The main focus of this review is on the provision of folate (with or without other vitamins and minerals) before pregnancy and in early pregnancy (before 12 weeks’ gestation) to reduce the occurrence of neural tube defects (NTDs) and cleft lip and palate. Other Cochrane reviews and protocols focus on related topics such as folate supplementation throughout pregnancy (Haider 2008); oral iron with or without folate during pregnancy (Peña Rosas 2006); treatment for iron deficiency and anaemia (Reveiz 2007); and the use of various vitamin and multivitamin/micronutrient supplements during pregnancy to improve pregnancy outcomes (Haider 2006). This review updates and expands the scope of a previously published Cochrane review on periconceptional supplementation with folate and/or multivitamins for preventing NTDs (Lumley 2001).
Folate is a water-soluble B vitamin present in legumes, leafy green vegetables (such as spinach and turnip greens) and some fruits (such as citrus fruits and juices). Folic acid is the synthetic and most stable form of folate and the form often used in supplements and in fortified foods. The bioavailability of folic acid is approximately 1.7 that of folate naturally contained in foods, although there are wide variations depending on the methodology used (McNulty 2004).
Folate status in populations is generally assessed using static biochemical tests that directly measure folate levels in serum and in erythrocytes or red blood cells (WHO 2008). Cut-off points to assess folate status have been proposed with a new approach which relies on the combination of the concentrations in blood of vitamins and their respective functional indicators of folate in populations (Selhub 2008). The cut-off suggested to define deficiency is < 10 nmol/L for serum folate (< 4 ng/mL) an indicator sensitive to recent usual intake; and < 340 nmol/L for red blood cell folate (< 151 ng/L would be considered deficiency, and it is an indicator of folate storage) (WHO 2008). There are no universally accepted cut-off points to define deficiency during pregnancy, as concentrations decline over gestation and recover at delivery (WHO 2008), probably due to haemodilution. It is important to highlight that folate concentrations can differ depending on the method used for assessment including radioimmunoassay and microbiological assays, particularly at the lower range of concentrations (CDC 2008; Life Sciences Research Office 1994).
The increasing interest in folate relates to the fact that the status of this vitamin in many populations is less than adequate (insufficient and not necessarily deficient) and that such inadequacy may have adverse public health effects.
Description of the condition
Periconceptional folate deficiency is associated with a number of birth defects that may also relate to genetic and environmental factors (IOM 2003) operating before conception or during pregnancy. Birth defects can cause lifelong problems with health, growth, and learning and may be immediately apparent after birth, or manifest later in life (Murray 1997; WHO 1999; WHO 2000). Environmental factors, which include nutritional status, are thought to contribute to about 5% to 10% of total birth defects (IOM 2003).
NTDs, which include anencephaly, spina bifida and encephalocele, are congenital malformations that arise during the structural development of the spinal cord, a process that is completed within 28 days after conception. In 1991 one randomized controlled trial (RCT) demonstrated that periconceptional folic acid supplementation prevented the recurrence of NTDs (MRC 1991) and in 1992 another RCT demonstrated that a multivitamin containing folic acid prevented the occurrence of NTDs (Czeizel 1992). While maternal intake of folate and folic acid are specifically associated with a decreased risk for NTD they may also provide protection for other selected birth defects. There is evidence of protection for cardiovascular defects, limb defects, cleft lip with or without cleft palate, urinary tract anomalies and congenital hydrocephalus (Goh 2006; Wilcox 2007); although this remains controversial. However, such controversy may possibly be explained by the differences in dosage and type of supplementation (i.e. folic acid alone or with other micronutrients) used among studies (Botto 2004; Botto 2006). No effects have been shown in preventing Down syndrome, pyloric stenosis, undescended testis or hypospadias. Approximately half of birth defects are limited to a single organ and the other half frequently present additional birth defects, such as heart malformations (Shibuya 1998).
Suspicion of a neural tube defect may be raised by a maternal serum screening test in the second trimester of pregnancy which detects an elevated concentration of alpha-feto-protein. In this setting, the diagnosis is confirmed by ultrasound examination during the second trimester of pregnancy. Cleft lip and palate can be identified on detailed ultrasound examination, but if there is only involvement of the palate, diagnosis by ultrasound can be difficult, and often not established until after birth. Unfortunately, these tests are not yet routinely done in most developing countries. Today, as infant mortality rates fall, birth defects are responsible for an increasing proportion of infant mortality and morbidity (Modell 1989; WHO 1997; WHO 2004). Affected infants have difficulty with feeding, and later with speech development, hearing, and tooth formation. Stigmatization and discrimination may pose lifelong problems. Malnutrition and infection resulting from cleft lip or cleft palate, or both, can lead to severe illness and, in some cases, death (Shibuya 1998).
The impact of folate insufficiency on birth defects in different populations varies with each healthcare system. It partly relates to the use and coverage of preventive strategies including education and awareness of the importance of folic acid intake among women of reproductive age, access to and/or distribution of pre-pregnancy folic acid supplements, and/or fortification of staple foods with folic acid, in some cases with mandatory regulations for some foods such as enriched wheat flour. Supplementation recommendations alone remain an ineffective approach in translating the known protective effect of folic acid into a population-wide decline in NTDs rates, even within six years of implementation in many countries. Recent evidence demonstrates that only public health policies including folic acid fortification of staple foods are likely to result in a large-scale prevention of NTDs (Botto 2005; Busby 2006). The additional intake from folic acid fortification of wheat flour in US and Canada has been estimated to be around 100 ug to 150 ug, and has successfully proven to reduce NTDs prevalence in both countries (Berry 2009). It would seem reasonbale to fully implement both interventions, especially in countries with a high prevalence of birth defects.
Several gene polymorphisms affect folate metabolism and are associated with reduced folate absorption and therefore, increased folate needs. Some of the most studied mutations are the methylene-tetrahydrofolate reductase (MTHFR) gene and the reduced folate carrier (RFC1) gene. The former affects 10% to 30% of various population groups studied, mostly in industrialzed countries. In the absence of a folate-rich diet, these mutations are associated with increased risk of NTDs and conotruncal defects in offspring (Chango 2000; Van der Put 1998; Wilcken 1997).
Folate deficiency may also affect fetal and child growth. Observational and controlled trials have showed a positive effect of periconceptional folic acid supplementation on fetal growth (Iyengar 1975; Relton 2005; Rolschau 1999). However, the evidence for this association remains controversial (Czeizel 1994).
Description of the intervention
After the first consistent evidence about the protective effect of folic acid supplementation against NTDs emerged, the United States Public Health Service in 1992 recommended daily supplementation with 400 ug of folic acid for all women that could get pregnant. The international recommendation today is the supplementation with 400 ug to women from the moment they are trying to conceive until 12 weeks of pregnancy. For women with a previous history of delivery of a baby with a NTD, who have diabetes, or who are receiving an anticonvulsant treatment, the recommended daily dose is 5 mg of folic acid in addition to dietary advice to increase food folate intake (IOM 2003; WHO 2006). Daily supplementation with 400 ug of folic acid in addition to iron is routinely recommended for all pregnant women in areas of high prevalence of malnutrition to prevent anaemia (WHO 2006). The World Health Organization also recommends weekly iron and folic acid supplementation in population groups where the prevalence of anaemia is above 20% among women of reproductive age, and where mass fortification programs of staple food-stuffs with iron and folic acid are unlikely to be implemented within the next one to two years (WHO 2009). The evidence of the effectiveness of the folic acid content of the supplements in a weekly regimen is limited, and little attention was given to the change in folic acid intake when supplements were given on a weekly as opposed to daily basis.
Recently, the use of 5-methyl-tetrahydrofolate (5-MTHF) has been proposed as an alternative to folic acid supplementation as most dietary folate, and folic acid in the diet, is metabolized to 5-MTHF during its passage across the intestinal mucosa. It may be an adequate alternative for supplementation in the presence of MTHFR gene mutation. Four controlled trials using different doses have shown that supplementation with 5-MTHF is at least as effective as folic acid in improving folate status in women of childbearing age (Houghton 2006; Lamers 2004; Venn 2002; Venn 2003). Information should be considered in relation to the formulation of folate (i.e. folic acid versus 5-MTHF, alone or with other micronutrients) and the rationale for the difference in effect with the varying formulations, the dosing interval, as well as the dose, as these are all areas of uncertainty and can have an impact on requirements (Czeizel 2009).
Furthermore, folic acid supplementation predicts failure of malaria treatment in African pregnant women (English 2006; Van Eijk 2008) and there is also evidence of positive effects with malaria treatment and the use of similar folic acid compounds (i.e. 5-methyl-tetrahydrofolate) that need to be explored (Nduati 2008).
How the intervention might work
The main function of folate is as coenzyme in one-carbon transfer during the methylation cycle; a process essential for the syntheses of nucleic acids, which form part of DNA and the neurotransmitters. From these reactions it is immediately apparent why folate is so important to gene expression. Folate also plays an important role in protein synthesis and metabolism and other processes related to cell multiplication and tissue growth (WHO 2008). Therefore, lack of folate, or folate deficiency, has severe consequences throughout life and especially for the unborn child. The main consequence of folate deficiency in adults is megaloblastic anaemia, characterized by abnormally large red-cell precursors in the bone marrow and larger than normal red cells in the peripheral blood.
The methylation of homocysteine to produce methionine (both essential amino acids) uses 5-MTHF as the methyl donor in the reaction. In folate deficiency, homocysteine accumulates in the serum resulting in negative effects for health. Elevated circulating homocysteine concentrations have been associated with an increased risk in cardiovascular disease (Refsum 2008) and late pregnancy complications such as pre-eclampsia (Makedos 2007; Patrick 2004; Tamura 2006), and maybe NTDs. Therefore, elevated plasma homocysteine may be a risk factor or, alternatively, merely a marker that needs to be determined (WHO 2008).
High levels of folic acid during pregnancy have been associated with some adverse effects. Recently, research on the fetal origins of disease has proposed that the susceptibility to type 2 diabetes originates in intrauterine life by environmental fetal programming. Normal to high maternal folate status coupled with low vitamin B
Additional potentially undesired effects of pre-pregnancy folic acid supplementation come from the possible association of the use of multivitamins containing folic acid and an increase in twin pregnancies found in the Swedish study (Ericson 2001). However, these findings could be strongly confounded by in vitro fertilization (Berry 2005; Vollset 2005). Further studies have confirmed no impact of suplementation with 400 ug of folic acid (Li 2003) and folic acid fortification (Kucik 2004; Shaw 2003; Signore 2005), on twinning.
Why it is important to do this review
There seems to be sufficient evidence of known benefits of folic acid supplemetation on neural tube defects but not on other birth defects or on benefits to the mother. However, the best scheme (daily versus weekly supplementation), dose and/or form (5-MTHF versus folic acid) for providing folate supplements to women of childbearing age or during the periconceptional period are not completely known. Additionally, concerns raised by some authors about the potential harm associated with high levels of folic acid supplementation, merit exploration (Cole 2007; Mason 2007).
In countries where the prevalence of NTDs is high, the cost-effectiveness of an integrated approach with supplementation and fortification may be more favourable. However, it is important to consider that countries with mandatory fortification have achieved a significant increase in folate intake and a significant decline in the prevalence of NTDs, but the relative decline depends on the initial NTD rate: countries with lower NTD prevalence may have much smaller reductions in NTD rates with folic acid fortification (Heseker 2008).
This review updates the previous systematic review assessing the effects of periconceptional supplementation of folic acid to reduce neural tube defects (Lumley 2001) and aims to examine whether folate supplementation before and during early pregnancy can reduce the prevalence of neural tube and other birth defects (including cleft palate) without causing adverse outcomes for mothers or babies.
Criteria for considering studies for this review
Types of studies
We will include both randomized and quasi-randomized trials. Crossover trials are not an appropriate study design for examining the interventions considered in this review and we will not include them. We will include cluster-randomized trials if they are otherwise eligible. Other levels of evidence (e.g. cohort or case-control studies) will not be included in meta-analyses nor will contribute to the results or conclusions, but will be addressed in the discussion where relevant.
Types of participants
All women who become pregnant or pregnant women during the first 12 weeks of pregnancy independent of age and parity. We will include studies focusing on women who have had a previous pregnancy affected by a neural tube defect.
Types of interventions
We will include a range of interventions including supplementation with folic acid alone (FA) and with other micronutrients. We will include studies where supplementation is offered before pregnancy and during early pregnancy. Where data are available we plan to compare:
- supplementation with FA alone versus no treatment/placebo;
- supplementation with FA + other micronutrients versus no treatment/placebo;
- supplementation with FA + other micronutrients versus other micronutrients (without folic acid);
- supplementation with 5-methyl-tetrahydrofolate (5MTHF) versus supplementation with FA;
- supplementation with 5MTHF alone versus no treatment/placebo;
- supplementation with 5MTHF + micronutrients versus no treatment/placebo;
- supplementation with 5MTHF + other micronutrients versus other micronutrients (without 5MTHF).
Where data are available and study populations are similar we will combine results from studies examining FA and 5MTHF to produce an overall effect of supplementation.
Types of outcome measures
- Neural tube defects
- Cleft lip
- Cleft palate
- Congenital cardiovascular defects
- Other birth defects
- Maternal anaemia at term (defined as Hb < 110 g/L)
- Red blood cell folate at term
- Serum folate at term
- Neonatal deaths
- Pregnancy termination for fetal abnormality
- Low birthweight (< 2500 g)
- Very low birthweight (< 1500 g)
- Infant optimal health status at birth (as defined by trialists)
- Admission to special care
- Macrosomia (> 4000 g)
- Infant insulin resistance (defined by trialists)
- Apgar at one and five minutes (> eight)
- Preterm delivery (< 37 weeks)
- Long-term outcomes (as defined by trialists)
- Multiple pregnancy
- Maternal concentration of homocystein at term (continuous)
- Maternal concentration of vitamin B
6at term (continuous)
- Maternal concentration of vitamin B
12at term (continuous)
- Any side effects
Search methods for identification of studies
We will contact the Trials Search Co-ordinator to search the Cochrane Pregnancy and Childbirth Group’s Trials Register.
The Cochrane Pregnancy and Childbirth Group’s Trials Register is maintained by the Trials Search Co-ordinator and contains trials identified from:
- quarterly searches of the Cochrane Central Register of Controlled Trials (CENTRAL);
- weekly searches of MEDLINE;
- handsearches of 30 journals and the proceedings of major conferences;
- weekly current awareness alerts for a further 44 journals plus monthly BioMed Central email alerts.
Details of the search strategies for CENTRAL and MEDLINE, the list of handsearched journals and conference proceedings, and the list of journals reviewed via the current awareness service can be found in the ‘Specialized Register’ section within the editorial information about the Cochrane Pregnancy and Childbirth Group.
Trials identified through the searching activities described above are each assigned to a review topic (or topics). The Trials Search Co-ordinator searches the register for each review using the topic list rather than keywords.
Searching other resources
We will contact the World Health Organization, Centers for Disease Control and Prevention (CDC), the International Clearinghouse for Birth Defects Monitoring Systems (ICBDMS) and March of Dimes to identify ongoing studies and unpublished reports.
We will not apply any language restrictions.
Data collection and analysis
Selection of studies
Two review authors will independently assess for inclusion the abstracts of all the studies we identify as a result of the search strategy. We will resolve any disagreement through discussion or, if required, we will consult a third author.
If studies are published only as abstracts, or study reports contain little information on methods, we will attempt to contact the authors to obtain further details of study design and results; if there is insufficient information for us to be able to assess risk of bias, studies will await assessment until further information is published, or made available to us.
Data extraction and management
We will design a form to extract data. For eligible studies, two review authors will extract the data using the agreed form. We will resolve discrepancies through discussion or, if required, we will consult a third person. We will enter data into Review Manager software (RevMan 2008) and carry out checks for accuracy.
When information regarding any of the above is unclear, we will attempt to contact authors of the original reports to provide further details.
Assessment of risk of bias in included studies
Two review authors will independently assess risk of bias for each study using the criteria outlined in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2008). We will resolve any disagreement by discussion or by involving a third assessor.
(1) Sequence generation (checking for possible selection bias)
We will describe for each included study the method used to generate the allocation sequence.
We will assess the method as:
- adequate (any truly random process, e.g. random number table; computer random number generator);
- inadequate (any non-random process, e.g. odd or even date of birth; hospital or clinic record number);
(2) Allocation concealment (checking for possible selection bias)
We will describe for each included study the method used to conceal the allocation sequence and will assess whether intervention allocation could have been foreseen in advance of, or during recruitment, or changed after assignment.
We will assess the methods as:
- adequate (e.g. telephone or central randomization; consecutively numbered sealed opaque envelopes);
- inadequate (open random allocation; unsealed or non-opaque envelopes, alternation; date of birth);
(3) Blinding (checking for possible performance bias)
We will describe for each included study the methods used, if any, to blind study participants and personnel from knowledge of which intervention a participant received. For interventions involving the provision of nutritional supplements it may be possible to blind women, clinical staff and outcome assessors to group allocation by providing placebo preparations. Blinding will be assessed separately for different outcomes or classes of outcomes, and we will indicate where there has been attempt at partial blinding (e.g. of outcome assessors).
We will assess the methods as:
- adequate, inadequate or unclear for participants;
- adequate, inadequate or unclear for personnel;
- adequate, inadequate or unclear for outcome assessors.
(4) Incomplete outcome data (checking for possible attrition bias through withdrawals, dropouts, protocol deviations)
We will describe for each included study, and for each outcome or class of outcomes, the completeness of data including attrition and exclusions from the analysis. We will state whether attrition and exclusions were reported, the numbers included in the analysis at each stage (compared with the total randomized participants), reasons for attrition or exclusion where reported, and whether missing data were balanced across groups. Where sufficient information is reported, or can be supplied by the trial authors, we will re-include missing data in the analyses which we undertake. We will assess methods as:
(5) Selective reporting bias
We will describe for each included study how we investigated the possibility of selective outcome reporting bias and what we found.
We will assess the methods as:
- adequate (where it is clear that all of the study’s prespecified outcomes and all expected outcomes of interest to the review have been reported);
- inadequate (where not all the study’s prespecified outcomes have been reported; one or more reported primary outcomes were not prespecified; outcomes of interest are reported incompletely and so cannot be used; study fails to include results of a key outcome that would have been expected to have been reported);
(6) Other sources of bias
We will describe for each included study any important concerns we have about other possible sources of bias.
We will assess whether each study was free of other problems that could put it at risk of bias:
(7) Overall risk of bias
We will make explicit judgements about whether studies are at high risk of bias, according to the criteria given in the Handbook (Higgins 2008). With reference to (1) to (6) above, we will assess the likely magnitude and direction of the bias and whether we consider it is likely to impact on the findings. We will explore the impact of the level of bias through undertaking sensitivity analyses - see 'Sensitivity analysis'.
Measures of treatment effect
For dichotomous data, we will present results as summary risk ratio with 95% confidence intervals.
For continuous data, we will use the mean difference if outcomes are measured in the same way between trials. We will use the standardized mean difference to combine trials that measure the same outcome (e.g. serum folate levels) but use different methods.
Unit of analysis issues
We will include cluster-randomized trials in the analyses along with individually-randomized trials. We will adjust the standard error of the effect estimate from cluster trials using the methods described in the Handbook (Higgins 2008). Meta-analyses will be carried out using the generic inverse-variance method available in Revman (RevMan 2008). We will use an estimate of the intracluster correlation co-efficient (ICC) derived from the trial (if possible), or from another source. If ICCs from other sources are used, we will report this and conduct sensitivity analyses to investigate the effect of variation in the ICC. If we identify both cluster-randomized trials and individually-randomized trials, we plan to synthesise the relevant information. We will consider it reasonable to combine the results from both if there is little heterogeneity between the study designs, and the interaction between the effect of intervention and the choice of randomization unit is considered to be unlikely.
We will also acknowledge heterogeneity in the randomization unit and perform a separate meta-analysis.
Crossover trials are not an appropriate study design for the interventions considered in this review. If we identify any such trials we will exclude them.
Studies with more than two treatment groups
If we identify studies with more than two intervention groups (multi-arm studies) where possible we will combine groups to create a single pair-wise comparison or use the methods set out in the Handbook to avoid double-counting study participants (Higgins 2008).
Dealing with missing data
For included studies, we will note levels of attrition. We will explore the impact of including studies with high levels of missing data in the overall assessment of treatment effect by using sensitivity analysis.
For all outcomes analyses will be carried out, as far as possible, on an intention-to-treat basis, i.e. we will attempt to include all participants randomized to each group in the analyses. The denominator for each outcome in each trial will be the number randomized minus any participants whose outcomes are known to be missing.
Assessment of heterogeneity
We will examine the forest plots from meta-analysis to look for heterogeneity among studies, and will use the I² statistic to quantify the level of heterogeneity among the trials in each analysis. If we identify moderate or substantial heterogeneity (I
Assessment of reporting biases
Where we suspect reporting bias (see 'Selective reporting bias' above), we will attempt to contact study authors asking them to provide missing outcome data. Where this is not possible, and the missing data are thought to introduce serious bias, the impact of including such studies in the overall assessment of results will be explored by a 'Sensitivity analysis'.
We will carry out statistical analysis using the Review Manager software (RevMan 2008). We will use fixed-effect meta-analysis for combining data where trials are examining the same intervention, and the trials’ populations and methods are judged sufficiently similar. Where we suspect clinical or methodological heterogeneity between studies sufficient to suggest that treatment effects may differ between trials we will repeat the analysis using a random-effects model (as a sensivtivity analysis).
Subgroup analysis and investigation of heterogeneity
Where data are available we will carry out subgroup analysis:
- by scheme: daily supplementation versus weekly supplementation;
- by dose: 400 ug or less of folic acid versus more than 400 ug folic acid;
- by start of supplementation: before pregnancy versus, during first trimester versus mixed;
- by assisted reproduction: assisted versus non-assisted reproduction;
- by mandatory fortification: places with mandatory flour fortification versus non-flour fortification or not mandatory;
- by personal history of neural tube defect (recurrence): yes versus no.
The primary outcomes will be used in subgroup analysis.
For fixed-effect meta-analyses we will conduct planned subgroup analyses classifying whole trials by interaction tests as described by Deeks 2001. For both fixed- and random-effects meta-analyses we will examine differences between subgroups by inspection of the subgroups’ confidence intervals; non-overlapping confidence intervals indicate a statistically significant difference in treatment effect between the subgroups.
We will carry out sensitivity analysis to examine the effects of removing studies at high risk of bias (studies with poor or unclear allocation concealment) from the analysis. If cluster trials are included we will carry out sensitivity analysis using a range of intracluster correlation values.
As part of the pre-publication editorial process, this protocol has been commented on by three peers (an editor and two referees who are external to the editorial team), a member of the Pregnancy and Childbirth Group's international panel of consumers and the Group's Statistical Adviser.
Protocol first published: Issue 3, 2009
Contributions of authors
All four review authors contributed to drafting the text of the protocol and commenting on drafts.
Declarations of interest
There are no known conflicts of interest.
Sources of support
- The University of Liverpool, UK.
- TD is supported by a grant from the National Institute for Health Research (NIHR), UK.UKNIHR NHS Cochrane Collaboration Programme Grant Scheme award for NHS prioritised, centrally managed, pregnancy and childbirth systematic reviews: CPGS02
- Department of Nutrition for Health and Development and Department of Reproductive Health and Research, World Health Organization, Switzerland.