Approximately one third of pregnancies are lost after implantation, many before clinical recognition,1 making pregnancy loss the most frequent adverse pregnancy outcome.2 Although chromosomal abnormalities are implicated in approximately 50% of all spontaneous abortions, the remaining 50% may be preventable and related to environmental factors.3 Research on the role of dietary factors in human reproduction is limited, but there is reason to believe that intake of certain nutrients, particularly folate, could positively influence reproductive success.4
Folic acid prevents neural tube defects.5 The American College of Obstetricians and Gynecologists recommends that all women planning or capable of pregnancy take 400 micrograms per day of folic acid.6 However, folic acid supplementation may have reproductive benefits beyond the prevention of neural tube defects. Folic acid supplementation in animals promotes embryo and fetal survival rates throughout gestation,7–9 yet the association between folate intake and fetal survival in humans is less clear.
The objective of this study was to evaluate the relationship between prepregnancy folate intake and risks of spontaneous abortion and stillbirth in a large, prospective cohort of women with a wide range of folate intake. We aimed to expand on previous studies by examining dose–response relationships comparing food with supplemental folate. We hypothesized that higher intake of folate, particularly supplemental folate (as a result of higher absorption and bioavailability and wider range of intake), is associated with reduced risk of pregnancy loss.
MATERIALS AND METHODS
The Nurses' Health Study II is an ongoing prospective cohort of 116,480 female nurses ages 24–44 years at the study's inception in 1989. Questionnaires are distributed every 2 years to update lifestyle and medical characteristics and capture incident health outcomes. Response rates for each questionnaire cycle have exceeded 90%. Diet was first assessed in 1991 and updated every 4 years thereafter. Women were eligible for this analysis if they had no history of pregnancy loss in 1991 and reported at least one pregnancy during 1992–2009. Eligible participants contributed pregnancies until their first pregnancy loss or the end of follow-up. Women were censored after their first pregnancy loss to prevent reverse causation, that is, behavioral changes in response to an adverse outcome. Of the 19,451 eligible pregnancies, we excluded those with missing data on diet (n=2,475), implausible or missing gestational age (n=111), missing year of pregnancy (n=619), and diagnosis of type II diabetes (n=69), cardiovascular disease (n=86), or cancer (n=141) before the pregnancy. The final sample consisted of 15,950 pregnancies from 11,072 women. This study was approved by the institutional review board of Partners Health Care, Boston, Massachusetts.
Diet was evaluated using a validated 131-item food frequency questionnaire.10 Women reported how often, on average, they consumed specified amounts of each food during the previous year. Participants were also asked whether they used multivitamins and other nutrient supplements and, if so, the brand, dose, and frequency of use. Nutrient intakes were estimated by summing nutrient contributions of each food item and supplement. Nutrient contents of each item were obtained from a nutrient database derived from the U.S. Department of Agriculture and additional information from manufacturers.11 The database was updated in 1998 to reflect the universal folic acid supplementation of flour and cereals. Folate intake with this questionnaire has been validated against prospectively collected diet records (r=0.71),12 red blood cell folate (r=0.51),13 and plasma folate levels (r=0.63).14 Nutrient intakes were adjusted for total energy intake using the nutrient residual method.15 We used diet information from 1991 for pregnancies in 1992, 1993, 1994, and 1995; the 1995 diet for pregnancies in 1996, 1997, 1998, and 1999; and so forth. If a woman was missing diet, less than 5% of women, the most recent dietary data were carried forward.
Women reported their pregnancies in 1989 and in each biennial follow-up questionnaire. In the 2009 questionnaire, women also reported information on the year, length, complications, and outcomes of all previous pregnancies. Options for pregnancy outcomes were a singleton live birth, multiple birth, miscarriage or stillbirth, tubal or ectopic pregnancy, or induced abortion. Gestational lengths were reported in categories: less than 8 weeks, 8–11 weeks, 12–19 weeks, 20–27 weeks, 28–31 weeks, 32–36 weeks, 37–39 weeks, 40–42 weeks, and 43+ weeks of gestation. Self-reported pregnancy outcome and gestation length have been shown to be validly reported.16 Spontaneous abortion was defined as a fetal loss under 20 completed weeks of gestation. Stillbirth was defined as a fetal loss at 20+ completed weeks of gestation. The validity of maternal recall of spontaneous abortion has not been assessed in this population; the sensitivity is estimated to be approximately 75%.17 , 18 Noncases were all pregnancies that did not end in fetal loss (live births, induced abortions, and tubal or ectopic pregnancies).
Information on covariates was assessed in 1989 and during follow-up. For variables updated over follow-up, the most recent value before that pregnancy was used. Age was computed as the difference between year of birth and year of pregnancy. Physical activity was ascertained in 1991, 1997, 2001, and 2005. The questionnaire-based estimates correlated well with detailed activity diaries in a validation study (r=0.56).19 Smoking status, multivitamin use, oral contraceptive pill (OCP) use, and history of infertility were self-reported in 1989 and updated every 2 years. History of ovulation-inducing medication use was self-reported starting in 1993 and updated every 2 years. Marital status was reported in 1989, 1993, and 1997. Weight was self-reported in 1989 and updated every 2 years thereafter. Race and height were reported in 1989. Body mass index (BMI) is calculated as weight (kg)/[height (m)]2. In a validation study, self-reported weight was highly correlated with weight measured by a technician (r=0.97).20
Baseline characteristics were derived from the 1991 questionnaire for all women contributing eligible pregnancies. We divided women into groups according to quintiles of calorie-adjusted total folate intake and categories of supplemental folate intake. Differences in baseline characteristics by prepregnancy total and supplemental folate intake were compared using a χ2 test for categorical variables and Kruskal–Wallis nonparametric tests for continuous variables.
The relative risk (RR) of spontaneous abortion and stillbirth in relation to prepregnancy folate intake was estimated using log-binomial regression. Generalized estimating equations with an exchangeable working correlation structure were used to account for the within-person correlation between pregnancies. Tests for linear trend were conducted by using the median values in each category as a continuous variable. In addition to age-, calorie-, and year-adjusted models, multivariable models were further adjusted for a priori-selected prepregnancy covariables: BMI, smoking status, physical activity, history of infertility, marital status, and race. Categorical covariables included an indicator for missing data, if necessary.
We assessed whether the other B vitamins (thiamin, riboflavin, niacin, pantothenic acid, vitamin B6, or vitamin B12) were associated with spontaneous abortion, because these B vitamins come from similar dietary sources and are highly correlated with folate intake. In addition, we investigated whether the relation of folate with spontaneous abortion differed by gestational age at loss (less than 8 weeks, 8–11 weeks, and 12–19 weeks of gestation). Relative risks for specific gestational windows were estimated using log-binomial regressions. The reference group for fetal losses less than 8 weeks of gestation was all initiated pregnancies, for fetal losses 8–11 weeks was all pregnancies lasting beyond 8 weeks of gestation, and for fetal losses 12–19 weeks of gestation was all pregnancies lasting beyond 12 weeks of gestation. P values for heterogeneity were derived from the cross-product interaction term added to the main-effects multivariable model.
To address the potential of residual confounding by factors strongly related to risk of pregnancy loss, we performed sensitivity analyses restricted to pregnancies from women 40 years or younger, pregnancies with no history of infertility, and first eligible pregnancies. To capture uncontrolled confounding by behaviors related to pregnancy planning and pregnancy recognition, we performed analyses restricted to married women not using OCPs. To address the potential of misclassification of exposure resulting from the interval between diet assessments, we restricted analyses to pregnancies in the years closest to diet assessment (1992, 1996, 2000, and 2004). Effect modification by prepregnancy BMI (less than 25 compared with 25 or greater), smoking status (current compared with never or former smokers), and maternal age (younger than 35 years compared with 35 years or older) was tested using cross-product terms in the final multivariable model. All data were analyzed using SAS 9.1.
Of the 15,950 eligible pregnancies, 2,756 (17.3%) ended in spontaneous abortion and 120 (0.8%) in stillbirth. Women in the cohort had a mean (standard deviation) age of 31.6 (3.4) years and BMI of 23.3 (4.3) in 1991. The majority were white (93%), married (71%), never smokers (71%), and nulliparous (46%) in 1991. On average, women with higher folate intake were slightly heavier, reported more physical activity, were less likely to be current smokers and current users of OCPs, and reported higher calorie intake and more frequent multivitamin consumption. These women were also more likely to be parous, white women who were married and had a history of infertility (Table 1).
Higher intake of total folate before pregnancy was associated with reduced risk of spontaneous abortion (Table 2). Compared with women in the lowest quintile of prepregnancy folate intake (less than 285 micrograms/d), those in the highest quintile (greater than 851 micrograms/d) had a RR of spontaneous abortion of 0.91 (95% confidence interval [CI] 0.82–1.02) after adjusting for energy intake, maternal age, BMI, physical activity, year of pregnancy, history of infertility, marital status, and race (P trend=.04). This association was driven solely by folate from supplements. Specifically, after multivariable adjustment, women in the highest category of supplemental folate intake (greater than 730 micrograms/d) had an RR of spontaneous abortion of 0.80 (95% CI 0.71–0.90) compared with women who did not consume supplemental folate (P trend <.001). The adjusted absolute risks of spontaneous abortion by quintile of folate intake and category of supplemental folate (Fig. 1 ) suggest that 42 women would need to go from 400 to 729 micrograms per day of supplemental folate (quintile 3) to greater than 730 micrograms per day (quintile 4) of supplemental folate to prevent one spontaneous abortion.
After multivariable adjustment, higher intake of vitamin B12 was associated with lower risk of spontaneous abortion (P trend=.04) (Table 3). With further adjustment for supplemental folate intake, however, intake of B12 was no longer related to spontaneous abortion (P trend=.93), whereas the inverse dose–response relation between supplemental folate intake and risk of spontaneous abortion remained statistically and clinically significant. In this model, the RR (95% CI) of spontaneous abortion for increasing categories of supplemental folate intake were 1.00 (reference), 0.94 (0.86–1.04), 0.91 (0.81–1.02), 0.78 (0.68–0.90) (P trend <.001).
Overall, the magnitude of association between prepregnancy total and supplemental folate intake with risk of spontaneous abortion was fairly consistent across different gestational window (P interaction=.92 and .71, respectively) (Table 4). Furthermore, total and supplemental folate intake had an inverse association with stillbirth, which fell short of conventional significance (P trend=.06 and .14, respectively). The adjusted absolute risks of stillbirth in quintile 1 and quintile 5 of total folate intake were 0.0072 and 0.0040 and in category 1 and category 4 of supplemental folate were 0.0064 and 0.0041.
In sensitivity analyses (see the Appendix, available online at http://links.lww.com/AOG/A522) limited to pregnancies in women younger than 40 years of age, pregnancies without a history of infertility, and pregnancies among married women who were not using OCPs, the results remained similar. Results became slightly stronger when analyses were restricted to the first eligible pregnancy and pregnancies closest to diet assessments. No substantial differences in effect estimates were seen when assessing consumption of folate and risk of spontaneous abortion in overweight compared with nonoverweight women, in current compared with never or former smokers, and in younger compared with older women (younger than 35 years compared with 35 years or older).
In this prospective cohort of 15,950 pregnancies, we found that the risk of spontaneous abortion was 20% lower among women in the highest category of supplemental folate intake (greater than 730 micrograms/d) than in the lowest (0 micrograms/d) category.
Since 1992, the U.S. Preventive Services Task Force and Centers for Disease Control and Prevention recommend that all women planning or capable of pregnancy take 400 micrograms of folic acid daily to prevent neural tube defects.6 In the mid-1990s, the safety of folic acid supplementation was called into question on the basis of three papers,21–23 which suggested folic acid supplementation increased the risk of miscarriage. These findings were subsequently challenged as a result of methodologic errors24,25 (eg, using a one-tailed test) and incongruent conclusions. Specifically, the Hungarian trial found that folic acid increased fertility and multiple birth rates (in addition to miscarriage rates), which seemed implausible. Furthermore, the Medial Research Council Vitamin Study, which used a dose of folic acid approximately five times greater the Hungarian trial, found no detrimental effect of folic acid supplementation on miscarriage when the analysis was limited to only women receiving folic acid. Two follow-up studies from China26 and Brazil27 also provided evidence that periconceptional folic acid use did not increase miscarriage rates. Two recent cohort studies reported that the use of folic acid or multiple vitamins during pregnancy was associated with a 50–60% reduced risk of miscarriage.28,29 Our study allowed us to improve on previous studies by contrasting the relationship between prepregnancy folate from food and supplements and spontaneous abortion and to examine a dose–response relationship across this broad range of intake.
Lower folate intake has been linked to reduced cell division, disrupted methylation reactions, increased inflammatory cytokine production, oxidative stress levels, and apoptosis, all of which could subsequently affect the developing embryo.30 Folate deficiency has also been suggested as a risk factor for abruptio placentae and preeclampsia.31 Thus, the vascular effects related to folate deficiency might also increase the risk of spontaneous abortion and stillbirth. Another explanation is that low folate levels increase the incidence of neural tube defects, and fetuses affected with neural tube defects are more commonly aborted spontaneously.32 Although plausible, neural tube defects are rare conditions and this could only explain a fraction of the association between low folate levels and spontaneous abortion.
Supplemental folate was more strongly related to spontaneous abortion than was folate from foods. This difference could be attributable partly to the greater absorption rates of synthetic folate.33 Relative to folic acid, natural food folate has a lower proportion of folate that is absorbed and available for metabolic reactions and storage. Several luminal factors also hinder the absorption of natural food folate.33 In addition, the range of supplemental folate intake was wider than the range of intakes from foods, increasing our ability to detect an association with supplemental folate intake.
We did not have information on diet during pregnancy and were thus unable to discern whether the association of prepregnancy folate intake with spontaneous abortion is independent of pregnancy folate intake. Nevertheless, prepregnancy folate intake is likely the more relevant time window because most spontaneous abortions occur early in pregnancy. Male partner data were also lacking. Previous studies have reported positive associations between folate and male fertility34,35; however, it is unlikely that supplemental folate consumption is highly correlated among partners, thus reducing the likelihood that male diet would be a strong confounder. Second, there is some concern about differential misclassification of fetal loss by gestational age and pregnancy intention. However, our subanalyses addressing these issues confirmed a robust association. Third, it is possible that many spontaneous abortions were unrecognized and thus not reported. Although plausible, underreporting of early losses is likely nondifferential with respect to folate intake (as a result of the prospective design) and would be expected to attenuate the association. Fourth, misclassification of folate intake is likely, particularly because diet information was updated only every 4 years. As expected, when we limited our analyses to pregnancies in the years closest to diet assessment, the results were stronger (Appendix, http://links.lww.com/AOG/A522). Fifth, despite our adjustment and stratification for a variety of potential confounders, we cannot rule out the possibility that there may be residual or unmeasured confounding. However, differences between unadjusted and multivariate-adjusted effect estimates were small suggesting that any residual confounding is unlikely to have a large effect on our results. Our analyses of folate and risk of stillbirth were limited by low number of cases, which reduced statistical power. Finally, our study does not distinguish chromosomally normal from abnormal miscarriages.
The strengths of this study are the large, prospective design, nearly complete follow-up over 18 years, inclusion of early pregnancy losses, ability to examine a dose–response association across a broad range of folate intake, and the ability to contrast supplemental folate with food folate.
Our results have important public health and clinical implications. The American College of Obstetricians and Gynecologists and the World Health Organization recommend that women of childbearing age in many countries, including the United States, take prenatal folic acid supplements. Despite this recommendation, the majority of U.S. reproductive-aged women consume far below the recommendation of 400 micrograms per day.36 In addition, food fortification with folic acid has been introduced in many countries and is being considered in others.37–39 Our results provide reassurance that higher intake of supplemental folate is not associated with increased risk of pregnancy loss; rather, it may be an effective strategy to prevent spontaneous abortion. Given that the first prenatal visit is likely too late to initiate a discussion on the importance of folic acid supplements, annual obstetrician–gynecologist visits might be the best opportunity to talk to women about the importance of folic acid.
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