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Open Access

Peer-reviewed

Research Article

Maternal vitamin B12 deficiency and perinatal outcomes in southern India

  • Julia L. Finkelstein ,

    Roles Conceptualization, Formal analysis, Funding acquisition, Writing – original draft, Writing – review & editing

    jfinkelstein@cornell.edu

    Affiliations Division of Nutritional Sciences, Cornell University, Ithaca, NY, United States of America, St. John’s Research Institute, Bangalore, Karnataka, India

    ⨯
  • Amy Fothergill,

    Roles Writing – review & editing

    Affiliation Division of Nutritional Sciences, Cornell University, Ithaca, NY, United States of America

    ⨯
  • Jesse T. Krisher,

    Roles Formal analysis

    Affiliation Division of Nutritional Sciences, Cornell University, Ithaca, NY, United States of America

    ⨯
  • Tinku Thomas,

    Roles Project administration

    Affiliation St. John’s Research Institute, Bangalore, Karnataka, India

    ⨯
  • Anura V. Kurpad,

    Roles Conceptualization, Funding acquisition, Project administration

    Affiliation St. John’s Research Institute, Bangalore, Karnataka, India

    ⨯
  • Pratibha Dwarkanath

    Roles Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing

    Affiliation St. John’s Research Institute, Bangalore, Karnataka, India

    ⨯

Abstract

Background

Vitamin B12 deficiency during pregnancy has been associated with adverse maternal and infant health outcomes. Few prospective studies have investigated vitamin B12 status early in pregnancy, and its links to infant vitamin B12 status, particularly in India where the burden of vitamin B12 deficiency is estimated to be the highest globally. The objective of this study was to examine the associations of maternal vitamin B12 biomarkers with neonatal vitamin B12 status.

Methods

Pregnant women (~12 weeks’ gestation) were enrolled in a perinatal cohort study in Bangalore, India. Total vitamin B12, methylmalonic acid (MMA), and homocysteine concentrations were evaluated in maternal samples at enrollment and in neonates at birth using cord blood. Linear and binomial regression models were used to evaluate the associations of maternal vitamin B12 biomarkers with neonatal vitamin B12 status and perinatal outcomes.

Results

A total of 63.2% of women had vitamin B12 deficiency (<148 pmol/L), 87.2% had vitamin B12 insufficiency (<221 pmol/L), and 47.3% had impaired vitamin B12 status (vitamin B12<148 pmol/L and MMA>0.26μmol/L) at enrollment; 40.8% of neonates had vitamin B12 deficiency, 65.6% were insufficiency, and 38.1% had impaired vitamin B12 status at birth. Higher maternal vitamin B12 concentrations at enrollment were associated with increased neonatal vitamin B12 concentrations (β(SE): 0.40 (0.05); p<0.0001) and lower risk of neonatal vitamin B12 deficiency (Risk Ratio [RR]: 0.53; 95% CI: [0.43, 0.65]; p<0.0001). Maternal vitamin B12 deficiency (RR: 1.97 [1.43, 2.71]; p<0.001), insufficiency (RR: 2.18 [1.23, 3.85]; p = 0.007), and impaired vitamin B12 status (RR: 1.49 [1.13, 1.97]; p = 0.005) predicted a two-fold increase in the risk of neonatal vitamin B12 deficiency at birth.

Conclusions

The prevalence of vitamin B12 deficiency was high early in pregnancy and predicted neonatal vitamin B12 status. Future research is needed to determine the role of vitamin B12 in the development of pregnancy and infant outcomes, and to inform screening and interventions to improve maternal and child health.

Citation: Finkelstein JL, Fothergill A, Krisher JT, Thomas T, Kurpad AV, Dwarkanath P (2021) Maternal vitamin B12 deficiency and perinatal outcomes in southern India. PLoS ONE 16(4): e0248145. https://doi.org/10.1371/journal.pone.0248145

Editor: Pal Bela Szecsi, Holbaek Sygehus, DENMARK

Received: October 25, 2020; Accepted: February 19, 2021; Published: April 6, 2021

Copyright: © 2021 Finkelstein et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The data is publicly available at: https://doi.org/10.7910/DVN/HIOUQN (Contact Dr. Pratibha Dwarkanath at: Pratibha@sjri.res.in).

Funding: This research was funded by the Division of Nutrition, St. John’s Research Institute and the Division of Nutritional Sciences, Cornell University. J.L.F. was supported by the ILSI North America Future Leader Award. A.F. was supported by the National Institutes of Health under award 5 T32 HD087137. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, or the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Vitamin B12 deficiency (vitamin B12 <148 pmol/L) is an important public health problem worldwide [1–3]. Although there is limited population-level data, vitamin B12 deficiency affects individuals across the life cycle, with the highest prevalence in the elderly, pregnant women, and young children [1, 2, 4–12]. The burden of vitamin B12 deficiency in India is estimated to be among the highest in the world [1, 13–23]. Inadequate vitamin B12 status during pregnancy has been associated with increased risk of adverse maternal and infant health outcomes [1–3, 24, 25], and linked to long-term impairments in child growth and development which may be irreversible [3, 24, 26–30].

Previous cross-sectional research has noted associations between maternal and infant vitamin B12 status at delivery in studies in Belgium, Canada, Norway, Germany, United Kingdom, Turkey, Serbia, and Brazil [3, 31–40]. Findings from prospective studies have reported significant associations of maternal vitamin B12 status during pregnancy with infant vitamin B12 status at birth (i.e., in cord blood or serum) [41–46] and at six weeks of age [23], in studies in the Netherlands, Norway, Turkey, Spain, India, and the United States. In contrast, findings regarding the associations of maternal vitamin B12 status with other child health outcomes or vitamin B12 status later in childhood have been heterogeneous. Inadequate maternal vitamin B12 status during gestation has also been associated with risk of pregnancy complications, such as spontaneous abortion, preterm delivery, intrauterine growth restriction, low birth weight, and neural tube defects [3, 7, 47–66]. However, most studies to date have been cross-sectional or case-control in design, constrained by limited sample sizes, and relied on a single biomarker of maternal total vitamin B12 concentrations, evaluated at mid-gestation or delivery.

Few prospective studies have been conducted to date to examine the impact of maternal vitamin B12 status during pregnancy on vitamin B12 status early in life, or the role of vitamin B12 in the development of adverse birth or infant outcomes [3]. There is limited prospective data, particularly early in gestation, and from settings with the highest burden of vitamin B12 deficiency and adverse pregnancy outcomes, such as India. Further research is needed to determine the burden of vitamin B12 deficiency in pregnant women and their infants in high-risk populations, and its implications for maternal and child health outcomes.

We conducted a prospective observational analysis to: 1) determine the prevalence of vitamin B12 deficiency in pregnant women and their infants; 2) examine the associations of maternal vitamin B12 biomarkers with neonatal vitamin B12 status; and 3) examine the associations of maternal vitamin B12 biomarkers with perinatal outcomes, in women participating in a cohort study in Bangalore, India.

Methods

Ethics statement

The research protocol and study procedures were approved by the Institutional Ethical Board of St. John’s Medical College (Reference number: IEC 42/2001). Written signed informed consent was obtained from all study participants at enrollment.

Study population

This study was a prospective observational cohort study of pregnant women conducted at St John’s Medical College Hospital (SJMCH) in Bangalore, India. St. John’s Medical College is a 1350-bed teritary care hospital located in Bangalore, India. Established with a commitment to serve poor and vulnerable populations, its catchment includes a patient population from diverse social and economic backgrounds. The overall study design of the perinatal cohort and recruitment [67], inclusion criteria, and sampling for the biomarker sub-study [68] have been previously published. Pregnant women were eligible for this study if they were at least 17 years of age, <14 weeks of gestation at enrollment, healthy, and carrying a single fetus. Women who had multiple fetuses (e.g., twins, triplets), reported clinical diagnoses of a chronic condition (e.g., diabetes mellitus, hypertension, cardiovascular disease, thyroid disease), tested positive for HIV, hepatitis B, or syphilis infections, were taking medications, or planned to move outside of Bangalore prior to delivery were excluded from the study. Pregnant women recruited to the perinatal cohort study from 2008 to 2014 who had a venous blood sample collected at enrollment, delivered a live baby at SJCMH, and had cord blood collected at the time of delivery were eligible for this analysis (n = 419). A flow chart of study participants is presented in Fig 1.

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Fig 1. Participant flow chart.

https://doi.org/10.1371/journal.pone.0248145.g001

All women received routine antenatal supplements in accordance with the National guidelines of India. As per standard of care, all women received folic acid at recruitment until the end of the first trimester, followed by iron-folic acid and calcium supplementation from the second trimester through delivery. Participants did not take vitamin B12 supplements or multivitamins containing vitamin B12, and no additional supplements or counseling were provided. All study procedures were reviewed and approved by the institutional ethical review board at SJCMH, and all participants provided written and signed consent at enrollment.

Data collection

Validated structured questionnaires were administered by trained research staff to collect sociodemographic, clinical, and dietary data, and standardized forms were used to record anthropometric and biochemical data prospectively beginning at enrollment. Gestational age in weeks was calculated from the day of last menstrual period and confirmed through ultrasonographic measurements (GE Volusun 730 Expert, probe 4C-A, Via Del Rio, Yorba Linda, CA, USA) within two weeks of enrollment; ultrasonographic measurements were collected again before delivery.

At each antenatal visit, maternal weight was recorded to the nearest 100 grams using a digital balance (Soehnie, Reutilngen, Germany); maternal height was recorded to the nearest 0.1 centimeter using a stadiometer; mid-upper arm circumference (MUAC; cm) was recorded using a plastic tape; and skinfolds thickness (biceps, triceps, subscapular; mm) were recorded to the nearest 0.2 millimeters using Holtain skinfold calipers (Holtain Limited, Crosswell, Wales, UK). Maternal body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared (kg/m2). Infant birth weight was measured on an electronic weighing scale (Salter Housewares 914 Electronic Baby and Toddler Scale, NY, USA) immediately after birth to the nearest 10 grams. Infant length (cm), circumferences (MUAC, head, chest; cm), and skinfolds (biceps, triceps, subscapular; mm) were measured within 72 hours of birth.

Laboratory analyses

The laboratory procedures and analyses have been previously described [67, 68]. Briefly, maternal venous whole blood and cord blood samples were collected at enrollment and delivery, respectively, in ethylenediaminetetraacetic acid (EDTA)-coated anticoagulant tubes and plain vacutainers (Becton Dickenson, NJ, USA). Maternal venous whole blood and cord blood samples were processed and analyzed in batch, using identical protocols, and instrument specific calibrators were used for all biochemical estimations. Whole blood was treated with 1% ascorbic acid, and hemolysate was stored <–80°C. Plasma, serum, and red blood cells were separated, processed, and stored <–80°C until batch analysis. A total of 399 participants had blood samples at enrollment available for laboratory analyses.

Hemoglobin and complete blood count (CBC) were analyzed using an automated cyanmethemoglobin technique analyzer (ABX Pentra 60 C+, Horiba ABX Diagnostics, Montpellier, France). The measuring range was between 8 and 18 g/dL with a within run precision of <1.0%. Plasma vitamin B12 concentrations were measured via electrochemiluminescence (Elecsys 2010, Roche Diagnostics Mannheim, USA). Quality-control samples pertaining to low, middle, and high ranges of vitamin B12 were analyzed along with the samples. Intra- and inter-assay CVs were 0.5% and 2.4%, respectively. Plasma methylmalonic acid (MMA) and total homocysteine (tHcy) concentrations were assessed by gas chromatography-mass spectrometry (GCMS-SQ, 5975, Agilent Technologies, CA, USA) [69].

Definitions of exposures and outcomes

Primary analyses were based on continuous vitamin B12 biomarkers (i.e., total vitamin B12, MMA) in pregnant women and neonates (i.e., cord blood). We also used conventional cut-offs from adult (non-pregnant) populations to describe categorical vitamin B12 variables. Vitamin B12 deficiency and insufficiency were defined as total vitamin B12 concentrations <148 and <221 pmol/L, respectively [7, 70]. Elevated methylmalonic acid concentrations were defined as MMA >0.26 and >0.37 μmol/L, to reflect depleted and deficient vitamin B12 status, respectively [7]. Impaired vitamin B12 status was defined as total vitamin B12 concentrations <148 pmol/L and MMA >0.26 μmol/L. Combined vitamin B12 (cB12), a composite indicator of vitamin B12 status, modified for three biomarkers (i.e., vitamin B12, MMA, tHcy), was calculated and defined using the methods developed by Fedosov et al. [71]. Elevated homocysteine concentrations were defined as tHcy >15.0 and >10.0 μmol/L [7]. Maternal anemia was defined as hemoglobin <11.0 g/dL [72].

Preterm delivery was defined as <37 weeks of completed gestation. Low birth weight was defined as birth weight <2,500 grams [73]. Small for gestational age (SGA) was defined as birth weight <10th percentile for gestational age and sex, using the INTERGROWTH reference [73]. Infant ponderal index was defined as weight in grams divided by length in centimeters cubed (g/cm3). World Health Organization (WHO) standards were used to calculate length-for-age (LAZ), weight-for-age (WAZ), and weight-for-length (WLZ) z-scores. Stunting was defined as LAZ <–2, underweight as WAZ <–2, and wasting as WLZ <–2 [74–76]. Neonatal anemia was defined as hemoglobin <11.0 g/dL [72].

Birth outcomes (e.g., birth weight, low birth weight, gestational age at delivery, preterm delivery); neonatal vitamin B12 status (e.g., vitamin B12 concentrations, MMA concentrations, impaired vitamin B12 status) [77–79]; and neonatal anthropometric outcomes (e.g., ponderal index, LAZ, WAZ, WLZ, MUAC) were evaluated.

Statistical analyses

Variables were defined using conventional cutoffs wherever available; medians were used to describe variables based on distributions in this population. Non-normally distributed variables were natural-logarithmically transformed to ensure normality before analysis. Non-transformed data are presented in Tables 1 and 2 for interpretation purposes.

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Table 1. Characteristics of the study population.

https://doi.org/10.1371/journal.pone.0248145.t001

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Table 2. Maternal vitamin B12 status at enrollment and neonatal vitamin B12 status at delivery.

https://doi.org/10.1371/journal.pone.0248145.t002

Primary analyses were based on continuous vitamin B12 biomarkers (i.e., total vitamin B12, MMA) in pregnant women and neonates (i.e., cord blood). We also used conventional cut-offs from adult (non-pregnant) populations to define categorical vitamin B12 variables. Linear and binomial regression models were used to evaluate the associations of maternal vitamin B12 biomarkers at enrollment with neonatal vitamin B12 status and perinatal outcomes, for continuous and categorical outcomes, respectively. Binomial regression models were used to obtain risk ratio estimates for dichotomous variables, and Poisson regression models were used when binomial regression models did not converge [77, 79]. All models were adjusted for gestational age at sample collection, in order to account for timing of sample collection. In order to adjust for multiple hypothesis testing, significance was evaluated after applying the Bonferroni correction. All p-values presented are the original (unadjusted) p-values for interpretation purposes, and the threshold used to determine statistical significance was α/n, where α is the level of significance (α = 0.05) and n is the number of comparison tests conducted. If the results remained significant after applying the Bonferroni correction, this is reported in the text.

The Rothman and Greenland approach was used to evaluate and adjust for confounding, in which all known or suspected risk factors for the outcome which led to a >10% change-in-estimate were included in the model [59]. Final linear models were assessed for normality, using the Kolmogorov-Smirnoff test; collinearity, using variance inflation factors; and homoscedasticity, using plots of residuals versus predicted values. Final binomial models were examined for goodness of fit, using Hosmer-Lemeshow tests. Statistical analyses were conducted using SAS software, version 9.4 (SAS Institute, Inc., Cary, NC, USA).

Results

Participant characteristics

A flowchart of participants in this study is presented in Fig 1. The design of the perinatal cohort study [67] and inclusion criteria and sampling for the biomarker sub-study [68] have been previously published. Briefly, a total of 1,625 pregnant women were initially contacted regarding the perinatal cohort study, of which 1,272 provided informed consent. There were a total of 1,059 live births (n = 103 lost to follow-up; n = 110 fetal loss), of which 745 participants delivered at the study site, SJCMH. A total of 419 participants were selected for the biomarker sub-study [68]; of these, vitamin B12 status was analyzed in 399 maternal enrollment samples (n = 20 insufficient sample/sample volume). Birth outcome data was available for 399 neonates, and 374 cord blood samples were available for vitamin B12 analyses.

The characteristics of participants in this study are presented in Table 1. Women selected for the biomarker sub-study (n = 419) were similar compared to women in the overall cohort (n = 1,272 consented) with respect to baseline characteristics, including age, gestational age at enrollment, socioeconomic status (e.g., education), parity, and nutritional indicators (e.g., weight, hemoglobin). At enrollment, women had a median age of 24.0 (IQR: 21.0, 26.0) years, and median gestational age of 12.0 (IQR: 9.6, 13.3) weeks; 55.4% were nulliparous and 35.8% had received a university degree. A total of 82.2% were non-vegetarian (i.e., consumed poultry, meat, and/or fish), 17.8% of women were vegetarian (i.e., consumed milk and/or eggs), and 0% were vegan (i.e., no animal source foods); 70.4% of women reported taking prenatal supplements at enrollment. Participants did not take vitamin B12 supplements or multivitamins containing vitamin B12. At delivery, 13.5% of neonates were low birthweight (<2,500 g), 6.0% were preterm (<37 weeks’ gestation), and 21.6% were small for gestational age (Table 1).

Maternal vitamin B12 status

Vitamin B12 status in pregnant women at enrollment is presented in Table 2. At enrollment (median [IQR]: 12.0 [9.6, 13.3] weeks), 63.2% of women had vitamin B12 deficiency (vitamin B12 <148 pmol/L), 87.2% had vitamin B12 insufficiency (vitamin B12 <221 pmol/L), 71.3% had elevated methylmalonic acid (MMA >0.26 μmol/L) levels (MMA >0.37 μmol/L: 51.6%), and 47.3% had impaired vitamin B12 status (vitamin B12 <148 pmol/L and MMA >0.26 μmol/L). Associations between maternal biomarkers of vitamin B12 status (i.e., MMA, tHcy) and maternal vitamin B12 deficiency are presented in S1 Table. Maternal MMA levels (RR: 1.12; 95% CI: [0.43, 0.65]; p = 0.04) and elevated MMA (>0.26 μmol/L; RR: 1.26 [1.03, 1.53]; p = 0.02) were associated with maternal vitamin B12 deficiency.

Neonatal vitamin B12 status

Vitamin B12 status in neonates at birth is presented in Table 2. A total of 40.8% of neonates had vitamin B12 deficiency (vitamin B12 <148 pmol/L), 65.6% had vitamin B12 insufficiency (vitamin B12 <221 pmol/L), 95.7% had elevated MMA (MMA >0.26 μmol/L) concentrations (MMA >0.37 μmol/L: 87.4%), and 38.1% had impaired vitamin B12 status (vitamin B12 <148 pmol/L and MMA >0.26 μmol/L) at birth. Associations between neonatal biomarkers of vitamin B12 status (i.e., MMA, tHcy) and neonatal vitamin B12 deficiency are presented in S1 Table. Neonatal MMA levels (RR: 1.59 [1.25, 2.02]; p = 0.0001 and tHcy concentrations (RR: 1.37 [1.06, 1.76]; p = 0.01) were associated with neonatal vitamin B12 deficiency.

Maternal vitamin B12 status at enrollment and neonatal vitamin B12 status at birth

Vitamin B12 status in pregnant women at enrollment and in neonates at birth is shown in Table 2. Neonatal vitamin B12 concentrations were significantly higher compared to maternal vitamin B12 concentrations (172.4 [109.7, 265.1] vs. 127.0 [IQR: 89.6, 172.3]; p<0.0001). However, neonatal MMA (0.63 [IQR: 0.47, 0.86] vs. 0.38 [IQR: 0.24, 0.59]; p<0.0001) and homocysteine (17.6 [IQR: 13.5, 24.8] vs. 15.7 [IQR: 12.4, 20.7]; p<0.0001) levels were significantly higher in neonates. The prevalence of neonatal vitamin B12 deficiency (40.8% vs. 63.2%; p<0.0001), vitamin B12 insufficiency (65.6% vs. 87.2%; p<0.0001), and impaired vitamin B12 status (vitamin B12 <148 pmol/L and MMA >0.26 μmol/L; (38.1% vs. 47.3%; p = 0.01) were lower at delivery compared to pregnant women at enrollment.

The associations of maternal vitamin B12 biomarkers at enrollment with neonatal vitamin B12 concentrations at delivery are presented in Table 3. Higher maternal vitamin B12 concentrations were associated with increased neonatal vitamin B12 concentrations at delivery (β [SE]: 0.40 [0.05]; p<0.0001), in multivariate models adjusting for gestational age at enrollment, maternal age, parity, educational level, and BMI at enrollment. Maternal vitamin B12 deficiency (vitamin B12 <148 pmol/L; β [SE]: -0.30 [0.07]; p<0.0001) and vitamin B12 insufficiency (vitamin B12 <221 pmol/L; β [SE]: -0.54 [0.10]; p<0.0001) also predicted significantly lower neonatal vitamin B12 concentrations at delivery. Impaired maternal vitamin B12 status (vitamin B12 <148 pmol/L and MMA >0.26 μmol/L) at enrollment was associated with significantly lower neonatal vitamin B12 concentrations at delivery (β [SE]: –0.21 [0.07]; p<0.002) in multivariate analyses. Higher maternal cB12 was associated with increased neonatal vitamin B12 concentrations (β [SE]: 0.20 [0.05]; p<0.0001). Findings from these analyses remained statistically significant after correcting for multiple hypothesis testing (p<0.006). However, individually, maternal MMA, or homocysteine were not significantly associated with neonatal vitamin B12 concentrations at delivery.

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Table 3. Associations between maternal vitamin B12 status at enrollment and neonatal vitamin B12 concentrations.

https://doi.org/10.1371/journal.pone.0248145.t003

Maternal vitamin B12 biomarkers at enrollment and their associations with risk of neonatal vitamin B12 deficiency (vitamin B12 <148 pmol/L) are presented in Table 4. Higher maternal vitamin B12 concentrations predicted lower risk of neonatal vitamin B12 deficiency at birth (RR: 0.53 95% CI: [0.43, 0.65]; p<0.0001), in multivariate models adjusting for gestational age at enrollment, maternal age, parity, educational level, and BMI at enrollment. Impaired maternal vitamin B12 status (RR: 1.49 95% CI: [1.13, 1.97]; p = 0.005) and maternal vitamin B12 deficiency (RR: 1.97 95% CI: [1.43, 2.71]; p<0.0001) predicted a 1.49 to 1.97-fold higher risk of neonatal vitamin B12 deficiency at birth. Findings from these analyses remained statistically significant after correcting for multiple hypothesis testing (p<0.006). However, maternal MMA or homocysteine concentrations were not significantly associated with risk of neonatal vitamin B12 deficiency.

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Table 4. Associations between maternal vitamin B12 status at enrollment and neonatal vitamin B12 deficiency (<148 pmol/L).

https://doi.org/10.1371/journal.pone.0248145.t004

The associations of maternal vitamin B12 biomarkers at enrollment with neonatal methylmalonic acid concentrations at delivery are shown in Table 5. Maternal vitamin B12 deficiency (β [SE]: 0.16 [0.05]; p = 0.002) at enrollment was associated with increased neonatal MMA concentrations at birth, in multivariate models adjusting for gestational age at enrollment, maternal age, parity, educational level, and BMI at enrollment. However, maternal vitamin B12 concentrations or impaired vitamin B12 status were not significantly associated with neonatal MMA concentrations at birth, after correcting for multiple hypothesis testing (p<0.006).

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Table 5. Associations between maternal vitamin B12 status at enrollment and neonatal methylmalonic acid concentrations.

https://doi.org/10.1371/journal.pone.0248145.t005

The associations of maternal vitamin B12 biomarkers at enrollment with impaired neonatal vitamin B12 status at birth are presented in Table 6. Higher maternal vitamin B12 concentrations were associated with lower risk of impaired neonatal vitamin B12 status (RR: 0.52 95% CI: [0.42, 0.65]; p<0.0001), in multivariate analyses adjusting for gestational age at enrollment, maternal age, parity, educational level, and BMI at enrollment. Similarly, maternal vitamin B12 deficiency (RR: 2.08 95% CI: [1.46, 2.97]; p<0.0001) and impaired maternal vitamin B12 status (RR: 1.54 95% CI: [1.15, 2.06]; p = 0.004) predicted increased risk of impaired neonatal vitamin B12 status. Higher maternal cB12 (RR: 0.73 95% CI: [0.60, 0.90]; p = 0.004) was associated with lower risk of impaired neonatal vitamin B12 status at birth. Findings from these analyses remained statistically significant after correcting for multiple hypothesis testing (p<0.006).

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Table 6. Associations between maternal vitamin B12 status at enrollment and impaired neonatal vitamin B12 status.

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Maternal vitamin B12 status at baseline and its associations with neonatal homocysteine levels at birth are summarized in Table 7. Maternal vitamin B12 insufficiency (vitamin B12 <221 pmol/L) at enrollment was associated with higher neonatal homocysteine concentrations (β [SE]: 0.22 [0.07]; p = 0.003) at birth, in multivariate analyses adjusting for gestational age at enrollment, parity, maternal age, BMI, and educational level at enrollment.

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Table 7. Associations between maternal vitamin B12 status at enrollment and neonatal homocysteine concentrations.

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Maternal vitamin B12 status at enrollment and perinatal outcomes

The associations between maternal vitamin B12 biomarkers at enrollment with perinatal outcomes are presented in supplemental tables (S2–S4 Tables). After adjusting for gestational age at enrollment, parity, maternal age, BMI, and educational level at enrollment, maternal vitamin B12 concentrations were not significantly associated with perinatal outcomes (S2 Table). Similarly, maternal MMA concentrations at enrollment were not associated with perinatal outcomes in multivariate analyses (S3 Table). After adjusting for gestational age at enrollment, parity, maternal age, BMI, and highest level of education attained, impaired maternal vitamin B12 status (vitamin B12 <148 pmol/L and MMA >0.26 μmol/L) at enrollment was not associated with any perinatal outcomes (S4 Table). Other maternal vitamin B12 biomarkers were not significantly associated with perinatal outcomes, after correcting for multiple hypothesis testing (p<0.002).

Discussion

In this prospective analysis among pregnant women participating in a cohort study, maternal vitamin B12 deficiency was common early in pregnancy and predicted neonatal vitamin B12 status at birth. Maternal vitamin B12 status at enrollment–including vitamin B12 deficiency (<148 pmol/L), insufficiency (<221 pmol/L), and impaired vitamin B12 status (vitamin B12 <148 pmol/L and MMA >0.26 μmol/L)—predicted risk of neonatal vitamin B12 deficiency. Higher maternal vitamin B12 concentrations at enrollment were associated with increased neonatal vitamin B12 concentrations. Maternal vitamin B12 status at enrollment was not associated with risk of other perinatal outcomes.

This is among the largest prospective studies conducted to date to examine the burden of vitamin B12 deficiency in pregnancy and its associations with neonatal vitamin B12 status. Vitamin B12 deficiency was common early in pregnancy: 63.2% of women had vitamin B12 concentrations <148 pmol/L, 87.2% had vitamin B12 levels <221 pmol/L, and 47.3% had impaired vitamin B12 status (i.e., vitamin B12 <148 pmol/L and MMA >0.26 μmol/L) at enrollment. Findings are consistent with previous studies conducted among pregnant women in India (vitamin B12 <148 pmol/L or <162 pmol/L: 51–73%; ≤18 weeks’ gestation) [20, 23, 82, 83], and higher than studies in Bangladesh (vitamin B12 <150 pmol/L: 35.4%; <13 weeks’ gestation), Spain (vitamin B12 <150 pmol/L: 0%; ≤221 pmol/L: 6.3%; <12 weeks’ gestation), and Canada (vitamin B12 <148 pmol/L: 17%; 12–16 weeks’ gestation) [12, 39, 43, 84, 85].

The prevalence of vitamin B12 deficiency and insufficiency was also high in neonates at birth in this study. A total of 41% of neonates had vitamin B12 deficiency, 66% had vitamin B12 insufficiency, and 38% had impaired vitamin B12 status at birth. Findings are consistent with previous studies conducted among young infants in India (vitamin B12 <150 pmol/L: 44%, 6 weeks; <150 pmol/L: 62%, 1–3 months of age) [23, 86] and higher than in studies conducted in the UK (vitamin B12 <140.9 pmol/L: 29%; cord blood at delivery) and the United States (<148 pmol/L: 0%; cord blood at delivery) [45]. In the current study, neonatal vitamin B12 concentrations were 1.3-fold higher than maternal vitamin B12 concentrations early in pregnancy. Findings are consistent with previous studies that reported higher vitamin B12 concentrations in infants compared to mothers, ranging from 13 to 43% at mid-gestation [23, 42, 44, 46, 87, 88] or delivery [31–40, 43, 45, 89] to two-fold higher than maternal vitamin B12 concentrations at delivery [35, 36, 43, 45].

In the current study, maternal vitamin B12 status early in pregnancy–including vitamin B12 concentrations, vitamin B12 deficiency and insufficiency, and impaired vitamin B12 status–predicted neonatal vitamin B12 status at birth. For example, neonates born to women who had vitamin B12 deficiency at enrollment had a 2-fold greater risk of vitamin B12 deficiency at birth. Findings are consistent with studies of the associations of maternal vitamin B12 concentrations during gestation [23, 42, 44, 87, 88] and at delivery [31–38, 40, 43, 45, 89] with neonatal vitamin B12 levels at birth and in the first six weeks of life.

Maternal vitamin B12 deficiency at enrollment also predicted neonatal MMA concentrations and elevated neonatal MMA concentrations at birth. Few studies to date have evaluated MMA concentrations or other functional biomarkers of vitamin B12 status in young infants, and findings have been divergent. For example, in a study in Ireland, maternal vitamin B12 levels at 8 weeks of gestation were associated with maternal MMA concentrations during labor, but not with MMA levels in cord blood [43]. However, this study was constrained by limited range of maternal vitamin B12 status (i.e., no participants had vitamin B12 deficiency or elevated MMA concentrations during pregnancy; and ~26% reported taking cobalamin containing supplements during pregnancy) and smaller sample size (n = 92). In a study in Norway among 169 mother-infant pairs postpartum, maternal vitamin B12 levels were significantly correlated with infant MMA levels (r = -0.38, p<0.001) [90]; however, this study did not report MMA levels during pregnancy to which our findings can be directly compared.

In the current study, maternal vitamin B12 status during pregnancy was not significantly associated with risk of adverse perinatal outcomes. Findings are in contrast to previous research which identified vitamin B12 as a risk factor for adverse birth outcomes [3]: inadequate maternal vitamin B12 status in pregnancy has been associated with increased risk of spontaneous abortion or early miscarriage [54, 55, 91, 92], low birth weight (LBW, <2,500 g) [57, 83, 93, 94], intrauterine growth restriction (IUGR) [93], small for gestational age (SGA) [94], and neural tube defects [48–52, 59, 63, 95–97]. However, most of these studies have been case-control (or cross-sectional) in design. In previous prospective analyses in this perinatal cohort, lower maternal vitamin B12 concentrations in pregnancy were associated with increased risk of IUGR [57], and lower tertiles of maternal vitamin B12 levels throughout pregnancy were associated with increased risk of SGA [67], although vitamin B12 deficiency (<148 pmol/L) was not significantly associated with risk of SGA [58]. However, these analyses used the previous definition of SGA and IUGR as <10th percentile of birth weight for gestational age, which constrains comparability of findings. In contrast, other studies have reported that inadequate maternal vitamin B12 status during pregnancy was not associated with risk of adverse birth outcomes, such as gestational age at birth [98–100], birth weight [12, 28, 60, 82, 87, 98–111], LBW (<2,500 g) [12, 67], or SGA [60, 98, 105, 106, 109, 111]. In a systematic review and meta-analysis of vitamin B12 in pregnancy and preterm birth and low birth weight, maternal vitamin B12 levels during pregnancy were not significantly associated with birth weight [58]. In overall meta-analyses, vitamin B12 levels were associated with lower risk of preterm birth (ARR 0.89 (95% CI 0.82, 0.97); and low vitamin B12 status (<148 pmol/L) was associated with increased risk of low birth weight (<2,500; ARR: 1.15, 95% CI: 1.01, 1.31), although these findings were not significant in the individual studies [58]. Randomized trials are needed to determine the efficacy of vitamin B12 supplementation on birth outcomes, including preterm birth, low birth weight, and small-for-gestational age.

In this study, maternal vitamin B12 status was not associated with other neonatal outcomes, including WHO z-scores, ponderal index, mid-upper arm circumference, or head circumference. Although few studies have evaluated the association of vitamin B12 status during pregnancy and infant anthropometric outcomes, most studies to date have focused on neonatal head circumference, and findings have been heterogenous [61, 88, 99, 102]. Findings regarding the association of maternal vitamin B12 status with other infant health outcomes have been divergent.

This study is among the largest prospective studies to date to examine the burden of vitamin B12 deficiency in pregnancy and its associations with neonatal vitamin B12 status and perinatal outcomes. Maternal vitamin B12 biomarkers were evaluated early in pregnancy (~12 weeks’ gestation), and vitamin B12 status was evaluated with both circulating (i.e., vitamin B12) and functional (i.e., MMA, tHcy) biomarkers. We also considered additional indicators of vitamin B12 status, including cB12 and impaired vitamin B12 status, which combine circulating and functional biomarkers.

This study has several limitations. Maternal and neonatal vitamin B12 status were assessed at a single time point at enrollment and birth, respectively. The assessment of maternal vitamin B12 status ~12 weeks of gestation may not reflect vitamin B12 status periconceptionally or throughout pregnancy. Assessment of neonatal vitamin B12 status using cord blood at birth is an important study limitation–and limits interpretations of the associations between maternal vitamin B12 and infant status early in life. Participants in this biomarker sub-study were similar to the overall perinatal cohort (n = 1,272), in terms of sociodemographic status (e.g., age, educational level), gestational age at enrollment, parity, nutritional variables (e.g., weight, BMI, hemoglobin), and birth outcomes (e.g., live birth, preterm delivery, birth weight, LBW, SGA); however, they may differ on other unmeasured variables. In terms of vitamin B12 biomarkers, in addition to total vitamin B12, MMA, and tHcy, inclusion of holo-transcobalamin may be an important circulating biomarker of vitamin B12 status during gestation, although it has not been validated in pregnancy [112]. The role of vitamin B12 needs to be examined in the context of folate-mediated one-carbon metabolism. Vitamin B12 deficiency may co-occur with other micronutrient deficiencies (e.g., folate) that may influence vitamin B12 status and are independent risk factors for adverse perinatal outcomes. An important limitation of this study is the use of vitamin B12 biomarker cut-offs from adult non-pregnant populations–these vitamin B12 biomarker cut-offs have not been validated in pregnancy or in infancy. Further research is needed to develop and validate vitamin B12 biomarker cut-offs in pregnant women and young infants. Although findings provide evidence of an association of maternal and neonatal vitamin B12 status, the interpretations of these associations are not causal. Importantly, our study was not powered to detect differences in birth outcomes, including low birth weight and small for gestational age. Randomized trials are needed to examine the effects of vitamin B12 (periconceptionally and throughout pregnancy) on the development of maternal and infant health outcomes.

In summary, in this cohort of pregnant women, the prevalence of vitamin B12 deficiency was high early in pregnancy and predicted risk of infant vitamin B12 deficiency. This is one of the largest studies to date to evaluate the burden of vitamin B12 deficiency in pregnant women and their neonates. Findings suggest that vitamin B12 deficiency is an important public health problem in this population, and vitamin B12 status early in pregnancy has an important role in determining vitamin B12 status early in life. Future research, including randomized trials, is needed to determine the independent effects of vitamin B12 on the development of perinatal outcomes, to inform screening and interventions to improve maternal and child health.

Supporting information

S1 Table. Associations of vitamin B12 biomarkers with vitamin B12 deficiency in pregnant women and their neonates.

https://doi.org/10.1371/journal.pone.0248145.s001

(DOCX)

S2 Table. Associations between maternal vitamin B12 concentrations at enrollment and perinatal outcomes.

https://doi.org/10.1371/journal.pone.0248145.s002

(DOCX)

S3 Table. Associations between maternal methylmalonic acid concentrations at enrollment and perinatal outcomes.

https://doi.org/10.1371/journal.pone.0248145.s003

(DOCX)

S4 Table. Associations between maternal impaired vitamin B12 status at enrollment and perinatal outcomes.

https://doi.org/10.1371/journal.pone.0248145.s004

(DOCX)

Acknowledgments

We are grateful to Nancy Nanditha M., Roopashree C., Aruna B.S. and Arogya Mary for their assistance in data collection and Sarita Devi in laboratory analyses. We thank the mothers and children, and field teams, including physicians, nurses, midwives, and research, laboratory and administrative staff who made this study possible; and St. John’s Medical College, Bangalore, India, for its institutional support.

References

  1. 1. Allen LH. How common is vitamin B-12 deficiency? Am J Clin Nutr. 2009;89(2):693S–6S. Epub 2009/01/01. pmid:19116323.
  2. 2. McLean E, Benoist B, Allen L. Review of the magnitude of folate and vitamin B12 deficiencies worldwide. Food and Nutrition Bulletin. 2008;29(2):S38–S51. pmid:18709880
  3. 3. Finkelstein JL, Layden AJ, Stover PJ. Vitamin B-12 and Perinatal Health. Adv Nutr. 2015;6(5):552–63. Epub 2015/09/17. pmid:26374177; PubMed Central PMCID: PMC4561829.
  4. 4. Allen LH, Miller JW, de Groot L, Rosenberg IH, Smith AD, Refsum H, et al. Biomarkers of Nutrition for Development (BOND): Vitamin B-12 Review. J Nutr. 2018;148(suppl_4):1995S–2027S. Epub 2018/12/01. pmid:30500928; PubMed Central PMCID: PMC6297555.
  5. 5. Allen L, Rosenberg IH, Oakley GP, Omenn GS. Considering the case for vitamin B12 fortification of flour. Food and Nutrition Bulletin. 2010;31:S36–S46. pmid:20629351
  6. 6. Stabler SP, Allen RH. Vitamin B12 deficiency as a worldwide problem. Annu Rev Nutr. 2004;24:299–326. Epub 2004/06/11. pmid:15189123.
  7. 7. Green R, Allen LH, Bjorke-Monsen AL, Brito A, Gueant JL, Miller JW, et al. Vitamin B12 deficiency. Nat Rev Dis Primers. 2017;3:17040. Epub 2017/07/01. pmid:28660890.
  8. 8. Ramirez-Velez R, Correa-Bautista JE, Martinez-Torres J, Meneses-Echavez JF, Lobelo F. Vitamin B12 concentrations in pregnant Colombian women: analysis of nationwide data 2010. BMC Pregnancy Childbirth. 2016;16:26. Epub 2016/02/03. pmid:26832149; PubMed Central PMCID: PMC4736095.
  9. 9. Baik HW, Russel RM. Vitamin B12 Deficiency in the Elderly. Annual Review of Nutrition. 1999;19:357–77. pmid:10448529
  10. 10. Stover PJ. Vitamin B12 and older adults. Curr Opin Clin Nutr Metab Care. 2010;13(1):24–7. Epub 2009/11/12. pmid:19904199; PubMed Central PMCID: PMC5130103.
  11. 11. Sukumar N, Saravanan P. Investigating vitamin B12 deficiency. BMJ. 2019;365:l1865. Epub 2019/05/12. pmid:31076395.
  12. 12. Sukumar N, Rafnsson SB, Kandala NB, Bhopal R, Yajnik CS, Saravanan P. Prevalence of vitamin B-12 insufficiency during pregnancy and its effect on offspring birth weight: a systematic review and meta-analysis. Am J Clin Nutr. 2016;103(5):1232–51. Epub 2016/04/15. pmid:27076577.
  13. 13. Refsum H, Yajnik C, Milind G, Schneede J, Vollset SE, Orning L, et al. Hyperhomocysteinemia and elevated methylmalonic acid indicate a high prevalence of cobalamin deficiency in Asian Indians. American Journal of Clinical Nutrition. 2001;74:233–41. pmid:11470726
  14. 14. Bhardwaj A, Kumar D, Raina SK, Bansal P, Bhushan S, Chander V. Rapid Assessment for Coexistence of Vitamin B12 and Iron Deficiency Anemia among Adolescent Males and Females in Northern Himalayan State of India. Anemia. 2013;2013:959605. Epub 2013/08/24. pmid:23970962; PubMed Central PMCID: PMC3736489.
  15. 15. Taneja S, Bhandari N, Strand TA, Sommerfelt H, Refsum H, Ueland PM, et al. Cobalamin and folate status in infants and young children in a low-to-middle income community in India. Am J Clin Nutr. 2007;86:1302–9. pmid:17991639
  16. 16. Naik S, Mahalle N, Bhide V. Identification of vitamin B12 deficiency in vegetarian Indians. Br J Nutr. 2018;119(6):629–35. Epub 2018/02/16. pmid:29446340.
  17. 17. Chakraborty S, Chopra M, Mani K, Giri AK, Banerjee P, Sahni NS, et al. Prevalence of vitamin B12 deficiency in healthy Indian school-going adolescents from rural and urban localities and its relationship with various anthropometric indices: a cross-sectional study. J Hum Nutr Diet. 2018;31(4):513–22. Epub 2018/02/23. pmid:29468754.
  18. 18. Singla R, Garg A, Surana V, Aggarwal S, Gupta G, Singla S. Vitamin B12 Deficiency is Endemic in Indian Population: A Perspective from North India. Indian J Endocrinol Metab. 2019;23(2):211–4. Epub 2019/06/05. pmid:31161105; PubMed Central PMCID: PMC6540890.
  19. 19. Duggan C, Srinivasan K, Thomas T, Samuel T, Rajendran R, Muthayya S, et al. Vitamin B-12 supplementation during pregnancy and early lactation increases maternal, breast milk, and infant measures of vitamin B-12 status. J Nutr. 2014;144(5):758–64. Epub 2014/03/07. pmid:24598885; PubMed Central PMCID: PMC3985831.
  20. 20. Katre P, Bhat D, Lubree H, Otiv S, Joshi S, Joglekar C, et al. Vitamin B 12 and folic acid supplementation and plasma total homocysteine concentrations in pregnant Indian women with low B12 and high folate status. Asia Pac J Clinical Nutrition. 2010;19(3):335–43.
  21. 21. Pathak P, Kapil U, Yajnik C, Kapoor S, Dwivedi S, Singh R. Iron, folate, and vitamin B12 stores among pregnant women in a rural area of Haryana State, India. Food and Nutrition Bulletin. 2007;28(4):435–8. pmid:18274171
  22. 22. Samuel TM, Duggan C, Thomas T, Bosch R, Rajendran R, Virtanen SM, et al. Vitamin B(12) intake and status in early pregnancy among urban South Indian women. Ann Nutr Metab. 2013;62(2):113–22. Epub 2013/01/25. pmid:23344013; PubMed Central PMCID: PMC4742783.
  23. 23. Finkelstein JL, Kurpad AV, Thomas T, Srinivasan K, Duggan C. Vitamin B12 status in pregnant women and their infants in South India. Eur J Clin Nutr. 2017. pmid:28402324.
  24. 24. Black M. Effects of vitamin B12 and folate deficiency on brain development in children. Food and Nutrition Bulletin. 2008;29. pmid:18709887
  25. 25. Molloy AM, Kirke PN, Brody LC, Scott JM, Mills JL. Effects of folate and vitamin B12 deficiencies during pregnancy on fetal, infant, and child development. Food Nutr Bull. 2008;29(2 Suppl):S101–11; discussion S12-5. Epub 2008/08/20. pmid:18709885.
  26. 26. Venkatramanan S, Armata IE, Strupp BJ, Finkelstein JL. Vitamin B-12 and Cognition in Children. Advances in Nutrition. 2016;7. pmid:27633104
  27. 27. Pepper MR, Black MM. B12 in fetal development. Semin Cell Dev Biol. 2011;22(6):619–23. Epub 2011/06/15. pmid:21664980.
  28. 28. Bhate VK, Joshi SM, Ladkat RS, Deshmukh US, Lubree HG, Katre PA, et al. Vitamin B12 and folate during pregnancy and offspring motor, mental and social development at 2 years of age. J Dev Orig Health Dis. 2012;3(2):123–30. Epub 2012/04/01. pmid:25101923.
  29. 29. Lai JS, Mohamad Ayob MN, Cai S, Quah PL, Gluckman PD, Shek LP, et al. Maternal plasma vitamin B12 concentrations during pregnancy and infant cognitive outcomes at 2 years of age. Br J Nutr. 2019;121(11):1303–12. Epub 2019/04/03. pmid:30935438; PubMed Central PMCID: PMC6660314.
  30. 30. Thomas S, Thomas T, Bosch RJ, Ramthal A, Bellinger DC, Kurpad AV, et al. Effect of Maternal Vitamin B12 Supplementation on Cognitive Outcomes in South Indian Children: A Randomized Controlled Clinical Trial. Matern Child Health J. 2018. Epub 2018/07/14. pmid:30003521.
  31. 31. Obeid R, Morkbak AL, Munz W, Nexo E, Herrmann W. The cobalamin-binding proteins transcobalamin and haptocorrin in maternal and cord blood sera at birth. Clin Chem. 2006;52(2):263–9. Epub 2005/12/31. pmid:16384893.
  32. 32. Balci YI, Ergin A, Karabulut A, Polat A, Dogan M, Kucuktasci K. Serum vitamin B12 and folate concentrations and the effect of the Mediterranean diet on vulnerable populations. Pediatr Hematol Oncol. 2014;31(1):62–7. Epub 2013/10/04. pmid:24088029.
  33. 33. Radunovic N, Lockwood CJ, Stanojlovic O, Steric M, Kontic-Vucinic O, Sulovic N, et al. Fetal and maternal plasma homocysteine levels during the second half of uncomplicated pregnancy. J Matern Fetal Neonatal Med. 2014;28(11):1244–9. Epub 2014/08/28. pmid:25162468.
  34. 34. Guerra-Shinohara EM, Paiva A, Rondo P, Yamasaki K, Terzi C, D’Almeida V. Relationship between total homocysteine and folate levels in pregnant women and their newborn babies according to maternal serum levels of vitamin B12. BJOG. 2002;109:784–91. pmid:12135215
  35. 35. Koc A, Kocyigit A, Soran M, Demir N, Sevinc E, Erel O, et al. High frequency of maternal vitamin B12 deficiency as an important cause of infantile vitamin B12 deficiency in Sanliurfa province of Turkey. Eur J Nutr. 2006;45(5):291–7. Epub 2006/04/08. pmid:16601915.
  36. 36. Lindblad B, Zaman S, Malik A, Martin H, Ekstrom AM, Amu S, et al. Folate, vitamin B12, and homocysteine levels in South Asian women with growth-retarded fetuses. Acta Obstet Gynecol Scand. 2005;84:1055–61. pmid:16232172
  37. 37. Frery N, Huel G, Leroy M, Moreau T, Savard R, Blot P, et al. Vitamin B12 among parturients and their newborns and its relationship with birthweight. European Journal of Obstetrics and Gynecology. 1992;42:155–63. pmid:1511760
  38. 38. Adaikalakoteswari A, Vatish M, Lawson A, Wood C, Sivakumar K, McTernan PG, et al. Low maternal vitamin B12 status is associated with lower cord blood HDL cholesterol in white Caucasians living in the UK. Nutrients. 2015;7(4):2401–14. Epub 2015/04/08. pmid:25849948; PubMed Central PMCID: PMC4425151.
  39. 39. 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(5):1035–42. Epub 2016/04/15. pmid:27075906.
  40. 40. Jacquemyn Y, Alaji M, Karepouan N, Jacquemyn N, Van Sande H. Vitamin B12 and folic acid status of term pregnant women and newborns in the Antwerp region, Belgium. Clin Exp Obstet Gynecol. 2014;2:141–3. pmid:24779238
  41. 41. Relton CL, Pearce MS, Parker L. The influence of erythrocyte folate and serum vitamin B12 status on birth weight. British Journal of Nutrition. 2007;93(05). pmid:15975157
  42. 42. Muthayya S, Dwarkanath P, Mhaskar M, Mhaskar R, Thomas A, Duggan C, et al. The relationship of neonatal serum vitamin B12 status with birth weight. Asia Pac J Clinical Nutrition. 2006;15:538–43. pmid:17077072
  43. 43. Murphy MM, Molloy AM, Ueland PM, Fernandez-Ballart J, Schneede J, Arija V, et al. Longitudinal Study of the Effect of Pregnancy on Maternal and Fetal Cobalamin Status in Healthy Women and Their Offspring. Journal of Nutrition. 2007.
  44. 44. Kalay Z, Islek A, Parlak M, Kirecci A, Guney O, Koklu E, et al. Reliable and powerful laboratory markers of cobalamin deficiency in the newborn: plasma and urinary methylmalonic acid. J Matern Fetal Neonatal Med. 2014;29(1):60–3. Epub 2014/11/12. pmid:25385266.
  45. 45. Finkelstein JL, Guillet R, Pressman EK, Fothergill A, Guetterman HM, Kent TR, et al. Vitamin B12 Status in Pregnant Adolescents and Their Infants. Nutrients. 2019;11(2). Epub 2019/02/20. pmid:30781902; PubMed Central PMCID: PMC6413223.
  46. 46. Bergen NE, Schalekamp-Timmermans S, Jaddoe VW, Hofman A, Lindemans J, Russcher H, et al. Maternal and Neonatal Markers of the Homocysteine Pathway and Fetal Growth: The Generation R Study. Paediatr Perinat Epidemiol. 2016;30(4):386–96. Epub 2016/06/09. pmid:27271101.
  47. 47. Molloy AM. Should vitamin B12 status be considered in assessing risk of neural tube defects? Ann N Y Acad Sci. 2018;1414(1):109–25. Epub 2018/01/30. pmid:29377209; PubMed Central PMCID: PMC5887889.
  48. 48. 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. Epub 2009/03/04. pmid:19255021; PubMed Central PMCID: PMC4161975.
  49. 49. Ratan SK, Rattan KN, Pandey RM, Singhal S, Kharab S, Bala M, et al. Evaluation of the levels of folate, vitamin B12, homocysteine and fluoride in the parents and the affected neonates with neural tube defect and their matched controls. Pediatr Surg Int. 2008;24(7):803–8. Epub 2008/05/09. pmid:18463884.
  50. 50. Wilson A, Platt R, Wu Q, Leclerc D, Christensen B, Yang H, et al. A common variant in methionine synthase reductase combined with low cobalamin (Vitamin B12) increases risk for spina. Molecular Genetics and Metabolism. 1999;67(4). pmid:10444342
  51. 51. Gu Q, Li Y, Cui ZL, Luo XP. Homocysteine, folate, vitamin B12 and B6 in mothers of children with neural tube defects in Xinjiang, China. Acta Paediatr. 2012;101(11):e486–90. Epub 2012/08/07. pmid:22860981.
  52. 52. Ray JG, Wyatt PR, Thompson MD, Vermeulen MJ, Meier C, Wong P-Y, et al. Vitamin B12 and the Risk of Neural Tube Defects in a Folic-Acid-Fortified Population. Epidemiology. 2007;18(3):362–6. pmid:17474166
  53. 53. Rowland AS, Baird DD, Shore DL, Weinberg CR, Savitz DA, Wilcox AJ. Nitrous oxide and spontaneous abortion in female dental assistants. American Journal of Epidemiology. 1995;141(6). pmid:7900720
  54. 54. Hubner U, Alwan A, Jouma M, Tabbaa M, Schorr H, Herrmann W. Low serum vitamin B12 is associated with recurrent pregnancy loss in Syrian women. Clin Chem Lab Med. 2008;46(9):1265–9. Epub 2008/07/19. pmid:18636794.
  55. 55. Reznikoff-Etievant MF, Zittoun J, Vaylet C, Pernet P, Milliez J. Low Vitamin B12level as a risk factor for very early recurrent abortion. European Journal of Obstetrics, Gynecology, & Reproductive Biology. 2002;104(2). pmid:12206930
  56. 56. Hogeveen M, Blom HJ, den Heijer M. Maternal homocysteine and small-for-gestational-age offspring: systematic review and meta-analysis. Am J Clin Nutr. 2012;95(1):130–6. Epub 2011/12/16. pmid:22170376.
  57. 57. Muthayya S, Kurpad AV, Duggan CP, Bosch RJ, Dwarkanath P, Mhaskar A, et al. Low maternal vitamin B12 status is associated with intrauterine growth retardation in urban South Indians. Eur J Clin Nutr. 2006;60(6):791–801. Epub 2006/01/13. pmid:16404414.
  58. 58. Rogne T, Tielemans MJ, Chong MF, Yajnik CS, Krishnaveni GV, Poston L, et al. Associations of Maternal Vitamin B12 Concentration in Pregnancy With the Risks of Preterm Birth and Low Birth Weight: A Systematic Review and Meta-Analysis of Individual Participant Data. Am J Epidemiol. 2017;185(3):212–23. Epub 2017/01/22. pmid:28108470; PubMed Central PMCID: PMC5390862.
  59. 59. Senousy SM, Farag MK, Gouda AS, El Noury MA, Dabbous OA, Gaber KR. Association between biomarkers of vitamin B12 status and the risk of neural tube defects. J Obstet Gynaecol Res. 2018;44(10):1902–8. Epub 2018/07/26. pmid:30043514.
  60. 60. Sukumar N, Venkataraman H, Wilson S, Goljan I, Selvamoni S, Patel V, et al. Vitamin B12 Status among Pregnant Women in the UK and Its Association with Obesity and Gestational Diabetes. Nutrients. 2016;8(12). Epub 2016/12/06. pmid:27916927; PubMed Central PMCID: PMC5188423.
  61. 61. Jiang HL, Cao LQ, Chen HY. Blood folic acid, vitamin B12, and homocysteine levels in pregnant women with fetal growth restriction. Genet Mol Res. 2016;15(4). Epub 2016/12/22. pmid:28002587.
  62. 62. Thompson MD, Cole DE, Ray JG. Vitamin B-12 and neural tube defects: the Canadian experience. Am J Clin Nutr. 2009;89(2):697S–701S. Epub 2009/01/01. pmid:19116334.
  63. 63. Nasri K, Ben Fradj MK, Touati A, Aloui M, Ben Jemaa N, Masmoudi A, et al. Association of maternal homocysteine and vitamins status with the risk of neural tube defects in Tunisia: A case-control study. Birth Defects Res A Clin Mol Teratol. 2015;103(12):1011–20. Epub 2015/09/20. pmid:26386249.
  64. 64. Fofou-Caillierez MB, Gueant-Rodriguez RM, Alberto JM, Chery C, Josse T, Gerard P, et al. Vitamin B-12 and liver activity and expression of methionine synthase are decreased in fetuses with neural tube defects. Am J Clin Nutr. 2019;109(3):674–83. Epub 2019/03/09. pmid:30848279.
  65. 65. Kouroglou E, Anagnostis P, Daponte A, Bargiota A. Vitamin B12 insufficiency is associated with increased risk of gestational diabetes mellitus: a systematic review and meta-analysis. Endocrine. 2019;66(2):149–56. Epub 2019/08/30. pmid:31463884.
  66. 66. Peker E, Demir N, Tuncer O, Ustyol L, Balahoroglu R, Kaba S, et al. The levels of vitamin B12, folate and homocysteine in mothers and their babies with neural tube defects. J Matern Fetal Neonatal Med. 2016;29(18):2944–8. Epub 2015/10/20. pmid:26479326.
  67. 67. Dwarkanath P, Barzilay J. R., Thomas T., Thomas A., Bhat S., & Kurpad A. V. High folate and low vitamin B12 intakes uring pregnancy are associated with small-for-gestational age infants in South Indian women: a prospective observational cohort study. American Journal of Clinical Nutrition. 2013. pmid:24108785
  68. 68. Dwarkanath P, Vinotha P, Thomas T, Joseph S, Thomas A, Shirley G, et al. Relationship of Early Vitamin D Concentrations and Gestational Diabetes Mellitus in Indian Pregnant Women. Front Nutr. 2019;6:116. Epub 2019/08/27. pmid:31448279; PubMed Central PMCID: PMC6691186.
  69. 69. Windelberg A, Arseth O, Kvalheim G, Ueland PM. Automated assay for the determination of methylmalonic acid, total homocysteine, and related amino acids in human serum or plasma by means of methylchloroformate derivatization and gas chromatography-mass spectrometry. Clin Chem. 2005;51(11):2103–9. Epub 2005/08/27. pmid:16123148.
  70. 70. Yetley EA, Pfeiffer CM, Phinney KW, Bailey RL, Blackmore S, Bock JL, et al. Biomarkers of vitamin B-12 status in NHANES: a roundtable summary. Am J Clin Nutr. 2011;94(1):313S–21S. Epub 2011/05/20. pmid:21593512; PubMed Central PMCID: PMC3127527.
  71. 71. Fedosov SN, Brito A, Miller JW, Green R, Allen LH. Combined indicator of vitamin B12 status: modification for missing biomarkers and folate status and recommendations for revised cut-points. Clin Chem Lab Med. 2015;53(8):1215–25. pmid:25720072.
  72. 72. WHO. Iron deficiency anemia: assessment, prevention and control: a guide for programme managers. Geneva, Switzerland: UNICEF, United Nations University, WHO, 2001.
    • 73. Villar J, Cheikh Ismail L, Victora CG, Ohuma EO, Bertino E, Altman DG, et al. International standards for newborn weight, length, and head circumference by gestational age and sex: the Newborn Cross-Sectional Study of the INTERGROWTH-21st Project. Lancet. 2014;384(9946):857–68. pmid:25209487.
    • 74. Organization WH. WHO Anthro for Personal Computers; Software for assessing growth and development of the world’d children. In: Development DoNfHa, editor. Geneva: World Health Organization; 2011.
      • 75. Group WMGRS. WHO Child Growth Standards: growth velocity based on weight, length and head circumference: methods and development. Geneva2009.
        • 76. Group WMGRS. WHO Child Growth Standards: methods and development: length/height-forage, weight-for-age, weight-for-length, weight-for-height and body mass index-for-age. Geneva: World Health Organization; 2006.
          • 77. Spiegelman D, Hertzmark E. Easy SAS calculations for risk or prevalence ratios and differences. Am J Epidemiol. 2005;162(3):199–200. Epub 2005/07/01. pmid:15987728.
          • 78. Wacholder S. Binomial regression in GLIM: estimating risk ratios and risk differences. Am J Epidemiol. 1986;123(1):174–84. pmid:3509965.
          • 79. Zou G. A modified poisson regression approach to prospective studies with binary data. Am J Epidemiol. 2004;159(7):702–6. pmid:15033648.
          • 80. Report WHOT. Obesity: preventing and managing the global epidemic. Report of a WHO consultation. 2000.
            • 81. Consultation WE. Appropriate body-mass index for Asian populations and its implications for policy and intervention strategies. Lancet. 2004. pmid:14726171
            • 82. 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 Pune Maternal Nutrition Study. Diabetologia. 2008;51(1):29–38. Epub 2007/09/14. pmid:17851649; PubMed Central PMCID: PMC2100429.
            • 83. Mishra J, Tomar A, Puri M, Jain A, Saraswathy KN. Trends of folate, vitamin B12, and homocysteine levels in different trimesters of pregnancy and pregnancy outcomes. Am J Hum Biol. 2020:e23388. Epub 2020/01/04. pmid:31898383.
            • 84. Schulze KJ, Mehra S, Shaikh S, Ali H, Shamim AA, Wu LS, et al. Antenatal Multiple Micronutrient Supplementation Compared to Iron-Folic Acid Affects Micronutrient Status but Does Not Eliminate Deficiencies in a Randomized Controlled Trial Among Pregnant Women of Rural Bangladesh. J Nutr. 2019;149(7):1260–70. Epub 2019/04/23. pmid:31006806; PubMed Central PMCID: PMC6602890.
            • 85. Sole-Navais P, Salat-Batlle J, Cavalle-Busquets P, Fernandez-Ballart J, Ueland PM, Ballesteros M, et al. Early pregnancy folate-cobalamin interactions and their effects on cobalamin status and hematologic variables throughout pregnancy. Am J Clin Nutr. 2018;107(2):173–82. Epub 2018/03/13. pmid:29529156.
            • 86. Mittal M, Bansal V, Jain R, Dabla PK. Perturbing Status of Vitamin B12 in Indian Infants and Their Mothers. Food Nutr Bull. 2017;38(2):209–15. Epub 2017/05/18. pmid:28513265.
            • 87. Hay G, Clausen T, Whitelaw A, Trygg K, Johnston C, Henriksen T, et al. Maternal folate and cobalamin status predicts vitamin status in newborns and 6-month-old infants. J Nutr. 2010;140(3):557–64. Epub 2010/01/15. pmid:20071650.
            • 88. Youssry MA, Radwan AM, Gebreel MA, Patel TA. The Impact of Third Trimester Maternal Serum Vitamin B12 and Folate Status on Fetal Birth Weight. Is Maternal Serum Homocysteine a Predictor of Low Birth Weight Infants? Open Journal of Obstetrics and Gynecology. 2017;07(11):1102–15.
            • 89. Pisal H, Dangat K, Randhir K, Khaire A, Mehendale S, Joshi S. Higher maternal plasma folate, vitamin B12 and homocysteine levels in women with preeclampsia. J Hum Hypertens. 2019;33(5):393–9. Epub 2019/01/17. pmid:30647465.
            • 90. Bjorke Monsen AL, Ueland PM, Vollset SE, Guttormsen AB, Markestad T, Solheim E, et al. Determinants of Cobalamin Status in Newborns. Pediatrics. 2001;108:624–30. pmid:11533328
            • 91. Rowland AS, Baird DD, Shore DL, Weinberg CR, Savitz DA, Wilcox AJ. Nitrous oxide and spontaneous abortion in female dental assistants. American journal of epidemiology. 1995;141(6):531–8. pmid:7900720.
            • 92. Reznikoff-Etievant MF, Zittoun J, Vaylet C, Pernet P, Milliez J. Low Vitamin B(12) level as a risk factor for very early recurrent abortion. Eur J Obstet Gynecol Reprod Biol. 2002;104(2):156–9. Epub 2002/09/11. pmid:12206930.
            • 93. Muthayya S, Kurpad AV, Duggan CP, Bosch RJ, Dwarkanath P, Mhaskar A, et al. Low maternal vitamin B12 status is associated with intrauterine growth retardation in urban South Indians. European journal of clinical nutrition. 2006;60(6):791–801. Epub 2006/01/13. pmid:16404414.
            • 94. Hogeveen M, Blom HJ, den Heijer M. Maternal homocysteine and small-for-gestational-age offspring: systematic review and meta-analysis. The American journal of clinical nutrition. 2012;95(1):130–6. Epub 2011/12/16. pmid:22170376.
            • 95. Adams MJ Jr., Khoury MJ, Scanlon KS, Stevenson RE, Knight GJ, Haddow JE, et al. Elevated midtrimester serum methylmalonic acid levels as a risk factor for neural tube defects. Teratology. 1995;51(5):311–7. pmid:7482352.
            • 96. Wilson A, Platt R, Wu Q, Leclerc D, Christensen B, Yang H, et al. A common variant in methionine synthase reductase combined with low cobalamin (vitamin B12) increases risk for spina bifida. Molecular genetics and metabolism. 1999;67(4):317–23. pmid:10444342.
            • 97. Ray JG, Wyatt PR, Thompson MD, Vermeulen MJ, Meier C, Wong PY, et al. Vitamin B12 and the risk of neural tube defects in a folic-acid-fortified population. Epidemiology (Cambridge, Mass). 2007;18(3):362–6. Epub 2007/05/03. pmid:17474166.
            • 98. Chen LW, Lim AL, Colega M, Tint MT, Aris IM, Tan CS, et al. Maternal folate status, but not that of vitamins B-12 or B-6, is associated with gestational age and preterm birth risk in a multiethnic Asian population. J Nutr. 2015;145(1):113–20. Epub 2014/12/21. pmid:25527665.
            • 99. Yang T, Gu Y, Wei X, Liang X, Chen J, Liu Y, et al. Periconceptional folic acid supplementation and vitamin B 12 status in a cohort of Chinese early pregnancy women with the risk of adverse pregnancy outcomes. 2017;60(2):136–42.
            • 100. Krikke GG, Grooten IJ, Vrijkotte TG, van Eijsden M, Roseboom TJ, Painter RC. Vitamin B12 and folate status in early pregnancy and cardiometabolic risk factors in the offspring at age 5–6 years: findings from the ABCD multi-ethnic birth cohort. BJOG. 2016;123(3):384–92. Epub 2016/01/27. pmid:26810674.
            • 101. Zhu X, Wei L, Cao D, Liu C, Tian J, Long Y, et al. Low serum folate status in the second trimester increase the risk of low birthweight in Chinese women. J Obstet Gynaecol Res. 2018;44(11):2037–44. Epub 2018/07/19. pmid:30019799.
            • 102. Halicioglu O, Sutcuoglu S, Koc F, Ozturk C, Albudak E, Colak A, et al. Vitamin B12 and folate statuses are associated with diet in pregnant women, but not with anthropometric measurements in term newborns. J Matern Fetal Neonatal Med. 2012;25(9):1618–21. Epub 2011/12/22. pmid:22185230.
            • 103. Riaz M, Shaikh F, Fawwad A, Hakeem R, Shera AS, Hitman GA, et al. Maternal Nutrition during Early Pregnancy and Cardiometabolic Status of Neonates at Birth. J Diabetes Res. 2018;2018:7382946. Epub 2018/06/01. pmid:29850608; PubMed Central PMCID: PMC5941780.
            • 104. McCullough LE, Miller EE, Mendez MA, Murtha AP, Murphy SK, Hoyo C. Maternal B vitamins: effects on offspring weight and DNA methylation at genomically imprinted domains. Clin Epigenetics. 2016;8:8. Epub 2016/01/26. pmid:26807160; PubMed Central PMCID: PMC4722751.
            • 105. Baker PN, Wheeler SJ, Sanders TA, Thomas JE, Hutchinson CJ, Clarke K, et al. A prospective study of micronutrient status in adolescent pregnancy. Am J Clin Nutr. 2009;89(4):1114–24. Epub 2009/02/27. pmid:19244368.
            • 106. Bergen NE, Jaddoe VW, Timmermans S, Hofman A, Lindemans J, Russcher H, et al. Homocysteine and folate concentrations in early pregnancy and the risk of adverse pregnancy outcomes: the Generation R Study. BJOG. 2012;119(6):739–51. Epub 2012/04/12. pmid:22489763.
            • 107. Kaymaz C, Demir A, Bige O, Cagliyan E, Cimrin D, Demir N. Analysis of perinatal outcome by combination of first trimester maternal plasma homocysteine with uterine artery Doppler velocimetry. Prenat Diagn. 2011;31(13):1246–50. Epub 2011/11/29. pmid:22120509.
            • 108. 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. Epub 2007/07/12. pmid:17623226.
            • 109. Sukumar N, Bawazeer N, Patel V, Saravanan P. Low B12 level is associated with maternal obesity and higher birthweight in gestational diabetes. Journal of Developmental Origins of Health and Disease. 2011;2:128–9.
            • 110. Dayaldasani A, Ruiz-Escalera J, Rodriguez-Espinona M, Rueda I, Perez-Valero V, Yahyaoui R. Serum Vitamin B12 Levels During the First Trimester of Pregnancy Correlate with Newborn Screening Markers of Vitamin B12 Deficiency. International Journal for vitamin and nutrition research. 2014;84:92–7. pmid:25835239
            • 111. Krishnaveni GV, Veena SR, Karat SC, Yajnik CS, Fall CH. Association between maternal folate concentrations during pregnancy and insulin resistance in Indian children. Diabetologia. 2014;57(1):110–21. Epub 2013/10/29. pmid:24162586; PubMed Central PMCID: PMC3855580.
            • 112. Nexo E, Hoffmann-Lucke E. Holotranscobalamin, a marker of vitamin B-12 status: analytical aspects and clinical utility. Am J Clin Nutr. 2011;94(1):359S–65S. Epub 2011/05/20. pmid:21593496; PubMed Central PMCID: PMC3127504.