1. Objectives

To determine whether periconceptional folate supplementation reduces the risk of neural tube defects and other congenital anomalies, such as cleft palate, without causing adverse outcomes in mothers or their infants

2. How studies were identified

The Cochrane Pregnancy and Childbirth Group's Trials Register was searched to August 2015, which includes studies identified from the following databases:

• CENTRAL (The Cochrane Library)
• MEDLINE
• EMBASE
• CINAHL

In addition, handsearches of relevant journals and conference proceedings were performed, and relevant organizations, including the WHO, UNICEF, International Clearinghouse for Birth Defects Surveillance and Research, March of Dimes, World Food Programme, the United States CDC, Micronutrient Initiative, Global Alliance for Improved Nutrition, USAID, Helen Keller International, and Sight and Life were contacted

3. Criteria for including studies in the review

3.1 Study type

Randomized and quasi-randomized controlled trials, including cluster-randomized trials

3.2 Study participants

Women who became pregnant, or were ≤12 weeks’ pregnant, at the time of the intervention, with no restrictions on maternal age, parity, or history of having a previous pregnancy affected by a neural tube defect

(Studies or intervention groups in which supplementation was continued throughout pregnancy were excluded)

3.3 Interventions

Oral supplementation with folate alone, or in combination with other micronutrients, on a daily or intermittent basis in comparison to a placebo, no supplementation, or other micronutrients without folate

(Oral folate supplementation was defined as folic acid, 5-methyl-tetrahydrofolate (5MTHF) or folinic acid, delivered directly to the oral cavity, either as a tablet, capsule, dispersible tablet or liquid)

(Studies examining folic acid fortification of food, or oral contraceptives containing folic acid were excluded)

(Intermittent supplementation was defined as a supplementation regimen of one, two or three times a week on non-consecutive days)

3.4 Primary outcomes

Infant

• Neural tube defects (all)
• Cleft lip
• Cleft palate
• Congenital cardiovascular anomalies
• Other congenital anomalies (excluding those listed above)
• Neonatal death (death within the first 28 days of life)

Maternal

• Anaemia at or near term (defined as haemoglobin <110 g/L at ≥34 weeks’ gestation)
• Red blood cell folate at or near term (nmol/L at ≥34 weeks’ gestation)
• Serum folate at term or near term (nmol/L at ≥34 weeks’ gestation)
• Miscarriage (defined by the trialists)

Secondary outcomes for the infant included: spina bifida, anencephaly, encephaloceles, hydranencephaly, iniencephaly, schizencephaly, stillbirth, low birth weight (<2500 g), very low birth weight (<1500 g), infant optimal health status at birth, admission to special care for any cause, macrosomia (>4000 g), infant insulin resistance, Apgar score at one minute after birth (>8), Apgar score at five minutes after birth (>8), and preterm birth (<37 weeks' gestation). Secondary outcomes for the mother included: multiple pregnancy, homocysteine at term or near term (µmol/L at ≥34 weeks’ gestation), serum vitamin B6 concentration at or near term (nmol/L at ≥34 weeks’ gestation), serum vitamin B12 concentration at or near term (pmol/L at ≥34 weeks’ gestation), pre-eclampsia (defined as gestational blood pressure higher than 140/90 mmHg and proteinuria of more than 300 mg of protein in a 24-hour urine sample), pregnancy termination for fetal anomaly, and any side effects

4. Main results

4.1 Included studies

Five trials involving 7391 women and their pregnancies were included in this review:

• Four trials investigated neural tube defect recurrence, and one trial assessed the first occurrence of neural tube defects
• All participants were blinded to the intervention, although blinding was unclear for outcome assessors in all five trials
• All studies employed folic acid, and doses ranged from <400 μg/day to 4000 μg/day. No studies used intermittent supplementation, although two studies used divided daily doses (two and three times per day)
• In all trials women began supplementation prior to pregnancy and ceased supplementation after 12 weeks of pregnancy
• One trial compared folic acid alone to placebo
• Two trials compared folic acid plus multiple micronutrients to multiple micronutrients without folic acid, with copper, manganese, zinc, vitamin C used in one trial; and in the other, iron and calcium
• One trial had three arms: i) folic acid only, ii) vitamin A, vitamin D, thiamine, riboflavin, vitamin B6, niacin, vitamin C, calcium, iron; and iii) folic acid plus the same micronutrients
• One trial had four arms: i) folic acid plus iron and calcium; ii) folic acid plus iron, calcium and multiple micronutrients; iii) iron, calcium and multiple micronutrients without folic acid; and iv) the control group received iron and calcium, but without multiple micronutrients or folic acid

4.2 Study settings

• Four studies were conducted in high-income countries: Hungary, Ireland, the United Kingdom of Great Britain and Northern Ireland, and multi-country (Australia, Canada, France, Hungary, Israel, Russian Federation and the United Kingdom of Great Britain and Northern Ireland)
• One study was conducted in a lower-middle-income country: India
• Studies were conducted in the community, in hospitals, in family planning clinics, and in human genetics or paediatrics centres

4.3 Study settings

How the data were analysed
Four comparisons were made: i) supplementation with any folate versus no treatment/placebo or other micronutrients; ii) supplementation with any folate alone versus no treatment/placebo; iii) supplementation with folic acid plus other micronutrients versus other micronutrients without folic acid; and iv) supplementation with folate plus other micronutrients versus the same micronutrients without folate. The trials were assessed for methodological quality using the standard Cochrane criteria. Random effects meta-analysis was used to combine dichotomous data to produce pooled risk ratios (RR), with corresponding 95% confidence intervals (CI). Denominators only included women who became pregnant. Outcomes may not have been mutually exclusive for total neural tube defects and terminations of pregnancy due to fetal anomaly. Heterogeneity was planned to be explored in the following subgroup analyses:

• By supplementation regimen: daily supplementation, intermittent supplementation
• By daily dose: ≤400 μg (0.4 mg) of folic acid per day, >400 μg (0.4 mg) of folic acid per day
• By timing of supplementation: before pregnancy, during first trimester only, periconceptional period
• By history of a pregnancy affected by a neural tube defect (recurrence): yes, no, or unknown/unreported/mixed
• By folate compound: folic acid, 5MTHF, other compound, or unknown/unreported/mixed
• By micronutrient composition: folate alone, folate plus iron, folate plus other micronutrient (except iron), or folate plus multiple micronutrients;
• By malaria endemicity at the time of the study: yes, no, or unknown/unreported/mixed

Results
Supplementation with any folate versus no treatment, placebo or other micronutrients without folate (5 trials)
Infant outcomes
Neural tube defects (all)
The risk of having a neural tube defect-affected pregnancy was reduced by more than two-thirds among women receiving folic acid supplementation (RR 0.31, 95% CI [0.17 to 0.58], p=0.00025; 5 trials/6708 births). Among women who had a previous pregnancy affected by a neural tube defect, the effect of folic acid was similar (RR 0.34, 95% CI [0.18 to 0.64], 4 trials/1846 births). In the single study in women without a history of having a neural tube defect-affected pregnancy, no cases occurred among those receiving folic acid supplements, resulting in a greater reduction in risk that was not statistically significant (RR 0.07, 95% CI [0.00 to 1.32], 1 trial/4862 births). In the trial in which supplementation was <400 μg folic acid/day (360 μg/day), the results favoured the intervention group, but were not statistically significant (RR 0.17, 95% CI [0.01 to 4.21], 261 births), while pooled analysis of the other four trials in which the dose was >400 μg folic acid/day produced a statistically significant effect (RR 0.32, 95% CI [0.17 to 0.60], 6447 births).

Other primary outcomes for the infant
In three trials including 5612 births, the risk of cleft lip was not significantly different between treatment groups (RR 0.79, 95% CI [0.14 to 4.36], p=0.79), and nor was cleft palate (RR 0.73, [0.05 to 10.89], p=0.82). Additionally, there were no significant differences detected between groups for the outcomes congenital cardiovascular anomalies (RR 0.57, [0.24 to 1.33], p=0.19; 3 trials/5612 births) and other birth defects (RR 0.94, 95% CI [0.53 to 1.66], p=0.82; 3 trials/5612 births).

Additional infant outcomes
Spina bifida and anencephaly were both reduced by approximately two-thirds in the treatment group, although this was not statistically significant (spina bifida: RR 0.32, 95% CI [0.06 to 1.62], 3 trials/4546 births/6 cases; anencephaly: RR 0.36, 95% CI [0.13 to 1.03], 4 trials/4807 births/17 cases). There was no evidence of a difference between groups in stillbirths, low birth weight infants, macrosomia or preterm births. The risk of pregnancy termination for fetal anomaly was reduced by 71% with folic acid supplementation (RR 0.29, 95% CI [0.15 to 0.56], p=0.00025; 4 trials/7110 pregnancies/50 cases).

Maternal outcomes
There was no evidence of a difference between treatment groups in the risk of miscarriage (RR 1.10, 95% CI [0.94 to 1.28], p=0.22; 5 trials/7391 pregnancies), multiple pregnancy (RR 1.38, 95% CI [0.89 to 2.14], 4 trials/7280 pregnancies), or side effects. No trials reported on other maternal outcomes.

Supplementation with folic acid alone versus no treatment or placebo (1 trial)
Infant outcomes
There was no evidence of a significant difference in the risk of having a neural tube defect-affected pregnancy between those receiving folic acid alone and those receiving placebo in one trial including 111 births (RR 0.43, 95% CI [0.08 to 2.23], p=0.31). In addition, no evidence of a significant difference was found between groups for spina bifida (RR 0.43, 95% CI [0.04 to 4.55]), or anencephaly (RR 0.43, 95% CI [0.04 to 4.55]).

Maternal outcomes
No significant difference between treatment groups was detected in the number of miscarriages (RR 0.85, 95% CI [0.05 to 13.25], p=0.91; 1 trial/111 pregnancies), or termination pregnancy due to fetal anomaly (RR 0.28, 95% CI [0.01 to 6.83]), although no cases of occurred in the treatment group.

Supplementation with folic acid plus other micronutrients versus other micronutrients without folic acid (4 trials)
Infant outcomes
Neural tube defects (all)
Women receiving folic acid in addition to other micronutrients were at a 69% reduced risk of having a neural tube defect-affected pregnancy in comparison to those who received micronutrients without folic acid (RR 0.31, 95% CI [0.16 to 0.60], p=0.00055; 4 trials/6512 births). Among the three trials in which the recurrence of neural tube defects was assessed, there were fewer cases among women receiving folic acid (RR 0.33, 95% CI [0.17 to 0.66], 1650 births), while in the single trial assessing the first occurrence of neural tube defects, no significant difference between treatment groups was found (RR 0.07, 95% CI [0.00 to 1.32], 4862 births). In the trial in which supplementation was <400 μg folic acid/day (360 μg/day), results were not statistically significant (RR 0.34, 95% CI [0.01 to 8.26], 176 births), while for the three trials in which the dose was >400 μg folic acid/day, the risk of neural tube defects was statistically significantly reduced (RR 0.31, 95% CI [0.15 to 0.61], 6336 births).

Other outcomes for the infant
No statistically significant difference between treatment groups was found for cleft lip (RR 1.02, 95% CI [0.26 to 3.96], p=0.98), cleft palate (RR 0.73, 95% CI [0.05 to 10.89], p=0.82; 3 trials/5527 births/3 cases), congenital cardiovascular anomalies (RR 0.60, 95% CI [0.25 to 1.41], p=0.24), or other birth defects (RR 0.98, 95% CI [0.54 to 1.77], p=0.94; 3 trials/5527 births). The risk of having a pregnancy terminated for fetal anomaly was significantly reduced with folic acid supplementation (RR 0.29, 95% CI [0.15 to 0.57], p=0.00034; 3 trials/6999 pregnancies). Spina bifida (RR 0.26, 95% CI 0.03 to 2.31], 2 trials/4435 births) and anencephaly (RR 0.38, 95% CI [0.12 to 1.22], 3 trials/4611 births) were not statistically significantly reduced with folic acid supplementation, and no difference between treatment groups was detected for stillbirth, low birth weight, macrosomia, or preterm birth.

Maternal outcomes
In four trials including 7187 confirmed pregnancies, the risk of miscarriage was not different between treatment groups (RR 1.08, 95% CI [0.87 to 1.32], p=0.49). The risk of multiple pregnancy was not statistically significantly different between women supplemented with folic acid and those that were not (RR 1.39, 95% CI [0.90 to 2.17], 4 trials/7187 pregnancies).

Supplementation with folate plus other micronutrients versus the same other micronutrients without folate (2 trials)
Infant outcomes
In two trials involving 1371 births to women with a history of having a neural tube defect-affected pregnancy, the risk of having a neural tube defect recurrence was reduced by 71% (RR 0.29, 95% CI [0.12 to 0.70], p=0.0055) with folic acid supplementation. However, in these two trials there was no evidence of a significant difference between treatment groups for the outcomes cleft lip (RR 0.34, 95% CI [0.01 to 8.26], p=0.51), cleft palate (RR 3.05, 95% CI [0.12 to 74.61], p=0.49), congenital cardiovascular anomalies (RR 0.50, 95% CI [0.11 to 2.31], p=0.37), or other birth defects (RR 1.39, 95% CI [0.74 to 2.62], p=0.31). Pregnancy termination for fetal anomaly was significantly reduced among women receiving folic acid (RR 0.35, 95% CI [0.15 to 0.83], p=0.017; 1 trial/1362 pregnancies). The risk of stillbirth and anencephaly were not statistically significantly different between treatment groups.

Maternal outcomes
The risk of miscarriage did not differ significantly according to folic acid supplementation (RR 1.08, 95% CI [0.82 to 1.41], p=0.58; 2 trials/1550 pregnancies), and nor did the risk of multiple pregnancy (RR 2.04, 95% CI [0.19 to 22.15], 2 trials/1550 pregnancies).

5. Additional author observations*

Overall, the included trials were rated as being at unclear or low risk of bias, with the majority of studies failing to adequately report methods of randomization or allocation concealment. The evidence for the main comparison (supplementation with folate versus no intervention/placebo or other micronutrients without folate) judged as being high quality for neural tube defects, moderate quality for miscarriage, and low quality for cleft lip, cleft palate, congenital cardiovascular anomalies, and other congenital anomalies. The women included in this review were of Caucasian and Indian origin, and gene polymorphisms of folate metabolism may result in variable outcomes in other ethnicities. The trials included in this review were performed before many countries introduced mandatory flour fortification with folic acid, and with this in mind, some authors have suggested that the current recommendation for women planning to become pregnant to take 400 μg/day of supplemental folic acid in addition to dietary folate requires revision.

Folic acid alone or in combination with other micronutrients prevents the recurrence of neural tube defects. The evidence reviewed here does not support a beneficial effect of folic acid supplementation on other congenital anomalies or maternal outcomes. Although the risk of pregnancy termination due to fetal anomaly was reduced with folic acid supplementation in most analyses, there was potential for overlap whereby the same fetus counted in the figure for pregnancy termination may have also been included in the figure for neural tube defects.

Future research could examine whether intermittent weekly dosing of folic acid is effective in the prevention of birth defects, and further trials investigating the effect of current internationally recommended folic acid doses on birth defects are warranted.