Research

www. AJOG.org

OBSTETRICS

Folic acid supplementation in early second trimester

and the risk of preeclampsia
Shi Wu Wen, MB, PhD; Xi-Kuan Chen, MD, PhD; Marc Rodger, MD; Ruth Rennicks White, RN, BScN;
Qiuying Yang, MD, PhD; Graeme N. Smith, MD, PhD; Ronald J. Sigal, MD, MPH;
Sherry L. Perkins, PhD; Mark C. Walker, MD, MSc
OBJECTIVE: The objective of the study was to evaluate the association
between folic acid supplementation in early second trimester and the
risk of developing preeclampsia.
STUDY DESIGN: We carried out a prospective cohort study between

October 2002-December 2005. We recruited women who had their prenatal care visit (12-20 weeks gestation) at the Ottawa Hospital and
Kingston General Hospital. All charts for participants with a diagnosis
of preeclampsia were audited and blindly adjudicated by 4 study investigators to validate the diagnosis.

RESULTS: A total of 2951 pregnant women were included in the final analysis.
Supplementation of multivitamins containing folic acid was associated with increased serum folate (on average 10.51 mol/L), decreased plasma homocysteine (on average 0.39 mol/L), and reduced risk of preeclampsia (adjusted
odds ratio, 0.37; 95% confidence interval, 0.18-0.75).
CONCLUSION: Supplementation of multivitamins containing folic acid in the
second trimester is associated with reduced risk of preeclampsia.

Key words: 5, 10-methylenetetrahydrofolate reductase, folic acid,

homocysteine, preeclampsia, supplementation

Cite this article as: Wen SW, Chen X-K, Rodger M, et al. Folic acid supplementation in early second trimester and the risk of preeclampsia. Am J Obstet
Gynecol 2008;198:45.e1-45.e7.

reeclampsia is hypertension and

proteinuria that develop during
pregnancy, affecting at least 5% of pregnancies worldwide.1 It is a leading cause
of maternal and neonatal morbidity and
mortality.1 Women with a history of preeclampsia are at increased risk of cardiovascular disease in later life.2 Preeclampsia may also increase the risks of

cardiovascular disease and diabetes in

the offspring of the affected mothers
through fetal origins of adult diseases.3
The current hypothesis for the pathogenesis of preeclampsia is that factors
produced by the poorly perfused placenta enter the systemic circulation and
alter vascular sensitivity to circulating
pressors, activate coagulation, and re-

From the OMNI Research Group, Department of Obstetrics and Gynecology (Drs Wen,
Chen, Yang, and Walker and Ms White), and the Department of Epidemiology and
Community Medicine (Dr Wen), University of Ottawa Faculty of Medicine; the Clinical
Epidemiology Program, Ottawa Health Research Institute (Drs Wen, Chen, Rodger, Yang,
Sigal, and Walker and Ms White); and the Division of Hematology, Department of
Medicine (Dr Rodger), the Department of Medicine (Dr Sigal), and the Division of
Biochemistry (Dr Perkins); Queens Perinatal Research Unit, Kingston General Hospital,
and the Department of Obstetrics and Gynecology, Queens University School of Medicine,
Kingston, ON (Dr Smith); and the Division of Endocrinology, University of Calgary,
Calgary, AB (Dr Sigal), Canada.
Received Nov. 20, 2006; revised April 17, 2007; accepted June 29, 2007.
Supported in part by a grant from the Canadian Institute for Health Research (Grant MOP
53188). S.W.W., G.N.S., R.J.S., and M.C.W. are recipients of a New Investigators Award from
the Canadian Institute for Health Research. M.R. is a Clinical Investigator of the Heart and Stroke
Foundation of Canada, and X.-K.C. and Q.Y. are Canadian Institute for Health
Research/Strategic Training Initiatives of Research in Reproductive Health Sciences postdoctor
fellows.
Reprints: Shi Wu Wen, MB, PhD, OMNI Research Group, Department of Obstetrics and
Gynecology, University of Ottawa, Faculty of Medicine, 501 Smyth Rd, Box 241, Ottawa,
Ontario, Canada K1H 8L6; swwen@ohri.ca
0002-9378/$34.00 2008 Mosby, Inc. All rights reserved. doi: 10.1016/j.ajog.2007.06.067

duce vascular integrity, resulting in the

pathophysiologic changes of preeclampsia.4 However, which factors produced
by the poorly perfused placenta are responsible for the development of preeclampsia and how they interact with
maternal predisposing factors to induce
the clinical syndrome of preeclampsia
remain elusive.4
Recent studies have found that supplementation of multivitamins containing
folic acid was associated with reduced
risk of preeclampsia.5 Folic acid may reduce the risk of preeclampsia by improving placental and systemic endothelial
functions and directly or indirectly by
lowering blood homocysteine levels.6-10
The objective of this study was to comprehensively evaluate the association between folic acid supplementation, serum
folate, homocysteine, and 5, 10-methylenetetrahydrofolate reductase (MTHFR)
thermolabile variant gene with the risk of
preeclampsia.

M ATERIALS AND M ETHODS

The Ottawa and Kingston (OaK) Birth
Cohort recruited nontransferred, consenting women between 12-20 weeks
gestation during their prenatal visit at

JANUARY 2008 American Journal of Obstetrics & Gynecology

45.e1

Research

Obstetrics

the Ottawa Hospital and Kingston General Hospital. The current analysis included subjects from phase I of the OaK
Birth Cohort, which started in October
2002 and ended in December 2005. The
research nurses explained to pregnant
women the purpose of the study, what
would be expected from them, and what
they could expect from the study. For
participants who gave signed informed
consent, blood was drawn for genetic
and biochemical analyses. Twins or
higher order of multiples or subjects
with missing information on gestational
age or birth weight were excluded.
Demographic and clinical data were
collected by structured interview and
chart review. Additional chart review or
participant contact was performed if ambiguities or missing data were encountered. Information on supplementation
of folic acid and other vitamins, including brand name, date of initiation, and
date of discontinuation, was collected
both at recruitment and at delivery. Participants were told that this study was
observation only and that during the
study we did not want them to change
anything regarding their daily life or
health care. Only women with regular
(daily) supplementation were counted.
These same questions were asked again
at the time of delivery to determine
whether there was any change in
supplementation.
Laboratory testing was performed to
determine serum folate levels and
plasma homocysteine levels and for the
presence of the MTHFR thermolabile
variant gene. Blood for MTHFR genotyping and homocysteine measurement
was collected in K2EDTA Vacutainer
tubes (Becton Dickinson, Lincoln Park,
NJ). Homocysteine specimens were put
on ice immediately after collection,
transported to the laboratory within 30
minutes, and centrifuged at 4C for 10
minutes at 3000 g. Plasma was removed
and stored at -20C until analysis. Samples were assayed in batches. Blood for
serum folate was collected in serum separator tubes (SST; Becton Dickinson).
The specimens were allowed to clot and
then be centrifuged for 10 minutes at
3000 g to separate serum, which was
stored at -20C until analysis. Serum fo45.e2

www.AJOG.org
late was measured on the Beckman
Coulter Access II using manufacturers
reagents (Beckman Coulter Inc, Fullerton, CA). Homocysteine was measured
on the Abbott Ax Sym II (Abbott Laboratories, Abbott Park, IL) using fluorescence polarization immunoassay technology. MTHFR genotyping was
conducted using the method of Donnelly and Rock.11
Preeclampsia was defined as having a
blood pressure of 140/90 mm Hg or
30/15 mm Hg above baseline with proteinuria of 2 on dipstick or 300 mg in
24-hour urine collection in women
greater than 20 weeks gestation. All
charts for participants with a diagnosis of
preeclampsia were audited and blindly
adjudicated by 4 study investigators
(S.W.W., M.R., R.W., and M.W.) to validate the diagnosis.
The patterns of supplementation of
folic acid and other vitamins in pregnancy and the distribution of maternal
demographic and clinical characteristics
of the study participants were ascertained. The effects of folic acid supplementation on serum folate and plasma
homocysteine levels was then determined overall and stratified by gestational age at recruitment and by MTHFR
genotype. Finally, the effect of folic acid
supplementation on preeclampsia was
determined. Adjusted odds ratios
(aORs) and 95% confidence intervals
(CIs) for folic acid supplementation
were estimated by multiple logistic regression analysis, with no supplementation as the reference.
Potential confounding variables included in the regression models were
maternal age, ethnic background, educational level, parity, previous preeclampsia, chronic hypertension, diabetes,
prepregnancy body mass index, household income, gestational age at recruitment, and cigarette smoking during
pregnancy. The effects of low serum folate, hyperhomocystinemia, and the
presence of the MTHFR thermolabile
variant gene on preeclampsia were also
examined. Low serum folate was defined
as folate concentration below the 10th
percentile, and hyperhomocystinemia
was defined as concentration higher than
the 90th percentile of the study popula-

American Journal of Obstetrics & Gynecology JANUARY 2008

tion, after stratification by gestational

week at which the blood sample was
taken.
Additional analyses assessing the effect
of supplementation initiation time (before vs after conception) and discontinuation of supplementation in the third
trimester (yes vs no) were conducted. All
analyses were performed using SAS (version 9.1; SAS Institute Inc, Cary, NC).

R ESULTS
A total of 4024 women were approached
to participate in the study; 3134 (78%)
agreed and were recruited. Among them,
70 women were excluded because of twin
births and 113 women were excluded because of missing information such as
gestational age at delivery, birthweight,
or sex (59) and lost to follow-up because
of the participants relocation outside
the study center (54), leaving 2951 subjects for final analysis.
Ninety-two percent of the study subjects were taking folic acid supplementation in the early second trimester, most
by taking multivitamins containing folic
acid at a dose of 1.0 mg or higher (Table
1). More than half of the women initiated supplementation before conception
and approximately 20% discontinued
supplementation in the third trimester
partly or completely (Table 1).
The majority of the study participants
were white with high socioeconomic status. Women with no supplementation
were more likely to be younger, multiparous, non-whites; to have lower education level and household income; and to
smoke cigarettes during pregnancy than
women with supplementation (Table 2).
Folic acid supplementation was associated with increased serum folate and
decreased plasma homocysteine (Table
3). The association between supplementation and serum folate and plasma homocysteine was stronger in blood samples taken at later gestation and
(especially) in participants with MTHFR
thermolabile variant genes (Table 3).
The rate of preeclampsia was lower in
the supplementation group than in the
no supplementation group, with an aOR
of 0.37 and 95% CIs 0.18-0.75 (Table 4).
Women with supplementation of folic

Obstetrics

www.AJOG.org

TABLE 1

Folic acid supplementation in pregnant women of the

OaK Birth Cohort Study, October 2002-December 2005a
Number

Percent

Category of supplementation

.....................................................................................................................................................................................................................................

Multivitamins containing folic acid

2016

74.30

Folic acid alone

250

9.22

Two or more types of vitamins

447

16.48

.....................................................................................................................................................................................................................................
.....................................................................................................................................................................................................................................
..............................................................................................................................................................................................................................................

Dose of folic acid (mg)

.....................................................................................................................................................................................................................................

0.10.9

114

4.20

2152

79.32

1.11.9

178

6.56

2.0

269

9.92

Initiated before conception

1682

62.00

Initiated after conception

1031

38.00

544

20.10

.....................................................................................................................................................................................................................................

1.0

.....................................................................................................................................................................................................................................
.....................................................................................................................................................................................................................................
..............................................................................................................................................................................................................................................
..............................................................................................................................................................................................................................................
..............................................................................................................................................................................................................................................

Discontinued in third trimester (partly or completely)

..............................................................................................................................................................................................................................................
a

Doses of folic acid were derived from vitamin brands; doses of other vitamins in multivitamins were not presented in the
table; the ranges (medians) of doses for these vitamins were: vitamin A, 1000-4000 IU (1800 IU); vitamin C, 60-150 mg
(95.83 mg); vitamin B1, 1.4 to 3.0 mg (2.23 mg); vitamin B2, 1.4 to 3.75 mg (2.78 mg); vitamin B3, 10-40 mg ( 25 mg);
vitamin B5, 0.03 to 10.0 mg (5.02 mg); vitamin B6, 1.9 to 10 mg (4.98 mg); vitamin B12, 2.6 to 14 g (9.77 g); vitamin
D, 200-400 IU (360 IU); vitamin E, 25-30 IU (25.58 IU); beta-carotene,1050-3000 IU (2270 IU); biotin, 30-45 g (36 g);
niacin, 15-20 mg (18.25 mg); pantothenic acid, 6-10 mg (9 mg). Minerals were: calcium carbonate, 175-450 mg (250 mg);
copper, 1-2 mg (1.6 mg); chromium, 25-30 g (27 g); iodine, 0.15 to 0.22 mg (0.16 mg); iron, 10-30 mg (22 mg); lutein,
250-300 g (275 g); magnesium, 50-100 mg (60 mg); manganese, 2-50 mg ( 16 mg); molybdenum, 25-50 g (38 g);
phosphorous, 125 mg (125 mg); selenium, 25-30 g (28 g); zinc, 7.5-25 mg (16.88 mg).
Wen. Folic acid supplementation and the risk of preeclampsia. Am J Obstet Gynecol 2008.

acid alone also had a lower rate of preeclampsia than those women who had
no supplementation, although the difference was not statistically significant
(Table 4). No significant associations between low serum folate level, hyperhomocystinemia, and MTHFR thermolabile variant genes with risk of
preeclampsia were found (Table 4).
Whether women initiated supplementation before or after conception or
whether women discontinued supplementation in the third trimester, the rate
of preeclampsia was similarly lower than
those women who did not have any supplementation (Table 4).

C OMMENT
Our prospective cohort study in a cohort
of Canadian women found that 92% had
supplementation with folic acid or multivitamins containing folic acid in the
early second trimester, and among them,
most (95%) had a supplementation of
1.0 mg or higher, twice the recommended level for the prevention of neural tube defects by Health Canada.12

Supplementation of multivitamins
containing folic acid was associated with
increased serum folate, lowered plasma
homocysteine, and reduced risk of preeclampsia (by 63%). In our study,
women with no supplementation were
of lower socioeconomic status, which
may lead to increased risk of preeclampsia.1 On the other hand, women with no
supplementation were more likely to be
younger and multiparous and to smoke
cigarettes during pregnancy, which may
lead to a decreased risk of preeclampsia.1,13 Thus, the potential confounding
effects of maternal age, parity, socioeconomic status, and cigarette smoking may
have canceled each other and may have
limited impact on the observed association between supplementation and preeclampsia. Our study did not observe an
association between MTHFR gene mutation and preeclampsia, which is consistent with the study by Powers et al.14 We
speculate that high-level supplementation of folic acid in our study population
may have suppressed the genetic effect
on preeclampsia.

Research

A previous study reported an association between supplementation of multivitamins containing folic acid and reduced risk of preeclampsia.5 Bodnar et
al5 conducted a prospective cohort study
in 1835 women in Pittsburgh, PA, between 1997-2001 and found that regular
use of multivitamins containing folic
acid at less than 16 weeks gestation was
associated with a 45% reduction in preeclampsia risk, compared with nonusers
(OR, 0.55; 95% CI, 0.32-0.95). This finding was consistent with ours. However, a
previous study on this issue has several
weaknesses. The Pittsburgh study did
not conduct tests for genetics or blood
folate and homocysteine levels.5 Our
study has the largest sample size with detailed information on supplementation
patterns such as type, duration, and dose
of supplementation. We managed to
measure several aspects of folate metabolism, including genetics, supplementation, and blood folate and homocysteine
levels simultaneously. We used a prospective cohort study design and blindly
adjudicated the outcome, which helped
to minimize bias.
There are compelling biologic rationale to believe that folic acid may reduce
the risk of developing preeclampsia. Preeclampsia is likely a 2-stage disorder: at
stage I (most likely at late first trimester
or early second trimester), a decreased
placental perfusion, secondary to abnormal placental developments, develops;
and at stage II (most likely at the early
third trimester), the maternal syndrome
of preeclampsia, secondary to systemic
endothelial dysfunction, develops.4
Supplementation of large doses of folic acid in early gestation may work at
both stages of preeclampsia development. Folic acid, or folate, is one of the B
vitamins. It is a coenzyme in the production of nucleic acids and therefore is required by all cells for growth. The placenta develops from a single cell to a
complex entity with a weight of about
500 g during pregnancy. An adequate
cellular folate supply may play an important role in the implantation and development of the placenta. Folate may also
reduce the risk of developing preeclampsia by improving endothelial function at
both placental and systemic levels, di-

JANUARY 2008 American Journal of Obstetrics & Gynecology

45.e3

Research

Obstetrics

www.AJOG.org

TABLE 2

Demographic and clinical characteristic of the pregnant women who participated

in the OaK Birth Cohort Study, October 2002-December 2005a
Variables

Overall

No supplementation

Supplementation

Number of subjects

2951

238

2713

P value

................................................................................................................................................................................................................................................................................................................................................................................

Maternal age (y)

.......................................................................................................................................................................................................................................................................................................................................................................

25

6.40

13.45

5.79

25-29

21.82

23.11

21.71

30-34

40.09

36.55

40.40

35

31.68

26.89

32.10

.0001

.......................................................................................................................................................................................................................................................................................................................................................................
.......................................................................................................................................................................................................................................................................................................................................................................
.......................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................

Maternal background

.......................................................................................................................................................................................................................................................................................................................................................................

Aboriginal

0.75

0.42

0.77

83.46

74.38

84.26

Middle Eastern

5.29

7.56

5.09

African

3.42

10.08

2.84

Asian

7.08

7.56

7.04

.0001

.......................................................................................................................................................................................................................................................................................................................................................................

White

.......................................................................................................................................................................................................................................................................................................................................................................
.......................................................................................................................................................................................................................................................................................................................................................................
.......................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................
2

Prepregnancy body mass index (kg/m )

.......................................................................................................................................................................................................................................................................................................................................................................

18.5 (underweight)

6.03

5.46

6.08

60.01

59.24

60.08

.1492

.......................................................................................................................................................................................................................................................................................................................................................................

18.5-24 (normal)

.......................................................................................................................................................................................................................................................................................................................................................................

25-29 (overweight)

20.40

17.23

20.68

30 (obesity)

13.55

18.07

13.16

.......................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................

Education level

.......................................................................................................................................................................................................................................................................................................................................................................

High school and below

13.15

27.97

11.85

.0001

.......................................................................................................................................................................................................................................................................................................................................................................

College/university not completed

9.34

10.17

9.27

77.51

61.86

78.88

34.46

23.53

35.42

.......................................................................................................................................................................................................................................................................................................................................................................

College/university completed

................................................................................................................................................................................................................................................................................................................................................................................

Nulliparous

.0002

................................................................................................................................................................................................................................................................................................................................................................................

Household income ($,CAD)

.......................................................................................................................................................................................................................................................................................................................................................................

25,000

.0001

6.76

18.50

5.75

25,000-49,999

14.07

22.47

13.35

50,000-79,999

27.76

27.31

27.80

80,000

51.41

31.72

53.10

Smoking during pregnancy

7.73

13.03

7.27

.0014

Chronic hypertension

1.58

2.14

1.53

.4761

Type 1 diabetes

0.82

1.29

0.78

.4151

Type 2 diabetes

0.86

0.86

0.86

.9990

Previous preeclampsia

3.96

4.62

3.91

.5879

.......................................................................................................................................................................................................................................................................................................................................................................
.......................................................................................................................................................................................................................................................................................................................................................................
.......................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................

Gestational age at recruitment (wks)

.......................................................................................................................................................................................................................................................................................................................................................................

12

33.82

27.31

34.39

13-15

45.21

49.58

44.82

16-20

20.98

23.11

20.79

.2079

.......................................................................................................................................................................................................................................................................................................................................................................
.......................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................
a

Because of missing information in some variables: smoking (2), chronic hypertension (39), type 1 diabetes (40), type 2 diabetes (40), numbers in this table did not add up.

Wen. Folic acid supplementation and the risk of preeclampsia. Am J Obstet Gynecol 2008.

45.e4

American Journal of Obstetrics & Gynecology JANUARY 2008

Obstetrics

www.AJOG.org

Research

TABLE 3

Serum folate and plasma homocysteine levels in subjects with folic acid supplementation vs those without,
by gestational age at recruitment and MTHFR genotype, OaK Birth Cohort Study, October 2002-December 2005
Variables

No supplementation

Supplementation

P value

Folate level (mean SD, mol/L)a

25.79 8.60

36.30 7.99

.0001

................................................................................................................................................................................................................................................................................................................................................................................

Folate level stratified by gestational weeks at recruitment (mean SD, mol/L)

.......................................................................................................................................................................................................................................................................................................................................................................

12

25.93 8.78

37.00 7.71

.0001

13-15

26.42 8.69

36.63 7.82

.0001

16-20

24.27 8.15

34.35 8.52

.0001

.......................................................................................................................................................................................................................................................................................................................................................................
.......................................................................................................................................................................................................................................................................................................................................................................

................................................................................................................................................................................................................................................................................................................................................................................
b

Folate level stratified by MTHFR genotype (mean SD, mol/L)

.......................................................................................................................................................................................................................................................................................................................................................................

677CC

26.93 8.47

36.24 8.16

.0001

677CT

24.77 7.96

36.25 7.79

.0001

677TT

24.29 10.43

36.63 8.14

.0001

4.69 1.24

4.32 1.01

.0001

.......................................................................................................................................................................................................................................................................................................................................................................
.......................................................................................................................................................................................................................................................................................................................................................................

................................................................................................................................................................................................................................................................................................................................................................................
c

Homocysteine level (mean SD, mol/L)

................................................................................................................................................................................................................................................................................................................................................................................

Homocysteine level stratified by gestational weeks at recruitment (mean SD, mol/L)

.......................................................................................................................................................................................................................................................................................................................................................................

12

4.61 1.02

4.43 1.02

.1868

13-15

4.85 1.36

4.36 1.02

.0002

16-20

4.43 1.13

4.04 0.94

.0043

.......................................................................................................................................................................................................................................................................................................................................................................
.......................................................................................................................................................................................................................................................................................................................................................................

................................................................................................................................................................................................................................................................................................................................................................................
b

Homocysteine level stratified by MTHFR genotype (mean SD, mol/L)

.......................................................................................................................................................................................................................................................................................................................................................................

677CC

4.58 1.07

4.28 0.99

.0019

677CT

4.59 1.15

4.31 1.04

.0199

677TT

5.43 1.83

4.48 0.96

.0112

.......................................................................................................................................................................................................................................................................................................................................................................
.......................................................................................................................................................................................................................................................................................................................................................................

................................................................................................................................................................................................................................................................................................................................................................................
a

Thirty-five subjects missing information on serum folate level.

Forty-four subjects missing information on genotype

36 subjects missing information on serum homocysteine

Wen. Folic acid supplementation and the risk of preeclampsia. Am J Obstet Gynecol 2008.

rectly or indirectly by its effect on lowering blood homocysteine level.7,15-17

Endothelial dysfunction is demonstrable within the myometrial arteries of
women with preeclampsia, and the incubation of healthy vessels within plasma
obtained from women with preeclampsia induces similar endothelial changes.6
Young women with folate deficiency or
hyperhomocystinemia may be prone to
not only systemic endothelial dysfunction but also placental endovasculature.9
In our study, whether women initiated
folic acid supplementation before or after conception or whether the women
discontinued supplementation in the
third trimester, the rate of preeclampsia
was similarly lower than in those women
who did not have supplementation, suggesting that for preeclampsia prevention, folic acid supplementation in the
late first trimester or early second trimester, the most critical time window for

preeclampsia development, may be the

most important.
We did not observe an association between serum folate or plasma homocysteine level with preeclampsia. In largescale epidemiologic studies, it is difficult
to tightly control for factors that may affect the measured values of folate or homocysteine. Variations in gestational age
and timing at which blood samples were
taken, folate consumption by the growing fetus, and other physical and pathological processes that may have an impact on the metabolism of folate/
homocysteine during pregnancy may
introduce variability in the measurement of folate/homocysteine.18
It is rather difficult to distinguish
cause and effect for biomarkers that are
the intermediate steps of a health problem occurring in a rather short period of
time, such as preeclampsia. Most previous studies that observed an association

between serum folate and plasma homocysteine levels with preeclampsia collected blood sample in late gestation or at
delivery.19,20 For those studies that collected blood samples in the early second
trimester, the prediction of hyperhomocystinemia on preeclampsia was
poor.19,2,22
These findings suggest that variation
by gestational period is a major obstacle
in the study of the cause-and-effect relationship between a particular metabolite
and a particular pregnancy outcome. Increasing study samples to precisely measure gestation period specific values in
future studies may help determine the
association between metabolites and
pregnancy outcomes. Using biomarkers
with longer half-life biomarkers (eg, red
blood cell folate) may be helpful as well.
These markers measure not only the current status but also the previous status.
Because the development of specific

JANUARY 2008 American Journal of Obstetrics & Gynecology

45.e5

Research

Obstetrics

www.AJOG.org

TABLE 4

Occurrences of pregnancy complications according to folic acid supplementation status, serum folate level,
plasma homocysteine level, and MTHFR genotype, OaK Birth Cohort Study, October 2002-December 2005
Number of
subjects

Variables

Preeclampsia

ORs (95% CIa)

Folic acid supplementation

.......................................................................................................................................................................................................................................................................................................................................................................

No

238

12 (5.04)

Reference

Yes

2713

59 (2.17)

0.37 (0.18-0.75)

.......................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................

Supplementation of folic acid alone

.......................................................................................................................................................................................................................................................................................................................................................................

No supplementation

238

12 (5.04)

Reference

Yes

421

12 (2.85)

0.46 (0.16-1.31)

.......................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................

Initiation time of supplementation

.......................................................................................................................................................................................................................................................................................................................................................................

Before conception

1031

24 (2.33)

Reference

After conception

1682

35 (2.08)

1.04 (0.59-1.82)

.......................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................

Discontinuation of supplementation in late gestation

.......................................................................................................................................................................................................................................................................................................................................................................

Yes

544

14 (2.57)

Reference

No

2169

45 (2.07)

0.77 (0.41-1.46)

.......................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................
b

Serum folate level

.......................................................................................................................................................................................................................................................................................................................................................................

Normal (10th percentile for each gestational week)

2649

64 (2.42)

302

7 (2.32)

Reference

.......................................................................................................................................................................................................................................................................................................................................................................

Low (10th percentile for each gestational week)

1.22 (0.52-2.90)

................................................................................................................................................................................................................................................................................................................................................................................
c

Serum homocysteine level (mol/L)

.......................................................................................................................................................................................................................................................................................................................................................................

Normal (90th percentile for each gestational week)

2529

56 (2.21)

Reference

422

15 (3.55)

1.25 (0.66-2.36)

.......................................................................................................................................................................................................................................................................................................................................................................

High (90th percentile for each gestational week)

................................................................................................................................................................................................................................................................................................................................................................................
d

Genotype

.......................................................................................................................................................................................................................................................................................................................................................................

677CC

1282

29 (2.26)

Reference

677CT

1286

35 (2.72)

1.27 (0.75-2.15)

677TT

339

6 (1.77)

0.70 (0.28-1.77)

.......................................................................................................................................................................................................................................................................................................................................................................
.......................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................

OR, odds ratio.

a

Adjusted for maternal age, ethnic background, education level, parity, history of preeclampsia, chronic hypertension, diabetes, prepregnancy body mass index, household income, gestational age
at recruitment, and cigarette smoking.

Thirty-five subjects missing information on serum folate level.

Thirty-six subjects missing information on serum homocysteine.

Forty-four subjects missing information on genotype.

Wen. Folic acid supplementation and the risk of preeclampsia. Am J Obstet Gynecol 2008.

health problem is often a process rather

than a point effect, biomarkers of accumulation measures may be better measures of the cause-and-effect relationship than biomarkers of point measures.
Most women in our study had supplementation of multivitamins containing
folic acid. There are several reasons for us
to believe that folic acid may have played
a more important role in preeclampsia
than other vitamins. First, there is strong
biologic rationale to believe that folic
acid may reduce the risk of developing
preeclampsia. No similar biologic mechanisms could be found for other vita45.e6

mins. Second, recent randomized

controlled trials found that supplementation with vitamins C and E (without
folic acid) during pregnancy at doses
many times higher than the ones in our
study had no protective effect on preeclampsia.23,24 Third, in the subgroup of
women who had supplementation of folic acid alone (n 421), we did observe a
statistically nonsignificant trend toward
similar protective effect on preeclampsia. However, because the effect of supplementation by folic acid alone (odds
ratio, 0.46) was smaller than supplementation dominated by multivitamins con-

American Journal of Obstetrics & Gynecology JANUARY 2008

taining folic acid (odds ratio, 0.37), other

vitamins (eg, vitamin B6) may also play
some role in the prevention of
preeclampsia.
Our study has several weaknesses.
The sample sizes for women with no
supplementation or with supplementation at the doses of 1.0 mg or 1.0
mg folic acid or with folic acid alone
were small. As a result, we could not
find statistically significant association
of supplementation with folic acid
alone or perform a dose-response analysis. Although we adjusted for several
obstetric, socioeconomic, and lifestyle

Obstetrics

www.AJOG.org
factors in our analysis, residual confounding may exist. We did not collect
data on folic acid intake from food.
Women with folic acid supplementation were of a higher socioeconomic
status and therefore may have higher
folic acid from food intake. Ray and
Mamdani25 studied hospital discharge
data in the Canadian province of Ontario before (1990-1997) and after
(1998-2000) mandatory folic acid food
fortification and found no change in
the rate of preeclampsia, suggesting
that folic acid from food intake was too
low to make any impact on the risk of
preeclampsia.
The findings of our study and others
give hope of a new prevention strategy
for preeclampsia, which needs further
evaluation. Randomized, controlled trials could provide definitive evidence regarding the relationship between folic
acid supplementation and the risk of
preeclampsia. Findings from observational studies such as the current one can
help the design of future randomized trials (eg, when the supplementation
should be initiated and what dose should
be used, etc) and to establish the equipoise for future randomized trials.21 f
ACKNOWLEDGMENTS
We acknowledge the following people because
without their commitment and expertise, this
project would not have been possible: the
OMNI Research Nurse Group (Ottawa and Lucy
Chura, RN, BScN, study coordinator, Queens
Perinatal Research Unit research team
(Queens Perinatal Research Unit Kingston and
Lizy Kodiattu, MD); Dr. George Tawagi and
team (Ottawa HospitalCivic Campus); and
Carol Ann Jodouin, Department of Laboratory
Medicine, Ottawa Hospital.

REFERENCES
1. ACOG practice bulletin. Diagnosis and management of preeclampsia and eclampsia. Number 33, January 2002. American College of Ob-

stetricians and Gynecologists. Int J Gynaecol

Obstet 2002;77:67-75.
2. Wilson BJ, Watson MS, Prescott GJ, et al.
Hypertensive diseases of pregnancy and risk of
hypertension and stroke in later life: results from
cohort study. BMJ 2003;326:845.
3. Barker DJP. Fetal programming and public
health. In: OBrien PMS, Wheeler T, Barker
DJP, eds. Fetal programming: influences on development and disease in later life. London:
RCOG Press; 1999. p. 3-11.
4. Roberts JM, Speer P. Antioxidant therapy to
prevent preeclampsia. Semin Nephrol 2004;
24:557-64.
5. Bodnar LM, Tang G, Ness RB, Harger G,
Roberts JM. Periconceptional multivitamin use
reduces the risk of preeclampsia. Am J Epidemiol 2006;164:470-7.
6. Ashworth JR, Warren AY, Johnson IR, Baker
PN. Plasma from pre-eclamptic women and
functional change in myometrial resistance arteries. Br J Obstet Gynecol 1998;105:459-61.
7. Graham IM, Daly LE, Refsum HM, et al.
Plasma homocysteine as a risk factor for vascular disease. JAMA 1997;277:1775-81.
8. Ray JG, Laskin CA. Folic acid and homocysteine metabolic defects and the risk of placenta
abruption, pre-eclampsia and spontaneous
pregnancy loss: a systematic review. Placenta
1999;20:519-29.
9. Roberts JM, Taylor RM, Goldfein A. Clinical
and biochemical evidence of endothelial cell
dysfunction in the pregnancy syndrome preeclampsia. Am J Hypertens 1991;4:700-8.
10. Olthof MR, Bots ML, Katan MB, Verhoef P.
Effect of folic acid and betaine supplementation
on flow-mediated dilation: a randomized, controlled study in healthy volunteers. PLoS Clin
Trials 2006;1:e10.
11. Donnelly JG, Rock GA. Genetic determinants of heritable venous thrombosis: genotyping methods for factor V(Leiden)A1691G, methylenetetrahydrofolate
reductase
C677T,
prothrombin G20210A mutation, and algorithms for venous thrombosis investigations.
Clin Biochem 1999;32:223-8.
12. Health Canada. Canadian Perinatal Health
Report 2003. Ottawa, Canada: Ministry of Public Works and Government Services Canada;
2003.
13. Conde-Agudelo A, Belizan JM, Lammers C.
Maternal-perinatal morbidity and mortality associated with adolescent pregnancy in Latin
America: cross-section study. Am J Obstet Gynecol 2005;192:342-9.
14. Powers RW, Dunbar MS, Gallaher MJ,
Roberts JM. The 677 C-T methylenetetrahydro-

Research

folate reductase mutation does not predict

increased maternal homocysteine during
pregnancy. Obstet Gynecol 2003;101:762-6.
15. Anothiades C, Shirodaria C, Warrick N, Cai
S, de Bono J, Lee J. 5-Methyltetrahydrofolate
rapidly improves endothelial function and decreases superoxide production in human vessels: effects on vascular tetrahydrobiopterin
availability and endothelial nitric oxide synthase
coupling. Circulation 2006;114:1193-201.
16. Rimm EB, Willett WC, Hu FB, et al. Folate
and vitamin B6 from diet and supplements in
relation to risk of coronary heart disease among
women. JAMA 1998;279:359-64.
17. Title LM, Ur E, Giddens K, McQueen MJ,
Nassar BA. Folic acid improves endothelial dysfunction in type 2 diabetesan effect independent of homocysteine-lowering. Vasc Med
2006;11:101-9.
18. Walker MC, Smith GN, Perkins SL, Keely
EJ, Garner PR. Changes in homocysteine levels
during normal pregnancy. Am J Obstet Gynecol
1999;180:660-4.
19. Hogg BB, Tamura T, Johnston KE, Dubard
MB, Goldenberg RL. Second-trimester plasma
homocysteine levels and pregnancy-induced
hypertension, preeclampsia, and intrauterine
growth restriction. Am J Obstet Gynecol
2000;83:805-9.
20. Powers RW, Evans RW, Majors AK, et al.
Plasma homocysteine concentration is increased in preeclampsia and is associated with
evidence of endothelial activation. Am J Obstet
Gynecol 1998;179:1605-11.
21. Hietala R, Turpeinen U, Laatikainen T. Serum homocysteine at 16 weeks and subsequent preeclampsia. Obstet Gynecol 2001;
97:527-9.
22. Zuckerman B, Frank DA, Hingson R, et al.
Effects of maternal marijuana and cocaine use
on fetal growth. N Engl J Med 1989;320:762-8.
23. Poston L, Briley AL, Seed PT, Kelly FJ,
Shennan AH. Vitamins in pre-eclampsia (VIP)
Trial Consortium. Vitamin C and vitamin E in
pregnant women at risk for pre-eclampsia (VIP
trial): randomized placebo-controlled trial. Lancet 2006;367:1145-54.
24. Rumbold AR, Crowther CA, Haslam RR,
Dekker GA, Robinson JS. Vitamins C and E and
the risks of preeclampsia and perinatal complications. N Engl J Med 2006;354:1796-806.
25. Ray JG, Mamdani MM. Association between folic acid food fortification and hypertension or preeclampsia in pregnancy. Arch Intern
Med 2002;162:1776-7.

JANUARY 2008 American Journal of Obstetrics & Gynecology

45.e7