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Folate and human reproduction^1,2,3

¹ From the Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL (TT) and the Office of Dietary Supplements, National Institutes of Health, Bethesda, MD (MFP)

² Supported in part by the Intergovernmental Personnel Act (TT) from the National Institutes of Health.

³ Address reprint requests to T Tamura, Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, Alabama 35294. E-mail: tamurat{at}uab.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION FOLATE STRUCTURE AND FUNCTION FOLATE METABOLISM IN PREGNANCY FOLATE INTAKE AND REQUIREMENT... FOLATE DEFICIENCY IN PREGNANCY FOLATE AND PREGNANCY... FOLATE AND FETAL GROWTH FOLATE AND FETAL DEVELOPMENT FOLATE AND FETAL MALFORMATIONS FOLATE METABOLISM DURING... FOLATE AND MALE REPRODUCTION SUMMARY AND FUTURE STUDIES REFERENCES

 
The influence of folate nutritional status on various pregnancy outcomes has long been recognized. Studies conducted in the 1950s and 1960s led to the recognition of prenatal folic acid supplementation as a means to prevent pregnancy-induced megaloblastic anemia. In the 1990s, the utility of periconceptional folic acid supplementation and folic acid food fortification emerged when they were proven to prevent the occurrence of neural tube defects. These distinctively different uses of folic acid may well be ranked among the most significant public health measures for the prevention of pregnancy-related disorders. Folate is now viewed not only as a nutrient needed to prevent megaloblastic anemia in pregnancy but also as a vitamin essential for reproductive health. This review focuses on the relation between various outcomes of human reproduction (ie, pregnancy, lactation, and male reproduction) and folate nutrition and metabolism, homocysteine metabolism, and polymorphisms of genes that encode folate-related enzymes or proteins, and we identify issues for future research.

Key Words: Folate • folic acid • pregnancy • complications • fetal growth • malformations • lactation • male reproduction

	INTRODUCTION

TOP ABSTRACT INTRODUCTION FOLATE STRUCTURE AND FUNCTION FOLATE METABOLISM IN PREGNANCY FOLATE INTAKE AND REQUIREMENT... FOLATE DEFICIENCY IN PREGNANCY FOLATE AND PREGNANCY... FOLATE AND FETAL GROWTH FOLATE AND FETAL DEVELOPMENT FOLATE AND FETAL MALFORMATIONS FOLATE METABOLISM DURING... FOLATE AND MALE REPRODUCTION SUMMARY AND FUTURE STUDIES REFERENCES

 
The main objective of the present article was to review the evidence for the role of folate nutrition in human reproductive health. The term folate represents all forms of this B vitamin, including the many derivatives found in biological systems; folic acid (pteroylmonoglutamic acid) is the synthetic form found in dietary supplements and fortified foods. The effect of folate status on pregnancy outcomes has long been recognized (1). Since Wills (2) successfully treated megaloblastic anemia in pregnancy with a yeast extract (Marmite; Marmite Food Company Ltd, London, United Kingdom) in 1931, researchers have studied the prevalence and treatment of pregnancy-related folate deficiency and megaloblastic anemia (1). Studies conducted in the 1950s and 1960s led to the recognition that supplementing with folic acid reduced the prevalence of folate deficiency in pregnancy, and prenatal folic acid supplementation in the second and third trimesters became a common public health measure. In 1970, the US Food and Nutrition Board (3) recommended folic acid supplementation (200–400 µg/d) for pregnant women, and this became a common practice in developed countries and substantially reduced pregnancy-induced severe folate deficiency, which can lead to megaloblastic anemia. Prenatal folic acid, along with iron, supplementation reduced the prevalence of 2 of the most common pregnancy-related deficiencies.

The second major achievement with the use of folic acid occurred in the 1990s. For years, researchers suspected an association between maternal folate status and fetal malformations, particularly neural tube defects (NTDs) (4, 5). However, this relation was not confirmed until the early 1990s, when periconceptional folic acid supplementation was found to reduce both the recurrence (6) and occurrence (7) of NTDs. This periconceptional folic acid supplementation no longer aims to treat or prevent pregnancy-induced severe folate deficiency, but to correct abnormal folate metabolism or a subtle folate inadequacy that is possibly present in a certain segment of the population. These discoveries led to mandated folic acid food fortification in several countries (8-11). These distinctively different uses of folic acid—prenatal folic acid supplementation, periconceptional folic acid supplementation, and folic acid fortification of staple foods—may well be ranked among the most significant public health measures for the prevention of pregnancy-related disorders.

In the present review, we focus on the relation between human reproductive outcome and folate nutrition and metabolism, homocysteine metabolism, and polymorphisms of folate-related genes. We conducted a Medline literature search for the terms "folate, folic acid, pregnancy, and lactation." Over 2500 articles were identified after limiting the search to English language articles and studies conducted in humans, and our final update was in May 2005. However, despite our attempts at completeness, important publications may have been excluded from the review.

	FOLATE STRUCTURE AND FUNCTION

TOP ABSTRACT INTRODUCTION FOLATE STRUCTURE AND FUNCTION FOLATE METABOLISM IN PREGNANCY FOLATE INTAKE AND REQUIREMENT... FOLATE DEFICIENCY IN PREGNANCY FOLATE AND PREGNANCY... FOLATE AND FETAL GROWTH FOLATE AND FETAL DEVELOPMENT FOLATE AND FETAL MALFORMATIONS FOLATE METABOLISM DURING... FOLATE AND MALE REPRODUCTION SUMMARY AND FUTURE STUDIES REFERENCES

 
Folic acid consists of a pteridine ring, p-aminobenzoic acid, and glutamic acid (Figure 1). Naturally occurring folates are generally reduced to tetrahydrofolate with hydrogen at the 5, 6, 7, and 8 positions or to dihydrofolate with hydrogen at the 7 and 8 positions, and they have a one-carbon unit (methyl, methylene, methenyl, formyl, or formimino) at the N-5 or N-10 positions, or both. Most folates exist as polyglutamyl folates with a -linked glutamic acid chain (12).

View larger version (5K): [in this window] [in a new window]   FIGURE 1. Structure of folic acid. Natural folates are generally reduced to either tetrahydrofolate with hydrogen at the 5,6,7, and 8 positions or dihydrofolate with hydrogen at the 7 and 8 positions, have a one-carbon unit (methyl, methylene, methenyl, formyl, or formimino) at the 5 or 10 positions or bridging the 5 and 10 positions, and exist as polyglutamyates with a glutamyl chain (R).

Figure 2

Figure 2

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Folate and homocysteine metabolism. H4folate, tetrahydrofolate; 5-CH3-H4folate, 5-methyltetrahydrofolate; 5,10-CH2-H4folate, 5,10-methylenetetrahydrofolate; H2folate, dihydrofolate; 10-HCO-H4folate, 10-formyltetrahydrofolate; 5,10-CH=H4folate, 5,10-methenylterahydrofolate; B-12, methylcobalamin; B-6, pyridoxal phosphate; dUMP, deoxyuridylic acid; dTMP, thymidylate. Numbers represent enzymes. 1: methionine synthase; 2: 5,10-CH2-H4folate reductase; 3: dihydrofolate reductase; 4: thymidylate synthase; 5: serine hydroxymethyltransferase; 6: glycinamide and aminoimidazolecarboxamide ribotide transformylases; 7: 10-HCO-H4folate synthetase and 10-HCO-H4folate dehydrogenase; 8: 5,10-CH=H4folate cyclohydrolase; 9: 5,10-CH2-H4folate dehydrogenase; 10: cystathionine ß-synthase; and 11, cystathionase; 12, various methyltransferases.

	FOLATE METABOLISM IN PREGNANCY

TOP ABSTRACT INTRODUCTION FOLATE STRUCTURE AND FUNCTION FOLATE METABOLISM IN PREGNANCY FOLATE INTAKE AND REQUIREMENT... FOLATE DEFICIENCY IN PREGNANCY FOLATE AND PREGNANCY... FOLATE AND FETAL GROWTH FOLATE AND FETAL DEVELOPMENT FOLATE AND FETAL MALFORMATIONS FOLATE METABOLISM DURING... FOLATE AND MALE REPRODUCTION SUMMARY AND FUTURE STUDIES REFERENCES

Blood folate concentrations in pregnancy
Circulating folate concentrations decline in pregnant women who are not supplemented with folic acid (1, 19, 21-28). Chanarin (1) reported an average decline in serum folate of 10 nmol/L (from 20 to 10 nmol/L) during the 40-wk gestation. This decline may represent a physiologic response to pregnancy, but the mechanism is unknown. The pattern of changes in erythrocyte folate varies, with a decline observed in early pregnancy followed by a slight increase in midpregnancy (1, 25, 26). Possible causes for the declines in blood folate include increased folate demand for the growth of the fetus and uteroplacental organs (1), dilution of folate due to blood volume expansion (27), increased folate catabolism (15-18), increased folate clearance and excretion (19, 20), decreased folate absorption (1), hormonal influence on folate metabolism as a physiologic response to pregnancy (1), and low folate intake (1). Although the techniques used in the studies that were conducted in the 1950s and 1960s may be different from those used in recent days, the fundamental conclusions derived from the results are generally reasonable. It is apparent that the first and last causes mentioned above lead to a decrease in folate stores, but it is less apparent how much of the observed decline is due to the other factors. For example, Bruinse et al (24) measured plasma volume by a dye dilution method and estimated the total circulating amount of folate during both pregnancy and lactation (Figure 3). They found that serum folate declined 42% between 16 and 34 wk of gestation, and this decline was markedly greater than the decline in total circulating folate (28% in the same period), suggesting that the decline in serum folate cannot be explained by hemodilution.

View larger version (10K): [in this window] [in a new window]   FIGURE 3. Total serum folate in circulation during pregnancy and lactation (24). Serum folate concentrations declined by 42% between 16 and 34 wk of gestation, and this decline was markedly greater than the 28% decline in total circulating folate in the same period. Serum folate was measured with a radiobinding assay. Numbers in parentheses represent the number of subjects. Data from this unique study suggest that the decline in serum folate could not be explained by hemodilution. The lack of recovery during lactation suggests that folate nutrition is a continuing burden.

Results on plasma folate clearance after folic acid administration in pregnancy are consistent. Chanarin et al (32) found that folate clearance after an injection of folic acid was higher in pregnant than nonpregnant women, accelerated as pregnancy progressed, and was greater in pregnant women with megaloblastic anemia than in those without. Landon and Hytten (19) estimated 24-h urinary folate serially during pregnancy and postpartum and reported that the mean urinary folate was 32 and 8 nmol/d, respectively. Fleming (20) also reported that mean folate clearance and urinary folate excretion was higher in pregnancy than in the nonpregnant state. Collectively, administered folic acid is more rapidly incorporated into cells and excreted in urine in pregnant than in nonpregnant women.

Whether a decrease in folate absorption contributes to an increased folate requirement in pregnancy is less certain. Chanarin et al (32) found that the peak serum folate concentration after an oral folic acid dose was significantly lower in pregnant than nonpregnant women, which suggested a decrease in folate absorption. However, Landon and Hytten (33) measured plasma folate after an oral folic acid dose in pregnant women, postpartum women, and adult men and found no difference between the 3 groups, which indicated that folate absorption is not altered in pregnancy. McLean et al (34) reported that oral loading with either folic acid or polyglutamyl folate (yeast) resulted in similar increases in serum folate in pregnant women, which suggested that malabsorption of polyglutamyl folate does not occur. The differences in the quantity of folate administered and the methods used to assess folate absorption may explain the discrepancies between these studies.

Several mechanisms, probably in combination, may explain the decline in blood folate in pregnancy. Whatever the reasons for the decline, it is essential that plasma folate be kept above a critical level (>7.0 nmol/L; 1) because plasma folate is the main determinant of transplacental folate delivery to the fetus. Adequate plasma folate is likely to be achieved if prenatal folic acid supplementation or folic acid fortification of foods is practiced. However, in countries without such measures, the risk for gestational folate deficiency remains a public health problem.

Placental folate transfer and metabolism
Although nutrient transfer via the placenta from the maternal plasma pool must be effective to satisfy the demand for fetal growth, information on placental folate transfer is scarce (35-38). Landon et al (35) measured the placental transport of an intravenous dose of [³H]folic acid in women who were scheduled for pregnancy termination. Tritium uptake was greatest in the fetal liver, and an analysis indicated that a peak of reduced folates in the placenta was detected shortly after the dose was intravenously administered, which suggested that folic acid was rapidly metabolized before or at the time of placental transfer. Baker et al (36) found a strong positive association between maternal plasma, cord plasma, and placental folate concentrations, suggesting that transplacental folate delivery depends on maternal plasma folate concentrations.

In placental perfusion studies, Henderson et al (37) found that 5-methyltetrahydrofolate (the main form of folate found in plasma) is extensively and rapidly bound in the placenta but transferred to the fetus in reduced amounts at a slower pace, and that the transfer is bidirectional and saturable. The placental folate receptor (FR) favors the binding of 5-methyltetrahydrofolate and can transfer folate against a concentration gradient; hence, the fetal perfusate is about 3-fold that of the maternal perfusate, which indicates that folate is concentrated during placental transport. Bisseling et al (38) found that the transfer of 5-methyltetrahydrofolate from the maternal to the fetal perfusate was not saturable in a range well above typical physiologic concentrations.

The placenta is rich in FRs and is one of the tissues (along with the choroid plexus and renal proximal tubules) that expresses the -isoform of FR (FR-) in abundance. FR- is a membrane-bound glycosylphosphatidylinositol-linked glycoprotein and the primary form of FR in epithelial cells. The importance of FR- to placental folate transfer is inferred from the fact that an FR- knockout mouse is embryo-lethal, whereas the FR-ß knockout is not (39). Placental folate transport may be mediated by FR- via a 2-step process (40), which includes the binding of 5-methyltetrahydrofolate to placental FR- to produce an intravillous concentration 3 times that of maternal plasma and transporting folate to the fetus against a concentration gradient. Maternal folate status should be kept adequate to maintain plasma folate above a certain concentration for placental transfer. High-affinity binding proteins in the maternal circulation, cord blood, and newborns are derived from membrane-associated precursors (41-43).

The activities of dihydrofolate reductase (Figure 2, reaction 3; 44), folylpoly -glutamate carboxypeptidase II (folate conjugase; 45), methionine synthase (46), MTHFR (47), and serine hydroxymethyltransferase (Figure 2, reaction 5; 48) were detected in human placenta. mRNA expression of mitochondrial C₁-tetrahydrofolate synthase [5,10-methylenetetrahydrofolate dehydrogenase (Figure 2, reaction 9); 5,10-methenyltetrahydrofolate cyclohydrolase (reaction 8); and 10-formyltetrahydrofolate synthetase (reaction 7)] was detected, although the activity was not measured (49). Daly et al (47) reported that placental MTHFR activities were related to C677T MTHFR variants, which suggests a possible association with NTD development. The biochemical and physiologic implications of placental folate metabolism and transport require additional studies, and the use of folates labeled with stable isotopes may make such human studies feasible.

Folate metabolism in the fetus
Many researchers have evaluated the relations between folate concentrations in maternal, cord, and neonatal blood at or shortly after delivery (50-55). They reported that blood folate is markedly elevated in fetuses and newborns, which indicates an effective placental folate transport against a concentration gradient. Despite a several-fold elevation of blood folate in cord or newborn blood over maternal blood, total fetal folate stores do not appear to be large, because fetal hepatic folate content is lower than that in adults. Fetal hepatic folate concentrations ranged from 1.5 to 4.0 µg/g (56-58), whereas adult hepatic folate concentrations were >5.0 µg/g (59, 60). These data suggest that fetal folate acquisition and utilization differ from those of adults. Amniotic fluid folate concentrations range between 3 and 33 nmol/L (61-63), but the metabolic significance of folate in amniotic fluid is unknown.

The ontogeny of folate-dependent enzymes in humans has not been extensively studied due to the obvious difficulty, with a few exceptions. Gaull et al (64) reported that the activities of methionine synthase in fetal tissues are higher than in adult tissues, whereas those of serine hydroxymethyltransferase were similar. Kalinsky et al (65) reported that the activities of hepatic MTHFR and methionine synthase in preterm infants were higher than those in full-term infants or young children, whereas the activities of hepatic formiminotransferase and 5,10-methylenetetrahydrofolate dehydrogenase (Figure 2, reaction 9) were just the opposite. These results suggest dynamic changes in folate-dependent reactions late in fetal life and in neonatal life. In studies conducted in animals, the data indicated that specific activities of some of the folate-dependent enzymes also changed during the perinatal period (66-68). Furthermore, Xiao et al (69) elucidated the effect of maternal folate status on the regulation of fetal FR in mice. However, it is unclear to what extent the findings from the animal studies can be extrapolated to human conditions.

Homocysteine metabolism in pregnancy
Homocysteine metabolism is regulated by the nutritional status of folate, vitamin B-12, and vitamin B-6; and folate status has the strongest influence on plasma tHcy concentration (13). Even though blood folate is generally low in pregnant women, plasma tHcy is low. Kang et al (70) first reported that plasma tHcy is significantly lower in pregnant than nonpregnant women. Subsequently, Andersson et al (71) reported that the decline in tHcy started in the first trimester with a nadir reached in the second trimester. Research interest in homocysteine metabolism intensified in the area of obstetrics in the 1990s (28, 54, 55, 72-77), because hyperhomocysteinemia could lead to altered placental circulation. The interest in this association was further strengthened by the finding that periconceptional folic acid supplementation prevented NTDs (78-83).

Possible mechanisms for the decline in plasma tHcy in pregnancy include increased methionine requirement for fetal growth (70, 71), hemodilution due to plasma volume expansion (73, 75), changes in endocrine functions (70, 71), increased renal homocysteine clearance (77), and decreased plasma albumin to which homocysteine is bound (75). Of these, endocrine changes are likely the major reason for the observed decline. As shown in Table 1, maternal plasma tHcy concentrations at delivery are slightly higher than those in cord plasma and are several-fold those in amniotic fluid (54, 55, 72, 73). Malinow et al (73) found large tHcy differences between umbilical vein and artery blood, indicating fetal homocysteine uptake and metabolism. These findings are consistent with elevated fetal methionine synthase activity (64). In the fetal liver, no cystathionase (Figure 2, reaction 11) activity was detected and cystathionine ß-synthase (Figure 2, reaction 10) activity was only 20% of adult levels (84), which indicated that transmethylation is more active than transsulfuration in the fetus. Whether already low tHcy concentrations in pregnant women decline further after folic acid fortification remains to be seen.

	FOLATE INTAKE AND REQUIREMENT IN PREGNANCY

TOP ABSTRACT INTRODUCTION FOLATE STRUCTURE AND FUNCTION FOLATE METABOLISM IN PREGNANCY FOLATE INTAKE AND REQUIREMENT... FOLATE DEFICIENCY IN PREGNANCY FOLATE AND PREGNANCY... FOLATE AND FETAL GROWTH FOLATE AND FETAL DEVELOPMENT FOLATE AND FETAL MALFORMATIONS FOLATE METABOLISM DURING... FOLATE AND MALE REPRODUCTION SUMMARY AND FUTURE STUDIES REFERENCES

These 2 factors—the new food folate assay and DFEs—make the interpretation of folate intake data challenging. Furthermore, only extremely limited information on the folate bioavailability of individual foods exists (90-92). This difficulty will remain until food composition tables incorporate reliable data and more information on food folate bioavailability is attained. Achieving these goals will take a lot of work, but knowledge of the composition and bioavailability of food folate is fundamental to understanding the role of folate in human nutrition.

Folate intake in pregnancy
Chanarin et al (93) measured folate content in individually prepared meals collected from pregnant women and found that mean folate intake was 676 µg/d, which significantly correlated with erythrocyte folate concentrations. However, this value is considered extremely high. Moscovitch and Cooper (94) measured the folate content of meals consumed by women who were in the second trimester of pregnancy and who prepared duplicate diets and found the mean folate intake was 242 µg/d. The large difference between the 2 groups may be due to differences in food selection and folate assay methods. Since these reports >30 y ago, there have been no reports on direct folate analyses of self-selected diets consumed by pregnant women. Instead, investigators have estimated dietary intakes by dietary recalls or food-frequency questionnaires and calculated the values for folate intake from food tables (95-101). In these reports, the mean folate intakes of pregnant women varied widely from 85 to 668 µg/d. These data were obtained without trienzyme extraction, and most were obtained before the initiation of folic acid fortification of foods. The results of the 2 studies that included fortified values in the calculation indicated that the mean folate intake of pregnant women was 600 DFEs/d (99, 101). Stark et al (101) reported that >50% of inner-city black pregnant women did not meet the recommended 600 DFEs/d.

Folate requirement for pregnant women
In 1970, the US Food and Nutrition Board (3) set the recommended folate intake for pregnant women at 400 µg/d; this was reduced to 270 µg/d in 1989 mainly because of data showing that this amount was typically ingested by healthy folate-replete adults (102). The third National Health and Nutrition Examination Survey dietary data (1989–1991) indicated that the mean folate intake of US women of childbearing age was 230 µg/d (103). The recommendation was increased to 600 DFEs/d in 1999, after the bioavailability of food folate and folic acid was considered (88). Caudill et al (104) monitored blood folate and urinary 5-methyltetrahydrofolate excretion in a metabolic study conducted in pregnant and nonpregnant women who consumed a diet containing only 120 µg folate/d with additional supplements of folic acid (330 or 730 µg/d). They concluded that 450 µg folate/d (600 DFEs/d) was sufficient to maintain adequate folate status in pregnant women. As reviewed above, most of the estimated dietary folate intakes were <400 µg/d.

Studies were conducted in the 1960s to determine the quantity of folic acid required, in addition to regular dietary intake, to maintain adequate folate status in pregnancy (1, 105-107). Willoughby and Jewell (105) measured the dose-response effect of prenatal folic acid (0–530 µg/d) on serum folate concentrations in the postpartum period and found that serum folate increased linearly with the amount of folic acid supplemented, which was given from 3 mo of gestation to delivery (Figure 4). To keep postpartum serum folate >7.0 nmol/L, they concluded that the minimum dose of folic acid needed during late pregnancy, in addition to a dietary folate intake of 50 µg/d, was close to 300 µg/d. Hansen and Rybo (106) conducted a similar study by monitoring blood folate concentrations in late pregnancy. Plasma folate increased linearly when folic acid was given at 200–500 µg/d. They suggested that an oral dose of 200 µg folic acid/d is close to the minimum requirement to maintain normal blood folate concentrations, although dietary folate intake was not reported in this study. Colman et al (108) conducted a pioneering study providing evidence that the folic acid fortification (300–1000 µg/d) of foods (maizemeal) improved folate status late in pregnancy. They found that erythrocyte folate responded linearly to the amount of folic acid added and suggested that maize containing 300 µg/d of fortified folic acid is effective in preventing folate depletion late in pregnancy.

View larger version (8K): [in this window] [in a new window]   FIGURE 4. Dose-response effect of prenatal folic acid on serum folate concentrations 2–4 d after delivery (105). In addition to a mean dietary folate intake of 50 µg/d, subjects were given oral folic acid doses ranging from 0 to 450 µg/d from 3 mo of gestation to delivery. Numbers in parentheses represent the number of subjects. Serum folate at 2–4 d postpartum was measured with an L. rhamnosus assay. The data suggest that the minimum folic acid dose needed during pregnancy to keep postpartum serum folate concentrations >7.0 nmol/L was 300 µg/d (in addition to dietary intake).

	FOLATE DEFICIENCY IN PREGNANCY

TOP ABSTRACT INTRODUCTION FOLATE STRUCTURE AND FUNCTION FOLATE METABOLISM IN PREGNANCY FOLATE INTAKE AND REQUIREMENT... FOLATE DEFICIENCY IN PREGNANCY FOLATE AND PREGNANCY... FOLATE AND FETAL GROWTH FOLATE AND FETAL DEVELOPMENT FOLATE AND FETAL MALFORMATIONS FOLATE METABOLISM DURING... FOLATE AND MALE REPRODUCTION SUMMARY AND FUTURE STUDIES REFERENCES

 
In addition to the blood folate assay, various biochemical (ie, formiminoglutamic acid analysis after a histidine load or deoxyuridine-suppression test) and hematologic (ie, neutrophil lobe count, mean corpuscular volume, or bone marrow test) tests were used to diagnose folate deficiency, assess the degree of folate deficiency, or measure responses to folic acid therapy in pregnancy (50, 93, 105, 109-113). In the 1990s, the plasma tHcy assay was added as a tool to assess folate adequacy. Of these tests, assays of folate and tHcy concentrations are the most extensively used; the other tests noted are used less because they lack sensitivity and specificity.

Before prenatal folic acid supplementation effectively reduced the prevalence of folate deficiency in developed countries, many cases of folate deficiency or megaloblastic anemia in pregnancy were reported (50, 114, 115). However, folate deficiency was prevalent worldwide in the 1970s. For example, >30% of women with pregnancy-related anemia in Venezuela were folate deficient (116), and a prevalence of folate deficiency of >10% was reported in pregnant women in Australia and the United States (117, 118). The presence of folate deficiency with or without megaloblastic anemia is still a public health problem for pregnant women in developing countries (119-121). A short interpregnancy interval associated with inadequate folate status was found to lead to unfavorable pregnancy outcome (96, 122, 123).

	FOLATE AND PREGNANCY COMPLICATIONS

TOP ABSTRACT INTRODUCTION FOLATE STRUCTURE AND FUNCTION FOLATE METABOLISM IN PREGNANCY FOLATE INTAKE AND REQUIREMENT... FOLATE DEFICIENCY IN PREGNANCY FOLATE AND PREGNANCY... FOLATE AND FETAL GROWTH FOLATE AND FETAL DEVELOPMENT FOLATE AND FETAL MALFORMATIONS FOLATE METABOLISM DURING... FOLATE AND MALE REPRODUCTION SUMMARY AND FUTURE STUDIES REFERENCES

 
Various pregnancy complications have been associated with folate deficiency, but findings are equivocal. Discrepancies have resulted because many studies, out of necessity, were performed with a limited number of patients that yielded weak statistical power to provide firm conclusions, and because criteria for the evaluation of folate status varied between the studies. We review the relation of folate deficiency to each complication independently and discuss the findings on homocysteine metabolism or polymorphisms of genes encoding folate-related proteins.

Placental abruption
In the 1960s and 1970s, many studies evaluated the association of folate deficiency with placental abruption, a premature detachment of the placenta (124-133). Only 4 studies, which involved >600 cases, found folate deficiency to be associated with an increased risk of placental abruption (124, 125, 128, 129); the remaining studies, which involved 300 cases, found no association (126, 127, 130-133). These findings indicate that the association is possible, but not certain, and a mechanism for the possible association is unknown.

Because of the possible vasculotoxicity attributed to hyperhomocysteinemia (14), interest in studying the relation between tHcy and placental abruption was renewed in the 1990s. Most of the studies indicated an association of hyperhomocysteinemia with an increased risk for placental abruption (134-139). However, plasma tHcy analysis in these studies was made after the onset of symptoms; thus, the causal effect of tHcy cannot be established. Steegers-Theunissen et al (138) reported that an association between elevated tHcy and placental abruption was no longer significant after adjustment for the time between actual postpartum tHcy analysis and delivery.

The prevalence of placental abruption is reported to be associated with polymorphisms of folate-related genes. The abbreviations of these genes are shown in Table 2. A few research groups showed associations of placental abruption with maternal variants of the MTHFR gene (C677T, A1298C, or both; 140, 141), whereas others reported no such association (142, 143). Parle-McDermott et al (143) reported that the 1958AA variant of the gene encoding 10-formyltetrahydrofolate synthetase (Figure 2, reaction 7), a part of the C₁-tetrahydrofolate synthase, was an independent risk factor for placental abruption. Associations between placental abruption and altered folate or homocysteine metabolism appear to be weak. Possible associations between placental abruption and altered folate or homocysteine metabolism or polymorphisms of folate-related genes require additional study with attention to environmental factors, such as maternal folate status, that may exert an influence on these relations.

Of >30 studies reviewed, 11 included values for both plasma tHcy and folate (146-149, 153, 154, 156, 158, 159, 161-163) (Table 3). Most indicated that plasma folate concentrations were similar between women with and without preeclampsia. One showed decreased plasma folate in women with preeclampsia (148), whereas 3 indicated increased plasma folate (146, 158, 159). The reason for this discrepancy is unknown. Folic acid supplementation in pregnancy decreases plasma tHcy (165), but whether such a reduction decreases the risk of preeclampsia is unknown. In a comparison of the rate of preeclampsia before (1990–1997) and after (1998–2000) folic acid fortification of food in Canada, Ray et al (166) reported no effect of increased folate intake on the risk of preeclampsia. Evidence appears to indicate that poor folate status is not responsible for the risk of preeclampsia; thus, improvement in folate status by folic acid supplementation or fortification may not be effective in preventing preeclampsia.

Spontaneous abortion and stillbirth
The causes of spontaneous abortion (loss before 20 wk of gestation) or stillbirths (baby born dead after 20 wk of gestation) are considered to be multifactorial and are often unclear.

Spontaneous abortion
In the 1960s, Martin et al (179) reported that serum folate was low in women who had a history of spontaneous abortion and that folic acid supplementation prevented recurrent abortion, whereas Chanarin et al (107) reported that women had similar erythrocyte folate concentrations regardless of their history of miscarriage. Researchers later reported no association between folate status and spontaneous abortion, but the statistical power was not sufficient due to small sample sizes (180-183). In a large Swedish cohort with and without a history of spontaneous abortion, George et al (184) reported that women with lower plasma folate (<4.9 nmol/L) had a greater risk for miscarriage than did those with higher plasma folate, particularly when fetal chromosomal anomalies were present. Gindler et al (185) evaluated the effect of folic acid supplementation on the risk of NTDs in China and reported that the supplementation did not alter the risk of miscarriage (186). Similarly, Czeizel et al (187) reported no effect of folic acid supplementation on the rates of spontaneous abortion or stillbirth.

After Steegers-Theunissen et al (134) provided the first evidence of an association between hyperhomocysteinemia and miscarriage in 1992, many researchers performed similar evaluations (188-190). These studies and a meta-analysis indicated that elevated plasma tHcy may be related to an increased risk of spontaneous abortion (191).

On the basis of the hypothesis that abnormal procoagulant activity has a potential role in the etiology of recurrent abortion due to impaired placental function, researchers examined whether the risk was associated with maternal polymorphisms of MTHFR (C677T, A1298C, or C776G) along with various coagulation factor genes (189, 192-195). Except for 2 reports (189, 194), these studies suggested that variants of MTHFR alone do not increase the risk of spontaneous abortion. A meta-analysis of data from all published studies should be performed to confirm or refute the association. Isotalo et al (196) found that the fetal 677CT/1298CC or 677TT/1298CC variants increased the risk of spontaneous abortion. Zetterberg et al (197) also reported an increased risk of spontaneous abortion for the combination of fetal 677TT/TC and 776GG/CG MTHFR variants, although Volcik et al (198) reported that the 677CT/1298CC variants did not affect fetal viability. These inconsistencies warrant additional studies, and the risk of miscarriage associated with maternal and fetal polymorphisms may have important implications for genetic counseling.

Stillbirth
Giles (50) and Ainley (115) reported that the stillbirth rate was higher in women with megaloblastic anemia than in those without, whereas Varadi et al (199) found no such association. In a large Norwegian female population with a history of stillbirth, Vollset et al (137) reported that women in the higher quartile for plasma tHcy had a significantly higher risk of stillbirth. However, the analysis of tHcy in this study was made 25 y after the index pregnancy. Whether it is reasonable to associate plasma tHcy with an incident that took place years before is uncertain (200). Only a few studies tested whether the risk of stillbirth was associated with MTHFR polymorphisms (141, 201, 202), and the findings are equivocal. Additional studies are needed to clarify whether such an association exists.

Other pregnancy complications
Other possible associations of abnormal folate nutrition and metabolism with pregnancy complications include relations between low blood folate, elevated tHcy, or variants of folate-related genes and threatened abortion (22), vaginal bleeding (22, 128, 131, 203), placental infarction (135, 151), or premature rupture of the membrane (204, 205). Conclusions about these associations cannot be reached because few cases have been examined and additional investigation is needed.

	FOLATE AND FETAL GROWTH

TOP ABSTRACT INTRODUCTION FOLATE STRUCTURE AND FUNCTION FOLATE METABOLISM IN PREGNANCY FOLATE INTAKE AND REQUIREMENT... FOLATE DEFICIENCY IN PREGNANCY FOLATE AND PREGNANCY... FOLATE AND FETAL GROWTH FOLATE AND FETAL DEVELOPMENT FOLATE AND FETAL MALFORMATIONS FOLATE METABOLISM DURING... FOLATE AND MALE REPRODUCTION SUMMARY AND FUTURE STUDIES REFERENCES

 
Folate status and fetal growth
Birth weight is probably the most important pregnancy outcome, because fetal growth restriction (FGR; birth weight <10th percentile of a given population) is highly related to high mortality and morbidity (206). Many researchers examined the relations between birth weight and the rates of FGR, low-birth weight (<2500 g), or very-low-birth weight (<1500 g) and maternal folate status (207-211), folate intake or folic acid supplementation (93, 95, 212, 213), or megaloblastic anemia in pregnancy (111). Conclusions as to whether maternal folate nutrition and metabolism affect fetal growth could not be made because of the lack of consistency between the studies and the insufficient statistical power due to small sample sizes. It is essential to understand that potential deficiencies in nutrients other than folate acting as confounding variables make it difficult to draw a solid conclusion, and this issue applies to interpreting data on the association between folate status and other pregnancy outcomes.

In 1992, Burke et al (214) first noted the possible relation between elevated tHcy and FGR. In a large Norwegian cohort, Vollset et al (137) later reported that the risk of FGR infants was significantly increased in women who were in the higher quartiles of tHcy than those in the lower quartiles, and others reported similar findings (139, 215). However, in other studies, elevated tHcy did not increase the risk of having an FGR infant (138, 149, 216-218). The relation of FGR risk with maternal or fetal MTHFR polymorphisms is also controversial (140-143, 170, 172, 219-222). Kupferminc et al (170) reported an increased risk of FGR in women who had the 677TT variant. In a large Norwegian cohort, Nurk et al (141) found that associations between the risk of FGR, low-birth weight, or very-low-birth weight and the C677T or A1298C variants were marginally significant. In contrast, Gebhardt et al (140) reported that C677T, A1298C, or both, variants were not related to FGR, and similar findings were reported by others (172, 219-221). Wisotzkey et al (222) reported that fetal growth was not related to fetal MTHFR polymorphisms. It appears that no firm consensus can be drawn about whether maternal folate nutrition and metabolism influences fetal growth.

Folic acid supplementation and fetal growth
Twelve studies (Table 4) evaluated the effect of prenatal folic acid supplementation on birth weight (187, 223-233). In 7 of the 12 studies, supplementation increased birth weight (223, 225, 226, 228, 229, 230-232). In contrast, no such effect was found in the remaining studies, probably due to sufficient maternal folate status early in pregnancy and the time of supplementation. Possible reasons for the discrepancy include race, maternal size, initial folate status, socioeconomic status, and dietary habits, including the intake of folate and other nutrients. For example, an impressive birth weight increase (300 g) was seen in Bantu women, whose diet consisted mainly of maizemeal with infrequent vegetable consumption, whereas no effect was seen in white women, whose diet habitually contained vegetables and fruit (223). The overall findings of these studies indicate that adequate folate status promotes fetal growth. This is supported by the recent report of an analysis of >5 million birth records in California that showed small but significant reductions in the rates of low-birth weight and very-low-birth weight infants and preterm delivery after folic acid fortification (234).

	FOLATE AND FETAL DEVELOPMENT

TOP ABSTRACT INTRODUCTION FOLATE STRUCTURE AND FUNCTION FOLATE METABOLISM IN PREGNANCY FOLATE INTAKE AND REQUIREMENT... FOLATE DEFICIENCY IN PREGNANCY FOLATE AND PREGNANCY... FOLATE AND FETAL GROWTH FOLATE AND FETAL DEVELOPMENT FOLATE AND FETAL MALFORMATIONS FOLATE METABOLISM DURING... FOLATE AND MALE REPRODUCTION SUMMARY AND FUTURE STUDIES REFERENCES

 
Maternal folate status and child neurodevelopment
Mental retardation is one of the clinical features of inborn errors of folate metabolism, although the mechanisms by which an altered folate metabolism causes retardation are unknown (239). Studies of the consequences of inadequate prenatal folate status on the neurodevelopment of infants and children are scarce, although prenatal folate deficiency is known to be detrimental to neurodevelopment in animals (240-243). Two studies that evaluated this connection yielded conflicting data (244, 245). This may be due to differences in the degree of maternal folate deficiency, the age of children at assessment, and the sensitivity and specificity of the assessment tools used.

Folate and Down syndrome
Cystathionine ß-synthase activity is high in patients with Down syndrome (trisomy 21) because the gene encoding for cystathionine ß-synthase resides on chromosome 21 (246), and this leads to increased transsulfuration and reduced plasma tHcy (247). The distribution of the C677T variant was reported to be higher in mothers of children with Down syndrome than in mothers of non-Down syndrome children, which suggests that this variant is a risk factor for Down syndrome (248, 249). The T allele of the C677T variant was transmitted at a higher rate to children with Down syndrome than to children without Down syndrome (250), whereas no such increase was reported in the variant in mothers of children with Down syndrome (251-254). O'Leary et al (255) reported that the frequency of the A66G variant of the methionine synthase reductase gene was higher in mothers of children with Down syndrome than in mothers of children without Down syndrome. Fillon-Emery et al (256) analyzed polymorphisms of genes including MTHFR (C677T and A1298C), methionine synthase (A2756G), methionine synthase reductase (A66G), and reduced-folate carrier (RFC1; A80G) in adults with Down syndrome and found that only the distribution of the variant in the RFC gene was different from that of control subjects.

Because of a possible influence of folate inadequacy on genetic expression, the effect of folic acid fortification on the chromosomal anomalies was examined. No changes in the prevalence of chromosomal abnormalities were found after fortification (257, 258), and the risk for autosomal trisomy was not affected by maternal periconceptional multivitamin use (259). The possible association of folate-dependent enzyme gene polymorphisms with the increased risk of Down syndrome is attractive. Whether these positive data withstand additional scrutiny remains to be seen.

	FOLATE AND FETAL MALFORMATIONS

TOP ABSTRACT INTRODUCTION FOLATE STRUCTURE AND FUNCTION FOLATE METABOLISM IN PREGNANCY FOLATE INTAKE AND REQUIREMENT... FOLATE DEFICIENCY IN PREGNANCY FOLATE AND PREGNANCY... FOLATE AND FETAL GROWTH FOLATE AND FETAL DEVELOPMENT FOLATE AND FETAL MALFORMATIONS FOLATE METABOLISM DURING... FOLATE AND MALE REPRODUCTION SUMMARY AND FUTURE STUDIES REFERENCES

 
Folate and NTDs
In 1976, Smithells et al (5) suggested that folate deficiency was a cause of NTDs because women with an NTD infant had low blood folate; later, Smithells et al (260) reported that periconceptional vitamin supplementation, which included folic acid, reduced the recurrence of NTD pregnancies. Others also reported the same effectiveness of periconceptional supplementation with folic acid alone or in combination with multivitamins (261, 262). Although the nonrandomized nature of these trials was criticized, the apparently clear and positive findings became a powerful driving force for the Medical Research Council to launch a large-scale, randomized trial to evaluate the effect of multivitamin supplementation with and without folic acid on the recurrence of NTDs (6). Several studies evaluated the association between folate status in early pregnancy and the risk of NTDs and provided conflicting data (263-266), indicating the difficulty of identifying NTD pregnancies by a single blood folate analysis early in pregnancy. Similarly, epidemiologic studies conducted in the 1980s provided mixed results (267-269), whereas additional studies conducted in the 1990s were consistent with the effectiveness of folate suppplementation (270-272).

Periconceptional folic acid supplementation and NTD prevention
In 1991, the Medical Research Council group (6) performed a randomized daily periconceptional folic acid (4.0 mg) supplementation trial to evaluate the effect on the recurrence of infants born with NTDs in women who had a history of infants born with NTDs (high-risk population) and found that the recurrence was only 5 in 593 women who received folic acid supplements and 21 in 602 women who did not. The mean risk of recurrence was 0.28 (95% CI: 0.12, 0.71) for the women who received folic acid, which showed the benefit of folic acid given before the critical period for neural-tube closure (4 wk of gestation). The outcome of the trial may be the most significant for disease prevention in the folate research area, and provided support for the hypothesis put forward by Smithells (5, 260). Periconceptional folic acid supplementation is a clear departure from the prenatal supplementation that was established earlier for the prevention of folate deficiency.

After 1991, research on the mechanisms by which folic acid prevents NTDs intensified. In the 10 y before the trial (1981–1990), there were 4 articles per year on "NTDs and folic acid;" the rate increased to 67 articles per year in the next 10 y (1992–2001). The topics included the relation between the risk of NTDs and altered folate or homocysteine metabolism and polymorphisms of folate-related genes. Interest in homocysteine and polymorphisms was strong, because these coincided with the recognition of possible vasotoxicity of elevated tHcy (14) and rapid advances in molecular genetics (273, 274).

In 1992, Kirke et al (275) reported that periconceptional folic acid supplementation (0.36 mg/d) reduced the recurrence of NTDs in a small group of Irish women who had an NTD infant, which provided supporting evidence for the protective effect of folic acid. In 1992, Czeizel and Dudás (7) reported on a large-scale trial of periconceptional folic acid (0.8 mg/d) in Hungarian women without a history of NTDs (first occurrence). None of the 2394 women who received folic acid supplements had an NTD infant, whereas 6 of the 2310 women who did not receive supplementation had an NTD infant. The prevention of first NTD occurrence by periconceptional folic acid supplementation was thus established. The importance of this finding cannot be overemphasized, because most NTDs are first occurrences.

Berry et al (186) conducted a study in 2 areas of China between 1993 and 1995. Although this was not a randomized trial, the NTD occurrence rate was compared between 130 142 women who elected to receive folic acid supplements (0.4 mg/d) starting at their premarital examination until the end of the first trimester and 117 689 women who elected not to receive folic acid supplementation. Overall, 102 fetuses or infants of women who received folic acid supplementation and 173 of those who did not receive folic acid supplementation had NTDs, a significant difference. In northern China, where the prevalence of NTDs was high, folic acid supplementation reduced the rate from 4.8 to 1.0 per 1000 births (80% reduction), and in the southern region, folic acid supplementation reduced the rate from 1.0 to 0.6 per 1000 births (40% reduction). Periconceptional supplementation of a relatively low dose of folic acid reduced the risk for NTDs in areas with high and low NTD prevalence.

Against seemingly solid scientific evidence of folic acid supplementation for the prevention of NTDs, Kalter (276) cautioned that trials tend to have unavoildable methodologic uncertainties, such as subject selection and recruitment, type of supplements, and unexplained reasons for high or low NTD risk in certain populations. However, with endorsements from scientific communities, governments moved to implement policies for periconceptional folic acid supplementation and folic acid fortification of foods.

Awareness of the importance of folate intake for NTD prevention
The above studies provided firm scientific evidence of the importance of folic acid supplementation for the prevention of NTDs. Although folic acid supplementation was encouraged by prenatal health care workers, the awareness and practice of supplementation by women of childbearing age was often unsatisfactory. In the past decade, the reported rates of knowledge of the importance of adequate folate intake were 17–77% in young women worldwide (277-281). Reports by the Centers for Disease Control and Prevention indicated that the rate improved from 48% to 77% in the past decade (278, 280). Ray et al (282) reviewed 34 studies on the use of periconceptional folic acid by young women and found that the rate varied from 0.9% to 50%. The connection between awareness and practice depended on the women's socioeconomic status, education, race, location of residence, and the presence of an NTD-affected child within the family. Efforts to educate young women on the importance of high folate intake before conception should be intensified.

Results of periconceptional folic acid supplementation
The transfer of a successful intervention to community programs is not always straightforward. Not surprisingly, the prevalence of NTDs either declines or remains unchanged in areas of the world that have programs promoting folic acid supplementation (283-286). Botto et al (286) analyzed >13 million birth records from 10 countries and found no detectable reduction in the NTD prevalence between 1988 and 1998. Busby et al (287) reported that the NTD prevalence declined by only 0.9% in European countries<