Epidemiology

DOI: 10.1111/j.1471-0528.2009.02150.x
www.blackwellpublishing.com/bjog

Intake of vitamin C and E in pregnancy and risk

of pre-eclampsia: prospective study among
57 346 women
AK Klemmensen,a,b A Tabor,c ML sterdal,a VK Knudsen,a TI Halldorsson,a TB Mikkelsen,a
SF Olsena,d
a
Maternal Nutrition Group, Danish Epidemiology Science Centre, Statens Serum Institut, Copenhagen, Denmark b Department of Obstetrics
and Gynaecology, Region-H Hvidovre Hospital, University of Copenhagen, Copenhagen, Denmark c Department of Fetal Medicine, Juliane
Marie Center, Region-H Rigshospitalet, Copenhagen, Denmark d Department of Nutrition, Harvard School of Public Health, Boston, MA,
USA
Correspondence: AK Klemmensen, MD, PhD, Department of Obstetrics and Gynaecology, Rigshospitalet, University of Copenhagen,
Blegdamsvej 9, DK-2100 Copenhagen , Denmark. Email klem@dadlnet.dk

Accepted 4 February 2009.

Objective It has been suggested that vitamin C, alone or in

combination with vitamin E, may protect against pre-eclampsia,

whereas the safety of high-dose vitamin E supplements has
been questioned. We investigated dietary intakes of vitamins C
and E to see if they correlated with the incidence of
pre-eclampsia.
Design Prospective cohort study.
Setting The Danish National Birth Cohort; a population-based

pregnancy cohort; analyses were based on 57 346 pregnancies.

Methods Vitamin intake was estimated from a food frequency
questionnaire completed in gestational week 25, recording
intake from diet and supplements during the previous four
weeks. Pre-eclampsia diagnoses were obtained from the Danish
National Patient Registry; we worked with two entities,
pre-eclampsia (all types) and severe pre-eclampsia/eclampsia/
HELLP. We adjusted for confounding factors by logistic
regression.

Results The incidence of pre-eclampsia (all types) did not

correlate with dietary vitamin C and E intake. There was a
decreasing trend (P = 0.01) in the incidence of severe preeclampsia/eclampsia/HELLP with increasing dietary vitamin C
intake; with an intake of 130170 mg/day as reference, odds ratios
ranged from 1.21 (95% confidence interval 0.83 to 1.75) for an
intake below 70 mg/day to 0.70 (0.40 to 1.23) for an intake
exceeding 275 mg/day (total n = 57 346). For vitamin E intake
aggregated from diet and supplements (n = 49 373), with an
intake of 10.513.5 mg/day as reference, the severe pre-eclampsia/
eclampsia/HELLP odds ratio was 1.46 (1.02 to 2.09) for an intake
exceeding 18 mg/day.
Conclusions Low dietary intake of vitamin C was associated

with a trend towards an increased incidence of either severe

pre-eclampsia, eclampsia or HELLP. A small increase in the
incidence of severe disease was also seen in the group of
women with a high intake of vitamin E from supplements and
dietary sources.

Main outcome measures A small increase in the incidence

Keywords Antioxidants, pre-eclampsia, prevention, vitamin C,

of severe disease was also seen in the group of women (64,

n = 49 373) with a high intake of vitamin E from supplements
and dietary sources.

vitamin E.

Please cite this paper as: Klemmensen A, Tabor A, sterdal M, Knudsen V, Halldorsson T, Mikkelsen T, Olsen S. Intake of vitamin C and E in pregnancy
and risk of pre-eclampsia: prospective study among 57 346 women. BJOG 2009;116:964974.

Introduction
Pre-eclampsia is one of the most important causes of
maternal and perinatal morbidity and mortality.1 Preeclampsia is a syndrome that belongs to the group of
hypertensive disorders of pregnancy, which is defined as
hypertension in pregnancy appearing after the 20th gesta-

964

tional week while the definition of pre-eclampsia also

includes proteinuria. Pre-eclampsia is a multi-organ disease
and can affect the kidney, liver, brain and the blood
clotting system.2,3 The severe complications associated
with pre-eclampsia are rare, although more frequent in
developing parts of the World, possibly because of insufficient antenatal care, ethnic variations and the fact that in

2009 The Authors Journal compilation RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology

Associations between antioxidants and pre-eclampsia

some areas preventive medication, such as antihypertensive

medicine and magnesium sulphate is scarce.4
The aetiology of pre-eclampsia is largely unknown, but
several theories have been put forward and several studies
suggest that oxidative stress may be involved in the pathogenesis.57 Whether intake of antioxidants can protect
against pre-eclampsia8,9 is less certain. Although a small
randomised controlled trial showed a lower occurrence of
pre-eclampsia in high-risk women who were supplemented
with high doses of the antioxidative vitamins C and E,10
recently, two large randomised studies combining high
doses of vitamin C and E showed no difference in the incidence of pre-eclampsia in the treated women compared to
the controls.11,12 There was also an increase in the rates of
low birthweight in the intervention group in the first of
these trials.11 On the other hand, an observational study
showed an increased risk of pre-eclampsia among women
with an intake of vitamin C below the US recommended
dietary allowance.9
We examined the relationship between estimated intakes
of the vitamins C and E and incidence of pre-eclampsia in
a large prospective cohort of pregnant women, the Danish
National Birth Cohort (DNBC).

participated in the first telephone interview and filled in

the FFQ. For analyses including information on food supplements, the study sample was reduced to 49 373 women,
as only the latter versions of the FFQ had available data on
supplement doses (Figure 1).
Data on vitamin C and E intake was extracted from the
FFQ. The amount of food intake (grams per day) was estimated using standard portion sizes and recipes. Data on
nutrient content in the food items were derived from the
Danish Food tables16 and intakes of energy and macronutrients and micronutrients were calculated using FoodCalc.17 Only FFQs with reported energy intake in the
interval between 4200 KJ (1,000 kcal) and 16 700 KJ
(3980 kcal) per day were included. Intake levels of vitamin
C and E were adjusted for total energy intake with the
residual method, as described by Willett et al.,18 as higher
energy intake may be linked to obesity, which itself is

Materials and methods

The DNBC enrolled 101 045 pregnant women from 1996
2002. Enrolment was primarily undertaken by general practitioners in week 610 of gestation and about 35% of the
total pregnant population was included.13 The Danish
Committee of Ethics and the Danish Data Protection
Agency approved the study. The women gave written
consent and were interviewed by telephone twice antenatally (overall response rate 92 and 86% respectively) and
again 6 and 18 months after delivery (overall response rate
70 and 62% respectively). The median (2.5th percentile,
97.5th percentile) gestational age at the interview was 16
(10, 27) completed weeks for the first antenatal interview
and 31 (26, 37) completed weeks for the second antenatal
interview. The questions covered a broad variety of topics
including data on elevated blood pressure and pre-eclampsia. Additionally, a self-administered food frequency questionnaire (FFQ) was mailed in gestational week 25,
recording frequencies of intake during the previous 4 weeks
for approximately 360 food and drink items. Intake of
dietary supplements (product name, number of daily
doses) during the past 4 weeks was recorded as well. The
questionnaire was a modified version of a questionnaire
used at the Danish Cancer Registry.14 The overall response
rate for the FFQ was 70%. Intakes estimated in the FFQ of
protein, n-3 fatty acids and folate have been validated.15
The main study population of 57 346 women consisted
of all singleton pregnancies in DNBC where the mother

Figure 1. Flowchart illustrating how the Danish National Birth Cohort

is reduced in accordance with the requirements for the adjusted logistic
regression analyses.

2009 The Authors Journal compilation RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology

965

Klemmensen et al.

associated with pre-eclampsia. By using the residual

method, variation in vitamin intake because of differences
in total energy intake is taken into account; the method is
described in detail elsewhere.18 Information on vitamin
contents in the various food supplement brands was
collected from the Danish Veterinary and Food Administration, the Danish Medicine Agency, the manufacturer or
the internet.
The definition of pre-eclampsia was in accordance with
the American College of Obstetricians2 and Gynecologists,
Royal College of Obstetricians and Gynaecologists and The
National obstetric guideline group in Denmark2,19 and is
shown in (Figure 2). All pre-eclampsia diagnoses were
obtained from the Danish National Patient Registry.
Subgroups of pre-eclampsia included severe pre-eclampsia,
Haemolysis, Elevated Liver enzymes and Low Platelet
count (HELLP) and eclampsia. A priori we had decided to
pool these subgroups into one group (in the following
referred to as severe pre-eclampsia/eclampsia/HELLP) and
study this as a separate entity and outcome, in addition to
studying the entity pre-eclampsia (all types). A large validation study (n = 3039), which we conducted in a subcohort of DNBC, found a specificity of 100% and a
sensitivity of 43.6% for severe pre-eclampsia/eclampsia/
HELLP; for pre-eclampsia (all types) the specificity was
99.3% and the sensitivity was 69.3%.20
Covariate information was extracted from the first and
second telephone interview. Parity was included as a

covariate since Primiparity is a risk factor for the development of pre-eclampsia.21,22 Smoking which is known to
protect against pre-eclampsia23 was included as a categorical variable (nonsmokers according to both interviews,
occasional smokers (either not smoking every day or quit
smoking from the first to the second interview), daily
smokers (<15 cigarettes per day), daily smokers (15 cigarettes per day reported in both interviews). If smoking
data was only available from the first interview, smoking
status was based on this information only. Maternal age
(years) at conception was included as a categorical variable
(20, 2139, 40).24 Maternal height (cm) has been shown
to influence the risk of pre-eclampsia25 and was divided
into quartiles (<165, 165168, 169172, 173). Heights
<140 cm were excluded. Body Mass Index (BMI) was
grouped as suggested by the National Board of Health,26
with reference to the World Health Organization recommendations.27 Socio-economic position was defined
according to the classification used by Statistics Denmark
called DISCO-88, a Danish version of the international
ISCO-88,28 which divides individuals into ten groups
according to their job description. Some of the groups
were collapsed, leaving a total of six groups, and those still
in education were placed between the group of skilled
workers and the group of unskilled workers rather than
below the group of unemployed. The womans social
status was coded as the maximum of her own and her
partners status (if the latter was available) and included

Figure 2. Definition of pre-eclampsia.

966

2009 The Authors Journal compilation RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology

Associations between antioxidants and pre-eclampsia

as a categorical variable. Additionally we adjusted for the

womens connubial position (single or cohabiting) and
also for their type of accommodation (rented, private residence and those without own accommodation (categorical
variable)). As casecontrol studies have suggested that
physical activity protects against pre-eclampsia, we also
adjusted for this factor (categorised as 0 minute/week,
144 minutes/week, 4574 minutes/week, 75149 minutes/
week, 150269 minutes/week, 270419 minutes/week, 420+
minutes/week); unexpectedly; however, we found that a
high physical activity was associated with increased risk of
severe pre-eclampsia, which justified further analyses
presented in another study.29

Statistical methods
Initially, total vitamin intake (diet plus supplements) was
divided into five intervals with approximately the same
number of women in each interval. The lower intake group
was furthermore separated into two groups, according to
whether the intake was above or below the Nordic Recommended Dietary Allowance (NRDA) for pregnant women
(70 mg/day for vitamin C and 7 mg/day for vitamin E
(Nordic nutrient recommendations 1996,30), giving a total

of six groups. The same cut-off points were used regarding

intake from diet only.
Multivariate logistic regression was used to assess confounder adjusted odds ratios comparing the vitamin intake
groups, the reference group was the third lowest intake
group. Vitamin intake was also modelled with restricted,
cubic splines31 (knots at the 5, 35, 65 and 95 percentiles).
Analyses of vitamin C intake were adjusted for intake of
vitamin E (as a categorical variable) and vice versa. Furthermore, adjustment was made for all other covariates.
Overall, we used generalised estimating equations (GEE,
Proc Genmod in sas 9.1 [SAS Institute Inc., Cary, NC,
USA]) with independent working correlation and robust
variance estimates to allow for inclusion of more than one
pregnancy in the same woman.

Results
Mean intakes of vitamin C and E from the diet (energyadjusted) were estimated to be 131.3 mg (SD 74) and
6.87 mg (SD 1.54) (Table 1). The mean intakes from supplements of all types were estimated as 85.6 mg (SD 116.2)
and 10.8 (SD 21.0) for vitamin C and E respectively

Table 1. Intake levels of vitamin C and E

Vitamin C
Mean
SD
Median

Dietary intake (energy

adjusted) n = 57 346

Intake from supplements n = 49 373

Dietary intake (energy

adjusted) + intake from
supplements
n = 49 373

131.3
74.0
113.2

85.6
116.2
70.0

217.3
139.2
188.9

mg
mg
mg
n

<70
)130
)170
)210
)275
>275
Vitamin E
Mean
SD
Median

mg
mg
mg
mg
mg
mg

9292
25 512
10 243
5160
4275
2864

mg
mg
mg

mg
mg
mg
mg
mg
mg

16.2
44.5
17.7
9
7.5
5

20 761
21 969
3131
1645
569
1298

42
44.5
6.3
3.3
1.2
2.6

1297
7388
10 929
10 068
9985
9706

2.6
15
22.1
20.4
20.2
19.7

6.87
1.54
6.72
n

<7
)10.5
)13.5
)15.5
)18
>18

33 451
22 696
993
125
38
43

10.8
21.0
7.0
%
58.3
39.6
1.7
0.2
0.1
0.1

17.7
21.1
14.4

11 706
30 574
265
317
502
6009

23.7
61.9
0.5
0.6
1
12.2

4759
4165
10 350
10 700
10 083
9316

9.6
8.4
21
21.7
20.4
19

2009 The Authors Journal compilation RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology

967

Klemmensen et al.

Table 2. Crude associations between covariates and pre-eclampsia and covariates and vitamin intake
Pre-eclampsia

Mothers age (years)

20
853
2139
55 946
40
547
Pre-pregnancy BMI (kg/m2)
18.5
2367
18.624.9
39 078
25.029.9
11 197
30.034.9
3418
35.039.9
937
40.0
349
Smoking in pregnancy
Non-smokers
43 531
Occasional smokers
7205
Daily smokers (<15 cig./day)
5597
Daily smokers (15 cig./day)
1013
Height (cm)
<165
13 352
165168
14 479
169172
14 278
173
15 237
Parity
Nullipara
28 286
Multipara
29 060
Socio-economic position
High level proficiencies
13 853
Medium level proficiencies
18 243
Skilled
15 522
Students
3017
Unskilled
5824
Unemployed
887
Ownership of residence
Owner
41 416
Rent
15 659
Without a residence
271
Marital status
Couple
56 367
Single
979
Physical activity (minutes/week)
0
35 670
1144
2861
4574
5331
75149
6560
150269
4515
270419
1596
420
813

Severe pre-eclampsia/
eclampsia/ HELLP

Mean

Cases

OR (95% CI)

Cases

OR (95% CI)

Dietary
vitamin C
intake (mg)*

Dietary
vitamin E
intake (mg)*

21
1442
24

0.95 (0.62,1.48)
1 (ref.)
1.73 (1.15,2.62)

7
327
3

1.41 (0.66,2.98)
1 (ref.)
0.94 (0.30,2.93)

124.1
131.3
140.3

6.31
6.87
7.51

33
802
363
196
57
36

0.67 (0.47,0.96)
1 (ref.)
1.60 (1.41,1.81)
2.90 (2.47,3.41)
3.09 (2.34,4.08)
5.49 (3.86,7.80)

9
209
68
37
10
4

0.71 (0.36,1.38)
1 (ref.)
1.14 (0.86,1.50)
2.04 (1.43,2.89)
2.01 (1.06,3.80)
2.16 (0.80,5.82)

128.0
133.2
129.0
123.4
119.2
113.6

7.22
6.94
6.67
6.61
6.58
6.67

1187
178
102
20

1 (ref.)
0.90 (0.77,1.06)
0.66 (0.54,0.81)
0.72 (0.46,1.12)

288
26
20
3

1 (ref.)
0.54 (0.36,0.81)
0.54 (0.34,0.85)
0.45 (0.14,1.39)

134.1
130.8
115.6
98.8

6.88
6.91
6.80
6.70

387
387
373
340

1 (ref.)
0.92 (0.80,1.06)
0.90 (0.78,1.04)
0.76 (0.66,0.89)

105
78
85
69

1 (ref.)
0.68 (0.51,0.92)
0.76 (0.57,1.01)
0.57 (0.42,0.78)

129.3
130.5
132.2
132.8

6.83
6.86
6.89
6.90

1074
413

2.74 (2.44,3.07)
1 (ref.)

263
74

3.68 (2.83,4.77)
1 (ref.)

141.5
121.4

6.82
6.92

1 (ref.)
(1.07,1.43)
(1.09,1.47)
(0.91,1.52)
(1.27,1.84)
(0.65,1.69)

67
108
95
19
44
4

1.23
1.27
1.30
1.57
0.93

1 (ref.)
(0.90,1.66)
(0.93,1.73)
(0.79,2.16)
(1.07,2.29)
(0.34,2.56)

139.9
133.2
125.6
132.6
120.5
123.1

7.06
6.90
6.74
6.82
6.71
6.87

1060
420
7

1 (ref.)
1.05 (0.94,1.18)
1.01 (0.48,2.14)

240
95
2

1 (ref.)
1.05 (0.82,1.33)
1.28 (0.32,5.16)

130.0
134.4
136.9

6.88
6.84
6.83

1462
25

1 (ref.)
0.98 (0.66,1.47)

333
4

1 (ref.)
0.69 (0.26,1.85)

131.3
131.1

6.87
6.94

1 (ref.)
(0.80,1.30)
(1.01,1.42)
(0.86,1.20)
(0.79,1.18)
(0.74,1.39)
(0.90,1.95)

200
10
35
37
24
18
13

0.62
1.17
1.01
0.95
2.02
2.88

1 (ref.)
(0.33,1.18)
(0.82,1.68)
(0.71,1.43)
(0.62,1.45)
(1.25,3.29)
(1.64,5.07)

125.7
137.3
136.7
139.6
143.5
148.2
151.2

6.83
6.88
6.86
6.94
7.01
6.97
7.18

298
483
421
76
189
20

904
74
161
169
111
41
27

1.24
1.27
1.18
1.53
1.05

1.02
1.20
1.02
0.97
1.01
1.32

For pre-eclampsia associations, generalised estimating equations with independent working correlation were used. n = 57 346.
*Energy adjusted with the residual method.

968

2009 The Authors Journal compilation RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology

Associations between antioxidants and pre-eclampsia

Table 3. Associations between risk of pre-eclampsia and intake levels of vitamin C and E
Pre-eclampsia
Cases n

Vitamin C from diet (mg)

70
)130
)170
)210
)275
>275
Trend test**
Test for curvature***
Test for overall significance****
Test for linear trend*****
Vitamin E from diet (mg)
7
)10.5
)13.5
>13.5
Trend test**
Test for curvature***
Test for overall significance****
Test for linear trend*****
Total vitamin C intake (mg)
70
)130
)170
)210
)275
>275
Trend test**
Test for curvature***
Test for overall significance****
Test for linear trend*****
Total vitamin E intake (mg)
7
)10.5
)13.5
)15.5
)18
>18
Trend test**
Test for curvature***
Test for overall significance****
Test for linear trend*****

Severe pre-eclampsia/eclampsia/ HELLP

OR (95% CI)
crude

OR (95% CI)
adjusted*

255
626
253
128
129
96

1.11 (0.93,1.33)
0.99 (0.86,1.15)
1 (ref.)
1.00 (0.81,1.25)
1.23 (0.99,1.52)
1.37 (1.08,1.73)

1.17 (0.98,1.41)
1.03 (0.89,1.20)
1 (ref.)
0.97 (0.78,1.20)
1.14 (0.92,1.42)
1.18 (0.93,1.51)
P = 0.91
P = 0.03/P = 0.05
P = 0.06/P = 0.09
P = 0.80/P = 0.56

872
593
20
2

1.30 (0.83,2.04)
1.31 (0.83,2.05)
1 (ref.)
0.48 (0.11,2.06)

32
193
276
261
253
279

126
111
270
287
236
264

Cases n

OR (95% CI)
crude

OR (95% CI)
adjusted*

58
150
62
31
21
15

1.03 (0.72,1.48)
0.97 (0.72,1.31)
1 (ref.)
0.99 (0.64,1.53)
0.81 (0.49,1.33)
0.86 (0.49,1.52)

1.21 (0.83,1.75)
1.08 (0.80,1.45)
1 (ref.)
0.92 (0.60,1.42)
0.72 (0.44,1.19)
0.70 (0.40,1.23)
P = 0.01
P = 0.71/P = 0.58
P = 0.04/P = 0.04
P = 0.01/P = 0.004

1.20 (0.77,1.88)
1.34 (0.85,2.11)
1 (ref.)
0.45 (0.10,1.96)
P = 0.33
P = 0.29/P = 0.71
P = 0.44/P = 0.74
P = 0.53/P = 0.40

181
149
6
1

0.89 (0.40,1.02)
1.09 (0.48,2.47)
1 (ref.)
0.80 (0.10,6.70)

0.78 (0.34,1.77)
1.07 (0.47,2.43)
1 (ref.)
0.70 (0.08,5.76)
P = 0.01
P = 0.51/P = 0.52
P = 0.23/P = 0.23
P = 0.05/P = 0.08

0.98 (0.67,1.43)
1.04 (0.86,1.25)
1 (ref.)
1.03 (0.87,1.22)
1.00 (0.84,1.19)
1.14 (0.97,1.35)

0.98 (0.64,1.49)
1.05 (0.86,1.28)
1 (ref.)
1.01 (0.85,1.20)
0.95 (0.80,1.13)
1.01 (0.85,1.21)
P = 0.75
P = 0.83/P = 0.34
P = 0.94/P = 0.53
P = 0.90/P = 0.86

9
48
65
66
53
60

1.17 (0.58,2.35)
1.09 (0.75,1.59)
1 (ref.)
1.10 (0.78,1.55)
0.89 (0.62,1.28)
1.04 (0.73,1.48)

1.27 (0.58,2.79)
1.14 (0.76,1.70)
1 (ref.)
1.06 (0.75,1.49)
0.80 (0.56,1.15)
0.85 (0.59,1.21)
P = 0.08
P = 0.05/P = 0.16
P = 0.11/P = 0.13
P = 0.64/P = 0.20

1.02 (0.82,1.26)
1.02 (0.81,1.28)
1 (ref.)
1.03 (0.87,1.22)
0.89 (0.75,1.07)
1.09 (0.92,1.29)

1.15 (0.90,1.48)
1.28 (1.01,1.63)
1 (ref.)
1.09 (0.92,1.29)
1.00 (0.84,1.20)
1.19 (1.00,1.42)
P = 0.86
P = 0.12/P = 0.71
P = 0.09/P = 0.87
P = 0.14/P = 0.90

30
29
56
69
53
64

1.17 (0.75,1.82)
1.29 (0.82,2.02)
1 (ref.)
1.19 (0.84,1.70)
0.97 (0.67,1.42)
1.27 (0.89,1.82)

1.26 (0.76,2.10)
1.53 (0.95,2.47)
1 (ref.)
1.27 (0.89,1.82)
1.10 (0.75,1.61)
1.46 (1.02,2.09)
P = 0.53
P = 0.45/P = 0.65
P = 0.60/P = 0.35
P = 0.27/P = 0.20

Vitamin intake is measured either as intake from diet alone (n = 57 346) or as total intake (diet plus supplements, n = 49 373). Generalised estimating equations with independent working correlation were used.
*Adjusted for maternal age, pre-pregnancy BMI, smoking, height, parity, socio-economic position, ownership of residence, marital status, physical
activity and intake level of either vitamin C or vitamin E.
**Intake levels coded 0,1,2,3,4,5 (vitamin C) or coded 0,1,2,3 (vitamin E).
***Spline model (restricted, cubic splines, knots at 5, 35, 65, 95 percentiles) versus linear model. Original scale/log scale.
****Spline model versus model including confounders only. Original scale/log scale.
*****Linear model versus model including confounders only. Original scale/log scale.

2009 The Authors Journal compilation RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology

969

Klemmensen et al.

Table 4. Associations between risk of pre-eclampsia and intake levels of fruit and selenium
Pre-eclampsia (all subgroups)
Cases n

OR (95% CI)
adjusted*

Fruit intake (g/day)

Quintile 1
Quintile 2
Quintile 3
Quintile 4
Quintile 5
1 (ref.)
Fruit minus citrus (g/day)
Quintile 1
Quintile 2
Quintile 3
Quintile 4
Quintile 5
1 (ref.)
Selenium intake (mg/day), energyadjusted
Quintile 1
Quintile 2
Quintile 3
Quintile 4
Quintile 5
1 (ref.)

Severe pre-eclampsia/eclampsia/ HELLP

OR (95% CI)
adjusted**

1.19
0.95
1.07
1.15

1.07
1.06
0.93
1.11

0.96
0.86
0.87
0.99

Cases n

(0.99,1.42)
(0.79,1.14)
(0.89,1.28)
(0.97,1.36)
1 (ref.)
(0.90,1.27)
(0.89,1.26)
(0.78,1.10)
(0.94,1.31)
1 (ref.)
(0.81,1.13)
(0.73,1.02)
(0.74,1.03)
(0.84,1.16)
1 (ref.)

OR (95% CI)
adjusted*

OR (95% CI)
fruit adjusted**

1.28
0.96
1.12
1.41

(0.86,1.90)
(0.64,1.44)
(0.75,1.65)
(1.00,2.00)
1 (ref.)

1.02
1.06
0.82
1.09

(0.71,1.46)
(0.75,1.50)
(0.56,1.18)
(0.79,1.52)
1 (ref.)

0.85
0.98
0.79
0.91

(0.61,1.19)
(0.71,1.36)
(0.56,1.12)
(0.65,1.26)
1 (ref.)

1 (ref.)

1 (ref.)

1 (ref.)

n = 57 346. Generalised estimating equations with independent working correlation were used.
*Adjusted for maternal age, pre-pregnancy BMI, smoking, height, parity, socio-economic position, ownership of residence, marital status, physical
activity.
**Adjusted for maternal age, pre-pregnancy BMI, smoking, height, parity, socio-economic position, ownership of residence, marital status, physical activity and dietary intake of vitamin C and E.

(Table 1). When nutrient contributions from diet and supplements were aggregated, 2.6% (1297) and 9.6% (4759) of
the women had an intake of vitamin C and E below the
NRDA, respectively (Table 1). Only 0.52% of the women
had an intake of vitamin C at or above 1000 mg and only
two women had a vitamin E intake above 600mg, which
were the amounts given in recent trials.1012 Mean energy
intake was 10 269 kJ/day (SD 2431) (2454 kcal/day (SD
581)).
Dietary vitamin intake was clearly associated with age,
pre-pregnancy BMI, smoking, parity and socio-economic
status, whereas associations with mothers height, ownership of residence and marital status were weaker or nonexistent (Table 2). In univariate analyses, several of these
factors were associated with an increased incidence of preeclampsia and severe pre-eclampsia/eclampsia/HELLP. This
applied particularly to primiparity, non-smoking, low
height and pre-pregnant overweight or obesity (Table 2).
In the main study population of 57 346 women, there were
1487 (2.6%) cases of pre-eclampsia and 337 (0.6%) cases
of severe pre-eclampsia/eclampsia/HELLP.
When all types of pre-eclampsia were included as the
outcome variable, there was no evidence of an elevated risk
with decreasing levels of vitamin C intake. For severe

970

pre-eclampsia/eclampsia/ HELLP, there was a trend indicating a protective effect of dietary intake of vitamin C
(OR ranging from 1.21(0.83,1.75) to 0.70(0.40,1.23), trend
test P = 0.01) (Table 3). This trend became less clear,
when intake of vitamin C from supplements was included
(OR ranging from 1.27(0.58,2.79) to 0.85(0.59,1.21), trend
test P = 0.08) (Table 3). For total vitamin E intake, high
levels (>18 mg/day) were associated with a significantly
increased incidence of all types of pre-eclampsia (OR 1.19
(1.00, 1.42)) and an increased incidence of severe
pre-eclampsia/eclampsia/HELLP (OR 1.46 (1.02, 2.09))
(Table 3).
The trend in risk of severe pre-eclampsia/eclampsia/
HELLP versus intake levels of vitamin C from diet did not
depend on parity (interaction test P = 0.38) or BMI (interaction test with BMI dichotomised at 25.0 yielded a
P-value of 0.90). Potential interaction between vitamin C
and E was also explored; when total intake of vitamin C
and E were dichotomised at 275 mg and 18 mg respectively, there was no significant interaction between the two
vitamins, either for pre-eclampsia as a whole (P = 0.69) or
severe pre-eclampsia/eclampsia/HELLP (P = 0.90). When
dietary intake of vitamin C and E were dichotomised at
275 mg and 10.5 mg respectively, there was also no signifi-

2009 The Authors Journal compilation RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology

Associations between antioxidants and pre-eclampsia

cant interaction (P = 0.70 for pre-eclampsia as a whole,

P = 0.72 for severe pre-eclampsia/eclampsia/HELLP).
We made adjustments for both fruit and for fruits minus
citrus fruits (because citrus fruits are the main contributors
of vitamin C in the diet), which is shown in Table 4. Our
conclusion was that neither the adjustment for fruit nor
the adjustment for selenium intake (Tables S1 and S2 ) can
explain the trend of association that we observe with vitamin C.
The exclusion of women who in the first antenatal interview reported that they had been diagnosed earlier with
hypertension or diabetes did not affect the observed associations.

Discussion
Our data could not confirm that a simultaneous, high
intake of both vitamin C and E reduces the risk of preeclampsia and this finding agrees with Poston et al.s recent
trial and Rumbold et al.s trial.11,12 Conversely, our data
suggest that, within the intake range of Danish women, the
risk of severe pre-eclampsia/eclampsia/HELLP may decrease
with increasing intake of vitamin C.
Strengths of our study include its size, its prospective
design and the general quality of data. The DNBC is so far
the largest birth cohort where dietary intake has been
assessed in pregnancy and the assessment was performed
prior to the typical onset of pre-eclampsia.
We have previously found good validity of estimated
intakes of some other vitamins (folate and retinol).32 The
FFQ was evaluated with a 7-day-weighed food diary and
the correlation ranged from 0.20 for retinol intake to 0.57
for folic acid intake. Moreover, the erythrocyte folate correlated significantly with the estimated total intake from
the FFQ (r = 0.55, P < 0.0001). This suggests that our estimations of vitamin C and E intake also have acceptable
validity.
Selection bias is always a potential problem in a study
such as this and may arise at recruitment or follow up.
During the recruitment period, approximately 35% of all
pregnant women in Denmark were enrolled into the
DNBC.13 Women who were enrolled were likely to have a
higher educational level and socio-economic status and
therefore perhaps a lower risk adverse pregnancy outcomes
such as pre-eclampsia. Such selection bias is likely to have
resulted in narrower exposure distributions and consequently lower statistical power. The prospective design of
this study should reduce this selection bias, however, as it
does not seem plausible that the likelihood of a woman
enrolling in the study in her first trimester is simultaneously associated with her vitamin intake in midpregnancy
and her risk of developing pre-eclampsia in the latter half
of pregnancy.

The use of a clinical diagnosis of pre-eclampsia in large

cohort studies of this sort is not ideal. We considered
undertaking a casecontrol study where clinical charts for
cases and controls were reviewed to confirm the diagnosis,
but we were unable to accomplish this because we would
have needed access to patient records from all hospitals in
the country. Instead we elected to review clinical charts for
3084 women who were consecutively recruited to the
DNBC and who delivered at three hospitals (different sizes)
in Denmark. The conclusion was that information available
from the national patient registry had a specificity and
sensitivity, which could justify the use of this source to
identify cases with pre-eclampsia or the severe pre-eclampsia/eclampsia/HELLP.20 Given that misclassification of the
pre-eclampsia related diagnoses is non-differential relative
to the exposures we have studied here, that is vitamin C
and E, this misclassification would tend to weaken any true
association, but would not create spurious associations. We
adjusted the analysis for a number of potential confounding factors, such as maternal smoking, parity and body
mass index. We were also able to adjust for some specific
nutritional factors; neither fruit nor selenium intake could
explain the associations that we observed with vitamin C
intake.
Our data indicated a decline in the incidence of severe
pre-eclampsia/eclampsia/HELLP across the intake range of
vitamin C. As we employed food frequency methodology,
absolute nutrient intakes should be interpreted with care.
Nevertheless, our findings are compatible with the results
of the casecontrol study by Zhang et al.,9 who observed
an increased risk of pre-eclampsia among women with
intakes below 85 mg vitamin C per day, which defines the
lower border of the recommended dietary intake for pregnant women in the US. They included 109 cases and 259
controls. At the time of birth, they collected information
on nutrition, reflecting the previous 12 months food
intake and measured plasma ascorbic acid. The latter confirmed that low plasma ascorbic acid, presumably reflecting
a low intake of vitamin C, is associated with an increased
risk of pre-eclampsia.
The optimal gestation of pregnancy at which to measure vitamin C intake is uncertain; although as trophoblast invasion is completed by the second half of
pregnancy, information collected around this gestation
will reflect vitamin C intake during this important period
of placental development. It is not generally considered
that the time at which placentation is complete is the
gestational time-point most likely to be associated with
oxidative stress.33
We had the opportunity to estimate the intake during
this critical period of pregnancy by using data on the
nutrients with antioxidative characteristics, collected in gestational week 2025. We can only speculate whether our

2009 The Authors Journal compilation RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology

971

Klemmensen et al.

information on vitamin intake in this period of gestation

also reflects the intake previously and thereby is present at
the time of early placentation.34
Antioxidants were consumed at a much lower level by
the women in our study population compared to the
doses used in the randomised controlled trial by Chappell et al.10 They proposed a protective effect on the risk
of developing pre-eclampsia by giving 1000 mg vitamin
C in combination with 400 iu (268 mg) vitamin E. It
was also suggested that there may be an interactive effect
from the two vitamins, which has been show in other
cardio-vascular studies.35 Our results did not support a
positive interactive effect between vitamin C and E.
Indeed, the same group and others could not reproduce
these findings in later studies using similar protocols.11,12,36 In our cohort of 49 373 pregnant women,
there were too few women, who consumed such large
doses of even one of the vitamins to assess this effect.
However, we made an attempt to evaluate whether the
higher amounts, which could be reasonably studied in
our data (above 275 mg vitamin C per day and above
18 mg vitamin E per day), were associated with a lower
incidence of pre-eclampsia. We were not able to detect
any such association and thus not able to support the
contention of a beneficial interaction of high doses of
these two nutrients in relation to pre-eclampsia risk.
An explanation for the increased incidence of severe preeclampsia/eclampsia/HELLP among women with a low
vitamin C intake would be entirely speculative. Whether
inadequate intake of vitamin C is enough to compromise
maternal anti-oxidant defence or, through oxidative stress,
to heighten maternal inflammatory responses is not known.
The multifactorial origin of this syndrome suggests that it
might be both.37 An explanation of the increased incidence
of severe pre-eclampsia/eclampsia/HELLP among women
with a high intake of vitamin E deriving from diet and
food supplements would be equally speculative. However,
it is noteworthy that recent evidence suggests that supplementation with high doses of vitamin E may increase the
risk of cardiovascular diseases and even be associated with
an increased risk of mortality.38
The public health implications of our findings are that
the data substantiate the current dietary recommendations
with respect to intakes of vitamin C in pregnancy. Our
findings may also indicate that greater caution is needed
when issuing recommendations regarding the consumption
of vitamin supplements in pregnancy, as high doses of
some vitamins may be deleterious.
One advantage of a large data set like ours is that it will
have a high statistical power to detect associations that
might exist. However, precision of exposure and outcome
measures in cohorts of this size are never ideal and,
because of the large size of the data set, even relatively

972

weak associations, which may be clinically unimportant or

spuriously created by some source of bias may become
significant. Indeed, the associations that we observed are
not strong.

Conclusions
Among 57 346 women from The Danish National Birth
Cohort, we had information on relevant confounders and
data on womens vitamin C and E intake over a 4-weekperiod in midpregnancy. Although overall there was no
correlation of vitamin C intake with the incidence of
pre-eclampsia, when the severe complications associated
with pre-eclampsia were considered, there was a trend
towards an increased incidence in these conditions in
women with low dietary vitamin C intake. We saw no such
trend for vitamin E; on the contrary, the incidence of severe
pre-eclampsia, HELLP or eclampsia was unexpectedly
increased in women consuming high amounts of vitamin E.

Disclosure of interests
There are no conflicts of interest (financial, personal, political, intellectual or religious interests).

Contribution to authorship
Ase K Klemmensen: Main author, contributed with validation of pre-eclampsia data from patient files and the general maintenance of data, contributed with main parts of
the text and revising the final paper.
Marie Louise sterdal: Main statistical management and
the general maintenance of data, contributed with parts of
the text and revising the final paper.
Ann Tabor: Contributed with parts of the text and revising the final paper.
Vibeke Kildegaard Knudsen: Contributed nutritional
expertise and wrote specific parts of the text.
Thorhallur Ingi Halldorsson: Statistical support and
management of the data sets.
Tina Broby Mikkelsen: Contributed nutritional
expertise.
Sjurdur Frodi Olsen: Contributed nutritional and epidemiological expertise and wrote parts of the text and
contributed with revising the final paper. Initiated the
study. Guarantor of study.

Details of ethics approval

Details of ethics approval was obtained from The Danish
National Committee on Biomedical Research Ethics and
The Danish Data Protection Agency.

Funding
The study was supported by grants from the Danish Hospital Foundation for Medical Research in the Region of

2009 The Authors Journal compilation RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology

Associations between antioxidants and pre-eclampsia

Copenhagen, The Faeroe Islands and Greenland, the

Copenhagen Medical Society and by a grant from the University of Copenhagen. In addition the Maternal Nutrition
Group of the Danish National Birth Cohort has been supported by the March of Dimes Birth Defects Foundation
(6-FY-96-0240, 6-FY97-0553, 6-FY97-0521, 6-FY00-407),
EU (QLK1-CT-2000-00083), Danish National Research
Foundation, Danish Medical Research Council (9601842
and 22-03-0536), Danish Health Foundation (11/263-96)
and Danish Heart Foundation (96-2-4-83-22450). Financial
support for DNBC was obtained from the Danish National
Research Foundation, Pharmacy Foundation, Ergmont
Foundation and Augustinus Foundation.

Acknowledgements
The study was supported by grants from The Danish Hospital Foundation for Medical Research in the Region of
Copenhagen, The Faeroe Islands and Greenland, the
Copenhagen Medical Society and by a grant from The
University of Copenhagen. In addition the Maternal Nutrition Group of the Danish National Birth Cohort has been
supported by the March of Dimes Birth Defects Foundation (6-FY-96-0240, 6-FY97-0553, 6-FY97-0521, 6-FY00407), EU (QLK1-CT-2000-00083), Danish National Research Foundation, Danish Medical Research Council
(9601842 and 22-03-0536), Danish Health Foundation (11/
263-96) and Danish Heart Foundation (96-2-4-83-22450).
Financial support for DNBC was also obtained from the
Danish National Research Foundation, Pharmacy Foundation, Egmont Foundation and Augustinus Foundation. The
Managerial Team of The Danish National Birth Cohort
consists of Jrn Olsen (Chair), Mads Melbye, Anne Marie
Nybo Andersen, Sjurdur F Olsen, Thorkild I.A. Srensen
and Peter Aabye.

Supporting information
The following supplementary materials are available for this
article:
Table S1. Sensitivity analysis. Women with chronic
hypertension and diabetes mellitus excluded. Associations
between risk of preeclampsia and intake levels of vitamin C
and E. Vitamin intake is measured either as intake from
diet alone (n = 56 448) or as total intake (diet plus supplements, n = 48 604). Generalised esimating equations with
independent working correlation were used.
Tables S2. Associations between risk of preeclampsia and
intake levels of vitamin C. Vitamin intake is measured either
as intake from diet alone (n = 57 346) or as total intake
(diet plus supplements, n = 49 373). Generalised esimating
equations with independent working correlation were used.
Additional Supporting Information may be found in the
online version of this article.

Please note: Wiley-Blackwell are not responsible for the

content or functionality of any supporting information
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author. j

References
1 Roberts JM, Speer P. Antioxidant therapy to prevent preeclampsia.
Semin Nephrol 2004;24:55764.
2 ACOG practice bulletin. Diagnosis and management of preeclampsia
and eclampsia. Number 33, January 2002. American College of
Obstetricians and Gynecologists. Int J Gynaecol Obstet 2002;77:67
75.
3 Roberts JM, Pearson GD, Cutler JA, Lindheimer MD. Summary of
the NHLBI Working Group on research on hypertension during pregnancy. Hypertens Pregnancy 2003;22:10927.
4 Geographic variation in the incidence of hypertension in pregnancy.
World Health Organization International Collaborative Study of
Hypertensive Disorders of Pregnancy. Am J Obstet Gynecol
1988;158:803.
5 Poston L, Chappell LC. Is oxidative stress involved in the aetiology of
pre-eclampsia? Acta Paediatr Suppl 2001;90:35.
6 Hubel CA. Oxidative stress in the pathogenesis of preeclampsia. Proc
Soc Exp Biol Med 1999;222:22235.
7 Raijmakers MT, Dechend R, Poston L. Oxidative stress and preeclampsia: rationale for antioxidant clinical trials. Hypertension
2004;44:37480.
8 Chappell LC, Seed PT, Kelly FJ, Briley A, Hunt BJ, Charnock-Jones
DS, et al. Vitamin C and E supplementation in women at risk of
preeclampsia is associated with changes in indices of oxidative stress
and placental function. Am J Obstet Gynecol 2002;187:77784.
9 Zhang C, Williams MA, King IB, Dashow EE, Sorensen TK, Frederick
IO, et al. Vitamin C and the risk of preeclampsiaresults from dietary
questionnaire and plasma assay. Epidemiology 2002;13:40916.
10 Chappell LC, Seed PT, Briley AL, Kelly FJ, Lee R, Hunt BJ, et al. Effect
of antioxidants on the occurrence of pre-eclampsia in women at
increased risk: a randomised trial. Lancet 1999;354:8106.
11 Poston L, Briley AL, Seed PT, Kelly FJ, Shennan AH. Vitamin C and
vitamin E in pregnant women at risk for pre-eclampsia (VIP trial):
randomised placebo-controlled trial. Lancet 2006;367:114554.
12 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;354:1796806.
13 Olsen J, Melbye M, Olsen SF, Sorensen TI, Aaby P, Andersen AM,
et al. The Danish National Birth Cohortits background, structure
and aim. Scand J Public Health 2001;29:3007.
14 Overvad K, Tjonneland A, Haraldsdottir J, Ewertz M, Jensen OM.
Development of a semiquantitative food frequency questionnaire to
assess food, energy and nutrient intake in Denmark. Int J Epidemiol
1991;20:9005.
15 Mikkelsen TB, Osler M, Olsen SF. Validity of protein, retinol, folic
acid and n-3 fatty acid intakes estimated from the food-frequency
questionnaire used in the Danish National Birth Cohort. Public
Health Nutr 2006;9:7718.
16 The Composition of Foods. The Danish Food Administration, 2000.
17 Foodcalc. ibtku. 2005 [www.ibt.ku.dk/jesper/foodcalc/]. Accessed 26
March 2009.
18 Willett WC, Howe GR, Kushi LH. Adjustment for total energy intake
in epidemiologic studies. Am J Clin Nutr 1997;65(Suppl):1220S8S.
19 The Danish Society of Obstetrics and Gynaecology (DSOG). Hypertension hos gravide. 2003 [www.dsog.dk/files/hypertension_%20
hos_gravide.pdf]. Accessed 26 March 2009.

2009 The Authors Journal compilation RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology

973

Klemmensen et al.

20 Klemmensen AK, Olsen SF, sterdal ML, Tabor A. Validity of diagnoses related to preeclampsia based on two registries and a review of
3039 patient charts. Am J Epidemiol 2007;166:11724.
21 Dekker G, Sibai B. Primary, secondary, and tertiary prevention of
pre-eclampsia. Lancet 2001;357:20915.
22 Saftlas AF, Levine RJ, Klebanoff MA, Martz KL, Ewell MG, Morris
CD, et al. Abortion, changed paternity, and risk of preeclampsia in
nulliparous women. Am J Epidemiol 2003;157:110814.
23 Xiong X, Wang FL, Davidge ST, Demianczuk NN, Mayes DC, Olson
DM, et al. Maternal smoking and preeclampsia. J Reprod Med
2000;45:72732.
24 Esplin MS, Fausett MB, Fraser A, Kerber R, Mineau G, Carrillo J,
et al. Paternal and maternal components of the predisposition to
preeclampsia. N Engl J Med 2001;344:86772.
25 Basso O, Wilcox AJ, Weinberg CR, Baird DD, Olsen J. Height and risk
of severe pre-eclampsia. A study within the Danish National Birth
Cohort. Int J Epidemiol 2004;33:85863.
26 SST. BMI recommendations from Sundheds Styrrelsen. Sundheds Styrrelsen. 2005 [www.sst.dk/Forebyggelse/Mad_og_motion/Overvaegt/
Fakta_om_overvaegt.aspx?lang=da]. Accessed 26 March 2009.
27 WHO. BMI WHO definition. 2005 [www.euro.who.int/nutrition/
20030507_1]. Accessed 26 March 2009.
28 ISCO-88. [http://ideas.repec.org/p/oec/elsaaa/20-en.html]. Accessed
26 March 2009.
29 Osterdal ML, Strom M, Klemmensen AK, Knudsen VK, Juhl M,
Halldorsson TI, et al. Does leisure time physical activity in early
pregnancy protect against pre-eclampsia? Prospective cohort in
Danish women. BJOG 2009;116:98107.

974

30 Nordiska naringsrekommendationer. In: Sandstrom B, editor. Nordiska Ministerradet, 1996.

31 Durrleman S, Simon R. Flexible regression-models with cubic-splines.
Stat Med 1989;8:55161.
32 Mikkelsen TB. Validation of the Food frequency Questionnaire used
in the Danish national Birth Cohort. Unpublished PhD Thesis,
76100, Maternal Nutrition Group, Danish Epidemiology Science
Center, Statens Serum Institut, Copenhagen, 2004.
33 Jauniaux E, Poston L, Burton GJ. Placental-related diseases of pregnancy: involvement of oxidative stress and implications in human
evolution. Hum Reprod Update 2006;12:74755.
34 Sebire NJ, Goldin RD, Regan L. Term preeclampsia is associated with
minimal histopathological placental features regardless of clinical
severity. J Obstet Gynaecol 2005;25:1178.
35 Hamilton K. Antioxidantes and cardioprotection. Med Sci Sports
Exerc 2007;39:154453.
36 Beazley D, Ahokas R, Livingston J, Griggs M, Sibai BM. Vitamin C
and E supplementation in women at high risk for preeclampsia: a
double-blind, placebo-controlled trial. Am J Obstet Gynecol
2005;192:5201.
37 Chaouat G, Ledee-Bataille N, Zourbas S, Dubanchet S, Sandra O,
Martal J, et al. Implantation: can immunological parameters of
implantation failure be of interest for pre-eclampsia? J Reprod
Immunol 2003;59:20517.
38 Miller ER III, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ,
Guallar E. Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann Intern Med 2005;142:
3746.

2009 The Authors Journal compilation RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology