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Int. J. Environ. Res. Public Health 2013, 10, 590-605; doi:10.3390/ijerph10020590
International Journal of
Environmental Research and
Public Health
ISSN 1660-4601
www.mdpi.com/journal/ijerph
Article
High Dosage Folic Acid Supplementation, Oral Cleft
Recurrence and Fetal Growth
George L. Wehby 1, Têmis Maria Félix 2, Norman Goco 3, Antonio Richieri-Costa 4,
Hrishikesh Chakraborty 5, Josiane Souza 6, Rui Pereira 7, Carla Padovani 8,
Danilo Moretti-Ferreira 9 and Jeffrey C. Murray 10,*
1 Department of Health Management and Policy, University of Iowa, Iowa City, IA 52242, USA;
E-Mail: george-wehby@uiowa.edu
2 Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul 90035-903, Brazil;
E-Mail: tfelix@hcpa.ufrgs.br
3 RTI International, Research Triangle Park, NC 27709, USA; E-Mail: ngoco@rti.org
4 Hospital de Reabilitação de Anomalias Craniofaciais, Bauru, Sao Paulo 17.043-900, Brazil;
E-Mail: richieri@usp.br
5 Department of Epidemiology and Biostatistics, University of South Carolina, Columbia, SC 29208,
USA; E-Mail: rishic@mailbox.sc.edu
6 Centro de Atendimento Integral ao Fissurado Lábio Palatal, Curitiba, Paraná 81.050-000, Brazil;
E-Mail: jositwin@yahoo.com.br
7 Instituto Materno Infantil Prof. Fernando Figueira-CADEFI/IMIP, Recife, Pernambuco 50070-550,
Brazil; E-Mail: ruimrpereira@terra.com.br
8 Hospital Santo Antônio: Obras Sociais Irmã Dulce, Salvador, Bahia 40.415-000, Brazil;
E-Mail: carla.mca@uol.com.br
9 Genetic Counseling Service, São Paulo State University, Botucatu, Sao Paulo 18618-000, Brazil;
E-Mail: danilo@ibb.unesp.br
10 Department of Pediatrics, University of Iowa, Iowa City, IA 52242, USA
* Author to whom correspondence should be addressed; E-Mail: jeff-murray@uiowa.edu;
Tel.: +1-319-335-6897; Fax: +1-319-335-6970.
Received: 8 October 2012; in revised form: 25 January 2013 / Accepted: 25 January 2013 /
Published: 4 February 2013
Abstract: Objectives: To evaluate the effects of folic acid supplementation on isolated oral
cleft recurrence and fetal growth. Patients and Methods: The study included 2,508 women
who were at-risk for oral cleft recurrence and randomized into two folic acid
OPEN ACCESS
Int. J. Environ. Res. Public Health 2013, 10 591
supplementation groups: 0.4 and 4 mg per day before pregnancy and throughout the first
trimester. The infant outcome data were based on 234 live births. In addition to oral cleft
recurrence, several secondary outcomes were compared between the two folic acid groups.
Cleft recurrence rates were also compared to historic recurrence rates. Results: The oral
cleft recurrence rates were 2.9% and 2.5% in the 0.4 and 4 mg groups, respectively. The
recurrence rates in the two folic acid groups both separately and combined were
significantly different from the 6.3% historic recurrence rate post the folic acid fortification
program for this population (p = 0.0009 when combining the two folic acid groups). The
rate of cleft lip with palate recurrence was 2.9% in the 0.4 mg group and 0.8% in the 4 mg
group. There were no elevated fetal growth complications in the 4 mg group compared to
the 0.4 mg group. Conclusions: The study is the first double-blinded randomized clinical
trial (RCT) to study the effect of high dosage folic acid supplementation on isolated oral
cleft recurrence. The recurrence rates were similar between the two folic acid groups.
However, the results are suggestive of a decrease in oral cleft recurrence compared to the
historic recurrence rate. A RCT is still needed to identify the effect of folic acid on oral
cleft recurrence given these suggestive results and the supportive results from previous
interventional and observational studies, and the study offers suggestions for such future
studies. The results also suggest that high dosage folic acid does not compromise fetal growth.
Keywords: oral clefts; cleft lip; cleft palate; birth defects; folic acid; vitamins; prevention;
pregnancy; nutrition; Brazil
ClinicalTrials.gov Identifier: NCT00397917
1. Introduction
Oral clefts are among the most common birth defects worldwide, with incidence ranging from
1/200 births in Amerindian populations to about ½,500 births in African populations and an
intermediate incidence for European populations at about 1/700 births [1]. In addition to the short-term
health consequences and healthcare costs due to reduced fetal growth [2] and the need for surgical
repair, oral clefts have long-term adverse effects on health requiring dental, speech, and psychosocial
interventions and increase hospitalization risks up to adulthood [3]. Oral clefts significantly reduce the
quality of life of affected children and their families [4,5].
The etiology of oral clefts is complex involving genetic and environmental factors with both
independent and interactive effects. Although much of the genetic etiology, particularly functional and
developmental pathways, remain unknown, several genes/loci have been implicated in oral clefts [6–9],
with most compelling evidence for IRF6 and loci near 8q24, MAFB, ABCA4 and VSX1 [8,10].
Several environmental/behavioral factors may play a role particularly smoking, which may have both
independent [9] and interactive effects with genetic risk factors [11], and folate/multivitamin use [12].
There has been a wide interest in evaluating the role of maternal nutrition particularly the
consumption of folate rich diets and use of folic acid supplements in oral cleft recurrence and
occurrence. A detailed review of this literature is presented elsewhere [12]. Several observational
Int. J. Environ. Res. Public Health 2013, 10 592
studies provide supportive evidence for a protective effect of folate and folic acid supplementation on
oral cleft occurrence. However, there is still a large uncertainty about the role of folic acid
supplementation on occurrence because these observational studies might not entirely account for bias
resulting from maternal self-selection into vitamin use and nutritional behaviors, which may be
correlated with unobserved health or behavioral characteristics that affect oral cleft risks.
The risk of oral cleft recurrence, defined as the occurrence of oral clefts in already affected families
(i.e., mother is affected or has had a previous child with oral clefts) is higher by about 40 times than
overall prevalence in the general population (4–5% versus 1/1,000, respectively). Several studies have
evaluated the effects of folic acid supplementation, particularly high versus low doses on oral cleft
recurrence. Most of these studies found significant protective effects of high doses—on average a
decrease of 50% in recurrence (see detailed review in [12]). However, unlike the evidence for a
preventive effect on neural tube defect (NTD) recurrence [13], the evidence for a preventive effect on
oral cleft recurrence is still controversial, mainly because none of the previous interventional studies
used a randomized design and as a result were still prone to bias by lacking an appropriate control
group. Another limitation of some studies was the challenge involved in evaluating folic acid effects
independent of other vitamins.
The Oral Cleft Prevention Program (OCPP) was as a double blinded RCT to assess the effect of
high-dose versus low-dose folic acid supplementation on oral cleft recurrence among children of
Brazilian women [14]. We describe the OCPP’s design and report the findings as of December 2009
after the trial was halted due to lower than expected enrollment and pregnancy rates among study
participants. We also compare the recurrence rates in the OCPP to a historic recurrence rate.
In addition, we report data on fetal growth outcomes, compliance, and adverse events. The
comparisons we present on fetal development outcomes between high and low folic acid
supplementation groups are the first from a randomized study in humans. Finally, we provide insights
based on the lessons learned from the OCPP for a future study that can provide a definitive answer
about the effects of high dose folic acid on oral cleft recurrence.
2. Methods
2.1. Design
This study aimed at testing the effect of 4 mg folic acid taken daily before pregnancy throughout the
first trimester by women who were themselves born with a cleft or had had a prior child with a cleft on
recurrence of clefting in a subsequent child compared to taking the currently recommended 0.4 mg
dose [14]. A placebo control group was not used, as a low dose of folic acid had already been
recommended as a standard vitamin therapy for women during preconception and pregnancy period
for prevention of NTDs.
The study had a double-blinded randomized design, where both investigators and participants were
blinded to the study group assignment [14]. Participants were randomly assigned before pregnancy to
the two study groups of taking 4 mg or 0.4 mg pills of folic acid that were identical in appearance.
Randomization occurred at the participant level and was stratified by the study site to ensure a
balanced representation in both treatment groups. The study Data Center at RTI International oversaw
the randomization and generated the randomization sequence using permuted blocks of random size.
Int. J. Environ. Res. Public Health 2013, 10 593
The randomization sequence linked the treatment assignment (0.4 mg or 4 mg) to a sequential list of
serial numbers that were affixed to the study pill boxes under the supervision of the Data Center. The
pillboxes were also numbered in order of dispensing (1, 2, 3, etc.). The folic acid pills were
specifically manufactured for the study by ATIVUS Pharmaceutical Industries in Brazil. The
manufacturer conducted quality control tests for each batch of pills including identification test of the
raw folic acid material used in the pills (prior to production), assay tests for the folic acid dosage in the
pills, content uniformity tests (to ensure that the dosage is consistent across pills), and dissolution tests to
ensure that the pills dissolve adequately in a standard time.
Participants who had a delay in menstruation of 14 days or more at baseline underwent a pregnancy
test before randomization; only those with a negative test were randomized [14]. After randomization,
all participants were asked to take a single pill of 4 mg or 0.4 mg of folic acid daily throughout the first
trimester of pregnancy if they became pregnant or until end of participation in the study if they did not
become pregnant. Participants were followed up every two months to check on their health status,
provide new pill boxes for the next two months, and collect old pillboxes with any unused pills to
measure compliance. During the first two years of the study (between 2004 and 2006), bimonthly
follow-ups were completed in person. In later years, bimonthly follow-ups were mostly completed
over the phone and study pills delivered to participants through express mail. When participants were
available, periodic in-person follow-ups were conducted every six months. Compliance was evaluated
by counts of unused pills from returned pillboxes. An average compliance rate per participant was
generated from all the returned pillboxes for that participant. Serum and RBC folate levels, evaluated
periodically after randomization in 2004–2006 and after 12 months of randomization (or at pregnancy,
whichever occurred first) beginning in 2007 also provided another measure of compliance. Folate
levels were not communicated with the subjects and were seen and entered by staff not directly
involved with the study subjects.
Participants confirmed to be pregnant were advised by the study staff to stop taking the study pills
at the end of the first trimester of pregnancy [14]. There were no restrictions on participants taking
other supplements and vitamins before or during pregnancy as recommended by their physicians. The
study staff attempted to follow pregnant participants periodically (usually every two months) over the
phone to check on pregnancy progress. Furthermore, the study staff attempted to maintain contact with
the participants’ prenatal care providers to check on pregnancy progress.
After delivery, all live births were followed in-person by the study staff to measure oral cleft status.
Mothers also completed an interview with the study staff after delivery either in-person or over the
phone to obtain data on perinatal and infant health outcomes [14]. All interviews after delivery
occurred within 2 years after birth; ~90% occurred within 6 months after delivery; another 9%
occurred before the end of first year of life. The participants’ physicians were also interviewed after
delivery when possible to obtain data on the occurrence of oral clefts and birth defects and abnormal
delivery events. Participants and physicians were also interviewed after miscarriages/stillbirths to
obtain data on birth defects. However, no data were available to measure the occurrence of birth
defects and oral clefts in most miscarriage/stillbirth cases (there were only two stillbirth cases in the
study). The study was designed from the beginning to focus on oral cleft occurrence in live births
given the challenges of measuring these outcomes in miscarriages/stillbirths.
Int. J. Environ. Res. Public Health 2013, 10 594
The OCPP aimed at observing 1,582 births (791 births in each folic acid group) in order to achieve
80% power for a 50% reduction in a baseline rate of 5% recurrence. The study assumed a birth rate of
0.0957 births per participant-year, which implied that about 16,531 participant-years were required to
observe the 1,582 births.
2.2. Recruitment and Ethics Approvals
All potential participants were screened for eligibility using the same data forms and inclusion
criteria across all sites by trained and qualified study staff before they were enrolled [14]. After
confirming eligibility, the study staff asked the women to consent. For illiterate participants,
confirmation was obtained as a thumbprint in the presence of a witness (only 10 participants reported
no formal schooling). The study staff assured all women that refusal to participate in the study would
in no way affect further treatment or care at the clinic. All interviews, informed consents, and data
collection forms were in Portuguese. The study protocol, informed consent, manual of operations, and
data collection forms were approved by the ethics committees of the study sites and by CONEP, the
national committee of research ethics in Brazil.
2.3. Study Population
The study was conducted at six craniofacial clinics in Brazil (see detailed list in online Appendix).
The clinics were selected based on their long experience in providing care to patients with oral clefts
and most of them are considered referral centers for oral cleft care. Participants were identified from
the population of patients served by these clinics. Enrollment was first initiated in 2004 using an
outreach model for the patient pool served by the first study clinic. In that model, the study staff
conducted meetings with the study participants in their local communities during which data were
collected and study pills provided. In 2005, a clinic-based recruitment model was initiated at two new
sites, where participants were recruited and interviewed at the study clinic. The clinic-based model
collected data from participants over phone interviews and mostly delivered study pills via express
mail; in-person follow-ups were completed with the participant every 6 months when possible.
In 2007, the first site switched from the outreach model to a clinic-based model, and two new sites
were added under the clinic-based model. A sixth site following the clinic-based model was added in 2008.
2.4. Inclusion Criteria
The study participants were women 16–45 years of age who have nonsyndromic/isolated oral clefts
or had had at least one natural child of any age with nonsyndromic/isolated oral clefts who and who
had received care at the study clinics [14]. The study participants also resided in the study catchment
area consisting of the state where the clinic is located and surrounding states. During the first two years
of the study (2004–2006) in the outreach model, both cleft lip with/without palate as well as cleft
palate only were included in the study. Beginning in 2006, new recruitment was limited to cases of
cleft lip with or without cleft palate, and cases with cleft palate only were no longer enrolled. The
reason was to avoid any potential confounding effects of cleft palate only which is commonly
separated from cleft lip with/without cleft palate in studies of cleft etiology, even though there may be
overlap in etiology and recurrence prevention.
Int. J. Environ. Res. Public Health 2013, 10 595
2.5. Exclusion Criteria
Participants who met any of the following criteria at the time of screening were excluded from
participation [14]:
1. Syndromic/non-isolated cleft status including cases with recognized syndromes, cases with a
chromosome abnormality, cases with one or more other major structural anomaly, cases with
cognitive delay (IQ or equivalent less than 80), or cases exposed to phenytoin or valproic
acid in utero.
2. Any first degree relative (that is a parent, sibling or child) who has cleft palate only (this was
added in 2007 when new enrollment was limited to participants who are themselves affected
or have affected children with cleft lip with/without palate).
3. The woman or her husband/partner was sterilized (such as tubal ligation).
4. Using intrauterine devices or injectible contraceptives (added in 2008).
5. Using anti-epileptic drugs (since the metabolism of anti-epileptic drugs requires a great deal
of folic acid).
6. Using drugs that contain benzodiazepines (as these may increase the risk for birth defects
and oral clefts).
7. Women who were pregnant at screening.
8. Women who were planning to move outside of the catchment area of the study within the
next year.
9. Women who had B12 deficiency as determined from testing participants’ blood samples in
the study before supplementation (B12 level below 174 pg/mL or 134.328 pmol/L), which
may be masked by folic acid.
10. Women who were allergic to folic acid.
Participants who did not meet these criteria at enrollment because these were missed during the
initial screening or the subject met these criteria after enrollment but were later found to meet the
following exclusion criteria were discontinued from participation: definite sterilization of the woman
and/or husband/partner, detecting B12 after randomization and a hematologist recommended that the
woman be fully withdrawn from the study (no participants met this criterion), and taking epileptic
drugs unless the participant is pregnant. B12 levels were assessed every 4 months between 2004 and
2006. After that, B12 levels were assessed once every 12 months.
2.6. Study Outcomes
The primary outcome of the study was the recurrence of isolated oral clefts, defined as the birth of a
child with an oral cleft to the study women during their participation [14]. This is considered
recurrence since only women who are themselves affected with oral clefts or have had a previous child
with an oral cleft are enrolled in the study. The study infants were examined in person by a trained
study staff to check and document oral clefting status and look for evidence of associated syndromes.
The study evaluated several secondary outcomes. The cleft recurrence rates in the OCPP were
compared to historic recurrence rates. We calculated these rates by surveying women who are
themselves affected or have had children affected with isolated cleft lip with/without cleft palate who
Int. J. Environ. Res. Public Health 2013, 10 596
were obtaining care from the same clinics involved in the OCPP but who did not enroll in the OCPP
for reasons unrelated to cleft recurrence risk (such as sterilization and refusal to participate). Only
cases with cleft lip with/without cleft palate were included in the historic recurrence survey; cases with
cleft palate only were excluded. The study staff interviewed these women in 2011–2012 for their
complete pregnancy history. We calculated the historic recurrence rates for the period after the
initiation of the folic acid fortification program (after 2004) since the OCPP infants were born during
this period and fortification may affect oral cleft recurrence. For affected women, the recurrence rate
was defined as the proportion of affected children among all live births. For unaffected women with an
affected child, the recurrence rate was defined as the proportion of affected children among all live
births after the affected child.
The severity of oral clefts, birth weight, gestational age, and preeclampsia were also evaluated as
secondary outcomes. In addition, we report here a comparison of head circumference, length at birth,
and Apgar scores between the study births in the two folic acid groups.
2.7. Statistical Analysis
Outcomes were compared between the two doses of folic acid to which the women were assigned
randomly at enrollment. Data for all women and infants were analyzed in the treatment arm to which
the women were randomized, even when the women did not comply fully with the assigned treatment
regimen as described above. We recognize that the sample size does not provide adequate power to
identify moderate effects for small frequency outcomes when analyzing the live birth sample. Given
the randomized design of the study, we compared the outcomes between the two folic acid groups
using two-group comparison tests including a Fisher’s exact test for binary outcomes and a Student’s
t-test for continuous outcomes. A Wilcoxon rank-sum (or Mann-Whitney) test was also used for the
continuous outcomes since they did not meet normality tests (except for head circumference). Fisher
exact tests and student’s t-tests were also used to compare the baseline characteristics between the two
folic acid groups. We compared the OCPP recurrence rates to the historic rates using a one-sample
z-test for proportions.
At the beginning of the study, there was no limit on how long a woman may participate.
Participants were allowed to enroll again in the study in the same treatment group after their pregnancy
if they wished to do so and if they still met all the eligibility criteria. Nine women participated a
second time and had live births. In 2008, participation time was limited to three years. The study
participants participated in the study until their delivery if they became pregnant or until they had
completed three years of participation without becoming pregnant. After delivery, the participants
were allowed to participate again if they had not completed a total of three years of participation in the
study. We first report the analyses only for first-enrollments and first pregnancies. In additional
analyses of cleft recurrence, we add women who participated a second time.
3. Results
3.1. Sample Description
Between January 2004 and May 2009, a total of 3,821 women were screened for eligibility into the
study. Of these, 851 (22.3%) met one or more of the study exclusion criteria. Of the 2,970 eligible
Int. J. Environ. Res. Public Health 2013, 10 597
women, 2,508 women gave consent and were randomized into the study treatment and control groups;
the 462 who were not randomized did not consent to participate in the study. Of the randomized
women, 273 became pregnant during their first participation in the study. Of these pregnancies, there
were 269 verifiable pregnancy outcomes (miscarriage, still birth or live birth); 234 were live births,
33 were miscarriages, and two were still births. Of these live births, 225 live births occurred in families
where the previous affected child or affected mother had cleft lip with/without cleft palate (but not
cleft palate only). Of the randomized women, 913 chose to discontinue their participation in the study
before pregnancy, and five pregnant women were lost to follow up. On average, these women
withdraw 20 months after randomization (ranging from 0 to 57). Figure 1 shows a flow diagram for
study screening, enrollment/randomization, withdrawal, and pregnancy occurrence and outcome status.
The average participation length was 20 months and ranged from 0 to 72 months.
Figure 1. Study flow diagram.
Notes: Pregnancy and outcome counts are based on first time enrollments only. The pregnancy outcome
count in the 0.4 mg group includes twin births.
In 2009, the DSMB overseeing the study with the funding institute (NIDCR) recommended that all
new recruitment be halted and enrollment of non-pregnant women participating in the study be ended
due to the lower than anticipated enrollment and pregnancy rates, which implied that the sample of
infants needed to achieve 80% power could not be reached over the planned project period. Therefore,
the enrollment of all non-pregnant participants was terminated at all sites after that. All pregnant
Int. J. Environ. Res. Public Health 2013, 10 598
women were followed according to the protocol and their babies examined in person by a trained study
staff while maintaining the blinding of the subject and study staff to the folic acid group assignment.
Table 1 compares the distributions of baseline clinical, demographic and socioeconomic
characteristics of all randomized women between the two folic acid groups. There were no statistically
significant differences in these variables between the two folic acid groups. Of the randomized sample,
about 41% were women who are themselves affected with oral clefts and had no children at the time of
enrollment; about 59% were unaffected mothers with affected children; affected mothers with affected
children were less than 1%. About 77% of the affected patients (either participants or their children)
had cleft lip with palate, and about 21% had cleft lip only. The sample had overall low socioeconomic
status with about 40% of the participants having only fundamental or no education and 51% having
intermediate education; about 49% of the sample were employed. There were no significant
differences between the two groups in baseline serum and RBC folate levels measured at enrollment
before supplementation.
Table 1. Baseline confounders by treatment.
Demographic Variable 0.4 mg
Folic Acid
4.0 mg
Folic Acid p-Value Total
Subjects, N 1,251 1,257 2,508
Cleft status group, n (%) 1,251 1,257 0.6437 2,508
Affected mother without children 502 (40.1) 518 (41.2) 1,020 (40.7)
Unaffected mother of affected children 742 (59.3) 729 (58.0) 1,471 (58.7)
Affected mother with affected children 7 (0.6) 10 (0.8) 17 (0.7)
Family cleft type, n (%) 1,233 1,238 0.4733 2,471
Cleft lip 243 (19.7) 268 (21.6) 511 (20.7)
Cleft palate 36 (2.9) 33 (2.7) 69 (2.8)
Cleft lip with cleft palate 954 (77.4) 937 (75.7) 1,891 (76.5)
Age, n (%) 1,243 1,253 0.5682 2,496
<20 229 (18.4) 204 (16.3) 433 (17.3)
20–29 625 (50.3) 646 (51.6) 1,271 (50.9)
30–39 356 (28.6) 370 (29.5) 726 (29.1)
≤40 33 (2.7) 33 (2.6) 66 (2.6)
Marital status, n (%) 1,247 1,257 0.6058 2,504
Single 422 (33.8) 414 (32.9) 836 (33.4)
Married or stable relationship 801 (64.2) 812 (64.6) 1,613 (64.4)
Divorced or widowed 24 (1.9) 31 (2.5) 55 (2.2)
Highest level of schooling, n (%) 1,174 1,176 0.1423 2,350
Fundamental or none 432 (36.8) 427 (36.3) 859 (36.6)
Intermediate 616 (52.5) 592 (50.3) 1,208 (51.4)
University 126 (10.7) 157 (13.4) 283 (12.0)
Employed in the past month, n (%) 1,174 1,176 0.4336 2,350
Yes 579 (49.3) 561 (47.7) 1,140 (48.5)
No 595 (50.7) 615 (52.3) 1,210 (51.5)
Smoke cigarettes, n (%) 1,247 1,257 0.8070 2,504
Yes 139 (11.1) 144 (11.5) 283 (11.3)
No 1,108 (88.9) 1,113 (88.5) 2,221 (88.7)
Int. J. Environ. Res. Public Health 2013, 10 599
Table 1. Cont.
Demographic Variable 0.4 mg
Folic Acid
4.0 mg
Folic Acid p-Value Total
Drink alcoholic beverages, n (%) 1,246 1,257 0.1103 2,503
Yes 145 (11.6) 173 (13.8) 318 (12.7)
No 1,101 (88.4) 1,084 (86.2) 2,185 (87.3)
Multivitamin Use, n (%) 876 888 0.6271 1,764
Did not take multivitamin 801 (91.4) 825 (92.9) 1,626 (92.2)
1 to 3 times a week 7 (0.8) 5 (0.6) 12 (0.7)
4 to 6 times a week 2 (0.2) 3 (0.3) 5 (0.3)
Every day of the week 66 (7.5) 55 (6.2) 121 (6.9)
Baseline Serum Folate (ng/mL) 1,211 1,211 2,422
Mean (SD) 11.4 (5.1) 11.9 (8.8) 0.0650 11.6 (7.2)
Median 10.7 10.7 0.8838 10.7
Baseline Red Cell Folate (ng/mL) 1,150 1,156 2,306
Mean (SD) 606.7 (451.6) 595.9 (387.2) 0.5390 601.3 (420.5)
Median 573.5 581.0 0.8658 577.5
Note: Some observations had missing data on certain measures.
3.2. Cleft Recurrence and Secondary Outcomes
Table 2 shows the cleft recurrence and type in the OCPP focusing first on families where the
previously affected child or affected mother had cleft lip with/without cleft palate but not cleft palate
only and on first-time enrollments. The cleft recurrence rate was 2.9% (three affected out of 105 births)
in the 0.4 mg folic acid group and 2.5% (three affected out of 120 births) in the 4 mg group (p = 0.59
based on a one-sided Fisher’s exact test). The total recurrence rate combining the two folic acid groups
was 2.7% (six affected out of 225 infants). One of the live births in the 4 mg group was diagnosed with
Van-der-Woude syndrome (VWS), which is the most common syndromic form of oral clefting with a
dominant genetic inheritance model [15] and about 50% recurrence risk.
Table 2. Cleft recurrence and types in OCPP.
Outcomes 0.4 mg
Folic Acid
4.0 mg
Folic Acid Total
Infants delivered, n 105 120 225
Recurrence of oral clefts, n (%) 3 (2.9) 3 (2.5) 6 (2.7)
Cleft type, n (%)
Cleft lip only 0 (0.0) 1 (0.8) 1 (0.44)
Cleft palate only 0 (0.0) 1 (0.8) 1 (0.4)
Cleft lip with palate 3 (2.9) 1 (0.8) 4 (1.8)
Notes: This is based on first time enrollment births only where previous
affected child or affected mother had cleft lip with/without cleft palate
(but not cleft palate only). One case with cleft lip only in the 4 mg group
had Van-der-Woude syndrome (missed at maternal screening);
excluding this case results in recurrence of 1.6%.
Int. J. Environ. Res. Public Health 2013, 10 600
VWS was not diagnosed when the mother of this case was screened for eligibility before enrollment
and should have been excluded. Excluding the VWS case results in an isolated oral cleft recurrence
rate of 1.6% (p = 0.44). All three cleft cases in the 0.4 mg group were cleft lip with palate compared to
one case in the 4 mg group; the rates of cleft lip with palate rates were 2.9% versus 0.8% in the 0.4 and
4 mg groups, respectively. Adding the births from families where the previously affected child or
affected mother had cleft palate only or including the babies of mothers who participated a second time
(i.e., only including cases that qualified because of the first affected child or mother having cleft lip
with or without cleft palate) has virtually no effect on the estimates of oral cleft recurrence as only a few
cases are added as shown in Supplementary online Table 1.1 (the same 6 affected cases were observed).
Table 3 shows the calculated recurrence rates in the historic control group. Data were obtained on 1,238
at-risk children born subsequent to an affected child (722 children) or to an affected mother (516 children).
Table 3. Historic recurrence rates in Brazil post-fortification.
Group Total
births Affected Rate (%)
Any period
Sibling affected 722 48 6.65
Mother affected 516 36 6.98
Post-fortification period
Sibling affected 278 18 6.47
Mother affected 50 3 6.00
We compared the OCPP rates first to the recurrence rate in the total historic group, and then to the
recurrence rate in children exposed to the fortification program during the first trimester of pregnancy
in order to account for any change in recurrence with fortification since women were randomized into
the OCPP after the beginning of the fortification. The overall recurrence rate was 6.65% for having a
prior affected sibling and 6.98% for affected mothers; these rates were fairly similar when restricting
the sample to children exposed to the fortification program (6.47% for a prior affected sibling and
6.0% for affected mothers). Based on these historic recurrence rates and the proportions of affected
women versus unaffected mothers of affected children, we calculated the “expected” historic
recurrence rates for the OCPP groups for the births from the first time enrollments and whose
previously affected sibling or mother had cleft lip with/without cleft palate (but not cleft palate only)
and compared them to the observed rates in Table 4. The expected recurrence rate among OCPP live
births was about 6.8% when using the overall historic recurrence rate, and about 6.3% when using the
post-fortification historic recurrence rate. These expected rates were more than twice as the observed
ones for both folic acid groups and were significantly different from the observed rates in the OCPP
folic acid groups both separate and combined (p = 0.0009 when comparing the post-fortification
expected recurrence rate to the observed recurrence rate in the combined group).
Int. J. Environ. Res. Public Health 2013, 10 601
Table 4. Expected versus observed recurrence rates in OCPP.
RCT group Expected historical
recurrence rate (%)
Observed
recurrence rate (%) p-value
Compared to overall historic recurrence rate
0.4 folic acid group 6.8 a 2.9 0.0172
4 mg folic acid group 6.8 b 2.5 0.0026
Both 0.4 and 4 mg groups 6.8 c 2.7 0.0001
Compared to historic recurrence rate post-fortification
0.4 folic acid group 6.3 e 2.9 0.0379
4 mg folic acid group 6.3 f 2.5 0.0077
Both 0.4 and 4 mg groups 6.3 g 2.7 0.0009
Note: Expected historical rates are calculated based on the historical rates from the recurrence
study weighted by the proportions of babies born into each group (affected and unaffected
mothers). Observed rates are based on first time enrollment births only with previous affected
child or affected mother with cleft lip with/without palate (but not cleft palate only).
a ((37 × 6.98) + (68 × 6.65))/105; b ((54 × 6.98) + (66 × 6.65))/120; c ((91 × 6.98) + (134 ×
6.65))/225; e ((37 × 6.00) + (68 × 6.47))/105; f ((54 × 6.00) + (66 × 6.47))/120; g ((91 × 6.00) +
(134 × 6.47))/225.
Table 5 compares the other secondary outcomes between the two folic acid groups. There are no
significant differences in infant’s mean birth weight, gestational age, length at birth, head circumference,
and apgar scores between the two folic acid groups. Virtually similar results for differences in birth
weight, length at birth, head circumference, and apgar scores between the two folic acid groups were
observed when adjusted for gestational age using regression analysis, which is expected since there is
no significant difference in gestational age between the two groups (detailed regression results
available from the authors upon request). There was no significant difference between the two folic
acid groups in preeclampsia (4.8% versus 3.7% in the 4 and 0.4 mg groups, respectively).
Table 5. Secondary outcomes by treatment.
Outcomes 0.4 mg
Folic Acid
4.0 mg
Folic Acid p-value Total
Birth weight (g), n 108 125 233
Mean (SD) 3,228.8 (443.7) 3,159.9 (508.2) 0.2753 (0.4287) 3,191.8 (479.6)
Gestational age (weeks), 108 123 231
Mean (SD) 38.5 (1.6) 38.6 (2.1) 0.6590 (0.3929) 38.5 (1.9)
Head Circumference (cm), n 87 102 189
Mean (SD) 34.1 (1.4) 34.1 (1.8) 0.9075 (0.8427) 34.1 (1.6)
Length (cm), n 107 120 227
Mean (SD) 48.2 (2.5) 48.3 (2.6) 0.9036 (0.4521) 48.3 (2.6)
Apgar score, n 73 86 159
Median 9 9 (0.0868) 9
Interquartile range 9–10 9–10 9–10
Min-Max 2–10 8–10 2–10
Preeclampsia, n 108 125 0.755 233
Yes n (%) 4 (3.7) 6 (4.8) 10 (4.3)
No n (%) 99 (96.3) 107 (95.2) 206 (95.7)
Notes: The outcomes are for first time enrollment births only. Some observations had missing data on certain
outcomes. The p values from the Wilcoxon rank-sum test are in brackets.
Int. J. Environ. Res. Public Health 2013, 10 602
3.3. Compliance
Based on the pill counts, median compliance was about 74% in both groups. The changes in blood
folate levels also suggest good compliance with the study interventions. Based on a subgroup of
participants with reviewed laboratory tests conducted at the laboratory at the study site at the Hospital
de Clínicas de Porto Alegre for participants enrolled in the clinic-based model (total of 1,312 tests),
mean post-supplementation serum folate levels were about 13.0 and 14.3 ng/mL in the 0.4 and 4 mg
groups, respectively, representing an increase by about 1.7 and 3.1 ng/mL compared to the baseline
levels (about 15% and 28%). The post-supplementation mean serum folate level was significantly
higher in the 4 mg than 0.4 mg group (p < 0.0001). Similarly, post-supplementation mean RBC folate
levels were about 716 and 793 ng/mL in the 0.4 and 4.0 mg groups (increase from baseline mean
values of 712 ng/mL and 717 ng/mL, respectively, in this subgroup). Similar to serum folate, the
post-supplementation mean RBC folate level was significantly higher in the 4 mg than 0.4 mg group
(p = 0.0021).
4. Discussion
The study observes no difference in recurrence rates between the 4 mg and 0.4 mg groups. The
small sample limits our ability to draw formal statistical inference on the effect of high dosage folic
acid (4 mg) relative to low dosage (0.4 mg) on oral cleft recurrence based on comparing the recurrence
rates between these two groups. With an observed recurrence rate of 2.9% in the 0.4 mg group, the
current sample size provides 37% power to detect a 100% decrease—the maximum possible effect
size —in recurrence in the 4 mg group based on a one-sided test (assuming that recurrence in the 4 mg
group is equal to or less than that in the 0.4 mg) and a 5% type-1 error. However, at this sample size,
the study has 80% power to detect a 6 percentage-point difference in recurrence between the two
groups (i.e., 8.5% recurrence in the 0.4 mg group relative to 2.5% recurrence in the 4 mg group) based
on a two-sided chi-square test and 5% type-1 error. Therefore at the current sample size, the study does
not have statistical power to detect a smaller difference (<6 percentage-points) in recurrence rates
between the two folic acid groups; a larger sample is needed to achieve acceptable power. In contrast,
there was a decline in recurrence in both OCPP groups separate and combined compared to historic
control groups. The study has reasonable power (65%) to detect the observed difference in recurrence
in the combined sample of 0.4 and 4 mg groups compared to the post-fortification historic recurrence
rate reported in Table 4 using a two-sided test and a 5% type-1 error (power of 80% using a one-sided
test). The recurrence rates that can be detected in the individual folic acid groups as different from the
post-fortification historic recurrence rate (at 80% power and based on a one-sided test and 5% type-1
error) and can therefore be ruled out in this analysis are 1.6% in the 4 mg group and 1.4% in the 0.4 mg.
The decrease in recurrence in the 0.4 mg group compared to the historic rate (similar to the 4 mg
group) suggests that this dose may be effective in reducing cleft recurrence risk. However, more
research is needed to test this hypothesis and quantify the effect. The relatively similar recurrence rates
between the two folic acid groups may be due to the potential ineffectiveness of the higher dose in
further reducing recurrence relative to the lower dose. This may also be complicated by the
participants’ use of prenatal vitamins and folic acid supplements on their own as well as their dietary
folate consumption. About 40% of the participants (based on data for 207 live births) reported using
Int. J. Environ. Res. Public Health 2013, 10 603
multivitamins and/or folic acid supplements other than those supplied by the study during the month
before pregnancy and/or during the first trimester (37% in the 0.4 mg and 43% in the 4.0 mg). Even
though these additional folate sources would elevate the folate levels in both the 0.4 and 4 mg folic
acid groups, they may result in an average folate intake in the lower dose group that is markedly higher
than the 0.4 mg per day.
One limitation of the study is introducing some changes in recruitment strategies (such as limiting
length of participation to 3 years) and inclusion/exclusion criteria (such as not including cleft palate
only and excluding women using injectable contraceptives) while the study was ongoing. Another
limitation is the potential over-estimation of population recurrence risk based on the historic control
groups since these were identified through craniofacial clinics providing care to patients with oral
clefts. Therefore, women who have experienced a recurrence may have been overrepresented. This
highlights the need to calculate these rates using population-based registries; however, these resources
are relatively underdeveloped in Brazil and are currently not available for such efforts. These
limitations should be considered when evaluating the generalizability of the study results.
The study is the first to shed some light using a double-blinded randomized design on effects of
high dosage folic acid on fetal development. The results suggest that high dosage folic acid does not
compromise fetal growth or increase perinatal risks. These findings are consistent with previous
observational study results suggesting positive effects of prenatal folic acid supplementation on
development [16,17], and are opposite to recent results from a mice study reporting adverse effects on
fetal growth. Furthermore, there were no significant differences in the rate and types of adverse events
between the two groups (detailed results available upon request). Adverse events were regularly
reported to the DSMB and IRBs and a member of the DSMB routinely reviewed all adverse events.
There were no meaningfully elevated rates of adverse events in either group compared to expected
population rates. These results suggest that high periconceptional folic acid supplementation may be a
generally safe intervention for future studies.
Acknowledgements
This work has been funded by NIH/NIDCR grant U01 DE017958 and NIH/NICHD grant U01
HD040561. Its contents are solely the responsibility of the authors and do not necessarily represent the
official views of the NIH, NIDCR, or NICHD.
The authors would like to thank all the staff who have contributed to this study especially Pricila
Copedê Frascareli Freitas, Priscila Padilha Moura, Cinthia Carolina Dalastti, Juliana Mercado Santos,
Renata Belmonte Ramalho, Daniela Vera Cruz dos Santos, Kenzo Martins Matuzawa, Mariza Branco,
Fernanda Queiros, Camilla Vila Nova Guimaraes, Lorena Silva de Argolo, Lorene Lins, Eduardo
Varella, Daniela Bezerra de Melo, Stela Brandao, Amanda Oliveira, Cirlei Ribeiro dos Santos, Denise
de Souza, Rita Tonochi, Miriam Neis, Ana Paula Vanz, Fabiane Dresch, Liliane Todeschini de Souza,
Nichole Nidey, and Kwame Nyarko. We also thank the following co-investigators: José Alberto de
Souza Freitas, Alain Viegas, and Lucildo Drebes and collaborators: João Henrique Nogueira Pinto,
Regina Célia Bortoleto Amantini, Zeus Moreira, and Lauro Consentino Filho, for their contributions.
Int. J. Environ. Res. Public Health 2013, 10 604
Conflict of Interest
None of the authors has any competing interests in this work.
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Appendix
List of Study Clinics
Hospital de Reabilitação de Anomalias Craniofaciais, Bauru, Sao Paulo
Hospital Santo Antônio: Obras Sociais Irmã Dulce, Salvador, Bahia
Instituto Materno Infantil Prof. Fernando Figueira, Recife, Pernambuco
Centro de Atendimento Integral ao Fissurado Lábio Palatal, Curitiba, Paraná
Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul
Fundação para Reabilitação das Deformidades Crânio-faciais, Lajeado, Rio Grande do Sul
Table 1.1. Oral cleft recurrence outcomes for selected groups.
Outcomes 0.4 mg
Folic Acid
4.0 mg
Folic Acid Total
Adding births from families where previously affected child or mother had cleft palate only
Recurrence of oral clefts, n (%) 109 125 234
Yes 3 (2.8) 3 (2.4) * 6 (2.6)
No 107 (97.2) 122 (97.6) 228 (97.3)
Including births from first and second time enrollments
Recurrence of oral clefts, n (%) 113 130 243
Yes 3 (2.7) 3 (2.3) 6 (2.5)
No 110 (97.3) 127 (97.7) 237 (97.5)
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