Land and Food Systems, Faculty of
DSpace
UBCV
Cochrane, Kelsey M.
Doctor of Philosophy - PhD
University of British Columbia
Folic acid supplementation is recommended to support fetal development. However, folic acid is an oxidized form that must undergo reduction for use in the body, and unmetabolized folic acid (UMFA) is widely detected in circulation. (6S)-5-methyltetrahydrofolic acid (5-MTHF) is an alternative to folic acid which is reduced; however, the effect of (6S)-5-MTHF on maternal folate status during pregnancy is unknown. Thus, we conducted a pilot study to estimate the impact of supplementation with (6S)-5-MTHF versus folic acid on maternal folate status. Pregnant individuals (n=60) were randomized to 0.6 mg/day folic acid or (6S)-5-MTHF x 16-weeks, with the option to continue until 1-week postpartum. Fasting (3-hr) blood specimens were collected at baseline (8-21-weeks’ gestation) and after 16-weeks (endline); at 1-week postpartum, non-fasting blood and human milk specimens were collected. Red blood cell (RBC) and serum folate were quantified via microbiological assay and UMFA (plasma and human milk) via LC-MS/MS. Differences in blood folate markers were evaluated using multivariable linear/quantile regression, adjusting for dietary folate, baseline concentrations (endline concentrations for postpartum estimates), and weeks of gestation (total weeks’ supplementing for postpartum estimates). At endline, mean ± SD (or median, IQR) RBC folate, serum folate, and plasma UMFA (nmol/L) in the (6S)-5-MTHF and folic acid groups, respectively, were: 1826 ± 471 and 1998 ± 421 (adjusted difference: 161, 95% CI: -79, 400); 70 ± 13 and 78 ± 17 (adjusted difference: 7.4, 95% CI: -1.4, 16); 0.5 (0.4, 0.8) and 1.3 (0.9, 2.1) (adjusted difference: 0.6, 95% CI: 0.2, 1.1). Postpartum median (IQR) RBC folate concentrations (nmol/L) were 1724 (1474, 1877) and 1962 (1576, 2284) (adjusted difference: 11, 95% CI: -272, 294). The mean ± SD proportion of human milk UMFA as part of total milk folate was 2% (±2%) and 28% (±14%) in the (6S)-5-MTHF and folic acid groups, respectively. In conclusion, concentrations of RBC and serum folate were comparable between groups, although confidence intervals were not sufficiently precise to conclusively rule out a potential difference. The clinical significance of higher UMFA following folic acid supplementation has yet to be elucidated, including whether it has adverse consequences for the mother or baby.
A COMPARISON OF (6S)-5-METHYLTETRAHYDROFOLIC ACID VS FOLIC ACID AND THE EFFECTS ON MATERNAL FOLATE STATUS AND THE HUMAN MILK FOLATE PROFILE: EVIDENCE FROM A RANDOMIZED CLINICAL TRIAL by Kelsey M Cochrane B.Sc., University of Saskatchewan, 2014 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Human Nutrition) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2023 © Kelsey M Cochrane, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: A comparison of (6S)-5-methyltetrahydrofolic acid vs folic acid and the effects on maternal folate status and the human milk folate profile: Evidence from a randomized clinical trial submitted by Kelsey M Cochrane in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Human Nutrition Examining Committee: Crystal D Karakochuk, Assistant Professor, Department of Food, Nutrition and Health, UBC Supervisor Angela M Devlin, Professor, Department of Pediatrics, UBC Supervisory Committee Member Deborah L O’Connor, Professor, Temerty Faculty of Medicine, University of Toronto External Examiner Joel Singer, Professor Emeritus, School of Population and Public Health, UBC University Examiner David Kitts, Professor, Department of Food Science, UBC University Examiner Additional Supervisory Committee Members: Jennifer A Hutcheon, Associate Professor, Department of Obstetrics & Gynaecology, UBC Supervisory Committee Member Rajavel Elango, Associate Professor, Department of Pediatrics, UBC Supervisory Committee Member iii Abstract Folic acid supplementation is recommended to support fetal development. However, folic acid is an oxidized form that must undergo reduction for use in the body, and unmetabolized folic acid (UMFA) is widely detected in circulation. (6S)-5-methyltetrahydrofolic acid (5-MTHF) is an alternative to folic acid which is reduced; however, the effect of (6S)-5-MTHF on maternal folate status during pregnancy is unknown. Thus, we conducted a pilot study to estimate the impact of supplementation with (6S)-5-MTHF versus folic acid on maternal folate status. Pregnant individuals (n=60) were randomized to 0.6 mg/day folic acid or (6S)-5-MTHF x 16-weeks, with the option to continue until 1-week postpartum. Fasting (3-hr) blood specimens were collected at baseline (8-21-weeks’ gestation) and after 16-weeks (endline); at 1-week postpartum, non-fasting blood and human milk specimens were collected. Red blood cell (RBC) and serum folate were quantified via microbiological assay and UMFA (plasma and human milk) via LC-MS/MS. Differences in blood folate markers were evaluated using multivariable linear/quantile regression, adjusting for dietary folate, baseline concentrations (endline concentrations for postpartum estimates), and weeks of gestation (total weeks’ supplementing for postpartum estimates). At endline, mean ± SD (or median, IQR) RBC folate, serum folate, and plasma UMFA (nmol/L) in the (6S)-5-MTHF and folic acid groups, respectively, were: 1826 ± 471 and 1998 ± 421 (adjusted difference: 161, 95% CI: -79, 400); 70 ± 13 and 78 ± 17 (adjusted difference: 7.4, 95% CI: -1.4, 16); 0.5 (0.4, 0.8) and 1.3 (0.9, 2.1) (adjusted difference: 0.6, 95% CI: 0.2, 1.1). Postpartum median (IQR) RBC folate concentrations (nmol/L) were 1724 (1474, 1877) and 1962 (1576, 2284) (adjusted difference: 11, 95% CI: -272, 294). The mean ± SD proportion of human milk UMFA as part of total milk folate was 2% (±2%) and 28% (±14%) in the (6S)-5-MTHF and folic acid groups, respectively. In conclusion, concentrations of RBC and iv serum folate were comparable between groups, although confidence intervals were not sufficiently precise to conclusively rule out a potential difference. The clinical significance of higher UMFA following folic acid supplementation has yet to be elucidated, including whether it has adverse consequences for the mother or baby. v Lay Summary Folic acid is recommended to support a healthy pregnancy. However, our bodies are only able to process small amounts of folic acid, leaving some “un-used” folic acid (UMFA) in blood and breastmilk. “5-MTHF” may be a good alternative to folic acid because it can be used by the body directly, but its ability to raise blood folate levels during pregnancy is unknown. To address this gap, we enrolled 60 pregnant women to supplement with 5-MTHF or folic acid; folate status was measured in early and late pregnancy, after delivery, and in a breastmilk sample. Maternal folate status appeared similar following supplementation with each folate form; however, folic acid significantly increased UMFA in blood and breastmilk by ~2x and ~14x, respectively. The results of the study should be confirmed in a larger sample size; the impact of UMFA on human health is unknown, but should be determined in future studies. vi Preface This research centered around a randomized clinical pilot trial which took place in Vancouver, BC, Canada. The trial was originally designed by Co-Principal Investigators Dr. Crystal Karakochuk and Dr. Jennifer Hutcheon, Co-Investigator Dr. Rajavel Elango, and collaborator Dr. Chantal Mayer. Myself and my committee members, Dr. Karakochuk, Dr. Hutcheon, Dr. Elango, and Dr. Angela Devlin, and collaborator Dr. Mayer finalized the study design and protocol. I led the preparation and submission of study materials to the UBC Children and Women’s Research Ethics Board (approval obtained September 16, 2019; H18-02635) and the Clinical Trials Application to the Natural and Non-Prescription Health Products Directorate of Health Canada (approval obtained July 26, 2019; Submission No. 244456). The trial was registered at ClinicalTrials.gov (NCT04022135). Once approved, I led all aspects of the trial implementation, including recruitment, enrollment, facilitating study visits, blood and human milk processing in Dr. Karakochuk’s NUTRI-HEME lab at the University of British Columbia, and data management. Other laboratory analyses were completed at Bevital AS, Bergen, Norway (microbiological assay of red blood cell and serum folate), the Analytical Core for Metabolomics and Nutrition (ACMaN) at the BC Children’s Hospital Research Institute (plasma vitamin B12, pyridoxal phosphate, total homocysteine, cysteine, methionine, free choline, and betaine), the UBC Nutritional Biomarker Laboratory (plasma UMFA, 5-MTHF and human milk folate forms), and the UBC Center for Molecular Medicine and Therapeutics (genotyping of MTHFR 677 C > T variant, rs1801133). vii As a result of my PhD work, I have published three manuscripts and have two manuscripts under review: A version of Chapter 2 has been published. Cochrane KM, Mayer C, Devlin AM, Elango R, Hutcheon JA, Karakochuk CD. Is natural (6S)-5-methyltetrahydrofolic acid as effective as synthetic folic acid in increasing serum and red blood cell folate concentrations during pregnancy? A proof-of-concept pilot study. Trials 2020; 21(1):380. The study team (KMC, CM, AMD, RE, JAH, and CDK) finalized the study protocol. KMC drafted the manuscript and responded to peer reviews. All authors contributed to manuscript revisions. CDK had primary responsibility for the content. All authors read and approved the final manuscript. A version of Chapter 3 is under review for publication (portions of this manuscript are also included in Chapter 5: Conclusion, Discussion, & Future Research Directions). Cochrane KM, Mayer C, Devlin AM, Elango R, Hutcheon JA, Karakochuk CD. Supplementation with (6S)-5-methyltetrahydrofolic acid appears as effective as folic acid in maintaining maternal folate status while reducing unmetabolized folic acid in maternal plasma: A randomized trial of pregnant women in Canada. KMC led the implementation of the trial, conducted statistical analyses, and drafted the manuscript. All authors contributed to data interpretations and manuscript revisions. CDK had primary responsibility for the content. All authors read and approved the final manuscript. A version of Chapter 4 is under review for publication (portions of this manuscript are also included in Chapter 5: Conclusion, Discussion, & Future Research Directions). Cochrane KM, viii Mayer C, Devlin AM, Elango R, Hutcheon JA, Karakochuk CD. Human milk unmetabolized folic acid is increased following supplementation with synthetic folic acid as compared to (6S)-5-methyltetrahydrofolic acid. KMC led the implementation of the trial, conducted statistical analyses, and drafted the manuscript. All authors contributed to data interpretations and manuscript revisions. CDK had primary responsibility for the content. All authors read and approved the final manuscript. A version of Appendix A has been published. Cochrane KM, Williams BA, Elango R, Barr SI, Karakochuk CD. Pregnancy-induced alterations of one-carbon metabolism and significance for maternal nutrition requirements. Nutr Rev 2022; 80(9):1985-2001. KMC and CDK conceived the project. KMC and BAW conducted the literature review and developed the tables. KMC drafted the manuscript, figures, and responded to peer reviews. All authors contributed to data interpretations and manuscript revisions. CDK had primary responsibility for the content. All authors read and approved the final manuscript. A version of Appendix B has been published. Cochrane KM, Hutcheon JA, Karakochuk CD. Strategies for improving recruitment of pregnant women to clinical research: An evaluation of social media versus traditional offline methods in Vancouver, Canada. Digital Health 2022; 8:1-10. KMC conceived the project, completed the data analyses, drafted the manuscript, and responded to peer reviews. All authors contributed to data interpretations and manuscript revisions. CDK had primary responsibility for the content. All authors read and approved the final manuscript. ix Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ......................................................................................................................... ix List of Tables .............................................................................................................................. xiv List of Figures ...............................................................................................................................xv List of Abbreviations ................................................................................................................. xvi Acknowledgements .................................................................................................................. xviii Dedication .....................................................................................................................................xx Chapter 1: Introduction ........................................................................................................... 1 1.1 Overview of folate ...................................................................................................... 1 1.1.1 Chemical structure & folate forms ...................................................................... 1 1.1.2 Sources & bioavailability .................................................................................... 2 1.1.3 Absorption, transport, storage & excretion ......................................................... 4 1.1.4 Unmetabolized folic acid .................................................................................... 8 1.1.5 Folate handling in pregnancy .............................................................................. 9 1.1.6 Human milk folate ............................................................................................ 11 1.2 One carbon metabolism ............................................................................................ 13 1.3 Folate assessment ...................................................................................................... 17 1.3.1 Biochemical markers of folate status ................................................................ 17 1.3.2 Analytical methods ........................................................................................... 18 1.3.3 Biochemical reference ranges ........................................................................... 21 x 1.3.4 Genetic variations & folate status ..................................................................... 22 1.4 Perinatal folate recommendations & status ............................................................... 24 1.4.1 Dietary Reference Intakes & perinatal nutrition recommendations ................. 24 1.4.2 Folic acid food fortification .............................................................................. 28 1.4.3 Estimates of perinatal folate intake & status..................................................... 29 1.5 Supplemental folate forms ........................................................................................ 31 1.5.1 Folic acid versus 5-methyltetrahydrofolate ...................................................... 33 1.5.2 Concerns of excess folic acid supplementation ................................................ 35 1.6 Rationale & significance of the research program .................................................... 37 1.7 Research objectives & hypotheses ............................................................................ 38 Chapter 2: Study protocol ...................................................................................................... 40 2.1 Summary ................................................................................................................... 40 2.2 Introduction ............................................................................................................... 41 2.3 Study overview & objectives .................................................................................... 43 2.4 Participants & eligibility ........................................................................................... 45 2.4.1 Sample size ....................................................................................................... 46 2.4.2 Recruitment ....................................................................................................... 46 2.5 Informed consent ...................................................................................................... 47 2.6 Study visits ................................................................................................................ 47 2.7 Study supplements .................................................................................................... 48 2.7.1 Double-blinding, randomization & provision of supplements ......................... 49 2.7.2 Concomitant medications.................................................................................. 50 2.7.3 Strategies to enhance adherence ....................................................................... 50 xi 2.8 Biological specimen collection, processing & analysis ............................................ 51 2.8.1 Blood collection & processing .......................................................................... 51 2.8.2 Human milk collection & processing ............................................................... 52 2.8.3 Laboratory analyses .......................................................................................... 52 2.9 Summary of data to be collected ............................................................................... 53 2.9.1 Demographic, medical & nutrition data............................................................ 53 2.9.2 Anthropometrics ............................................................................................... 53 2.9.3 Dietary assessment ............................................................................................ 54 2.9.4 Postpartum data ................................................................................................. 54 2.10 Withdrawal criteria ................................................................................................... 54 2.11 Data handling & privacy ........................................................................................... 54 2.12 Statistical analyses .................................................................................................... 55 2.13 Safety ........................................................................................................................ 57 2.14 Discussion & study significance ............................................................................... 57 Chapter 3: Maternal folate status ......................................................................................... 59 3.1 Summary ................................................................................................................... 59 3.2 Introduction ............................................................................................................... 60 3.3 Methods..................................................................................................................... 61 3.3.1 Study participants & design .............................................................................. 61 3.3.2 Blood collection & processing .......................................................................... 62 3.3.3 Biochemical analyses ........................................................................................ 63 3.3.4 Statistical analyses ............................................................................................ 64 3.4 Results ....................................................................................................................... 66 xii 3.4.1 Participant enrollment & baseline characteristics ............................................. 66 3.4.2 Maternal folate status ........................................................................................ 70 3.5 Discussion & conclusions ......................................................................................... 76 Chapter 4: Human milk folate profile .................................................................................. 80 4.1 Summary ................................................................................................................... 80 4.2 Introduction ............................................................................................................... 81 4.3 Methods..................................................................................................................... 83 4.3.1 Study participants & design .............................................................................. 83 4.3.2 Human milk collection ...................................................................................... 83 4.3.3 Maternal blood specimen collection & processing ........................................... 84 4.3.4 Quantification of folate forms ........................................................................... 85 4.3.5 Statistical analyses ............................................................................................ 85 4.4 Results ....................................................................................................................... 86 4.4.1 Participant enrollment & baseline characteristics ............................................. 86 4.4.2 Human milk folate profile at 1 week postpartum ............................................. 89 4.4.3 Association of UMFA in maternal plasma and human milk ............................ 93 4.5 Discussion & conclusions ......................................................................................... 93 Chapter 5: Conclusion, Discussion, & Future Research Directions .................................. 98 5.1 Discussion of key findings ........................................................................................ 98 5.1.1 Effect of (6S)-5-MTHF and folic acid on RBC and serum folate levels .......... 98 5.1.2 High maternal folate status ............................................................................. 100 5.1.3 Increased plasma UMFA due to folic acid supplementation .......................... 102 5.1.4 Human milk folate profile ............................................................................... 104 xiii 5.2 Strengths & limitations ........................................................................................... 107 5.3 Significance & contributions of the research .......................................................... 109 5.4 Future research directions ....................................................................................... 110 References ...................................................................................................................................116 Appendices ..................................................................................................................................152 Appendix A: Pregnancy-induced alterations of 1-carbon metabolism ........................................... 152 Appendix B: Recruitment of pregnant women to clinical research ................................................ 200 Appendix C: Other considerations for the interpretation of change in maternal folate status ........ 226 xiv List of Tables Table 3-1 Participant characteristics (maternal folate status) ....................................................... 69 Table 3-2 Crude and adjusted differences between groups in measures of folate status at endline and postpartum .............................................................................................................................. 71 Table 3-3 Full multivariable regression model outputs for adjusted differences in markers of folate status between groups ......................................................................................................... 72 Table 3-4 Estimates of exploratory biochemical outcomes at baseline and endline .................... 75 Table 4-1 Participant characteristics (human milk folate profile) ................................................ 88 Table 4-2 Human milk folate profile following supplementation with (6S)-5-MTHF or folic acid....................................................................................................................................................... 90 Table 4-3 Full multivariable regression model outputs for adjusted differences in human milk folate profile between groups........................................................................................................ 91 xv List of Figures Figure 1-1 The chemical structure of folate .................................................................................... 2 Figure 1-2 Metabolism of folate to 5-MTHF .................................................................................. 5 Figure 1-3 The cycles of 1C metabolism ...................................................................................... 15 Figure 2-1 Chemical structure of folic acid and (6S)-5-methyltetrahydrofolic acid .................... 42 Figure 2-2 Standard protocol items: recommendation for interventional trials (SPIRIT) schedule of enrollment, intervention, and assessments ............................................................................... 44 Figure 3-1 Participant flow diagram (maternal folate status) ....................................................... 67 Figure 4-1 Participant flow diagram (human milk folate profile) ................................................ 87 Figure 4-2 Human milk folate profile among 43 participants supplemented with (6S)-5-MTHF or folic acid........................................................................................................................................ 92 Figure 4-3 Association of maternal plasma UMFA and human milk UMFA among 36 participants at 1 week postpartum................................................................................................. 93 xvi List of Abbreviations 1C One carbon 5-MTHF 5-methyltetrahydrofolate 10-formylTHF 10-formyltetrahydrofolate AI Adequate Intake apABG p-acetamidobenzoyl-glutamate BMI Body mass index CI Confidence interval CV Coefficient of variation DFEs Dietary folate equivalents DHF Dihydrofolate DHFR Dihydrofolate reductase DRI Dietary Reference Intake EAR Estimated Average Requirement EFSA European Food Safety Authority FASST Folic Acid Supplementation in the Second and Third Trimester FBP Folate binding protein FFQ Food frequency questionnaire FPGS Folylpolyglutamate synthetase FR Folate receptor GGH γ-glutamyl-hydrolase GPI Glycosylphosphatidylinositol HPLC High performance liquid chromatography xvii IQR Interquartile range LC-MS/MS Liquid chromatography-tandem mass spectrometry MeFox 4α-hydroxy-5-MTHF MIREC Maternal-Infant Research on Environmental Chemicals MTHFR Methylenetetrahydrofolate reductase NHANES National Health and Nutrition Examination Survey NTD Neural tube defect pABA p-aminobenzoic acid pABG p-aminobenzoyl-glutamate PBMCs Peripheral blood mononuclear layer cells PCFT Proton-coupled folate transporter PLP Pyridoxal phosphate PREFORM Prenatal Folic Acid Exposure on DNA Methylation in the Newborn Infant RBC Red blood cell RDA Recommended Dietary Allowance REDcap Research Electronic Data Capture RFC Reduced folate carrier SAH S-adenosylhomocysteine SAM S-adenosylmethionine SD Standard deviation THF Tetrahydrofolate UL Tolerable Upper Intake Level UMFA Unmetabolized folic acid xviii Acknowledgements First, thank you to my supervisor and mentor, Dr. Crystal D Karakochuk, for your unwavering support throughout the past (almost) 5 years. Crystal, you have provided me with so many amazing opportunities during my time at UBC, with countless reference letters, support to do research abroad, and conferences all over the world. Above all that, thank you for trusting me with this research, which has enabled me to set a foundation for a future career in an area that I am deeply passionate about, optimizing nutrition in pregnancy for healthy starts in life. You have always encouraged my curious mind and have allowed me to explore several different research interests (probably too many!). You are a true advocate for your students; thank you for always believing in me, even when my own self-confidence was lacking. Your brilliant mind, proactive and efficient work ethic, generosity, and collaborative approach are so inspiring, and I hope to approach my future work in the same way. Here’s to many years ahead of research together! Thank you to my committee members for your mentorship and unique perspectives which have shaped my learning over the years. Dr. Jennifer A Hutcheon, I have enjoyed working with you so much; I appreciate your positivity, thorough answers to my many questions about statistical methods and pregnancy-related outcomes, and for helping me to stay grounded by always coming back to the clinical and practical importance of my findings. Dr. Rajavel Elango, I have enjoyed our many laughs and I truly appreciate the mentorship you’ve provided to help me in successfully completing a clinical trial with pregnant women in Vancouver; as you know, it is no easy feat! Dr. Angela M Devlin, you are a brilliant scientist and have challenged and expanded my learning in many ways; you’ve helped me to always dig a little deeper and to always question the underlying why. I will take these lessons with me and hope to work with you all in the future. xix To all my lab mates, past and present, Brock Williams, Jordie Fischer, Tebby Leepile, Lulu Pei, Shannon Steele, and Kaitlyn Samson – we did it! Kaela Barker, thank you for your patience and training in the lab. Brock, we’ve had countless laughs and amazing adventures, from navigating Phnom Penh and that “unamed” road in Siem Reap, to la crème de la crème, Tokyo. I have learnt so much from you; you’re amazing and I can’t wait to see what you do next. Thank you to the funding agencies; I received two CIHR Frederick Banting and Charles Best Canadian Graduate Scholarships (Masters and Doctoral), a CIHR Michael Smith Foreign Study Supplement, a BC Graduate Scholarship, and a Trainee Travel Fund award from the International Society for Research in Human Milk & Lactation and Family Larsson Rosenquist Foundation; special thanks to Dr. Lars Bode for hosting me at UC San Diego! It was such a pleasure working with you, and I hope we have many more collaborations in the years to come. Finally, thank you to BCCHR Healthy Starts for funding the trial and making this work possible. To all my friends and family, thank you for always believing in me and for pretending to be interested in folate (I know some of you really do find it interesting!). A special thank you to Evan Taylor, my husband, who took this leap of faith with me to move from Saskatoon to Vancouver so that I could pursue this PhD. Evan, you are the most supportive partner; thank you for your patience, being my constant cheerleader, and for helping me to not sweat the small stuff (even though that can be hard for me!). Finally, a sincerest thank you to the participants. Science cannot advance without people like you, and your participation helps to improve care for all pregnant individuals and their babies. xx Dedication For my beloved, witty, smart, caring, unbelievably strong grandma – Yvonne Lucille Cochrane – you’re with me in everything I do. 1 Chapter 1: Introduction 1.1 Overview of folate 1.1.1 Chemical structure & folate forms Folate is a water-soluble B-vitamin (vitamin B9) that is present in numerous chemically related forms.1 Folate forms share a common parent structure with three distinct parts required for vitamin activity; this includes 2-amino-4-hydroxypteridine (pteridine), p-aminobenzoic acid (pABA), and glutamic acid.2 Folate is formed when pteridine is conjugated with a methylene group (—CH2—) to pABA at position C6, forming pteroic acid, and the carboxyl group of pABA is peptide-bound to the amino group (—NH—) of glutamic acid.2 While humans can synthesize all three components of folate, mammalian cells lack the complete pathway for folate synthesis, specifically the enzyme required for the coupling of pteridine to pABA; thus, folate is essential in the human diet.3,4 Folate forms differ based on the oxidation state of pteridine, addition of one carbon groups at positions N-5 and/or N-10, and the number of glutamic acid residues (Figure 1.1).5,6 Folates found naturally in foods are mostly reduced and polyglutamated (containing γ-peptide linked chains of up to ~9 glutamic acid residues).4 Food folates exist mainly as 5-methyltetrahydrofolate (5-MTHF) and 10-formyltetrahydrofolate (10-formylTHF),4 with 5-MTHF as the predominant form.6 Folic acid is a synthetic form of folate which is fully oxidized at the pteridine ring and contains one glutamic acid residue5; folic acid is not found in nature, but is commonly used in supplements and fortified foods as it is highly stable and bioavailable.1,7 2 Figure 1-1 The chemical structure of folate 1.1.2 Sources & bioavailability Food sources rich in folate include leafy green vegetables, legumes, citrus fruits, and organ meats (e.g., liver, kidney).4,6 Folic acid fortified foods are also a significant dietary source of folate (present in >80 countries globally).8 In Canada, the level of fortification is 0.15 mg per 100 g of white flour and cornmeal, and 0.20-0.27 mg per 100 g of pasta,9 aiming to increase population-level folic acid intake by ~0.1 mg/day.10 However, a Canadian study found that the folic acid content in fortified foods were on average ~50% greater than the listed content.11 Folic acid food fortification is further discussed in section 1.4.2. 3 The gut microbiota is also recognized as a source of folate, as certain bacterial species in the human gastrointestinal tract are capable of folate biosynthesis.12–15 Potential for de novo folate synthesis was recently evaluated using a reference set of 512 gastrointestinal bacterial reference genomes15; the capacity for at least some portion of folate synthesis was present ubiquitously, with 68 strains (13%) capable of complete tetrahydrofolate (THF) synthesis.15 The size of the colonic depot of folate is substantial (>400 µg),16 and may increase with the provision of dietary fiber (prebiotics).17 However, the extent to which endogenously synthesized folate contributes to human serum folate concentrations (e.g., as opposed to being used directly by folate-producing bacteria) is unclear and remains speculative at this time.18 Folate bioavailability refers to the amount ingested that is absorbed and available for use in metabolic reactions or storage.19 Food folates must undergo hydrolysis to the monoglutamate form prior to intestinal absorption; this occurs via a brush border membrane enzyme (glutamate carboxypeptidase II).20 Incomplete deconjugation of the polyglutamate chain, destruction in the gastrointestinal tract, or inability to fully release from food matrixes may all hinder bioavailability of food folates upon ingestion.19 Dietary folate equivalents (DFEs) are used to adjust for differences in bioavailability of folate forms. While bioavailability of food folates may vary greatly (~44-80%) based on a number of factors (e.g., genetic variations, the food matrix),19,21 the DFEs assume a bioavailability of 50% for folates derived from a mixed diet.22 Conversely, it is assumed that folic acid is 100% bioavailable when consumed on an empty stomach, and 85% bioavailable when consumed with a meal.22 Thus, a multiplier of 1.7 is generally used to convert µg folic acid to DFEs, based on a ratio of 85:50. Alternatively, 1 µg of 4 DFE = 1 µg food folate, 0.6 µg folic acid (taken with meals), or 0.5 µg folic acid (taken on an empty stomach).22 1.1.3 Absorption, transport, storage & excretion Monoglutamate folates are primarily absorbed in the proximal small intestine (mainly in the jejunum).20 Folate specific transporters on the apical surface of the brush border of enterocytes include: the reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT), the latter being the predominant mechanism by which folates are absorbed in the intestine.23 PCFT is a saturable transporter with activity that increases in low pH environments (maximum activity at a pH of ~5.5)7,23; thus, PCFT folate transport in the intestine is supported by an acidic environment that is maintained at the mucosal surface by Na+/H+ exchangers.2,23 PCFT has a similar affinity for reduced folates and oxidized folic acid.7,20,23 Conversely, RCF has a pH optimum of 7.4 (making it an ineffective transporter in the proximal small intestine), and has a very low affinity for folic acid.20,23 Folate absorption may also occur in the large intestine. In vivo evidence in humans was obtained following an infusion of labeled [13C]-5-formylTHF into the cecum of healthy adults during a screening colonoscopy (n=6),24 and following oral provision of [13C5]-5-formylTHF to healthy adults in enteric-coated capsules (n=6),25 with subsequent detection of the labeled folate in blood specimens. Following oral intake of the enteric-coated capsules, ~46% of the labeled folate was detected in circulation.25 Exact transport mechanisms for folate across the human colon are not well-described; while both PCFT and RFC are present, the pH in the colon varies and is generally not ideal to support transport.23 However, the presence of lactic acid producing bacteria 5 in the colon (e.g., Lactobacillus reuteri), which lower the pH at the intestinal mucosa, may help to facilitate local folate absorption.15 Once folate is absorbed into the intestinal mucosa, it is metabolized to 5-MTHF (Figure 1.2).1,7 For folic acid, this includes a two-step reduction, first to dihydrofolate (DHF) and then to THF, catalyzed by the enzyme dihydrofolate reductase (DHFR).7 Reduced THF is ultimately metabolized to 5-MTHF by methylenetetrahydrofolate reductase (MTHFR).7 Food folates are mainly present as 5-MTHF, but if not, similarly undergo metabolism to this form.6,7 Folic acid and reduced folates may bypass metabolism to 5-MTHF in the enterocyte, and instead undergo metabolism in the liver prior to release into systemic circulation.7 Figure 1-2 Metabolism of folate to 5-MTHF A version of this figure was published in Cochrane KM, et al., (2020)26 Food folates are hydrolyzed to the monoglutamate form by glutamate carboxypeptidase II; 2) Folic acid is reduced to THF in a two-step reaction facilitated by DHFR; 3) all folate forms may undergo metabolism to 5-MTHF by MTHFR prior to efflux from the enterocyte. 6 Folate (predominantly 5-MTHF) exits the basolateral membrane of the enterocyte and is delivered to the hepatic sinusoids via the hepatic portal vein.20 Net intestinal folate absorption is influenced by enterohepatic circulation (e.g., the flow of folate to the liver, into bile, and back to the small intestine).23 Folate that reaches the liver has three possible fates: 1) polyglutamation by folylpolyglutamate synthetase (FPGS) for storage7,20; 2) entering the bile duct (at the bile canalicular membrane) for reabsorption in the intestine20,23; and 3) folate monoglutamates (either delivered directly from the portal vein or following hydrolysis of stored folate polyglutamates by γ-glutamyl-hydrolase; GGH27) can exit the liver via the hepatic vein and enter systemic circulation.20,23 It is noted that 5-MTHF is a poor substrate for FPGS28; thus, for effective polyglutamylation and retention, 5-MTHF is first metabolized to THF by methionine synthase.7 Folate circulates predominantly as monoglutamate 5-MTHF either freely (accounting for ~one third of 5-MTHF) or loosely bound to proteins.2,4,6 A variety of transmembrane carriers and folate binding proteins (FBP) are present in mammalian tissues.2 Transport of folate into peripheral tissues is mainly facilitated by the transmembrane carrier RFC (pH in arterial system = 7.4),23,29 which is expressed ubiquitously throughout human tissue.30 The RFC is an organic phosphate antiporter29; organic phosphate is highly concentrated in cells where it is synthesized in ATP-dependent reactions; this creates a transmembrane anion gradient (an organic phosphate gradient), allowing RFC to transport folate into cells.20,29 Folate receptors (FR) are high affinity FBPs embedded in the cell membrane of epithelial tissues by a glycosylphosphatidylinositol (GPI) anchor.29,31 High levels of FR-α (the predominant FR species32) are expressed in epithelial cells of various tissues, including the kidney, choroid 7 plexus, lung, uterus, placenta, ovaries, and breast.2,20,29 Unlike RFCs which have a low binding affinity for folic acid, the affinity of FR-α for folic acid is ~6-10 fold higher than for reduced folate forms.32,33 FRs transport folate into cells using receptor-mediated endocytosis; folate binds to the FR and is internalized by an area of the cell membrane, which buds off and subsequently releases folate intracellularly.29,32 While FBPs may remain membrane bound during transport, the GPI tail is susceptible to cleavage by a phosphatidylinositol-specific phospholipase34; soluble FBP (a cleavage product of the membrane-associated folate receptor) can be determined in circulation and other body fluids (e.g., human milk).34,35 Total body folate stores are estimated to range from 12-28 mg, with ~50% being stored in the liver.22,36 Within mammalian cells, the cytosol and mitochondria have distinct folate pools, and FPGS is located in both compartments to facilitate metabolism to the polyglutamate form for retention and use in folate-related metabolism.2,37 Homeostatic mechanisms prevent excessive tissue folate retention; when tissue folate levels are elevated, metabolism of incoming 5-MTHF monoglutamates are impeded, resulting in their efflux from cells and preventing retention, even when circulating concentrations are elevated.2,7 Clearance of folate from plasma occurs rapidly, based on continuous uptake into tissues and filtration at the glomerulus (with most folates reabsorbed in the proximal renal tubule).2,38 The rate of reabsorption varies based on folate status, with greater urinary excretion as intake and status increase.7,39 Whole-body folate turnover occurs slowly, with an estimated half-life of 100-200 days.2,7 Folate is excreted mainly as cleavage products following catabolism.2 While the exact cellular pathways of folate catabolism are not well-characterized,5 cleavage of folate 8 occurs at the C9-N10 bond, resulting in p-aminobenzoyl-glutamate (pABG) and a pterin moiety.2,7 The pABG is hydrolyzed to the monoglutamate form by a lysosomal glutamylhydrolase, followed by N-acetylation and urinary excretion as p-acetamidobenzoyl-glutamate (apABG), the major urinary catabolite.7,40,41 The pterin moiety is excreted into bile and then the feces.1,7 1.1.4 Unmetabolized folic acid In the two-step reduction of folic acid to THF via DHFR, the first step (reduction to DHF) is rate limiting and may become saturated when folic acid is consumed above a certain threshold.7 If DHFR saturation occurs in the enterocyte, unmetabolized folic acid (UMFA) may escape the intestine and enter the liver via the portal vein.7 Evidence from a small study (n=6) in which blood was collected from the portal vein in humans with a transjugular intrahepatic portosystemic shunt 15 minutes after the provision of 0.22 mg isotopically-labelled folic acid found that 80% of folic acid reached the portal vein unmetabolized42; 30 minutes after intake, labeled folic acid was detected in systemic circulation.42 While UMFA reduction occurs in the liver, an in vitro investigation found that the capacity of DHFR in human liver was low and variable43; using folic acid as a substrate, the highest rate of DHFR activity in human livers (n=6) was 0.02 nmol/min/g wet weight liver (~56 times lower than rodent hepatic DHFR rate).43 Thus, saturation of both intestinal and hepatic DHFR may occur in humans following folic acid intake, and results in the release of UMFA into systemic circulation.42 While older studies (conducted in settings lacking mandatory folic acid food fortification) report an acute appearance of UMFA in plasma following intake of folic acid >0.2 mg,44–46 newer 9 population-wide studies using more sensitive analytical methods (National Health and Nutrition Examination Survey; NHANES; 2007-2008 and 2011-2012) report plasma UMFA detected in almost all individuals (>95%), regardless of fasting state or folic acid intake.47,48 Mechanisms to clear UMFA from plasma may include tissue uptake, enhanced excretion, and upregulation of DHFR activity.49–51 The use of folic acid supplements and fasting state were the strongest predictors of plasma UMFA concentrations >1 nmol/L, a cut-off representing the 75th percentile for fasting individuals in NHANES 2007-2008.47 While plasma UMFA concentrations cannot be entirely explained by these factors,48 other cohort studies also report a positive association between reported use of folic acid supplements and plasma UMFA concentrations, regardless of fasting state.52–54 1.1.5 Folate handling in pregnancy Pregnancy is a unique physiological state that is associated with higher folate needs and altered folate handling to accommodate growth of the fetus and uteroplacental organs.55 As such, longitudinal studies of pregnant individuals not consuming supplemental folate have reported declining plasma folate concentrations with advancing gestational age.56,57 Various underlying mechanisms may contribute to decreasing folate concentrations in plasma55; this includes folate transfer to the placenta and fetus,58–60 pregnancy-related hemodilution,61 and increased folate catabolism and excretion62,63; these are reviewed in further detail below. Overall, adequate folate consumption, generally requiring supplementation (as per evidence obtained in countries without folic acid food fortification56,57), is required to maintain maternal plasma folate concentrations throughout pregnancy.55,64 10 Hemodilution does not appear to fully explain declining plasma folate concentrations,65 as the decrease in serum folate in un-supplemented women throughout pregnancy prior to folic acid food fortification has been reported as greater (42%) than the rate of hemodilution (28%).66 Glomerular filtration rate and urinary output increase during pregnancy,62 and the presence of placental N-acetyltransferase type I (catalyzes N-acetylation of pABG to apABG) may facilitate increased folate catabolism.67 In two Irish studies,62,63 concentrations of urinary folate catabolites (pABG and apABG) increased significantly with advancing pregnancy (n=662 and n=2463 pregnant women), with total outputs over two-times greater than in non-pregnant controls (n=662 and n=2563). Conversely, findings from a controlled feeding trial with provision of isotopically labeled folic acid reported no differences in urinary folate catabolites during the second trimester versus non-pregnant controls,40 or in the excretion of labeled folates.68 Further research is required to elucidate folate catabolism and excretion in pregnancy, but it appears that differences as compared to the non-pregnant state may be limited to, or are most significant in, later pregnancy. Mobilization of folate from maternal plasma to the placenta and fetus is essential to meet folate demands in these compartments.55 Folate concentrations in maternal plasma, the placenta, and cord blood are strongly correlated,55,69,70 with higher folate measured in cord blood than maternal plasma59,60,71 suggesting a high degree of transfer which is dependent on maternal status. Folate receptors (FRα, RFC, and PCFT) are highly expressed in the placenta, with establishment in early pregnancy20,58; while the role of each transporter is not well-described, lack of placental FRα in pregnant mice resulted in severe defects in embryonic development and fetal death.72 In a placental perfusion study, folate bound rapidly to FBPs on the maternal surface and accumulated 11 in the intervillous space at concentrations 3-fold that of maternal plasma, followed by transport to fetal circulation against a concentration gradient.73 In an ex vivo study of 28 placentas from uncomplicated pregnancies, transfer of folate from maternal to fetal perfusate was non-saturable when exposed to very high folate conditions (50-500 nmol/L).74 Within the placenta, folate is thought to be involved in active metabolism, given the presence of folate related enzymes, including DHFR, MTHFR, and methionine synthase.65,75,76 It is proposed that folic acid is mostly reduced in the placenta prior to fetal transfer65,77; however, UMFA has been detected in umbilical cord blood.60,71,78 In the Canadian Prenatal Folic Acid Exposure on DNA Methylation in the Newborn Infant cohort (PREFORM; n=368), UMFA was present in almost all (>90%) cord blood specimens.60 However, as fetal liver capacity for folate storage is limited, UMFA that does reach the fetus is hypothesized to be rapidly metabolized.55,65 Overall, studies have reported that cord blood UMFA is only weakly or not associated with total serum cord blood folate or maternal plasma UMFA.60,71 Additionally, concentrations of UMFA in cord blood appear to remain relatively low, making up ~1% of total serum cord blood folate as opposed to ~2-5% in maternal plasma, regardless of reported maternal folic acid supplementation, in countries with both mandatory60 and voluntary71 folic acid food fortification. 1.1.6 Human milk folate Folate in human milk is present in numerous reduced forms and is mainly polyglutamated65,79; the predominant folate species present is widely noted as 5-MTHF,80,81 with recent large cohort studies estimating that 5-MTHF constitutes ~50%82 to ~70%83 of folate in mature milk. As folate circulates in plasma predominantly as 5-MTHF in the monoglutamate form (representing >90% 12 of plasma folate7), it is proposed that folates may be interconverted and polyglutamylated within the mammary epithelium.55,84,85 Folate concentrations in human milk are the lowest in colostrum, increasing in the weeks after delivery and peaking at ~2-3 months postpartum.80,83,86 Concentrations may vary by time of day and throughout a single feed, with higher folate reported in hindmilk than foremilk.84,87 Recent estimates of folate content in mature human milk range from ~120-200 nmol/L82,83,88,89; an average concentration 193 nmol/L (85 µg/L) was used to establish the Adequate Intake (AI) of 65 µg/day folate for infants 0-6 months (assuming an intake of 0.78 L/day human milk).22 Concentrations of human milk folate are maintained at the expense of maternal stores unless severe deficiency occurs.81,90,91 Most reports indicate that milk folate content is unaffected by maternal diet and supplementation83,88,92; however, in the era of folic acid food fortification and high maternal folate status, supplementation may increase human milk folate concentrations and shift the human milk folate profile.82 Recent findings from the multi-province Canadian Maternal-Infant Research on Environmental Chemicals cohort (MIREC; n=561) report that folic acid supplementation >0.4 mg/day resulted in an 18% increase (~115 nmol/L versus ~135 nmol/L; P <0.001) in total milk folate at ~1 month postpartum as compared no folic acid supplementation.82 Further, the proportion of UMFA as part of total milk folate was ~50% in those who reported supplementation with >0.4 mg/day folic acid, as opposed to ~25% in those consuming <0.4 mg/day.82 As such, a dose-dependent effect of folic acid on increasing UMFA in human milk was proposed.82 13 Human milk folates are bound to soluble FBP34,55,85; exact transport mechanisms, such as when binding occurs (upon entry/within the lactating cell or following secretion into human milk, or both), are unclear.85 A soluble FBP (a cleavage product of the membrane-bound receptor) and FBP with a GPI anchor have been identified in human milk,34 and FBPs in human milk are proposed to serve various physiological roles.55,85 First, binding to FBP may increase the stability of folate in milk, improving its bioavailability.85,93,94 Further, FBPs regulate folate sequestration from maternal plasma, ultimately controlling the folate content in human milk.65,85 Older studies have reported that FBPs exceeded folate concentrations in human milk by ~68 nmol/L55; human milk folate concentrations at this time (~200 nmol/L) were ~5-10x higher than in maternal plasma.55,85 However, population-wide folate status has increased since the onset of folic acid food fortification,95,96 and while total human milk folate concentrations appear similar to pre-fortification estimates, mean milk FBP concentrations have been reported as substantially lower (~40 nmol/L).88 Recent studies have reported that total human milk folate concentrations among folic acid supplement users is ~1.5 to 2x higher than in maternal plasma.54,82,88,97 It was proposed that the decrease in milk FBP is due to a down-regulation of folate receptor synthesis, possibly as a protective mechanism to prevent excessive folate accumulation in human milk, despite increased maternal folate status.88 1.2 One carbon metabolism One carbon (1C) metabolism refers to a series of interlinked cellular pathways including the folate cycle, methionine cycle, and trans-sulfuration pathway.98 Folate participates in 1C metabolism by serving as a methyl donor, in which 1C units are transferred from one molecule to another.99 The cycles of 1C metabolism support numerous physiological processes including 14 cellular proliferation, re-methylation of homocysteine to methionine, nucleic acid synthesis, and methylation of DNA, RNA, and proteins.99–101 As such, 1C metabolism, supported by sufficient intake of methyl nutrients (e.g., folate and other B-vitamins), is critical during periods of rapid growth such as pregnancy and early life (see Appendix A).64 Key pathways of 1C metabolism are summarized below and in Figure 1.3. 15 Figure 1-3 The cycles of 1C metabolism Schematic representation of pathways in the liver. This figure is published in Cochrane KM, et al., 202264 (see Appendix A). 5-MTHF: 5-methyltetrahydrofolate; BHMT: betaine-homocysteine methyltransferase; CBS: cystathionine β-synthase; CTH: cystathionine γ-lyase; DHF: dihydrofolate; DHFR: dihydrofolate reductase; DMG: dimethylglycine; GCS: glycine cleavage system (a multienzyme complex, including glycine decarboxylase encoded by GLDC, and amino-methyltransferase encoded by AMT); HCY: homocysteine; MAT: methionine adenosyltransferase; MET: methionine; MTHFD1: a trifunctional, cytosolic protein containing activities of: 5,10-methyleneTHF dehydrogenase, 5,10-methenylTHF cyclohydrolase, and 10-formylTHF synthetase; MTHFD1L: mitochondrial 10-formylTHF synthetase; MTHFD2L: a bifunctional, mitochondrial protein containing activities of: 5,10-methyleneTHF dehydrogenase, and 5,10-methenylTHF cyclohydrolase; MTHFR: methylene-THF reductase; MTR: methionine synthase; MTRR: methionine synthase reductase; SAH: S-adenosylhomocysteine; SAHH: S-adenosylhomocysteine hydrolase; SAM: S-adenosylmethionine; SHMT: serine hydroxy-methyltransferase; SHMT2: mitochondrial serine hydroxy-methyltransferase; THF: tetrahydrofolate; TYMS: thymidylate synthase Folate cycle: The flux of the folate cycle depends on availability of THF (the 1C “carrier”) and supply of 1C units.99,102 Reactions within the folate cycle are critical for purine and thymidylate synthesis and to facilitate the re-methylation of homocysteine to methionine.7,102 Purines are 16 synthesized from 10-formylTHF, which can be generated from THF using formate as the 1C donor, or via multi-step oxidation of 5,10-methyleneTHF.98,99 THF can also be converted to 5,10-methyleneTHF in a reversible reaction via the vitamin B6-dependent enzyme serine hydroxy-methyltransferase; in this reaction, serine donates its β-carbon, generating glycine upon transfer of this 1C unit.98,99 5,10-methyleneTHF can be used for thymidylate synthesis upon conversion to DHF, or it can be converted to 5-MTHF via MTHFR (a vitamin B2-dependent enzyme).98 5-MTHF donates a methyl group to homocysteine, forming methionine and THF; this reaction is catalyzed by methionine synthase, an enzyme that requires co-factors vitamin B12 and methionine synthase reductase.1,98 Methionine cycle: The methionine cycle consists of transmethylation and remethylation. Transmethylation begins when the sulfur atom of methionine is adenylated by methionine adenosyltransferase, forming S-adenosylmethionine (SAM).98,103 SAM, the “universal” methyl donor, serves as a substrate for >300 methyltransferases, forming key outputs including (but not limited to): sarcosine, creatine, phosphatidylcholine, and methylation of DNA, proteins, and RNA.102–105 Upon methyl donation, SAM is converted to S-adenosylhomocysteine (SAH), which is then hydrolyzed to homocysteine and adenosine by SAH-hydrolase.98,106 Homocysteine may undergo trans-sulfuration to cysteine or remethylation to methionine.98 Remethylation can occur as previously described (methyl donation from 5-MTHF) or via a parallel pathway in which betaine serves as a methyl donor, catalyzed by betaine-homocysteine methyltransferase, yielding methionine and dimethylglycine.103 Action of betaine-homocysteine methyltransferase is tissue-specific, and is expressed predominantly in the liver and kidneys.103 Betaine- and folate-17 dependent pathways are considered equally responsible for homocysteine remethylation in the mammalian liver.103,107 Trans-sulfuration pathway: In this pathway, homocysteine is irreversibly oxidized following transfer of its sulfur atom for the synthesis of cysteine.106 Cystathionine β-synthase catalyzes the condensation of homocysteine with serine, forming cystathionine, which is then converted to cysteine by cystathionine γ-lyase.103 Both enzymes in this pathway require vitamin B6 as a co-factor and exert tissue-specific activity in the liver, pancreas, kidney, and intestine.103,108 Cysteine can contribute to protein synthesis or be further metabolized to end-products such as taurine and glutathione.98,108 1.3 Folate assessment 1.3.1 Biochemical markers of folate status Folate status is most commonly evaluated using red blood cell (RBC) and serum folate concentrations.109,110 Serum folate provides information on short-term folate status and is highly sensitive to recent intake; the response is higher following consumption of folic acid from supplements or fortified foods as compared to food folates.110–112 RBC folate concentrations reflect the folate incorporated into RBCs during erythropoiesis.110 Folates within RBCs are inactive and stable, only being released upon hemolysis (RBC lifespan ~120 days).113 Thus, RBC folate is more reflective of long-term status.110,113 Further, RBC folate is used as a proxy for total tissue folate, as it parallels liver folate concentrations (which account for ~50% of total body stores).110,114 Fasting is generally not required for the assessment of RBC folate concentrations, but is essential for the accurate assessment of an individual’s serum folate status; however, in 18 large population-based studies, serum folate concentrations generally only vary by ~10% in non-fasted versus fasted groups.110 1.3.2 Analytical methods Methods for folate quantification in biological specimens were initially developed >80 years ago; over the years, these methods have been continuously improved to increase the generalizability of results across assays and laboratories.110,115 However, many challenges and differences between methodologies remain, which require careful consideration in the interpretation of folate results. Folate is the least stable B-vitamin and must be handled with care during all phases of sample collection, storage, and analysis110; for example, reduced folate forms are susceptible to oxidative degradation or interconversions when exposed to light, extreme pH values and elevated temperatures, or oxygen.115–117 Various pre-analytical factors can influence folate stability in biological specimens. Serum is generally preferred over plasma for the quantification of circulating folate, as EDTA (an anticoagulant in vacutainers used for plasma collection) may cause accelerated folate degradation118; however, results using both matrices are generally comparable and highly correlated.110,119 For serum folate analysis, care should be taken to avoid hemolysis of the blood specimen upon collection, as a release of folate from RBCs will inflate results.110 RBC folate concentrations are typically calculated using concentrations of whole blood folate, serum folate, and hematocrit.120 The processing of whole blood includes RBC hemolysis, followed by deconjugation of polyglutamate folates.115 Slightly acidic whole blood hemolysate is prepared via the addition of ascorbic acid (1% wt:vol) to whole blood, activating endogenous GGH for 19 deconjugation,121 followed by incubation at 37°C (~30 minutes is sufficient for analysis by microbiological assay)110; the addition of ascorbic acid also acts as an antioxidant to protect labile folates and improves stability during storage.120,122 Immediate processing of biological specimens followed by storage at -80°C is recommended to reduce folate degradation; if whole blood is refrigerated, processing within four days generally results in <10% folate loss.110,123 Folates appear stable for several years in serum or whole blood hemolysate at -80°C110,120; freeze-thaw cycles with these mediums should be avoided, but only minimal degradation seems to occur for at least three cycles.110,124 Human milk specimens also require careful preparation for folate analysis.125 Addition of a reducing agent (e.g., ascorbate, ascorbic acid) prior to freezing protects reduced milk folates during storage.55,125 Further, the application of a tri-enzyme (protease, amylase, and conjugase) extraction prior to folate analysis is essential to deconjugate glutamate residues and to release protein-bound folates in human milk, enabling total folate quantification.125,126 Various analytical techniques exist for folate quantification in biological specimens; the choice of assay depends on the research goals and laboratory setting. For example, protein-binding assays are generally best suited to clinical settings which require a fast turn-around and use fully automated analyzers with commercially prepared assay kits; however, these methods are limited by lot-to-lot kit variability and may have questionable accuracy due to different binding affinities of each folate form to the binding protein.110,120 In research or public health settings, the microbiological assay and chromatography-based methods are considered best practice.110 20 Microbiological assay: generally regarded as the gold-standard.110,127,128 The basic principle is that when a biological specimen is added to a culture medium inoculated with a folate-dependent microorganism (chloramphenicol-resistant Lactobacillus rhamnosus), growth of the microorganism is proportional to the amount of folate present in the biological specimen.120,124,129 Quantification of total folate is calculated using a calibration curve, generated with 5-MTHF or folic acid (the calibrator).124,127 The choice of calibrator may impact assay results, as the growth response of L. rhamnosus is greater following 5-MTHF calibration than with folic acid.110 While folic acid has historically been used given its greater stability and ease of use, 5-MTHF is recommended in recent guidelines as it is the predominant folate form in human specimens.120 Calibration with folic acid versus 5-MTHF appears to increase calculated folate concentrations by ~12% and ~15% in serum and whole blood, respectively.127 Advantages of the microbiological assay include a similar response to all biologically active forms (with ≤3 glutamate residues), high sensitivity (requiring only a small volume of the biological specimen), and it is relatively inexpensive to run, requiring only simple instrumentation.110,115,121 Limitations include that only total folate is calculated (as opposed to individual folate forms), there is potential for low generalizability across laboratories based on the protocol and reagents used, and the methods require skilled technicians to mitigate any risk of contamination while conducting the assay.110,115,130 Chromatography-based assays: used to quantify individual folate forms, the sum of which equate to total folate in biological specimens.115 These methods can achieve the highest level of precision, but are technically complex and require expensive instrumentation.110,120 Tandem mass 21 spectrometry (MS/MS) is the preferred detector for liquid-chromatography (LC) based folate assays.110 Methods for folate quantification in serum, whole blood lysates, and human milk via LC-MS/MS are published and have been continually modified to improve their sensitivity and precision.82,119,131–133 While there is generally good agreement between the microbiological assay and LC-MS/MS for total serum folate,109 RBC folate results obtained from the two methods vary, with results from the microbiological assay reported as up to 25% higher.109,134 Lower concentrations of RBC folate via chromatography-based assays may be attributed to the longer incubation time required for deconjugation (up to 4 hours), increasing the risk of folate degradation.135 While recent methods have explored the addition of exogenous GGH as solution to this concern,136 RBC folate analysis (calculated using whole blood folate) via the microbiological assay continues to be recommended for population wide investigations (e.g., NHANES) to allow for consistent evaluation of folate status trends over time.121 1.3.3 Biochemical reference ranges Various cut-offs for folate deficiency have been proposed using serum and RBC folate, which were derived using various analytical methods.110,137 Careful attention is required when choosing a suitable cut-off, to ensure that the assay used to derive said cut-off produces comparable results to the assay used to derive the data in question.120,138 First, cut-offs based on increasing plasma homocysteine (<10 nmol/L for serum folate; <340 nmol/L for RBC folate) were derived via a radioimmunoassay (Bio-Rad radioprotein binding assay), which is no longer commercially available.120,137 As it is unknown how most modern assays compare to this method, use of these 22 cut-offs for population-based folate assessment is not recommended.120,138 Alternatively, cut-offs based on macrocytic anemia (<7 nmol/L for serum folate; <305 nmol/L for RBC folate) were derived using the microbiological assay (wild-type microorganism and folic acid calibration).120,138 These cut-offs are deemed suitable for data quantified via more recent microbiological assay methods (e.g., chloramphenicol-resistant L. rhamnosus and 5-MTHF calibration).120,138 For risk reduction of neural tube defects (NTDs), a cut-off of 906 nmol/L was derived using the microbiological assay (chloramphenicol-resistant L. rhamnosus and folic acid calibration).129,139 Given the calibrator-effect of the microbiological assay, a cut-off of 748 nmol/L has been proposed when 5-MTHF calibration is used as compared to folic acid.120,138,140 Serum folate concentrations which correspond to this RBC folate cut-off for NTD risk reduction have also been proposed; this includes concentrations of 25.5 nmol/L, based on modeling of data from a population-based trial in China (n=1673 women of reproductive age),141 and concentrations between 28-30 nmol/L, as suggested in the 2022 Society of Obstetricians and Gynaecologists of Canada folic acid clinical practice guidelines.142,143 1.3.4 Genetic variations & folate status Polymorphisms of various genes involved in folate metabolism may influence folate status; this includes genes regulating the action of MTHFR, which catalyze the irreversible reduction of 5,10-methyleneTHF to 5-MTHF.1 23 The MTHFR 677C>T polymorphism (SNP rs1801133) is the most extensively studied variant in folate metabolism.144 This transition (C>T) results in an alanine to valine substitution and consequently a thermolabile variant of the MTHFR enzyme with reduced activity.145 Prevalence of the TT genotype varies geographically and by region of ancestry.144 In European populations, prevalence varies from ~5-25%, with higher rates reported in southern Europe (~15-25%) as compared to northern Europe (~5-15%)144,146; prevalence in Mexico and other Hispanic regions is higher (~15-35%), and in African regions is lower (~2%).146–148 In Asia, the prevalence has been reported as ~10-18%.144,149 Prevalence of the CT genotype is more common than the TT, with rates of the CT genotype up to ~40% in those from European or Asian regions and up to ~50% in those from Mexican regions.148,150 Among Canadians (MIREC cohort; n=549), ~9% and 48% had the TT and CT genotypes, respectively.151 Folate status may be impacted by MTHFR 677 C>T genotype. In a systematic review and meta-analysis among non-pregnant women, RBC folate (n=908) and serum folate (n=1538) concentrations quantified via the microbiological assay decreased consistently across MTHFR genotypes (CC>CT>TT).152 On average, the median RBC folate concentrations in those with the CC and CT genotypes were 16% and 9% higher than those with the TT genotype; similarly, median serum folate concentrations were 13% and 6% higher in those with the CC and CT genotype as compared to the TT.152 In a recent genome wide association study among young adults in Ireland (n=2232), MTHFR (rs1801133) was the most significant gene associated with RBC and serum folate concentrations153; however, it is noted that this genotype only accounted for ~3% (R2=0.03) and ~2% (R2=0.02) of the variation in RBC and serum folate concentrations, respectively.153 24 The MTHFR 1298 A>C (rs1801131) polymorphism results in a glutamate to alanine substitution.144 Prevalence rates of the homozygous CC genotype range from ~6-11% in those of European ancestry, and ~2-4% in those of African, Mexican, and Asian ancestry.144,147 While a mild reduction in MTHFR enzyme activity has been reported with the homozygous 1298C polymorphism as compared to the 1298AA (retaining ~60-70% of enzyme activity),144,154 genotypes do not seem to significantly alter folate status.153 Of note, the MTHFR 677 C>T and 1298 A>C polymorphisms are in linkage disequilibrium, such that the compound homozygous genotype is rarely reported in the same individual.144,155 1.4 Perinatal folate recommendations & status 1.4.1 Dietary Reference Intakes & perinatal nutrition recommendations The Dietary Reference Intakes (DRIs) are used by Health Canada and the National Academy of Medicine (former Institute of Medicine; United States) to provide nutrient recommendations throughout the lifecycle.156 Recommendations include the Estimated Average Requirement (EAR) and the Recommended Dietary Allowance (RDA), which represent the median intake that meets requirements for 50% and 97-98% of healthy individuals at a particular life stage, respectively. Alternatively, an AI is established when there is insufficient evidence to estimate the EAR and RDA, and represents the average level of intake in a population with no indication of inadequacy.22,156 Finally, the Tolerable Upper Intake Level (UL) represents the maximum intake of a nutrient that is likely to pose no risk of adverse health effects in the general population.156 25 Folate recommendations increase during pregnancy to accommodate increased growth and metabolic demands, and to reduce the risk folate-related birth defects.156,157 This includes NTDs, which are severe, irreversible congenital anomalies which occur 3-4 weeks after conception.158 Although the underlying etiology remains unclear, numerous studies have demonstrated that folic acid supplementation is effective in reducing the occurrence of NTDs.143,159–161 In two landmark clinical trials, supplementation with 4 mg/day folic acid reduced NTD recurrence by 72%,159 and no first occurrence NTDs occurred in n=2104 supplemented with 0.8 mg/day, as compared to six occurrences in n=2052 consuming no folic acid.160 An observational study following a population-based public health campaign in China found that 0.4 mg/day folic acid was sufficient for reducing the rate of first occurrence NTDs in both higher risk regions (northern China: reduction of 4.8 to 1.0/1000) and lower risk regions (southern China: reduction of 1.0 to 0.6/1000).161 Perinatal DRIs: The RDA in pregnancy is 0.6 mg/day DFE, as compared to 0.4 mg/day DFE in non-pregnant women >14 years of age.22 This is based on a controlled feeding trial, in which 0.6 mg/day DFE during pregnancy was sufficient to maintain RBC folate concentrations above the cut-off for NTD risk reduction (906 nmol/L).157,162 During lactation, the RDA is 0.5 mg/day DFE; this is based on the estimated intake needed to replace the folate secreted into human milk (~0.1 mg/day DFE), while meeting maternal folate needs (~0.4 mg/day DFE).22,157 The UL for folate applies only to folic acid, and is 1.0 mg/day for adults >19 years of age22,157; this is based on case reports which demonstrated the progression of neurological disorders in those with vitamin B12 deficiency when supplemented with ≥5 mg/day folic acid (this is further discussed in section 1.5.2).22 26 As it is difficult to obtain the RBC folate concentrations for NTD risk reduction from food sources alone, public health agencies including Health Canada and the Society of Obstetricians and Gynaecologists of Canada recommend supplementation with a folic acid containing multivitamin at least 2-3 months prior to conception and continued throughout pregnancy and lactation.142,163 To ensure folate adequacy in the event of an unplanned pregnancy, any person who could become pregnant is advised to maintain a folate rich diet, including supplementation with a folic acid-containing multivitamin.142,156 The recommended dose of folic acid is determined by an individual’s risk for the development of an NTD-affected pregnancy or other folate-sensitive congenital anomaly. Some key identified risk factors include142: lifestyle factors (e.g., a poor or restricted diet, alcohol use disorder, smoking, recreational drug use), maternal type I or type II pre-gestational diabetes mellitus, maternal obesity (pre-pregnancy body mass index [BMI] >30 kg/m2 or pre-pregnancy weight ≥80 kg), maternal intake of folate-inhibiting medications (Chloramphenicol, Methotrexate, Metformin, Sulfasalazine, Phenobarbital, Phenytoin, Primidone, Triamterene, Barbiturates), maternal gastrointestinal or malabsorption conditions (e.g., Crohn’s, active Celiac disease, gastric bypass surgery, advanced liver disease), chronic kidney disease (receiving dialysis), a personal or family history in the female or male partner of folate-sensitive birth defects (e.g., anomalies for cardiac, limb, cleft palate, urinary tract, congenital hydrocephaly) or a previous NTD-affected pregnancy. 27 The 2022 Society of Obstetricians and Gynaecologists of Canada clinical practice guidelines suggest the following risk classifications and folic acid recommendations142: Low risk: No identified risk factors; 0.4 mg/day folic acid is recommended 2-3 months prior to conception until the end of lactation (or until 4-6 weeks postpartum if not breastfeeding). Moderate risk: As determined via a detailed assessment of medical, surgical, and lifestyle risk factors, or history of a non-NTD folate-sensitive anomaly in a previous pregnancy; 1.0 mg/day folic acid is recommended beginning 2-3 months prior to conception until 12 weeks of gestation, followed by 0.4 mg/day folic acid until the end of lactation (or until 4-6 weeks postpartum if not breastfeeding). High risk: As determined via a detailed assessment of medical, surgical, and lifestyle risk factors, or history of a previous NTD-affected pregnancy. Two pre-conception care options have been proposed for high-risk individuals: 1) Standard care: 4.0 mg/day folic acid is recommended beginning 2-3 months prior to conception until 12 weeks of gestation, followed by 0.4 mg/day folic acid until the end of lactation (or until 4-6 weeks postpartum if not breastfeeding); 2) Personalized care: 0.4-1.0 mg/day folic acid is initiated 2-3 months prior to conception, with an assessment of fasting serum folate concentrations within the first 4-6 weeks of the ~3 month pre-conception period; if serum folate concentrations are ≥28 nmol/L, then 0.4-1.0 mg/day folic acid is recommended until 12 weeks of gestation; if serum folate concentrations are <28 nmol/L, 1.0-4.0 mg/day folic acid is recommended (exact dosing is based on the risk assessment and serum folate concentrations) until 12 weeks of gestation; in either scenario, 0.4 mg/day folic acid is 28 recommended after 12 weeks of gestation until the end of lactation (or until 4-6 weeks postpartum if not breastfeeding). The personalized care approach is a new addition to the Society of Obstetricians and Gynaecologists of Canada clinical practice guidelines (updated in 2022 from 2015)142,164; this approach acknowledges that evidence for the risk reduction of NTD-recurrence following supplementation with 4.0 mg/day folic acid was established prior to folic acid food fortification.159 However, limitations of this approach include unknown clinical acceptance of the newly proposed serum folate cut-off (≥28 nmol/L) and identification of the lowest effective folic acid dose for risk reduction of NTD recurrence.142 1.4.2 Folic acid food fortification Despite recommendations for initiation of folic acid supplementation prior to conception, many only begin supplementing once their pregnancy is confirmed.10,165 This is concerning as neural tube closure occurs early in pregnancy, before many women know they are pregnant. Thus, the Government of Canada initiated mandatory folic acid fortification of white flour, enriched pasta, and cornmeal in 1998 (folic acid fortification programs are present in >80 countries globally).8–10 Fortification in Canada aims to increase folic acid intake by ~0.1 mg/day, to increase blood folate levels in women of child-bearing age by 30-70% without posing a risk to the remainder of the population.9,10 Again, there is likely a discrepancy between actual folic acid added to fortified foods and mandated values (with higher values added as compared to label claims).11 29 Folic acid food fortification has been greatly successful in reducing NTD rates.9 In the 3 years following Canadian food fortification, NTDs decreased by ~46% overall (1.58/1000 births to 0.86/1000 births)9; decreases were the greatest for spina bifida (53%), followed by anencephaly (38%) and encephalocele (31%).9 Prior to fortification in Canada, NTD rates varied geographically, with higher rates in eastern provinces; these differences were resolved following fortification, with a greater impact in provinces that initially had higher rates.9 The overall NTD prevalence rate in Canada was estimated at 5.66 per 10,000 births in 2021.143 1.4.3 Estimates of perinatal folate intake & status Estimates for population-wide folate status have increased since the onset of folic acid food fortification95,166,167; however, cut-offs for “high” RBC folate and serum folate concentrations are not established.168 RBC folate >1090 nmol/L has been reported as indicative of intakes >2.1 mg DFEs/day, and 1360 nmol/L was reflective of the 97th percentile as per NHANES 1999-2004.96,166,167 As per the Canadian Health Measures Survey (2007-2009, 6-79 years, n=5248; non-pregnant), RBC folate deficiency (<305 nmol/L) was virtually non-existent (<1%) and 40% of the population had RBC folate concentrations >1360 nmol/L.96,166 However, among non-pregnant women of childbearing age (n=1162), 22% were below the cut-off for NTD risk reduction (<906 nmol/L).96 Folic acid supplement use has been reported as the strongest predictor for RBC folate concentrations >906 nmol/L and higher serum folate concentrations (>25.5 nmol/L).169,170 Among non-supplement users in the Canadian Health Measures Survey, younger age was the strongest characteristic associated with lower folate status171; lower education in adolescents and young adults (15-24 years) and lower income in middle-aged adults (25-49 years) were the strongest risk factors among age-specific groups.171 30 While guidelines suggest supplementation with 0.4 mg/day folic acid for those at low risk for an NTD-affected pregnancy (and for all women after 12 weeks of gestation), this is generally not feasible as prenatal vitamins on the Canadian market contain higher doses (0.6-1.0 mg).163 Among those in the MIREC cohort who provided data on folic acid supplement use (n=1476), 95% reported supplementation with ≥0.4 mg/day54; the median (interquartile range; IQR) folic acid dose consumed in those who reported supplementation with 0.4-1.0 mg/day was 1.0 mg/day (1.0, 1.0).54 A report from the Public Health Agency of Canada indicated that ~58% and ~90% of Canadians consume a folic acid-containing prenatal vitamin three months prior to becoming pregnant and during the first three months of pregnancy, respectively165; among those supplementing during pregnancy, >90% report daily supplementation.165 Thus, Canadians who are pregnant generally consume excess folic acid, with total intakes very commonly exceeding the UL (1.0 mg/day folic acid) due to the combination of food fortification and prenatal multivitamins.54,170,172,173 In the PREFORM cohort, 85% of participants reported doses of folic acid at or exceeding the UL through supplements alone.172 Overall, RBC and serum folate concentrations among pregnant Canadians are higher than necessary for NTD-risk reduction.163 In the Alberta Pregnancy Outcomes and Nutrition (APrON) study (n=599), median (95% confidence interval; CI) RBC folate concentrations in the first, second, and third trimesters were: 1280 nmol/L (1114, 1393), 1504 nmol/L (1450, 1568), and 1462 nmol/L (1421, 1529)174; among those who reported supplementing with ≤1.0 mg/day folic acid (median dose=1.0 mg/day), mean RBC folate concentrations in the first (n=387) and second (n=347) trimesters were ~1400 nmol/L.174 In two Ontario-based studies (including the 31 PREFORM cohort) the mean RBC folate concentrations were >2000 nmol/L in early and late pregnancy (12-16 and 36 weeks of gestation) and at delivery, regardless of folic acid supplementation regimen.60,97 Among those in the MIREC cohort, over 99% had serum folate concentrations (non-fasted) ≥25.5 nmol/L, regardless of supplemental folic acid dose54; in the first and third trimesters (n=1893), median (IQR) serum folate concentrations were 96 nmol/L (78, 118) and 98 nmol/L (74, 133), respectively.54 1.5 Supplemental folate forms Health Canada uses a Multi-Vitamin/Mineral Supplement Monograph which outlines regulations on approved source materials, nomenclature, doses, and label claims for each vitamin and mineral added to nutritional supplements.175 Approved source ingredients which can accompany the term “folate” or “vitamin B9” on a product label include folic acid and salts of 5-MTHF.175 Supplemental 5-MTHF are analogues of naturally occurring 5-MTHF, which are reduced and can be used by the body directly.176 Minimally 0.4 mg of folate (folic acid of 5-MTHF) must be included in supplements which are marketed for use in pregnancy; such products may include the following claims on their packaging: “helps to reduce the risk of NTDs when taken daily at least three months prior to becoming pregnant and during early pregnancy” and “helps to support normal early fetal development (brain and spinal cord)”.175 A maximum of 1.0 mg folate may be included in over-the-counter supplements intended for adults (>19 years of age).175 Of note, the UL does not seem to apply to salts of 5-MTHF, as this form is unlikely to mask symptoms of vitamin B12 deficiency176,177; however, there is some confusion on this matter as the folate DRIs were established after salts of 5-MTHF were made commercially available.178 32 Supplements of 5-MTHF are stabilized with calcium or other salts (e.g., glucosamine, monosodium)179 and are available as (6S) and (6R) isomers, as determined by the configuration of C6 on pteridine180; (6S) is the biologically active diastereoisomer (also referred to as L-5-MTHF, a chemically equivalent form).7 Doses of 5-MTHF which are equimolar to folic acid are calculated using molecular weights. For example: the molecular weights of folic acid and Metafolin® (calcium salt of (6S)-5-methyltetrahydrofolic acid; (6S)-5-MTHF), respectively, are 441.4 g/mol and 459.5 g/mol; thus, to obtain a dose equivalent to 1.0 mg folic acid, 1.04 mg of (6S)-5-MTHF is required (data obtained from Merck & Cie, Switzerland). Currently, there is no established conversion factor to convert doses of supplemental 5-MTHF to DFEs178; however, supplement manufacturers are currently permitted to apply the conversion factor used for folic acid (1 µg folic acid to 1.7 µg DFEs).176,178 The appropriateness of this is uncertain, as the bioavailability of folic acid versus 5-MTHF may differ.178 The European Food Safety Authority (EFSA) recently published a report (2022) addressing this concern181; following a systematic review and dose-response meta-analysis, they concluded that at doses <0.4 mg/day, the same conversion factor as used for folic acid is acceptable.181 However, at doses >0.4 mg, a conversion factor of 2 was suggested, given that the evidence in totality suggests an increased response on folate status at higher doses of 5-MTHF as compared to equimolar doses of folic acid.181 The rationale provided by the EFSA as to why (6S)-5-MTHF may elicit a greater response as compared to folic acid is based on the limited capacity of DHFR for folic acid reduction, resulting in circulating UMFA and subsequently, increased urinary folic acid excretion (the rate of which, they propose, increases with increased doses of folic acid).89,181 It is 33 noted that pregnant and lactating women were not included in this meta-analysis,181 due to minimal data in these populations. 1.5.1 Folic acid versus 5-methyltetrahydrofolate Multiple human trials have compared the effect of folic acid and salts of 5-MTHF on RBC and serum folate concentrations; this includes one trial in chronically-ill patients,182 six trials in healthy adults (men and women),50,183–188 six trials in women of reproductive age,189–195 one trial in lactating women,97 and one in pregnant women with recurrent pregnancy loss.196 Most of these trials evaluated both RBC and serum folate concentrations (however, six studies measured only serum folate50,185–187,193,196) with intervention periods up to 24 weeks,188–191,195 providing a range of doses equivalent to 0.1 mg/day188,189 to 1.0 mg/day192,196 folic acid; in one study, 7.5 mg (1x or 2x per day) x three days was provided, followed by lower doses (0.4-0.8 mg/day) x two weeks.193 Studies used calcium salts of (6S)-5-MTHF, except for two trials which did not specify the form,182,196 one trial which evaluated both calcium (Metafolin®) and monosodium (Arcofolin®) salts of (6S)-5-MTHF,187 and one trial which used a racemic mixture calcium salt containing 50% each of (6S) and (6R) isomers.194 In healthy, non-pregnant adults, studies have generally reported that (6S)-5-MTHF is equally as effective50,183,184,188–190,195 or more effective187,191–193 than folic acid in raising blood folate status (as per RBC and/or serum folate concentrations); doses ≤0.4 mg/day were provided in the aforementioned trials which reported equal effectiveness, whereas higher doses (≥0.4 mg) were provided in trials which found increased effectiveness. This supports conclusions by the EFSA that bioavailability of (6S)-5-MTHF as compared to folic acid is dose-dependent.181 34 One trial of 5-MTHF supplementation during pregnancy has been conducted, which took place in Iran (2015) and included those aged 20-45 years with ≥3 pregnancy losses.196 Women who were trying to conceive were recruited from Recurrent Abortion Clinics; those who did not become pregnant within 6 months were excluded. Of 220 women enrolled and randomized to 1.0 mg/day folic acid or 5-MTHF (the salt of 5-MTHF and whether an equimolar dose was used is not specified), 135 (~60%) became pregnant. While the study notes that serum folate concentrations were analyzed (via an immunoassay kit, fasting state not specified) at baseline, after 8 weeks (preconceptionally), and at 4, 8, 12, and 20 weeks of gestation, it does not state the number of individuals included at each time point, and only baseline serum folate concentrations are reported; baseline mean ± standard deviation (SD) serum folate concentrations in the folic acid and 5-MTHF groups were 17.5 ± 2.1 nmol/L and 17.9 ± 1.5 nmol/L, respectively.196 For other time points, it only states that serum folate concentrations increased significantly over time, with a greater change observed following 5-MTHF supplementation as compared to folic acid.196 Thus ultimately, the effect of 5-MTHF on folate status during pregnancy remains insufficiently characterized. In the study conducted during lactation,97 n=72 were recruited at 36 weeks’ gestation, and were advised to supplement with 1.0 mg/day folic acid until study baseline (~1 week postpartum). At baseline, participants were randomized to 0.416 mg/day (6S)-5-MTHF or a placebo; a reference group provided 0.4 mg/day folic acid was also included.97 RBC folate concentrations were evaluated via microbiological assay at 4 and 16 weeks postpartum.97 At 16-weeks, RBC folate concentrations (mean, 95% CI) in the (6S)-5-MTHF, folic acid, and placebo groups, respectively, 35 were: 2178 nmol/L (1854-2559), 1967 nmol/L (1628-2377) and 1390 nmol/L (1198-1613).97 Differences in the human milk folate profile were also evaluated88; total human milk folate concentrations (via microbiological assay) and milk UMFA (via high performance [HP]-LC) were quantified.88 No significant differences in total human milk folate or concentrations of milk UMFA were found between the three groups, with UMFA making up ~8% of total folate in the human milk specimens. 1.5.2 Concerns of excess folic acid supplementation The original concern related to excess folic acid supplementation was its potential to mask vitamin B12 deficiency, leading to exacerbation of B12 deficiency-related neuropathy; this was the basis for the current UL (1.0 mg/day folic acid).22 The underlying premise is that when concentrations of vitamin B12 are low, folate remains trapped as 5-MTHF, as methionine synthase (facilitates conversion of 5-MTHF to THF) is vitamin B12 dependent; this is considered functional folate deficiency, and may result in macrocytic anemia.1 Consumption of folic acid may resolve the anemia, as it can be metabolized to THF without vitamin B12 as a co-factor, allowing the vitamin B12 deficiency to progress unnoticed.1 However, the extent to which folic acid exacerbates vitamin B12 deficiency has been questioned in recent years, due to the use of case-studies to inform the UL which do not demonstrate causality.197 While a revision of the UL may be warranted, sufficient new evidence supporting safety at higher doses is needed before an update in the DRIs will be considered.177 Excessive maternal folic acid supplementation has been associated with a wide array of other adverse health outcomes.33,163,176 Outcomes related to offspring health and development which 36 have been investigated in human epidemiological studies and animal models include neurodevelopmental disorders,198–204 allergic diseases,205–209 and metabolic outcomes.210–213 However, results in human studies are mixed, with some reporting a harmful,199,204–206,211,212 null,198,200,207–209 or a protective201–203 association with maternal folic acid supplementation and folate status. These concerns and future research priorities were addressed at a 2019 National Institute of Health expert workshop,33 and among Canadian representatives in perinatal research, industry, and policy development in 2018.163 The concerns may be related to an elevated folate status (particularly in pregnant women, given high intakes from both prenatal supplements and via fortified foods) or an apparent saturable capacity to metabolize folic acid, resulting in the appearance of UMFA in biological fluids, the consequence of which is unclear.33,163 Various mechanisms for the biological effect of UMFA have been proposed.33 First, UMFA is not metabolically active, and only weakly binds to folate-related enzymes7; thus, it likely is not efficiently stored or able to participate effectively in 1C metabolism.33 However, UMFA may still interfere with the transport and/or use of reduced folates in non-direct ways.33,176 Due to the greater affinity of some FBPs (e.g., FR-α) for folic acid, it has been proposed that UMFA may tightly bind to cell surfaces and block or impair the uptake of reduced folates; uptake of 5-MTHF into human umbilical vein endothelial cells exposed to folic acid (20-100 nmol/L) was reduced by ~50%,214 and folic acid exposure (100 µmol/L) to cultured human-derived intestinal (Caco-2) and renal (HK-2) epithelial cells resulted in a down-regulation of folate transporters.215 Overall, little is known about the accumulation of UMFA at cell surfaces in-vivo.33 FBPs exert effects beyond folate transport, as they also act as signaling proteins and transcription factors216,217; these activities may be altered and/or activated following interaction with folic acid 37 (UMFA).33,218 Ultimately, more research is needed to elucidate whether there are any meaningful biological effects of UMFA exposure in humans. Mechanisms by which a high folate status may impact offspring development and disease risk includes hyperproliferation of cells facilitated by increased nucleotide synthesis (e.g., promoting growth of neoplastic cells),219 pro-inflammatory effects,220,221 and disturbances in 1C metabolism.222,223 In an epigenome-wide meta-analysis of two European pregnancy cohorts (n= ~2000 total), the association of maternal plasma folate concentration and DNA methylation in cord blood was evaluated (Illumina’s Human Methyl450 Beadchip); 443 CpGs (320 genes) were identified as differentially methylated based on maternal folate status (48 CpGs remained significant after correction for multiple comparisons).224 Ultimately, as maternal folate status increases, the fetus is also exposed to elevated levels of the vitamin; it is proposed that increased folate exposure in early life may impact fetal programming (in deleterious or beneficial ways) via epigenetic mechanisms, leading to changes in gene expression and subsequently phenotypic characteristics.33,101,225,226 1.6 Rationale & significance of the thesis research While the potential adverse consequences associated with folic acid supplementation remain uncertain,33,163,227–229 experts in a national call to re-evaluate perinatal folic acid supplementation guidelines urge implementation of the precautionary principle163; this aims to anticipate, monitor, and prevent unintended adverse consequences of public health interventions. Caution regarding over-supplementation is urged given the potential for risk combined with a very low risk of deficiency in the Canadian context (given folic acid food fortification). Further, concerns 38 have led to greater use of alternative folate forms.176,178 Supplements containing 5-MTHF may have advantages over folic acid, as this form does not require metabolism via DHFR, thus is not predicted to be metabolically limited or produce UMFA. However, the effect of 5-MTHF on folate status during pregnancy is not established, and only folic acid has been shown to reduce the risk of NTDs in randomized controlled trials (as such, only folic acid is recommended in current Health Canada perinatal nutrition guidelines). Despite this, salts of 5-MTHF are approved for use in prenatal supplement formulations and are widely consumed by pregnant women.176,178 Pregnancy is a demanding physiological state with unique folate requirements and handling; as such, it is imperative to determine the effect of supplementation with 5-MTHF versus folic acid on maternal folate status. This research program centered around a randomized clinical pilot trial with the primary aim of estimating maternal folate status following supplementation with 0.625 mg/day (6S)-5-MTHF versus 0.6 mg/day folic acid during pregnancy. The effect of (6S)-5-MTHF versus folic acid supplementation on maternal folate status after delivery and on the human milk folate profile was also evaluated. 1.7 Research objectives & hypotheses i. Conduct a pilot study of supplementation with (6S)-5-MTHF versus folic acid during pregnancy. A pilot study is needed to determine estimates of maternal folate status following supplementation with (6S)-5-MTHF versus folic acid; these estimates are not currently described in humans and are needed to inform the design of an adequately powered definitive trial. Maternal folate status will include concentrations of RBC folate, 39 total serum folate, and plasma UMFA. To address this objective, a detailed protocol for this randomized, double-blind, clinical trial is outlined in Chapter 2 and the evaluation of folate status following supplementation during pregnancy and after delivery is addressed in Chapter 3. Hypothesis: Supplementation with (6S)-5-MTHF will result in comparable concentrations of RBC and serum folate, and lower concentrations plasma UMFA, as compared to supplementation with folic acid. ii. Evaluate the human milk folate profile. The human milk folate profile includes the quantification of folate forms (including absolute concentrations of reduced folates and UMFA) and the proportion of reduced folates and UMFA as part of total milk folate. We examined differences in human milk folate following (6S)-5-MTHF or folic acid supplementation. This objective is addressed in Chapter 4. Hypothesis: Supplementation with both forms will result in comparable concentrations of total human milk folate; however, folic acid supplementation will result in higher UMFA (absolute concentrations and proportionally) than supplementation with (6S)-5-MTHF. 40 Chapter 2: Study protocol Acknowledgement: A version of this chapter has been published. Cochrane KM, Mayer C, Devlin AM, Elango R, Hutcheon JA, Karakochuk CD. Is natural (6S)-5-methyltetrahydrofolic acid as effective as synthetic folic acid in increasing serum and red blood cell folate concentrations during pregnancy? A proof-of-concept pilot study. Trials 2020; 21:380. 2.1 Summary North American health authorities recommend 0.4 mg/day folic acid before conception and throughout pregnancy to reduce the risk of NTDs and support increased growth and development. Folic acid is a synthetic form of folate that must be reduced by DHFR and then further metabolized. Recent evidence suggests that the maximal capacity for this process is limited and UMFA has been detected in circulation. The biological effects of UMFA are unknown. A synthetic analogue of natural folate, (6S)-5-MTHF (Metafolin®), may be a superior alternative because it does not need to be reduced in the small intestine. (6S)-5-MTHF is currently used in some prenatal multivitamins; however, its effect on folate status during pregnancy is unknown. This double-blind, randomized trial will recruit 60 pregnant women aged 19-42 years. Participants will receive either 0.6 mg/day folic acid or an equimolar dose (0.625 mg/day) of (6S)-5-MTHF for 16 weeks. The trial will be initiated at 8-21 weeks of gestation (after neural tube closure) to reduce the risk of harm should (6S)-5-MTHF prove less effective. All participants will also receive a prenatal multivitamin (not containing folate) to ensure adequacy of other nutrients. Baseline and endline blood specimens will be collected to assess primary outcome measures, including serum folate, RBC folate, and plasma UMFA. Participants 41 will have the option to continue supplementing until 1-week postpartum to provide a human milk and/or postpartum blood specimen. This proof-of-concept trial is needed to obtain estimates of the effect of (6S)-5-MTHF 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-MTHF is as effective as folic acid in raising blood folate concentrations during pregnancy. Ultimately, these findings will inform folate supplementation guidelines and policies for pregnant women. 2.2 Introduction Folic acid is a synthetic form of folate which is oxidized and contains one glutamic acid residue; this is the most stable form.230 Therefore, folic acid is used in food fortification and most supplements.1,7 North American guidelines recommend that all women at low risk for a NTD-affected pregnancy supplement with 0.4 mg/day folic acid, starting 2-3 months before conception and throughout pregnancy.142 (6S)-5-MTHF is approved by Health Canada as a source material of folate,175 but its effect on folate status during pregnancy as compared to folic acid is unknown; see the chemical structures of folic acid and (6S)-5-MTHF in Figure 2.1. 42 Figure 2-1 Chemical structure of folic acid and (6S)-5-methyltetrahydrofolic acid This figure was submitted with the manuscript: Cochrane KM, et al., (2022), Supplementation with (6S)-5-methyltetrahydrofolic acid appears as effective as folic acid in maintaining maternal folate status while reducing unmetabolized folic acid in maternal plasma: A randomized trial of pregnant women in Canada (presented in Chapter 3). 1a) Folic acid; a synthetic analogue of the folate parent structure. Folic acid is fully oxidized, increasing its stability in supplements and fortified foods, and requires reduction via dihydrofolate reductase for co-enzymatic function. 1b) Calcium salt of (6S)-5-methyltetrahydrofolic acid (Metafolin®); a synthetic analogue of natural 5-methyltetrahydrofolate. It is critical to ensure that (6S)-5-MTHF can maintain serum and RBC folate concentrations throughout pregnancy to adequately supply the growing fetus. Additionally, when 0.4 mg/day folic acid was compared to (6S)-5-MTHF during lactation, no difference in total human milk folate or in the proportion of UMFA in milk was found.88 However, given recent findings that 0.4 mg/day folic acid may be a threshold for increasing the proportion of folic acid in milk from 25 to 50%,82 further investigation is warranted. Thus, we aim to address these gaps by conducting a pilot study which will inform the design of a definitive trial investigating whether 43 (6S)-5-MTHF is as effective as synthetic folic acid in increasing serum and RBC folate concentrations during pregnancy and following delivery, while resulting in lower maternal plasma and human milk UMFA. 2.3 Study overview & objectives In this two-arm, double-blind, randomized trial, we will recruit 60 pregnant women aged 19-42 years living in Vancouver, 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-MTHF. The 0.6 mg dose will match levels found in leading commercial prenatal multivitamin brands (Materna®), thus increasing the generalizability of our findings. The 16 week intervention was based on the estimated lifespan of RBC folate and intends to enable measurement of long-term folate status in RBCs (past ~12-16 weeks).113 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 specimens will be collected at baseline and endline to measure primary outcome measures, including RBC folate, serum folate, and plasma UMFA. Participants will have the option to continue to the postpartum phase of the study, where at their endline visit, they will be given more supplements to take until ~1 week postpartum. On day 5-7 postpartum, participants will provide a small (3 mL) human milk specimen and a non-fasting venous blood specimen. Outcomes evaluated in human milk will include folate forms (UMFA, THF, 5-methyl-THF, 5-formylTHF, and 5,10-methenylTHF) and milk FBP. Outcomes evaluated in postpartum blood specimens will include RBC folate and plasma UMFA. The study protocol has been developed in accordance with the 2013 SPIRIT guidelines (see Figure 2.2).231 44 Figure 2-2 Standard protocol items: recommendation for interventional trials (SPIRIT) schedule of enrollment, intervention, and assessments 45 The primary objective of this pilot study is to gain estimates for RBC folate, serum folate, and plasma UMFA following supplementation with (6S)-5-MTHF and folic acid for 16 weeks of pregnancy to inform the design of a definitive trial which is adequately powered to detect a statistically significant and clinically meaningful difference in folate status following supplementation with each folate form during pregnancy. The secondary objective is to obtain recruitment and participation rates to assist in the design and feasibility of a definitive trial. Exploratory objectives of the postpartum phase are to quantify the proportion of total human milk folate as UMFA in each group, evaluate correlation of maternal postpartum plasma UMFA and human milk UMFA, and to evaluate RBC folate concentrations following delivery in each group. 2.4 Participants & eligibility Inclusion criteria: 1) pregnant woman (singleton pregnancy); 2) living in the greater Vancouver area; 3) ≤21 weeks gestation at time of consent; 4) 19-42 years of age; 5) able to provide informed consent. Exclusion criteria: 1) 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)164; 2) lifestyle factors known to impact maternal folate status (current smoking, alcohol consumption, recreational drug use)164; 3) 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 46 history or a previous NTD-affected pregnancy)164; 4) medications known to interfere with B-vitamin metabolism (Chloramphenicol, Methotrexate, Metformin, Sulfasalazine, Phenobarbital, Phenytoin, Primidone, Triamterene, Barbiturates)164; 5) pre-pregnancy BMI ≥30 kg/m2; or 6) allergy to any study supplement ingredients. 2.4.1 Sample size The sample size was calculated based on the primary objective to estimate the distribution (mean ± SD) of primary outcomes (concentrations of serum folate, RBC folate, and plasma UMFA) following 16 weeks of supplementation. To accomplish this, we will require 50 women (25 in each group); a sample of at least n=50 is generally recognized as adequate for clinical research pilot studies.232 A 20% inflation will be added to account for dropouts or loss to follow up, thus a total of 60 women (30 in each group) will be recruited. 2.4.2 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. 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 area. 47 2.5 Informed consent Those interested in participating will contact the research coordinator directly. At this time, study details, eligibility will be described, and the informed consent form will be shared. Eligible participants who would like to be enrolled will be given a study ID and their baseline visit will be scheduled. Participants will be instructed to continue any current prenatal multivitamin/folate supplementation until their baseline visit. The optional postpartum phase of the study will be described in the informed consent form; however, participants who choose to continue past the primary 16 weeks will be given a separate consent form to sign. 2.6 Study visits Baseline (8-21 weeks gestation): Both the participant and the research coordinator will sign the informed consent form (including confirmation of eligibility via a checklist); 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 (FFQ) will be used to calculate DFEs.233 Weight and height will be taken using an electronic scale and stadiometer and recorded to 0.1 kg and 0.10 m, respectively. A 3-hour fasting venous blood specimen (12 mL) will be collected. Participants will receive their study supplements (either folic acid or (6S)-5-MTHF) and be provided with a supplement diary to record daily intake. Midpoint: After 8 weeks, all participants will be contacted to check-in on compliance, address any questions or concerns, and to schedule an endline visit. 48 Endline (24-37 weeks gestation): A 3-hour fasting venous blood specimen (12 mL) will be collected, weight will be measured 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 their supplement diary and all remaining capsules will be counted. Postpartum: At the endline visit, participants who are planning to breastfeed and who have a working household freezer may opt to continue in postpartum phase of the study. Once consent is obtained in a second informed consent form, participants will be given supplements to continue taking until ~1 week postpartum and a new supplement diary. Participants will be trained to collect a small (3 mL) human milk specimen in their home at day 5-7 postpartum and given all supplies needed (described further in section 2.8.2). Participants will store their human milk specimen in the freezer and notify the research coordinator when it is ready for pick up. Pick up will occur within 4 days of notification and a 12 mL venous blood specimen (non-fasting) will be taken at this time. Participants will be instructed to take their folate supplement 2 hours prior to collection of both human milk and blood specimens to standardize any peak in plasma UMFA (and potentially uptake of UMFA into human milk; however, the timing of this process not well described); following folic acid intake, UMFA increases in plasma, peaking after ~1-2 hours, followed by clearance over the next several hours.44,187,234 2.7 Study supplements Folic acid and (6S)-5-MTHF will be provided separately from the prenatal multivitamin. This decision was made because some women report intolerance when consuming prenatal vitamins; this is typically due to the high iron content and resolves after the first trimester.235 Should 49 temporary intolerance arise, participants may skip or take a half dose of their prenatal vitamin, while continuing to supplement with the full dose of folate. Bulk ingredients for the folic acid and prenatal vitamins have been provided by Natural Factors (Coquitlam, Canada). Bulk (6S)-5-MTHF (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-MTHF 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 quality overall summary was completed for all study supplements by Natural Factors (Coquitlam, Canada) and verified by Health Canada, with confirmation of folic acid and (6S)-5-MTHF doses via HPLC. 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. 2.7.1 Double-blinding, randomization & provision of supplements An independent research assistant from Natural Factors (Coquitlam, Canada) will assign blinding codes (A or B) to the folic acid and (6S)-5-MTHF 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 permuted 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 50 coordinator will unblind the allocation (A or B) for that study ID only, to provide the allocated study supplements. At baseline, participants will receive a supplement pack containing all capsules for the 16-week intervention; if participants choose to continue to the postpartum phase of the study, they will be given more supplements at their endline visit to continue taking until ~1 week postpartum. 2.7.2 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. 2.7.3 Strategies to enhance adherence Pregnant women tend to be highly motivated and are generally interested in participating in clinical research trials.236 As per a survey from the Public Health Agency of Canada, 94% of women in British Columbia supplement with prenatal multivitamins during pregnancy.165 Therefore, participants will likely be accustomed to daily supplementation upon enrollment. The research coordinator will contact each participant at midpoint (8 weeks after their baseline visit) to address any questions or concerns. Additionally, the supplement pack will allow for organized storage of supplements at home and 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. 51 2.8 Biological specimen collection, processing & analysis 2.8.1 Blood collection & processing Venous blood specimens (12 mL total) will be collected in a 6 mL EDTA tube, 2 mL EDTA tube, and 4 mL serum tube (BD Biosciences, Franklin Lakes, NJ, USA) at baseline, endline, and postpartum (if applicable) visits. Baseline and endline blood draws will occur following a 3-hour fast; this timeframe was chosen to reduce the confounding influence of recent folate intake on plasma UMFA, as concentrations tend to decrease after ~2-3 hours, with no clear pattern for up to 10 hours after intake.43,44,53 The postpartum blood sample (~1 week postpartum) will be non-fasting, as this will not influence RBC folate and will reduce burden on participants. After collection, tubes will be shielded from light, inverted gently as per manufacturers recommendations,237 placed in a cooler and transported to the laboratory for processing (within 2 hours of collection). For preparation of the whole blood hemolysate, whole blood (0.3 mL) will be removed from the 6 mL EDTA tube and diluted 1/11 by adding 3 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) using the SepMate PBMC protocol (Stemcell technologies, Vancouver, Canada). The 2 mL 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. 52 2.8.2 Human milk collection & processing Participants will be instructed to collect the human milk specimen on day 5-7 postpartum between 1300 and 1450 hours (as folate concentrations during this time appear representative of mean human milk folate concentrations over a 24-h period),87,88 2-3 hours after their last full expression. Participants will completely express breastmilk (manually or by electric pump) from the right breast. Once breastmilk is expressed, women will be instructed to gently swirl the milk to mix it, and then using a sterile falcon pipette, they will transfer 0.5 ml into two amber cryovials (each containing 0.005 g ascorbic acid for a 1% dilution) and 1 ml into two amber cryovials. All cryovials will be clearly marked at the 0.5- and 1-ml levels. Participants will place all vials in a freezing box, which will be stored in the freezer for up to 4 days. Frozen milk specimens will be picked up and transferred to the laboratory for storage at -80°C until further analyzed. 2.8.3 Laboratory analyses Serum folate (nmol/L) and RBC folate (nmol/L) concentrations will be analyzed by microbiological assay using the microtitre plate method outlined by Molloy et.al.129 RBC folate (nmol/L) will be calculated with the following formula:124 RBC Folate = (Whole blood hemolysate folate * 11) – Serum folate (1-Hematocrit/100) Hematocrit/100 Plasma biomarkers including UMFA (nmol/L), SAM (µmol/L), SAH (µmol/L), total homocysteine (µmol/L), cysteine (µmol/L), methionine (µmol/L), free choline (µmol/L), betaine 53 (µmol/L), and pyridoxal phosphate (PLP; nmol/L) will be analyzed using LC-MS/MS.131,238–240 Vitamin B12 (pmol/L) will be analyzed using an immunoanalyzer. Gene variants (MTHFR [677 C>T] rs1801133 and [1298 A>C] rs1801131, and DHFR rs1643649 and rs70991108) will be genotyped by using taqman SNP genotyping assays. A complete blood count will be performed using an automated hematology analyzer (Sysmex XNL-550). Human milk folate forms (including UMFA, THF, 5-MTHF, 5-formylTHF, and 5,10-methenylTHF) will be quantified via LC-MS/MS; total folate will represent the sum of these forms.82,131 Human milk FBP concentrations will be measured using a competitive binding radioassay procedure.85 2.9 Summary of data to be collected 2.9.1 Demographic, medical & nutrition data Baseline data collection will include age, ethnicity, parity, education, 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. 2.9.2 Anthropometrics Pre-pregnancy BMI will be calculated using self-reported pre-pregnancy weight and measured height. Participant measurements will be taken at baseline (weight and height) and endline (weight only) for calculation of gestational weight gain throughout the intervention period. 54 2.9.3 Dietary assessment Total DFEs (µg/day) from food folates and supplemental folic acid will be calculated using the Block Folic Acid/Dietary Folate Equivalents Screener (NutritionQuest, Berkeley, USA).233 2.9.4 Postpartum data Total weeks of supplementation, weeks of gestation at delivery, compliance to the human milk collection protocol, and timing of folate supplementation prior to collection of human milk and postpartum blood specimens will be recorded. 2.10 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). 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. Participants who give birth prior to their endline visit will have the option to continue in the postpartum phase of the study. 2.11 Data handling & privacy All data and biological specimens will be identified using each participant’s study ID. Data with personal identifiers will be stored on an encrypted, password-protected computer in a secure server space with the BC Children’s Hospital Research Institute. Data (with personal identifiers de-linked) will be cleaned, and double entry will be completed by the research coordinator and 55 an independent research assistant into REDCap (Research Electronic Data Capture) hosted by BC Children’s Hospital Research Institute. Paper documents will be stored in a locked filing cabinet and biological specimens 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/human milk (if applicable) folate results (including DFEs, serum and RBC folate at baseline, endline and postpartum, and total human milk folate concentrations) after final statistical analyses are completed. 2.12 Statistical analyses Descriptive statistics will be used for participant baseline data. Continuous variables will be reported with a mean ± SD (or median and IQR if not normally distributed) and categorical variables as absolute numbers per group and percentages. Capsule counts will be used to calculate participant adherence and the supplement diary will be used to provide descriptive insight into adherence barriers. Data will be assessed for normality, and transformations or use of equivalent non-parametric tests will be considered if data do not follow a normal distribution. Statistical analyses will be performed using Stata (Stata Corp, Texas, USA). All analyses 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. Multiple imputation will be used to correct for missing data as appropriate in sensitivity analyses.241 56 Primary analyses: We will calculate the mean ± SD for primary outcomes (serum folate, RBC folate, and plasma UMFA) in each group at baseline and endline, as well as the mean ± SD within-woman change from baseline to endline in these outcomes in each group. These estimates can be used to inform the sample size calculation of the definitive trial. Secondary analyses: Exploratory analyses of primary outcomes will be conducted using a multivariable linear regression model to investigate if the change from baseline to endline differed according to treatment status, by adjusting for baseline values and explanatory variables (DFEs, exploratory biomarkers, weeks of gestation at supplement initiation). Covariates were selected as potentially important modifiers of folate status, to gain an unbiased assessment of the treatment effect and increase effect size sensitivity. The proportion of women with UMFA ≥0.2 nmol/L (considered a detectable concentration) at baseline and endline in each group will also be calculated. To inform our secondary objective, the 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. For each recruitment strategy (including posters, social media, word of mouth), total reach (social media only), inquiries, enrollments and cost will be evaluated. The odds of enrollment after inquiring from each strategy will be evaluated with an odds ratio. As available, reasons for not enrolling will be recorded. 57 Postpartum analyses: We will calculate the mean ± SD for the proportion of UMFA in human milk (dividing concentrations of human milk UMFA by the sum of all milk folate forms) and for maternal postpartum RBC folate. Linear regression will be used to estimate the association between human milk folic acid concentrations and maternal postpartum plasma UMFA. Postpartum outcomes will be further analyzed using multivariable linear regression, adjusting for endline values and explanatory variables (DFEs, total weeks supplementing, adherence to the human milk collection protocol). 2.13 Safety Folic acid is provided at a dose that meets Canadian recommendations during pregnancy (0.4-1.0 mg/day); thus, is considered low risk. Calcium salt of (6S)-5-MTHF is recognized as safe and is included in Canadian prenatal multivitamins with no established UL (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 and qualified investigator 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. 2.14 Discussion & study significance These pilot data are critical to inform the design a definitive trial which will be powered to assess whether (6S)-5-MTHF supplementation is as effective as synthetic folic acid in increasing serum and RBC folate concentrations during pregnancy, while resulting in lower UMFA. Although 58 analyses in the postpartum phase will be exploratory, no other study to our knowledge has assessed long-term supplementation of folic acid as compared to (6S)-5-MTHF starting in mid-pregnancy and subsequent influence on folate concentrations in human milk and lactating blood specimens; thus, these postpartum investigations are also warranted and may be further explored in the definitive trial. Ultimately, a definitive trial will help to inform the safest and most effective form of folate supplementation for pregnant women and their babies. 59 Chapter 3: Maternal folate status Acknowledgement: A version of this chapter has been submitted for publication and is currently under review. Cochrane KM, Elango R, Devlin AM, Mayer C, Hutcheon JA, Karakochuk CD. Supplementation with (6S)-5-methyltetrahydrofolic acid appears as effective as folic acid in maintaining maternal folate status while reducing unmetabolized folic acid in maternal plasma: A randomized trial of pregnant women in Canada. 3.1 Summary Folic acid supplementation is recommended before and during pregnancy to support healthy fetal development; (6S)-5-MTHF is available in some commercial prenatal vitamins as an alternative to folic acid, but its effect on blood folate status during pregnancy is unknown. To address this, we randomized 60 pregnant individuals at 8-21 weeks of gestation to 0.6 mg/day folic acid or 0.625 mg/day (6S)-5-MTHF. Fasting (3-hr) blood specimens were collected at baseline and after 16 weeks (endline), and at 1-week postpartum (non-fasting). RBC and serum folate concentrations were quantified via microbiological assay and plasma UMFA via LC-MS/MS. Differences in biochemical folate markers between folate intervention groups were explored using multivariable linear/quantile regression, adjusting for baseline concentrations, dietary folate intake, and gestational weeks. At endline (n=54), mean ± SD (or median, IQR) concentrations of RBC folate, serum folate, and plasma UMFA (nmol/L) in those supplemented with (6S)-5-MTHF versus folic acid, respectively, was: 1826 ± 471 and 1998 ± 421; 70 ± 13 and 78 ± 17; 0.5 (0.4, 0.8) and 1.3 (0.9, 2.1). At 1-week postpartum (n=36), median (IQR) RBC folate concentrations (nmol/L) in those supplemented with (6S)-5-MTHF and folic acid were 60 1724 (1474, 1877) and 1962 (1576, 2284), respectively. In regression analyses, RBC and serum folate did not differ significantly by treatment group at endline or postpartum; however, concentrations of plasma UMFA in pregnancy were 0.6 nmol/L higher (95% CI: 0.2-1.1) in those supplementing with folic acid as compared to (6S)-5-MTHF. In conclusion, supplementation with (6S)-5-MTHF resulted in plasma UMFA concentrations that were ~50% lower than in those supplemented with folic acid, the biological relevance of which is unclear. As folate is currently available for purchase in both forms, the impact of circulating maternal UMFA on perinatal outcomes needs to be determined. 3.2 Introduction An increasing number of prenatal vitamins contain (6S)-5-MTHF as an alternative to folic acid.175,178 In 2019, 32% of prescription and 25% of non-prescription prenatal vitamins commercially available in the United States (as per the National Institute of Health Dietary Supplement Label and DailyMed databases) contained (6S)-5-MTHF; prior to 2015, none were listed.178 Supplements in Canada which are marketed for use in pregnancy may contain folic acid or (6S)-5-MTHF, so long as a minimum dose 0.4 mg is provided.175 While supplementation with (6S)-5-MTHF has proven more effective than folic acid in increasing folate status in non-pregnant women,97,191–193 pregnancy is a unique physiological state associated with numerous metabolic changes, including increased hemodilution, urinary excretion, and micronutrient needs to support growth of maternal and fetal tissue.55,64 Maternal plasma supplies folate to the fetus and uteroplacental organs to accommodate growth within these compartments.55 As (6S)-5-MTHF supplements are currently widely consumed by pregnant individuals,178 despite 61 a lack of evidence in pregnancy, it is imperative to ensure that (6S)-5-MTHF can maintain folate status to the same extent as folic acid during pregnancy. This study aimed to generate estimates of folate status, including RBC folate, serum folate, and plasma UMFA, following supplementation with 0.625 mg/day (6S)-5-MTHF or 0.6 mg/day folic acid x 16 weeks of pregnancy. These estimates are a critical first step in understanding any differences between commercially available folate forms during pregnancy. 3.3 Methods 3.3.1 Study participants & design Individuals aged 19-42 years with singleton pregnancies in Vancouver, Canada were recruited via printed posters and social media advertisements; full details on the recruitment methods and study aims related to recruitment are published (see Appendix B).242 The study took place between Sept 2019 and Sept 2021. Exclusion criteria included medical conditions, medications, and behavioral factors (current smoking, alcohol consumption, or recreational drug use) associated with altered folate status,164 a pre-pregnancy BMI ≥30 kg/m2, and being medium to high risk for development of an NTD-affected pregnancy.164 Eligible participants were scheduled for a baseline visit between 8-21 weeks’ gestation, with instructions to continue any folate supplementation until the day before this visit. At baseline, informed consent was obtained, weight and height were measured, and demographic information and usual intake of DFEs (as per a validated FFQ; NutritionQuest, Berkeley, CA, USA233) were collected. Participants were randomized to 0.6 mg/day folic acid or an equimolar dose (0.625 62 mg/day) of (6S)-5-MTHF (Metafolin®); a prenatal vitamin (NPN: 80025456) with folic acid removed was also provided to all participants. Participants were given all supplements needed for the 16-week intervention period and were instructed to begin supplementation immediately following baseline visits. Following 16 weeks, participants were scheduled for a second visit (endline). Participants had the option to continue supplementation for the postpartum study phase, to explore the effect on folate status after delivery (~1 week postpartum); new informed consent was obtained, and more study supplements were provided to those interested. Capsule counts were completed at endline and postpartum visits to assess adherence to daily supplementation. Participants were also given a supplement diary to record daily intake or reasons for missing a dose. 3.3.2 Blood collection & processing Venous blood specimens were collected at baseline, endline, and postpartum visits in a 4 mL serum tube, 6 mL EDTA tube, and 2 mL EDTA tube (Beckton Dickinson, Franklin Lakes, NJ, USA). At baseline and endline visits, participants were instructed to fast for 3 hours to mitigate the influence of recent folic acid intake on plasma UMFA.43,44 Blood specimen collections at postpartum visits were non-fasting to reduce participant burden, with instruction to consume the folate supplement 2 hours prior to the visit (to standardize and capture any peak in plasma UMFA concentrations following intake); RBC folate is less sensitive to recent intake.110 After collection, tubes were shielded from light, inverted gently, and transported to the laboratory for immediate processing (within 2 hours of collection). 63 Whole blood (0.3 mL) was removed from the 6 mL EDTA tube and diluted 1/11 with a 3 mL 1% ascorbic acid solution, followed by incubation at 37°C for 30 minutes.124 Remaining whole blood in the 6 mL EDTA tube was centrifuged at 3000 rpm for 15 minutes at 4°C; plasma was separated into aliquots. Remaining contents in the 6 mL EDTA tube were processed for PBMC isolation (SepMateTM STEMCELL Technologies, Vancouver, Canada). The 2 mL EDTA tube was used for a complete blood count, including hematocrit (L/L) determination via an automated hematology analyzer (Sysmex XNL-550, Sysmex Corp., Japan). Serum tubes were left at room temperature to clot for minimally 30 minutes and then centrifuged at 3000 rpm for 10 minutes at 4°C; serum was separated into aliquots. All specimens were frozen immediately after processing at -80°C until analysis. 3.3.3 Biochemical analyses Total serum and whole blood folate (nmol/L) were determined as per the microbiological assay (Bevital AS, Bergen, Norway) with chloramphenicol-resistant Lactobacillus rhamnosus and folic acid calibration, using previously described methods.127 The inter-assay coefficient of variation (CV) ranged from 3.1 to 5.6% based on three quality controls that were analyzed in duplicate. RBC folate (nmol/L) was subsequently calculated as previously described (using whole blood folate, serum folate, and hematocrit).124 Plasma UMFA (nmol/L) was determined via LC-MS/MS as previously described.82,131,132,136,243 The inter-assay CV for plasma UMFA was 8%, as determined using internal quality control samples (NetCad internal plasma control). Exploratory outcomes included other methyl nutrients and MTHFR 677 C > T variant genotyping. Plasma vitamin B12 (pmol/L) was determined via an 64 immunoanalyzer (Abbott Architect i1000, IL, USA; CV = 3.3%). PLP (nmol/L; CV = 8%), total homocysteine (µmol/L; CV = 1.7%), cysteine (µmol/L; CV = 1.4%), methionine (µmol/L; CV = 1.1%), free choline (µmol/L; CV = 5.4%), and betaine (µmol/L; CV = 2.7%) were determined using HPLC-MS/MS.244,245 Genotyping of MTHFR 677 C > T variant (rs1801133) was determined via pyrosequencing using PyromarkTM Q96 MD Pyrosequencer (Qiagen, Hilden,Germany), as previously described.246 Plasma vitamin B12, PLP, total homocysteine, cysteine, methionine, free choline, and betaine were quantified at baseline and endline visits only. Post-hoc amendments to biochemical outcomes and processing: Some biological outcomes which were outlined in our protocol were not determined due to lack of available funds for these analyses; this included plasma SAM, SAH, and genotyping of MTHFR 1298 A>C and DHFR rs1643649 and rs70991108. Genotyping of MTHFR 677 C>T genotype (rs1801133) was determined via pyrosequencing,246 as opposed to a TaqMan SNP genotyping assay. The limit of detection for UMFA as per LC-MS/MS was 0.105 nmol/L (as opposed to 0.2 nmol/L). 3.3.4 Statistical analyses Participant demographic data and biochemical outcomes were summarized descriptively. Folate status in each group (RBC folate, serum folate, and plasma UMFA) was summarized using means ± SD or medians with IQRs if not normally distributed. The median (IQR) within-person change in folate status (RBC folate, serum folate, and plasma UMFA) from baseline to endline was calculated. 65 The change in concentrations of RBC folate, serum folate, and plasma UMFA in each intervention group was further explored by evaluating differences in endline concentrations, adjusting for baseline concentrations, dietary folate intake, and weeks of gestation at supplement initiation, using multivariable linear regression (or multivariable quantile regression for non-normally distributed outcomes). Lack of collinearity among covariates was confirmed with variance inflation factors (<10) and by evaluation of correlation between covariates (≤ 0.3). Quantile regression was chosen over data transformations as it requires fewer assumptions about the underlying distribution and leads to more straightforward biological interpretations of the model estimates.247 Differences in postpartum RBC folate concentrations was explored using quantile regression, adjusting for endline concentrations, dietary folate intake, and total weeks supplementing. Postpartum concentrations of serum folate and plasma UMFA were not explored in adjusted models given their limited interpretability due to the non-fasting state. Covariates were chosen a priori26; however, in a post-hoc protocol change, we opted not to adjust for exploratory biomarkers (plasma concentrations of other methyl nutrients), as no clinically meaningful differences between groups were observed, or MTHFR genotype, due to few women with the homozygous (TT) polymorphism (n=2 in the folic acid group). All analyses were completed on an intention-to-treat basis according to group allocation. Due to high adherence to the study protocol throughout the trial (median [IQR] adherence to daily supplementation = 98% [96%, 100%], as per capsule counts), per-protocol analyses were deemed unnecessary. A P value < 0.05 was considered statistically significant. 66 3.4 Results 3.4.1 Participant enrollment & baseline characteristics Overall, n=60 participants completed baseline visits, n=54 completed endline visits (retention rate of 90%), and n=37 provided a postpartum blood specimen (retention rate of 69%). One participant delivered prematurely and could not participate in the endline visit; however, they provided a postpartum blood specimen. See Figure 3.1 for the participant flow diagram. Adherence to daily supplementation as per capsule counts was high, with rates of daily supplementation in 93% (n=50) of participants ≥90%. At postpartum visits, 92% (n=34) of participants had rates of daily adherence ≥90%. Per the supplement diary, primary reasons for a missed dose included: fatigue, forgetting, illness (gastrointestinal distress), being on vacation, and lack of intake while admitted to hospital for delivery. 67 Figure 3-1 Participant flow diagram (maternal folate status) In the folic acid group, n=19 at the postpartum visit includes: n=18 from the n=25 at endline (n=7 chose to not participate in the postpartum visit) and n=1 participant who delivered prematurely (they did not participate in the endline visit, but provided a postpartum blood specimen) Participant characteristics are presented in Table 3.1. Participants appeared similar between groups, and were ~33 years of age, highly educated (97% with post-secondary education), and predominantly nulliparous (73%). All participants reported a pre-pregnancy BMI <30 kg/m2; median (IQR) gestational weight gain from baseline to endline in the (6S)-5-MTHF and folic acid groups, respectively, was 0.44 kg/week (0.36 to 0.56) and 0.46 kg/week (0.39 to 0.56) (missing n=3 endline weights); recommended gestational weight gain ranges from 0.3-0.5 kg/week in the second and third trimesters for those with a pre-pregnancy BMI <30 kg/m2.248 The intervention period from baseline to endline was 16 weeks (± 6 days); however, due to 68 COVID-19 pandemic countermeasures, endline visits for n=5 overall were moved earlier or delayed; the min/max intervention period was 13/17 weeks in the (6S)-5-MTHF group and 14/20 weeks in the folic acid group. The postpartum visit was planned for ~1 week after delivery; however, this varied slightly based on participant availability; the median (IQR) days postpartum in the (6S)-5-MTHF and folic acid groups, respectively, was day 9 (7 to 11 days) and day 8 (7 to 10 days); overall, weeks of supplementing from baseline to postpartum was 24 ± 4 weeks. 69 Table 3-1 Participant characteristics (maternal folate status) (6S)-5-MTHF (n=30) Folic acid (n=30) Age, years (mean ± SD) 33 ± 3.1 33 ± 3.8 Ethnicity, n (%) European 16 (53) 18 (60) South, East, & Southeast Asian 6 (20) 7 (23) Hispanic/Latino 5 (17) 2 (7) Middle Eastern 2 (7) 0 (0) Mixed ethnicity 1 (3) 3 (10) Education, n (%) Highschool 1 (3) 1 (3) College 7 (23) 2 (7) Undergraduate 10 (34) 15 (50) Graduate 12 (40) 12 (40) 1Household income per year, CAD$, n (%) <20,000 1 (3) 0 (0) 20,000-50,000 2 (7) 4 (14) 50,000-100,000 10 (34) 11 (39) >100,000 17 (56) 13 (47) Nulliparous, n (%) 24 (80) 20 (67) Dietary folate intake (FFQ), mg DFE/day 2From foods (mean ± SD) 0.53 ± 0.17 0.45 ± 0.16 3From supplements, median (IQR) 0.7 (0.7, 0.7) 0.7 (0.7, 0.7) Total intake, median (IQR) 1.2 (1.1, 1.4) 1.2 (1, 1.3) 4Special diet followed, n (%) 4 (13) 4 (13) Weeks’ gestation, median (IQR) 18 (15, 20) 17 (13, 20) min/max weeks 9/21 8/21 1Missing n=2 household income (participants chose not to disclose); 2From natural food sources and fortified foods; 3Supplementation prior to study enrollment; all participants reported folate supplementation prior to study enrollment, which was discontinued and replaced with the study vitamins at baseline. 4Lacto-ovo-vegetarian (n=1), pescatarian (n=1), vegan (n=1), gluten-free (n=3), other (n=2). Abbreviations: DFE: dietary folate equivalents; FFQ: food frequency questionnaire 70 3.4.2 Maternal folate status Median (IQR) concentrations of RBC folate, serum folate, and plasma UMFA at baseline in the (6S)-5-MTHF and folic acid groups, respectively, were: 1574 nmol/L (1380, 1811) and 1656 nmol/L (1342, 1957), 65 nmol/L (60, 73) and 68 nmol/L (61, 85), and 1.2 nmol/L (0.8, 1.5) and 1.1 nmol/L (0.9, 1.5). Folate status at endline and postpartum, and the estimated difference between groups, is presented in Table 3.2. No participants had RBC folate (<305 nmol/L) or serum folate (<7 nmol/L) concentrations indicative of deficiency at any point.138 Further, RBC folate remained >906 nmol/L in all participants throughout the study (a cut-off associated with maximal risk reduction of NTDs139,163). Plasma UMFA was detectable in all participants at all timepoints. 71 Table 3-2 Crude and adjusted differences between groups in measures of folate status at endline and postpartum Results are median (IQR); mean ± SD is also presented if normally distributed. n=54 participants included for all endline analyses (crude and adjusted); n=36 participants included for crude estimates of postpartum RBC folate and serum folate (missing n=1 due to insufficient blood sample); n=35 participants included for adjusted estimates of postpartum RBC folate (missing n=2 due to [1] insufficient blood sample and [2] missed endline visit due to premature delivery, participated in postpartum visit only); n=37 participants included for crude estimates of postpartum plasma UMFA. 1Endline outcomes adjusted for: folate form ([6S]-5-MTHF as the reference group), baseline values (nmol/L), weeks of gestation at baseline, and dietary folate intake (mg dietary folate equivalents/d). Postpartum RBC folate adjusted for: folate form ([6S]-5-MTHF as the reference group), endline values (nmol/L), total weeks supplementing, and dietary folate intake (mg dietary folate equivalents/d). Full multivariable linear/quantile regression model outputs are presented in Table 3.3. (6S)-5-MTHF Crude concentrations Folic acid Crude concentrations Crude difference (95% CI) 1Adjusted difference (95% CI) RBC folate (nmol/L) Endline 1851 (1555, 2004) 1826 ± 471 2078 (1654, 2336) 1998 ± 421 171 (-74, 417) 161 (-79, 400) Postpartum 1724 (1474, 1877) 1962 (1576, 2284) 239 (-159, 636) 11 (-272, 294) Serum folate (nmol/L) Endline 68 (65, 73) 70 ± 13 74 (69, 91) 78 ± 17 7.7 (-0.6, 16) 7.4 (-1.4, 16) Postpartum 88 (74, 114) 96 ± 39 109 (73, 133) 104 ± 34 8.9 (-16, 33) -- Plasma UMFA (nmol/L) Endline 0.5 (0.4, 0.8) 1.3 (0.9, 2.1) 0.7 (0.3, 1.1) 0.6 (0.2, 1.1) Postpartum 0.8 (0.7, 1.2) 13 (7, 23) 12 (6.1, 19) -- 72 Table 3-3 Full multivariable regression model outputs for adjusted differences in markers of folate status between groups Difference in endline RBC folate (nmol/L) n=54 Difference in endline serum folate (nmol/L) n=54 Difference in endline plasma UMFA (nmol/L) n=54 Difference in postpartum RBC folate (nmol/L) n=35 β 95% CI β 95% CI β 95% CI β 95% CI 1Folate form Folic acid 161 -79, 400 7.4 -1.4, 16 0.6 0.2, 1.1 11 -272, 294 Baseline values nmol/L 0.3 0.08, 0.5 0.07 -0.2, 0.3 0.002 -0.02, 0.02 -- -- Weeks’ gestation at baseline -4.8 -37, 27 -0.2 -1.4, 1 -0.04 -0.09, 0.02 -- -- Dietary folate mg DFE/day -315 -1065, 435 0.6 -28, 29 0.06 -1.3, 1.4 -424 -1415, 567 Endline values nmol/L -- -- -- -- -- -- 0.9 0.6, 1.2 Total weeks supplementing -- -- -- -- -- -- 10 -25, 45 1Compared with the reference group of (6S)-5-MTHF. Abbreviations: DFE: Dietary folate equivalents 73 Concentrations of RBC and serum folate at endline and postpartum did not appear clinically different between intervention groups, as both groups were well-above cut-offs for deficiency and NTD risk reduction, with a high degree of overlap and variability in crude concentrations (SD ± ~450 nmol/L for RBC folate and SD ± ~15 nmol/L for serum folate, at endline); there was also no evidence of a statistically significant difference in RBC and serum folate concentrations, as the 95% CI for all crude and adjusted differences crossed zero. Conversely, there appeared to be a meaningful difference in plasma UMFA at endline and postpartum, as there was no overlap in the IQRs of crude estimates, and concentrations were significantly higher at endline (adjusted difference = 0.6 nmol/L, 95% CI: 0.2-1.1) and postpartum (crude difference = 12 nmol/L, 95% CI: 6.1-19) in those supplemented with folic acid as compared to (6S)-5-MTHF. A high degree of inter-individual variability of within-person change in folate status during pregnancy was noted; the median (IQR) within-person change from baseline to endline in RBC folate and serum folate concentrations, respectively, in those supplemented with (6S)-5-MTHF was 88 nmol/L (26, 346) and 2.5 nmol/L (-9.8, 15), and in those supplemented with folic acid, was 368 nmol/L (-81, 549) and 0.8 nmol/L (-15, 15). In those supplemented with (6S)-5-MTHF, plasma UMFA concentrations decreased by a median (IQR) of 0.5 nmol/L (0.9, 0.2), representing a ~50% decrease from baseline to endline. Conversely, in the folic acid group, plasma UMFA concentrations remained mostly consistent from baseline to endline (median [IQR] within-person change = 0.03 nmol/L [-0.2, 0.9]). Other considerations for the interpretation of within-person change are presented in Appendix C. 74 Concentrations of exploratory biochemical outcomes (other methyl nutrients) at baseline and endline are presented in Table 3.4. While cut-offs during pregnancy are not established, median concentrations of vitamin B12 and PLP remained above the cut-offs for deficiency in non-pregnant women (vitamin B12 <148 pmol/L and PLP <20 nmol/L), and total homocysteine remained <13 µmol/L (cut-off for hyperhomocysteinemia) in all participants, at all visits.249,250 Of note, a cut-off for vitamin B12 of >221 pmol/L upon entering pregnancy is proposed to reduce the risk of NTDs251; however, the relevance of this cut-off in later pregnancy is unclear, as concentrations of plasma vitamin B12 are reported to decline by up to 50% as pregnancy progresses in women with healthy pregnancies, despite consumption of adequate dietary vitamin B12 (RDA = 2.6 µg/day).249 Significantly lower plasma vitamin B12 concentrations in pregnant women versus non-pregnant controls were also observed in a controlled feeding trial providing ~8.6 µg/day vitamin B12252; additionally, a greater conversion of vitamin B12 to holotranscobalamin was observed during pregnancy.252 See Appendix A for further discussion regarding the flux of 1C metabolism and plasma methyl nutrients during pregnancy. 75 Table 3-4 Estimates of exploratory biochemical outcomes at baseline and endline Baseline Endline (6S)-5-MTHF (n=30) Folic acid (n=30) (6S)-5-MTHF (n=29) Folic acid (n=25) Vitamin B12 (pmol/L) 288 (210, 349) 299 (222, 349) 204 (159, 239) 206 (166, 300)2 Pyridoxal phosphate (nmol/L) 55 (38, 94)1 88 (42, 101) 26 (20, 35) 32 (18, 46) Total homocysteine (µmol/L) 4.7 (4, 4.9)1 5 (4.5, 5.6) 4.9 (4.5, 5.6) 5.2 (4.7, 5.8)2 Cysteine (µmol/L) 236 (217, 254)1 256 (233, 271) 231 ± 20 237 ± 242 Methionine (µmol/L) 25 (24, 29)1 25 (23, 27) 27 (26, 29) 27 (25, 29)2 Free choline (µmol/L) 7.2 (5.9, 8.2)1 7.1 (6.1, 8.1) 9.1 (7.7, 10) 9.5 (7.9, 11) Betaine (µmol/L) 15 (14, 18)1 16 (13, 20) 15 ± 3 14 ± 3.8 MTHFR 677 C>T genotype, n (%) CC 16 (53) 16 (53) -- -- CT 8 (27) 12 (40) -- -- TT 6 (20) 2 (7) -- -- Results are mean ± SD if normally distributed, median (IQR) if not normally distributed. 1Missing n=1 due to insufficient blood sample (n=29); 2Missing n=1 due to insufficient blood sample (n=24).76 3.5 Discussion & conclusions Investigations in non-pregnant191–193 and lactating97 women have reported that (6S)-5-MTHF is more effective than folic acid in raising RBC and/or serum folate concentrations. Bailey et al.193 conducted an investigation of 7.5 mg/day (6S)-5-MTHF or folic acid x three days, followed by 0.4 mg/day x two weeks; those supplemented with (6S)-5-MTHF more quickly achieved the target cut-off (serum total folate >50 nmol/L), but by day 12, there were no differences between groups.193 Henderson et al.192 and Lamers et al.191 investigated (6S)-5-MTHF and folic acid supplementation at 1.0 mg/day x 12 weeks192 and 0.4 mg/day x 24 weeks191 in non-pregnant women of childbearing age; both reported greater increases in RBC folate concentrations following (6S)-5-MTHF supplementation, and a similar190 or increased192 effect on plasma folate concentrations. Similarly, Houghton et al.97 reported significantly higher RBC folate concentrations in those supplementing with (6S)-5-MTHF versus folic acid at 0.4 mg/day x 16 weeks during lactation, with a similar effect on plasma folate concentrations. While we did not investigate pharmacokinetic action, we propose that the lack of a stronger effect of (6S)-5-MTHF as compared to folic acid on RBC and serum folate concentrations found in the current study may be attributed to changes in folate handling during pregnancy55; perhaps any increased effect of (6S)-5-MTHF on folate status as compared to folic acid was nullified due to the abundant transfer of folate from maternal plasma to the placenta and fetus, or due to increased folate catabolism and excretion, particularly in later pregnancy.62,63 While the difference in plasma concentrations of UMFA between groups was modest after fasting, there did appear to be a meaningful effect driven by supplemental folate form. All participants reported supplementation with folate prior to study enrollment; as per the type of 77 folate or brand of prenatal vitamin, >90% consumed folic acid at doses ranging from 0.4-1.0 mg/day. At baseline visits, plasma UMFA was ~1 nmol/L in both groups; we did not observe continued increases of UMFA in the folic acid group (UMFA concentrations remained at ~1 nmol/L at endline). However, concentrations in the (6S)-5-MTHF group decreased by ~50%. Fasting state and supplementation with folic acid have previously been identified as the strongest predictors of circulating UMFA.47,48 We do suspect a high degree of UMFA clearance following the (minimum) 3-hour fast, given comparison to the non-fasting postpartum UMFA values (see Table 3.2). However, as per NHANES 2007-2008,47 plasma UMFA concentrations remain significantly higher after fasting ≥8 hours in folic acid supplement users (~1 nmol/L) versus non-supplement users (~0.7 nmol/L). Overall, it seems that a significant reduction of UMFA in plasma can be effectively achieved by supplementation with (6S)-5-MTHF. Ultimately, it is noted that while plasma UMFA is statistically different between groups, concentrations after fasting are low overall; as such, the biological and clinical implications are uncertain. Excess folic acid intake has been associated with various adverse health outcomes for both the mother and child.33,163,176 However, the evidence is very mixed, and is mainly limited to preclinical and observational research. Further, there is a lack of clarity as to whether adverse outcomes are due to folic acid specifically (UMFA), or more generally, a “high” folate status. While a cut-off for “high” RBC folate is not established, 1360 nmol/L was reflective of the 97th percentile as per NHANES 1999-2004.96,167 Overall, it is very difficult for pregnant individuals in Canada to consume 0.4 mg/day folic acid, due to food fortification and as the vast majority of prenatal vitamins contain 0.6-1.0 mg folic acid54,163,172,173; this has contributed to RBC folate levels ~1500-2500 nmol/L among pregnant and lactating individuals.60,97,174,253 High RBC and 78 serum folate concentrations were similarly found in the current study. Those consuming folic acid from fortified foods and prenatal vitamins are very likely to exceed the UL of 1.0 mg/day folic acid.54,172,173,254,255 At this time, the UL does not apply to (6S)-5-MTHF178; this would require the conversion of (6S)-5-MTHF doses to DFEs.178,181 While the EFSA has started this work,181 findings of the current trial suggest that bioavailability of folate forms during pregnancy may differ from non-pregnant individuals, in that a greater effect of (6S)-5-MTHF on folate status was not observed at a 0.6 mg dose. There are limitations to consider in the interpretation of this study. First, the sample size was small, as the trial was designed as a pilot which aimed to generate estimates for a definitive trial. While a 3-hour fast was selected to mitigate the effect of folic acid on plasma UMFA,43,44,53 total serum folate concentrations may still have been influenced by recent intake within this time frame.47,110 Estimation of dietary folate intake was only assessed once (at baseline); thus, its relevance in later pregnancy and after delivery is uncertain. Dietary folate assessment was conducted via an FFQ, which can be useful when assessing a single nutrient over a longer period, as it captures food sources that may be consumed only occasionally and thus may be missed via other methods.256 However, previous research indicates that FFQs may overestimate folate intake.256,257 Further, DFEs of folate supplementation prior to the trial as per the FFQ is likely underestimated; the median (IQR) mg DFEs from supplements was 0.7 (0.7, 0.7 mg DFEs), assuming a folic acid dose of 0.4 mg (x 1.7 = 0.7 mg DFE). While the dose of folate provided (0.6 mg/day) was chosen to improve generalizability, given that it matches folic acid content in leading Canadian brands (Materna®), over-the-counter prenatal vitamins contain varying quantities of folate (often 1 mg in Canada). Finally, timing of supplement initiation varied widely 79 (8-21 weeks). While folate recommendations in Canada and the United States do not differ based on gestational weeks, we note a potential limitation when comparing folate status of those across different stages of pregnancy. Results of this study should be confirmed in a definitive trial; however, the current findings are very timely and of interest immediately, as both folate forms are currently widely available and consumed in pregnancy at a dose of 0.6 mg/day or greater. More research is needed to confirm whether there is any risk associated with folic acid supplementation in pregnancy (particularly at doses >0.4 mg) and the presence of plasma UMFA. This should be followed by the establishment of high-risk cut-offs for maternal folate status, which can be incorporated into perinatal clinical practice guidelines, and re-formulation of prenatal vitamins available in North America (perhaps by lowering the dose to 0.4 mg), to support achievement of optimal maternal folate status. 80 Chapter 4: Human milk folate profile Acknowledgement: A version of this chapter has been submitted for publication and is currently under review. Cochrane KM, Elango R, Devlin AM, Hutcheon JA, Karakochuk CD. Human milk unmetabolized folic acid is increased following supplementation with synthetic folic acid as compared to (6S)-5-methyltetrahydrofolic acid 4.1 Summary Folic acid supplementation is recommended perinatally, but may increase UMFA in human milk in a dose-dependent fashion. Increased UMFA in human milk is concerning as it is an inactive form of folate which may be less bioavailable for the infant. The objective of this investigation was to determine the human milk folate profile following maternal supplementation with (6S)-5-MTHF versus folic acid. Pregnant individuals (n=60) were enrolled at 8-21 weeks of gestation and randomized to 0.6 mg/day folic acid or 0.625 mg/day (6S)-5-MTHF. At ~1 week postpartum, participants provided a human milk and blood specimen. Human milk folate forms (nmol/L), including UMFA, THF, 5-methylTHF, 5,10-methenylTHF, 5-formylTHF, and 4α-hydroxy-5-MTHF (MeFox; an oxidation product of 5-MTHF), and plasma UMFA (nmol/L) were quantified via LC-MS/MS. Differences in milk folate forms and total milk folate between supplement groups were evaluated using multivariable quantile/linear regression, adjusting for dietary folate intake, total weeks of supplementing, and milk collection methods. Concentrations of UMFA in human milk and maternal plasma were pooled, and quantile regression was used to examine their association. No significant difference in total human milk folate was found between intervention groups; however, median milk UMFA concentrations were 11 nmol/L 81 higher in those receiving folic acid versus (6S)-5-MTHF (95% CI = 6.4-17 nmol/L). The mean ± SD proportion of human milk UMFA as part of total milk folate was 2% (±2%) and 28% (±14%) in those receiving (6S)-5-MTHF and folic acid, respectively. Concentrations of UMFA in maternal plasma and human milk were positively associated, with maternal plasma UMFA explaining ~40% of the variability in milk UMFA (pseudo R2=0.4). In conclusion, the form of supplemental folate had markedly differential effects on the human milk folate profile, with folic acid increasing the mean proportion of human milk UMFA by ~14-fold. Investigation of whether increased UMFA impacts folate-related metabolism and infant health outcomes is required. 4.2 Introduction Human milk provides optimal nutrition for infants and has many established health benefits for both the mother and child.258,259 Short term benefits of human milk for infants are clear (reducing the risk of all-cause mortality and infectious diseases), and increasing evidence suggests long-lasting protection against chronic diseases and increased intelligence in adulthood.260–262 Both biological and environmental factors contribute to human milk composition.80,259 Consumption of prenatal multivitamins (providing 0.4 mg/day folic acid) is recommended preconceptionally until the end of lactation to support healthy development of the fetus and infant (via human milk), while meeting maternal nutrient needs.156,157,163,263 Understanding how modifiable factors, such as maternal micronutrient intake, influences human milk composition is imperative to supporting optimal breastfeeding practices and thus, infant health. Most studies suggest that total human milk folate is not substantially affected by maternal diet unless severe deficiency occurs81,83,88,89; however, the impact of supplemental folic acid, an 82 oxidized, synthetic form of folate, on the human milk folate profile is unclear. Folate is transported across the mammary epithelium via FR-α32; the affinity of FR-α for oxidized folates (UMFA) is ~6-10 fold greater than for reduced forms, potentially leading to the preferential uptake of UMFA into human milk.20,32,264 Exact transport mechanisms including the potential for interconversion of folate forms at the mammary epithelium and the correlation of maternal plasma UMFA with the human milk folate profile remains poorly described.55,85,88 However, as compared to those who reported supplementing with <0.4 mg/day folic acid in the MIREC cohort, supplementation with >0.4 mg/day increased the proportion of UMFA as part of total milk folate by 2-fold82; in those consuming >0.4 mg/day folic acid, the human milk folate profile shifted so that UMFA became the predominant folate species (representing 50% of total human milk folate).82 Evidence regarding the effect of supplemental folic acid versus (6S)-5-MTHF on the human milk folate profile is scarce. Houghton et al.88 randomized lactating individuals to 0.416 mg/day (6S)-5-MTHF or a placebo starting at 1 week postpartum, and included a folic acid (0.4 mg/day) reference group. Detectable UMFA was reported in almost all (96%) human milk specimens, with no differences in UMFA concentrations between groups.88 However, findings from the MIREC cohort suggest that the impact of folic acid on the human milk folate profile is dose-dependent.82 Thus, the lack of differences reported by Houghton et al. may have been attributed to the lower dose (0.4 mg/day)88; commercial prenatal vitamins in North America contain doses of folic acid >0.4 mg (generally 0.6 to 1.0 mg).163,178 83 The objectives of this study were to determine the impact of supplementation with 0.6 mg/day folic acid or an equimolar dose (0.625 mg/day) of (6S)-5-MTHF on the human milk folate profile, and to evaluate the association of UMFA in maternal plasma and human milk. 4.3 Methods 4.3.1 Study participants & design The study design and participants included in the clinical trial (e.g., inclusion/exclusion criteria) are described in the previous chapters. All participants who were enrolled in the trial (n=60 pregnant individuals who were randomized to supplementation with 0.6 mg/day folic acid or 0.625 mg/day (6S)-5-MTHF x 16 weeks during pregnancy, starting at 8-21 weeks of gestation) were eligible for continuation in the postpartum study phase, which included the collection of a human milk and/or blood specimen at ~1 week postpartum. Demographic characteristics and dietary folate intake (DFEs per day; FFQ; NutritionQuest, Berkeley, CA, USA233) were collected at baseline visits. Those interested in the postpartum phase of the study provided consent in a new informed consent form at the endline visit, and were given more of their allocated supplements to continue daily supplementation until ~1 week postpartum. Adherence to daily folate supplementation throughout the study was assessed with capsule counts at both endline and postpartum visits. 4.3.2 Human milk collection At endline visits, participants were trained and were provided with the required supplies to collect a human milk specimen in their home at day 5-7 postpartum. Instructions indicated collection from the right breast, between 1300-1450 hours, at the start of a new feed (2-3 hours after the last 84 expression), and intake of the study supplements 2 hours prior to collection.265 Supplement intake timing was selected to standardize the peak in maternal plasma UMFA concentrations,44,53 and reduce any subsequent bias on milk UMFA. Participants were instructed to fully express milk manually or by electric pump; however, if a partial expression was preferred, participants were instructed to express milk at the start and end of the feed. Participants were given an amber-colored collection jar to protect the labile folate from light, or if using an electric pump, tinfoil was provided to wrap the bottle. Once the milk was expressed, participants were instructed to gently swirl it, and then using a sterile plastic falcon pipette, 0.5 mL milk was transferred into two 2-mL amber cryovials (containing 0.005 g ascorbic acid for a 1% dilution). The vials were gently inverted and stored in a provided box in their household freezer (aiming for pick-up in ≤4 days); frozen milk samples were transported to the laboratory for storage at -80°C. 4.3.3 Maternal blood specimen collection & processing Upon human milk specimen pick-up, participants opted to provide a non-fasting blood specimen for quantification of plasma UMFA and 5-MTHF concentrations, collected in a 6-mL EDTA tube (BD Biosciences, Franklin Lakes, NJ, USA). To match conditions at the human milk collection, participants were instructed to consume their folate supplements 2 hours prior to the blood draw. Vacutainers were shielded from light, inverted gently, and transported to the laboratory for immediate processing (within 2 hours of collection). Vacutainers were centrifuged at 3000 rpm x 15 minutes at 4°C. Plasma was aliquoted and stored at -80°C. 85 4.3.4 Quantification of folate forms Human milk folate forms (nmol/L), including UMFA, 5-MTHF, THF, 5,10-methenylTHF, 5-formylTHF, and MeFox, and maternal plasma UMFA (nmol/L) and plasma 5-MTHF concentrations, were determined via LC-MS/MS; detailed description of quantification methods are published.82,131,132,136,243 Total milk folate (nmol/L) equalled the sum of all folate forms. Inter-assay variability of each folate form was determined using internal quality control samples; the CV ranged from 3-6% for human milk folates and was 8% for plasma UMFA and 4% for plasma 5-MTHF (NetCad internal plasma control, pooled breast milk internal control, and NIST SRM 1849a external control sample). Post-hoc amendments to biochemical outcomes and processing: Some biological outcomes which were outlined in our protocol were not determined due to lack of available funds for these analyses; this included human milk FBP. Human milk folate forms analysis also included quantification of MeFox (nmol/L), and postpartum plasma 5-MTHF (nmol/L) was quantified. 4.3.5 Statistical analyses The effect of folic acid versus (6S)-5-MTHF on human milk folate forms and total folate concentrations, adjusting for dietary folate intake, total weeks of supplementing, and adherence to the human milk collection protocol (a full or partial milk expression), was evaluated with multivariable linear (total folate) or quantile (folate forms) regression. Quantile regression was chosen over data transformations to account for the non-normal distribution of the human milk folate forms, as it leads to more straightforward interpretations of the model estimates.247 Covariates were chosen a priori.26 Concentrations of UMFA in human milk and plasma in each 86 group were pooled and quantile regression was used to examine their association; pooling of data from both groups was chosen to examine the overall association, while preserving the largest sample size possible. All analyses were completed on an intention-to-treat basis. P <0.05 was considered significant. 4.4 Results 4.4.1 Participant enrollment & baseline characteristics A total of n=43 participants provided a human milk specimen; of those, 84% (n=36) also provided a postpartum blood specimen (see Figure 4.1). 87 Figure 4-1 Participant flow diagram (human milk folate profile) Participant characteristics are presented in Table 4.1. Most participants (n=39; 91%) were full term at delivery. The mean ± SD total weeks of supplementing was 24 ± 4 weeks. The median (IQR) daily supplementation rate at the human milk collection was 98% (94 to 100%). 88 Table 4-1 Participant characteristics (human milk folate profile) (6S)-5-MTHF (n=22) Folic acid (n=21) Age, years 32 ± 3 33 ± 3.2 Ethnicity, n (%) European 11 (50) 12 (57) South, East, & Southeast Asian 5 (22) 5 (24) Hispanic/Latino 4 (18) 2 (10) Middle Eastern 1 (5) 0 (0) Mixed ethnicity 1 (5) 2 (9) Education, n (%) Highschool 1 (5) 0 (0) College 4 (18) 1 (5) Undergraduate 8 (36) 12 (57) Graduate 9 (41) 8 (38) 1Household Income per year, CAD$, n (%) <20,000 1 (5) 0 (0) 20,000-50,000 1 (5) 3 (15) 50,000-100,000 6 (27) 7 (35) >100,000 14 (63) 10 (50) Nulliparous, n (%) 18 (82) 12 (57) 2Dietary folate intake (FFQ), mg DFEs/day 0.52 ± 0.17 0.46 ± 0.15 3Total weeks supplementing 24 ± 3.6 24 ± 5.2 Weeks of gestation at delivery 39 ± 1.5 39 ± 1.6 4Full term (≥37 wks) at delivery, n (%) 20 (91) 19 (90) Results are mean ± SD unless otherwise noted; 1Missing n=1 household income (participant chose not to disclose); 2DFEs from food sources only; 3Total weeks supplementing from baseline (8-21 weeks of gestation) to the milk collection (~1-week postpartum). 4Those who delivered prematurely were between 35-36 weeks of gestation. Abbreviations: DFEs: Dietary folate equivalents; FFQ: food frequency questionnaire The mean ± SD day postpartum at the human milk collection was day 7 ± 3, and frozen milk samples were picked up within 4 days for 93% (n=40) of participants; for n=3 participants, pick-up occurred after 5 days (n=2) and 10 days (n=1). The blood specimen collection (as applicable) took place upon human milk pick-up (except for 1 participant; due to COVID-19 pandemic countermeasures, the blood draw occurred 10 days after a contactless human milk pick-up). Most participants collected the human milk specimen from the right breast (88%) and between 1300-89 1450 hours (84%). Most participants fully expressed their breast upon milk collection (67%) and most used an electric pump (77%). However, rates of fully expressing were 50% (n=11) and 86% (n=18) in the (6S)-5-MTHF and folic acid groups, respectively. The median (IQR) hours of maternal folate supplementation prior to the human milk and blood collections, respectively, were 2.25 hours (2 to 3 hrs) and 2 hours (2 to 2.5 hrs); n=2 (1 in each group) did not record the timing of supplementation prior to the human milk collection. 4.4.2 Human milk folate profile at 1 week postpartum Biochemical outcomes are summarized in Table 4.2. Mean levels of total milk folate were not significantly different between groups, with a high degree of variability in the crude estimates. Further, no significant differences were found between any of the reduced folate forms in the human milk specimens (5-MTHF, THF, 5,10-methylTHF, 5-formylTHF) or MeFox. Conversely, the median concentration of milk UMFA in those supplemented with folic acid was 11 nmol/L (adjusted 95% CI = 6.4-17 nmol/L) higher than in those supplemented with (6S)-5-MTHF. 90 Table 4-2 Human milk folate profile following supplementation with (6S)-5-MTHF or folic acid (6S)-5-MTHF Crude concentrations Folic acid Crude concentrations Crude difference (95% CI) 1Adjusted difference (95% CI) Human milk (n=43) Participants, n 22 21 -- -- UMFA (nmol/L) 0.6 (0.3, 1.1) 12 (9.9, 18) 11 (6.5, 16) 11 (6.4, 17) 5-MTHF (nmol/L) 29 (17, 36) 21 (18, 27) -7.2 (-16, 1.5) -9 (-20, 1.8) THF (nmol/L) 13 (7.8, 20) 12 (8.0, 24) -1.3 (-11, 8) -0.8 (-9.4, 7.9) 5,10-methenylTHF (nmol/L) 2.1 (1.2, 2.6) 1.6 (1.2, 3.3) -0.5 (-1.7, 0.8) -0.6 (-1.8, 0.6) 5-formylTHF (nmol/L) 0.7 (0.5, 1.2) 0.9 (0.5, 1.1) 0.1 (-0.4, 0.6) 0.3 (-0.2, 0.8) MeFox (nmol/L) 0.6 (0.5, 0.8) 0.6 (0.5, 0.8) 0.02 (-0.2, 0.2) 0.07 (-0.1, 0.3) 2Total folate (nmol/L) 45 (29, 63) 47 ± 20 53 (40, 70) 61 ± 28 13 (-1.8, 28) 13 (-2.9, 29) Postpartum maternal plasma (n=36) Participants, n 17 19 -- -- UMFA (nmol/L) 0.8 (0.7, 1.2) 13 (7, 23) 12 (5.9, 19) -- 5-MTHF (nmol/L) 72 (64, 96) 74 ± 27 68 (53, 84) 71 ± 24 -2.6 (-20, 15) -- Results are median (IQR); mean ± SD is also presented if normally distributed. 1Milk folate forms and total folate adjusted for: supplemental folate form ([6S]-5-MTHF as the reference group), milk expression (partial expression as the reference group), total weeks of supplementing, dietary folate intake (mg dietary folate equivalents/d). 2Total folate = sum of all forms. Abbreviations: 5-MTHF: 5-methyltetrahydrofolate; THF: tetrahydrofolate; UMFA: unmetabolized folic acid. Full multivariable linear/quantile regression model outputs are presented in Table 4.3. 91 Table 4-3 Full multivariable regression model outputs for adjusted differences in human milk folate profile between groups Human milk folate form β-coefficient (95% CI) UMFA (nmol/L) Supplemental folate group (folic acid) 11 (6.4, 17) Milk expression (full expression) -0.6 (-6, 4.9) Total weeks of supplementing 0.02 (-0.6, 0.6) Dietary folate (mg DFE/day) -0.9 (-17, 15) 5-MTHF (nmol/L) Supplemental folate group (folic acid) -9 (-20, 1.8) Milk expression (full expression) 2.8 (-8.7, 14) Total weeks of supplementing 0.5 (-0.7, 1.7) Dietary folate (mg DFE/day) 0.4 (-33, 34) THF (nmol/L) Supplemental folate group (folic acid) -0.8 (-9.4, 7.9) Milk expression (full expression) 1.3 (-8, 11) Total weeks of supplementing 0.5 (-0.5, 1.5) Dietary folate (mg DFE/day) 3.5 (-23, 30) 5,10-methenylTHF (nmol/L) Supplemental folate group (folic acid) -0.6 (-1.8, 0.6) Milk expression (full expression) -0.3 (-1.6, 1) Total weeks of supplementing 0.04 (-0.1, 0.2) Dietary folate (mg DFE/day) 1.4 (-2.4, 5.1) 5-formylTHF (nmol/L) Supplemental folate group (folic acid) 0.3 (-0.2, 0.8) Milk expression (full expression) -0.2 (-0.7, 0.4) Total weeks of supplementing 0.04 (-0.01, 0.1) Dietary folate (mg DFE/day) 0.7 (-0.8, 2.2) MeFox (nmol/L) Supplemental folate group (folic acid) 0.07 (-0.1, 0.3) Milk expression (full expression) -0.1 (-0.3, 0.07) Total weeks of supplementing 0.02 (-0.002, 0.04) Dietary folate (mg DFE/day) 0.6 (0.04, 1.3) 1Total folate (nmol/L) Supplemental folate group (folic acid) 13 (-2.9, 29) Milk expression (full expression) -0.8 (-18, 16) Total weeks of supplementing 1.9 (0.09, 3.8) Dietary folate (mg DFE/day) 4.1 (-46, 54) n=43 Milk folate forms and total folate adjusted for: supplemental folate form ([6S]-5-MTHF as the reference group), milk expression (partial expression as the reference group), total weeks of supplementing, dietary folate intake (mg dietary folate equivalents/d). 1Total folate = sum of all forms. Abbreviations: 5-MTHF: 5-methyltetrahydrofolate; DFE: Dietary folate equivalents; UMFA: unmetabolized folic acid; THF: tetrahydrofolate. 92 Differences in the human milk folate profile are further visualized in Figure 4.2; mean ± SD proportion of milk UMFA as part of total milk folate was 2% (±2%) and 28% (±14%) in (6S)-5-MTHF and folic acid groups, respectively. Figure 4-2 Human milk folate profile among 43 participants supplemented with (6S)-5-MTHF or folic acid “5-MTHF” includes: 5-MTHF and MeFox (an oxidation product of 5-MTHF); “Other reduced folates” include: THF, 5,10-methenyl-THF, and 5-formyl-THF. Abbreviations: 5-MTHF: 5-methyl-tetrahydrofolate; THF: tetrahydrofolate; UMFA: unmetabolized folic acid 93 4.4.3 Association of UMFA in maternal plasma and human milk Concentrations of UMFA in maternal plasma were positively associated with human milk UMFA (β-coefficient = 0.5 nmol/L, 95% CI = 0.3-0.7 nmol/L); maternal plasma UMFA explained ~40% of the variability in human milk UMFA (pseudo R2 = 0.4); see Figure 4.3. Figure 4-3 Association of maternal plasma UMFA and human milk UMFA among 36 participants at 1 week postpartum A two-way linear prediction scatterplot. “A”: (6S)-5-MTHF (n=17) and “B”: folic acid (n=19). Abbreviations: UMFA: unmetabolized folic acid 4.5 Discussion & conclusions This investigation provides evidence that the human milk folate profile can be altered by maternal folate supplementation; at a dose of 0.6 mg/day, folic acid resulted in significantly higher UMFA in human milk as compared to (6S)-5-MTHF. While 5-MTHF has historically been regarded the predominant folate form in human milk,80,81 this may be shifting in the modern 94 era where most pregnant individuals in Canada165 and the United States178 supplement with folic acid-containing prenatal multivitamins and consume folic acid fortified foods.8 The effect of folic acid supplementation on human milk UMFA was fairly congruent with previous evidence, and further supports that the impact of maternal folic acid supplementation on the human milk folate profile is dose-dependent. In the current study, UMFA represented ~28% of total milk folate following supplementation with 0.6 mg/day folic acid; in other reports, UMFA represented ~8% of total human milk folate following a lower dose (0.4 mg/day)88, and ~40-50% following higher doses (0.75 mg/day89 and >0.4 mg/day82). Of note, participants of the MIREC cohort82 reported folic acid dose consumed in the past 30 days prior to milk sampling and were classified as consuming >0.4 mg/day; it was later reported that most MIREC participants consumed 1.0 mg/day,54 given available prenatal vitamins on the Canadian market. This may explain why the proportion of UMFA in those supplementing with >0.4 mg/day was higher (~50%) as compared to the current findings at 0.6 mg/day (~28%) and West et al.89 at 0.75 mg/day (~40%). Conversely, milk UMFA following (6S)-5-MTHF supplementation (~2% proportion) was lower than previous similar reports. In studies where participants reported no supplemental folate of any form, the proportion of UMFA was ~23%82 and ~18%89; in a randomized trial of 0.4 mg/day (6S)-5-MTHF or placebo, UMFA represented ~8% of total milk folate in both groups.88 Reasons for higher human milk UMFA noted in previous studies are unknown, but may include differences in the study design (self-reported folic acid supplementation82,89 versus clinical trial participation88), assay for the quantification of folate forms, or the human milk 95 collection/processing methods. Further, maternal plasma UMFA and human milk UMFA were positively associated in the current study; with folic acid intake, UMFA increases in plasma, followed by clearance over the next several hours.44 Thus, if folic acid-containing foods were consumed habitually, particularly in the hours prior to the milk collection, it may have contributed to an increase in milk UMFA, despite no folic acid supplementation. Previous evidence suggests that UMFA may be incorporated into human milk at the expense of 5-MTHF.82 In the current trial, median milk 5-MTHF concentrations were 7.2 nmol/L lower in those supplementing with folic acid as compared to (6S)-5-MTHF, despite similar concentrations of maternal plasma 5-MTHF in each group; while the 95% CI for the difference in human milk 5-MTHF between groups crossed zero, non-significance may have been due to a lack of power, given that differences in crude estimates appear meaningful with minimal overlap in IQRs. The underlying mechanism may include a stronger affinity of FRα for UMFA as compared to reduced forms. It is worth noting that once UMFA is incorporated into human milk via binding to FBP, it likely cannot be cleared via mechanisms observed in plasma (e.g., increased metabolism to 5-MTHF or excretion). It has been hypothesized that UMFA in human milk could negatively impact infant folate status.82,266 Folates in human milk are bound to soluble FBP (a cleavage product of the mammary epithelial-associated FRα).34,267 In vitro studies report that UMFA remains more tightly bound to soluble FBP than 5-MTHF during gastric passage, reducing the bioavailability of UMFA upon ingestion.266,268,269 However, evidence from suckling rats note that the folic acid-FBP complex can attach at the intestinal brush-border for absorption without dissociation.270 Further, the 96 human intestine is permeable in early life, enabling the absorption of proteins.271 In adults, UMFA is cleared from plasma via increased metabolism and excretion50,272; the rate at which this occurs in infants is not established, but UMFA has been detected in plasma and feces of breastfed infants.79,273 Increased folate excretion due to an influx of UMFA into circulation may be an alternative mechanism by which infant folate status is impacted. Ultimately, the biological and clinical consequences of the capacity to absorb protein-bound folate and the subsequent influx of UMFA into infant circulation remains unclear. Current studies for the effect of folic acid intake on human health have produced mixed results and fail to demonstrate a causal association with UMFA specifically. However, it is feasible that continuous UMFA exposure while breastfeeding may influence folate-related metabolism due to competition with reduced folates for transporters, binding, and folate-related enzymes,33,176 and it is hypothesized that the potential adverse effects of UMFA may be proportional to the level of exposure.33 Elucidating the role of maternal folic acid supplementation and UMFA in predisposition to future health and disease risk has been deemed a research priority by experts in the field of folate and perinatal nutrition.33,163 Limitations of this investigation include the small sample size, which was powered to generate estimates of folate status during pregnancy. Assessment of the association between human milk UMFA and maternal plasma UMFA may be limited by sample collection on separate days; however, it is unlikely that this significantly biased results, as human milk and blood collections occurred within ≤4 days for almost all participants, with standardized folate supplementation prior to each collection. It is possible that the difference in milk UMFA between groups would 97 have been less apparent if folate supplementation had not occurred 2 hours prior to the milk collection; however, our findings represent the impact of folic acid consumption on the human milk folate profile when maternal plasma UMFA may be at its peak. Finally, human milk folate concentrations increase with advancing lactation, with lowest concentrations in colostrum, peaking at ~2-3 months postpartum81,274,275; thus, generalizability of our results (transition milk) to other studies examining human milk sampled at different stages (colostrum or mature milk) may be limited. As human milk may serve as the sole source of nutrition for infants, it is imperative to understand factors which impact its composition. While others have shown that supplemental folic acid at recommended doses (0.4 mg/day) did not increase concentrations of human milk UMFA as compared to (6S)-5-MTHF or a placebo,88 we demonstrate that at higher doses (0.6 mg/day), there was a differential impact. While the downstream effects of elevated UMFA are unclear, current practices of folic acid supplementation may be shifting the “normal” human milk folate profile, given that commercially available prenatal multivitamins contain higher than recommended doses (0.6-1.0 mg). Our results suggest that reduction of UMFA in human milk may be effectively achieved by supplementation with (6S)-5-MTHF as opposed to folic acid, or by lowering the content of folic acid in prenatal vitamins to the recommended dose (0.4 mg). In the interim, re-evaluation of the human milk folate profile and infant folate status in the post-fortification era, and prospective monitoring to determine any associations with adverse outcomes in childhood and beyond, should be prioritized. 98 Chapter 5: Conclusion, Discussion, & Future Research Directions This section includes further discussions of the key findings from a pilot trial of (6S)-5-MTHF versus folic acid supplementation during pregnancy and lactation. The overall strengths and limitations are further discussed. This is followed by a discussion of the overarching significance to the field of perinatal nutrition, concluding with a discussion of future research directions to advance this field, including suggestions for a future definitive trial. 5.1 Discussion of key findings 5.1.1 Effect of (6S)-5-MTHF and folic acid on RBC and serum folate levels In this pilot study, we did not find a statistically significant or clinically meaningful difference in maternal RBC and serum folate concentrations following supplementation with 0.6 mg/day folic acid or 0.625 mg/day (6S)-5-MTHF. However, to conclude that (6S)-5-MTHF is non-inferior to folic acid in increasing maternal blood folate status during pregnancy, the upper bound of the 95% CI for the difference between treatment groups should be within the boundaries of a non-inferiority margin.276 A non-inferiority margin should be chosen at the study design stage and represent a potentially clinically important difference276; as this was a pilot study, a non-inferiority margin was not pre-specified in our protocol. While there is no published cut-off which represents a clinically important difference in RBC folate concentrations, 300 nmol/L may be a suitable non-inferiority margin for the definitive trial. A margin of 300 nmol/L would be a reasonable and conservative estimate, given that RBC folate status among pregnant Canadians is generally high, but with concentrations reported as low as ~1200 nmol/L in the first trimester (APrON trial174). A difference of 300 nmol/L from this estimate (1200 nmol/L) could equate to 99 levels of ~900 nmol/L, a cut-off of clinical importance during pregnancy (RBC folate >906 nmol/L is recommended to reduce the risk of NTDs). In this pilot trial, the adjusted mean difference in RBC folate concentrations between groups was 161 nmol/L; as per the aforementioned post-hoc non-inferiority margin of 300 nmol/L, this point estimate suggests that (6S)-5-MTHF is non-inferior to folic acid in increasing RBC folate concentrations during pregnancy. However, the upper end of the 95% CI for the adjusted difference between groups at endline was 400 nmol/L; thus, using a 300 nmol/L margin, we cannot rule out the possibility of a stronger response following folic acid supplementation. We hypothesize that with a larger sample size, the 95% CI for the difference between groups would tighten around the null, confirming a similar effect of both folate forms at a 0.6 mg dose; this is based on the fact that the 95% CIs were wide and crossed zero, there was high degree of variability overall in crude estimates (which would likely become less variable with a larger sample size), and considering the body of evidence on the dose-response to folic acid versus (6S)-5-MTHF (which generally reports a stronger effect of (6S)-5-MTHF).181,187,193 Ultimately, this further substantiates that a definitive trial is needed to confirm the effect of each folate form at a 0.6 mg dose on maternal folate status during pregnancy. The overall change in blood folate status during pregnancy and after delivery generally coincided with estimates among folate-supplement users reported in other Canadian studies.54,97,174,277 Overall, RBC folate concentrations increased throughout pregnancy, while serum folate concentrations remained mostly consistent. After delivery, RBC folate concentrations tended to decrease, while serum folate concentrations increased (likely driven by the non-fasting state). 100 However, a high degree of variability for within-person change in folate status (RBC and serum folate levels) was observed in the current trial. We note that the change in folate status may have been influenced by an overall high folate status in our study population; a greater change may be expected as baseline status decreases.7 Thus, a greater effect size following supplementation with folic acid or (6S)-5-MTHF in earlier pregnancy, when status is likely lower, may occur. However, kinetics of RBC folate plateau following supplementation is not well-described, with previous studies reporting that steady state is not achieved following folic acid supplementation at 0.14 and 1.0 mg/day x 40 weeks in non-pregnant women,278 and 1.0 and 5.0 mg/day x 40-50 weeks during pregnancy.279 5.1.2 High maternal folate status Across the full study group, a “high” maternal folate status was apparent (with RBC folate concentrations nearing ~2000 nmol/L by endline visits). Similar high blood folate levels have been reported in other Canadian pregnancy cohorts (~1.5-2.5-fold the cut-off for NTD risk reduction: RBC folate = 906 nmol/L), regardless of folate supplementation practices.60,174 It is difficult to characterize the clinical significance of these levels, as a cut-off for high folate status is not established.168 Further, use of various analytical methods for folate quantification limits the comparison across studies. While ensuring that RBC folate concentrations are >906 nmol/L is important preconceptionally and in the first trimester (for neural tube closure), it is unclear whether RBC folate maintenance >906 nmol/L provides any additional benefit in later pregnancy, or if prevention of deficiency is sufficient (>305 nmol/L). Overall, folate needs remain increased throughout the duration of pregnancy and lactation, and declining plasma folate 101 concentrations have been observed when there is a lack of supplementation55–57; however, does this matter, so long as deficiency is prevented? While continued supplementation with 0.4 mg/day folic acid starting preconceptionally until the end of lactation is based on the premise that folate requirements remain increased as compared to the non-pregnant state, it is very difficult for pregnant individuals in North America to consume only 0.4 mg/day folic acid, due to folic acid food fortification and as the vast majority of prenatal vitamins contain higher doses of folic acid.54,163,172,173 There is some evidence that supplement manufacturers are becoming more aware of excessive doses of folic acid in prenatal supplements; Materna® reduced the folic acid content in their prenatal vitamin from 1.0 mg to 0.6 mg in 2018, given concerns raised by experts in the field.163 However, several prenatal vitamins in Canada still contain 1.0 mg.54 Whether supplementation is beneficial beyond the first trimester in countries with folic acid food fortification is unclear, and has recently been questioned.280 There is some evidence that at recommended folic acid doses (0.4 mg/day), continuation of folic acid supplementation throughout all of pregnancy is beneficial for offspring development.281,282 In the Folic Acid Supplementation in the Second and Third Trimester trial (FASST), pregnant women in Ireland (a country with voluntary folic acid food fortification) were randomized to 0.4 mg/day folic acid (n=59) or a placebo (n=60) starting in the 2nd trimester.283 In FASST follow-up studies, improved cognition at 3 years (Bayley’s Scale of Infant and Toddler Development), 7 years (Wechsler Preschool and Primary Scale of Intelligence), and 11 years (Wechsler Intelligence Scale for Children, magnetoencephalographic brain imaging) was reported in children whose mothers 102 were randomized to supplementation with 0.4 mg/day folic acid as compared to a placebo throughout the 2nd and 3rd trimesters.281,282 Ultimately, while this investigation concludes with support for continued folic acid supplementation throughout pregnancy (at recommended dosing; 0.4 mg/day), relevance to countries with mandatory folic acid fortification and prenatal vitamins which contain >0.4 mg/day is unclear. A better understanding of optimal maternal folate status (balancing potential benefits and risks) throughout pregnancy is required and would aid in the establishment of cut-offs during pregnancy which are associated with beneficial health outcomes. RBC folate concentrations (quantified via microbiological assay) among FASST participants at baseline (14 weeks’ gestation) were ~1150 nmol/L (SD ± ~700 nmol/L); by 36 weeks’ gestation, RBC folate concentrations were ~1750 nmol/L (SD ± ~700 nmol/L) in the folic acid group and ~900 nmol/L (SD ± ~400 nmol/L) in the placebo group.283 However, “optimal status” is difficult to ascertain from the estimates obtained in the FASST trial, given a high degree of variability in RBC folate concentrations in both intervention groups. Finally, “optimal status” may include the identification of windows of opportunity, in which a higher or lower maternal folate status during pregnancy is particularly beneficial or harmful for offspring development. 5.1.3 Increased plasma UMFA due to folic acid supplementation Results of this pilot study suggest that concentrations of plasma UMFA are altered by supplemental folate form, with higher plasma UMFA in those who supplement with folic acid. Almost all participants (>95%) were folic acid supplement users prior to study initiation and the median (IQR) plasma UMFA concentrations at baseline were similar in both groups: 1.2 nmol/L 103 (0.8, 1.5) and 1.1 nmol/L (0.9, 1.5) in (6S)-5-MTHF and folic acid groups, respectively. In those randomized to folic acid supplementation, the median (IQR) UMFA concentration stayed relatively consistent at endline (1.3 nmol/L; 0.9, 2.1), whereas concentrations in those who were randomized to (6S)-5-MTHF decreased by ~50% (median, IQR = 0.5 nmol/L; 0.4, 0.8). At endline, this equated to a mean ± SD proportion of UMFA as part of total serum folate of 2.4% (±1.8%) and 0.9% (±0.4%) in the folic acid and (6S)-5-MTHF groups, respectively. Concentrations at baseline and endline were quantified following a 3-hour fast; this time frame was chosen to mitigate the influence of folic acid intake on plasma UMFA43,44,53 and to increase study feasibility (minimizing the burden of fasting on pregnant participants). However, as per NHANES 2007-2008, UMFA concentrations of those who fasted for <3 hours or 3 to ≤8 hours were significantly higher than in those who fasted for >8 hours, in both folic acid supplement users and non-users.47 Thus, it is possible that concentrations of plasma UMFA (in both intervention groups) would have continued to decrease following a longer fast. The importance of fasting state on concentrations of plasma UMFA is further highlighted in our study design, in which postpartum visits were non-fasting with folate supplementation ~2-hours prior to the blood collection. Median (IQR) plasma UMFA concentrations at postpartum visits were 0.8 nmol/L (0.7, 1.2) and 13 nmol/L (7, 23) in (6S)-5-MTHF and folic acid groups, respectively; as compared to the values obtained at endline, we estimate that ~0.3 nmol/L and ~12 nmol/L of UMFA was cleared from plasma following the 3-hour fast. Thus, greater absolute clearance is observed when concentrations of plasma UMFA are increased. A similar comparison was made using data from NHANES 2007-200847,49; non-fasting plasma UMFA 104 concentrations in folic acid supplement users versus non-users were 2.1 nmol/L and 0.9 nmol/L, respectively; as compared to concentrations in those who fasted ≥8 hours, a clearance of ~1.2 nmol/L and ~0.3 nmol/L was estimated in each group, respectively.47,49 Of note, plasma UMFA concentrations as per NHANES 2007-2008 remained significantly higher (P <0.0001) in folic acid supplement users (~1 nmol/L) versus non-users (~0.7 nmol/L) after fasting.47 It does not appear that increased doses of folic acid result in increased residual levels of UMFA in plasma after fasting. In a non-fasted state, higher doses of folic acid result in higher concentrations of plasma UMFA, as observed in an ancillary study of the Folic Acid Clinical Trial284; following randomization to ~5 mg/day or ~1 mg/day folic acid in early pregnancy, median (IQR) non-fasting plasma UMFA concentrations at 24-26 week’s gestation were 4.6 nmol/L (2.5, 34) and 1.9 nmol/L (0.9, 4.1), respectively.284 Alternatively, fasting concentrations of plasma UMFA were evaluated in a study of non-pregnant women randomized to 5.0 or 1.1 mg/day folic acid51; no significant differences in plasma UMFA were found between groups after 6, 12, and 30 weeks of supplementation, and while plasma UMFA increased after 12 weeks in both groups, concentrations by week 30 were similar to baseline.51 Thus, the mechanisms which clear UMFA from plasma seem to increase as UMFA concentrations (and folic acid doses) rise, particularly after long term supplementation. 5.1.4 Human milk folate profile We observed markedly differential effects of supplemental folate form on the human milk folate profile at a 0.6 mg/day dose of (6S)-5-MTHF versus folic acid. While the significance of folate forms present in human milk for future offspring health is unknown, it is important to understand 105 modifiable factors which influence human milk composition. Ultimately, our findings build on landmark evidence from Houghton et al.88 and Page et al.82 regarding the effect of folic acid dose on human milk folate profile and concentrations of human milk UMFA. Average concentrations of total folate in human transition milk (~2-14 days postpartum) are not very well described; however, the concentrations we observed (~50-60 nmol/L) were similar to a recent cohort study in China which reported human milk folate via LC-MS/MS from days 1-15 postpartum.83 Although the AI for infants 0-6 months assumes total folate concentrations of almost 200 nmol/L in human milk,22 it is reported that folate levels are low in colostrum, increasing until ~2-3 months postpartum.80 We suspect that concentrations of total milk folate in our study group continued to increase over the coming weeks, as total milk folate concentrations are homeostatically regulated and are maintained at the expense of maternal nutrition status.80,81 Finally, it remains unclear whether MeFox (an oxidation product of 5-MTHF) is naturally present in human milk82; it is possible that the low levels observed in the human milk specimens of the current trial (median = 0.6 nmol/L) were due to sample processing and/or storage. There remain many unknowns regarding the underlying mechanism of folate transportation from maternal plasma into human milk; of note for the current trial is the impact of folate supplementation 2 hours prior to the human milk collection (and an overall non-fasting state) on the resultant human milk folate profile. We opted to have participants supplement ~2 hours prior to the milk collection, to observe the impact of folic acid versus (6S)-5-MTHF supplementation on the human milk folate profile when UMFA may be at its peak in maternal plasma.44,53 The impact of this timing on resultant human milk UMFA concentrations is unknown, but we suspect 106 that it increased UMFA transfer in the folic acid group. However, West et al.89 quantified human milk UMFA after 10 weeks of supplementation with 0.75 mg/day folic acid as part of a controlled feeding trial; human milk specimens were collected after an overnight fast, yet UMFA represented ~40% of total milk folate.89 In the current trial, concentrations of maternal plasma UMFA explained ~40% of the variability in human milk UMFA (pseudo R2=0.4); ultimately, other factors which influence the transfer of UMFA into human milk (perhaps concentrations of FBP) warrant further investigation. Once incorporated into human milk, folate forms are not thought to undergo further metabolism or interconversions, as they are bound to soluble FBP.85 This means that the UMFA incorporated into human milk can only undergo metabolism once ingested by the infant, the rate of which is not well described. Infant folate status, including plasma UMFA, was evaluated in a recent trial of healthy term infants who were randomized at ~1 month of age to formula containing (6S)-5-MTHF (n=91) or folic acid (n=95); a reference group of breastfed infants (n=112) was also included.273 Blood draws took place ≥3 hours after the last feed. Plasma UMFA concentrations at ~3.5 months of age were significantly higher in infants who received folic acid formula (median, IQR = 1.2 nmol/L; 0.92, 1.4) than in the (6S)-5-MTHF formula group (median, IQR = 0.7 nmol/L; 0.6, 1.0) or the breastfed group (median, IQR = 0.7 nmol/L; 0.6, 0.9)273; however, UMFA was only detectable in ~40% of the infants overall (n=124). While plasma UMFA in the breastfed infants was presumably derived from maternal folic acid intake, the origin of plasma UMFA detected in those fed the (6S)-5-MTHF-containing formula is unclear. However, there was some mixed feeding reported (e.g., occasional human milk provided to infants in the 107 formula intervention groups) and it is noted that “great care was taken by the study team to promote and protect breastfeeding”.273 Interestingly, concentrations of plasma UMFA reported in infants from the above trial273 were comparable to those that we observed in maternal plasma at baseline and endline in the current trial. While metabolism should not be inferred from plasma levels alone, perhaps UMFA is cleared in infant plasma at a similar rate as observed in adults, given that the fasting time prior to UMFA quantification was consistent in both studies (≥3 hours). Of note, while the differences in plasma UMFA between the infant groups (folic acid versus (6S)-5-MTHF containing formula or human milk) were statistically significant, absolute concentrations remained low overall273 (similar to maternal levels in our pilot trial). Ultimately, the biological implications of increased plasma UMFA concentrations, by any degree, in both infants and adults remains unclear. 5.2 Strengths & limitations Strengths of this research program include the rigorous study design: a double-blind, randomized clinical trial, with provision of folic acid or (6S)-5-MTHF at a 0.6 mg dose (currently used in common prenatal vitamin formulations in Canada). We observed very high adherence to daily supplementation (median rate = supplementation on 98% of the study days). We had planned for an 80% retention rate, with the aim of generating reliable estimates of maternal folate status from minimally 50 participants; in practice, our retention rate was 90% (54/60) following 16 weeks of supplementation. Following randomization, groups were generally equal in terms of demographic characteristics, weeks of gestation at study enrollment, and total weeks of supplementing. We used gold standard analytical methods to quantify RBC and serum folate 108 concentrations (microbiological assay) which improves the comparison of our findings to pregnancy-related cut-offs: RBC folate >906 nmol/L for risk reduction of NTDs (also determined via microbiological assay). Further we quantified numerous other related biological outcomes, including plasma total homocysteine, vitamin B12, free choline and betaine; again, while metabolism cannot be inferred by these measures alone, a lack of differences in these outcomes between groups further supports the comparable nature of the two folate forms in their effect on maternal folate status during pregnancy. Overall, this pilot study has provided essential data to inform the design of an adequately powered definitive trial. Limitations for our research aims were previously outlined in Chapters 3 and 4; some final thoughts on the overarching limitations of the trial are discussed here. First, substantive findings have limited interpretability given the small sample size and pilot study design; thus, results must be confirmed in a definitive trial. However, we did reach the target sample size for the purpose of the pilot trial, thus in that sense, the sample size is not a limitation. Some of the strengths and limitations associated with the methods for dietary assessment and the use of an FFQ were previously discussed in Chapter 3; to expand, the FFQ used (the Block Folic Acid/Dietary Folate Equivalents Screener; NutritionQuest) is an American screener which was developed using the top food contributors to dietary folate as per NHANES 1999-2000. Both countries generally fortify foods with folic acid at similar rates (0.14 mg and 0.15 mg per 100 g white flour and cereals in the United States and Canada, respectively), aiming to provide ~0.1 mg/day.285 However, ready-to-eat cereals are permitted to be fortified with up to 0.4 mg folic acid in the United States.286 While dietary patterns of those in Canada versus the United States may differ, green salads, orange juice, bread products, and cold cereals are reported as top folate contributors 109 for pregnant and lactating Canadians,172,287 all of which are included on the NutritionQuest folate screener. A separate issue is that the folic acid content in fortified foods may actually be much higher than the fortification rate or what is listed on the label.11 Thus, an accurate assessment of dietary folate intake, regardless of the tool used, is limited. 5.3 Significance & contributions of the research Although (6S)-5-MTHF is approved for use in prenatal vitamins, its effect on maternal folate status as compared to folic acid has never been rigorously evaluated during pregnancy. The ability to maintain adequate blood folate concentrations during pregnancy is critical, given increased requirements to support growth in maternal, placental, and fetal tissue.55 Despite data on the use of (6S)-5-MTHF in non-pregnant adults, which has shown that it is equally or more effective than folic acid (depending on the dose provided),181 pregnancy is a unique physiological state; thus, confirmation of its effectiveness in this population directly is valuable. Overall, this gap in perinatal nutrition knowledge has led to the current situation, in which pregnant Canadians are currently supplementing with (6S)-5-MTHF, despite no evidence-based guidance from public health agencies. This problem is exacerbated by the disconnect between current guidelines (which only recommend folic acid, given its proven association with a reduced risk of NTDs159,160), and the reality that prenatal vitamins available for purchase can contain either (6S)-5-MTHF or folic acid (but may state simply “folate” on the label).175 While there was no evidence for a statistically significant difference in effect of (6S)-5-MTHF and folic acid on maternal RBC and serum folate concentrations at a 0.6 mg/day dose, 110 confirmation of non-inferiority requires investigation in an adequately powered definitive trial, particularly given the variability observed in our estimates and wide 95% CIs for the differences between groups. The main difference between folate forms was in relation to UMFA concentrations, which were significantly higher in both maternal plasma and human milk following supplementation with folic acid as compared to (6S)-5-MTHF; again, results should be confirmed in a definitive trial. While the impact of UMFA on human health outcomes is unclear and controversial, it has been proposed that any negative consequences may exhibit a dose-dependent effect.33 Thus, even if absolute concentrations remain low, there may be a benefit to reducing UMFA by as much as possible. At this time, implementation of the precautionary principle for the impact of excess folic acid intake and UMFA has been proposed.163,176 This means that the scientific uncertainty about unintended adverse health effects associated with a public health measure is acknowledged, while continuing to prioritize research which can fill said uncertainty gaps. 5.4 Future research directions The natural next step for this research is the implementation of a definitive trial. As the impact of (6S)-5-MTHF on maternal folate levels has never been evaluated in a clinical trial among pregnant individuals in Canada, it was uncertain if women would be willing to participate, or what concerns they may have about the study design. Successful completion of this pilot trial provides proof-of-concept of the feasibility for conducting a definitive trial; further, there were many lessons learned which can be used to improve the feasibility and rigour of a future definitive trial. 111 First, the pilot study was intentionally designed with a wide range of gestational weeks included in the eligibility criteria (between 8-21 weeks of gestation). The low end of this range (8 weeks) was deemed long enough after neural tube closure (3-4 weeks) to reduce any risk of harm should (6S)-5-MTHF prove less effective in pregnancy; the higher end (21 weeks) was chosen to increase the eligibility range as much as possible, without nearing too close to delivery after 16 weeks of supplementation. The level of risk that a premature delivery would occur was deemed acceptable to enhance recruitment feasibility. We had one participant who enrolled at 21 weeks of gestation and delivered prior to their endline visit (at 35 weeks of gestation). The average gestational weeks upon initial contact was 13 ± 5 weeks (reported by n=251 prospective participants upon inquiry about the trial; Appendix B).242 For a definitive trial, this data can be used to inform a feasible range of gestational weeks for eligibility, which is perhaps less wide to prevent missing outcome data due to a premature delivery and to ensure a more homogenous starting point for supplementation. Reasons for declining to participate were recorded as available to help reduce potential barriers for future recruitment. Some potential participants raised concerns related to the study supplements. These included a preference for either (6S)-5-MTHF or folic acid only, preference for a chewable vitamin option, preference (or recommendation from a doctor) for a higher dose of folate (1.0 to 5.0 mg), and unwilling to change their current supplementation regimen. These are interesting insights, but are difficult to address for a definitive trial, as a double-blind, randomized approach is essential for scientific rigour. While a preference for a higher dose of folate was only reported by a small number of inquiries (n=5), it is possible that others also had this concern but did not report it, as many individuals who inquired did not disclose a reason for 112 not enrolling (n=279). This is concerning, particularly if recommended by a physician, as there is mounting evidence that excessive maternal folic acid intake (>1.0 mg/day) is associated with adverse pregnancy outcomes.33,163,176 Further, regardless of NTD risk classification, a dose of 0.4 mg/day folic acid is recommended from 12 weeks of gestation onward.142 Perhaps further public health campaigns targeted at health care professionals in family medicine, obstetrics, and midwifery are needed to ensure that folate supplementation recommendations are aligned with current evidence; this may include written editorials in practitioner-focused journals or presentations at clinician meetings. Other data which will be useful for informing the design and feasibility of a definitive trial includes the rate of recruitment, compliance estimates, and the expected rate of loss to follow-up. We were able to recruit 4 participants/week via use of social media and printed posters (Appendix B).242 However, it is noted that this rate was negatively impacted by the COVID-19 pandemic.242 Without this barrier, a recruitment rate of 5-6 participants/week is likely attainable. We observed very high compliance (mostly >90%) to daily supplementation as per capsule counts; similar rates could be expected in a definitive trial. The retention rate was 90% (54/60) for the primary phase of the study (16 weeks of supplementation during pregnancy); for the postpartum phase, retention was 80% for the human milk collection (43/54) and 69% for the postpartum blood collection (37/54). While the blood collection occurred upon pick-up of the human milk specimen for most participants, there was one participant who opted to provide a blood specimen only. For a definitive trial, later timing of the postpartum visit should be considered (e.g., perhaps at 4 weeks postpartum as opposed to 1 113 week postpartum). While timing of this visit in the pilot study (1 week postpartum) was limited by the number of remaining folate capsules for continued supplementation, a visit so soon after delivery is likely more difficult for parents than at ~1 month (or later) postpartum. Of note, most participants were interested in providing a human milk specimen, with 91% (n=49 of 54) initially indicating willingness to continue; however, n=6 opted out after delivery due to either a reported difficult labour or difficulty breastfeeding. We speculate that retention would be higher for the human milk collections if visits were held further from delivery. Estimates from the pilot study which could be used to calculate the sample size for an adequately powered future trial include endline concentrations of RBC folate, serum folate, and plasma UMFA, which represent the effect of supplementation with 0.6 mg/day folic acid or (6S)-5-MTHF x 16 weeks during pregnancy. Two separate research objectives could be addressed in a definitive trial: 1) to establish non-inferiority of supplementation with (6S)-5-MTHF versus folic acid at a 0.6 mg/day dose on maternal folate status during pregnancy (RBC and/or serum folate), and 2) to determine if supplementation with (6S)-5-MTHF reduces UMFA (in maternal plasma, human milk, and potentially other biological fluids such as umbilical cord blood) as compared to folic acid. Addressing these two research gaps would be a starting point for understanding differences of these folate forms on maternal folate status during pregnancy; as many Canadians and other pregnant women globally choose to supplement with (6S)-5-MTHF, it is imperative to ensure that recommendations with this form are evidenced-based. Establishing the effect of (6S)-5-MTHF versus folic acid on maternal folate status will also aid in the development of a conversion factor for (6S)-5-MTHF to DFEs that is appropriate for a 114 pregnant population. This is necessary to ensure adequate dose recommendations in perinatal nutrition public health guidelines and to ensure that doses are accurately listed on supplement packaging for prenatal vitamins. As previously discussed, the EFSA has started this work,181 and suggested a conversion factor of 1.7 and 2 in doses ≤0.4 mg and >0.4 mg, respectively. However, this report did not include any studies during pregnancy, and again, results from the current trial suggest that (6S)-5-MTHF is not more effective than folic acid in increasing maternal folate status at a 0.6 mg dose. Measures evaluating differences in folate metabolism could be considered for a definitive trial. This may include quantification of urinary folate catabolites (pABG and apABG) following supplementation with (6S)-5-MTHF versus folic acid. Placental folate stores and placental folate-related metabolism may also be of interest, given a high degree of folate transfer from maternal plasma, followed by active 1C metabolism in the placenta, and the proposition that a high degree of UMFA may undergo reduction in the placenta prior to fetal transfer.65,77 These aims would be best explored via the use of stable isotopes, which could quantify changes in absorption, distribution, metabolism, and excretion of each supplemental folate form.288 Various clinical outcomes associated with maternal folate supplementation may be considered. Maternal folate status is a significant predictor of infant birth weight289,290; while folic acid supplementation at recommended doses (0.4 mg/day) is associated with a reduced risk of small-for-gestational-age births,291,292 intakes >1.0 mg have been associated with an increased risk.293 In a cohort of 786 pregnant women (Spain), the rate of weight and height <10th percentile for sex (using Spanish standardized growth reference charts) was 16.7% and 11.5%, respectively, in 115 those who reported folic acid supplementation in doses >1 mg/day, as compared to 9.5% and 4.6% in those who reported no folic acid supplementation.293 Thus, measures of fetal growth and birth weight may be of interest. In childhood, measures of cognitive testing could build on evidence from the FASST trial, to determine whether improved cognition is observed in the Canadian context, or whether the effect is further differentiated by folate form (folic acid versus (6S)-5-MTHF) or dose (0.6 mg/day versus 0.4 mg/day). Finally, given the mounting evidence of a high folate status among pregnant women in Canada,54,60 a follow-up trial may consider inclusion of a placebo group, alongside (6S)-5-MTHF and folic acid arms, starting after neural tube closure; this design would help disentangle any biological or clinical effects of UMFA versus a high folate status on adverse pregnancy outcomes, and would help to establish whether continued folate supplementation is warranted past the first trimester in the Canadian context.116 References 1. Shane B. Folate and vitamin B12 metabolism: Overview and interaction with riboflavin, vitamin B6, and polymorphisms. Food Nutr Bull. 2008;29(2):S5–16. 2. Shane B. Folate Chemistry and Metabolism. In: Bailey LB, editor. Folate in Health and Disease. 2nd ed. Taylor & Francis; 2009. p. 1–24. 3. Hanson AD, Gregory JF. Synthesis and turnover of folates in plants. Curr Opin Plant Biol. 2002;5:244–9. 4. Gropper S, Smith J, Groff J. Chapter 9 Water-Soluble Vitamins. In: Advanced Nutrition and Human Metabolism. 5th Ed. Belmont, CA: Wadsworth Cengage Learning; 2009. p. 348–69. 5. Zheng Y, Cantley LC. Toward a better understanding of folate metabolism in health and disease. J Exp Med. 2019;216(2):253–66. 6. Lucock M. Folic acid: Nutritional biochemistry, molecular biology, and role in disease processes. Mol Genet Metab. 2000;71(1–2):121–38. 7. Pietrzik K, Bailey L, Shane B. Folic acid and L-5-methyltetrahydrofolate: Comparison of clinical pharmacokinetics and pharmacodynamics. Clin Pharmacokinet. 2010;49(8):535–48. 8. Garrett GS, Bailey LB. A public health approach for preventing neural tube defects: Folic acid fortification and beyond. Ann N Y Acad Sci. 2018;1414:47–58. 9. De Wals P, Tairou F, Van Allen M, Uh S, Lowry B, Sibbald B, et al. Reduction in neural-tube defects after folic acid fortification in Canada. N Engl J Med. 2007;357:135–42. 10. Ami N, Bernstein M, Boucher F, Rieder M, Parker L. Folate and neural tube defects: The role of supplements and food fortification. Paediatr Child Heal. 2016 Apr 1;21(3):145–9. 117 11. Shakur YA, Rogenstein C, Hartman-Craven B, Tarasuk V, O’Connor DL. How much folate is in Canadian fortified products 10 years after mandated fortification? Can J Public Heal. 2009;100(4):281–4. 12. O’Connor EM. The role of gut microbiota in nutritional status. Curr Opin Clin Nutr Metab Care. 2013;16:509–16. 13. Kok DE, Steegenga WT, Smid EJ, Zoetendal EG, Ulrich CM, Kampman E. Bacterial folate biosynthesis and colorectal cancer risk: More than just a gut feeling. Crit Rev Food Sci Nutr. 2020;60(2):244–56. 14. Magnúsdóttir S, Ravcheev D, De Crécy-Lagard V, Thiele I. Systematic genome assessment of B-vitamin biosynthesis suggests cooperation among gut microbes. Front Genet. 2015;6:148. 15. Engevik MA, Morra CN, Röth D, Engevik K, Spinler JK, Devaraj S, et al. Microbial metabolic capacity for intestinal folate production and modulation of host folate receptors. Front Microbiol. 2019;10:2305. 16. O’Keefe SJD, Ou J, Aufreiter S, O’Connor D, Sharma S, Sepulveda J, et al. Products of the colonic microbiota mediate the effects of diet on colon cancer risk. J Nutr. 2009;139:2044–8. 17. Aufreiter S, Kim JH, O’Connor DL. Dietary oligosaccharides increase colonic weight and the amount but not concentration of bacterially synthesized folate in the colon of piglets. J Nutr. 2011;141:366–72. 18. Malinowska AM, Schmidt M, Kok DE, Chmurzynska A. Ex vivo folate production by fecal bacteria does not predict human blood folate status: Associations between dietary patterns, gut microbiota, and folate metabolism. Food Res Int. 2022;156:111290. 118 19. Caudill MA. Folate bioavailability: Implications for establishing dietary recommendations and optimizing status. Am J Clin Nutr. 2010;91:1455S-60S. 20. Zhao R, Matherly L, Goldman D. Membrane transporters and folate homeostasis; Intestinal absorption, transport into systemic compartments and tissues. Expert Rev Mol Med. 2009;11:e4. 21. McNulty H, Pentieva K. Folate bioavailability. Proc Nutr Soc. 2004;63:529–36. 22. Institute of Medicine, Food and Nutrition Board. “Folate” in Dietary Reference intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Washington, DC, USA; 1998. 23. Visentin M, Diop-Bove N, Zhao R, Goldman D. The intestinal absorption of folates. Annu Rev Physiol. 2014;76:251–74. 24. Aufreiter S, Gregory JF, Pfeiffer CM, Fazili Z, Kim YI, Marcon N, et al. Folate is absorbed across the colon of adults: Evidence from cecal infusion of 13C-labeled [6S]-5-formyltetrahydrofolic acid. Am J Clin Nutr. 2009;90:116–23. 25. Lakoff A, Fazili Z, Aufreiter S, Pfeiffer CM, Connolly B, Gregory JF, et al. Folate is absorbed across the human colon: Evidence by using enteric-coated caplets containing 13C-labeled [6S]-5-formyltetrahydrofolate. Am J Clin Nutr. 2014;100:1278–86. 26. Cochrane KM, Mayer C, Devlin AM, Elango R, Hutcheon JA, Karakochuk CD. Is natural (6S)-5-methyltetrahydrofolic acid as effective as synthetic folic acid in increasing serum and red blood cell folate concentrations during pregnancy? A proof-of-concept pilot study. Trials. 2020;21:380. 27. Shafizadeh TB, Halsted CH. γ-glutamyl hydrolase, not glutamate carboxypeptidase II, hydrolyzes dietary folate in rat small intestine. J Nutr. 2007;137(5):1149–53. 119 28. Chen L, Qi H, Korenberg J, Garrow TA, Choi YJ, Shane B. Purification and properties of human cytosolic folylpoly-γ-glutamate synthetase and organization, localization, and differential splicing of its gene. J Biol Chem. 1996;271(22):13077–87. 29. Zhao R, Diop-Bove N, Visentin M, Goldman ID. Mechanisms of membrane transport of folates into cells and across epithelia. Annu Rev Nutr. 2011;31:177–201. 30. Whetstine JR, Flatley RM, Matherly LH. The human reduced folate carrier gene is ubiquitously and differentially expressed in normal human tissues: Identification of seven non-coding exons and characterization of a novel promoter. Biochem J. 2002;367:629–40. 31. Chancy CD, Kekuda R, Huang W, Prasad PD, Kuhnel JM, Sirotnak FM, et al. Expression and differential polarization of the reduced-folate transporter-1 and the folate receptor α in mammalian retinal pigment epithelium. J Biol Chem. 2000;275(27):20676–84. 32. Kamen BA, Smith AK. A review of folate receptor alpha cycling and 5-methyltetrahydrofolate accumulation with an emphasis on cell models in vitro. Adv Drug Deliv Rev. 2004;56:1085–97. 33. Maruvada P, Stover PJ, Mason JB, Bailey RL, Davis CD, Field MS, et al. Knowledge gaps in understanding the metabolic and clinical effects of excess folates/folic acid: A summary, and perspectives, from an NIH workshop. Am J Clin Nutr. 2020;112(5):1390–403. 34. Holm J, Hansen SI. Ligand binding characteristics of two molecular forms, one equipped with a hydrophobic glycosyl phosphatidylinositol tail, of the folate binding protein purified from human milk. Biosci Rep. 2002;22(3–4):455–63. 35. Antony AC. Folate receptors. Ann Rev Nutr. 1996;16:501–21. 36. Hoppner K, Lampi B. Folate levels in human liver from autopsies in Canada. Am J Clin 120 Nutr. 1980;33(4):862–4. 37. Lawrence SA, Titus SA, Ferguson J, Heineman AL, Taylor SM, Moran RG. Mammalian mitochondrial and cytosolic folylpolyglutamate synthetase maintain the subcellular compartmentalization of folates. J Biol Chem. 2014;289(42):29386–96. 38. Lin Y, Dueker SR, Follett JR, Fadel JG, Arjomand A, Schneider PD, et al. Quantitation of in vivo human folate metabolism. Am J Clin Nutr. 2004;80:680–91. 39. Wolfe JM, Bailey LB, Herrlinger-Garcia K, Theriaque DW, Gregory JF, Kauwell GPA. Folate catabolite excretion is responsive to changes in dietary folate intake in elderly women. Am J Clin Nutr. 2003;77:919–23. 40. Caudill MA, Gregory JF, Hutson AD, Bailey LB. Folate catabolism in pregnant and nonpregnant women with controlled folate intakes. J Nutr. 1998;128(2):204–8. 41. Gregory JF, Williamson J, Liao JF, Bailey LB, Toth JP. Kinetic model of folate metabolism in nonpregnant women consuming [2H2]folic acid: Isotopic labeling of urinary folate and the catabolite para-acetamidobenzoylglutamate indicates slow, intake-dependent, turnover of folate pools. J Nutr. 1998;128(11):1896–906. 42. Patanwala I, King MJ, Barrett DA, Rose J, Jackson R, Hudson M, et al. Folic acid handling by the human gut: Implications for food fortification and supplementation. Am J Clin Nutr. 2014;100:593–9. 43. Bailey SW, Ayling JE. The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake. PNAS. 2009;106(36):15424–9. 44. Sweeney MR, McPartlin J, Weir DG, Daly L, Scott JM. Postprandial serum folic acid response to multiple doses of folic acid in fortified bread. Br J Nutr. 2006;95(1):145–51. 121 45. Sweeney MR, Mcpartlin J, Scott J. Folic acid fortification and public health: Report on threshold doses above which unmetabolised folic acid appear in serum. BMC Public Health. 2007;7(41):1–7. 46. Kelly P, McPartlin J, Goggins M, Weir DG, Scott JM. Unmetabolized folic acid in serum: Acute studies in subjects consuming fortified food and supplements. Am J Clin Nutr. 1997;65:1790–5. 47. Pfeiffer CM, Sternberg MR, Fazili Z, Yetley EA, Lacher DA, Bailey RL, et al. Unmetabolized folic acid is detected in nearly all serum samples from US children, adolescents, and adults. J Nutr. 2015;145:520–31. 48. Pfeiffer CM, Sternberg MR, Fazili Z, Lacher DA, Zhang M, Johnson CL, et al. Folate status and concentrations of serum folate forms in the US population: National Health and Nutrition Examination Survey 2011-2. Br J Nutr. 2015;113(12):1965–77. 49. Obeid R. Serum unmetabolized folic acid: The straw that broke dihydrofolate reductase’s back? J Nutr. 2015;387–90. 50. de Meer K, Smulders YM, Dainty JR, Smith DEC, Kok RM, Stehouwer CDA, et al. [6S]5-methyltetrahydrofolate or folic acid supplementation and absorption and initial elimination of folate in young and middle-aged adults. Eur J Clin Nutr. 2005;59(12):1409–16. 51. Tam C, O’Connor D, Koren G. Circulating unmetabolized folic acid: Relationship to folate status and effect of supplementation. Obstet Gynecol Int. 2012;(485179):1–17. 52. Palchetti CZ, Paniz C, de Carli E, Marchioni DM, Colli C, Steluti J, et al. Association between serum unmetabolized folic acid concentrations and folic acid from fortified foods. J Am Coll Nutr. 2017;36(7):572–8. 122 53. Bailey RL, Mills JL, Yetley EA, Gahche JJ, Pfeiffer CM, Dwyer JT, et al. Unmetabolized serum folic acid and its relation to folic acid intake from diet and supplements in a nationally representative sample of adults aged ≥60 y in the United States. Am J Clin Nutr. 2010;92:383–9. 54. Patti MA, Braun JM, Arbuckle TE, Macfarlane AJ. Associations between folic acid supplement use and folate status biomarkers in the first and third trimesters of pregnancy in the Maternal – Infant Research on Environmental Chemicals (MIREC) Pregnancy Cohort Study. Am J Clin Nutr. 2022;116(6):1852–63. 55. Tamura T, Picciando MF. Folate and human reproduction. Am J Clin Nutr. 2006;83:993–1016. 56. Cikot RJLM, Steegers-Theunissen RPM, Thomas CMG, de Boo TM, Merkus HMWM, Steegers EAP. Longitudinal vitamin and homocysteine levels in normal pregnancy. Br J Nutr. 2001;85:49–58. 57. Milman N, Byg KE, Hvas AM, Bergholt T, Eriksen L. Erythrocyte folate, plasma folate and plasma homocysteine during normal pregnancy and postpartum: A longitudinal study comprising 404 Danish women. Eur J Haematol. 2006;76:200–5. 58. Solanky N, Requena Jimenez A, D’Souza SW, Sibley CP, Glazier JD. Expression of folate transporters in human placenta and implications for homocysteine metabolism. Placenta. 2010;31:134–43. 59. Obeid R, Munz W, Jäger M, Schmidt W, Herrmann W. Biochemical indexes of the B vitamins in cord serum are predicted by maternal B vitamin status. Am J Clin Nutr. 2005;82(1):133–9. 60. Plumptre L, Masih SP, Ly A, Aufreiter S, Sohn K-J, Croxford R, et al. High 123 concentrations of folate and unmetabolized folic acid in a cohort of pregnant Canadian women and umbilical cord blood. Am J Clin Nutr. 2015;102:848–57. 61. Carlin A, Alfirevic Z. Physiological changes of pregnancy and monitoring. Best Pract Res Clin Obstet Gynaecol. 2008;22(5):801–23. 62. McPartlin J, Weir D, Halligan A, Darling M, Scott J. Accelerated folate breakdown in pregnancy. Lancet. 1993;341:148–9. 63. Higgins J, Quinlivin E, McPartlin J, Scott J, Weir D, Darling M. The relationship between increased folate catabolism and the increased requirement for folate in pregnancy. Br J Obstet Gynaecol. 2001;108(7):772–3. 64. Cochrane KM, Williams BA, Elango R, Barr SI, Karakochuk CD. Pregnancy-induced alterations of 1-carbon metabolism and significance for maternal nutrition requirements. Nutr Rev. 2022;80(9):1985–2001. 65. Tamura T, Picciano M, McGuire M. Folate in Pregnancy and Lactation. In: Bailey LB, editor. Folate in Health and Disease. 2nd ed. Taylor & Francis; 2009. p. 111–31. 66. Bruinse HW, Berg H v.d., Haspels AA. Maternal serum folacin levels during and after normal pregnancy. Eur J Obs Gynec reprod Biol. 1985;20(3):153–8. 67. Upton A, Smelt V, Mushtaq A, Aplin R, Johnson N, Mardon H, et al. Placental arylamine N-acetyltransferase type 1: Potential contributory source of urinary folate catabolite p-acetamidobenzoylglutamate during pregnancy. Biochim Biophys Acta. 2000;1524:143–8. 68. Gregory JF, Caudill MA, Jeffery Opalko F, Bailey LB. Kinetics of folate turnover in pregnant women (second trimester) and nonpregnant controls during folic acid supplementation: Stable-isotopic labeling of plasma folate, urinary folate and folate catabolites shows subtle effects of pregnancy on turnover of . J Nutr. 2001;131:1928–37. 124 69. Baker H, Frank O, Deangelis B, Feingold S, Kaminetzky H. Role of placenta in maternal-fetal vitamin transfer in humans. Am J Obs Gynecol. 1981;141:792–6. 70. Suliburska J, Kocyłowski R, Grzesiak M, Gaj Z, Chan B, Von Kaisenberg C, et al. Evaluation of folate concentration in amniotic fluid and maternal and umbilical cord blood during labor. Arch Med Sci. 2019;15(6):1425–32. 71. Obeid R, Kasoha M, Kirsch SH, Munz W, Herrmann W. Concentrations of unmetabolized folic acid and primary folate forms in pregnant women at delivery and in umbilical cord blood. Am J Clin Nutr. 2010;92(6):1416–22. 72. Piedrahita JA, Oetama B, Bennett GD, Van Waes J, Kamen BA, Richardson J, et al. Mice lacking the folic acid-binding protein Folbp1 are defective in early embryonic development. Nat Genet. 1999;23:228–32. 73. Henderson G, Perez T, Schenker S, Mackins J, Antony A. Maternal-to-fetal transfer of 5-methyltetrahydrofolate by the perfused human placental cotyledon: Evidence for a concentrative role by placental folate receptors in fetal folate delivery. J Lab Clin Med. 1995;126:184–203. 74. Bisseling TM, Steegers EAP, van den Heuvel JJM, Siero HLM, van de Water FM, Walker AJ, et al. Placental folate transport and binding are not impaired in pregnancies complicated by fetal growth restriction. Placenta. 2004;25:588–93. 75. Utley CS, Marcell PD, Allen RH, Antony AC, Kolhouse JF. Isolation and characterization of methionine synthetase from human placenta. J Biol Chem. 1985;260(25):13656–65. 76. Daly SF, Molloy AM, Mills JL, Less YJ, Conley M, Kirke PN, et al. The influence of 5,10 methylenetetrahydrofolate reductase genotypes on enzyme activity in placental tissue. Br J Obs Gynaecol. 1999;106:1214–8. 125 77. Landon MJ. Transfer of folate to the fetus. Br J Obs Gynaecol. 1975;82:12–9. 78. Sweeney MR, McPartlin J, Weir DG, Daly S, Pentieva K, Daly L, et al. Evidence of unmetabolised folic acid in cord blood of newborn and serum of 4-day-old infants. Br J Nutr. 2005;94:727–30. 79. Kim TH, Yang J, Darling PB, O’Connor DL. A large pool of available folate exists in the large intestine of human infants and piglets. J Nutr. 2004;134(6):1389–94. 80. Dror DK, Allen LH. Overview of nutrients in human milk. Adv Nutr. 2018;9(23):278S-294S. 81. Allen LH. B vitamins in breast milk: Relative importance of maternal status and intake, and effects on infant status and function. Adv Nutr. 2012;3:362–9. 82. Page R, Robichaud A, Arbuckle TE, Fraser WD, MacFarlane AJ. Total folate and unmetabolized folic acid in the breast milk of a cross-section of Canadian women. Am J Clin Nutr. 2017;105(5):1101–9. 83. Su Y, Mao Y, Tian F, Cai X, Chen R, Li N, et al. Profile of folate in breast milk from Chinese women over 1-400 days postpartum. Nutrients. 2022;14:1–11. 84. Brown C, Smith A, Picciano M. Forms of human milk folacin and variation patterns. J Pediatr Gastroenterol Nutr. 1986;5(2):278–82. 85. Selhub J, Arnold R, Smith AM, Picciano MF. Milk folate binding protein (FBP): A secretory protein for folate? Nutr Res. 1984;4(2):181–7. 86. Han YH, Yon M, Han HS, Kim KY, Tamura T, Hyun TH. Folate contents in human milk and casein-based and soya-based formulas, and folate status in Korean infants. Br J Nutr. 2009;101:1769–74. 87. Udipi S, Kirksey A, Roepke J. Diurnal variations in folacin levels of human milk: Use of a 126 single sample to represent folacin concentration in milk during a 24-h period. Am J Clin Nutr. 1987;45:770–9. 88. Houghton LA, Yang J, O’Connor DL. Unmetabolized folic acid and total folate concentrations in breast milk are unaffected by low-dose folate supplements. Am J Clin Nutr. 2009;89(1):216–20. 89. West AA, Yan J, Perry CA, Jiang X, Malysheva O V, Caudill MA. Folate-status response to a controlled folate intake in nonpregnant, pregnant, and lactating women. Am J Clin Nutr. 2012;96(4):789–800. 90. Metz J. Folate deficiency conditioned by lactation. Am J Clin Nutr. 1970;23(6):843–7. 91. Smith A, Picciano M, Deering R. Folate supplementation during lactation: Maternal folate status, human milk folate content, and their relationsihp to infant folate status. J Pediatr Gastroenterol Nutr. 1983;2(4):622–8. 92. Tamura T, Yoshimura Y, Arakawa T. Human milk folate and folate status in lactating mothers and their infants. Am J Clin Nutr. 1980;33:193–7. 93. Picciano MF, West SG, Ruch AL, Kris-Etherton PM, Zhao G, Johnston KE, et al. Effect of cow milk on food folate bioavailability in young women. Am J Clin Nutr. 2004;80:1565–9. 94. Jones ML, Nixon PF. Tetrahydrofolates are greatly stabilized by binding to bovine milk folate-binding protein. J Nutr. 2002;132(9):2690–4. 95. Pfeiffer CM, Sternberg MR, Zhang M, Fazili Z, Storandt RJ, Crider KS, et al. Folate status in the US population 20 y after the introduction of folic acid fortification. Am J Clin Nutr. 2019;110:1088–97. 96. Colapinto CK, O’Connor DL, Tremblay MS. Folate status of the population in the 127 Canadian Health Measures Survey. CMAJ. 2011;183(2):E100–6. 97. Houghton LA, Sherwood KL, Pawlosky R, Ito S, O’Connor DL. [6S]-5-Methyltetrahydrofolate is at least as effective as folic acid in preventing a decline in blood folate concentrations during lactation. Am J Clin Nutr. 2006;83:842–50. 98. Clare CE, Brassington AH, Kwong WY, Sinclair KD. One-carbon metabolism: Linking nutritional biochemistry to epigenetic programming of long-term development. Annu Rev Anim Biosci. 2019;7:263–87. 99. Ducker G, Rabinowitz J. One-carbon metabolism in liver health and disease. Cell Metab. 2017;25:27–49. 100. Tibbetts AS, Appling DR. Compartmentalization of mammalian folate-mediated one-carbon metabolism. Annu Rev Nutr. 2010;30:57–81. 101. Guéant JL, Namour F, Guéant-Rodriguez RM, Daval JL. Folate and fetal programming: A play in epigenomics? Trends Endocrinol Metab. 2013;24(6):279–89. 102. Leung KY, Pai YJ, Chen Q, Santos C, Calvani E, Sudiwala S, et al. Partitioning of one-carbon units in folate and methionine metabolism is essential for neural tube closure. Cell Rep. 2017;21:1795–808. 103. Robinson JL, Bertolo RF. The pediatric methionine requirement should incorporate remethylation potential and transmethylation demands. Adv Nutr. 2016;7:523–34. 104. Petrossian TC, Clarke SG. Uncovering the human methyltransferasome. Mol Cell Proteomics. 2011;10:1–12. 105. Law JA, Jacobsen SE. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet. 2010;11(3):204–20. 106. Randunu RS, Bertolo RF. The effects of maternal and postnatal dietary methyl nutrients 128 on epigenetic changes that lead to non-communicable diseases in adulthood. Int J Mol Sci. 2020;21:1–17. 107. Finkelstein JD, Martin JJ. Methionine metabolism in mammals. Distribution of homocysteine between competing pathways. J Biol Chem. 1984;259(15):9508–13. 108. Glier MB, Green TJ, Devlin AM. Methyl nutrients, DNA methylation, and cardiovascular disease. Mol Nutr Food Res. 2014 Jan 1;58(1):172–82. 109. Yetley EA, Pfeiffer CM, Phinney KW, Fazili Z, Lacher DA, Bailey RL, et al. Biomarkers of folate status in NHANES: A roundtable summary. Am J Clin Nutr. 2011;94(suppl):303S-12S. 110. Bailey LB, Stover PJ, McNulty H, Fenech MF, Gregory JF, Mills JL, et al. Biomarkers of nutrition for development-Folate review. J Nutr. 2015;145:1636S-1680S. 111. Hopkins SM, Gibney MJ, Nugent AP, McNulty H, Molloy AM, Scott JM, et al. Impact of voluntary fortification and supplement use on dietary intakes and biomarker status of folate and vitamin B-12 in Irish adults. Am J Clin Nutr. 2015;101:1163–72. 112. Yeung L, Yang Q, Berry RJ. Contributions of total daily intake of folic acid to serum folate concentrations. JAMA. 2008;300(21):2486–7. 113. Pietrzik K, Lamers Y, Brämswig S, Prinz-Langenohl R. Calculation of red blood cell folate steady state conditions and elimination kinetics after daily supplementation with various folate forms and doses in women of childbearing age. Am J Clin Nutr. 2007;86:1414–9. 114. Wu A, Chanarin I, Slavin G, Levi AJ. Folate deficiency in the alcoholic—its relationship to clinical and haematological abnormalities, liver disease and folate stores. Br J Haematol. 1975;29:469–78. 129 115. Pfeiffer C, Fazili Z, Zhang M. Folate Analytical Methodology. In: Bailey LB, editor. Folate in Health and Disease. 2nd ed. Taylor & Francis; 2009. p. 517–74. 116. Gregory JF. Chemical and nutritional aspects of folate research: Analytical procedures, methods of folate synthesis, stability, and bioavailability of dietary folates. Adv Food Nutr Res. 1989;33:1–101. 117. De Brouwer V, Zhang GF, Storozhenko S, Van Der Straeten D, Lambert WE. pH stability of individual folates during critical sample preparation steps in prevision of the analysis of plant folates. Phytochem Anal. 2007;18:496–508. 118. Hannisdal R, Ueland PM, Eussen S, Svardal A, Hustad S. Analytical recovery of folate degradation products formed in human serum and plasma at room temperature. J Nutr. 2009;139:1415–8. 119. Fazili Z, Whitehead RD, Paladugula N, Pfeiffer CM. A high-throughput LC-MS/MS method suitable for population biomonitoring measures five serum folate vitamers and one oxidation product. Anal Bioanal Chem. 2013;405(13):4549–60. 120. Pfeiffer CM, Zhang M, Jabbar S. Framework for laboratory harmonization of folate measurements in low-and-middle-income countries and regions. Ann NY Acad Sci. 2018;1414(1):96–108. 121. Fazili Z, Paladugula N, Zhang M, Pfeiffer CM. Folate forms in RBC and whole-blood lysates appear stable when stored frozen for 2 years. J Nutr. 2021;151(9):2852–60. 122. Fazili Z, Sternberg MR, Pfeiffer CM. Assessing the influence of 5,10-methylenetetrahydrofolate reductase polymorphism on folate stability during long-term frozen storage, thawing, and repeated freeze/thawing of whole blood. Clin Chim Acta. 2012;413:966–72. 130 123. Drammeh BS, Schleicher RL, Pfeiffer CM, Jain RB, Zhang M, Phuong HN. Effects of delayed sample processing and freezing on serum concentrations of selected nutritional indicators. Clin Chem. 2008;54:1883–91. 124. Pirkle J. CDC Laboratory Procedure Manual, Total Folate, Serum/Whole Blood Microbiological Assay. 2012. 125. Lim HS, Mackey AD, Tamura T, Wong SC, Picciano MF. Measurable human milk folate is increased by treatment with α-amylase and protease in addition to folate conjugase. Food Chem. 1998;63(3):401–7. 126. Hyun TH, Tamura T. Trienzyme extraction in combination with microbiologic assay in food folate analysis: An updated review. Exp Biol Med. 2005;230:444–54. 127. Zhang M, Sternberg MR, Pfeiffer CM. Harmonizing the calibrator and microorganism used in the folate microbiological assay increases the comparability of serum and whole-blood folate results in a CDC round-robin study. J Nutr. 2018;148(5):807–17. 128. Hunt SE, Netting MJ, Sullivan TR, Best KP, Houghton LA, Makrides M, et al. Red blood cell folate likely overestimated in australian national survey: Implications for neural tube defect risk. Nutrients. 2020;12(1283):1–6. 129. Molloy AM, Scott JM. Microbiological assay for serum, plasma, and red cell folate using cryopreserved, microtiter plate method. Methods Enzym. 1997;281:43–53. 130. Pfeiffer CM, Zhang M, Lacher DA, Molloy AM, Tamura T, Yetley EA, et al. Comparison of serum and red blood cell folate microbiologic assays for national population surveys. J Nutr. 2011;141(7):1402–9. 131. Pfeiffer C, Fazili Z, McCoy L, Zhang M, Gunter E. Determination of folate vitamers in human serum by stable-isotope-dilution tandem mass spectrometry and comparison with 131 radioassay and microbiologic assay. Clin Chem. 2004;50(2):423–32. 132. Fazili Z, Pfeiffer CM. Accounting for an isobaric interference allows correct determination of folate vitamers in serum by isotope dilution liquid chromatography-tandem MS. J Nutr. 2013;143:108–13. 133. Fazili Z, Pfeiffer C. Measurement of folates in serum and conventionally prepared whole blood lysates: Application of an automated 96-well plate isotope-dilution tandem mass spectrometry method. Clin Chem. 2004;50(12):2378–81. 134. Stamm RA, Harper MJ, Houghton LA. Quantitation of whole-blood total folate within defined MTHFR C677T genotype groups by isotope dilution-liquid chromatography-tandem mass spectrometry differs from microbiologic assay. J Nutr. 2012;142(12):2154–60. 135. Fazili Z, Pfeiffer CM, Zhang M, Jain R. Erythrocyte folate extraction and quantitative determination by liquid chromatography-tandem mass spectrometry: Comparison of results with microbiologic assay. Clin Chem. 2005;51(12):2318–25. 136. Stamm RA, Fazili Z, Pfeiffer CM. Addition of exogenous γ-glutamyl hydrolase eliminates the need for lengthy incubation of whole-blood lysate for quantitation of folate vitamers by high-performance liquid chromatography-tandem mass spectrometry. Curr Dev Nutr. 2018;2:nx003. 137. World Health Organization. Serum and red blood cell folate concentrations for assessing folate status in populations. Vitam Miner Nutr Inf Syst. 2015;1–7. 138. Pfeiffer CM, Sternberg MR, Hamner HC, Crider KS, Lacher DA, Rogers LM, et al. Applying inappropriate cutoffs leads to misinterpretation of folate status in the US population. Am J Clin Nutr. 2016;104:1607–15. 132 139. Daly LE, Kirke P, Molloy A, Weir D, Scott J. Folate levels and neural tube defects. JAMA. 1995;274(21):1698–702. 140. Tinker S, Hamner H, Qi Y, Crider K. U.S. women of childbearing age who are at possible risk of a neural tube defect-affected pregnancy due to suboptimal red blood cell folate concentrations, National Health and Nutrition Examination Survey 2007 to 2012. Birth Defects Res A Clin Mol Teratol. 2015;103(6):517–26. 141. Chen M, Rose C, Qi YP, Williams J, Yeung LF, Berry RJ, et al. Defining the plasma folate concentration associated with the red blood cell folate concentration threshold for optimal neural tube defects prevention: A population-based, randomized trial of folic acid supplementation. Am J Clin Nutr. 2019;109:1452–61. 142. Douglas Wilson R, O’Connor DL. Guideline No. 427: Folic acid and multivitamin supplementation for prevention of folic acid–sensitive congenital anomalies. J Obstet Gynaecol Canada. 2022;44(6):707–19. 143. Douglas Wilson R, O’Connor DL. Maternal folic acid and multivitamin supplementation: International clinical evidence with considerations for the prevention of folate-sensitive birth defects. Prev Med Reports. 2021;24:101617. 144. Christensen K, Rozen R. Genetic Variation: Effect on folate metabolism and health. In: Bailey LB, editor. Folate in Health and Disease. 2nd ed. Taylor & Francis; 2009. p. 75–110. 145. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, et al. A candidate genetic risk factor for vascular disease: A common mutation in methylnetetrahydrofolate reductase. Nat Genet. 1995;10:111–3. 146. Amorin M, Lima M, Castilla E, Orioli I. Non-Latin European descent could be a 133 requirement for association of NTDs and MTHFR variant 677C>T: A meta-analysis. Am J Med Genet. 2007;143A:1726–32. 147. Ogino S, Wilson RB. Genotype and haplotype distributions of MTHFR 677C>T and 1298A>C single nucleotide polymorphisms: A meta-analysis. J Hum Genet. 2003;48:1–7. 148. Gueant-Rodriguez R, Gueant J-L, Debard R, Thirion S, Hong L, Bronowicki J, et al. Prevalence of methylenetetrahydrofolate reductase 677T and 1298C alleles and folate status: A comparitive study in Mexican, West African, and European populations. Am J Clin Nutr. 2006;83:701–7. 149. Botto L, Yang Q. 5,10-Methylenetetrahydrofolate reductase gene variants and congenital anomalies: A HuGE review. Am J Epidemiol. 2000;151(9):862–77. 150. Wilcken B, Bamforth F, Li Z, Zhu H, Ritvanen A, Redlund M, et al. Geographical and ethnic variation of the 677C>T allele of 5,10 methylenetetrahydrofolate reductase (MTHFR): Findings from over 7000 newborns from 16 areas world wide. J Med Genet. 2003;40:619–25. 151. Page R, Wong A, Arbuckle TE, Macfarlane AJ. The MTHFR 677C>T polymorphism is associated with unmetabolized folic acid in breast milk in a cohort of Canadian women. Am J Clin Nutr. 2019;110:401–9. 152. Tsang BL, Devine OJ, Cordero AM, Marchetta CM, Mulinare J, Mersereau P, et al. Assessing the association between the methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism and blood folate concentrations: A systematic review and meta-analysis of trials and observational studies. Am J Clin Nutr. 2015;101:1286–94. 153. Shane B, Pangilinan F, Mills JL, Fan R, Gong T, Cropp CD, et al. The 677C→T variant of MTHFR is the major genetic modifier of biomarkers of folate status in a young, healthy 134 Irish population. Am J Clin Nutr. 2018;108:1334–41. 154. Weisberg I, Tran P, Christensen B, Sibani S, Rozen R. A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Mol Genet Metab. 1998;64:169–72. 155. Parle-McDermott A, Mills JL, Kirke PN, O’Leary VB, Swanson DA, Pangilinan F, et al. Analysis of the MTHFR 1298A → C and 677C → T polymorphisms as risk factors for neural tube defects. J Hum Genet. 2003;48:190–3. 156. Health Canada. Dietary Reference Intakes [Internet]. Ottawa, ON, Canada; 2010 [cited 2022 Dec 1]. Available from: https://www.canada.ca/en/health-canada/services/food-nutrition/healthy-eating/dietary-reference-intakes/tables/reference-values-vitamins-dietary-reference-intakes-tables-2005.html 157. Lamers Y. Folate recommendations for pregnancy, lactation, and infancy. Ann Nutr Metab. 2011;59(1):32–7. 158. Beaudin A, Stover P. Insights into metabolic mechanisms underlying folate-responsive neural tube defects: A minireview. Birth Defects Res A Clin Mol Teratol. 2009;85(4):274–84. 159. MRC Vitamin Study Research Group. Prevention of neural tube defects: Results of the Medical Research Council Vitamin Study. Lancet. 1991;338(8760):131–7. 160. Czeizel AE, Dudás I. Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N Engl J Med. 1992;327(26):1832–5. 161. Berry R, Zhu L, Erickson J, Li S, Moore C, Wang H, et al. Prevention of neural-tube defects with folic acid in China. N Engl J Med. 1999;341(20):1485–90. 162. Caudill MA, Cruz AC, Gregory JF, Hutson AD, Bailey LB. Folate status response to 135 controlled folate intake in pregnant women. J Nutr. 1997;127:2363–870. 163. Lamers Y, Macfarlane AJ, O’Connor DL, Fontaine-Bisson B. Periconceptional intake of folic acid among low-risk women in Canada: Summary of a workshop aiming to align prenatal folic acid supplement composition with current expert guidelines. Am J Clin Nutr. 2018;108:1357–68. 164. Douglas Wilson R. SOGC Clinical Practice Guideline: Pre-conception folic acid and multivitamin supplementation for the primary and secondary prevention of neural tube defects and other folic acid-sensitive congenital anomalies. J Obs Gynaecol Can. 2015;37(6):534–49. 165. Public Health Agency of Canada. What Mothers Say: The Canadian Maternity Experiences Survey. 2009. 166. Macfarlane AJ, Greene-finestone LS, Shi Y. Vitamin B-12 and homocysteine status in a folate-replete population: Results from the Canadian Health Measures Survey. Am J Clin Nutr. 2011;94:1079–87. 167. Pfeiffer CM, Johnson CL, Jain RB, Yetley EA, Picciano MF, Rader JI, et al. Trends in blood folate and vitamin B-12 concentrations in the United States, 1988-2004. Am J Clin Nutr. 2007;86:718–27. 168. Colapinto CK, O’Connor DL, Sampson M, Williams B, Tremblay MS. Systematic review of adverse health outcomes associated with high serum or red blood cell folate concentrations. J Public Heal. 2015;38(2):e84–97. 169. Colapinto CK, O’Connor DL, Dubois L, Tremblay MS. Folic acid supplement use is the most significant predictor of folate concentrations in Canadian women of childbearing age. Appl Physiol Nutr Metab. 2012;37:284–92. 136 170. St-Laurent A, Plante A-S, Lemieux S, Robitaille J, Macfarlane AJ, Morisset A-S. Higher than recommended folic acid intakes is associated with high folate status throughout pregnancy in a prospective French-Canadian cohort. J Nutr. 2023;153(5):1347-58. 171. Shi Y, Groh M De, Macfarlane AJ. Socio-demographic and lifestyle factors associated with folate status among non-supplement-consuming Canadian women of childbearing age. Can J Public Heal. 2014;105(3):e166–71. 172. Masih SP, Plumptre L, Ly A, Berger H, Lausman AY, Croxford R, et al. Pregnant Canadian women achieve recommended intakes of one-carbon nutrients through prenatal supplementation but the supplement composition, including choline, requires reconsideration. J Nutr. 2015;145(8):1824–34. 173. Dubois L, Diasparra M, Bedard B, Colapinto C, Fontaine-Bisson B, Morisset A, et al. Adequacy of nutritional intake from food and supplements in a cohort of pregnant women in Québec, Canada: The 3D Cohort Study (Design, Develop, Discover). Am J Clin Nutr. 2017;106(2):541–8. 174. Fayyaz F, Wang F, Jacobs RL, O’Connor DL, Bell RC, Field CJ. Folate, vitamin B12, and vitamin B6 status of a group of high socioeconomic status women in the Alberta Pregnancy Outcomes and Nutrition (APrON) cohort. Appl Physiol Nutr Metab. 2014;39:1402–8. 175. Health Canada. Multi-vitamin/mineral supplements monograph [Internet]. Ottawa, ON, Canada; 2022 [cited 2022 Dec 1]. p. 1–71. Available from: https://webprod.hc-sc.gc.ca/nhpid-bdipsn/atReq.do?atid=multi_vitmin_suppl 176. Pannia E, Hammoud R, Simonian R, Kubant R, Anderson GH. Folate dose and form during pregnancy may program maternal and fetal health and disease risk. Nutr Rev. 137 2022;80(11):2187–97. 177. Yaktine AL. Assessing the Tolerable Upper Intake Level for folic acid: Interpreting the evidence from the DRI perspective. Am J Clin Nutr. 2019;110:550–1. 178. Saldanha LG, Dwyer JT, Haggans CJ, Mills JL, Potischman N. Perspective: Time to resolve confusion on folate amounts, units, and forms in prenatal supplements. Adv Nutr. 2020;11:753–9. 179. Lian Z, Chen H, Liu K, Jia Q, Qiu F, Cheng Y. Improved stability of a stable crystal form C of 6S-5-methyltetrahydrofolate calcium salt, method development and validation of an LC–MS/MS method for rat pharmacokinetic comparison. Molecules. 2021;26:6011. 180. EFSA (European Food Safety Authority). Opinion of the scientific panel on food additives, flavourings, processing aids and materials in contact with food on a request from the commission related to calcium L-methylfolate. EFSA J. 2004;135:1–20. 181. Turck D, Bohn T, Castenmiller J, Henauw S De, Hirsch-ernst KI, Knutsen HK, et al. Conversion of calcium-L-methylfolate and (6S)-5-methyltetrahydrofolic acid glucosamine salt into dietary folate equivalents. EFSA J. 2022;20(8):7452. 182. Khandanpour N, Armon MP, Jennings B, Finglas PM, Willis G, Clark A, et al. Randomized clinical trial of folate supplementation in patients with peripheral arterial disease. Br J Surg. 2009;96:990–8. 183. Wright AJA, King MJ, Wolfe CA, Powers HJ, Finglas PM. Comparison of (6S)-5-methyltetrahydrofolic acid v. folic acid as the reference folate in longer-term human dietary intervention studies assessing the relative bioavailability of natural food folates: Comparative changes in folate status following a 16-week placebo-controlled study in healthy adults. Br J Nutr. 2010;103:724–9. 138 184. Green TJ, Liu Y, Dadgar S, Li W, Böhni R, Kitts DD. Wheat rolls fortified with microencapsulated L-5-methyltetrahydrofolic acid or equimolar folic acid increase blood folate concentrations to a similar extent in healthy men and women. J Nutr. 2013;143:867–71. 185. Bayes J, Agrawal N, Schloss J. A pilot trial examining the absorption of oral forms of folate supplementation in a healthy population: A randomised control trial. Adv Integr Med. 2019;6:51–7. 186. Sicińska E, Brzozowska A, Roszkowski W, Finglas PM. Supplementation with [6S]-5-methyltetrahydrofolate or folic acid equally reduces serum homocysteine concentrations in older adults. Int J Food Sci Nutr. 2018;69(1):64–73. 187. Obeid R, Schön C, Pietrzik K, Menzel D, Wilhelm M, Smulders Y, et al. Pharmacokinetics of sodium and calcium salts of (6S)-5-methyltetrahydrofolic acid compared to folic acid and indirect comparison of the two salts. Nutrients. 2020;12:3623. 188. Venn BJ, Green T, Moser R, Mann JI. Comparison of low-dose supplementation with L-5-methylterahydrofolate or folic acid on plasma homocysteine: A randomized placebo controlled study. Am J Clin Nutr. 2003;77:658–62. 189. Venn BJ, Green TJ, Moser R, Mckenzie JE, Murray Skeaff C, Mann J. Increases in blood folate indices are similar in women of childbearing age supplemented with [6S]-5-methyltetrahydrofolate and folic acid. J Nutr. 2002;132:3353–5. 190. Lamers Y, Prinz-Langenohl R, Moser R, Pietrzik K. Supplementation with [6S]-5-methyltetrahydrofolate or folic acid equally reduces plasma total homocysteine concentrations in healthy women. Am J Clin Nutr. 2004;79:473–8. 191. Lamers Y, Prinz-langenohl R, Bramswig S, Pietrzik K. Red blood cell folate 139 concentrations increase more after supplementation with [6S]-5-methyltetrahydrofolate than with folic acid in women of childbearing age. Am J Clin Nutr. 2006;84:156–61. 192. Henderson AM, Aleliunas RE, Loh SP, Khor GL, Harvey-Leeson S, Glier MB, et al. L-5-methyltetrahydrofolate supplementation increases blood folate concentrations to a greater extent than folic acid supplementation in Malaysian women. J Nutr. 2018;148:855–90. 193. Bailey SW, Ayling JE. The pharmacokinetic advantage of 5-methyltetrahydrofolate for minimization of the risk for birth defects. Sci Rep. 2018;8:4096. 194. Fohr IP, Prinz-Langenohl R, Brönstrup A, Bohlmann AM, Nau H, Berthold HK, et al. 5,10-Methylenetetrahydrofolate reductase genotype determines the plasma homocysteine-lowering effect of supplementation with 5-methyltetrahydrofolate or folic acid in healthy young women. Am J Clin Nutr. 2002;75(2):275–82. 195. Diefenbach K, Trummer D, Ebert F, Lissy M, Koch M, Rohde B, et al. EE-drospirenone-levomefolate calcium versus EE-drospirenone + folic acid: Folate status during 24 weeks of treatment and over 20 weeks following treatment cessation. Int J Womens Health. 2013;5:149–63. 196. Hekmatdoost A, Vahid F, Yari Z, Sadeghi M, Eini-Zinab H, Lakpour N, et al. Methyltetrahydrofolate vs folic acid supplementation in idiopathic recurrent miscarriage with respect to methylenetetrahydrofolate reductase C677T and A1298C polymorphisms: A randomized controlled trial. PLoS One. 2015;10(12):e0143569. 197. Berry RJ. Lack of historical evidence to support folic acid exacerbation of the neuropathy caused by vitamin B12 deficiency. Am J Clin Nutr. 2019;110:554–61. 198. Husebye ESN, Wendel AWK, Gilhus NE, Riedel B, Bjørk MH. Plasma unmetabolized folic acid in pregnancy and risk of autistic traits and language impairment in antiseizure 140 medication-exposed children of women with epilepsy. Am J Clin Nutr. 2022;115:1432–40. 199. Raghavan R, Selhub J, Paul L, Ji Y, Wang G, Hong X, et al. A prospective birth cohort study on cord blood folate subtypes and risk of autism spectrum disorder. Am J Clin Nutr. 2020;112(5):1304–17. 200. Wang S, Wei J, Wang D, Hua L, Pan D, Fu L, et al. The association between folic acid supplementation, maternal folate during pregnancy and intelligence development in infants: A prospective cohort study. Food Sci Hum Wellness. 2021;10:197–204. 201. Levine SZ, Kodesh A, Viktorin A, Smith L, Uher R, Reichenberg A, et al. Association of maternal use of folic acid and multivitamin supplements in the periods before and during pregnancy with the risk of autism spectrum disorder in offspring. JAMA Psychiatry. 2018;75(2):176–84. 202. Surén P, Roth C, Bresnahan M, Haugen M, Hornig M, Hirtz D, et al. Association between maternal use of folic acid supplements and risk of autism in children. JAMA. 2013;309(6):570–7. 203. Schmidt RJ, Tancredi DJ, Ozonoff S, Hansen RL, Hartiala J, Allayee H, et al. Maternal periconceptional folic acid intake and risk of autism spectrum disorders and developmental delay in the CHARGE (CHildhood Autism Risks from Genetics and Environment) case-control study. Am J Clin Nutr. 2012;96:80–9. 204. Egorova O, Myte R, Schneede J, Hägglöf B, Bölte S, Domellöf E, et al. Maternal blood folate status during early pregnancy and occurrence of autism spectrum disorder in offspring: A study of 62 serum biomarkers. Mol Autism. 2020;11:7. 205. McGowan E, Hong X, Selhub J, Paul L, Wood R, Matsui E, et al. Association between 141 folate metabolites and the development of food allergy in children. J Allergy Clin Immunol Pr. 2020;8(1):132–40. 206. Whitrow MJ, Moore VM, Rumbold AR, Davies MJ. Effect of supplemental folic acid in pregnancy on childhood asthma: A prospective birth cohort study. Am J Epidemiol. 2009;170(12):1486–93. 207. Best KP, Green TJ, Sulistyoningrum DC, Sullivan TR, Aufreiter S, Prescott SL, et al. Maternal late-pregnancy serum unmetabolized folic acid concentrations are not associated with infant allergic disease: A prospective cohort study. J Nutr. 2021;151:1553–60. 208. Magdelijns FJH, Mommers M, Penders J, Smits L, Thijs C. Folic acid use in pregnancy and the development of atopy, asthma, and lung function in childhood. Pediatrics. 2011;128(1):e135–44. 209. Molloy J, Collier F, Saffery R, Allen KJ, Koplin JJ, Louise Ponsonby A, et al. Folate levels in pregnancy and offspring food allergy and eczema. Pediatr Allergy Immunol. 2020;31:38–46. 210. Henderson AM, Tai DC, Aleliunas RE, Aljaadi AM, Glier MB, Xu EE, et al. Maternal folic acid supplementation with vitamin B12 deficiency during pregnancy and lactation affects the metabolic health of adult female offspring but is dependent on offspring diet. FASEB J. 2018;32:5039–50. 211. Krishnaveni G V, Veena SR, Yajnik C, Fall C. Association between maternal folate concentrations during pregnancy and insulin resistance in Indian children. Diabetologia. 2014;57:110–21. 212. Yajnik CS, Deshpande SS, Jackson AA, Refsum H, Rao S, Fisher DJ, et al. Vitamin B12 and folate concentrations during pregnancy and insulin resistance in the offspring: The 142 Pune Maternal Nutrition Study. Diabetologia. 2008;51:29–38. 213. Xie RH, Liu YJ, Retnakaran R, MacFarlane AJ, Hamilton J, Smith G, et al. Maternal folate status and obesity/insulin resistance in the offspring: A systematic review. Int J Obes. 2016;40:1–9. 214. Smith D, Hornstra J, Rocha M, Jansen G, Assaraf Y, Lasry I, et al. Folic acid impairs the uptake of 5-methyltetrahydrofolate in human umbilical vascular endothelial cells. J Cardiovasc Pharmacol. 2017;70:271–5. 215. Ashokkumar B, Mohammed ZM, Vaziri ND, Said HM. Effect of folate oversupplementation on folate uptake by human intestinal and renal epithelial cells. Am J Clin Nutr. 2007;86:159–66. 216. Boshnjaku V, Shim KW, Tsurubuchi T, Ichi S, Szany E V., Xi G, et al. Nuclear localization of folate receptor alpha: A new role as a transcription factor. Sci Rep. 2012;2(980):srep00980. 217. Frigerio B, Bizzoni C, Jansen G, Leamon CP, Peters GJ, Low PS, et al. Folate receptors and transporters: Biological role and diagnostic/therapeutic targets in cancer and other diseases. J Exp Clin Cancer Res. 2019;38:1–12. 218. Mohanty V, Siddiqui MR, Tomita T, Mayanil CS. Folate receptor alpha is more than just a folate transporter. Neurogenesis. 2016;4(1):e1263717. 219. Kim Y. Folic acid supplementation and cancer risk: Point. Cancer Epidemiol Biomarkers Prev. 2008;17:2220–5. 220. Protiva P, Mason JB, Liu Z, Hopkins ME, Nelson C, Marshall JR, et al. Altered folate availability modifies the molecular environment of the human colon: implications for colorectal carcinogenesis. Cancer Prev Res. 2011;4(4):530–43. 143 221. Kelly KB, Kennelly JP, Ordonez M, Nelson R, Leonard K, Stabler S, et al. Excess folic acid increases lipid storage, weight gain, and adipose tissue inflammation in high fat diet-fed rats. Nutrients. 2016;8:nu8100594. 222. Bahous RH, Jadavji NM, Deng L, Cos M, Lu J, Malysheva O, et al. High dietary folate in pregnant mice leads to pseudo-MTHFR deficiency and altered methyl metabolism, with embryonic growth delay and short-term memory impairment in offspring. Hum Mol Genet. 2017;26(5):888–900. 223. Hammoud R, Pannia E, Kubant R, Wasek B, Bottiglieri T, Malysheva O V., et al. Choline and folic acid in diets consumed during pregnancy interact to program food intake and metabolic regulation of male Wistar Rat offspring. J Nutr. 2021;151:857–65. 224. Joubert BR, Dekker HT Den, Felix JF, Bohlin J, Ligthart S, Beckett E, et al. Maternal plasma folate impacts differential DNA methylation in an epigenome-wide meta-analysis of newborns. Nat Commun. 2016;7(10577):1–8. 225. Zhu Z, Cao F, Li X. Epigenetic programming and fetal metabolic programming. Front Endocrinol (Lausanne). 2019;10:764. 226. Ly A, Ishiguro L, Kim D, Im D, Kim S, Sohn K, et al. Maternal folic acid supplementation modulates DNA methylation and gene expression in the rat offspring in a gestation period-dependent and organ-specific manner. J Nutr Biochem. 2016;33:103–10. 227. Molloy AM, Mills JL. Folic acid and infant allergy: Avoiding rash judgments. J Nutr. 2021;151:1367–8. 228. Kim Y. Folate and cancer: A tale of Dr. Jekyll and Mr. Hyde? Am J Clin Nutr. 2018;107:139–42. 229. Mills JL, Molloy AM. Lowering the risk of autism spectrum disorder with folic acid: Can 144 there be too much of a good thing? Am J Clin Nutr. 2022;115:1268–9. 230. Blom HJ. Folic acid, methylation and neural tube closure in humans. Birth Defects Res A Clin Mol Teratol. 2009;85:295–302. 231. Chan A, Tetzlaff JM, Gøtzsche PC, Altman DG, Mann H, Berlin JA, et al. SPIRIT 2013 explanation and elaboration: Guidance for protocols of clinical trials. BMJ. 2013;346:e7586. 232. Sim J, Lewis M. The size of a pilot study for a clinical trial should be calculated in relation to considerations of precision and efficiency. J Clin Epidemiol. 2012;65:301–8. 233. Clifford AJ, Noceti EM, Block-Joy A, Block T, Block G. Erythrocyte folate and its response to folic acid supplementation is assay dependent in women. J Nutr. 2005;135:137–43. 234. Kelly P, McPartlin J, Scott J. A combined high-performance liquid chromatographic-microbiological assay for serum folic acid. Anal Biochem. 1996;238(2):179–83. 235. Campbell K, Rowe H, Azzam H, Lane CA. The management of nausea and vomiting of pregnancy. J Obstet Gynaecol Canada. 2016;38(12):1127–37. 236. Sutton EF, Cain LE, Vallo PM, Redman LM. Strategies for successful recruitment of pregnant patients into clinical trials. Obstet Gynecol. 2017;129(3):554–9. 237. Becton Dickson Biosciences. Preanalytical Systems, BD Life Sciences Product Catalogue [Internet]. Franklin Lakes, NJ, USA; 2018 [cited 2023 Jan 9]. Available from: https://www.bd.com/resource.aspx?IDX=34369 238. Finkelstein JD. The metabolism of homocysteine: Pathways and regulation. Eur J Pediatr. 1998;157(S2):252–62. 239. Arning E, Bottiglieri T. Quantitation of S-adenosylmethionine and S-145 adenosylhomocysteine in plasma using liquid chromatography-electrospray tandem mass spectrometry. In: Methods in Molecular Biology. 2016. p. 255–62. 240. Ubbink JB, Serfontein WJ, De Villiers LS. Stability of pyridoxal-5-phosphate semicarbazone: Applications in plasma vitamin B6 analysis and population surveys of vitamin B6 nutritional status. J Chromatogr B Biomed Sci Appl. 1985;342(C):277–84. 241. Little RJ, Agostino RD, Cohen ML, Dickersin K, Emerson SS, Farrar JT, et al. The prevention and treatment of missing data in clinical trials. N Engl J Med. 2012;367(14):1355–60. 242. Cochrane KM, Hutcheon JA, Karakochuk CD. Strategies for improving recruitment of pregnant women to clinical research: An evaluation of social media versus traditional offline methods. Digit Heal. 2022;8:1–10. 243. Fazili Z, Sternberg MR, Paladugula N, Pfeiffer CM. Two international round-robin studies showed good comparability of 5-methyltetrahydrofolate but poor comparability of folic acid measured in serum by different high-performance liquid chromatography-tandem mass spectrometry methods. J Nutr. 2017;147:1815–25. 244. Innis SM, Hasman D. Evidence of choline depletion and reduced betaine and dimethylglycine with increased homocysteine in plasma of children with cystic fibrosis. J Nutr. 2006;136:2226–31. 245. Dominguez-Salas P, Moore SE, Cole D, Da Costa KA, Cox SE, Dyer RA, et al. DNA methylation potential: Dietary intake and blood concentrations of one-carbon metabolites and cofactors in rural African women. Am J Clin Nutr. 2013;97:1217–27. 246. Clifford RL, Jones MJ, MacIsaac JL, McEwen LM, Goodman SJ, Mostafavi S, et al. Inhalation of diesel exhaust and allergen alters human bronchial epithelium DNA 146 methylation. J Allergy Clin Immunol. 2017;139(1):112–21. 247. Cook B, Manning W. Thinking beyond the mean: A practical guide for using quantile regression methods for health services research. Shanghai Arch Psychiatry. 2013;25(1):55–9. 248. Public Health Agency of Canada. Prenatal Nutrition Guidelines for Health Professionals: Gestational Weight Gain. 2010. 249. Visentin CE, Masih SP, Plumptre L, Schroder TH, Sohn KJ, Ly A, et al. Low serum vitamin B-12 concentrations are prevalent in a cohort of pregnant Canadian women. J Nutr. 2016;146:1035–42. 250. Plumptre L, Masih SP, Sohn KJ, Kim D, Visentin CE, Ly A, et al. Suboptimal maternal and cord plasma pyridoxal 5′ phosphate concentrations are uncommon in a cohort of Canadian pregnant women and newborn infants. Matern Child Nutr. 2018;14:e12467. 251. Molloy AM, Kirke PN, Troendle JF, Burke H, Sutton M, Brody LC, et al. Maternal vitamin B12 status and risk of neural tube defects in a population with high neural tube defect prevalence and no folic acid fortification. Pediatrics. 2009;123(3):917–23. 252. Bae S, West AA, Yan J, Jiang X, Perry CA, Malysheva O, et al. Vitamin B-12 status differs among pregnant, lactating, and control women with equivalent nutrient intakes. J Nutr. 2015;145:1507–14. 253. Stamm RA, March KM, Karakochuk CD, Gray AR, Brown RC, Green TJ, et al. Lactating Canadian women consuming 1000 mcg folic acid daily have high circulating serum folic acid above a threshold concentration of serum total folate. J Nutr. 2018;148:1–6. 254. Ledowsky C, Mahimbo A, Scarf V, Steel A. Women taking a folic acid supplement in countries with mandatory food fortification programs may be exceeding the upper 147 tolerable limit of folic acid: A systematic review. Nutrients. 2022;14:2715. 255. Bailey RL, Pac SG, Iii VLF, Reidy KC, Catalano PM. Estimation of total usual dietary intakes of pregnant women in the United States. JAMA Netw Open. 2019;2(6):e195967. 256. Park Y, Dodd KW, Kipnis V, Thompson FE, Potischman N, Schoeller DA, et al. Comparison of self-reported dietary intakes from the Automated Self-Administered 24-h recall, 4-d food records, and food-frequency questionnaires against recovery biomarkers. Am J Clin Nutr. 2018;107:80–93. 257. Green TJ, Allen OB, O’Connor DL. A three-day weighed food record and a semiquantitative food-frequency questionnaire are valid measures for assessing the folate and vitamin B-12 intakes of women aged 16 to 19 years. J Nutr. 1998;128:1665–71. 258. Victora CG, Bahl R, Barros AJD, AFrança G V, Horton S, Krasevec J, et al. Breastfeeding in the 21st Century: Epidemiology, mechanisms and lifelong impact. Lancet. 2016;387(10017):475–790. 259. Bode L, Raman AS, Simon H, Rollins NC, Gordon JI. Understanding the mother-breastmilk-infant “triad.” Sci. 2020;367(6482):1070–2. 260. Binns C, Lee M, Low WY. The long-term public health benefits of breastfeeding. Asia-Pacific J Public Heal. 2016;28(1):7–14. 261. Horta BL, De Sousa BA, De Mola CL. Breastfeeding and neurodevelopmental outcomes. Curr Opin Clin Nutr Metab Care. 2018;21(3):174–8. 262. Sankar MJ, Sinha B, Chowdhury R, Bhandari N, Taneja S, Martines J, et al. Optimal breastfeeding practices and infant and child mortality: A systematic review and meta-analysis. Acta Paediatr. 2015;104:3–13. 263. Health Canada. Prenatal Nutrition Guidelines for Health Professionals: Folate. 2009. 148 264. Trippett TM, Bertino JR. Therapeutic strategies targeting proteins that regulate folate and reduced folate transport. J Chemother. 1999;11(1):3–10. 265. Galante L, Vickers MH, Milan AM, Reynolds CM, Alexander T, Bloomfield FH, et al. Feasibility of standardized human milk collection in neonatal care units. Sci Rep. 2019;9:14343. 266. Verwei M, Arkbåge K, Mocking H, Havenaar R, Groten J. The binding of folic acid and 5-methyltetrahydrofolate to folate-binding proteins during gastric passage differs in a dynamic in vitro gastrointestinal model. J Nutr. 2004;134:31–7. 267. Nygren-Babol L, Jägerstad M. Folate-binding protein in milk: A review of biochemistry, physiology, and analytical methods. Crit Rev Food Sci Nutr. 2012;52(5):410–25. 268. Verwei M, Van Den Berg H, Havenaar R, Groten JP. Effect of folate-binding protein on intestinal transport of folic acid and 5-methyltetrahydrofolate across Caco-2 cells. Eur J Nutr. 2005;44:242–9. 269. Verwei M, Arkbåge K, Havenaar R, Van den Berg H, Witthöft C, Schaafsma G. Folic acid and 5-methyltetrahydrofolate in fortified milk are bioaccessible as determined in a dynamic in vitro gastrointestinal model. J Nutr. 2003;133:2377–83. 270. Mason JB, Selhub J. Folate-binding protein and the absorption of folic acid in the small intestine of the suckling rat. Am J Clin Nutr. 1988;48:620–5. 271. Rosenberg IH, Selhub J. Folate and vitamin B12 transport systems in the developing infant. J Pediatr. 2006;149:S62–3. 272. Niesser M, Demmelmair H, Weith T, Moretti D, Rauh-Pfeiffer A, van Lipzig M, et al. Folate catabolites in spot urine as non-invasive biomarkers of folate status during habitual intake and folic acid supplementation. PLoS One. 2013;8(2). 149 273. Troesch B, Demmelmair J, Gimpfl M, Hecht C, Lakovic G, Roehle R, et al. Suitability and safety of L-5-methyltetrahydrofolate as a folate source in infant formula: A randomized-controlled trial. PLoS One. 2019;14(8):e0216790. 274. Ford JE, Zechalko A, Murphy J, Brooke OG. Comparison of the B vitamin composition of milk from mothers of preterm and term babies. Arch Dis Child. 1983;58(5):367–72. 275. Karra M, Udipi S, Kirkset A, Roepke J. Changes extended in specific nutrients in breast milk during extended lactation. Am J Clin Nutr. 1986;43:495–503. 276. Piaggio G, Elbourne DR, Altman DG, Pocock SJ, Evans SJW. Reporting of noninferiority and equivalence randomized trials: An extension of the CONSORT statement. JAMA. 2006;295(10):1152–60. 277. Wu BTF, Innis SM, Mulder KA, Dyer RA, King DJ. Low plasma vitamin B-12 is associated with a lower pregnancy-associated rise in plasma free choline in Canadian pregnant women and lower postnatal growth rates in their male infants. Am J Clin Nutr. 2013;98(5):1209–17. 278. Houghton LA, Gray AR, Rose MC, Miller JC, Hurthouse NA, Gregory JF. Long-term effect of low-dose folic acid intake: Potential effect of mandatory fortification on the prevention of neural tube defects. Am J Clin Nutr. 2011;94:136–41. 279. Shere M, Nguyen P, Tam C, Stern S, Kapur B, O’Connor DL, et al. Pregnancy-induced changes in the long-term pharmacokinetics of 1.1 mg vs. 5 mg folic acid: A randomized clinical trial. J Clin Pharmacol. 2015;55(2):159–67. 280. Sulistyoningrum D, Green T, Palmer D, Sullivan T, Wood S, Makrides M, et al. Study protocol for a randomised controlled trial evaluating the effect of folic acid supplementation beyond the first trimester on maternal plasma unmetabolised folic acid in 150 late gestation. BMJ Open. 2020;10:e040416. 281. Mcnulty H, Rollins M, Cassidy T, Caffrey A, Marshall B, Dornan J, et al. Effect of continued folic acid supplementation beyond the first trimester of pregnancy on cognitive performance in the child: A follow-up study from a randomized controlled trial (FASSTT Offspring Trial). BMC Med. 2019;17(196). 282. Caffrey A, McNulty H, Rollins M, Prasad G, Gaur P, Talcott JB, et al. Effects of maternal folic acid supplementation during the second and third trimesters of pregnancy on neurocognitive development in the child: an 11-year follow-up from a randomised controlled trial. BMC Med. 2021;19:73. 283. McNulty B, McNulty H, Marshall B, Ward M, Molloy AM, Scott JM, et al. Impact of continuing folic acid after the first trimester of pregnancy: Findings of a randomized trial of folic acid supplementation in the second and third trimesters. Am J Clin Nutr. 2013;98:92–8. 284. Murphy MSQ, Muldoon KA, Sheyholislami H, Behan N, Lamers Y, Rybak N, et al. Impact of high-dose folic acid supplementation in pregnancy on biomarkers of folate status and 1-carbon metabolism: An ancillary study of the Folic Acid Clinical Trial (FACT). Am J Clin Nutr. 2021;113(5):1361–71. 285. Crider KS, Bailey LB, Berry RJ. Folic acid food fortification-its history, effect, concerns, and future directions. Nutrients. 2011;3:370–84. 286. Rader JI, Weaver CM, Angyal G. Total folate in enriched cereal-grain products in the United States following fortification. Food Chem. 2000;70:275–89. 287. Sherwood KL, Houghton LA, Tarasuk V, O’Connor DL. One-third of pregnant and lactating women may not be meeting their folate requirements from diet alone based on 151 mandated levels of folic acid fortification. J Nutr. 2006;136:2820–6. 288. Klatt KC, Smith ER, Barberio MD. Toward a more stable understanding of pregnancy micronutrient metabolism. Am J Physiol Endocrinol Metab. 2021;321:E260–3. 289. Scholl T, Hediger H, Schall J, Khoo C, Fischer R. Dietary and serum folate: Their influence on the outcome of pregnancy. Am J Clin Nutr. 1996;63:520–5. 290. Relton CL, Pearce MS, Parker L. The influence of erythrocyte folate and serum vitamin B12 status on birth weight. Br J Nutr. 2005;93:593–9. 291. Li N, Li Z, Ye R, Liu J, Ren A. Impact of periconceptional folic acid supplementation on low birth weight and small-for-gestational-age infants in China: A large prospective cohort study. J Pediatr. 2017;187:105–10. 292. McGee M, Bainbridge S, Fontaine-Bisson B. A crucial role for maternal dietary methyl donor intake in epigenetic programming and fetal growth outcomes. Nutr Rev. 2018;76(6):469–78. 293. Pastor-Valero M, Navarrete-Muoz EM, Rebagliato M, Iñiguez C, Murcia M, Marco A, et al. Periconceptional folic acid supplementation and anthropometric measures at birth in a cohort of pregnant women in Valencia, Spain. Br J Nutr. 2011;105(9):1352–60. 152 Appendices Appendix A: Pregnancy-induced alterations of 1-carbon metabolism Acknowledgement: A version of this appendix has been published. Cochrane KM, Williams BA, Elango R, Barr SI, Karakochuk CD. Pregnancy-induced alterations of one-carbon metabolism and significance for maternal nutrition requirements. Nutr Rev 2022; 80(9):1985-2001. Summary This narrative review summarizes the pregnancy-induced alterations in 1C metabolism, effects of advancing gestation on maternal plasma concentrations of methyl nutrients, and potential implications for maternal dietary intake and infant clinical outcomes. 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. 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 which assessed 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 reviewed. Adaptations in 1C metabolism occur throughout a healthy pregnancy to support this unique 153 period of accelerated growth. Studies report similar temporal changes among healthy pregnant women from diverse geographical regions of plasma concentrations of many methyl nutrients, including B-vitamins, choline, betaine, methionine, and cysteine. 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 novel findings that glycine may be conditionally essential in pregnancy. 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 which aim to prevent deficiency and associated adverse health outcomes for mom and baby. Introduction 1C metabolism includes a number of interlinked cellular pathways in which 1C units are transferred from one molecule to another.1 This network supports numerous physiological processes, including cellular proliferation, synthesis of purines and pyrimidines, and establishment of epigenetic marks, and as such, 1C metabolism is critical for the growth and development of fetal and maternal tissue.1,2 The folate cycle, methionine cycle, and trans-sulfuration pathway are the linked cycles of 1C metabolism.3 This complex network requires several nutrients as methyl donors and co-factors, including folate, vitamin B12, vitamin B6, choline, betaine, methionine, cysteine, glycine, and serine (the “methyl nutrients”).3,4 Understanding of the pregnancy-related changes in 1C metabolism continues to evolve, given novel discoveries from animal models and use of stable isotopes. Isotopically-labeled 154 micronutrients can be provided and tracked to understand their absorption, metabolism, and excretion, separately from other dietary sources or endogenous production.5 Additionally, measurement of methyl nutrients in both maternal plasma and venous and arterial umbilical cord blood has advanced the understanding of maternal-placental-fetal transfer of methyl nutrients, which supports growth and 1C metabolism in these compartments; maintaining maternal plasma concentrations at a level to support this process, without depleting maternal stores, is critical. Dietary recommendations in North America were established by an expert committee based on available evidence for each nutrient at different life stages. The DRIs used by Health Canada and the National Academy of Medicine include the EAR (estimated to meet the needs of 50% of healthy individuals at a particular life stage), RDA (aims to meet the needs of 97-98% of healthy individuals at a particular life stage) and the AI (established when there is insufficient evidence to estimate the EAR and RDA).6 Therefore, the strength of evidence for different nutrient recommendations is variable; evidence is particularly lacking for pregnancy-specific recommendations of certain methyl nutrients (e.g., choline, amino acids). Understanding the normal 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 which aim to prevent deficiency and associated adverse health outcomes for mom and baby. A recent international study suggested that fluctuations in methyl nutrient status may be highly generalizable among healthy pregnant women.7 Gilley et al.7 conducted a prospective longitudinal study of two distinct cohorts (from the Women First trial8), which included assessment of methyl nutrient status in the first and third trimester in pregnant 155 women from Guatemala (n=134) and Pakistan (n=179). They found very similar between-country patterns of change in most 1C metabolite concentrations throughout pregnancy, despite major differences in subject characteristics between the two cohorts, including diet, socio-economic status, ethnicity, and pre-pregnancy BMI.7 Whether consistent methyl nutrient changes are observed across a larger body of evidence warrants investigation. This review aims to summarize the pregnancy-induced alterations in 1C metabolism, effects of advancing gestation on maternal plasma concentrations of methyl nutrients, and potential implications for maternal dietary recommendations. Clinical outcomes associated with disturbances in 1C flux or insufficient dietary intake of methyl nutrients are also explored. Methods A literature review in MEDLINE was completed using the following MeSH terms: Pregnancy/, Folic Acid/, Vitamin B 12/, Vitamin B 6/, Choline, Betaine, Homocysteine, Methionine, Cysteine, Serine, Glycine, Blood/, fetal blood/, plasma/, and serum/. A grey literature search was also conducted in Google Scholar, using search terms related to 1C metabolism, each methyl nutrient, and nutrition requirements in pregnancy. English-language articles which assessed 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 reviewed. Studies which measured maternal methyl nutrient status at ≥2 timepoints in pregnancy were reviewed to summarize temporal changes across gestation. 156 Discussion B-vitamins: folate, vitamin B12, vitamin B6 Longitudinal studies of pregnant women not consuming prenatal vitamins have reported an inverse association between plasma B-vitamin concentrations and gestational age.9–12 Pregnancy-related hemodilution may play a role, but doesn’t appear to fully explain this decline given declining plasma vitamin B12 despite maintenance of hematocrit in pregnancy.13 Only one controlled feeding trial using stable isotopes has assessed B-vitamin (folate) metabolism in pregnancy.14 Gregory et al.14 reported that in a folate-replete population (United States), pregnant women (n=6) and non-pregnant controls (n=6) did not significantly differ with respect to body folate pool size, catabolism, and excretion.14 A previous study of n=27 pregnant women (Denmark) investigated intestinal absorption of vitamin B12 with advancing gestation (CobaSorb assay); no decline in absorption capacity was found.15 There does appear to be a high degree of B-vitamin transfer to the placenta and fetus.13,16 This is evidenced by numerous studies which report higher concentrations of folate, vitamin B12, and vitamin B6 in venous umbilical cord blood compared to maternal plasma.13,16–19 Further, various physiological changes occur in pregnancy to support maternal-placental-fetal transfer of B-vitamins, including establishment of placental folate transporters in early pregnancy20 and placental synthesis of transcobalamin (the primary transporter of vitamin B12 to the fetus).21 Vitamin B6 (as PLP; the co-enzymatically active form) appears to be actively transferred to the fetus, given a dose-dependent increase in cord blood PLP following maternal vitamin B6 intake,22–25 and higher concentrations of PLP in venous as compared to arterial cord blood.26 Folate and vitamin B12 are efficiently sequestered and stored in the placenta,27 which may be 157 used for placental 1C metabolism or redistribution to the fetus as required (as folate and B12 storage in fetal liver is limited).16,20 Cord blood concentrations of vitamin B12 were strongly associated with maternal plasma concentrations in the PREFORM cohort (n=368 Canadian women; r2=0.51), suggesting that maternal plasma concentrations are an important determinant of transfer to the fetus.13 Consumption of prenatal vitamins appears to prevent some, but not all, B-vitamin decline in maternal plasma.2,28 Previous studies in Canada,13,18,29,30 Curaçao31 and Spain32 have reported that in women consuming prenatal vitamins, plasma folate increased or remained consistent, while plasma vitamin B12 and PLP decreased by ~30-50% as pregnancy progressed. Prenatal supplements typically contain B-vitamins at levels greater than the RDA, yet supplementation appears to only prevent decline in concentrations of plasma folate, but not plasma vitamin B12 and vitamin B6. As such, optimal intakes of vitamins B12 and B6 among pregnant women have recently been questioned. Among pregnant women of the PREFORM cohort, most had intakes of vitamins B12 and B6 exceeding the RDA through diet and prenatal vitamins.13,18 Using reference ranges for non-pregnant women, prevalence of vitamin B6 deficiency (PLP <20 nmol/L) at delivery was uncommon (~5%),18 while 38% and 43% had deficient (<148 pmol/L) and marginal (148-220 pmol/L) serum vitamin B12 concentrations at delivery, respectively.13 Clinical significance of serum vitamin B12 decline and whether reference ranges for non-pregnant women are appropriate in pregnancy is unclear, but vitamin B12 metabolism may be altered in pregnancy.33 In a controlled feeding trial providing ~8.6 µg/day vitamin B12 (~3-times greater than the RDA) to pregnant (n=26; third trimester) and non-pregnant (n=21) women in the United 158 States, Bae et al.33 reported that the concentration of serum vitamin B12 was significantly lower in pregnant versus non-pregnant women after 10-12 weeks, despite no differences in baseline concentrations or vitamin B12 intake throughout the trial.33 However, no pregnant women had levels <148 pmol/L at any point.33 Concentrations of holotranscobalamin (the bioactive form of vitamin B12) and holotranscobalamin-to-vitamin B12 ratio increased in pregnant women and was not significantly different from non-pregnant controls. Although plasma levels do not necessarily reflect metabolism, this suggests a greater conversion of vitamin B12 towards the active form in pregnancy, which should be further explored using stable isotopes.33 Additionally, pregnant women had greater concentrations of vitamin B12 bound to transcobalamin (30% versus 20% in non-pregnant women), possibly for placental-fetal transfer. At this time, there is insufficient evidence to determine whether increased intake of vitamin B12 (similar to Bae et al.33) is warranted to prevent maternal vitamin B12 “deficiency”, as cut-offs are for non-pregnant women. It is possible that such decline is “normal”, and additional intake would be added to liver stores, converted to holotranscobalamin, or transferred to the placenta as transcobalamin for placental storage or to supply the fetus. Concerns regarding folate intake are related to excessive folic acid supplementation (the synthetic form of folate), above the RDA of 0.6 mg/day DFEs.34,35 Prenatal vitamins typically contain more folic acid than necessary (0.6-1.0 mg; equivalent to ~1.0-1.7 mg DFEs), and whether supplementation is beneficial past the first trimester, after neural tube closure, is unclear and currently under investigation.36,37 Concerns of over-supplementation with folic acid are related to the apparent limited and variable capacity of intestinal and hepatic DHFR to reduce folic acid to DHF, resulting in the appearance of UMFA in circulation.38,39 Consequences of 159 UMFA are unknown, but it is considered an indication of excess intake and UMFA is a co-enzymatically inactive form.19,36 Additionally, with high intakes and/or high folate status, folic acid may interfere with normal folate metabolism due to competition with DHF for DHFR and inhibition of MTHFR.39,40 Investigation of UMFA, including impact on biological pathways and 1C metabolism, and association with adverse clinical outcomes, has been identified as a research priority following the 2019 National Institutes of Health expert workshop.41 160 The summary of studies on B-vitamin concentrations across trimesters of a healthy pregnancy are presented in Table 1. Table 1: B-vitamins Reference Country/ Region Study Design (Time Points) n Micronutrient Supplement1 Fasted2 Outcome Variable Method of Measurement Trend Across Trimesters3 Cikot et al. (2001)9 Netherlands Prospective longitudinal (6, 10, 20, 32 GW) 102 No No Folate (serum) Radioassay Decrease Folate (RBC) Radioassay Slight increase B12 (serum) Radioassay Decrease B6 (serum PLP) HPLC Decrease Velzing-Aarts et al. (2005)31 Curaçao Prospective longitudinal (9, 16, 20, 24, 28, 32, 36 GW) 50 Yes Yes Folate (plasma) Immunoassay Increase B12 (plasma) Immunoassay Decrease Milman et al. (2006)11 Denmark Prospective longitudinal (18, 32, 39 GW) 404 No No Folate (serum) Immunoassay Decrease Folate (RBC) Immunoassay Decrease Úbeda et al. (2011)42 Spain Prospective longitudinal (15, 24, 32 GW) 154 Unknown Yes Folate (serum) Ion-capture assay Decrease B12 (serum) Ion-capture assay Decrease Parazzini et al. (2011)43 Italy Prospective longitudinal (8-12, 16, 22 GW) 291 Yes No Folate (serum) Immunoassay Increase Folate (RBC) Immunoassay Increase Fernàndez-Roig et al. (2013)32 Spain Prospective longitudinal (12, 15, 24-27, 34 GW) 574 Yes Yes* Folate (plasma) Microbiological assay Increase from 1st to 2nd trimester, decrease 2nd to 3rd Folate (RBC) Microbiological assay Increase from 1st to 2nd trimester, decrease 2nd to 3rd B12 (plasma) Microbiological assay Decrease Wu et al. (2013)30 Canada Case-control, prospective (16, 36 GW) 264 P, 88 NP Yes Yes Folate (plasma) Ion-capture assay Consistent; increased vs. NP B12 (plasma) Immunoassay Decrease; decreased vs. NP 161 1Majority (>50%) of participants consumed micronutrient supplements (methyl nutrients typically included in prenatal micronutrient supplements: folic acid, vitamin B12, vitamin B6, vitamin B2) 2Fasted for ≥8 hours prior to blood sample collection; *fasting time not specified 3Unless specified, “increase/decrease/consistent” is the trend throughout the full study period; trimesters defined as: 1st: ~1-12 weeks of gestation; 2nd: ~13-27 weeks of gestation; 3rd: ~28-40 weeks of gestation GW: gestational weeks; PLP: pyridoxal 5’phosphate; HPLC: high performance liquid chromatography; RBC: red blood cell; LC-MS/MS: liquid chromatography tandem mass spectrometry; P: pregnant; NP: non-pregnant Bae et al. (2015)33 USA Case-control, prospective (26-29, 36-40 GW) 26 P, 21 NP Yes Yes B12 (serum) Immunoassay Decrease; decreased in only 3rd trimester vs. NP Visentin et al. (2016)13 Canada (PREFORM Study) Prospective longitudinal (12-16, 28-42 [delivery] GW) 368 Yes No B12 (serum) Immuno-enzymatic assay Decrease Plumptre et al. (2018)18 Canada (PREFORM Study) Prospective longitudinal (12-16, 28-42 [delivery] GW) 368 Yes No B6 (plasma PLP) Enzymatic assay Decrease Varsi et al. (2018)12 Norway Case-control, prospective (18, 28, 36 GW) 114 P, 123 NP No No B12 (serum) Microbiological assay Decrease; decreased in only 3rd trimester vs. NP Mujica-Coopman et al. (2019)10 Brazil Prospective longitudinal (5-13, 20-26, 30-36 GW) 186 No Yes B6 (plasma PLP) HPLC Decrease Mujica-Coopman et al. (2019)29 Canada Retrospective (biobanked samples) (8-13, 14-20 GW) 723 Unknown No Folate (serum) Microbiological assay Increase B12 (serum) Immunoassay Decrease B6 (serum PLP) LC-MS/MS Decrease 162 Clinical significance - Fetal growth: Folate directly contributes to cellular proliferation, gene expression, and growth of the fetus and utero-placental organs via synthesis of thymidylates, purines, and remethylation of homocysteine for production of SAM, the universal methyl donor.44 Vitamins B12 and B6 are important co-factors in these reactions.3,44 Maternal folate and cord blood concentrations are significant predictors of birth weight,45,46 however, the influence of folate consumption on fetal growth is unclear. While most studies report that folic acid supplementation is associated with higher birth weight,46–48 there appears to be a threshold effect where high doses of folic acid (>1 mg/day) have either no effect or are negatively associated with birth weight.49–51 High folic acid intake may be particularly detrimental to fetal growth when combined with low vitamin B12 intake.52 Evidence for this is provided by two studies among pregnant women in India, which found that higher plasma folate to B12 ratio was associated with reduced birth weight (r=-0.5, P=0.009)53 and that low intake of vitamin B12 in those who consumed >1.0 mg/day folic acid was associated with a higher risk of infants born small-for-gestational-age (RR=2.73, 95% CI 1.17-6.37).54 Clinical significance - NTDs: Exact mechanisms of the role of folic acid in NTD reduction is unknown, and correction of deficiency may only prevent NTDs when combined with certain genetic predispositions.55,56 For instance, NTDs were significantly increased following dietary folate restriction and antibiotics in splotch mice, which carry a mutation in Pax3, a gene encoding transcription factors for DNA methylation and neural tube closure, as compared to wild-type mice who did not develop NTDs as a result of folate restriction.55 In mouse models, defects in the mitochondrial folate cycle, including loss-of-function of genes in the glycine cleavage system (Amt, Gldc) and mitochondrial 10-formyl-THF synthetase (Mthfd1l), resulted in 163 the occurrence of NTDs.57–61 Findings suggest that transfer of 1C units from the mitochondria (e.g., via formate) and subsequent flux through the folate and methionine cycles is essential for neural tube closure.59,62 Other methyl nutrients may also be implicated in NTDs. Observational trials report an association between low maternal vitamin B12,63,64 choline,65,66 betaine,65 and methionine67,68 concentrations and NTD-affected pregnancies, however, evidence is less well-established compared to the association with folate. Formate Formate (the smallest carboxylic acid) is a critical molecule for inter-organelle transport of 1C groups.69 It is produced via a variety of mechanisms, including endogenously via its precursors (various amino acids, dimethylglycine, and sarcosine) and in different cellular organelles, with predominant production occurring in the mitochondria.69 Mitochondrial formate enters the cytosol and serves as an important 1C donor for production of 10-formyl-THF and subsequent conversion to more reduced folate forms.2,62 Importance of formate during pregnancy is well-established from animal models,57–59,61,62,70,71 but investigation in pregnant women (PREFORM cohort) was only first reported in 2019 by Brosnan et al.72 Plasma formate concentrations were consistent from early pregnancy to delivery, and concentrations were significantly higher in cord blood than maternal plasma.72 Increased cord blood formate (~60% higher than maternal plasma) may suggest increased maternal-fetal transfer, however, the correlation between formate in maternal plasma and cord blood was weak (r2=0.11).72 Thus, Brosnan et al.72 suggest that these two formate pools may be independently regulated.72 Formate metabolism appears to be highly active in the placenta and fetus; this is 164 supported by recent findings in rats of increased abundance of 10-formyl-THF synthetase mRNA (Mthfd1l; enzyme which catalyzes the formation of mitochondrial formate) in the placenta and fetal liver, as compared to the liver of 3- and 7-day old neonates.62 Determinants of plasma formate during pregnancy and implications for maternal dietary recommendations requires further investigation. Plasma formate concentrations were higher in the PREFORM cohort than in a recent cross-sectional study of n=1701 non-pregnant adults (31.5 ± 14.4 µmol/L vs. 25.9 ± 7.7 µmol/L, respectively).72,73 Increased estrogen (increases with advancing gestation) may be an underlying factor, as formate was reported as ~10% higher in non-pregnant women than men.73 It has been suggested that both folate and vitamin B12 deficiency can impair formate production and turnover (given their roles as co-factors in the folate cycle).74 In the PREFORM cohort, plasma formate and red blood cell folate in early pregnancy were significantly (but weakly) associated (r=0.26, P<0.0001).72 Providing dietary formate in mouse models pre-disposed to NTDs results in reduced occurrence of NTDs,57,58,61,71 however, no similar studies have been conducted in humans, thus relevance is unknown. Choline, Betaine Plasma free choline increases with gestational age during a healthy pregnancy30–32,75 to support increased formation of cell membranes, lipoprotein synthesis, and acceleration of reactions requiring 1C donors.76 This occurs despite hemodilution, increased urinary excretion, and maternal-fetal transfer.75–77 Maternal choline is thought to serve as a critical source of choline for the fetus, given low expression of phosphatidylethanolamine N-methyltransferase (PEMT) in human placenta and fetal liver.76 Increases of free choline in maternal plasma may be partially 165 explained by increased expression of maternal PEMT, which is responsive to increased estrogen.78,79 This results in increased production of phosphatidylcholine, which may be incorporated into cellular membranes or lipoproteins, converted to other choline forms (glycerophosphocholine, sphinogomyelin), or catabolized to free choline (Figure 1).76,80 Increased catabolism of PEMT-derived phosphatidylcholine to free choline occurs in pregnant (n=26; third trimester) compared to non-pregnant women (n=21), as reported by Yan et al.79 using stable-isotope tracers (deuterium-labeled methyl-d9-choline). The rise in plasma free choline may be slightly reduced by other methyl nutrient deficiencies (e.g., vitamin B12).30 166 Figure 1: Phosphatidylcholine synthesis; schematic representation of pathways in the liver. Choline is derived from phosphatidylcholine, which is synthesized in two pathways: the cytidine diphospho-choline (CDP) pathway or following methyl donation from S-adenosylmethionine (SAM) to phosphatidylethanolamine (PE), catalyzed by phosphatidyl-ethanolamine N-methyltransferase (PEMT). PC: phosphatidylcholine; Gly-PCho: glycerophosphocholine; SM: sphingomyelin; MET: methionine; MAT: methionine adenosyltransferase; SAH: S-adenosylhomocysteine; SAHH: S-adenosylhomocysteine hydrolase; HCY: homocysteine; CK: choline kinase; PCho: phosphocholine; CCT: CTP:phosphocholine cytidylyltransferase; CPT: choline phosphotransferase; CHDH: choline dehydrogenase; BADH: betaine aldehyde dehydrogenase; BHMT: betaine-homocysteine S-methyltransferase; DMG: dimethylglycine. Maternal plasma betaine appears to decline with advancing gestation.29,31,32,75 Some reports also note that this decline occurs predominantly in early pregnancy, followed by the stabilization of levels.31,32 Decline cannot be fully explained by hemodilution, as maternal plasma betaine was not associated with hematocrit in a previous report.31 Other proposed explanatory causes include increased use of betaine for homocysteine remethylation, as betaine-homocysteine methyltransferase expression is also responsive to estrogen,81 and maternal-fetal transfer.75 Recommended intake of betaine is not established, but dietary betaine may reduce the 167 requirement for its synthesis from choline, sparing choline requirement by up to 50% (as previously observed in chicks).82 Optimal dietary choline intake and normal concentrations of plasma free choline in pregnancy is complicated and remains unclear. Visentin et al.75 found that dietary choline was not associated with plasma free choline concentrations at delivery in maternal plasma or infant cord blood, while Wu et al.83 has reported that at 16 weeks of gestation, dietary choline was positively (but weakly) correlated with maternal plasma free choline (r=0.2, P=0.013). Optimal status is further complicated by findings that those in the PREFORM cohort, who only consumed choline ad libitum (predominantly <450 mg/day), had similar plasma free choline concentrations at delivery as those in late pregnancy who had supplemented with 480-930 mg/day for 10-12 weeks in a controlled feeding trial.75,79 Authors of the PREFORM study suggested that plasma free choline concentrations are homeostatically regulated and/or that plasma free choline is rapidly incorporated into tissue, thus the effect of dietary intake is not apparent in plasma.75 Despite uncertainty around optimal choline intake, it appears that varying doses of choline may differentially alter choline flux during pregnancy.79 Using stable isotope methodology, Yan et al.79 reported that pregnant women who consumed 480 mg/day choline for 10-12 weeks had enhanced use of dietary choline for phosphatidylcholine synthesis, at the expense of oxidation to betaine, compared to those consuming 930 mg/day. They suggested that the current AI may be insufficient to meet demands of these competing pathways during pregnancy.79 The average dietary intake of choline in Canadian pregnant women has been reported as less than the AI (450 mg/day).83,84 Although choline-containing prenatal vitamins have become more available over 168 the past few years, most supplements in Canada and the United States do not contain choline or betaine. The summary of studies on choline, betaine concentrations across trimesters of a healthy pregnancy are presented in Table 2. 169 Table 2: Choline, betaine 1Majority (>50%) of participants consumed micronutrient supplements (methyl nutrients typically included in prenatal micronutrient supplements: folic acid, vitamin B12, vitamin B6, vitamin B2) 2Fasted for ≥8 hours prior to blood sample collection; *fasting time not specified 3Choline quantified in plasma = free choline 4Unless specified, “increase/decrease/consistent” is the trend throughout the full study period; trimesters defined as: 1st: ~1-12 weeks of gestation; 2nd: ~13-27 weeks of gestation; 3rd: ~28-40 weeks of gestation GW: gestational weeks; LC-MS/MS: liquid chromatography tandem mass spectrometry; P: pregnant; NP: non-pregnant Reference Country/ Region Study Design (Time Points) n Micronutrient Supplement1 Fasted2 Outcome Variable3 Method of Measurement Trend Across Trimesters4 Velzing-Aarts et al. (2005)31 Curaçao Prospective longitudinal (9, 16, 20, 24, 28, 32, 36 GW) 50 Yes Yes Choline (plasma) LC-MS/MS Increase Betaine (plasma) LC-MS/MS Decrease until 20 GW, then consistent Fernàndez-Roig et al. (2013)32 Spain Prospective longitudinal (12, 15, 24-27, 34 GW) 574 Yes Yes* Choline (plasma) LC-MS/MS Increase Betaine (plasma) LC-MS/MS Decrease until 2nd trimester, then consistent Wu et al. (2013)30 Canada Case-control, prospective (16, 36 GW) 264 P, 88 NP Yes Yes Choline (plasma) LC-MS/MS Increase; increased vs. NP Betaine (plasma) LC-MS/MS Consistent; decreased vs. NP Visentin et al. (2015)75 Canada (PREFORM Study) Prospective longitudinal (12-16, 28-42 [delivery] GW) 296 Yes No Choline (plasma) LC-MS/MS Increase Betaine (plasma) LC-MS/MS Decrease Mujica-Coopman et al. (2019)29 Canada Retrospective (biobanked samples) (8-13, 14-20 GW) 723 Unknown No Betaine (serum) LC-MS/MS Decrease Gilley et al. (2020)7 Guatemala Prospective longitudinal (12, 34 GW) 134 Yes No Choline (bloodspot) LC-MS/MS Decrease Betaine (bloodspot) LC-MS/MS Decrease 170 Clinical significance - Infant cognition: Choline is important for fetal central nervous development, brain health and development, and may influence offspring cognition as evidenced by both animal and human models.85,86 Possible mechanisms for how choline supports brain health and cognition include: improved nerve conduction due to greater availability of phospholipids (sphinogomyelin) for nerve fibre myelination,87 increased transport of docosahexaenoic acid to the fetal brain (via lysophosphatidylcholine-docosahexaenoic acid),88 and choline’s role in the provision of methyl groups for 1C reactions.76 Caudill et al.89 reported that supplementation with 930 mg/day choline for 10-12 weeks in late pregnancy resulted in improved infant information processing speed and visuospatial memory at 4, 7, 10, and 13 months, as per standardized visual attention tasks and reactive saccade testing, compared to infants of mothers supplemented with 480 mg/day (n=13 in each supplementation group). Similarly, Wu et al.83 reported that maternal plasma free choline at 16 and 36 weeks had a significant positive association with infant cognitive test scores (Bayley Scales of Infant Development, Third Edition) at 18 months, controlling for potential confounders including maternal intelligent quotient, ethnicity, age, infant sex, breastfeeding duration, and maternal red blood cell docosahexaenoic acid (n=154, β=6.054, P=0.009).83 These findings are promising, but revision of dietary recommendations are complicated due to a lack of understanding of dose-response to dietary choline intake and what is defined as optimal maternal choline status. Methionine, Homocysteine, Cysteine Rates of trans-sulfuration, transmethylation, and remethylation increase at various points in pregnancy to support accelerated growth and development of maternal and fetal tissue.2,90 Dasarathy et al.90 examined methionine kinetics using isotopic tracers ([1-13C]methionine, 171 [C2H3]methionine, and [2H5]phenylalanine) in healthy pregnant women (n=10) compared to non-pregnant women (n=8) in the United States. Pregnant women consumed standard prenatal vitamins, and all participants consumed a study diet containing 75-100 g/day protein for seven days prior to the tracer study to ensure a protein-replete status. Trans-sulfuration was increased in the first trimester, decreasing in the second and third trimesters to rates similar to non-pregnant women. Alternatively, rates of both transmethylation and remethylation were similar to non-pregnant women in the first trimester, but increased throughout pregnancy and were significantly higher in the third trimester.90 Although it is unclear why trans-sulfuration increased in early pregnancy only, trans-sulfuration in general is critical to supply cysteine to the fetus, given low fetal activity of cystathionine β-synthase and absence of cystathionine γ-lyase in human fetal liver throughout gestation.90,91 Additionally, increased cysteine and glycine are required in pregnancy to support increased demand for glutathione (γ-L-glutamyl-L-cysteinyl-glycine), the most abundant antioxidant that defends against reactive oxygen species (produced in pregnancy due to rapid cellular growth and differentiation).92–94 Rates of transmethylation and remethylation are thought to increase as pregnancy progresses, and the rate of fetal growth increases, to increase production of outputs via various methyltransferases (Figure 2).2,90 172 Figure 2: Remethylation and transmethylation; schematic representation of pathways in the liver. S-adenosylmethionine (SAM) serves as a substrate for numerous methyltransferases, forming key outputs. PE: phosphatidylethanolamine; PEMT: phosphatidylethanolamine N-methyltransferase; PC: phosphatidylcholine; GNMT: glycine N-methyltransferase; PRMT: protein arginine N-methyltransferase; MMA: monomethylarginine; DNMT: DNA methyltransferase; GAMT: guanidinoacetate methyltransferase; SAH: S-adenosylhomocysteine; BHMT: betaine-homocysteine S-methyltransferase; 5-MTHF: 5-methyltetrahydrofolate; MTR: methionine synthase; MTRR: methionine synthase reductase. Maternal plasma methionine and cysteine are decreased in pregnancy compared to non-pregnant controls.30,90 Methionine concentrations tend to remain consistent throughout gestation in healthy, well-nourished women.30,72,90,95,96 Remethylation is a significant contributor of methionine regeneration, but the extent to which this fulfills methionine requirements in pregnancy is unclear.97,98 Alternatively, plasma total cysteine is reported to decrease with advancing gestation, predominantly in early pregnancy.10,90,96,99 This may reflect increased trans-sulfuration (and possible subsequent transfer of cysteine to the fetus) in the first trimester.90 173 Plasma total homocysteine is also decreased in pregnancy compared to non-pregnant women.90,100 This may be explained by hemodilution, reduced albumin (which normally binds 70-80% of homocysteine in plasma), and increased urinary excretion of homocysteine (which was reported as ~75% higher among pregnant women compared to non-pregnant controls).33,101,102 However, the temporal changes of plasma total homocysteine throughout pregnancy are less clear. Of n=8 studies which evaluated plasma concentrations of total homocysteine throughout all trimesters in healthy pregnant women, most (n=5) reported that concentrations decrease from the first to the second trimester.9,10,31,32,102 Decreasing concentrations in early pregnancy may be explained by increased trans-sulfuration (and irreversible catabolism of homocysteine) at this time.90 Most studies report that plasma concentrations increase from the second to third trimester,10–13,30,32,42,100 despite some conflicting results.9,31,102,103 When interpreting clinical significance of increasing homocysteine into later pregnancy, it is important to note that concentrations >13 µmol/L (cut-off for hyperhomocysteinemia13) in the aforementioned studies were rare, and in those which studied healthy, non-pregnant controls, total plasma homocysteine remained lower in pregnant women than controls at all timepoints.12,30,100 Predictors of circulating homocysteine include methyl nutrients which catalyze remethylation of homocysteine to methionine via the folate (folate, vitamin B12) and betaine dependent pathways.104 Folic acid supplementation may decrease total homocysteine concentrations,105 however in a folate-replete population (typically countries with folic acid food fortification), vitamin B12 is a stronger determinant of total homocysteine than folate.106 Additionally, in a 2019 174 retrospective Canadian cohort (biobanked samples of n=723 healthy pregnant women in the first and second trimester), maternal serum betaine and vitamin B12, but not folate, were inversely associated with plasma total homocysteine.29 In countries without folic acid fortification and high rates of low folate and methionine status, betaine appears to be the strongest predictor of plasma total homocysteine, particularly into the later stages of pregnancy (>20 weeks of gestation).31,107 The summary of studies on plasma methionine, total homocysteine, and cysteine concentrations across trimesters of a healthy pregnancy are presented in Table 3. 175 Table 3: Methionine, total homocysteine, cysteine Reference Country/ Region Study Design (Time Points) n Micronutrient Supplement1 Fasted2 Outcome Variable Method of Measurement Trend Across Trimesters3 Cikot et al. (2001)9 Netherlands Prospective longitudinal (6, 10, 20, 32 GW) 16 No No tHcy (plasma) HPLC Decrease from 1st to 2nd trimester, then consistent Murphy et al. (2002)102 Spain Prospective longitudinal (8, 20, 32 GW) 93 No Yes tHcy (plasma) Immunoassay Decrease Kalhan et al. (2003)95 USA Case-control, prospective (~11, ~34 GW) 12 P, 7 NP Yes Yes Methionine (plasma) HPLC Consistent; not significantly different vs. NP Velzing-Aarts et al. (2005)31 Curaçao Prospective longitudinal (9, 16, 20, 24, 28, 32, 36 GW) 50 Yes Yes tHcy (plasma) Immunoassay Decrease from 1st to 2nd trimester, then consistent Milman et al. (2006)11 Denmark Prospective longitudinal (18, 32, 39 GW) 404 No No tHcy (plasma) LC-MS Increase Viskova et al. (2007)99 Czech Republic Prospective longitudinal (19-21, 30-32 GW) 62 Yes No tHcy (plasma) HPLC Consistent Cysteine (plasma) HPLC Decrease Dasarathy et al. (2010)90 USA Case-control, cross-sectional (~11, ~20, ~30 GW) 10 P, 8 NP Yes Yes tHcy (plasma) HPLC Consistent; lower in only 3rd trimester vs. NP Cysteine (plasma) HPLC Decrease from 1st to 2nd trimester, then consistent; lower vs. NP Methionine (plasma) HPLC Consistent; lower in all trimesters vs. NP 176 Úbeda et al. (2011)42 Spain Prospective longitudinal (15, 24, 32 GW) 154 Unknown Yes tHcy (serum) Ion-capture assay Decrease in the 2nd trimester, increase from 2nd to 3rd Parazzini et al. (2011)43 Italy Prospective longitudinal (8-12, 16, 22 GW) 291 Yes No tHcy (serum) Immunoassay Consistent Fernàndez-Roig et al. (2013)32 Spain Prospective longitudinal (12, 15, 24-27, 34 GW) 574 Yes Yes* tHcy (plasma) LC-MS/MS Decrease 1st to 2nd trimester, increase from 2nd to 3rd Wu et al. (2013)30 Canada Case-control, prospective (16, 36 GW) 264 P, 88 NP Yes Yes tHcy (plasma) LC-MS/MS Increase; decreased vs. NP Cysteine (plasma) LC-MS/MS Consistent; decreased vs. NP Methionine (plasma) LC-MS/MS Consistent; decreased vs. NP Lindsay et al. (2015)96 USA Prospective longitudinal (~13, ~20, ~30 GW) 160 Yes Yes Cysteine (plasma) LC-MS/MS Decrease from 1st to 2nd trimester, then consistent Methionine (plasma) LC-MS/MS Consistent Visentin et al. (2016)13 Canada (PREFORM Study) Prospective longitudinal (12-16, 28-42 [delivery] GW) 368 Yes No tHcy (plasma) Enzymatic assay Increase Choi et al. (2016)103 Korea Prospective longitudinal (1st, 2nd, 3rd trimesters) 278 Yes Yes tHcy (serum) LC-MS/MS Consistent Varsi et al. (2018)12 Norway Case-control, prospective (18, 28, 36 GW) 114 P, 123 NP No No tHcy (plasma) GC-MS Consistent in the 2nd trimester, increase from 2nd to 3rd; decreased vs. NP 177 1Majority (>50%) of participants consumed micronutrient supplements (methyl nutrients typically included in prenatal micronutrient supplements: folic acid, vitamin B12, vitamin B6, vitamin B2) 2Fasted for ≥8 hours prior to blood sample collection; *fasting time not specified 3Unless specified, “increase/decrease/consistent” is the trend throughout the full study period; trimesters defined as: 1st: ~1-12 weeks of gestation; 2nd: ~13-27 weeks of gestation; 3rd: ~28-40 weeks of gestation GW: gestational weeks; LC-MS/MS: liquid chromatography tandem mass-spectrometry; HPLC: high performance liquid chromatography; GC-MS, gas chromatography-mass spectrometry; tHcy: total homocysteine; P: pregnant; NP: non-pregnant Mujica-Coopman et al. (2019)10 Brazil Prospective longitudinal (5-13, 20-26, 30-36 GW) 186 No Yes tHcy (plasma) HPLC Decrease from 1st to 2nd trimester, increase from 2nd to 3rd Cysteine (plasma) HPLC Decrease from 1st to 2nd trimester, increase from 2nd to 3rd Methionine (plasma) LC-MS/MS Decrease from 1st to 2nd trimester, then consistent Mujica-Coopman et al. (2019)29 Canada Retrospective (biobanked samples) (8-13, 14-20 GW) 723 Unknown No Methionine (serum) LC-MS/MS Decrease tHcy (serum) LC-MS/MS Decrease Yang et al. (2019)100 China Case-control prospective (1st, 2nd, 3rd trimesters) 354 P, 120 NP Unknown Yes tHcy (serum) Enzymatic cycling Consistent from 1st to 2nd trimester, increase 2nd to 3rd; decreased vs. NP Brosnan et al. (2019)72 Canada (PREFORM Study) Prospective longitudinal (12-16, 28-42 [delivery] GW) 215 Yes Yes Methionine (plasma) HPLC Consistent Gilley et al. (2020)7 Guatemala Prospective longitudinal (12, 34 GW) 134 Yes No Cysteine (bloodspot) LC-MS/MS Decrease Methionine (bloodspot) LC-MS/MS Consistent 178 Clinical significance - Fetal growth: Adequate methionine intake is critical for fetal growth, as methionine partitioning between protein synthesis and transmethylation (which supports growth via production of numerous 1C outputs) is influenced by intake of total protein, methionine, and other methyl donors.108 As pregnancy progresses, inadequate protein intake prioritizes methionine for protein synthesis at the expense of transmethylation, thus altering the overall flux of this 1C pathway.109 In a previous cohort of n=24 Indian women, the prevalence of small-for-gestational-age and low birth weight was 37.5% and 25%, respectively; protein deficiency, resulting in reduced transmethylation, was considered a major contributing factor.109 Additionally, numerous observational trials in both nutritionally sufficient and insufficient populations have reported an association between elevated maternal plasma total homocysteine and low birth weight,43,110–114 including one trial using Mendelian randomization that suggested a causal role.115 In a large Canadian pregnancy cohort (Ottawa and Kingston Birth Cohort; n=7587), a 5 µmol/L increase in plasma total homocysteine concentration was associated with 76% increased odds of small-for-gestational-age (OR=1.76, 95% CI 1.25-2.46).114 As such, maternal plasma homocysteine may be a useful screening biomarker for fetal growth restriction in early pregnancy.116 Collectively, this highlights the importance of establishing pregnancy-specific plasma reference ranges and ensuring adequate intake of homocysteine predictors, including vitamin B12 and betaine (via choline). Serine, glycine Serine and glycine are dispensable amino acids which are significant contributors to the 1C pool, but temporal changes of plasma concentrations throughout pregnancy are unclear. Maternal plasma concentrations of serine and glycine may decrease during a healthy pregnancy 179 (predominantly in early pregnancy) in women both receiving96 and not receiving10 prenatal vitamins, however, there are also reports that levels remain consistent.72,90,95 Prospective case-control studies have found that plasma concentrations of serine and glycine are lower in pregnant women than non-pregnant controls.90,95 Contrary to findings in animal models (sheep), transport of serine and glycine from maternal plasma and placental-fetal cycling of serine and glycine appears to be minimal.117,118 Conversion of serine to glycine in human placenta is limited, as placental activity of serine hydroxy-methyltransferase in humans is low.117 In a recent study by Holm et al.118 (Norway), blood samples were collected from the incoming and outgoing vessels on the maternal and fetal sides of the placenta during planned caesarean deliveries in healthy pregnant women (n=179). After adjusting for water transfer across the placenta, no significant placental uptake of serine from the fetus or maternal plasma, or transfer of placental glycine to the fetus, was found.118 Further, Holm et al.118 reports a significant uteroplacental release of glycine into maternal circulation. Glycine is transported by System L, a placental exchanger which uses energy created by the outward movement of non-essential amino acids, which in turn drives uptake of essential amino acids by other placental transport systems (e.g., System A), against its concentration gradient; thus, output of glycine may be critical for uptake of essential amino acids into the placenta.118 Overall, it is hypothesized that fetal serine and glycine demands may be predominantly met by fetal serine synthesis and subsequent metabolism to glycine within the fetus (with excess glycine produced by the fetus being exported as described above).117,118 180 Only a few studies to date have investigated serine and glycine metabolism in pregnancy. Stable-isotope tracers were used by Hsu et al.119 to quantify kinetics of both serine (15N-serine) and glycine (2,2-2H2-glycine) in early and late pregnancy, in healthy adolescent (16-17 years, n=11) and adult (≥18 years, n=10) women in Jamaica. No change in maternal serine flux was found with advancing gestation; rate of glycine flux and synthesis of glycine from serine declined, but only in the adolescent group, suggesting age-related differences.119 These findings corroborate earlier evidence from this research group of decreased ability to maintain glycine flux in pregnant adolescents,120 and further suggest decreased de novo synthesis of glycine from serine in adolescents as the underlying mechanism.119 A recent study using the indicator amino acid oxidation method has reported novel findings regarding maternal glycine requirements.121 Following an overnight fast, n=17 healthy pregnant women (Canada) were randomized to dietary glycine intakes ranging from 5-100 mg/kg/day and were provided protein meeting the EAR in pregnancy (0.88 g/kg/day6). No significant responses were found at 20-29 weeks gestation, but at 30-40 weeks gestation, lower dietary glycine intake resulted in increased oxidation of the indicator amino acid (L-[1-13C]phenylalanine), suggesting reduced protein synthesis, and that glycine may be “conditionally indispensable” at this time.121 These are important findings, but the EAR is estimated to meet requirements of only 50% of the population. Whether these results are generalizable to those meeting the RDA (1.1 g/kg/day6) is uncertain. In populations where pregnant women consume much less protein (as observed in certain South Asian populations122), it is likely that glycine becomes conditionally essential in pregnancy. 181 The summary of studies on serine, glycine concentrations across trimesters of a healthy pregnancy are presented in Table 4. 182 Table 4: Serine, glycine 1Majority (>50%) of participants consumed micronutrient supplements (methyl nutrients typically included in prenatal micronutrient supplements: folic acid, vitamin B12, vitamin B6, vitamin B2); 2Fasted for ≥8 hours prior to blood sample collection 3Unless specified, “increase/decrease/consistent” is the trend throughout the full study period; trimesters defined as: 1st: ~1-12 weeks of gestation; 2nd: ~13-27 weeks of gestation; 3rd: ~28-40 of weeks gestation GW: gestational weeks; LC-MS/MS: liquid chromatography tandem mass-spectrometry; HPLC: high performance LC; P: pregnant; NP: non-pregnant Reference Country/ Region Study Design (Time Points) n Micronutrient Supplemented1 Fasted2 Outcome Variable Method of Measurement Trend Across Trimesters3 Kalhan et al. (2003)95 USA Case-control, prospective (~11, ~34 GW) 12 P, 7 NP Yes Yes Serine (plasma) HPLC Consistent; decreased vs. NP Glycine (plasma) HPLC Consistent; decreased vs. NP Dasarathy et al. (2010)90 USA Case-control, Cross-sectional (~11, ~20, ~30 GW) 10 P, 8 NP Yes Yes Serine (plasma) HPLC Consistent; decreased vs. NP Glycine (plasma) HPLC Consistent; decreased vs. NP Thame et al. (2010)120 Jamaica Prospective longitudinal (~13, ~28 GW) 16 (8 adolescents, 8 adults) Unknown Yes Serine (plasma) HPLC Increased (adults); consistent (adolescents) Glycine (plasma) HPLC Consistent (adults); decreased (adolescents) Lindsay et al. (2015)96 USA Prospective longitudinal (~13, ~20, ~30 GW) 160 Yes Yes Glycine (plasma) LC-MS/MS Decrease Serine (plasma) LC-MS/MS Decrease from 1st to 2nd trimester, then consistent Mujica-Coopman et al. (2019)10 Brazil Prospective longitudinal (5-13, 20-26, 30-36 GW) 186 No Yes Serine (plasma) LC-MS/MS Decrease from 1st to 2nd trimester, then consistent Glycine (plasma) LC-MS/MS Decrease from 1st to 2nd trimester, then consistent Brosnan et al. (2019)72 Canada (PREFORM Study) Prospective longitudinal (12-16, 28-42 [delivery] GW) 215 Yes Yes Serine (plasma) HPLC Consistent Glycine (plasma) HPLC Consistent Gilley et al. (2020)7 Guatemala Prospective longitudinal (12, 34 GW) 134 Yes No Serine (bloodspot) LC-MS/MS Decrease 183 Conclusion This narrative review presents a comprehensive summary of the normal 1C flux in pregnancy, corresponding temporal changes in maternal plasma methyl nutrients, and potential implications for dietary requirements and infant clinical outcomes. Similar temporal changes appear to occur among plasma concentrations of many methyl nutrients, including B-vitamins, choline, betaine, methionine, and cysteine. Given the diverse geographical regions summarized (as noted in Tables 1-4), these findings support conclusions from Gilley et al.7 that temporal changes are relatively generalizable for several methyl nutrients among healthy pregnant women. Future research is needed to understand the kinetics of maternal-placental-fetal transfer of methyl nutrients and dietary intake needed to support 1C metabolism in these three separate compartments. Simply preventing maternal plasma decline may not be necessary if ultimately, values remain within an “acceptable range” to support placental and fetal growth and no clinical harm is experienced by the mother due to deficiency. Thus, establishment and validation of reference ranges for plasma methyl nutrient concentrations in pregnancy is urgently required. Overall, clinical trials and large-scale epidemiological research are needed to determine the dose-response effect of methyl nutrient supplementation and clinical significance for maternal and child health, potentially necessitating revision of perinatal dietary recommendations or re-formulation of prenatal vitamin supplements. 184 References 1. Ducker G, Rabinowitz J. One-carbon metabolism in liver health and disease. Cell Metab. 2017;25:27–49. 2. Kalhan SC. One carbon metabolism in pregnancy: Impact on maternal, fetal and neonatal health. Mol Cell Endocrinol. 2016;435:48–60. 3. Clare CE, Brassington AH, Kwong WY, Sinclair KD. One-carbon metabolism: Linking nutritional biochemistry to epigenetic programming of long-term development. Annu Rev Anim Biosci. 2019;7:263–87. 4. Glier MB, Green TJ, Devlin AM. Methyl nutrients, DNA methylation, and cardiovascular disease. Mol Nutr Food Res. 2014 Jan 1;58(1):172–82. 5. Klatt KC, Smith ER, Barberio MD. Toward a more stable understanding of pregnancy micronutrient metabolism. Am J Physiol Endocrinol Metab. 2021;321:E260–3. 6. Health Canada. Dietary Reference Intakes [Internet]. Ottawa, ON, Canada; 2010 [cited 2022 Dec 1]. Available from: https://www.canada.ca/en/health-canada/services/food-nutrition/healthy-eating/dietary-reference-intakes/tables/reference-values-vitamins-dietary-reference-intakes-tables-2005.html 7. Gilley SP, Weaver NE, Sticca EL, Jambal P, Palacios A, Kerns ME, et al. Longitudinal changes of one-carbon metabolites and amino acid concentrations during pregnancy in the Women First Maternal Nutrition Trial. Curr Dev Nutr. 2019;4(1):1–11. 8. Hambidge KM, Westcott JE, Garcés A, Figueroa L, Goudar SS, Dhaded SM, et al. A multicountry randomized controlled trial of comprehensive maternal nutrition supplementation initiated before conception: The Women First trial. Am J Clin Nutr. 2019;109:457–69. 185 9. Cikot RJLM, Steegers-Theunissen RPM, Thomas CMG, de Boo TM, Merkus HMWM, Steegers EAP. Longitudinal vitamin and homocysteine levels in normal pregnancy. Br J Nutr. 2001;85:49–58. 10. Mujica-coopman MF, Farias DR, Franco-sena AB, Vaz JS, Kac G, Lamers Y. Maternal plasma pyridoxal-5-phosphate concentration is inversely associated with plasma cystathionine concentration across all trimesters in healthy pregnant women. J Nutr. 2019;149:1354–62. 11. Milman N, Byg KE, Hvas AM, Bergholt T, Eriksen L. Erythrocyte folate, plasma folate and plasma homocysteine during normal pregnancy and postpartum: A longitudinal study comprising 404 Danish women. Eur J Haematol. 2006;76:200–5. 12. Varsi K, Ueland PM, Torsvik IK, Bjørke-Monsen AL. Maternal serum cobalamin at 18 weeks of pregnancy predicts infant cobalamin status at 6 months - A prospective, observational study. J Nutr. 2018;148:738–45. 13. Visentin CE, Masih SP, Plumptre L, Schroder TH, Sohn KJ, Ly A, et al. Low serum vitamin B-12 concentrations are prevalent in a cohort of pregnant Canadian women. J Nutr. 2016;146:1035–42. 14. Gregory JF, Caudill MA, Jeffery Opalko F, Bailey LB. Kinetics of folate turnover in pregnant women (second trimester) and nonpregnant controls during folic acid supplementation: Stable-isotopic labeling of plasma folate, urinary folate and folate catabolites shows subtle effects of pregnancy on turnover of folate pools. J Nutr. 2001;131:1928–37. 15. Greibe E, Andreasen BH, Lildballe DL, Morkbak AL, Hvas AM, Nexo E. Uptake of cobalamin and markers of cobalamin status: A longitudinal study of healthy pregnant 186 women. Clin Chem Lab Med. 2011;49(11):1877–82. 16. Obeid R, Munz W, Jäger M, Schmidt W, Herrmann W. Biochemical indexes of the B vitamins in cord serum are predicted by maternal B vitamin status. Am J Clin Nutr. 2005;82(1):133–9. 17. Layden AJ, O’Brien KO, Pressman EK, Cooper EM, Kent TR, Finkelstein JL. Vitamin B12 and placental expression of transcobalamin in pregnant adolescents. Placenta. 2016;45:1–7. 18. Plumptre L, Masih SP, Sohn KJ, Kim D, Visentin CE, Ly A, et al. Suboptimal maternal and cord plasma pyridoxal 5′ phosphate concentrations are uncommon in a cohort of Canadian pregnant women and newborn infants. Matern Child Nutr. 2018;14:e12467. 19. Plumptre L, Masih SP, Ly A, Aufreiter S, Sohn K-J, Croxford R, et al. High concentrations of folate and unmetabolized folic acid in a cohort of pregnant Canadian women and umbilical cord blood. Am J Clin Nutr. 2015;102:848–57. 20. Solanky N, Requena Jimenez A, D’Souza SW, Sibley CP, Glazier JD. Expression of folate transporters in human placenta and implications for homocysteine metabolism. Placenta. 2010;31:134–43. 21. Schneider H, Miller RK. Receptor-mediated uptake and transport of macromolecules in the human placenta. Int J Dev Biol. 2010;54:367–75. 22. Contractor S, Shane B. Blood and urine levels of vitamin B6 in the mother and fetus before and after loading of the mother with vitamin B6. Am J Obs Gynecol. 1970;107(4):635–40. 23. Chang SJ. Adequacy of maternal pyridoxine supplementation during pregnancy in relation to the vitamin B6 status and growth of neonates at birth. J Nutr Sci Vitaminol. 187 1999;45(4):449–58. 24. Lumeng L, Cleary R, Wagner R, Yu P, Li T. Adequacy of vitamin B6 supplementation during pregnancy: A prospective study. Am J Clin Nutr. 1976;29(12):1376–83. 25. Schuster K, Bailey L, Mahan C. Effect of maternal pyridoxine-HCl supplementation on the vitamin B6 status of mother and infant and on pregnancy outcome. J Nutr. 1984;114(5):977–88. 26. Cleary R, Lumeng L, Li T. Maternal and fetal plasma levels of pyridoxal phosphate at term: Adequacy of vitamin B6 supplementation during pregnancy. Am J Obs Gynecol. 1975;121(1):25–8. 27. Giugliani E, Jorge S, Goncalves A. Serum vitamin B12 levels in parturients, in the intervillous space of the placenta and in full-term newborns and their interrelationships with folate levels. Am J Clin Nutr. 1985;41(2):330–5. 28. Health Canada. Prenatal Nutrition Guidelines for Health Professionals: Folate. 2009. 29. Mujica-Coopman MF, Tan A, Schroder TH, Sinclair G, Vallance HD, Lamers Y. Serum betaine and dimethylglycine are higher in South Asian compared with European pregnant women in Canada, with betaine and total homocysteine inversely associated in early and midpregnancy, independent of ethnicity. J Nutr. 2019;149(12):2145–55. 30. Wu BTF, Innis SM, Mulder KA, Dyer RA, King DJ. Low plasma vitamin B-12 is associated with a lower pregnancy-associated rise in plasma free choline in Canadian pregnant women and lower postnatal growth rates in their male infants. Am J Clin Nutr. 2013;98(5):1209–17. 31. Velzing-Aarts F V., Holm PI, Fokkema MR, Van Der Dijs FP, Ueland PM, Muskiet FA. Plasma choline and betaine and their relation to plasma homocysteine in normal 188 pregnancy. Am J Clin Nutr. 2005;81(6):1383–9. 32. Fernàndez-Roig S, Cavallé-Busquets P, Fernandez-Ballart JD, Ballesteros M, Berrocal-Zaragoza MI, Salat-Batlle J, et al. Low folate status enhances pregnancy changes in plasma betaine and dimethylglycine concentrations and the association between betaine and homocysteine. Am J Clin Nutr. 2013;97:1252–9. 33. Bae S, West AA, Yan J, Jiang X, Perry CA, Malysheva O, et al. Vitamin B-12 status differs among pregnant, lactating, and control women with equivalent nutrient intakes. J Nutr. 2015;145:1507–14. 34. Lamers Y, Macfarlane AJ, O’Connor DL, Fontaine-Bisson B. Periconceptional intake of folic acid among low-risk women in Canada: Summary of a workshop aiming to align prenatal folic acid supplement composition with current expert guidelines. Am J Clin Nutr. 2018;108:1357–68. 35. Schrott R, Murphy SK. Folic acid throughout pregnancy: Too much? Am J Clin Nutr. 2018;107(4):497–8. 36. Sulistyoningrum D, Green T, Palmer D, Sullivan T, Wood S, Makrides M, et al. Study protocol for a randomised controlled trial evaluating the effect of folic acid supplementation beyond the first trimester on maternal plasma unmetabolised folic acid in late gestation. BMJ Open. 2020;10:e040416. 37. McNulty B, McNulty H, Marshall B, Ward M, Molloy AM, Scott JM, et al. Impact of continuing folic acid after the first trimester of pregnancy: Findings of a randomized trial of folic acid supplementation in the second and third trimesters. Am J Clin Nutr. 2013;98:92–8. 38. Tam C, O’Connor D, Koren G. Circulating unmetabolized folic acid: Relationship to 189 folate status and effect of supplementation. Obstet Gynecol Int. 2012;(485179):1–17. 39. Bailey SW, Ayling JE. The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake. PNAS. 2009;106(36):15424–9. 40. Bahous RH, Jadavji NM, Deng L, Cos M, Lu J, Malysheva O, et al. High dietary folate in pregnant mice leads to pseudo-MTHFR deficiency and altered methyl metabolism, with embryonic growth delay and short-term memory impairment in offspring. Hum Mol Genet. 2017;26(5):888–900. 41. Maruvada P, Stover PJ, Mason JB, Bailey RL, Davis CD, Field MS, et al. Knowledge gaps in understanding the metabolic and clinical effects of excess folates/folic acid: A summary, and perspectives, from an NIH workshop. Am J Clin Nutr. 2020;112(5):1390–403. 42. Ubeda N, Reyes L, Gonzalez-Medina A, Alonso-Aperte E, Varela-Moreiras G. Physiologic changes in homocysteine metabolism in pregnancy: A longitudinal study in Spain. Nutrition. 2011;27:925–30. 43. Parazzini F, Chiaffarino F, Ricci E, Improta L, Monni G. Homocysteine, red cell, and plasma folate concentrations and birth weight in Italian women: Results from a prospective study. J Matern Neonatal Med. 2011;24(3):427–31. 44. Shane B. Folate and vitamin B12 metabolism: Overview and interaction with riboflavin, vitamin B6, and polymorphisms. Food Nutr Bull. 2008;29(2):S5–16. 45. Relton CL, Pearce MS, Parker L. The influence of erythrocyte folate and serum vitamin B12 status on birth weight. Br J Nutr. 2005;93:593–9. 46. Scholl T, Hediger H, Schall J, Khoo C, Fischer R. Dietary and serum folate: Their 190 influence on the outcome of pregnancy. Am J Clin Nutr. 1996;63:520–5. 47. Timmermans S, Jaddoe VWV, Hofman A, Steegers-Theunissen RPM, Steegers EAP. Periconception folic acid supplementation, fetal growth and the risks of low birth weight and preterm birth: The Generation R Study. Br J Nutr. 2009;102:777–85. 48. Fekete K, Berti C, Trovato M, Lohner S, Dullemeijer C, Souverein OW, et al. Effect of folate intake on health outcomes in pregnancy: A systematic review and meta-analysis on birth weight, placental weight and length of gestation. Nutr J. 2012;11:75. 49. McGee M, Bainbridge S, Fontaine-Bisson B. A crucial role for maternal dietary methyl donor intake in epigenetic programming and fetal growth outcomes. Nutr Rev. 2018;76(6):469–78. 50. Pastor-Valero M, Navarrete-Muoz EM, Rebagliato M, Iñiguez C, Murcia M, Marco A, et al. Periconceptional folic acid supplementation and anthropometric measures at birth in a cohort of pregnant women in Valencia, Spain. Br J Nutr. 2011;105(9):1352–60. 51. Alwan N, Greenwood D, Simpson N, McArdle H, Cade J. The relationship between dietary supplement use in late pregnancy and birth outcomes: A cohort study in British women. BJOG. 2010;117:821–9. 52. Behere R V., Deshmukh AS, Otiv S, Gupte MD, Yajnik CS. Maternal vitamin B12 status during pregnancy and its association with outcomes of pregnancy and health of the offspring: A systematic review and implications for policy in India. Front Endocrinol (Lausanne). 2021;12:619176. 53. Gadgil M, Joshi K, Pandit A, Otiv S, Joshi R, Brenna JT, et al. Imbalance of folic acid and vitamin B12 is associated with birth outcome: An Indian pregnant women study. Eur J Clin Nutr. 2014;68:726–9. 191 54. Dwarkanath P, Barzilay J, Thomas T, Thomas A, Bhat S, Kurpad V. High folate and low vitamin B-12 intakes during pregnancy are associated with small-for-gestational-age infants in South Indian women: A prospective observational cohort study. Am J Clin Nutr. 2013;98:1450–8. 55. Burren KA, Savery D, Massa V, Kok RM, Scott JM, Blom HJ, et al. Gene – environment interactions in the causation of neural tube defects: Folate deficiency increases susceptibility conferred by loss of Pax3 function. Hum Mol Genet. 2008;17(23):3675–85. 56. Blom HJ. Folic acid, methylation and neural tube closure in humans. Birth Defects Res A Clin Mol Teratol. 2009;85:295–302. 57. Pai YJ, Leung K, Savery D, Hutchin T, Prunty H, Heales S, et al. Glycine decarboxylase deficiency causes neural tube defects and features of non-ketotic hyperglycinemia in mice. Nat Commun. 2015;6:7388. 58. Sudiwala S, Castro SCP De, Leung K, Brosnan JT, Brosnan ME, Mills K, et al. Formate supplementation enhances folate-dependent nucleotide biosynthesis and prevents spina bifida in a mouse model of folic acid- resistant neural tube defects. Biochimie. 2016;126:63–70. 59. Leung KY, Pai YJ, Chen Q, Santos C, Calvani E, Sudiwala S, et al. Partitioning of one-carbon units in folate and methionine metabolism is essential for neural tube closure. Cell Rep. 2017;21:1795–808. 60. Momb J, Lewandowski JP, Bryant JD, Fitch R, Surman DR, Vokes SA. Deletion of Mthfd1l causes embryonic lethality and neural tube and craniofacial defects in mice. PNAS. 2013;110(2):549–54. 61. Bryant JD, Sweeney SR, Sentandreu E, Shin M, Momb J, Tiziani S, et al. Deletion of the 192 neural tube defect–associated gene Mthfd1l disrupts one-carbon and central energy metabolism in mouse embryos. J Biol Chem. 2018;293(16):5821–33. 62. Brosnan ME, Tingley G, MacMillan L, Harnett B, Pongnopparat T, Marshall JD, et al. Plasma formate is greater in fetal and neonatal rats compared with their mothers. J Nutr. 2020;150(5):1068–75. 63. Ray JG, Blom HJ. Vitamin B12 insufficiency and the risk of fetal neural tube defects. Q J Med. 2003;96:289–95. 64. Molloy AM, Kirke PN, Troendle JF, Burke H, Sutton M, Brody LC, et al. Maternal vitamin B12 status and risk of neural tube defects in a population with high neural tube defect prevalence and no folic acid fortification. Pediatrics. 2009;123(3):917–23. 65. Shaw GM, Carmichael SL, Yang W, Selvin S, Schaffer DM. Periconceptional dietary intake of choline and betaine and neural tube defects in offspring. Am J Epidemiol. 2004;160(2):102–9. 66. Shaw GM, Finnell RH, Blom HJ, Carmichael SL, Vollset SE, Yang W, et al. Choline and risk of neural tube defects in a folate-fortified population. Epidemiology. 2009;20(5):714–9. 67. Shoob HD, Sargent RG, Thompson SJ, Best RG, Drane JW, Tocharoen A. Dietary methionine is involved in the etiology of neural tube defect-affected pregnancies in humans. J Nutr. 2001;131:2653–8. 68. Graham A, Brender JD, Sharkey JR, Zhu L, Felkner M, Suarez L, et al. Dietary methionine intake and neural tube defects in Mexican-American women. Birth Defects Res. 2010;88(6):451–7. 69. Brosnan ME, Brosnan JT. Formate: The neglected member of one-carbon metabolism. 193 Annu Rev Nutr. 2016;36:369–88. 70. Washburn SE, Caudil MA, Malysheva O, Macfarlane AJ, Behan NA, Harnett B, et al. Formate metabolism in fetal and neonatal sheep. Am J Physiol - Endocrinol Metab. 2015;308(10):E921–7. 71. Kim J, Lei Y, Guo J, Kim S, Wlodarczyk BJ, Cabrera RM, et al. Formate rescues neural tube defects caused by mutations in Slc25a32. PNAS. 2018;115(18):4690–5. 72. Brosnan JT, Plumptre L, Brosnan ME, Pongnopparat T, Masih SP, Visentin CE, et al. Formate concentrations in maternal plasma during pregnancy and in cord blood in a cohort of pregnant Canadian women: Relations to genetic polymorphisms and plasma metabolites. Am J Clin Nutr. 2019;110(5):1131–7. 73. Brosnan JT, Mills JL, Ueland PM, Shane B, Fan R, Chiu CY, et al. Lifestyle, metabolite, and genetic determinants of formate concentrations in a cross-sectional study in young, healthy adults. Am J Clin Nutr. 2018;107(3):345–54. 74. Pietzke M, Meiser J, Vazquez A. Formate metabolism in health and disease. Mol Metab. 2020;33:23–37. 75. Visentin CE, Masih S, Plumptre L, Malysheva O, Nielsen DE, Sohn K, et al. Maternal choline status but not fetal genotype, influences cord plasma choline. J Nutr. 2015;145:1491–7. 76. Caudill MA. Pre- and postnatal health: Evidence of increased choline needs. J Am Diet Assoc. 2010;110:1198–206. 77. Yan J, Jiang X, West AA, Perry CA, Malysheva O V., Devapatla S, et al. Maternal choline intake modulates maternal and fetal biomarkers of choline metabolism in humans. Am J Clin Nutr. 2012;95(5):1060–71. 194 78. Resseguie M, Song J, Niculescu MD, Costa K-A, Randall TA, Zeisel SH. Phosphatidylethanolamine N-methyltransferase (PEMT) gene expression is induced by estrogen in human and mouse primary hepatocytes. FASEB J. 2007;21:2622–32. 79. Yan J, Jiang X, West AA, Perry CA, Malysheva O V., Brenna JT, et al. Pregnancy alters choline dynamics: Results of a randomized trial using stable isotope methodology in pregnant and nonpregnant women. Am J Clin Nutr. 2013;98(6):1459–67. 80. Wiedeman AM, Barr SI, Green TJ, Xu Z, Innis SM, Kitts DD. Dietary choline intake: Current state of knowledge across the life cycle. Nutrients. 2018;10:1513. 81. Park EI, Garrow TA. Interaction between dietary methionine and methyl donor intake on rat liver betaine-homocysteine methyltransferase gene expression and organization of the human gene. J Biol Chem. 1999;274(12):7816–24. 82. Dilger RN, Garrow TA, Baker DH. Betaine can partially spare choline in chicks but only when added to diets containing a minimal level of choline. J Nutr. 2007;137:2224–8. 83. Wu BTF, Dyer RA, King DJ, Richardson KJ, Innis SM. Early second trimester maternal plasma choline and betaine are related to measures of early cognitive development in term infants. PLoS One. 2012;7(8):1–8. 84. Masih SP, Plumptre L, Ly A, Berger H, Lausman AY, Croxford R, et al. Pregnant Canadian women achieve recommended intakes of one-carbon nutrients through prenatal supplementation but the supplement composition, including choline, requires reconsideration. J Nutr. 2015;145(8):1824–34. 85. Zeisel SH, Niculescu MD. Perinatal choline influences brain structure and function. Nutr Rev. 2006;64(4):197–203. 86. McKee SE, Reyes TM. Effect of supplementation with methyl-donor nutrients on 195 neurodevelopment and cognition: Considerations for future research. Nutr Rev. 2018;76(7):497–511. 87. Oshida K, Shimizu T, Takase M, Tamura Y, Shimizu T, Yamashiro Y. Effects of dietary sphingomyelin on central nervous system myelination in developing rats. Pediatr Res. 2003;53(4):589–93. 88. Klatt KC, Mcdougall MQ, Malysheva O V., Brenna JT, Roberson MS, Caudill MA. Reproductive state and choline intake influence enrichment of plasma lysophosphatidylcholine-DHA: A post hoc analysis of a controlled feeding trial. Br J Nutr. 2019;122(11):1221–9. 89. Caudill MA, Strupp BJ, Muscalu L, Nevins JEH, Canfield RL. Maternal choline supplementation during the third trimester of pregnancy improves infant information processing speed: A randomized, double-blind, controlled feeding study. FASEB J. 2018;32:2172–80. 90. Dasarathy J, Gruca LL, Bennett C, Parimi PS, Duenas C, Marczewski S, et al. Methionine metabolism in human pregnancy. Am J Clin Nutr. 2010;91:357–65. 91. Kalhan SC, Bier DM. Protein and amino acid metabolism in the human newborn. Annu Rev Nutr. 2008;28:389–410. 92. Toboła-Wróbel K, Pietryga M, Dydowicz P, Napierała M, Brązert J, Florek E. Association of oxidative stress on pregnancy. Oxid Med Cell Longev. 2020;6398520:1–12. 93. Lu SC. Glutathione synthesis. Biochim Biophys Acta. 2013;1830(5):3143–53. 94. Rook D, Te Braake FWJ, Schierbeek H, Longini M, Buonocore G, Van Goudoever JB. Glutathione synthesis rates in early postnatal life. Pediatr Res. 2010;67(4):407–11. 95. Kalhan SC, Gruca LL, Parimi PS, Brien AO, Dierker L, Burkett E, et al. Serine 196 metabolism in human pregnancy. Am J Physiol Endocrinol Metab. 2003;284:733–40. 96. Lindsay KL, Hellmuth C, Uhl O, Buss C, Wadhwa PD, Koletzko B, et al. Longitudinal metabolomic profiling of amino acids and lipids across healthy pregnancy. PLoS One. 2015;10(12):1–20. 97. Elango R. Methionine nutrition and metabolism: Insights from animal studies to inform human nutrition. J Nutr. 2020;150:2518S-2523S. 98. Robinson JL, Bartlett RK, Harding S V., Randell EW, Brunton JA, Bertolo RF. Dietary methyl donors affect in vivo methionine partitioning between transmethylation and protein synthesis in the neonatal piglet. Amino Acids. 2016;48:2821–30. 99. Viskova H, Vesela K, Janosikova B, Krijt J, Visek JA, Calda P. Plasma cysteine concentrations in uncomplicated pregnancies. Fetal Diagn Ther. 2007;22(4):254–8. 100. Yang Y, Jiang H, Tang A, Xiang Z. Changes of serum homocysteine levels during pregnancy and the establishment of reference intervals in pregnant Chinese women. Clin Chim Acta. 2019;489:1–4. 101. Walker MC, Smith GN, Perkins SL, Keely EJ, Garner PR. Changes in homocysteine levels during normal pregnancy. Am J Obs Gynecol. 1999;180:660–4. 102. Murphy MM, Scott JM, Mcpartlin JM, Fernandez-ballart JD. The pregnancy-related decrease in fasting plasma homocysteine is not explained by folic acid supplementation, hemodilution, or a decrease in albumin in a longitudinal study. Am J Clin Nutr. 2002;76:614–9. 103. Choi R, Choi S, Lim Y, Cho YY, Kim HJ, Kim SW, et al. A prospective study on serum methylmalonic acid and homocysteine in pregnant women. Nutrients. 2016;8:797. 104. Melse-Boonstra A, Holm PI, Ueland PM, Olthof M, Clarke R, Verhoef P. Betaine 197 concentration as a determinant of fasting total homocysteine concentrations and the effect of folic acid supplementation on betaine concentrations. Am J Clin Nutr. 2005;81:1378–82. 105. Holmes VA, Wallace JMW, Alexander HD, Gilmore WS, Bradbury I, Ward M, et al. Homocysteine is lower in the third trimester of pregnancy in women with enhanced folate status from continued folic acid supplementation. Clin Chem. 2005;51(3):629–34. 106. Macfarlane AJ, Greene-finestone LS, Shi Y. Vitamin B-12 and homocysteine status in a folate-replete population: Results from the Canadian Health Measures Survey. Am J Clin Nutr. 2011;94:1079–87. 107. Wallace JMW, Bonham MP, Strain JJ, Duffy EM, Robson PJ, Ward M, et al. Homocysteine concentration, related B vitamins, and betaine in pregnant women recruited to the Seychelles Child Development Study. Am J Clin Nutr. 2008;87(2):391–7. 108. Randunu RS, Bertolo RF. The effects of maternal and postnatal dietary methyl nutrients on epigenetic changes that lead to non-communicable diseases in adulthood. Int J Mol Sci. 2020;21:1–17. 109. Kurpad A V., Anand P, Dwarkanath P, Hsu JW, Thomas T, Devi S, et al. Whole body methionine kinetics, transmethylation, transulfuration and remethylation during pregnancy. Clin Nutr. 2014;33(1):122–9. 110. Yajnik CS, Deshpande SS, Panchanadikar A V., Naik SS, Deshpande JA, Coyaji KJ, et al. Maternal total homocysteine concentration and neonatal size in India. Asia Pac J Clin Nutr. 2005;14(2):179–81. 111. Vollset S, Refsum H, Irgens L, Emblem B, Tverdal A, Gjessing H, et al. Plasma total homocysteine, pregnancy complications, and adverse pregnancy outcomes: The 198 Hordaland Homocysteine Study. Am J Clin Nutr. 2000;71:962–8. 112. Lindblad B, Zaman S, Martin H, Ekstrom A, Amu S, Holmgren A, et al. Folate, vitamin B12, and homocysteine levels in South Asian women with growth-retarded fetuses. Acta Obs Gynecol Scand. 2005;84:1055–61. 113. Takimoto H, Mito N, Umegaki K, Ishiwaki A, Kusama K, Abe S, et al. Relationship between dietary folate intakes, maternal plasma total homocysteine and B-vitamins during pregnancy and fetal growth in Japan. Eur J Nutr. 2007;46(5):300–6. 114. Chaudhry SH, Taljaard M, Macfarlane AJ, Gaudet LM, Smith GN, Rodger M, et al. The determinants of maternal homocysteine in pregnancy: Findings from the Ottawa and Kingston Birth Cohort. BMC Pregnancy Childbirth. 2019;19(75). 115. Yajnik CS, Chandak GR, Joglekar C, Katre P, Bhat DS, Singh SN, et al. Maternal homocysteine in pregnancy and offspring birthweight: Epidemiological associations and Mendelian randomization analysis. Int J Epidemiol. 2014;43(5):1487–97. 116. Gaccioli F, Aye ILMH, Sovio U, Charnock-Jones DS, Smith GCS. Screening for fetal growth restriction using fetal biometry combined with maternal biomarkers. Am J Obstet Gynecol. 2018;218(2):S725–37. 117. Lewis RM, Godfrey KM, Jackson AA, Cameron IT, Hanson MA. Low serine hydroxymethyltransferase activity in the human placenta has important implications for fetal glycine supply. J Clin Endocrinol Metab. 2005;90(3):1594–8. 118. Holm MB, Bastani NE, Holme AM, Zucknick M, Jansson T, Refsum H, et al. Uptake and release of amino acids in the fetal-placental unit in human pregnancies. PLoS One. 2017;12:e0185760. 119. Hsu JW, Thame MM, Gibson R, Baker TM, Tang GJ, Chacko SK, et al. Unlike pregnant 199 adult women, pregnant adolescent girls cannot maintain glycine flux during late pregnancy because of decreased synthesis from serine. Br J Nutr. 2016;115:759–63. 120. Thame M, Fletcher H, Baker T, Jahoor F. Comparing the in vivo glycine fluxes of adolescent girls and adult women during early and late pregnancy. Br J Nutr. 2010;104:498–502. 121. Rasmussen BF, Ennis MA, Dyer RA, Lim K, Elango R. Glycine, a dispensable amino acid, is conditionally indispensible in late stages of human pregnancy. J Nutr. 2021;151(2):361–9. 122. Bandyopadhyay S, Shivakumar N, Kurpad A V. Protein intakes of pregnant women and children in India—protein quality implications. Matern Child Nutr. 2020;16(S3):1–11. 200 Appendix B: Recruitment of pregnant women to clinical research Acknowledgement: A version of this appendix has been published. Cochrane KM, Hutcheon JA, Karakochuk CD. Strategies for improving recruitment of pregnant women to clinical research: An evaluation of social media versus traditional offline methods in Vancouver, Canada. Digital Health 2022; 8:1-10. Summary The objective of this study was 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. 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, enrollments) were recorded. Linear regression was used to explore the association between metrics and dollars spent per campaign. In total, n=481 inquiries were received: n=51 (11%) via offline methods and n=430 (89%) via social media. Enrollees (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 enrollee 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% versus 8%). The amount spent per campaign was significantly associated with improved 201 clicks and inquiries, but not enrollments. In conclusion, 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 enrollments, which was more dependent on study-specific factors (e.g., time of year, study design). Introduction Participant recruitment is considered one of the greatest obstacles in conducting clinical research and has been reported as the most significant contributor to randomized trial delays.1,2 It has been estimated that nearly 80% of pharmaceutical drug trials in the United States fail to meet recruitment timelines, and in a review of 114 clinical trials in the United Kingdom, only 31% met their original recruitment goal.3,4 In addition to increased costs and potential loss of funding, inadequate recruitment may cause reduction in data quality, poor demographic diversity and generalizability, underpowered analyses and ultimately, failure to complete the trial.5 Strong recruitment planning, which is tailored for a specific population and utilizes evidence-based strategies, can greatly increase the likelihood of meeting enrollment targets.2,5 There are numerous unique challenges in recruiting pregnant women.6,7 Medical clinics are often the primary recruitment site, with strategies including physician referrals and distributing printed materials such as posters or brochures.7–9 These strategies (referred to hereinafter as ‘offline’ methods) are labour-intensive and require established rapport between research and clinic personnel. Additionally, it can be difficult to target women at the appropriate gestational age, 202 particularly those in very early pregnancy, as first prenatal appointments typically occur at 8-12 weeks gestation. Participant diversity is inherently limited by the clinic location and to those within relatively close proximity.10,11 As such, perinatal research often has poor geographical and demographic diversity, and those in earlier pregnancy are often underrepresented.7,12 Social media encompasses various websites and applications (e.g., Facebook, Instagram, Twitter), which allow users to create and share information and participate in social networking.13 Use of these platforms has grown tremendously in recent years, with 1.9 billion global daily Facebook users reported in 2021.14 This growth was exacerbated with onset of the global COVID-19 pandemic and a requirement to shift activities online (a change hypothesized to persist post-COVID-19).15,16 Thus, use of social media for participant recruitment is an increasingly viable and attractive option. In a 2020 systematic review and meta-analysis of 61 studies, social media was significantly more efficient than offline methods for participant recruitment (incidence rate ratio = 4.17, 95% CI 1.12-15.59).2 Shorter term (generally <30 days), intermittent campaigns appeared to be more successful, but optimal timing is not well described.2 However, offline recruitment had a significantly higher conversion rate (percentage of screened participants who ultimately enrolled).2 A 2021 systematic review found that social media reached more participants than offline methods, but only ~20% of those who inquired via social media enrolled.17 Cost estimates vary greatly by individual studies, but both reviews reported that recruitment via social media is generally more cost effective than use of offline methods.2,17 Facebook’s advertising platform allows researchers to create a customized advertisement, targeted to reach a specific population, while specifying the total dollars spent (lifetime budget) 203 over a select period of time (referred to as a ‘campaign’). An objective is specified for each campaign, with options that include: ‘reach’ (aims to show the advertisement to the maximum number of people), ‘messages’ (encourages people to interact with you via messaging), ‘engagement’ (increases the number of likes, page comments, etc.) and others. Placement of advertisements can be divided between various Facebook platforms, including Facebook and Instagram. ‘Campaign budget optimization’ and ‘automatic placement’ can be used to monitor the lifetime budget of a campaign and allocate the placement of advertisements to achieve the best results based on the specified objective. Use of social media for recruitment of pregnant women in various types of research (clinical trials, surveys and epidemiological studies) has been explored.12,18–24 In two of these studies, pregnant women were recruited for Canadian-based clinical trials.12,18 First, Shere et al. posted study information for free on various social media platforms (Facebook, Twitter) and found a 12-fold increase in recruitment rate as compared to offline methods.12 Alternatively, Adam et al. ran paid Facebook campaigns (26 non-consecutive days), while concurrently using offline methods over seven months.18 Facebook was more cost effective and efficient (rate of recruitment approximately nine times greater), and recruited women earlier in their pregnancy, than offline methods.18 Whether demographic characteristics of participants reached varies by recruitment method is unclear. Group differences by recruitment strategy is an important consideration, as this could bias results.25 However, improving diversity in research would improve generalizability of findings. Conflicting reports on how social media affects participant diversity have been published.17,19,25 204 We enrolled n=60 healthy pregnant women in a clinical trial in Vancouver, Canada, using a series of paid Facebook campaigns and traditional offline methods.26 The aims of this study were twofold: 1) to evaluate and compare results of recruitment via social media and offline methods (including demographic characteristics, rate of recruitment and direct costs); and 2) to explore optimization of intermittent online campaigns (including dollars spent, campaign duration, photo used and settings in Facebook ad design (e.g., attribution setting)). Methods Study population Full study details are published elsewhere,26 but in brief, n=60 pregnant women were enrolled to a proof-of-concept pilot study of supplementation with folic acid or (6S)-5-methyltetrahydrofolic acid for 16 weeks of pregnancy, starting at 8-21 weeks gestation. The study was randomized and double-blinded. Inclusion criteria included: singleton pregnancy, living in Vancouver, Canada, ≤21 weeks’ gestation and 19-42 years of age. Exclusion criteria included: having a medical condition, currently taking medications or lifestyle factors (current smoking, alcohol consumption, recreational drug use, a pre-pregnancy body mass index (BMI) ≥30kg/m2) which may interfere with B-vitamin metabolism,27 and those medium to high risk for development of a neural tube defect-effected pregnancy.27 Social media recruitment A Facebook account was created for the ‘UBC Folate in Pregnancy Study’ to run a series of nine campaigns throughout October 2019 to January 2021. Campaign timelines ranged from 10 to 18 days with various lifetime budgets (CA$50, CA$75 and CA$100). Overall, campaigns ran for 205 103 days (~15 weeks) and CA$675 was spent. Campaigns were targeted to reach women aged 19-42 years, living in Vancouver, Canada (within a 25-mile radius), who match the following interests: pregnancy, nutrition and pregnancy, prenatal nutrition, motherhood and fit pregnancy. Advertisements were automatically optimized by Facebook using campaign budget optimization and automatic placement. The default attribution setting was used for all campaigns. Attribution setting is a defined period of time in which interactions with the advertisement (clicks or views) are used to inform campaign optimization. For the first eight campaigns, this setting was 28-day click, 1-day view. In January 2021, Facebook’s default setting changed to 7-day click, 1-day view, so this was used for the final (‘9th’) campaign. The objective chosen for all campaigns was ‘messages’. Those who saw the ad were prompted to click ‘send message’, which would automatically connect them to study staff via Facebook Messenger. Messages were monitored by the study’s research coordinator, who provided study information, fielded questions, assessed eligibility and enrolled participants. The primary text in the advertisement indicated current recruitment for pregnant women less than 21 weeks gestation for a nutrition study at UBC. Ad text was approved by Facebook advertising prior to going live, meeting requirements of the privacy policy (e.g., to ensure that personal attributes, such as pregnancy state, are not directly inferred). Participants could inquire using the research coordinator’s contact information (displayed in the ad) or by clicking ‘send message’. See Figure 1a for an example of social media advertisements. Three different photos (Figure 1b: ‘A’, ‘B’ and ‘C’) were subbed into the advertisement throughout campaigns (purchased at Can Stock Photo). Photos similar to those used by Adam et al. were selected, as they recruited pregnant individuals for a similar Canadian clinical nutrition trial.18 Photos were 206 evaluated with A/B testing; this allows two options for a variable in a campaign (e.g., photo) to run simultaneously to determine which performs best. When running an A/B test, the total budget is allocated 50/50 to each ‘version’ of the advertisement (e.g., for a $100 lifetime budget, $50 is allocated to advertisements with each photo). Photos featured in each campaign were as follows: campaigns ‘1-5’, photo ‘A’; campaign ‘6’, photo ‘B’; campaign ‘7’, A/B test of photos ‘A’ and ‘B’; campaign ‘8’, A/B test of photos ‘A’ and ‘C’; campaign ‘9’, photo ‘C’. Figure 1: Social media campaigns and photos ‘A’, ‘B’ and ‘C’ 207 Offline recruitment Various offline recruitment methods were used throughout September 2019 to December 2020 (16 months or ~64 weeks total). The predominant strategy included distribution of posters (which featured Photo ‘A’) across several locations in Vancouver, including the University of British Columbia (UBC), the BC Children’s and Women’s Hospital and Research Institute, medical and prenatal clinics and various retail and community establishments for pregnant women (prenatal classes, fitness studios, maternity clothing stores). Posters were distributed monthly and refreshed as needed across ~50 different locations from September 2019 to March 2020. Overall, 350 posters (CA$300 printing costs) were distributed. Upon onset of COVID-19 (April 2020 to December 2020), study information was shared electronically with accepting medical clinics and community establishments. Finally, study details were presented at the BC Women’s Hospital Obstetrics & Gynaecology Grand Rounds and the Midwife Grand Rounds in November 2019 and September 2020, respectively, to inform clinicians about the study and how to refer interested patients. Statistical analyses Aim 1: Descriptive statistics (mean ± SD, median and IQR, or counts with percentages) were used to present characteristics of enrolled participants and gestational weeks (as available) of those who inquired. Differences between groups were assessed using a two-sample t-test (for continuous, normally distributed variables) or Pearson chi-square test (for categorial variables). Fisher’s exact test was used for categorical variables when cell frequencies were <5. Direct cost (not including labour) per enrolled participant was determined by dividing the total cost of each method by the number of participants enrolled. Rate of recruitment (inquiries/week and 208 enrollments/week) for each recruitment strategy was determined by dividing the total number of participants who inquired and enrolled by the total weeks. The conversion rate for each method (enrollments/inquiries) was calculated to determine the number of inquiries who enrolled. Odds of enrollment after inquiring in each group was evaluated with an odds ratio (OR) and 95% CI. Aim 2: The following metrics were recorded for each social media campaign: reach (number of different people who saw the ad), impressions (the number of times the ad was on a screen; unlike reach, this may include multiple views by the same person), clicks, cost per click, inquiries (sent within Messenger and via e-mail), cost per inquiry, enrollments and cost per enrollment. Reach, impressions and clicks were available via Facebook analytics, while inquiries and enrollments were recorded by the research coordinator. Linear regression was used to explore the association between campaign metrics, including association of lifetime budget per campaign (controlling for campaign duration) with clicks, inquiries and enrollments, and association of clicks and inquiries with enrollments. A/B photo tests were evaluated using metrics available via Facebook analytics (reach, impressions, clicks). P-values < .05 were considered statistically significant. Statistical analyses were performed using Stata 16.1 (Stata Corp, Texas, USA). Results Aim 1: Comparison of recruitment via social media versus offline methods In total, n=481 inquiries were received from September 2019 to January 2021 to participate in the clinical trial. See Figure 2 for participant flow chart. Briefly, n=430 (89%) inquired after seeing a social media ad and n=51 (11%) inquiries were a result of offline methods. 209 Figure 2: Participant flow chart Gestational weeks were reported by n=251 (52%) women upon inquiry. Of these, n=208 (83%) inquired via social media and n=43 (17%) via offline methods. Mean ± SD gestational weeks at inquiry was comparable between the social media and offline groups: 13.2 ± 5.6 and 13.3 ± 4.7 weeks gestation, respectively (P = .96). Demographic characteristics of enrolled participants (n=60) are presented in Table 1. Briefly, n=36 (60%) and n=24 (40%) enrolled following inquiries from social media and offline methods, respectively. For offline methods, n=15 (63%) inquired after seeing a study poster and n=9 (37%) inquired following other offline strategies (clinician recommendation, word of mouth). Although not statistically significant, a higher proportion of those recruited via offline methods had University-level education and an annual 210 household income of >CA$100,000 per year (88% and 58%, respectively), as compared to those recruited via social media (78% and 47%, respectively). Table 1. Demographic characteristics of enrolled participants by recruitment strategy Characteristic Recruitment Method P value Offline Social Media Number of participants (n, %) 24 (40) 36 (60) Age, mean (SD) 33 (3.2) 33 (3.6) .67 Ethnicity (n, %) .99 European 14 (58) 20 (56) South, East & Southeast Asian 5 (21) 8 (22) Hispanic/Latino 3 (13) 4 (11) Middle Eastern 1 (4) 1 (3) Biracial 1 (4) 3 (8) Education (n, %) .82 Highschool 0 (0) 2 (5) College 3 (12) 6 (17) Undergraduate 11 (46) 14 (39) Graduate 10 (42) 14 (39) Household Income (CA$)/year (n, %) .23 <20,000 0 (0) 1 (3) 20,000-50,000 4 (17) 2 (6) 50,000-100,000 6 (25) 15 (44) >100,000 14 (58) 16 (47) Pre-pregnancy BMI, mean (SD) 22.2 (2.6) 23.4 (2.8) .11 Weeks gestation at inquiry, mean (SD) 13.5 (4.7) 13.0 (5.0) .72 Parity (n, %) .81 Nulliparous 18 (75) 26 (72) Multiparous 6 (25) 10 (28) Age, pre-pregnancy BMI and weeks gestation at inquiry were compared with a two-sample t-test. Ethnicity, education and household income were compared with a Fisher’s exact test. Parity was compared with a Pearson chi-square test. Direct cost per enrolled participant was CA$19 (CA$675 / n=36) in those recruited via social media and CA$13 (CA$300 / n=24) in those recruited via offline methods. The overall rate of inquiries per week was 28.7 (n=430 / 15 wks) using social media and 0.80 (n=51 / 64 wks) using 211 offline methods. The overall rate of enrollments per week was 2.4 (n=36 / 15 wks) using social media and 0.38 (n=24 / 64 wks) using offline methods. The conversion rate (inquiries which enrolled) was 8% in the social media group (n=36 / n=430) and 47% in the offline methods group (n=24 / n=51). The odds of enrollment via offline methods was 5.6 times higher than after seeing an online ad (OR=5.6, 95% CI 2.9-10.5). Aim 2: Explore optimization of social media campaigns A full summary of metrics from the social media campaigns can be found in Table 2. Briefly, campaigns reached a median of 2970 women (IQR 2872-4025), made a median of 6130 (IQR 4951-7547) impressions and resulted in a median of 300 clicks (IQR 287-450). Impressions, reach and clicks are inherently correlated (P < .001), as impressions include all women who were reached, but also captures the number of times the advertisement was on a screen, and clicks are recorded following a click of the advertisement. 212 Table 2: Summary of social media campaign metrics. a7-day attribution setting used (all other campaigns used 28-day attribution setting) bPhoto A/B testing used: two campaigns run simultaneously, $50/campaign cReach: number of different people who saw the advertisements dImpressions: number of times the ad was on a screen (may include multiple views by the same person) eClicks: number of people who clicked on the advertisement fInquires: number of people who inquired via Facebook messaging or by contacting the research coordinator gConversion rate: enrollments / inquiries x 100 hCost per click: Lifetime budget (CAD) / clicks iCost per inquiry: Lifetime budget (CAD) / inquiries jCost per enroll: Lifetime budget (CAD) / enrollments Campaign Total days Lifetime budget (CAD) Reachc Impressionsd Clickse Inquiriesf Enrollments Conversion rate (%)g Cost per click (CAD)h Cost per inquiry (CAD)i Cost per enroll (CAD)j 1 (Oct 2019) 10 100 4025 7547 447 49 4 8 0.22 2.04 25 2 (Nov 2019) 10 50 2690 4730 200 18 5 27 0.25 2.77 10 3 (Dec 2019) 10 75 2900 4951 261 20 1 5 0.29 3.75 75 4 (Jan 2020) 10 50 2872 5027 291 29 5 17 0.17 1.72 10 5 (Feb 2020) 10 50 3463 6130 287 34 2 5 0.17 1.47 25 6 (Mar 2020) 15 50 4476 7967 300 30 0 n/a 0.17 1.66 n/a 7 (Jul 2020) 10 100b 6082 10,441 594 79 8 10 0.17 1.27 12 8 (Oct 2020) 10 100b 3189 6713 450 35 4 11 0.22 2.86 25 9 (Jan 2021)a 18 100 5914 12,029 1190 136 7 5 0.08 0.74 14 213 Total clicks, inquiries and enrollments per campaign, and the cost analysis of each metric, are presented in Figure 3. Lifetime budget (CA$50 to CA$100), controlling for campaign duration (10 to 18 days), was positively associated with the number of clicks (β = 7.01, 95% CI 2.25 to 11.77; P = .01) and inquiries (β = .82, 95% CI .08 to 1.5; P = .04), but not the number of enrollments (P = .19). Although increased clicks (P = .09) and inquiries (P = .06) tended to result in increased enrollments, the associations did not reach significance. Figure 3. Summary of clicks, inquiries and enrollments per campaign and cost analysis by each metric (dollar amounts in CA$) Metrics for photos ‘A’ and ‘B’ in the first A/B test (campaign ‘7’, July 2020) were: reach 3010 and 3072; impressions 5550 and 4891; clicks 314 and 280. Photo ‘A’ was more successful, with Facebook analytics predicting a 92% chance of achieving the same result upon test repeat. In the second A/B test (campaign ‘8’, October 2020), metrics for photos ‘A’ and ‘C’ were: reach 1542 214 and 1647; impressions 3305 and 3408; clicks 203 and 247. Photo ‘C’ was more successful, with Facebook analytics predicting a 68% chance of achieving the same result upon test repeat. Discussion Our findings support that using social media improves the efficiency (speed of recruitment) and overall effectiveness (number of participants enrolled) of recruitment compared to traditional offline methods. Additionally, we provide novel data regarding the demographic characteristics of pregnant women recruited for clinical research via social media compared to offline methods and strategies for optimizing online campaigns. Contrary to previous findings, we did not find a significant difference in gestational weeks among pregnant women recruited via social media versus offline methods. Adam et al. recruited pregnant women (n=70) for a similar clinical trial, and reported that those recruited via social media (n=25) were significantly earlier in their pregnancy than those recruited via offline methods (n=45), finding a mean ± SD gestational weeks of 12.6 ± 3.7 and 14.7 ± 3.8 in social media and offline methods groups, respectively.18 We examined gestational age in a much larger cohort (n=251) and did not find a difference between groups (13.2 ± 5.6 and 13.3 ± 4.7 via social media and offline methods, respectively). In agreement with Adam et al., it does appear that most women inquire for participation early in their first trimester (once >12 weeks gestation). National survey data in the United States among n=11,928 pregnant women reported that the mean gestational age of pregnancy awareness is 5.5 weeks.28 Exact reasons why women tended to wait until >12 weeks to inquire is unknown; however, it is unsurprising. As previously stated, first prenatal ultrasound appointments to confirm fetal viability typically occur at 8-12 215 weeks gestation, thus, it is reasonable that women would not inquire about pregnancy-related clinical research until after this time. This data provides evidence for feasible gestational age ranges for future research. We did not find significant differences in ethnicity between women enrolled via social media (n=36) versus online methods (n=24). This is contrary to findings from Admon et al., who recruited a more racially diverse group of pregnant women based in the United States for an online survey using social media (n=759) as compared to clinic-based recruitment (n=190).19 Differences in study design (recruitment for a clinical trial versus online survey) may have contributed to these findings. Overall, we note that members of a visible minority were moderately under-represented in our study. According to 2016 Canadian Census data, 52% of Vancouver’s population are members of a visible minority (predominantly of South, East and Southeast Asian ancestry)29; 43% (n=26) of participants in the current study identified as a visible minority. Although we selected photos showing only a pregnant abdomen, we note that the lack of racial diversity in the photos may have contributed to this. Future studies should include photos with diverse skin tones. In agreement with Admon et al., we found that women recruited via offline methods tended to have higher levels of University-level education and reported a higher annual household income (>CA$100,000 per year) than those recruited via social media. However, these findings were not statistically significant. Placement of study posters around the UBC campus and the BC Children’s and Women’s Hospital Research Institute (where study visits took place) may explain these findings, as many with University-level education work and study at these locations. These 216 are important findings, as universities and research institutes are often where clinical trials take place, thus, are a common location for poster distribution. As per 2016 Canadian Census data, 36% of females (ages 25-64) in Vancouver, Canada, have University-level education, and 20% have an annual household income below the national poverty line (~CA$40,000/year for a couple with one child).29,30 Thus, although diversity (as per education and income) was improved in those recruited via social media, overall, participants in both groups were above provincial averages. It is possible that social media did reach a more diverse range of individuals, but the effect was diluted due to the location of study visits and bias towards those who live and work nearby. Expanding geographical reach of offline methods, including recruitment at various non-profit organizations (e.g., food banks, community centers) and medical centers in lower-income neighborhoods, may be effective strategies to increase the reach of those with lower socioeconomic status.31 Additionally, clinical researchers should offer study visits at a variety of locations whenever feasible. Ultimately, social media was more effective than offline recruitment, resulting in n=36 versus n=24 participants enrolled, respectively. There was also a stark difference in efficiency, as the participants were recruited approximately six times faster via social media than offline methods. This is similar to previous findings.2,18 A critical and unique consideration in this study is the effect of COVID-19. Offline strategies were shifted from manual distribution of study materials throughout the community (September 2019 to March 2020) to sharing them electronically (April 2020 to December 2020). Enrollments per month decreased from approximately two per month to one per month upon shifting offline strategies after March 2020. A factor to consider for the efficiency of social media recruitment is that although social media ads were active for 217 only 15 weeks, they spanned 64 weeks in total (October 2019-January 2021). However, inquiries and enrollments only occurred during active social media campaigns phases. Regardless, it is unknown if we would have obtained the same rate of recruitment via social media if the 15 weeks were run continuously. Intermittent campaigns have been reported as more effective than continuous ones,2 possibly due to minimizing saturation of the target population, and in our case, access to a ‘new wave’ of women becoming pregnant. Spacing of our online campaigns was affected by COVID-19, as spacing increased from one per month to every three to four months after March 2020. These differences in spacing did not appear to alter campaign performance. Overall, optimal spacing between campaigns requires further research. Cost estimates require careful interpretation. Although the direct cost per enrolled participant was CA$19 and CA$13 via social media and offline methods, respectively, costs in the offline groups only contributed to those recruited via posters (n=15), while the remaining participants in this group (n=9) were recruited following other offline strategies. Thus, estimation of direct costs per enrolled participant via posters only may be more appropriate (CA$300 / n=15 = CA$20/participant). These are similar to direct cost estimates from Adam et al. to a similar nutrition trial: CA$20/participant (social media) and CA$24/participant (offline methods).18 To provide a more comprehensive cost evaluation, we have calculated a post hoc estimation of labour costs in each group: CA$2120 (offline methods) and CA$500 (social media); see breakdown in Table 3. Using these estimates, total cost for recruitment via offline methods and social media was CA$2420 ($2120 + $300) and CA$1175 ($500 + $675), and cost per participant was CA$101 ($2420 / n=24) and CA$33 ($1175 / n=36), respectively. Overall, 218 offline methods were much more labour intensive than social media recruitment; these differences are reflected in the total cost. Table 3: Breakdown of post-hoc estimation of labour costs. Recruitment Method Cost (CAD)a Offline Social Media Poster development & printing 4 hours -- 80 Manual distribution of postersb 80 hours -- 1600 Electronic distribution of postersc 16 hours -- 320 Grand rounds presentation preparation 4 hours -- 80 Presentations (Nov 2019; Sept 2020) 2 hours -- 40 Initial development of online ads -- 2 hours 40 Individual campaign set upd -- 5 hours 100 Responding to inquiriese -- 18 hours 360 Total hours 106 hours 25 hours Total cost (CAD) $2120 $500 aCost estimates are based on $20 CAD/hour compensation bSeptember 2019 to March 2020; estimation of 10 hours/month cApril 2020 to December 2020; estimation of 2 hours/month dApproximately 30-minutes of set-up per campaign (x 9 campaigns) eApproximately 10-minutes per active campaign day (x 103 days) We provide insight for the optimization of social media advertisements, which is lacking in current literature. Performance of campaigns (clicks, inquiries) consistently improved over time. Numerous factors likely contributed to this. First, automatic optimization was used for all campaigns, which continually improves the advertisement placement and budget allocation. Campaigns may also experience organic growth over time, as the Facebook page receives more ‘likes’ and ‘shares’. The final (‘9th’) campaign was also the longest (18 days), used the maximum budget (CA$100) and used Photo ‘C’, which was the most successful photo as per A/B testing. Facebook Advertising suggests choosing bright, eye-catching, high-resolution photos directly relevant to the brand which convey the message clearly, and avoiding photos with text or small 219 details. Finally, Facebook’s default attribution setting was changed from 28-day click, 1-day view to 7-day click, 1-day view in January 2021. This meant that clicks from a shorter period of time were used to inform campaign optimization, thus targeting those who appeared interested (e.g., clicked the advertisement) more frequently. This shorter attribution setting was likely more effective for our campaign durations, which ranged from 10-18 days. Overall, clicks, cost per click, inquiries and cost per inquiry were improved by ~50% in the final campaign. Although the factors discussed above contributed to optimization of clicks and inquiries, this was not associated with a significant increase in enrollments in social media campaigns. Enrollments per campaign appeared to be more influenced by the time of year. For example, number of enrollees in campaigns ‘3’ (December 2019; n=1) and ‘6’ (March 2020; n=0) were notably reduced. This was likely explained by reduced availability of participants around the holidays and onset of the COVID-19 pandemic. Additionally, the highest enrollments occurred in campaign ‘7’ (July 2020; n=8), which was a time in Vancouver when strict COVID-19 restrictions were lifted, and women may have felt more motivated to leave their homes. Thus, researchers should critically evaluate the time of year when planning social media campaigns. The lower conversion rate of social media versus offline methods (8% versus 47%) should not be used to conclude that offline methods were more effective than social media, given that ultimately, social media resulted in a greater number of enrollees, more quickly. However, improving the conversion rate would help to further optimize social media recruitment. A factor likely impacting conversion rate of social media is ease of inquiry. After seeing an online ad, one can immediately message the researcher on the device they are currently using. Additionally, 220 individuals may have clicked on the ad due to initial interest in the photo or campaign aesthetic, without reading the ad details and reflecting on whether they are truly interested in enrolling. Alternatively, inquiring following offline methods requires manually recording contact information and then later inquiring. Thus, these individuals are likely more serious about enrolling at the time of inquiry, presumably explaining the greater conversion rate in this group. We note various strengths and limitations of the current study. First, we provide numerous tangible strategies for researchers to implement in future online recruitment initiatives. Additionally, this study adds novel evidence which can inform recruitment of a traditionally hard-to-reach population: pregnant women. We had relatively large sample sizes, particularly for analyses of gestational weeks (n=251), inquiry analyses and calculation of conversion rate (n=481) and for assessing metrics of online campaigns (see Supplementary Table 2). However, the lack of significance found between groups for education and income may be due to lack of power (as these analyses included only those who enrolled: n=60), given that differences appear meaningful. The association of campaign budget with clicks and inquiries should be interpreted with caution. Although we found that lifetime budget per campaign was significantly positively associated with clicks and inquiries, this was likely inflated by the final campaign, which had the greatest performance for numerous reasons (e.g., optimization over time, organic growth, attribution setting), in addition to use of the maximum budget of CA$100. Thus, the association of budget on campaign performance (clicks, inquiries) may have been exaggerated, as we could not control for these other factors that improved this campaign's performance. Additionally, it is possible that this small range of dollars spent (CA$50 to CA$100) is not enough to contribute to, or to detect a true difference in, performance metrics by budget. 221 Conclusion Transitioning recruitment methods from offline to online may help researchers to meet recruitment targets more quickly, reducing the labour-intensive burden of traditional offline strategies. Additionally, cost-effectiveness can be improved in future online campaigns by implementing the optimization strategies discussed in this paper. These insights may help to increase clicks and inquiries in future online recruitment initiatives for various study designs. However, this may not be directly correlated to enrollments in clinical research, which appears to depend more on study-specific factors such as time of year and study design (e.g., where study visits are located). Optimization of participant recruitment is vitally important for research success, and ultimately, translation of science into real-world change. 222 References 1. Gul RB, Ali PA. Clinical trials: The challenge of recruitment and retention of participants. J Clin Nurs. 2010;19:227–33. 2. Brogger-Mikkelsen M, Zarqa M, Zibert J, Andersen A, Thomsen S. Online patient recruitment in clinical trials: Systematic review and meta-analysis. J Med Internet Res. 2020;22(11):e22179. 3. Mcdonald A, Knight R, Campbell M, Entwistle V, Grant A, Cook J, et al. What influences recruitment to randomised controlled trials? A review of trials funded by two UK funding agencies. Trials. 2006;7(9):1–8. 4. Johnson O. An evidence-based approach to conducting clinical trial feasibility assessments. Clin Invest. 2015;5(5):491–9. 5. Fogel D. Factors associated with clinical trials that fail and opportunities for improving the likelihood of success: A review. Contemp Clin Trials Commun. 2018;11:156–64. 6. Wada K, Evans MK, de Vrijer B, Nisker J. Clinical research with pregnant women: Perspectives of pregnant women, health care providers, and researchers. Qual Health Res. 2018;28(13):2033–47. 7. Frew PM, Saint-Victor DS, Isaacs MB, Kim S, Swamy GK, Sheffield JS, et al. Recruitment and retention of pregnant women into clinical research trials: An overview of challenges, facilitators, and best practices. Clin Infect Dis. 2014;59(Suppl 7):S400–7. 8. Denne SC. Including pregnant women in clinical research: Time to overcome the barriers. Pediatr Res. 2019;86:554–5. 9. Manca DP, O’Beirne M, Lightbody T, Johnston DW, Dymianiw DL, Nastalska K, et al. The most effective strategy for recruiting a pregnancy cohort: A tale of two cities. BMC 223 Pregnancy Childbirth. 2013;13(75):1–7. 10. Sutton EF, Cain LE, Vallo PM, Redman LM. Strategies for successful recruitment of pregnant patients into clinical trials. Obstet Gynecol. 2017;129(3):554–9. 11. Strommer S, Lawrence W, Rose T, Vogel C, Watson D, Bottell JN, et al. Improving recruitment to clinical trials during pregnancy: A mixed methods investigation. Soc Sci Med. 2018;200:73–82. 12. Shere M, Nguyen P, Tam C, Stern S, Kapur B, O’Connor DL, et al. Pregnancy-induced changes in the long-term pharmacokinetics of 1.1 mg vs. 5 mg folic acid: A randomized clinical trial. J Clin Pharmacol. 2015;55(2):159–67. 13. Whitaker C, Stevelink S, Fear N. The use of Facebook in recruiting participants for health research purposes: A systematic review. J Med Internet Res. 2017;19(8):e290. 14. Facebook. Facebook Q3 2021 results. Menlo Park, California; 2021. 15. Porpiglia F, Checcucci E, Autorino R, Amparore D, Cooperberg M, Fiacarra V, et al. Traditional and virtual congress meetings during the COVID-19 pandemic and the post-COVID-19 era: Is it time to change the paradigm? Eur Urol. 2020;78:301–3. 16. Barnes SJ. Information management research and practice in the post-COVID-19 world. Int J Inf Manage. 2020;55:102175. 17. Ellington M, Connelly J, Clayton P, Lorenzo CY, Collazo-Velazquez C, Trak-Fellermeier MA, et al. Use of Facebook, Instagram, and Twitter for recruiting healthy participants in nutrition-, physical activity–, or obesity-related studies: A systematic review. Am J Clin Nutr. 2021;00:1–20. 18. Adam LM, Manca DP, Bell RC. Can facebook be used for research? Experiences using facebook to recruit pregnant women for a randomized controlled trial. J Med Internet Res. 224 2016;18(9):e250. 19. Admon L, Haefner JK, Kolenic GE, Chang T, Davis MM, Moniz MH. Recruiting pregnant patients for survey research: A head to head comparison of social media-based versus clinic-based approaches. J Med Internet Res. 2016;18(12):e326. 20. Gelder MM, Belt TH, Engelen LJ, Hooijer R, Bredie SJ, Roeleveld N. Google AdWords and Facebook ads for recruitment of pregnant women into a prospective cohort study with long-term follow-up. Matern Child Health J. 2019;23:1285–91. 21. Ibarra JL, Agas JM, Lee M, Pan JL, Buttenheim AM. Comparison of online survey recruitment platforms for hard-to-reach pregnant smoking populations: Feasibility study. J Med Internet Res. 2018;7(4):e:101. 22. Wise LA, Rothman KJ, Mikkelsen EM, Stanford JB, Wesselink AK, McKinnon C, et al. Design and conduct of an internet-based preconception cohort study in North America: Pregnancy study online. Paediatr Perinat Epidemiol. 2015;29(4):360–71. 23. Arcia A. Facebook advertisements for inexpensive participant recruitment among women in early pregnancy. Heal Educ Behav. 2014;41(3):237–41. 24. Harris ML, Loxton D, Wigginton B, Lucke JC. Recruiting online: Lessons from a longitudinal survey of contraception and pregnancy intentions of young Australian women. Am J Epidemiol. 2015;181(10):737–46. 25. Benedict C, Hahn AL, Diefenbach MA, Ford JS. Recruitment via social media: advantages and potential biases. Digit Heal. 2019;5:1–11. 26. Cochrane KM, Mayer C, Devlin AM, Elango R, Hutcheon JA, Karakochuk CD. Is natural (6S)-5-methyltetrahydrofolic acid as effective as synthetic folic acid in increasing serum and red blood cell folate concentrations during pregnancy? A proof-of-concept pilot study. 225 Trials. 2020;21:380. 27. Douglas Wilson R. SOGC Clinical Practice Guideline: Pre-conception folic acid and multivitamin supplementation for the primary and secondary prevention of neural tube defects and other folic acid-sensitive congenital anomalies. J Obs Gynaecol Can. 2015;37(6):534–49. 28. Branum AM, Ahrens KA. Trends in timing of pregnancy awareness among US women. Matern Child Heal J. 2017;21(4):715–26. 29. City of Vancouver, Social Policy and Projects. Vancouver, City Social Indicators Profile. 2020. 30. First Call BC Child and Youth Advocacy Coalition. BC Child Poverty Report Card. 2020. 31. Côté M, Lapointe A, Laramée C, Lemieux S, Desroches S, Belanger-Gravel A, et al. Beliefs related to participation in a large web-based prospective survey on diet and health among individuals with a low socioeconomic status: Qualitative study. JMIR Form Res. 2019;3(4):e13854. 226 Appendix C: Other considerations for the interpretation of change in maternal folate status Whether the within-person change in folate status throughout the 16-week intervention period was influenced by prior folate supplementation was evaluated. All participants reported folate supplementation prior to the trial; ~60% reported that supplementation was initiated preconceptionally and ~40% reported that it was initiated after pregnancy confirmation. Of those who reported the type of folate or brand of prenatal vitamin (n=56), >90% consumed folic acid; doses of supplemental folate based on the reported brand were 1.0 mg/day (~55%), 0.6 mg/day (~40%), and 0.4 mg/day (~5%); rates of each dose in both intervention groups were similar. Although one may suspect that changing from a 1.0 mg/day dose to a 0.6 mg/day dose would result in a reduced change from baseline to endline, or even a decrease in status, there did not appear to be a pattern for change based on reported pre-study folate supplementation. RBC folate concentrations decreased from baseline to endline in 20-30% of women from each dosing group (1.0 mg, 0.6 mg, 0.4 mg), with no noticeable differences between groups. While this is a bit surprising, inferences for the effect of pre-study folate supplementation are limited, as the dose of folate is based on reported data/prenatal vitamin brands, and the frequency of supplementation prior to the study was not recorded. There were two participants who experienced a large, unexplained change from baseline to endline, both randomized to the (6S)-5-MTHF group. In the first participant, RBC folate concentrations at baseline were >5600 nmol/L, followed by 2004 nmol/L at endline. The lab verified this result in a second run, and no information reported 227 by the participant (supplementation prior to the study, usual diet, dietary folate intake, medical history) can explain this finding; further, their complete blood count (e.g., hematocrit, hemoglobin, RBC count) and serum folate results were unremarkable (serum folate = 88 nmol/L at baseline, increasing by 10 nmol/L from baseline to endline). Ultimately, we question the biological plausibility of this result, or whether there was an issue at the analysis phase, perhaps with sample preparation. In the second participant, baseline serum folate concentrations were >170 nmol/L, followed by a concentration of 71 nmol/L at endline. Again, no baseline information or other markers of folate status can explain or substantiate this large decrease from baseline to endline (RBC folate concentrations were 1204 nmol/L and 1298 nmol/L at baseline and endline, respectively). While fasting was verbally confirmed at each visit, a lack of 3-hour fasting is suspected, or possibly consumption of a large meal/supplementation 3 hours prior to the visit. This is further evidenced by this participant’s corresponding plasma UMFA concentration, which was 69 nmol/L at baseline, suggesting recent intake of a substantial dose of folic acid, and 0.8 nmol/L at endline, conversely suggesting that the recommended fasting took place at this timepoint. Both of these individuals were included in all statistical analyses, as an intention-to-treat approach was chosen, and “outliers” were not pre-defined. Ultimately, we do not believe that this impacted our conclusions, as estimates for a definitive trial are based on crude concentrations at endline, and endline values after 16 weeks of supplementation in each group were explored in multivariable regression models, adjusting for baseline concentrations.
Thesis/Dissertation
eng
Human Nutrition
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Karakochuk, Crystal D.
A comparison of (6S)-5-methyltetrahydrofolic acid vs folic acid and the effects on maternal folate status and the human milk folate profile : evidence from a randomized clinical trial
Text