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

Background Maternal vitamin status preconception and during pregnancy have important consequences for pregnancy outcome and offspring development. Changes in status from preconception to early and late pregnancy and postpartum have been inferred from cross-sectional data, with lower pregnancy concentrations often ascribed to plasma volume expansion, but without truly longitudinal data from preconception through pregnancy and post-delivery, and sparse data on the influence of supplementation. This study characterized longitudinal patterns of maternal vitamin status from preconception, through early and late pregnancy, to 6-months post-delivery, and determined the influence of supplementation. Methods and Findings Between 2015-2017, 1729 UK, Singapore and New Zealand women aged 18-38 years planning conception were recruited from the community to a double-blind controlled trial and randomized to a standard (control) or an intervention supplement preconception and throughout pregnancy. Vitamins common to both supplements were folic acid and β-carotene, with the intervention additionally including riboflavin, vitamins B6, B12 and D in amounts available in over-the-counter supplements, alongside iron, calcium and iodine (control and intervention) and myo-inositol, probiotics and zinc (intervention only). We measured maternal plasma concentrations of B-vitamins, vitamin D and insufficiency/deficiency markers (homocysteine, hydroxykynurenine-ratio, methylmalonic acid), at recruitment and 1-month after commencing intervention preconception, in early and late pregnancy, and post-delivery (6-months after supplement discontinuation). From all timepoint data, we derived standard deviation scores (SDS) to characterize longitudinal changes in controls and differences between control and intervention participants. At recruitment preconception, significant proportions had marginal or low plasma status for folate (29.2% <13.6 nmol/L), riboflavin (7.5% <5 nmol/L, 82.0% ≤26.5 nmol/L), vitamin B12 (9.1% <221 pmol/L) and vitamin D (48.7% <50 nmol/L). Among controls, plasma concentrations showed differing longitudinal patterns from preconception; riboflavin fell through early/late pregnancy, 25-hydroxyvitamin D was unchanged in early pregnancy, and vitamin B6 and B12 concentrations declined through pregnancy, becoming >1 SDS lower than baseline by 28 weeks gestation, with 54.2% developing a low late pregnancy vitamin B6 (pyridoxal 5-phosphate <20 nmol/L). Preconception, the control/intervention groups had similar baseline vitamin concentrations; 1-month after supplement commencement, plasma concentrations became substantially higher in intervention participants; riboflavin by 0.77 SDS (95%CI 0.68-0.87), vitamin B6 1.07 (0.99-1.14), vitamin B12 0.55 (0.46-0.64) and vitamin D 0.51 (0.43-0.60), with the higher levels maintained during pregnancy and marked reduction in insufficiency/deficiency markers (lower homocysteine, hydroxykynurenine-ratio, methylmalonic acid) and the late pregnancy prevalence of vitamin D <50 nmol/L reduced from 35.1% to 8.5%. Plasma vitamin B12 was still higher in the intervention group 6-months post-delivery. Conclusion Significant proportions of preconception women have marginal or low status of folate, riboflavin, vitamin B12 and vitamin D, and many develop markers of vitamin B6 deficiency in late pregnancy. In the absence of supplementation, maternal plasma vitamin concentrations show differing longitudinal patterns from preconception to early and late pregnancy, suggesting plasma volume expansion does not wholly account for lower gestational concentrations. Preconception/pregnancy supplementation in amounts available in over-the-counter supplements substantially reduces the prevalence of deficiency/depletion markers before and during pregnancy, and a higher maternal plasma vitamin B12 was maintained during the recommended lactational period. ### Competing Interest Statement SC, PNB, YSC, WC and KG report grants from Société Des Produits Nestlé S.A. during the conduct of the study, and are co-inventors on patent filings by Nestlé S.A. relating to the NiPPeR intervention or its components. SC, SB, PT, WC and KG are part of an academic consortium that has received grants from Abbott Nutrition, Nestlé S.A., Danone and Benevolent AI Bio Ltd outside the submitted work. SC has received reimbursement and honoraria into her research funds from Nestlé S.A. for speaking at a conference. KG has received reimbursement for speaking at conferences sponsored by companies selling nutritional products. All other authors declare no competing interests. ### Clinical Trial ClinicalTrials.gov - U1111-1171-8056 https://www.clinicaltrials.gov/ct2/show/[NCT02509988][1] ### Funding Statement Public good funding for this investigator-led study is through the UK Medical Research Council (as part of an MRC award to the MRC Lifecourse Epidemiology Unit (MC\_UU\_12011/4)) the Singapore National Research Foundation, National Medical Research Council (NMRC, NMRC/TCR/012-NUHS/2014) the National University of Singapore (NUS) and the Agency of Science, Technology and Research (as part of the Growth, Development and Metabolism Programme of the Singapore Institute for Clinical Sciences (SICS) (H17/01/a0/005) and as part of Gravida, a New Zealand Government Centre of Research Excellence. Funding for provision of the intervention and control drinks and to cover aspects of the fieldwork for the study has been provided by Société Des Produits Nestlé S.A under a Research Agreement with the University of Southampton, Auckland UniServices Ltd, SICS, National University Hospital Singapore PTE Ltd and NUS. KMG is supported by the National Institute for Health Research (NIHR Senior Investigator (NF-SI-0515-10042), NIHR Southampton 1000DaysPlus Global Nutrition Research Group (17/63/154) and NIHR Southampton Biomedical Research Center (IS-BRC-1215-20004)), British Heart Foundation (RG/15/17/3174) and the European Union (Erasmus+ Programme ImpENSA 598488-EPP-1-2018-1-DE-EPPKA2-CBHE-JP). SYC is supported by a Singapore NMRC Clinician Scientist Awards (NMRC/CSA-INV/0010/2016 MOH-CSAINV19nov-0002). The funders had no role in the data collection and analysis, and the decision to submit for publication. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The trial was approved by the United Kingdom, Singapore and New Zealand research ethics services (Southampton: Health Research Authority NRES Committee South Central Research Ethics Committee, reference 15/SC/0142l Singapore: the National Healthcare Group Domain Specific Review Board, reference 2015/00205 New Zealand: the Health and Disability Ethics Committee, reference 15/NTA/21). The relevant regulatory authorities confirmed that the formulation was not an investigational medicinal product. All participants gave written informed consent. Trial oversight and monitoring were provided by an independent data and safety monitoring committee. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Individual participant data may be shared upon reasonable requests and are subject to approval by the trial management group and the trial consultative panel. Application can be made through the corresponding author. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02509988&atom=%2Fmedrxiv%2Fearly%2F2023%2F06%2F20%2F2023.06.19.23291584.atom