Folic Acid Supplementation for the Prevention of Neural Tube Defects: An Update of the Evidence for the U.S. Preventive Services Task Force Prepared by:

Abstract

Research and Quality (AHRQ), Rockville, MD. The investigators involved have declared no conflicts of interest with objectively conducting this research. The findings and conclusions in this document are those of the authors, who are responsible for its content, and do not necessarily represent the views of AHRQ. No statement in this report should be construed as an official position of AHRQ or of the U.S. Department of Health and Human Services. The information in this report is intended to help clinicians, employers, policymakers, and others make informed decisions about the provision of health care services. This report is intended as a reference and not as a substitute for clinical judgment. This report may be used, in whole or in part, as the basis for the development of clinical practice guidelines and other quality enhancement tools, or as a basis for reimbursement and coverage policies. AHRQ or U.S. Department of Health and Human Services endorsement of such derivative products may not be stated or implied.

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Evidence Synthesis______________
Number 70
Folic Acid Supplementation for the Prevention of
Neural Tube Defects:
An Update of the Evidence for the U.S. Preventive
Services Task Force
Prepared by:
Tracy Wolff, MD, MPH
Catherine Takacs Witkop, MD MPH
Therese Miller, DrPH
Shamsuzzoha B. Syed, MD, MPH, DPH (Cantab)
Agency for Healthcare Research and Quality
U.S. Department of Health and Human Services
540 Gaither Road
Rockville, MD 20850
AHRQ Publication No. 09-05132-EF-1
May 2009

This report is based on research conducted by staff of the Agency for Healthcare
Research and Quality (AHRQ), Rockville, MD. The investigators involved have declared
no conflicts of interest with objectively conducting this research. The findings and
conclusions in this document are those of the authors, who are responsible for its content,
and do not necessarily represent the views of AHRQ. No statement in this report should
be construed as an official position of AHRQ or of the U.S. Department of Health and
Human Services.
The information in this report is intended to help clinicians, employers, policymakers,
and others make informed decisions about the provision of health care services. This
report is intended as a reference and not as a substitute for clinical judgment.
This report may be used, in whole or in part, as the basis for the development of clinical
practice guidelines and other quality enhancement tools, or as a basis for reimbursement
and coverage policies. AHRQ or U.S. Department of Health and Human Services
endorsement of such derivative products may not be stated or implied.
Suggested Citation: Wolff T, Witkop CT, Miller T. Syed SB. Folic Acid
Supplementation for the Prevention of Neural Tube Defects: An Update of the Evidence
for the U.S. Preventive Services Task Force. Evidence Synthesis No. 70. AHRQ
Publication No. 09-051132-EF-1. Rockville, Maryland: Agency for Healthcare Research
and Quality. May 2009.
ii

ABSTRACT
Background: Neural tube defects (NTDs) are among the most common birth defects in
the United States.
Purpose: To update the evidence on folic acid supplementation in women of
childbearing age for the prevention of neural tube defects in their offspring.
Data Sources: MEDLINE and Cochrane Library searches (from January 1995 through
November 2007), recent systematic reviews, reference lists of retrieved articles, and
expert suggestions.
Study Selection: English language studies were selected to answer the following two
questions: Does folic acid supplementation in women of childbearing age reduce the risk
of a pregnancy affected by a neural tube defect? Does folic acid supplementation in
women of childbearing age increase the risk of any harmful outcomes for either the
woman or the infant? The following study types were selected: for potential benefits of
folic acid—randomized, controlled trials (RCTs), case-control studies, cohort studies,
systematic reviews and meta-analyses; for potential harms of folic acid—RCTs, case-
control studies, systematic reviews, meta-analyses, and large observational studies.
Data Extraction: All studies were reviewed, abstracted, and rated for quality using
predefined U.S. Preventive Services Task Force criteria.
Data Synthesis: Four observational studies reported benefit, in reduction of risk of NTD
associated with folic acid-containing supplements. Differences in study type and methods
prevent the calculation of a summary of the reduction in risk. The one included study on
harms reported that the association of twinning with folic acid intake disappeared after
adjusting for in vitro fertilization and for underreporting of folic acid intake.
Limitations: There is limited evidence on dose. We found no evidence on the potential
harm of masking vitamin B12 deficiency in women of childbearing age. Our search
focused on NTDs and therefore does not provide a comprehensive review of the effects
of folic acid on all possible outcomes.
Conclusions: New observational evidence supports previous RCT evidence that folic
acid–containing supplements reduce the risk of NTD-affected pregnancies. The
association of folic acid use with twin gestation may be confounded by fertility
interventions including ovulation stimulation and in vitro fertilization.
iii

INTRODUCTION
Neural tube defects (NTDs) are among the most common birth defects in the United
States.(1) Estimates of disease burden are difficult to determine because affected
pregnancies are sometimes spontaneously or electively aborted and are underreported on
birth certificates.(2) The Centers for Disease Control and Prevention (CDC) estimates
that the rates in 2005 for two of the most common NTDs, spina bifida and anencephaly,
were 17.96 per 100 000 live births and 11.11 per 100 000 live births, respectively.(3)
NTDs cover a range of congenital malformations affecting the brain and spinal cord. This
spectrum of congenital defects has been defined as:
“Anencephaly (the total or partial absence of the cranial vault, the covering skin
and the brain tissue), spina bifida (non-closure of the spine resulting in herniation
or exposure of the spinal cord, the meninges or both; in some cases together with
hydrocephalus), encephalocoele (herniation of the meninges and brain tissue
outside the cranium, covered by normal or atrophic skin).”(6)
The embryological events leading to NTDs occur early in pregnancy. These events begin
at approximately the twenty-first day after conception, and neural tube closing occurs by
approximately 28 days after conception.(5)
Although neural tube defects (NTDs) span a range of disorders and possible etiologies, a
number of risk factors for the disorders have been identified. Two well-established risk
factors are having a history of a previous fetus or child with NTD and/or a first-, second-,
or third-degree relative with NTD.(7, 8) There are ethnic and racial variations in the
prevalence: Hispanics and non-Hispanic whites have higher rates of NTDs than African-
Americans and Asians.(9) In a large population-based study in California, the prevalence
in Hispanic women was found to be 1.12 per 1000 women screened (95% CI 1.04-1.21),
the prevalence in Caucasian women was 0.96 per 1000 (95% CI 0.89-1.04), in African-
American women 0.75 per 1000 (95% CI 0.59-0.91) and in Asian women 0.75 per 1000
(95% CI 0.60-0.90).(10) There are likely environmental or diet-related differences that
contribute to these differences in prevalence of NTDs; however, the prevalence of
mutations in certain enzymes may also differ among these population groups. Two
enzymes that have been linked to increased risk for certain NTDs are
methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTRR).(11)
Maternal medical conditions such as diabetes and obesity have been associated with an
increased risk of NTDs. Women with epilepsy who take certain anti-epileptic
medications, such as valproic acid or carbamazepine, are also at increased risk. The risk
of an open NTD in a woman taking these medications in the first trimester of pregnancy
is reportedly 1-2%. Other medications associated with NTDs include vitamin A and
Warfarin.(11)
1

The effectiveness of folate in preventing NTDs has been reported in multiple studies.(6)
The mechanism of action has not been established. However, a main function for folate is
in one-carbon transfers, which are important in methylation reactions and in purine and
pyrimidine synthesis. Folate is necessary for the regulation of DNA synthesis and
function and therefore affects important events in embryogenesis that may lead to NTDs.
There are three main approaches to achieving adequate levels of folate in women who are
capable of becoming pregnant in the United States: ensuring a healthy diet that includes
foods fortified with folic acid; providing folic acid supplements; and providing a
combination of supplements and a folic acid-rich diet. Despite the US recommendations
and efforts in the United States to increase folic acid intake, in 1997 intake of
supplements by women had not achieved desired levels: only about 30% of women
reported taking a daily supplement containing folic acid.(12) Overall, 26% of women
reported taking at least 400 micrograms of folic acid per day via supplements within the
previous month and 34.3% of women of reproductive age reported consuming over 400
micrograms per day via a combination of fortified foods and supplements. Recently, the
2007 March of Dimes Gallup survey used a random-digit dialed telephone interview in
women of childbearing age. Of this population, 40% reported taking folic acid daily, as
compared to 32% in 2003, 40% in 2004, and 33% in 2005. When stratified by age, 47%
women from 25-34 years of age reported taking a daily supplement with folic acid as
compared to 30% of women aged 18-24.(13)
In 1998, the U.S. Food and Drug Administration (FDA) mandated fortification of all
enriched grain products at the level of 0.14 mg/100 grams of grain.(1) In the United
States, fortification was expected to add approximately 100 µg of folic acid per day to the
average American diet and increase the percentage of women of childbearing age
consuming at least 400 µg/day from 29% to 50%.(1) In reality, the first expectation was
met but the second was not: the proportion of women of childbearing age who consume
more than 400 µg/day of folate varies by race/ethnicity from 23% to 33%.(1) According
to more recent data from the 2001-2002 National Health and Nutrition Examination
Survey (NHANES 2001-2002), limited to non-pregnant female participants between 15
and 49 years of age, the adjusted mean daily consumption of folic acid via fortified foods
was estimated at 128 micrograms and was found to be 18% lower in non-Hispanic black
women (109 micrograms/day) than in non-Hispanic white women (133 micrograms/day).
Consumption of more than 400 micrograms/day from fortified foods was reported by
only 8% of women.(14) There were differences by race/ethnicity in intake: 40.5% of
non-Hispanic white, 19% of non-Hispanic black women, and 21.0% of Hispanic women
reported consuming over 400 micrograms per day from all sources.
Despite less than ideal population intake, the prevalence of NTDs reported on birth
certificates in the United States decreased from 37.8 per 100 000 live births before
fortification to 30.5 per 100 000 live births conceived after mandatory folic acid
fortification, representing a 19% decline.(15) More recently, the CDC reported that 23
population-based surveillance systems showed an approximately 26% decline in NTD
prevalence rates in the United States before and after mandated fortification.(16)
2

The last U.S. Preventive Services Task Force (USPSTF) recommendation on the use of
folic acid in women of childbearing age was made in 1996. At that time the USPSTF
recommended that all women planning a pregnancy or capable of conception take a
supplement containing folic acid. They found insufficient evidence to recommend for or
against counseling women to increase their dietary folate consumption as an alternative to
taking a folic acid supplement.
The purpose of this review is to update the evidence on folic acid supplementation in
women of childbearing age. The USPSTF decided to focus its new review on folic acid
supplementation; therefore, this review does not include a review of the evidence on
fortification, counseling to increase dietary intake, or on screening for neural tube
defects. Because this current review is an update, it includes only new literature
published since 1995. The analytic framework developed for this review following
USPSTF methods is shown in Figure 1. The USPSTF developed 2 key questions (KQs)
from the analytic framework to guide its consideration of the evidence on folic acid
supplementation. The key questions are:
KQ1: Does folic acid supplementation in women of childbearing age reduce the
risk of a pregnancy affected by a neural tube defect?
KQ2: Does folic acid supplementation in women of childbearing age increase the
risk of any harmful outcomes for either the woman or the infant?
In addition to these key questions, which define the scope of this systematic review, the
USPSTF requested that information be gathered to answer 2 additional questions to
provide context for their recommendation. The 2 contextual questions are:
1. What is the current dietary intake of folic acid (average level of folate) in
women of childbearing age?
2. What is the most effective dose of folic acid supplementation?
The results of these two contextual questions are discussed in the Introduction and
Discussion sections of this report.
METHODS
Data Sources and Searches
We performed a systematic search for English language articles published between
January 1, 1995, and November 30, 2007, through a MEDLINE search using the terms
“neural tube defects,” “folic acid,” “pregnancy,” “twinning,” and “twins.” Additional
studies were identified through a search of the Cochrane database, through discussions
with experts, and by hand-searching of reference lists from included studies and major
review articles and studies.
3

Study Selection
Two reviewers independently reviewed the titles and abstracts and selected articles for
inclusion based on predetermined inclusion and exclusion criteria. In general, studies
were selected for benefits if they were RCTs, case-control studies, cohort studies or
systematic reviews; and for harms if they were RCTs, case-control studies, cohort
studies, systematic reviews or large observational studies; reported overall effect on
reduction of neural tube defects or effect on harms in association with folic-acid
containing supplements; and provided new evidence that was not in the 1996 USPSTF
report. Studies were excluded if they included no new evidence since the 1996 review;
were subanalyses of data without overall effect on NTD or harms; did not report effect of
supplements separate from dietary effects; were letters, editorials, or non-systematic
reviews; were performed in special or high-risk populations; or were performed in a
country or population with widespread malnutrition or otherwise not generalizable to the
United States. More details on search terms and inclusion/exclusion criteria are described
in Appendix 1. Disagreements about inclusion of an article were discussed and selected
based on consensus; if necessary a third reviewer was used for disagreements.
Data Extraction and Quality Assessment
For all citations that met initial eligibility criteria, the full articles were reviewed,
abstracted, and quality-rated independently by two reviewers. Studies were ultimately
included if they were rated fair or good based on USPSTF criteria. Consensus about
article abstraction data and quality was achieved through discussion by the two
reviewers; disagreements were resolved by the involvement of a third reviewer. Data on
the following items were extracted from the included studies: methods; exposure
assessment; case ascertainment; selection of participants; dose of folic acid; sample size;
size of effect on NTDs, other congenital abnormalities, and twinning; and information on
confounders. Quality-rating of articles for all KQs was performed using standard
USPSTF methodology on internal and external validity. We evaluated the quality of
RCTs and cohort studies on the following items: initial assembly of comparable groups,
maintenance of comparable groups, important differential loss to follow-up or overall
high loss to follow-up, measurements (equality, reliability, and validity of outcome
measurements), clear definition of the interventions, and appropriateness of outcomes.
We evaluated systematic reviews and meta-analyses on the following items:
comprehensiveness of sources considered, search strategy, standard appraisal of included
studies, validity of conclusions, recency, and relevance. More complete criteria and
definitions for USPSTF quality ratings are listed in the Appendix 2.
Data Synthesis and Analysis
Data from studies included for KQ1 and KQ2 were synthesized qualitatively in tabular
and narrative format. Synthesized evidence was organized by key question.
Role of the Funding Source
4

The general work of the USPSTF is supported by the Agency for Healthcare Research
and Quality. This specific review did not receive separate funding.
RESULTS
Our search for evidence from PubMed, Cochrane library, reference lists, and experts
returned 810 articles. Details on reasons for exclusions are in Figure 2. The most
common reason for exclusion was incorrect study type; most were letters, editorials, or
non-systematic reviews. Many were also excluded because the studies were not on folic
acid supplementation; most of these were on folic acid fortification of foodstuffs. There
were several additional common reasons for exclusion: the study was in a setting that was
not thought to be generalizable to the United States; the study was a subanalysis of
previously included data; or the study did not report overall outcomes as benefits in the
reduction of NTD or harms associated with folic acid. Four studies for the key question
on benefits and one study for the key question on harms ultimately met inclusion criteria
and were of appropriate methodological quality. Studies that initially met inclusion
criteria, and that were abstracted and quality-rated but ultimately excluded are discussed
in Table 1 (KQ1-benefits) and Table 2 (KQ2 - harms).
KQ1: Does folic acid supplementation in women of childbearing age reduce the risk
of a pregnancy affected by a neural tube defect?
Summary of Study Results
Observational studies on the benefits of folic acid supplementation provide generally
consistent evidence that folic acid supplementation in the periconceptional period reduces
the risk of neural tube defects in offspring. This evidence was provided by three fair- or
good-quality cohort, case-control, and meta-analytic studies that found statistically
significant benefit; one small, fair-quality case-control study reported benefit that was not
statistically significant. In addition to NTDs, the cohort and meta-analysis found
reductions in cardiovascular congenital abnormalities associated with folic acid-
containing multivitamins.
Study Characteristics
The search for literature to answer this question returned four articles that met the
inclusion/exclusion criteria, were published within the search timeframe, and were of
appropriate methodological quality. Detailed study characteristics and outcomes are
available in Table 3.
A 2004 cohort study by Czeizel and colleagues followed 3056 women considering
pregnancy who were recruited from the Hungarian Periconceptional Service. Women in
the supplemented group were given multivitamins containing 0.8 mg of folic acid one
month prior to planned conception. Women were recruited into the cohort as
unsupplemented controls and matched to the supplemented group by age, socio-economic
status, and employment status if they arrived for their first prenatal visit between 8 and 12
5

weeks gestation and had not taken folic acid–containing supplements in the
periconceptional period. The study design had some methodological problems that led to
a fair rating. There was potential for self selection bias as evidenced by the higher
maternal rate of personal, family, offspring congenital abnormalities and the higher rate
of prior fetal and infant loss in the supplemented group. Measurement of exposure to
folic acid was performed earlier in the pregnancy in the supplemented group than in the
unsupplemented group.
Two case-control studies were found in the literature search. These studies explored the
association between exposure to folic acid supplementation in the periconceptional period
and NTD in women residing in two geographic areas—California counties and the state
of South Carolina. Both studies ascertained cases in a comprehensive way through review
of records from hospitals and genetic clinics, and controls were randomly selected from
hospitals in proportion to their contribution to all births in the respective populations. The
first case-control study by Shaw and colleagues in 1995 was rated good because of
accurate ascertainment of cases, selection of cases and controls without obvious biases,
response rates of 88% in both cases and controls, exposure measurement applied equally
to cases and controls, and exploration of reporting bias by asking mothers whether they
believed folic acid had protective, causal, or no effect on birth defects. The second case-
control study, by Thompson and colleagues in 2003, had accurate ascertainment of cases
and selection of cases and controls without obvious biases, but had a small sample size,
differential measurement assessments, and differential response rates in the cases and
controls. These methodological problems led to a fair rating.
The fourth study was a meta-analysis of studies on pre- and periconceptional
multivitamin use and congenital malformations. This fair-quality meta-analysis was
recent and relevant and searched several resources for evidence; however, it did not
include expert consultations and did not report a standard appraisal of study
methodology. In addition, the applicability of this meta-analysis to the current review is
limited because it excluded studies on folate-only supplementation and included several
studies that were excluded by us: studies that were published prior to 1995, that were
performed in special populations or that did not report NTD as a separate outcome.
Study Results
The Czeizel cohort study reported that 1 NTD and 9 NTDs occurred in the supplemented
and unsupplemented women, respectively, for an adjusted odds ratio (aOR) of 0.11 (95%
CI, 0.01-0.91); the odds ratio (OR) was adjusted for birth order, chronic maternal
disorders, and history of previous fetal death or congenital abnormality. The meta-
analysis also found a protective effect of folic acid–containing multivitamins in NTDs
with an OR of 0.67 (95% CI, 0.58-0.77) in case-control studies and an OR of 0.52 (0.39-
0.69) in RCTs and cohort studies. Both the Czeizel study and the meta-analysis found a
statistically significant association between folic acid supplementation and a reduction in
cardiovascular congenital abnormalities. In addition, there was a significant effect of folic
acid–containing multivitamin use on congenital limb defects in the meta-analysis. No
6

consistent effect of folic acid–containing multivitamins, either on orofacial clefts or on
urinary tract congenital abnormalities, was seen in the Czeizel study or the meta-analysis.
The Shaw 1995 case-control study reported an OR of 0.65 (95% CI, 0.45-0.94) for use of
folic acid–containing supplements in the 3 months before conception, and an OR of 0.60
(95% CI, 0.46-0.79) for supplement use in the 3 months after conception. The 2003 study
by Thompson and colleagues reported an OR of 0.55 (0.25-1.22) for regular use, and an
OR of 0.92 (0.55-1.55) for some use of folic acid–containing supplements, but neither of
these findings was statistically significant.
Several differences in these case-control studies may explain differences in results. The
2003 Thompson study was smaller and adjusted for dietary folate intake. Additionally,
the exposure timeframes were different: the Shaw study measured exposure in 2 time
frames, 3 months before and 3 months after conception, while the Thompson study
combined these same 6 months of periconception time into one measure of exposure.
KQ2: Does folic acid supplementation in women of childbearing age increase the
risk of any harmful outcomes for either the woman or the infant?
Summary of Study Results
We found one study of fair quality on the harms of twinning that suggests that the
association of folic acid supplementation with twinning may be the result of confounding
by infertility treatment and by differential reporting of folic acid use. We found no clear,
consistent evidence that preconceptional folic acid use results in increased rate of
twinning. We did not find any studies on other previously suggested potential harms such
as masking of vitamin B12 deficiency.
Study Characteristics
We identified one study meeting the inclusion and quality criteria that addressed whether
folic acid supplementation in women of childbearing age increases the risk of harmful
outcomes for either the woman or the infant.(17) Details of this study are in Table 4. This
retrospective cohort study examined the association between risk of twinning in 176 042
women who gave birth in Norway between December 1998 and December 2001 and their
history of multivitamin or folic acid supplementation before or during pregnancy.
Assessment of exposure was by birth attendant at the time of delivery, thus introducing
potential problems with both measurement validity and differential recall. Overall, six
percent of women in this study reported folic acid supplementation preconceptionally;
however 24% of women who became pregnant through in vitro fertilization (IVF)
reported supplementation. Given the concern for underreporting of folic acid use
(calculated to be about 45% when the authors linked the pregnancies in this analysis to
another large cohort study with more accurate assessment of folic acid exposure) and
potential confounding by IVF, the authors included adjustment for these factors.
7

This study was rated fair, given its use of reasonable, albeit not the best, methods for
exposure assessments, and its consideration of and accounting for most confounders,
including IVF. Recall by mothers was likely imperfect, given that exposure was assessed
at delivery, and there may have been differential recall of exposure by mothers with or
without twin pregnancies. The exact dose, timing, and duration of the interventions were
not clear, but all important outcomes were assessed.
Study Results
After adjusting for age and parity, the authors reported an OR of 1.59 (95% CI 1.41-1.78)
for twin delivery after preconceptional folic acid supplementation. In a subgroup analysis
of women who did not report IVF, the risk of twinning was lower and non-significant
(OR 1.13, 95% CI 0.97-1.33), as expected given the increase in multiple gestation
associated with IVF and other assisted reproductive technologies. The odds of having
twins of unlike sex, an outcome used as a proxy for dizygotic twinning, were increased in
women taking folate, (OR 1.43, 95% CI 1.12-1.83). The authors then adjusted for both a
45% underreporting of supplementation as well as an estimated 12.7% of unidentified
IVF pregnancies. When the likely underreporting for folic acid use and IVF were
accounted for, the OR for twin delivery after preconceptional supplementation fell to
1.02, and was no longer statistically significantly greater than the risk for women who did
not take folic acid (95% CI, 0.85-1.24).
DISCUSSION
Dose of Folic Acid Supplementation
There are considerable difficulties in determining the most effective dose, form, and
timing of folic acid supplementation for prevention of first NTDs. RCTs provide the best
opportunity to make these determinations, but there has only been a single RCT assessing
women without a history of a previously affected child.(18) In this study, women who
were treated periconceptionally with 0.8 milligrams per day had a statistically
significantly lower risk of NTDs, but there was no opportunity to study other doses in the
setting of this RCT. Observational studies have also attempted to answer these questions
about dosage, but are plagued by difficulties of accurate exposure assessment (dose,
form, and timing); heterogeneity with respect to whether studies accounted for
supplements, fortified foods, and dietary intake of naturally-occurring folate; and
variability in bioavailability of various sources.
One of the studies cited in the 1996 USPSTF report presented findings that could serve as
an estimate of the minimal dose effective at reducing NTD risk.(19) This study derived
relative risks according to daily folic acid dose among 18 case mothers and 322 control
mothers, and found that patients taking 0.4 mg/day from one month before to one month
after the last menstrual period had a 70% reduction in risk of NTD (RR 0.3, 95% CI 0.1,-
0.6), while those taking less than 0.4 mg (but still taking some supplementation) had a
non-statistically significant reduced risk of 50% (RR 0.5, 95% CI 0.2-1.5); the latter
group consisted of only 53 women however, making definitive conclusions difficult.(19)
8

Due to the difficulty of assessing doses in observational studies, other studies have
attempted to answer this question by relating the risk of NTDs instead to serum or red
blood cell folate levels. In a case-control study in Dublin, Ireland, that examined maternal
red-cell and plasma folate levels and risk of NTD, the risk of NTD decreased from 6.6
per 1000 births when red-cell folate levels were below 150 nanograms/mL to 0.8 per
1000 births at a level of greater than 400 nanograms/mL.(20) Most importantly, NTD
risk was found to be inversely associated with red-cell folate levels in a continuous
relationship. In another study by Daly (21), data demonstrated that women living in a
country without mandatory fortification who take 200 micrograms of additional folic acid
per day achieve red cell folate levels that exceed 400 nanograms/mL, the level shown in
the previous study to be associated with decreased NTD risk. The authors estimated that
adding 400 micrograms, 200 micrograms, and 100 micrograms daily could reduce NTD
incidence by 47%, 41% and 22% respectively.(21)
More recently, Wald and colleagues (24) used published data on “dose” (folic acid
intake) and “response” (serum folate concentrations and risk of NTD) to develop a model
with which to predict responses to specific increases in folic acid intake. They used data
on the effects of folic acid supplementation on serum folate from 13 published studies
and then attempted to relate doses to outcomes of NTDs based on serum folate levels
from the Daly (20) study mentioned above. At an increased level of 0.2 mg/day, the %
risk reduction of NTDs was calculated to be 36% for a woman with a background serum
folate of 2.5 ng/mL, 23% with baseline folate of 5.0 ng/mL (typical western diet,
according to authors), and 13% with a background of 10 ng/mL. The authors of this study
assert that maximal benefit can be derived by taking an additional 5.0 mg per day (in the
form of a supplement), which would reduce risk of NTDs by 85% in women with a
background folate intake of 5.0 ng/mL. However, the authors discount concerns about
adverse effects of high levels of folate supplementation, despite a lack of studies
investigating harms of such doses.
Other investigators have also taken into account both folic acid supplementation and
dietary folate. In one study, dietary and supplement information from 23 228 women in
the northeastern United States was collected in the early second trimester of pregnancy
(25). Accounting for differing bioavailability of folate from varying sources, the authors
converted all sources of folate to dietary folate–equivalent units and estimated the
prevalence of NTDs according to individual and total sources of folate. They identified a
dose-response relationship between increasing folate equivalents per day and decreasing
prevalence of NTDs (p-value for linear trend of 0.016). Thompson and colleagues (26)
also attempted to relate the risk of NTDs to total intake. In a case-control study of 487
women, they demonstrated decreased risk of NTDs at the highest quartiles of total folate
intake (0.880-3.125 mg/day) (OR 0.35, 95% CI 0.17-0.72), but no significant dose-
response relationship with supplement dose alone.
Benefits of Folic Acid Supplementation
9

New evidence from observational studies provides weight to previous evidence from
controlled trials that folic acid supplementation provides benefit in reduction of risk from
NTD-affected pregnancies. We found four studies of the benefits of supplementation, of
fair or good quality, published since the previous 1996 USPSTF report. Odds ratios for
reductions in NTDs associated with periconceptional folic acid supplementation ranged
from 0.11 to 0.65 in cohort and case-control studies. A meta-analysis reported an OR for
NTDs inversely associated with multivitamin use of 0.67 in case-control studies and 0.52
in RCTs and cohort studies.
A study that was excluded from our review because it was performed in a population not
generalizable to the United States deserves discussion. This cohort study evaluated the
pregnancy outcomes of 130 142 women in 3 provinces in China who were asked during
their premarital medical examination to take a 0.4 mg daily folic acid supplement.(27)
Periconceptional use of a folic acid supplement was associated with an approximately 40-
80% reduction in risk of NTD-affected pregnancies; the reduction was greater in a region
with higher pre-study rates of NTDs. While the direct applicability of these specific rate
reductions to the United States population is limited by the differences in the two
countries’ nutritional levels, these results nevertheless lend additional strength to the
evidence on benefit.
Harms of Folic Acid Supplementation
The only RCT included in the 1996 USPSTF report on the prevention of first occurrence
NTDs noted an increase in the risk of twinning among multivitamin users (28). These
findings were not statistically significant when the data were re-analyzed and twin
deliveries were considered as the outcome instead of twin births (29). In our current
review, we attempted to identify all studies published since 1996 that examined twinning
as an outcome. The one fair-quality study that was included found no association between
preconceptional folic acid use and twinning; this study differed from previous studies
because it accounted for both the high rate of underreporting of folic acid use (seen in
many populations and studies) and the use of IVF. Another study, excluded based on
population, that found no association with twinning was the prospective study from
China discussed above, in which exposure assessment was likely fairly accurate and IVF
and ovulation induction were not prevalent confounding factors (39).
One additional study that warrants discussion here was excluded from the systematic
review because it was of a study type that did not meet inclusion criteria (31). The
authors of this study used Australian pregnancy and birth and morbidity and mortality
data as well as pooled estimates of relative risks for NTDs and twinning from previous
trials to model the impact of periconceptional folate on NTDs and twinning in a
hypothetical cohort of 100 000 pregnancies. They concluded that, although full
supplementation could result in 119 fewer NTDs (95% CI, -74-141), it would result in
approximately 1144 (95% CI, -200-3174) more twin births after 20 weeks’ gestation. The
estimated RR of twinning was from studies that did not address potential confounders
such as IVF or ovulation induction. Although this study offers a unique perspective, its
applicability to a United States population is limited.
10

Other potential concerns about folic acid supplementation include masking of vitamin
B12 deficiency. Our current review yielded no evidence to support or refute this possible
harm. However, given the low prevalence of vitamin B12 depletion in young women, it is
unlikely that folic acid supplementation in women of childbearing age would result in a
significant number of cases of neurological sequelae due to masking of vitamin B12
deficiency. In a study using data from NHANES and the Hispanic Health and Nutrition
Examination Survey, the CDC National Center for Health Statistics reported in 1998 that
under 1% of the total population between 4 and 50 years of age had serum vitamin B12
less than 100 pg/mL, the level below which vitamin B12 deficiency is likely.(32)
Furthermore, in an ecologic study comparing patients in pre- and post- folic acid
fortification periods, there was no evidence of an increase in low vitamin B12 levels
without anemia.(33) Finally, folic acid supplementation is often given in the form of a
multivitamin or prenatal vitamin that includes supplementation with B12, reducing the
likelihood of masking of B12 deficiency in this population.
Limitations
This review looked specifically for studies on NTDs and therefore does not include a
comprehensive picture of how folic acid–containing supplements may prevent other
congenital abnormalities. We did not review the evidence on counseling to increase
dietary intake of folic acid. We reviewed the overall effect of folic acid on NTDs and did
not comprehensively review the evidence on how the effect may differ among ethnic
groups and among groups with genetic differences that may affect the metabolism of
folic acid.
Acknowledgements
The authors would like to express their gratitude to Gloria Washington, Coordinator of
the USPSTF program, who spent many hours tracking down articles and keeping us
organized. We would also like to thank AHRQ librarian, Caryn McManus, for her expert
assistance in navigating PubMed and Endnote. We would especially like to acknowledge
Mary Barton, Scientific Director of AHRQ’s Center for Primary Care, Prevention and
Clinical Practice, and the following Task Force members for their expert guidance during
this review: Lucy Marion, Kimberly Gregory, and Tom DeWitt.
11

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26. Thompson SJ, Torres ME, Stevenson RE, Dean JH, Best RG.
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acid in China. China-U.S. Collaborative Project for Neural Tube Defect
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30. Berry R, Kihlberg R, Devine O. Impact of misclassification of in vitro
fertilisation in studies of folic acid and twinning: modelling using population
based Swedish vital records. BMJ. 2004;330:815-.
31. Lumley J, Watson L, Watson M, Bower C. Modelling the potential impact of
population-wide periconceptional folate/multivitamin supplementation on
multiple births. Bjog. 2001;108(9):937-42.
32. CDC. Blood folate and vitamin B12: United States: 1988-94. Vital Health
Statistics. 1998;11(243).
33. Mills J, Von Kohorn I, Conley M, et al. Low vitamin B-12 concentrations in
patients without anemia: the effect of folic acid fortification of grain. Am J Clin
Nutr. 2003;77:1474-7.
34. Czeizel AE, Toth M, Rockenbauer M. Population-based case control study of
folic acid supplementation during pregnancy. Teratology. 1996;53(6):345-51.
13

35. Locksmith GJ, Duff P. Preventing neural tube defects: the importance of
periconceptional folic acid supplements. Obstet Gynecol. 1998;91(6):1027-34.
36. Kallen BA, Olausson PO. Use of folic acid and delivery outcome: a prospective
registry study. Reprod Toxicol. 2002;16(4):327-32.
37. Lumley J, Watson L, Watson M, Bower C. Periconceptional supplementation
with folate and/or multivitamins for preventing neural tube defects. Cochrane
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38. Medveczky E, Puho E. Parental employment status and neural-tube defects and
folic acid/multivitamin supplementation in Hungary. Eur J Obstet Gynecol
Reprod Biol. 2004;115(2):178-84.
39. Shaw GM, Nelson V, Carmichael SL, Lammer EJ, Finnell RH, Rosenquist
TH. Maternal periconceptional vitamins: interactions with selected factors and
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40. Shaw GM, Rozen R, Finnell RH, Wasserman CR, Lammer EJ. Maternal
vitamin use, genetic variation of infant methylenetetrahydrofolate reductase, and
risk for spina bifida. Am J Epidemiol. 1998;148(1):30-7.
41. Shaw GM, Todoroff K, Carmichael SL, Schaffer DM, Selvin S. Lowered
weight gain during pregnancy and risk of neural tube defects among offspring. Int
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42. Shaw GM, Velie EM, Schaffer D. Risk of neural tube defect-affected
pregnancies among obese women. Jama. 1996;275(14):1093-6.
43. Suarez L, Hendricks KA, Cooper SP, Sweeney AM, Hardy RJ, Larsen RD.
Neural tube defects among Mexican Americans living on the US-Mexico border:
effects of folic acid and dietary folate. Am J Epidemiol. 2000;152(11):1017-23.
44. Ericson A, Kallen B, Aberg A. Use of multivitamins and folic acid in early
pregnancy and multiple births in Sweden. Twin Res. 2001;4(2):63-6.
45. Czeizel AE, Vargha P. Periconceptional folic acid/multivitamin supplementation
and twin pregnancy. Am J Obstet Gynecol. 2004;191(3):790-4.
46. Kallen B. Use of folic acid supplementation and risk for dizygotic twinning.
Early Hum Dev. 2004;80(2):143-51.
47. Czeizel AE, Dobo M, Vargha P. Hungarian cohort-controlled trial of
periconceptional multivitamin supplementation shows a reduction in certain
congenital abnormalities. Birth Defects Res A Clin Mol Teratol.
2004;70(11):853-61.
48. Goh YI, Bollano E, Einarson TR, Koren G. Prenatal multivitamin
supplementation and rates of congenital anomalies: a meta-analysis. J Obstet
Gynaecol Can. 2006;28(8):680-9.
49. Shaw GM, Schaffer D, Velie EM, Morland K, Harris JA. Periconceptional
vitamin use, dietary folate, and the occurrence of neural tube defects.
Epidemiology. 1995;6(3):219-26.
14

Figure 1. Analytic Framework for the USPSTF Review on Folate Supplementation for
the Prevention of Neural Tube Defects
KQ = Key Question
NTD = Neural tube defect
Folic acid
supplementation
Decreased NTD-
affected
pregnancies
KQ2
Serum folate
levels
KQ1
Harms
Women of
childbearing age
15

16
Figure 2. Search results and article flow.
805 Articles excluded
353 Incorrect study type
225 Not on folic acid supplementation
73 No outcomes of interest
52 In a setting not generalizable to US
48 High risk or special population
42 Not in women of childbearing age
6 Methodological issues
4 Duplicate or no new data
2 n<100
5 articles included in review
4 studies for KQ1 on bene its f
1 study for KQ2 on harms
Stage of exclusion
358 at title
181 at abstract
266 at full article
810 Potentially relevant articles identified

Table 1. Studies excluded after abstraction and quality rating - key question on benefits
Study Methods Notes/Reason for Exclusion
Czeizel, 1996 (34)
Case-control study of participants of the Hungarian Case Control Surveillance of Congenital Abnormalities from
1980 to 1991, exploring the relationship of folic acid with congenital anomalies
Cases: 17 300 mothers of infants with congenital anomalies
Population controls: 30 663 mothers of infants with no congenital abnormalities matched to cases by sex, birth
week, residence
Patient controls: 607 mothers of infants with Down Syndrome
Exposure is folic acid supplementation:
Dosing included folic acid (3 mg tablets, 1-3 times per day) or multivitamin (folic acid dose not reported)
Timing included preconception, 1st month of pregnancy, 2-3rd month of pregnancy, 4-9th month of pregnancy, and
unknown
Retrospective exposure assessment poses potential
recall bias.
Differential measurement of exposure causes
potential measurement bias.
Lower response rate in controls
No adjustment for smoking
Locksmith, 1998 (35) Review of studies examining the use of supplemental folic acid for prevention of NTDs Study type not included in review (not a systematic
review)
Kallen, 2002 (36)
Cohort study of 5331 infants registered in the Swedish Medical Birth Registry between 1995 and 2001 whose
mothers had reported use of folic acid in early pregnancy, examining the relationship of folic acid and congenital
malformations
Exposure is folic acid in early pregnancy (doses ranging from 0 to 5 mg of folic acid).
Outcome is congenital malformations including NTDs.
Subgroup analyses: women with subfertility problems and use of antiepileptic drugs
Used involuntary childlessness as proxy for
infertility
Exposure assessed by questionnaire at gestational
week 10-12: drugs taken “since she became
pregnant”
No information about dose, timing
Lumley, 2001 (37)
Systematic review of randomized and quasi-randomized studies published until April 2001 relating to whether
NTDs can be reduced by increased consumption of multivitamins or folate before pregnancy or in first two months
of pregnancy
Studies included were not recent (many published
prior to 1995 and included in USPSTF previous
evidence report).
Medveczky, 2004 (38)
Case control study of participants in the Hungarian Case Control Surveillance of Congenital Abnormalities from
1980 to 1996, to explore the association between socioeconomic status, periconceptional folic acid/multivitamin
supplementation and NTDs in Hungary
Cases: 1202 mothers of infants or fetuses with NTDs
Population controls: 38 151mothers of infants without congenital anomalies matched for sex, week of birth and
district of residence
Patient controls: 22 475 mothers of infants with congenital anomalies other than NTDs
Exposure is periconceptional or pregnancy folic acid use and employment status classification
No information on overall effect of folic acid on
NTDs
Moore, 2003 (25) Prospective cohort study of 23 228 women predominantly from northeastern U.S. who were in early second This was a study of dose-response re-examining
17

trimester and had either serum alpha-fetoprotein screening test or amniocentesis to examine the effect of folic acid
dose during early pregnancy
Exposure: folate from food, supplements, or fortified grains (converted to dietary folate−equivalents (DFEs):
13 431 women had intake of 0 DFEs/day
2489 women had intake of 1-399 DFEs/d
1812 women had intake of 400-799 DFEs/d
5,494 women had intake of ≥800 DFEs/d
Outcome: infant with NTD
data from study reviewed in 1996 USPSTF report;
no new information about overall benefits of folic
acid supplementation
Shaw, 2002 (39)
Case-control study of live births and fetal deaths (at >20 weeks) from January 1987 to December 1989 in most
California counties, to evaluate possible interactions of periconceptional vitamins with selected factors on
congenital anomalies
Cases: mothers of infants or fetuses with congenital anomalies (265 with NTDs)
Controls: 734 mothers of infants without any major anomalies, randomly selected in same geographic area and time
period
Exposure: preconceptional vitamins in addition to smoking, fever, alcohol, race/ethnicity, education, BMI
No information on overall effect of folic acid on
NTDs
Shaw, 1998
California (40)
Case-control study of live births and fetal deaths (at >20 weeks) and fetuses with NTDs that were electively
terminated from 1987-1991 who were included in two previous studies by the California Birth Defects Monitoring
Program to examine potential interaction between infant MTHFR C677T polymorphism and maternal use of
vitamin supplements with folic acid
Cases: mothers of infants or fetuses with NTDs
Controls: mothers of infants without any major anomalies
Exposure: Periconceptional or pregnancy use of folic acid and presence of MTHFR C677T polymorphism
No information on overall effect of folic acid on
NTDs
Shaw, 2001 (41)
Case-control study of fetuses, live births, and fetuses with NTDs that were electively terminated from 1989 to 1991
in most California counties to examine the potential relationship between weight gain during pregnancy and risk of
NTDs
Cases: mothers of infants or fetuses with NTDs
Controls: mothers of infants without any major anomalies
Exposure: Periconceptional or pregnancy use of folic acid and weight gain during pregnancy
No information on overall effect of folic acid on
NTDs
Shaw, 1996 (42)
Case-control study of fetuses, live births and fetuses with NTDs that were electively terminated from 1989 to 1991
in most California counties, to investigate the potential association between maternal obesity, folic acid
supplementation, and NTD risk
Cases: mothers of infants or fetuses with NTDs
Controls: mothers of infants without any major anomalies
Exposure: Periconceptional or pregnancy use of folic acid and prepregnancy BMI
No information on overall effect of folic acid on
NTDs
Suarez, 2000 (43)
Case-control study of infants and fetuses in 14 Texas counties along the U.S.-Mexico border between 1995-1999,
to examine the relationship between folic acid intake and NTDs
Study performed in high-risk population
18

Cases: 148 mothers of infants or fetuses with NTDs
Controls: 158 mothers of infants without any congenital abnormalities
Exposures are preconceptional supplement use (any use in 5.4% of cases and 3.2% of controls and daily use in 2%
of cases and 2.5% of controls) and estimated dietary folate
19

Table 2. Studies excluded after abstraction and quality rating - key question on harms.
Study Methods Notes/Reason for Exclusion
Lumley, 2001 (31)
Modeling study based on relative risks for NTDS and twins after folic acid
supplementation (in a hypothetical cohort of 100,000 women)
Sources of data from registries in Victoria and Western Australia
Hypothetical exposure: adequate folic acid supplementation in 100,000 women
Outcomes: absolute difference in overall NTDs and twin gestations, perinatal and
postnatal deaths
Study type not included in review
Ericson, 2001 (44)
Retrospective cohort study of 442 906 deliveries in Sweden between 1995 and 1999
Exposure: folic acid (n=2569) or multivitamin (n=1971) use since pregnancy reported at
10 weeks of gestation
Outcome: twin gestation identified at delivery
Subset analyses: women not reporting unwanted childlessness; unlike sex twin pairs
Potential confounding by patients undergoing IVF or ovulation stimulation;
subgroup analysis on women without “period of involuntary childlessness”, but
authors reported known underreporting of infertility history (40% of women who
underwent IVF or ovulation stimulation did not report involuntary childlessness.)
Measurement validity issues: exposure measured at 10 weeks; reported folic acid
use was 0.6% in this study based on Birth Registry, as compared to 8% in
concurrent study.
No information on doses or timing of initiation of folic acid
Potential differential recall based on knowledge of twin gestation by 8-10 weeks
Czeizel, 2004 (45)
Case-control study of 38 151 subjects from the Hungarian Case Control Surveillance of
Congenital Abnormalities (HCCSCA) study between 1980 and 1996 who did not have
any congenital abnormalities (the control group from the previous study)
Cases: 395 twins
Controls: 27 756 singleton pregnancies
Exposure: folic acid use in pregnancy reported in prenatal log books and in
questionnaire completed after delivery
Including: no supplement, folic acid alone (dose range from 3-9 mg per day),
multivitamin (folic acid dose range 0.1-1 mg per day), or folic acid and multivitamin
No adjustment for possible confounders: IVF, ovulation induction, smoking
No information on doses or timing of initiation of folic acid
Potential differential recall based on knowledge of twin gestation early in
pregnancy or twin delivery
Kallen, 2004 (46) Retrospective cohort study of 576 873 women registered in the Swedish Medical Birth
Registry between 1995 and 2001, examining the relationship of folic acid and dizygotic
twinning
Exposure: folic acid use before conception or before first appointment (usually between
8-10 weeks) (n=6953)
Outcome: unlike-sexed twin gestation identified at delivery
Incomplete information on doses (women likely took either 400 micrograms or 5
mg) or whether prenatal vitamins with folic acid were included in analysis
No information on timing of initiation or duration of exposure
Initial comparability of groups unknown
Potential differential recall based on knowledge of twin gestation by 8-10 weeks
20

Subset analysis: non-Swedish (by nationality or birth) women not reporting unwanted
childlessness, use of ovulation induction or use of gestagens,
Residual confounding possible if incomplete reporting of fertility treatments.
Unclear how many women were included in the final analysis.
21

Table 3. Characteristics and results of studies included for key question 1: Folic acid supplementation and NTD reduction
Study Methods Participants Interventions Outcomes Results
(95% CI) Notes
Czeizel,
2004 (47)
Cohort study of pregnant
women who received folic
acid– containing MVI
prior to conception and
women who did not take
any supplements
periconceptionally.
USPSTF Level: II-2
Hungary, May 1, 1993 to April 30,
1996
Supplemented group – 3981 women
considering pregnancy recruited from
Hungarian Periconceptional Service
(HPS) originally created for the RCT
of folate and NTD.
Exclusions: 7 Unable to use MVI, 186
didn’t want to use MVI, 15 had
induced abortion, 15 ectopic, 488 had
miscarriage prior to 14 weeks, 147
didn’t take MVI. Outcomes couldn’t
be clarified in 54 (1.7%)
Unsupplemented group – 3069 women
recruited from regional antenatal care
clinics between 8th and 12th gestational
week who had not taken supplements.
Outcomes couldn’t be clarified in 47
(1.5%). 15% used supplements.
Matched to supplemented group by
age, quality of schools, employment
status, and residence.
Supplemented group more highly
educated, higher employment status,
less likely to smoke (8% vs 18%).
Supplemented group more likely to
have prior fetal and infant deaths and
more likely to have family history of
congenital anomalies.
Multivitamin tablets
containing 0.8 mg of folic
acid 1 month prior to
planned conception and
supplied every 3rd month for
up to 12 months.
Compliance for
supplemented group was
assessed by personal
interview at 4 separate
visits, “tick-off” form for
basal body temperature prior
to conception, and counting
unused tablets.
Women were considered
“fully supplemented” if they
missed no more than one
day for 28 days prior to
conception and/or the 3rd
missed menstruation.
“Partially supplemented”
women missed 2 or more
tablets.
Unsupplemented group
received routine care.
Presence of NTD,
abnormalities of
urinary tract,
cardiovascular, limbs,
pyloric stenosis, and
orofacial clefts.
NTD
1 NTD in supplemented
and 9 NTDs in
unsupplemented group.
aOR = 0.11
(0.01-0.91)
Urinary tract CAs
aOR = 0.71
(0.33-1.50)
Cardiovascular CAs
aOR = 0.60
(0.38-0.96)
Orofacial clefts
aOR = 1.63
(0.31-28.8)
Adjusted ORs not
calculated for pyloric
stenosis or limb
deficiencies.
All ORs adjusted for
birth order, chronic
maternal disorder,
history of previous fetal
death or CAs.
Fair quality
Measurement of
exposure to
supplements was
different in the two
groups.
Women with
personal, family, or
offspring history of
congenital
abnormalities or
fetal/infant loss
likely self-selected
into supplemented
group.
Collected
information on
SES but did not
adjust for these and
other potential
confounders.
Goh, 2006
(48)
Meta-Analysis
Searched up to July 2005
in Medline, PubMed,
EMBASE, Toxline,
HealthSTAR, and
Cochrane in all languages.
Search terms:
Initial search returned 92 articles. 41
studies eligible based on inclusion
criteria: 27 case control studies, 4
RCTs, and 10 cohort studies.
Inclusion criteria: RCTs, case-control,
or cohort study; reported pre- and
periconceptional multivitamin intake;
Multivitamin use before or
in first trimester of
pregnancy
Risk of congenital
malformations,
including NTD,
associated with
multivitamin use
before and in first
trimester of
pregnancy
NTD
“Consistent protective
effect”
OR 0.67 (0.58-0.77) in
CCS
OR 0.52 (0.39-0.69) in
Fair quality
Searched multiple
databases and
reference lists but
did not include
experts.
22

“multivitamin,”
“pregnancy,” and
“malformation;”
Reviewed reference lists
of all collected articles for
potential studies.
Two reviewers assessed
articles for possible
inclusion.
contained a control group; reported
raw data of rates of outcomes.
Exclusion criteria: studies on specific
vitamins, exposures to known
teratogens; review article, letters,
abstracts.
RCTs/cohorts
Cleft palate
OR 0.76 (0.62-0.93)
in CCS
OR 0.42 (0.06-2.84)
in RCTs/cohorts
Urinary tract anomalies
OR 0.48 (0.30-0.76)
in CCS
OR 0.68 (0.35-1.31)
in RCTs/cohorts
Cardiovascular defects
OR 0.78 (0.67-0.92) in
CCS
OR 0.61 (0.40-0.92)
in RCTs/cohorts
Limb defects
OR 0.48 (0.30-0.76)
in CCS
OR 0.57 (0.38-0.85) in
RCTs/cohorts
Congenital
hydrocephalus
OR 0.37 (0.24-0.56)
in CCS
OR 1.54 (0.53-4.50)
in RCTs/cohorts
No standard
appraisal of
included studies
explicitly stated.
Studies on folate-
only
supplementation
were excluded.
Included several
studies that were
excluded by us
because of
publication prior to
1995, because the
studies were
performed in
special populations
or did not report
NTD as a specific
outcomes. This
limits applicability
to the current
review.
Shaw,
1995 (49)
Case Control Study
exploring exposure to
folic acid supplementation
in cases of NTD and
controls without a birth
defect
USPSTF Level: II-2
Pregnant women and their offspring in
California counties (except Los
Angeles, Riverside, and Ventura)
NTD cases: 665 ascertained; diagnosed
prenatally and elective abortions
during Feb 1989–Jan 1991 and NTD
births: June 1989–May 1991;
Ascertained from all hospitals and
genetic clinics.
Controls: – births without major
structural malformations: June 1989–
May 1991; selected in proportion to
hospital’s contribution to total births
Folic acid from supplements
or multivitamins in the 3
months before and the 3
months after conception.
Amount of folic acid
estimated from personal
interview on type, brand,
and frequency of use.
Prenatal vitamins assumed
to have 0.8 mg;
multivitamins assumed to
have 0.4 mg; if no
information on frequency or
type of supplement, folic
Risk of NTD
associated with folic
acid–containing
supplement
NTD risk and use of
folic acid in 3 months
before conception:
OR = 0.65 (0.45-0.94)
Use of folic acid in 3
months after
conception:
OR = 0.60 (0.46-0.79)
Good quality
Accurate
ascertainment of
cases; selection of
cases/controls
appears nonbiased
with exclusion
criteria applied
equally to both;
response rates
>80%; exposure
measurement
applied equally to
each group;
23

Exclusions:
Not English or Spanish speaking (29
cases, 32 controls); prior NTD (11
cases and 1 control).
Interviews conducted in 88.0% of
cases, mothers at an average of 4.9
months after actual/estimated (for
terminations) date of delivery and
88.2% of control mothers at an average
of 4.6 months after date of delivery.
6.9% of cases refused to be
interviewed and
5% could not be located; 6.2% of
controls refused to be interviewed and
5% could not be located. Non-
participants similar to study
participants in race/ethnicity.
Cases mothers more likely to be
Hispanic, less than 25 years old and
completed fewer years of school.
acid intake considered to be
zero.
attention to
appropriate
covariates and
confounding
variables.
Reporting bias
explored:
Women who
thought “no effect”
of vitamins on birth
defects had larger
reduction in risk
(OR = 0.51, 0.31-
0.85) for use 3
months prior to
conception.
Women who
thought vitamins
were “protective” =
OR = 0.89 (0.46-
1.7).
Women who
thought vitamins
“causal” to birth
defects = OR =
0.40 (0.04-7.6).
Thompson,
2003 (26)
Case Control Study
exploring multivitamin
folic acid use, dietary
folate intake, and risk of
NTDs.
USPSTF Level: II-2
Pregnant women residing in South
Carolina who delivered in October
1992 through September 1997.
Cases obtained through monitoring of
amniocentesis programs, perinatal
centers, all medical practitioners
providing care to pregnant women,
medical records from hospitals with
delivery/newborn units and vital
records. CDC surveillance team
verified completeness of case
ascertainment in years one and four of
the study.
Controls randomly selected from
hospital in proportion to the hospital’s
estimated contribution to the total
population of infants born. Selected
concurrently with cases.
Average daily folic acid
supplement intake in
periconceptional period (3
months before and 3 months
after conception)
By maternal interview
conducted within 2 weeks of
discharge (liveborn control
or NTD baby) and 4 weeks
of termination.
77% of women with NTD
affected pregnancies and
86% of controls interviewed
within 6 months of delivery
(means not given)
Assumed Prenatal MVI =
0.8 mg
Risk of NTD
associated with folic
acid–containing
supplement
Regular use: 16 cases
and 43 controls, a OR
0.55 (0.25-1.22)
Some use: 123 cases
and 188 controls, aOR
= 0.92 (0.55-1.55)
No use: 40 cases and 57
controls, reference
Adjusted for age, race,
BMI, ETS exposure and
dietary folate
Fair quality
Potential for
selection bias:
25/71 women with
NTD pregnancies
chose not to
participate, but
participation rate
similar for NTD
patients as
compared to
controls.
Measurement by
interview of
exposure assessed
at different times
for cases
24

First occurrence of singleton isolated
NTD = 312
3 excluded because taking
anticonvulsant medication.
2 women gave birth to twins excluded.
1 mother with NTD excluded.185
agreed to participate (72.3%)
Controls = 398 eligible.
289 (72.6%) agreed to participate.
1 excluded because taking
anticonvulsant medication.
Cases and controls similar with respect
to age, education, gravidity, month
began PNC, previous NTD, BMI,
smoking, ETOH, drug use, chronic
conditions, MVI use.
Higher proportion of cases were white
(79.3% vs. 69.1%) and reported having
been exposed to ETS (51.4% vs.
26.1%).
Regular Use = 0.4 – 0.8 mg
or more at least 3 times per
week in periconceptional
period.
Some intake = less than 3
times per week or in partial
months.
No use = none at any time in
periconceptional period.
terminated versus
controls/NTD
babies creating
potential for
differential
measurement bias.
Sample size was
small. Few took
MVI regularly in 6
months
periconceptionally.
6 month
periconceptional
period is not very
precise timeframe
for critical period
of NTD.
Abbreviations: aOR = adjusted odds ration; CCS = case control studies; CA = congenital abnormalities; CI = confidence interval; ETS = environmental tobacco smoke; MVI =
multivitamins; NTD = neural tube defects; RCT = randomized controlled trials
25

26
Table 4. Characteristics and results of studies included for key question 2: Folic acid supplementation and harms
Study Methods Participants Interventions Outcomes Results (95% CI) Notes
Vollset,
2005 (17)
Retrospective cohort
study exploring
association of twin
gestation with folate use
before or during
pregnancy.
Norway, December
1998 to December
2001.
All women giving
birth in Norway
between (n=
176,042).
Folic acid tablets contain either 0.2
mg or 0.4 mg.
Multivitamins contain 0-0.2 mg
folic acid per tablet.
Folic acid use assessed by birth
attendant who checks off use of
multivitamin or folic acid before or
during pregnancy .
Preconception folate use in 6%
(24% among IVF pregnancies.)
Risk of twin
gestation after
preconceptional
folate use
OR: 1.59 (1.41-1.78)
OR in subset of women who did
not report IVF: 1.13 (0.97-1.33)
OR for unlike sex pairs (proxy
for dizygotic): 1.43 (1.12-1.83)
When modeling the
underreporting (12.7%
unidentified IVF; 45%
unidentified folate use):
OR for twin delivery: 1.02
(0.85-1.24)
In unlike sex pairs:
1.26 (0.91-1.73)
Adjusted for age and parity
Fair quality
Exposure measured at
delivery; may be problems
with recall and potential
differential recall for twin
gestations as compared to
singletons; authors did
model for underreporting of
folate use and unidentified
IVF pregnancies.

Appendix 1: PubMed search terms and exclusion criteria
PubMed search terms and limits:
("neural tube defects"[MeSH Terms] OR "spina bifida"[All Fields] OR "neural tube damage"[All
Fields] OR "neural tube defect"[All Fields] OR "neural tube defects"[All Fields] OR "neural tube
disorders"[All Fields] AND (("1995/01/01"[PDAT] : "2007/11/30"[PDAT]) AND
English[lang])) AND (("folic acid"[MeSH Terms] OR folic acid[Text Word]) AND
(("1995/01/01"[PDAT] : "2007/11/30"[PDAT]) AND English[lang])) AND
(("pregnancy"[MeSH Terms] OR pregnancy[Text Word]) AND (("1995/01/01"[PDAT] :
"2007/11/30"[PDAT]) AND English[lang])) AND
Exclusion Criteria for Folic Acid in Pregnancy Review
1. A study not on folic acid supplementation
2. Incorrect study type
3. In a setting not generalizable to US
4. Not in women of child bearing age
5. No outcomes of interest
6. High risk or special population (ex. Prior NTD)
7. n < 100
8. Duplicate study
27

Appendix 2. USPSTF HIERARCHY OF RESEARCH DESIGN AND QUALITY RATING CRITERIA1,2
HIERARCHY OF RESEARCH DESIGN
I Properly conducted randomized controlled trial (RCT)
II-1: Well-designed controlled trial without randomization
II-2: Well-designed cohort or case-control analytic study
II-3: Multiple time series with or without the intervention; dramatic results from uncontrolled
experiments
III: Opinions of respected authorities, based on clinical experience; descriptive studies or
case reports; reports of expert committees
DESIGN-SPECIFIC CRITERIA AND QUALITY CATEGORY DEFINITIONS
Systematic Reviews
Criteria:
• Comprehensiveness of sources considered/search strategy used
• Standard appraisal of included studies
• Validity of conclusions
• Recency and relevance are especially important for systematic reviews
Definition of ratings from above criteria:
Good: Recent, relevant review with comprehensive sources and search strategies;
explicit and relevant selection criteria; standard appraisal of included studies; and valid
conclusions.
Fair: Recent, relevant review that is not clearly biased but lacks comprehensive sources and
search strategies.
Poor: Outdated, irrelevant, or biased review without systematic search for studies, explicit
selection criteria, or standard appraisal of studies.
Case-Control Studies
Criteria:
• Accurate ascertainment of cases
• Nonbiased selection of cases/controls with exclusion criteria applied equally to both
• Response rate
• Diagnostic testing procedures applied equally to each group
• Measurement of exposure accurate and applied equally to each group
• Measurement of exposure accurate and applied equally to each group
• Appropriate attention to potential confounding variables
Definition of ratings based on criteria above:
Good: Appropriate ascertainment of cases and nonbiased selection of case and control
participants; exclusion criteria applied equally to cases and controls; response rate
equally to or greater than 80 percent; diagnostic procedures and measurements accurate
and applied equally to cases and controls; and appropriate attention to confounding
variables.
28

Fair: Recent, relevant, without major apparent selection or diagnostic work-up bias but
with response rates less than 80 percent or attention to some but not all important
confounding variables.
Poor: Major section or diagnostic work-up biases, response rates less than 50 percent, or
inattention to confounding variables.
Randomized Controlled Trials and Cohort Studies
Criteria:
• Initial assembly of comparable groups
o -for RCTs: adequate randomization, including first concealment and whether potential
confounders were distributed equally among groups
o -for cohort studies: consideration of potential confounders with either restriction or
measurement for adjustment in the analysis; consideration of inception cohorts
• Maintenance of comparable groups (includes attrition, cross-overs, adherence,
contamination)
• Important differential loss to follow-up or overall high loss to follow-up
• Measurements: equal, reliable, and valid (includes masking of outcome assessment)
• Clear definition of the interventions
• All important outcomes considered
Definition of ratings based on above criteria:
Good: Evaluates relevant available screening tests; uses a credible reference standard;
interprets reference standard independently of screening test; reliability of test assessed;
has few or handles indeterminate results in a reasonable manner; includes large number
(more than 100 broad-spectrum of patients.
Fair: Evaluates relevant available screening tests; uses reasonable although not best
standard; interprets reference standard independent of screening test; moderate sample
size (50 to 100 subjects) and a “medium” spectrum of patients.
Poor: Has fatal flaw such as: Uses inappropriate reference standard; screening test improperly
administered; biased ascertainment of reference standard; very small sample size or very
narrow selected spectrum of patients.
Diagnostic Accuracy Studies
Criteria:
• Screening test relevant, available for primary care, adequately described
• Study uses a credible reference standard, performed regardless of test results
• Reference standard interpreted independently of screening test
• Handles indeterminate result in a reasonable manner
• Spectrum of patients included in study
• Sample size
• Administration of reliable screening test
Definition of ratings based on above criteria:
Good: Evaluates relevant available screening test; uses a credible reference standard;
interprets reference standard independently of screening test; reliability of test assessed;
has few or handles indeterminate results in a reasonable manner;
29

includes large number (more than 100) broad-spectrum patients with and without
disease.
Fair: Evaluates relevant available screening test; uses reasonable although not best standard;
interprets reference standard independent of screening test; moderate sample size (50-
100 subjects) and a “medium” spectrum of patients.
Poor: Has fatal flaw such as: Uses inappropriate reference standard; screening test improperly
administered; biased ascertainment of reference standard; very small sample size or very
narrow selected spectrum patients.
Appendix 2: Reference List
1. Harris R, Atkins D, Berg AO, Best D, Eden KB, Feightner JW et al. US Preventive Services Task
Force Procedure Manual. Rockville, MD: Agency for Healthcare Research and Quality, 2001.
2. Harris RP, Helfand M, Woolf SH, Lohr KN, Mulrow CD, Teutsch SM et al. Current methods of the US
Preventive Services Task Force: a review of the process. Am J Prev Med 2001; 20(3 Suppl):21-35.
30

Grant support
There was no grant support for this review.
Address for reprint requests
Reprints are available from the Agency for Healthcare Research and Quality Web site
(www.ahrq.gov/clinic/uspstfix.htm).
Current addresses for all authors:
Tracy Wolff, MD, MPH (Corresponding Author)
Medical Officer, USPSTF Program
Agency for Healthcare Research and Quality
540 Gaither Road
Rockville, Maryland 20850
email: Tracy.Wolff@ahrq.hhs.gov
Catherine Takacs Witkop, MD MPH
Preventive Medicine/OB/Gyn
10 AMDS
2355 Faculty Drive, Rm 2N286
USAFA, CO 80840
email: catherine.witkop@usafa.af.mil
Therese Miller, DrPH
Senior Program Coordinator, USPSTF Program
Agency for Healthcare Research and Quality
540 Gaither Road
Rockville, Maryland 20850
email: Therese.Miller@ahrq.hhs.gov
Shamsuzzoha B. Syed, MD, MPH, DPH (Cantab)
31