Preeclampsia is a common complication of pregnancy affecting 3%–10% of pregnancies worldwide.1 The etiology is unknown. Since preeclampsia is characterized by reduced perfusion of the placenta, oxidative stress, and endothelial dysfunction, numerous nutritional targets for intervention have been suggested.2 One controlled trial of supplementation with antioxidants among high-risk women showed a protective effect,3 but the finding could not be replicated in another controlled trial.4 Vitamin D has immunosuppressive effects and may influence the expression of target genes for implantation.5 It has been reported that the serum concentration of 25(OH)D in early pregnancy is reduced in women who subsequently develop preeclampsia.6 1,25(OH)2D may play a key role in maintaining immunologic tolerance in pregnancy, and adequate vitamin D may help in the prevention and management of preeclampsia.7
Protective effects of the long chain n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on preeclampsia development have also been suggested.8–10 Some studies suggest a certain beneficial effect of the long chain n-3 fatty acids,9–11 whereas other studies have found that these nutrients may increase the risk of preeclampsia.12,13 Unfortunately, the intake of the long chain n-3 fatty acids is highly correlated with the intake of vitamin D in the Norwegian diet because of frequent use of cod liver oil as a food supplement.14 Despite this inherent difficulty, our aim is to estimate the association between the intake of vitamin D (in the ordinary diet and as supplements) and preeclampsia in primiparous women, using a prospective design.
METHODS
Subjects
The data set is part of the Norwegian Mother and Child Cohort Study, initiated by and maintained at the Norwegian Institute of Public Health. This study uses version 3 of the data files made available for research in 2007. In brief, this is a nationwide pregnancy cohort that has recruited women since 1999 with the aim of including at least 100,000 pregnancies by 2008.15 Pregnant women are asked to participate through a postal invitation after they sign up for routine ultrasound examination in their local hospital. Participation rate is about 43%.15 The women are asked to provide biologic samples and to answer 3 questionnaires during pregnancy, in gestational week 15 (Q1), week 22 (Q2), and week 30 (Q3). Q2 is a food frequency questionnaire (FFQ), whereas Q1 and Q3 are more general questionnaires covering health, exposures, lifestyles, and background factors. The records of the participating women in the Medical Birth Registry of Norway16 are included in the data set. At the time of this analysis, 29,888 women had answered Q1. Of these, 28,472 (95%) had delivered singleton births. Eighty percent (n = 23,856) had answered both Q1 and version 2 of Q2.17 The final number of 23,423 (78%) had a registered food intake >4500 kJ and <20,000 kJ per day and were included in the analyses. Calculation of vitamin D supplementation in late pregnancy was based on information collected from Q3. For the analysis of supplementation in both early and late pregnancy, 22,057 women were included, ie, those who in addition had completed Q3.
This study has been approved by the Regional Committee for Ethics in Medical Research and the Data Inspectorate in Norway.
Dietary Information
The FFQ (www.fhi.no/dav/22CA50E0D7.pdf) was completed in the 22nd week of gestation. These data have been collected from February 2002 onwards. The FFQ is a semi-quantitative questionnaire designed to capture dietary habits and intake of dietary supplements during the first 4–5 months of pregnancy, as has been described in detail elsewhere.17 Nutrient calculations were performed with the use of FoodCalc18 and the Norwegian food composition table.19 A validation study demonstrated that, relative to a dietary reference method and several biologic markers, the FFQ produces a realistic estimate of the habitual intake and is a valid tool for ranking pregnant women according to high and low intakes of energy, nutrients, and food.20
In the FFQ, the women were asked to record the use of dietary supplements. A database including more than 1000 dietary supplements, with nutrient content per portion, has been created. For calculations of nutrient intake from supplements, we used name and brand name, together with frequency and amount. Supplement use registered in the FFQ has been validated, showing a good correlation between reported use and biomarkers.21
The total intake of vitamin D was calculated by combining information regarding diet and supplements. The daily intake was divided into 5 categories (<5, 5–9.9, 10–14.9, 15–20, >20 μg/d), which corresponds to the intake below the recommendation, the lowest levels of recommended intake, recommended intake plus 5 μg/d during pregnancy, and 2 intake levels higher than recommended.22 Vitamin D intake from supplements was categorized into the same categories as for diet, in addition to one representing no supplementation. Intake of the long chain omega-3 fatty acids EPA and DHA through diet and supplements was divided into 4 categories, according to energy contribution in percentages (<0.5, 0.5–0.9, 1.0–1.5, >1.5 E%). These categories represented, respectively, energy contributions below recommended levels of n-3 fatty acids, the lowest recommended intake level, the highest recommended intake level of long chain n-3 fatty acids, and intake level above recommendations of long chain n-3 fatty acids.22 The ratio of n-6 to n-3 was categorized into levels of below recommended intake, recommended intake and above recommended intake (≤3.0, 3.0–9.0, >9.0).22
Additional Dietary Supplement Use
In addition to the information about dietary supplement use in the FFQ, Q1 (http://www.fhi.no/dav/687c89b365.pdf), and Q3 (http://www.fhi.no/dav/7ff764a791.pdf) contain questions about the intake of nutrients from dietary supplements at certain time periods during pregnancy. The time intervals used in these analyses were as follows: from week 8 before pregnancy until last menstruation before pregnancy, from week 1 to week 12 of pregnancy, and from week 22 to week 29 of pregnancy.
Preeclampsia
The main outcome was preeclampsia as registered in the birth registry. Information provided to the registry is based on forms completed by the midwives after birth. The form has 5 checkoff boxes relevant to preeclampsia: hemolysis, elevated liver enzymes, and low platelet count (HELLP); eclampsia; early preeclampsia (diagnosed before 34 weeks); mild preeclampsia; and severe preeclampsia. The diagnostic criteria for preeclampsia in Norway, according to guidelines issued by the Society for Gynecology, are blood pressure >140/90 after 20 weeks’ gestation, combined with proteinuria greater than +1 dipstick on at least 2 occasions.23 We defined preeclampsia as any of the above-mentioned diagnoses.
Other Variables
Prepregnancy body mass index (BMI) was divided into 4 categories (<20, 20–24, 25–29, and 30+ kg/m2); maternal height was arbitrarily categorized into 4 approximately equal groups (<1.65, 1.66–1.68, 1.69–1.72, 1.73+ m); education in 4 categories (less than high school, high school, 3 years of college/university, or 4 or more years of college/university education); smoking in 3 categories (daily smokers, occasional smokers, and nonsmokers); maternal age in 4 categories (<20, 20–29, 30–39, 40+ years); and season of childbirth as winter (December–February), spring (March–May), summer (June–August), or autumn (September–November).
Statistical Methods
To test for group differences in continuous variables with non-normal distributions, we used the Mann-Whitney U test or the Kruskal-Wallis test. We estimated Spearman correlation coefficients (ρ) to describe correlation. Odds ratios were adjusted for confounding using multiple logistic regression. Potentially confounding variables (ie, variables that are known to be associated with preeclampsia and related to vitamin D intake) were maternal age, BMI, height, education, smoking in pregnancy, and season of childbirth. Because of the high degree of correlation between the intake of vitamin D and the long chain n-3 fatty acids, these variables were not included in the same model, but were analyzed separately using the same set of confounders. All analyses were performed using SPSS, version 14 (SPSS Inc, Chicago, Ill, USA).
RESULTS
Among 23,423 nulliparous women, 1267 (5.4%) developed preeclampsia. The intakes of vitamin D and the long chain n-3 fatty acids increased with increasing age, education, and height, and the intake was lower in smokers and in women with BMI above 25 kg/m2 (Table 1). The intake of vitamin D was highest for women giving birth during the summer season (ie, intake during winter), whereas the intake of the long chain n-3 fatty acids did not vary with season of childbirth. The total intake of vitamin D correlated with both the total intake of the long chain n-3 fatty acids (ρ = 0.74 [95% CI = 0.73–0.75]) and the n-6/n-3 ratio (ρ = −0.64 [−0.63 to −0.65]).
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TABLE 1: Intake of Vitamin D and Long Chain n-3 Fatty Acids (LCn-3) (Includes Diet and Supplements) During Pregnancy for 23,423 Nulliparous Women, According to Maternal Characteristics and Season of Childbirth. The Norwegian Mother and Child Cohort Study, 2002–2006
Women who developed preeclampsia did not have lower intake of vitamin D or of the long chain n-3 fatty acids from the diet compared with women without preeclampsia. However, they did have lower Vitamin D intake from food supplements (Table 2). Food supplements that did not contain vitamin D provided no protection from preeclampsia (Table 3). A daily intake of vitamin D between 15-20 μg/d reduced the risk for preeclampsia by 31% in the crude analysis and by 25% in the adjusted analysis (Table 4). The impact of vitamin D intake from supplements was more pronounced than from the total diet; an intake of 10-15 μg/d from supplements reduced the risk of preeclampsia by 29% after adjustments. No association was seen with vitamin D intake from diet alone, although only 106 women (0.5%) had an intake >15.0 μg/d of vitamin D from their diet. An energy intake of 1.0%–1.5% from the long chain n-3 fatty acids reduced the risk of preeclampsia by 24%, but the impact was attenuated by adjustment. There was no association between the n-6/n-3 ratio and preeclampsia.
TABLE 2: Intake of Vitamin D and the Long Chain n-3 Fatty Acids From Diet and Food Supplements During Pregnancy for Women With and Without Preeclampsia
TABLE 3: Association Between Use of Food Supplements With and Without Vitamin D and Preeclampsia
TABLE 4: Risk of Preeclampsia According to Intake of Vitamin D, the Long Chain n-3 Fatty Acids, and the Ratio of n-6/n-3 From Diet and Supplements.
The risk of preeclampsia was estimated according to the timing of supplementary vitamin D use in 22,057 women, of whom 1149 (5.2%) developed preeclampsia (Table 5). Women reporting supplementary intake of vitamin D at all 3 points (before pregnancy, in early pregnancy, and in late pregnancy) had 29% reduced risk of preeclampsia compared with those never taking vitamin D supplements.
TABLE 5: Risk of Preeclampsia According to Vitamin D Supplement Status Before and During Pregnancy
DISCUSSION
We investigated the risk of preeclampsia in relation to dietary and supplementary vitamin D and long chain n-3 fatty acid (EPA/DHA) intake. We found that a supplementary intake of vitamin D was associated with a reduced risk of preeclampsia, whereas the intake of long chain n-3 fatty acids had minor impact on preeclampsia risk.
The strength of this study is the large sample of nulliparous women representing wide ranges of maternal age, BMI, height, and socioeconomic status. The preeclampsia diagnosis was collected from the Medical Birth Registry in Norway,16 whose criteria were recently validated in a study from Denmark24; with just a few false positives, the authors concluded that the registry had acceptable validity. Furthermore, the information on dietary intake and the use of dietary supplements were collected before the occurrence of preeclampsia. Although a true picture of dietary intake is difficult to obtain using FFQs, such questionnaires provide a general picture of dietary intake.25 In a validation study of the FFQ used in this study, the intakes of energy, nutrients, and foods were ranked correctly20 and the reported intake of vitamin D from dietary supplements correlated with the plasma concentration of 25(OH)D.21 This latter observation adds strength to the present results because circulating 25(OH) D levels are directly correlated with dietary vitamin D intake and skin exposure to ultraviolet light. In a country such as Norway, far to the North, skin exposure to ultraviolet light of sufficient strength to promote the conversion of provitamin D to vitamin D is limited to the summer season. The concentration of plasma 25(OH) vitamin D has been found in middle-aged women to be positively associated with hours of UV-B radiation.26,27 Seasonal variation in preeclampsia occurrence, with the lowest occurrence in August, has been reported in an earlier study from Norway.28 Accordingly, we adjusted for season of birth. This adjustment did not influence the risk of preeclampsia according to total intake of vitamin D. However, season of birth did appear to have a slight influence on preeclampsia risk, and this risk remained unchanged after adjustment for vitamin D intake (results not shown). Thus, both vitamin D intake and season may influence the risk of preeclampsia. The 2 exposures may act through different mechanisms.
A protective effect from vitamin D supplements seemed to be most evident at doses of at least 10 μg/d. A weakness of this study is that we could not demonstrate any effect of vitamin D intake from diet alone. However, in Norway, the vitamin D intake from diet is low and the recommendation of 10.0 μg/d for pregnant women can hardly be met without supplementation.22 The main dietary vitamin D sources are oily fish, cod liver oil, and fortified margarine and milk. Fortification of other food items was not allowed before 2007, when most of the dietary information in this study was collected. A median dietary intake of 3 μg/d (with only 109 [0.5%] women having a dietary intake higher than 15 μg of vitamin D per day) may explain our inability to demonstrate an association between dietary vitamin D intake and preeclampsia. It has been suggested that an intake of vitamin D of 5–10 μg/d is grossly insufficient during pregnancy. Hollis has suggested that an intake of 150 μg/d might be required.29 Our data on preeclampsia prevention do not suggest any advantage of an intake above 20 μg/d. However, this is higher than the present recommended intake of 10 μg/d during pregnancy.22
An alternative explanation for our observation may be that it is not vitamin D that is related to preeclampsia but health behaviors associated with the intake of supplements in general. However, we failed to find a reduced risk of preeclampsia in women who took supplements not containing vitamin D (Table 3). Furthermore, we have adjusted our estimates for BMI, education, maternal age, smoking, and height, which together are likely to capture a major part of the variability in socioeconomic background and health behavior.
We demonstrated an effect of vitamin D supplementation both in early and late stages of pregnancy. Vitamin D is important for fetal and childhood bone development,30 and new research has shown that it plays a wider role in health and disease prevention.31,32 In normal pregnancies, circulating maternal concentration of 1,25(OH)2D is elevated from the first trimester onwards.33 Local production of 1,25(OH)2D may be important for its increased concentration in the early stage of pregnancy, this has been suggested to influence implantation, partly through the immunomodulatory effect and partly by regulation of the target genes associated with implantation.5 The elevation of 1,25(OH)2D in the early stage of pregnancy could reflect its role in implantation, as demands to meet increased calcium requirements for the fetal skeletal development come later in pregnancy.7 Vitamin D might also be important for the maintenance of a normal pregnancy through its impact on the maternal immune response to the fetus.5,34
The first study to show a direct correlation between vitamin D status and occurrence of preeclampsia demonstrated an increased risk of preeclampsia with reduced maternal concentration of 25(OH)D.6 That study was performed in nulliparous women with singleton pregnancies, and the concentration of 25(OH)D was analyzed in samples collected in week 16 of the pregnancies. Our results suggest that it may be possible to reduce the risk of preeclampsia through a sufficient vitamin D intake. However, in a newly published study from the United States, no effect of vitamin D intake on preeclampsia occurrence was found.11 The main difference between their study and ours is the sample size (1718 vs. 23,423) and the occurrence of preeclampsia (3% vs. 5.4%). The total intake of vitamin D was also higher in the US study, which might suggest that the upper level of vitamin D intake for impact on preeclampsia was reached.
A possible preventive effect of low-dose fish oil on preeclampsia was suggested in an early clinical study in London.9 Women in that study were given a mixture of halibut oil, vitamin D (22.5 μg/d), calcium (260 mg/d), and other vitamins and minerals. Fish oil purified of vitamin D failed to demonstrate any beneficial effect on preeclampsia risk in a randomized, double-blind, placebo controlled trial,35 whereas in a recently published study, indications of a potential effect of the n-3 fatty acids EPA and DHA was reported.11 The intake of long chain fatty acids in the Oken et al study was much lower than in our study—0.15 mg/d compared with 0.61 mg/d. Due to frequent use of cod liver oil in Norway, the intake of vitamin D correlates closely with the intake of the long chain n-3 fatty acids EPA and DHA, and hence we could not adjust for intake of these fatty acids. However, running the same analysis with the intake of EPA/DHA as for vitamin D, we found an association of these n-3 fatty acids and occurrence of preeclampsia, although the association was much weaker. The intercorrelation between vitamin D intake and n-3 intake leaves uncertainties regarding the effects of the 2 nutrients. Our results, however, suggest that vitamin D supplementation may reduce the risk of preeclampsia, whereas the association between the long chain n-3 fatty acids and preeclampsia is less certain.
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