydrofolic acid supplements. Folate supplement bottles will be labelled with “A” or “B” and indicate that “capsules are either folic acid or folate”. Both the participants and the primary research team will be blinded to the supplement allocations, which will be de-coded only once the final statistical analyses are completed. The randomization sequence will be computer-generated by an independent statistician, using blocks of four which each contain two participants per supplement group. The primary research team will be blinded to the randomization sequence. At each participant’s baseline visit, the research coordinator (KMC) will unblind the allocation (A or B) for that study ID only, in order to provide the allocated study supplements.
Concomitant Medications
Once enrolled, there will be no restrictions on medication intake throughout the intervention period, however all medications must be reported. The only exception will be folate/folic acid containing nutritional supplements, which will not be permitted throughout the intervention period.
Strategies to Enhance Adherence
Pregnant women tend to be highly motivated and are generally interested in participating in clinical research trials.41 As per a survey from the Public Health Agency of Canada, 94% of women in British Columbia supplement with prenatal multivitamins during pregnancy.42 Therefore, participants will likely be accustomed to daily supplementation upon enrollment. The research coordinator (KMC) will contact each participant at midline (8-weeks after their baseline visit) to address any questions or concerns. Additionally, the supplement diary will serve as a daily reminder to enhance adherence throughout the 16-weeks. For these reasons, we anticipate high adherence (>90%) to the study protocol.
Blood Sample Collection & Processing
Three-hour fasting venous blood samples (12 mL total) will be collected in a 6 mL EDTA tube, 2 mL EDTA tube and 4 mL serum tube (BD Diagnostics). After collection, tubes will be shielded from light, inverted gently as per manufacturers recommendations, placed in a cooler and transported to the laboratory for immediate processing.
For preparation of whole blood hemolysate, whole blood (0.3 mL) will be removed from the 6 mL EDTA tube and diluted 1/11 by adding 3.0 mL of a 1% ascorbic acid solution and subsequently incubated at 37°C for 30 minutes. The 6 mL EDTA tube will then be centrifuged at 3000 rpm for 15 minutes at 4°C. Plasma will be collected and remaining contents of the 6 mL EDTA tube will be processed for isolation of peripheral blood mononuclear layer cells (PBMCs) with the SepMate PBMC isolation protocol (Stemcell technologies, Vancouver, Canada). The 2mL EDTA tube will be used for a complete blood count. The 4 mL serum tube will be left at room temperature for ~30 minutes (until clotted) and then centrifuged at 3000 rpm for 10 minutes at 4°C; serum will be collected. All aliquots will be stored at –80°C until further analyzed.
Laboratory Analyses
Serum folate (nmol/L) and RBC folate (nmol/L) will be analyzed by microbiological assay, as globally recommended, using the microtitre plate method outlined by Molloy et.al.43 RBC folate (nmol/L) will be calculated with the following formula:44
RBC Folate = (Whole blood hemolysate folate * 11)—Serum folate (1-Hematocrit/100)
Hematocrit/100
Plasma biomarkers including UMFA (nmol/), S-adenosyl-methionine (µmol/L), S-adenosyl homocysteine (µmol/L), total homocysteine (µmol/L), total cysteine (µmol/L), methionine (µmol/L), free choline (µmol/L) and betaine (µmol/L) will be analyzed using liquid chromatography mass spectrometry.45–47 Vitamin B–12 (pmol/L) will be analyzed using an immunoanalyzer. Pyridoxal–5-phosphate (nmol/L) will be analyzed using liquid chromatography-tandom mass spectrometry.48 Gene variants will be genotyped by using taqman SNP genotyping assays.1,49 Complete blood count will be performed using an automated hematology analyzer (Sysmex XNL–550).
Additional Data to be Collected
Demographic, medical and nutrition data via structured questionnaires: Baseline data collection will include age, ethnicity, parity, education, occupation, household income, medical and medication history, reported pre-pregnancy weight, general diet (including vegan, vegetarian, ketogenic, gluten free, and any foods/food groups avoided for any reason) and supplement use. Endline data collection will include any changes in overall health or medication use since the baseline visit.
Anthropometrics: Pre-pregnancy BMI will be calculated using self-reported pre-pregnancy weight and measured height. Participant measurements (weight and height) will be taken at baseline and endline for calculation of gestational weight gain throughout the intervention period and total weight gain throughout pregnancy (using self-reported pre-pregnancy weight and measured endline weight).
Dietary assessment: Total DFEs (µg/day) will be calculated using the Block Folic Acid/Dietary Folate Equivalents Screener (NutritionQuest, Berkeley, USA), a validated food frequency questionnaire.50
Withdrawal Criteria
Withdrawal criteria includes spontaneous or planned termination of pregnancy, use of additional folate/folic acid containing nutritional supplements and following an adverse/serious adverse event associated with the supplements/trial intervention (as determined by the qualified investigator [CM]). Participants may also self-withdraw at any time and no further information will be collected. Regardless of the reason for withdrawal, all previously collected data will be retained for analysis.
Data Handling & Privacy
All data and blood samples will be identified using each participant’s study ID and stored on an encrypted, password-protected computer in a secure server space with the BC Children’s Hospital Research Institute. Data will be cleaned and double entry will be completed by the research coordinator (KMC) and an independent research assistant. KMC, JAH and CDK will have access to the final dataset. Paper documents will be stored in a locked filing cabinet and blood samples will be stored in a locked freezer housed inside the principal investigator’s laboratory (CDK) at the University of British Columbia. Participants may be told their folate supplement allocation and blood folate results (including serum and RBC folate at baseline and endline) after final statistical analyses are completed.
Data and Statistical Analysis
Data will be assessed for normality and transformations will be considered if data do not follow a normal distribution. Statistical analyses will be performed using Stata (Stata Corp, Texas, USA). Two-sided p-values less than 0.05 will indicate statistical significance. Descriptive statistics will be used for participant baseline data. All quantitative variables will be reported with a mean ± standard deviation (or median and interquartile range if not normally distributed) and qualitative variables as absolute numbers per group. Missing data will be evaluated to identify whether it is random or non-random, and multiple imputation will be used to correct for missing data as appropriate.51
Primary objective: We will calculate the mean ± standard deviation for serum folate, RBC folate and plasma UMFA in each group at baseline and endline. The change from baseline to endline in each group will be evaluated using a paired t-test. These estimates will be used to inform the sample size calculation of the definitive trial. As a secondary test, we will evaluate the difference in the mean change between groups using a two-sample-t-test. All tests will initially be completed on an intention-to-treat basis according to initial group allocation at baseline and without any imputation for missing data. Secondary per-protocol analyses will be conducted including those who fully completed the study and adhered to the study protocol (supplementation rate ≥90%). Exploratory analyses will be conducted using a multiple linear regression model, adjusting for explanatory variables (DFEs, exploratory biomarkers, demographic data).
Secondary objective: Overall study participation rate will be estimated by dividing the total number of women enrolled by the total number of women who were interested in participating. Weekly recruitment rate will be estimated by dividing the total number of women enrolled by the total number of weeks it took to recruit them. Participant retention rate will be estimated by dividing the number of women who complete the full trial by the total number of women enrolled. Most effective recruitment strategies will be determined by recording number of successful participant acquisitions per recruitment site. Capsule counts will be used to calculate participant adherence and the supplement diary will used to provide descriptive insight into adherence barriers.
Safety
Folic acid is provided at a dose that meets Canadian recommendations for pregnant women (0.4–1.0 mg/day); thus, is considered low risk. Calcium salt of (6S)–5-methyltetrahydrofolic acid is recognized as safe and is included in Canadian prenatal multivitamins with no established tolerable upper limit (no risk of harm is currently known). Randomization will occur between 8–21 weeks gestation (after neural tube closure). Considering the low risk of this pilot study, assembly of a data monitoring committee was not deemed necessary. All safety concerns and if necessary, the decision to terminate the trial, will be addressed by the principal investigators (CDK and JAH) and qualified investigator (CM) and reported to the research ethics board and Health Canada. Unblinding of the intervention codes may be undertaken at any time if deemed necessary for participant safety monitoring.