Plasma Folate Levels and Risk of Spontaneous Abortion FREE

Some studies,^1- 5 but not all,^6- 9 have found that folate deficiency is a risk factor for spontaneous abortion. These conflicting results could be due to small sample size, highly selected populations, or lack of control for potential confounders, such as age, smoking, and alcohol consumption. Moreover, folate levels change during pregnancy,¹⁰ and most studies have included nonpregnant women as controls.

Conversely, it has also been suggested that folic acid supplementation, by increasing folate levels, may increase the rate of or shift the timing of spontaneous abortion.^11- 17 This has been highly controversial, with other studies challenging these findings.^18- 21 None of these studies have included measurements of folate levels.

Folic acid supplementation has been reported to prevent neural tube defects.^22- 24 Since 1998, the US Food and Drug Administration has required food fortification with folic acid,²⁵ whereas in Sweden, folic acid fortification has not been introduced. The possibility of studying the relationship between folate deficiency and spontaneous abortion is hampered in populations with folic acid food fortification,²⁶ whereas the ability to study high exposure is enhanced. In this large, population-based, case-control study in Sweden, we investigated the association between levels of plasma folate and risk of early spontaneous abortion after controlling for possible confounders.

The methods of this matched, case-control study are described in detail elsewhere.²⁷ The study was conducted in Uppsala County, Sweden, from January 1996 through December 1998. Women who had spontaneous abortion of a fetus with a gestational age of 6 to 12 completed weeks and whose pregnancies had been confirmed by a positive human chorionic gonadotropin test result were identified at the Department of Obstetrics and Gynecology of Uppsala University Hospital, which is the only place in the county for the care of women with spontaneous abortions. During this period, we identified 652 women as potential case patients, of whom 562 (86%) agreed to participate.

Control patients were primarily selected from pregnant women seeking prenatal care in Uppsala County and were frequency-matched to the cases by gestational week. Among cases and controls, gestational age was calculated from the first day of the last menstrual period. Of 1037 patients receiving antenatal care and asked to participate as controls, 953 (92%) agreed. All potential controls underwent vaginal ultrasonography before the interview to verify the viability of the fetus. If a nonviable intrauterine pregnancy was detected, the woman was recruited as a case patient. This occurred in 53 of the case patients. Of women recruited as controls, 5 women had a spontaneous abortion before 13 completed weeks. Since these women now fulfilled the criteria of being recruited as cases, they were thus also included as such and subjected to a new interview.

In Uppsala County, there are approximately 3 legally induced abortions for every 10 completed pregnancies, and some of these terminated pregnancies would have resulted in spontaneous abortion if the pregnancy had continued. Women with induced abortions may differ from women continuing their pregnancies in terms of factors associated with risk of spontaneous abortions, such as age, smoking, and possibly other lifestyle-related factors. To avoid this potential bias in the selection of control patients, women planning to have induced abortions were added to the control group. In total, 310 women who would later undergo induced abortion were asked to participate and 274 (88%) agreed. Of these women, 75 were added to the control group, a number estimated according to the gestational age distribution of induced abortions in Uppsala County during the study period.

Three specially trained midwives conducted in-person interviews with the cases and the controls recruited among women receiving antenatal care. Two physicians conducted interviews with the control patients who would undergo induced abortions. Ninety percent of the cases were interviewed within 2 weeks after their miscarriage diagnosis, and the other 10% were interviewed within 7 weeks. All controls were interviewed within 6 days after their last completed week of gestation used in matching. A structured questionnaire was used to reduce bias because the interviewers could not be blinded to status (ie, pregnant or aborted). Interviewers did not know folate status or the results of karyotyping.

Women were asked about possible risk factors for spontaneous abortion, including age, previous reproductive history, smoking, caffeine and alcohol use, shift work, and a number of other potential risk factors. Women were also asked how many days each week they used prescription or nonprescription drugs, starting 4 weeks before their last menstrual period and ending in their last completed week of gestation. Daily folic acid supplementation was defined as intake of at least 400 µg/d during this period.²⁸ Women were asked to quantify mean daily intake of various caffeine sources on a weekly basis, which was used to calculate average daily caffeine intake during pregnancy (ie, from conception through the last completed week of pregnancy).

Since pregnancy symptoms are associated with fetal viability and caffeine consumption, we determined scores for the presence of nausea (0 [never], 1 [sometimes but not daily], 2 [daily but not all day], 3 [daily and all day]); vomiting (0 [never], 1 [sometimes but not daily], 2 [daily]); and fatigue (0 [no], 1 [yes, but unchanged sleeping habits], 2 [yes, slightly changed sleeping habits], 3 [yes, pronounced change in sleeping habits]). The average weekly score during pregnancy for each symptom was then calculated and averaged to a mean score for each symptom.²⁷

Oral informed consent was obtained from all the women, and the ethics committee of the medical faculty at Uppsala University approved the study.

Patients were asked to provide blood samples. From the cases, blood samples were obtained at the emergency ward at the time of miscarriage diagnosis and from the controls at the time they were interviewed. Blood samples were kept frozen at −80°C until assayed. A plasma blood sample for analysis of folic acid and cotinine concentrations was available from 468 (83%) of the cases, 852 (89%) of the controls from antenatal care, and 69 (92%) of the controls with induced abortions. There were virtually no differences in baseline maternal characteristics and exposures between the cases and the controls who agreed to participate in the study and the cases and the controls from whom blood samples for folic acid analysis were available (data available on request from the authors).

Folate analyses were performed with an immunoassay analyzer (AxSYM, Abbot Laboratories, Abbot Park, Ill), using ion capture reaction technology. The analysis is quantified by measuring the amount of unoccupied folate-specific binding sites bound to matrix using a conjugate of pteroic acid (a folate analog) and alkaline phosphatase as the signal-generating molecule, and a substrate, 4-methylumbelliferyl phosphate. Plasma folate levels were categorized before data analyses. Low plasma folate level was defined as 2.19 ng/mL (4.9 nmol/L) or lower, which corresponds to the cutoff for recommending folic acid supplements.²⁹ Because there is no standard definition of high serum folate levels in Sweden, the reference group was defined as women with levels of 2.20 to 3.95 ng/mL (5.0-8.9 nmol/L) (including most cases and controls), and high folate levels were stratified into 3.96 to 6.16 ng/mL (9.0-13.9 nmol/L) and 6.17 ng/mL (14.0 nmol/L) or more (including approximately 20% and 10% of cases and controls, respectively).

Plasma cotinine was measured by gas chromatography with use of N-ethylnorcotinine as an internal standard.³⁰ We defined smokers as women who had a plasma cotinine concentration of higher than 15 ng/mL (85.2 nmol/L).³¹ We allowed self-reported daily smoking during all weeks of pregnancy to override missing cotinine values for classification of smoking.

Curettage was performed in all cases with incomplete spontaneous abortion at diagnosis. Intrauterine tissue was collected under sterile conditions and routinely sent for karyotype analyses. Chorionic villi were identified in tissue obtained by curettage in 243 of the 468 cases, allowing successful karyotyping in 216 (89%). Cytogenetic analysis was performed using direct preparation, and the chromosomes were banded with Giemsa stain. Eleven cells in metaphase were routinely analyzed, and karyotyping was considered unsuccessful if fewer than 3 cells in metaphase were obtained.

Data were analyzed with the use of conditional logistic regression analysis using SAS PROC PHREG.³² The controls were frequency matched to the cases by week of gestation. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to estimate the relative risk. The overall effect of folate was tested by using a Wald χ² test. The test considers all strata in determining significance and not just pairwise comparisons with the reference group. Since the study was frequency matched, all controls were considered in the subanalyses of risks of spontaneous abortion according to fetal karyotype and gestational age. Variables were included in the multivariate analyses (maternal age, cigarette smoking, average caffeine intake during pregnancy, previous spontaneous abortions, education, parity, country of birth, body mass index, change of eating habits, and the pregnancy symptoms nausea, vomiting, and fatigue) if they were judged a priori to be potential confounders and if they were associated with risk of miscarriage, with low or high folic acid levels, or with both (P<.05). A χ² test was used to test homogeneity of folate level proportions and folic acid supplement proportions.

The mean (SD) serum folate values among cases and controls were 3.76 (2.41) ng/mL (8.54 [5.48] nmol/L) and 3.84 (2.29) ng/mL (8.71 [5.20] nmol/L), respectively (P = .56). Compared with folate levels of 2.20 to 3.95 ng/mL (5.0-8.9 nmol/L), low (≤2.19 ng/mL [≤4.9 nmol/L]) folate levels were associated with a nonsignificant increase in risk of spontaneous abortion in the univariate analysis. High folate level was not associated with increased risk of spontaneous abortion (Table 1a). The women with spontaneous abortion were older than the controls and were more likely to previously have had spontaneous abortions and births, to be born outside the Nordic countries, to have medium to high education, to be smokers, and to consume more coffee and alcohol. Pregnancy symptoms, such as nausea, vomiting, tiredness, change in eating habits, and aversion to food and coffee, were more prevalent and severe among the controls. There were no significant differences between cases and controls with respect to body mass index, shift work, or age at menarche.

Many possible risk factors for spontaneous abortion were also associated with plasma folate levels. The proportion of women with low (≤2.19 ng/mL [≤4.9 nmol/L]) folate levels was increased among women who were younger than 30 years, had less than 12 years' education (P<.001 for all), were smokers, were obese (body mass index ≥30 kg/m²) (P = .005 for all), and were born within the Nordic countries (P = .027) and increased nonsignificantly (P = .10) with caffeine consumption. The proportion of women with high (≥6.17 ng/mL [≥14.0 nmol/L]) folate levels was increased among women aged 30 years or older, who were nulliparous (P<.001 for all), had more than 12 years' education (P = .002), or were born outside the Nordic countries (P = .003). Other variables, including gestational age, did not influence plasma folate levels (data available on request from the authors).

After adjustment, low folate levels were significantly associated with an increase in risk of spontaneous abortion (OR, 1.47; 95% CI, 1.01-2.14) (Table 2). When we only adjusted for maternal age, the corresponding OR was 1.45 (95% CI, 1.04-2.01), suggesting that the increase in risk was mainly due to elimination of confounding by maternal age. Because there was no significant interaction between maternal age and folate levels with regard to the risk of spontaneous abortion (P = .09), we did not perform analyses of plasma folate values stratified by maternal age. The risk of spontaneous abortion was also increased among women aged 35 years or older, among women with previous spontaneous abortions, among smokers, and among women consuming at least 300 mg/d of caffeine. Compared with women in the reference group, women with high folate levels had a nonsignificant decrease in their risk of spontaneous abortion (Table 2).

In analyses stratified by gestational age (Table 3), low folate levels were not associated with a significant increase in risk of spontaneous abortion at 6 to 8 gestational weeks, at 9 to 10 weeks, or at 11 to 12 weeks. In addition, there were no significant interactions between plasma folate levels and maternal age, smoking, caffeine intake, or gestational age (data available on request from the authors).

Karyotype analysis revealed that 83 fetuses (50 male and 33 female) were chromosomally normal, and 133 were abnormal. Of spontaneous abortions with abnormal fetal karyotype, 88 had autosomal aberrations (84 trisomies and 4 double trisomies); 14 were 45,X; 8 were 47,XXY; 4 were 47,XXX; 16 were triploidy; and 6 were tetraploidy. When we performed analyses stratified by fetal karyotype, the overall tests of an association between folate levels and karyotype-specific abortions were statistically nonsignificant (Table 3). Compared with women in the reference group, women with low folate levels were at increased risk of spontaneous abortion of fetuses with abnormal karyotype, whereas there were no significant associations with risk of normal and unknown fetal karyotype abortion. To further investigate the association between plasma folate levels and risk of spontaneous abortion with abnormal fetal karyotype, we stratified the abnormal fetal karyotypes into lethal (triploidy, tetraploidy, and autosomal except trisomy 21) and others (trisomy 21; 45,X; 47,XXY; and 47,XXX). Among women with low (≤2.19 ng/mL [≤4.9 nmol/L]) plasma folate levels who had spontaneous abortions with abnormal fetal karyotype, 19 (79%) of 24 had a fetus with a lethal chromosomal abnormality. Similarly, among women with plasma folate levels of 2.21 ng/mL (5.0 nmol/L) or more, 86 (79%) of 109 had fetuses assessed as having lethal chromosomal abnormality.

Since the pairwise comparison between the reference category and the low folate level group is significant for spontaneous abortions with abnormal fetal karyotype but not for those with normal fetal karyotype, we also performed an analysis restricted to cases. This case-case study does not yield a significant difference in folate levels between abnormal and normal cases (P = .38). Thus, we cannot show a significant difference in folate-associated risk patterns for normal and abnormal karyotype abortions.

In all, 4.7% (30 cases and 35 controls) of the women in the study reported daily intake of at least 400 µg of a folic acid supplement, beginning at a minimum of 4 weeks before conception and continuing through the last completed gestational week. Compared with women not taking a supplement, women taking a daily supplement were significantly older (P = .003) and had significantly less nausea (P = .04). Among women taking a folic acid supplement, none had low (≤2.19 ng/mL [≤4.9 nmol/L]) plasma folate levels and 91% (n = 59) had values of 3.96 ng/mL (9.0 nmol/L) or higher, whereas among those not taking a folic acid supplement, 16% (n = 206) had low levels and 27% (n = 352) had values of 3.96 ng/mL (9.0 nmol/L) or more. Compared with women not taking a folic acid supplement, the risk of spontaneous abortion among women using a folic acid supplement was not significantly increased (adjusted OR, 1.3; 95% CI, 0.7-2.4).

The distribution of gestational age at abortion among cases differed between those taking and not taking a folic acid supplement. Among cases taking daily folic acid supplements (n = 30), 6 (20%) had a spontaneous abortion in weeks 6 to 8, 19 (63%) in weeks 9 to 10, and 5 (17%) in weeks 11 to 12, whereas among cases not taking daily folate supplements (n = 438), 127 (29%) had a spontaneous abortion in weeks 6 to 8, 163 (37%) in weeks 9 to 10, and 148 (34%) in weeks 11 to 12 (P = .02). Thus, women taking supplements experienced spontaneous abortion earlier than did women not taking supplements.

Our study found no excess in spontaneous abortion risk in women with high levels of plasma folate. Women with low folate levels were found to be at significantly increased risk of spontaneous abortion.

No mechanism by which low folate levels could cause spontaneous abortion has yet been identified. Folate deficiency has been tentatively associated with abruptio placentae and preeclampsia,³³ and early vascular effects related to folate deficiency might also increase the risk of spontaneous abortion. Low folate levels increase the incidence of neural tube defects, and fetuses affected with neural tube defects are more commonly aborted spontaneously.³⁴ Women who give birth to infants affected by neural tube defects more often have a history of previous miscarriages.³⁵ However, neural tube defects are rare conditions and could explain only a fraction of the association between low folate levels and spontaneous abortions. It is also known that folate plays a critical role in DNA synthesis and regulation, by providing carbon groups for synthesis of both purine and pyrimidine and by providing methyl groups required for DNA regulation by methylation. Thus, it has been suggested that the rapidly developing cells in the embryo may be damaged by lack of adequate folate.³⁶ Failure to produce sufficient DNA and to regulate DNA function could lead to spontaneous abortion.

When we stratified our analyses by fetal karyotype, we found that the risk was primarily confined to spontaneous abortions with abnormal karyotypes. Most chromosomally abnormal concepti are spontaneously aborted, and we found no differences in the severity of fetal karyotype abnormality by plasma folate levels. It is possible that low folate status causes a larger proportion of these losses to occur in the 6- to 12-week period. Although our study has far better data than previous studies of folate effects on chromosomal status and timing of losses, future studies will be required to resolve these issues.

Our results show that high folate levels are not associated with an increased risk of spontaneous abortion; in fact, there was a nonsignificant trend toward a protective effect associated with high folate levels. Given the concern that folic acid supplements, which produce high folate levels, may increase abortion rates,^13,17 this is an important finding. It is reassuring because today women of childbearing age in many countries are advised to take folic acid supplements daily to prevent neural tube defects, and food fortification has been introduced in other countries to raise folate levels in the population. We were able to study actual folate levels rather than having to rely on self-reported folic acid use as a surrogate for folate status. Few studies have been able to identify fetal losses occurring as early as 6 weeks' gestation. Including cases of early fetal loss in this study enabled us to show that high folate levels were, if anything, associated with a reduced risk of spontaneous abortion even in early pregnancy.

Our study has a number of strengths. Unlike most previous investigations, we were able to examine folate status within a large population-based study, where plasma folate levels were measured at the time of fetal loss in cases, and in pregnant controls matched by gestational age. All patients were interviewed in person using a structured questionnaire. Our results also clearly show the necessity to control for potential confounding factors, especially maternal age. Measurement of plasma cotinine allowed us to adjust for smoking as a confounding factor.

There are also limitations to our study. Only slightly less than 5% of the patients in our study used folic acid supplements, probably because there were no recommendations for pregnant women to use folic acid supplements in Sweden during the study period. The low rate of supplement use resulted in somewhat limited power to study this group separately. It is impossible to identify all early fetal losses since many occur before pregnancy is recognized and before 6 weeks' gestation.³⁷ Karyotyping was not possible for all samples, which is to be expected since all women with spontaneous abortion did not undergo curettage, and among some cases where curettage was performed, no chorionic villi were identified. The success rate of karyotyping increased, as expected, by week of gestation.²⁷

Our results have important public health and clinical implications. High folate status, as increasingly seen in the United States and many other Western countries because of food fortification and supplement use, is not associated with an increased risk of spontaneous abortion, whereas low folate levels are associated with an increased risk of spontaneous abortion. Countries that have fortified their food supplies with folic acid and those considering doing so can be reassured that fortification most likely will not increase spontaneous abortion rates and might even decrease them.