Vitamin B12 Supplementation During Pregnancy
Condition: Nutritional Anemia in Mothers.; Nutritional Anemia in Infants.
MeSH terms: Anemia
Study type: Interventional
Phase: Phase 2/Phase 3

Summary: 
      Nutritional anemia is a major public health problem among children and women in developing
      countries. Despite ongoing national program of supplementing pregnant women with iron-folate,
      prevalence of anemia is 39% among pregnant women and 78% among infants in Bangladesh. Vitamin
      B12 deficiency is a more prevalent cause of megaloblastic anemia than folate in many
      developing countries. This data raises the interest to address the role of vitamin B12
      deficiency in nutritional anemia. Low dietary intake of animal products, a predominant source
      of vitamin B12 may cause anemia. Besides maintaining normal erythropoiesis, B12 is essential
      for immune function. However, no studies have evaluated the effect of maternal B12
      supplementation on reduction of anemia and improving immunity of their infants. The
      investigators hypothesize that vitamin B12 supplementation plus iron-folate during pregnancy
      and 3-mo postpartum would: (a) Decrease anemia among mothers and infants; (b) Improve vaccine
      specific cellular and humoral immune responses among mothers; (c) Improve vaccine specific
      immunity in infants by passive transfer; (d) Improve DNA methylation and one-carbon
      metabolism in mother-child pairs; (e) Reduce antenatal/postnatal depression. Results from
      this study will guide and provide support to the policy makers to identify effective
      strategies to reduce nutritional anemia in population at risk.

      The investigators aim to conduct a double-masked placebo controlled trial to investigate the
      added effect of vitamin B12 on the iron-folate supplementation among pregnant women. Anemic
      (Hb level <11.0 g/dl) mothers at 11-14 weeks of gestation will be randomized into two groups:
      supplement group will receive 250 ug vitamin B12 plus 400 ug folate and 60 mg iron; placebo
      group will receive folate and iron only. This daily supplementation will continue up to 3-mo
      postpartum. At 26-28 wk of gestation mothers will be given inactivated influenza vaccine.
      Data on anthropometric indices of mothers and children, birth size, infant growth and
      morbidity (mothers and children) throughout the study period will be recorded. 24-h dietary
      recall data will be collected from the mothers bimonthly throughout the study. Biochemical
      indicators of anemia including Hb, vitamin B12, ferritin, folate and α-glycoprotein (AGP)
      will be assessed in plasma of mothers (pre- and post-supplementation) and infants (cord blood
      and 3-months). Additional measurements include serum transferrin receptor (sTfR) in plasma
      and methyl malonic acid (MMA) and total homocysteine (tHcy) in the urine of mothers. Plasma
      vaccine specific antibody responses will be measured in mothers (pre- and post
      supplementation) and in infants (cord blood and 3-months). In breast milk, B12, folate and
      s-IgA will be determined. Genetic polymorphism (one-carbon metabolism) and DNA methylation
      will be studied in mothers and in cord blood.
    

Detailed description: 
      Study Area: A maternity clinic in Mirpur with substantial numbers of pregnant women enrolling
      each month (about 300-400/month) will be selected for recruitment of local pregnant women.
      The community in Mirpur has a low out-migration rate. Pregnant women attending the clinic at
      11-14 weeks of gestation and planning to deliver at the clinic to ensure safe delivery will
      be selected. The structured service and standard antenatal care of the clinic for safe
      delivery and antenatal care will be provided to each study participant.

      Study Population: Pregnant women (n=120) will be screened during 11-14 weeks of gestation.
      Infants (n=120) at 3 months of age.

      Inclusion Criteria:

        -  Hb level in the range of 7.0-11 g/dL

        -  Age: 22-35 years

        -  Mothers at the beginning of second trimester (i.e. at 11-14 weeks of gestation)

        -  Willing to stay in Dhaka during pregnancy and willing to get admitted in the clinic for
           delivery

        -  Self-reported Last Menstrual Period (LMP) and urine pregnancy test will be used to
           determine the gestational age.

      Exclusion Criteria:

        -  Women with severe anemia; hemoglobin concentration <7.0 g/dL

        -  History or presence of systemic disease

        -  H/O of previous complicated pregnancies or of pre-term delivery, abortion

        -  Receipt of influenza vaccine Definition: According to WHO, anemia is defined as a
           hemoglobin concentration <12 g/dL in adult women. Anemia is further categorized as mild
           (10-11.9 g/dL), moderate (7.0-9.9 g/dL) and severe (<7.0 g/dL).

      Study design: A double-blind controlled trial; After informed consent is obtained, pregnant
      women will be randomly assigned either to Placebo group (N=60) that will receive 60 mg of Fe
      and 400µg of folate daily or Supplementation group (N=60) that will receive 250 µg of vitamin
      B12 in addition to 60 mg of Fe and 400µg of folate. A third party (GlaxoSmithKline,
      Bangladesh) will prepare and supply vitamin B12 or placebo capsule in bottles. The two
      capsules will be similar in appearance and taste. They will label the bottles as A and B. The
      responsible personnel from the Company will keep it confidential until the end of study. The
      capsule either A or B to be received by the 1st participant will be determined by a lottery,
      and thereafter every alternate participant will receive that capsule. The IDs of the
      participants will be given sequentially. The daily supplementation of iron-folate as well as
      of B12 will continue up to 3-months postpartum. At 26-28 weeks of gestation, all mothers will
      receive a single inactivated, trivalent influenza virus vaccine intramuscularly (IM).
      Standard inactivated trivalent influenza as recommended by WHO for the Northern Hemisphere
      will be obtained from commercial sources for this project (GlaxoSmithKline; Dresden,
      Germany). Field workers (FW) will deliver two weeks' dose of either placebo or supplements to
      mothers in person and will check the compliance by card and left over pills. The pregnant
      women will be clearly instructed about the times for their subsequent follow-up visits, and
      when followed up in respective homes they will be reminded of the dates.

      Routine Service: The routine care provided by the clinic includes Hb measurement, physical
      examination (weight, blood pressure, pulse rate, abdominal check-up, fetal heart rate,
      ultrasonographic examination as necessary), counseling on diet, descriptions of normal
      procedures during pregnancy, associated potential complications, how and when to go to the
      clinic during such events etc. This service is provided by the clinic physician with
      assistance from the nurses.

      Specimens: Fasting peripheral blood will be collected from mothers at 11 to 14 weeks of
      gestation, cord blood at delivery, colostrums within 24 hours and breast milk and peripheral
      blood from mothers after three months postpartum. Peripheral blood from infants will be
      collected at three months of age. Blood will be collected in anticoagulant (EDTA) coated
      tubes and layered on Ficoll-paque for density gradient centrifugation; plasma will be
      separated from the top and stored at -70ºC for later analysis. Peripheral blood mononuclear
      cells (PBMC) will be separated for studying cellular immune responses. Urine will be
      collected from mothers at 11 - 14 weeks of gestation and at three months. For gene based
      studies, the DNA samples will be delinked and the identity will be made anonymous according
      to the standard procedures.

      Methods: Total hemoglobin (Hb) in whole blood will be measured by spectrophotometry using
      Drabakins Reagent (Sigma Diagnostics). Standardized blood smear stain will be used for
      evaluating megaloblastic anemia (presence of macroovalocytes and hypersegmented neutrophils).
      Ferritin, serum transferrin receptor (sTfR) and α-glycoprotein (AGP) will be measured in
      plasma by enzyme immune assays. Vitamin B12 and folate in plasma and vitamin B12 in breast
      milk will be assayed in Roche autoanalyzer Cobas e411. Urinary methyl malonic acid (MMA) will
      be measured by fluorescence detector assisted HPLC method (Sigma Diagnostics, St. Louis, MO.
      Briefly MMA will be extracted from urine sample with ethyl acetate. The extract is dried and
      derivatized with monodansylcadaverine and dicyclohexyl-carbodiimide prior to injection on the
      HPLC. Ethylmalonic acid will be used as an internal standard. Influenza vaccine-specific
      antibody responses (IgA, IgG) in plasma and colostrum/ breast milk [secretory IgA (s-IgA)]
      will be measured by ELISA. PBMC will be stimulated with Flu vaccine for blastogensis
      response. Genomic DNA methylation will be measured by the methyl acceptance assay based on
      the ability of isolated DNA to "accept" radiolabeled methyl groups from S-[3H-methyl]
      adenosylmethionine, using the bacterial CpG methyltransferase SssI. Total homocysteine (tHcy)
      will be measured in urine samples by using HPLC with fluorimetric detection in which Thiols
      in the column effluent are detected by a single gold-mercury electrode. NaBH4 is used as a
      reductant, and no derivatization of sample is required. Mutations in the ALPL, MTHFR C677T
      and FUT2 genes will be determined by PCR- RFLP assay and DNA sequencing. All these assays
      will be performed in the Nutritional Biochemistry Lab which has the facilities to determine
      the biochemical markers of anemia, vaccine specific antibody responses, DNA methylation and
      polymorphism assays.

      Assessment of anthropometry and morbidity: Maternal anthropometrical measurements will be
      taken at the beginning of supplementation and monthly during the post supplementation period
      by trained FWs. Birth weight and recumbent length will be taken within 72 hours after birth
      and monthly measurements will be taken by FW up to 3 mo of age. At each visit (fortnightly),
      Field Research Assistant (FRA) will collect data from mothers on morbidity of mothers and
      their infants (e.g. diarrhoea, respiratory infections, skin diseases, assessment of severity
      of disease episodes) of the previous 7 days using structured questionnaires. The FRAs will
      inform the women about the times of the first and subsequent follow-up visits, and ensure if
      they have understood clearly. In addition, study participants will be contacted over cell
      phones when available to remind them about the visit dates. When followed up in respective
      homes, they will be reminded of the next visit dates.

      Depression score: Participants will be interviewed on their mental status using the Centre
      for Epidemiological Studies-Depression (CES-D) questionnaire. The questionnaire contains 20
      items comprising six major aspects of depression: depressed mood, hopelessness,
      worthlessness, fatigue, appetite and sleep disturbances. It was translated to Bengali and
      piloted in earlier studies and involves how the women felt during the preceding 7 days. It
      has been previously used in rural and urban Bangladeshi women (Hamadani, unpublished data)
      and found to correlate sensibly to children's growth and development. The FRAs will be
      extensively trained to interview the women and inter-observer reliabilities will be obtained
      before and during the study. The interview will be conducted twice at the homes of the women
      during FRAs' visits, first at baseline and at 3 mo postpartum. The data will be analyzed and
      interpreted by one of the co-investigators (JDH) who has knowledge and experience in dealing
      with this type of information.

      Food security and dietary diversity: The effect of food insecurity on food intake, health and
      nutritional status has been observed in many developing countries showing significantly lower
      total daily per capita food expenditures in food-insecure households than food-secure
      households. Food insecurity was associated with maternal anxiety and depression, which can
      have strong negative influence on infant care practices including breastfeeding duration as
      well as child nutritional status. About half of the Bangladeshi population is food-insecure.
      Hunger and childhood malnutrition in Bangladesh are among the highest in the world. About 31%
      of the rural population in Bangladesh suffers from "chronic poverty" and about 19% of rural
      households cannot have "full three meals" a day. Thus, household food insecurity in relation
      to its enormous impact on health and nutritional status on human is a growing concern in
      Bangladesh. Household food security status and dietary diversity of the study participants
      (including maternal dietary energy, protein, vitamin B-12 and folate intake) will be assessed
      first, at the baseline when the pregnant women will be enrolled in the study and thereafter
      fortnightly throughout the study. USDA food composition database will be used for assessing
      vitamin B-12 intake. Any seasonal variation of the food security status will be assessed that
      might have significant impact on the outcome measures. Structured questionnaire on food
      security module will be used to measure household food security status. The FRAs will be
      trained to interview the women; they will take interviews when they make visits during the
      antenatal and postnatal periods at the home. The data will be analyzed and interpreted by one
      of the co-investigators (KKS) who has knowledge and experience in dealing with this type of
      information.
    

Intervention name(s): Vitamin B12; Placebo
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention MeSH terms: Vitamin B 12; Hydroxocobalamin
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Triple (Participant, Care Provider, Investigator)
Primary purpose: Health Services Research
Keywords: Nutritional anemia; Vitamin B12; flu vaccine; immunity