Women Taking a Folic Acid Supplement in Countries with Mandatory Food Fortification Programs May Be Exceeding the Upper Tolerable Limit of Folic Acid: A Systematic Review

Background: In preconception and pregnancy, women are encouraged to take folic acid-based supplements over and above food intake. The upper tolerable limit of folic acid is 1000 mcg per day; however, this level was determined to avoid masking a vitamin B12 deficiency and not based on folic acid bioavailability and metabolism. This review’s aim is to assess the total all-source intake of folate in women of childbearing age and in pregnancy in high-income countries with folate food fortification programs. Methods: A systematic search was conducted in five databases to find studies published since 1998 that reported folate and folic acid intake in countries with a mandatory fortification policy. Results: Women of childbearing age do not receive sufficient folate intake from food sources alone even when consuming fortified food products; however, almost all women taking a folic acid-based supplement exceed the upper tolerable limit of folic acid intake. Conclusions: Folic acid supplement recommendations and the upper tolerable limit of 1000 mcg set by policy makers warrant careful review in light of potential adverse effects of exceeding the upper tolerable limit on folic acid absorption and metabolism, and subsequent impacts on women’s health during their childbearing years.


Introduction
Governments worldwide encourage women of childbearing age to take folic acid (FA) prior to pregnancy to avoid neural tube defects (NTDs) [1][2][3][4][5][6][7][8][9][10] and some governments (e.g., Australia, Canada and United States) have implemented a FA food fortification program to ensure that women who unintentionally conceive or are not using a folic acid supplement have sufficient folate to prevent neural tube defects [11,12]. Since the mandatory folic acid fortification program began in 2009, Australian policy makers have recommended 400 mcg of FA in addition to fortification for individuals considering conception [6].
While the fortification policy has prevented NTDs, emerging research has documented the presence of unmetabolized folic acid (UMFA) in the serum in people in countries that have a FA fortification program and/or take a FA supplement [13][14][15]. While the effect of this is unclear, some have raised concerns regarding UMFA and adverse health effects [16][17][18][19][20][21].

Folate Metabolism
Folate-dependent biochemical processes are influenced by the form and bioavailability of folate [22]. Folate is a generic term for vitamin B9 that includes several forms, including that naturally found in food (natural folate). All folate forms have a common structure but  Food folate is naturally present in foods such as leafy green vegetables and is in a reduced form [13]. This natural folate exists in a polyglutamate form and needs to be further hydrolyzed to a monoglutamate form by the intestinal lumen to be transported [24]. The intestinal absorption of polyglutamate derivatives of tetrahydrofolate (THF) into monoglutamates is carried out by the brush border enzyme glutamate carboxypeptidase II (GPCII) [25]. This crossing of the apical brush border is carried out via two transporters, the proton-coupled folate transporter (PCFT) and the reduced folate carrier (RFC) [26,27]. Most of the absorbed natural folate is metabolized to 5-methyltetrahydrofolate  in the intestine and/or liver [26] because the RFC has a higher affinity for reduced folates such as 5-MTHF compared to FA [26]. The fate of both natural folate and FA is to be metabolized to 5-MTHF [28]. The RFC transporter is found throughout the intestinal tract and is highly expressed in the liver and placenta [26]. The PCFT's key role is as the major transporter of dietary folate in the proximal small intestine, specifically in the brush border of the duodenum and jejunum and has an optimal function with an acidic PH but has a higher affinity to FA than it does to reduced folates such as 5-MTHF [16,26,29,30]. Once absorbed by the RFC or PCFT, the folate is transported into the hepatic portal vein by multidrug resistance-associated protein 3 (MRP3) and then to the liver, where it is taken up via hepatocytes [26]. The intestine can convert reduced folates to 5-MTHF quite well; however, it is the first-pass metabolism and intestinal absorption where the difference between the metabolism of FA and the reduced folate forms exists as its reduction and methylation are dose dependent [14,28].
While natural folates do not need to be converted to 5-MTHF to cross the mucosal cell ( Figure 3) and enter the portal vein to the blood, they do need to be metabolized to 5-MTHF in the liver or released into the blood or bile. [27]. One small study showed that the majority of FA (at a physiologic dose) passes into the portal vein in an unmodified form, whilst monoglutamates are almost all converted to 5-MTHF [31]. All forms of folate must be converted to 5-methyltetrahydrofolate (5-MTHF), which is the main form of folate Folate-dependent biochemical processes are influenced by the form and bioavailability of folate [22]. Folate is a generic term for vitamin B9 that includes several forms, including that naturally found in food (natural folate). All folate forms have a common structure but differ based on the pteridine ring and whether it is reduced or oxidized [23]. Folate derived from food such as leafy green vegetables are polyglutamates (Figure 1), whereas synthetic FA is a monoglutamate (Figure 2) [21].  Food folate is naturally present in foods such as leafy green vegetables and is in a reduced form [13]. This natural folate exists in a polyglutamate form and needs to be further hydrolyzed to a monoglutamate form by the intestinal lumen to be transported [24]. The intestinal absorption of polyglutamate derivatives of tetrahydrofolate (THF) into monoglutamates is carried out by the brush border enzyme glutamate carboxypeptidase II (GPCII) [25]. This crossing of the apical brush border is carried out via two transporters, the proton-coupled folate transporter (PCFT) and the reduced folate carrier (RFC) [26,27]. Most of the absorbed natural folate is metabolized to 5-methyltetrahydrofolate  in the intestine and/or liver [26] because the RFC has a higher affinity for reduced folates such as 5-MTHF compared to FA [26]. The fate of both natural folate and FA is to be metabolized to 5-MTHF [28]. The RFC transporter is found throughout the intestinal tract and is highly expressed in the liver and placenta [26]. The PCFT's key role is as the major transporter of dietary folate in the proximal small intestine, specifically in the brush border of the duodenum and jejunum and has an optimal function with an acidic PH but has a higher affinity to FA than it does to reduced folates such as 5-MTHF [16,26,29,30]. Once absorbed by the RFC or PCFT, the folate is transported into the hepatic portal vein by multidrug resistance-associated protein 3 (MRP3) and then to the liver, where it is taken up via hepatocytes [26]. The intestine can convert reduced folates to 5-MTHF quite well; however, it is the first-pass metabolism and intestinal absorption where the difference between the metabolism of FA and the reduced folate forms exists as its reduction and methylation are dose dependent [14,28].
While natural folates do not need to be converted to 5-MTHF to cross the mucosal cell ( Figure 3) and enter the portal vein to the blood, they do need to be metabolized to 5-MTHF in the liver or released into the blood or bile. [27]. One small study showed that the majority of FA (at a physiologic dose) passes into the portal vein in an unmodified form, whilst monoglutamates are almost all converted to 5-MTHF [31]. All forms of folate must be converted to 5-methyltetrahydrofolate (5-MTHF), which is the main form of folate Food folate is naturally present in foods such as leafy green vegetables and is in a reduced form [13]. This natural folate exists in a polyglutamate form and needs to be further hydrolyzed to a monoglutamate form by the intestinal lumen to be transported [24]. The intestinal absorption of polyglutamate derivatives of tetrahydrofolate (THF) into monoglutamates is carried out by the brush border enzyme glutamate carboxypeptidase II (GPCII) [25]. This crossing of the apical brush border is carried out via two transporters, the proton-coupled folate transporter (PCFT) and the reduced folate carrier (RFC) [26,27]. Most of the absorbed natural folate is metabolized to 5-methyltetrahydrofolate (5-MTHF) in the intestine and/or liver [26] because the RFC has a higher affinity for reduced folates such as 5-MTHF compared to FA [26]. The fate of both natural folate and FA is to be metabolized to 5-MTHF [28]. The RFC transporter is found throughout the intestinal tract and is highly expressed in the liver and placenta [26]. The PCFT's key role is as the major transporter of dietary folate in the proximal small intestine, specifically in the brush border of the duodenum and jejunum and has an optimal function with an acidic PH but has a higher affinity to FA than it does to reduced folates such as 5-MTHF [16,26,29,30]. Once absorbed by the RFC or PCFT, the folate is transported into the hepatic portal vein by multidrug resistance-associated protein 3 (MRP3) and then to the liver, where it is taken up via hepatocytes [26]. The intestine can convert reduced folates to 5-MTHF quite well; however, it is the first-pass metabolism and intestinal absorption where the difference between the metabolism of FA and the reduced folate forms exists as its reduction and methylation are dose dependent [14,28].
While natural folates do not need to be converted to 5-MTHF to cross the mucosal cell ( Figure 3) and enter the portal vein to the blood, they do need to be metabolized to 5-MTHF in the liver or released into the blood or bile [27]. One small study showed that the majority of FA (at a physiologic dose) passes into the portal vein in an unmodified form, whilst monoglutamates are almost all converted to 5-MTHF [31]. All forms of folate must be converted to 5-methyltetrahydrofolate (5-MTHF), which is the main form of folate found in plasma, accounting for approximately 98% of total serum folate [16,23]. Where natural folates can be converted directly to 5-MTHF directly, FA relies on the dihydrofolate reductase (DHFR) enzyme to be reduced [21,24]. The activity of DHFR is slow and easily saturated in the first step conversion to dihydrofolate and therefore when the dose exceeds the capacity of the enzyme or there is a DHFR polymorphism, an increase in free FA in plasma may be seen [27]. The main circulating form of folate in the blood, 5-MTHF, is in high demand during fetal development as DNA synthesis and cell division are increased [26]. The folate receptor-α (encoded by the folate receptor 1 (FOLR1) gene) is expressed on epithelial cells and ensures uptake of 5-MTHF by the placenta and the brain during development. This receptor is upregulated in pregnancy due to the rapid increase in folate-dependent activities such as tissue growth, placental development, and enlargement of the uterus [26,32]. 5-MTHF donates its methyl group to support DNA methylation via DNA methyltransferase enzymes and 5-MTHF is directly linked to the viability of the embryo and the ability to maintain DNA methylation patterns in replicating cells [26,33]. A lack of 5-MTHF may therefore compromise DNA methylation, cause uracil misincorporation, chromosomal breaks, impaired ovarian follicle development and increased risk of pregnancy loss [32]. Folate metabolism can be impaired in people who carry the methylenetetrahydrofolate gene (MTHFR) polymorphisms, resulting in reduced enzyme activity and therefore a reduction in 5-MTHF [34][35][36]. The MTHFR gene has been linked to infertility in both men and women [37][38][39][40][41][42][43]. This is particularly pertinent given that studies in mice show that excess FA reduces MTHFR activity irrespective of MTHFR polymorphisms thereby reducing methylation capacity. Research has identified 5-MTHF as a possible alternative for FA in those with MTHFR polymorphisms as doses of 800 mcg have been shown to bypass the MTHFR enzyme [23][24][25][44][45][46][47].
found in plasma, accounting for approximately 98% of total serum folate [16,23]. Where natural folates can be converted directly to 5-MTHF directly, FA relies on the dihydrofolate reductase (DHFR) enzyme to be reduced [21,24]. The activity of DHFR is slow and easily saturated in the first step conversion to dihydrofolate and therefore when the dose exceeds the capacity of the enzyme or there is a DHFR polymorphism, an increase in free FA in plasma may be seen [27]. The main circulating form of folate in the blood, 5-MTHF, is in high demand during fetal development as DNA synthesis and cell division are increased [26]. The folate receptor-α (encoded by the folate receptor 1 (FOLR1) gene) is expressed on epithelial cells and ensures uptake of 5-MTHF by the placenta and the brain during development. This receptor is upregulated in pregnancy due to the rapid increase in folate-dependent activities such as tissue growth, placental development, and enlargement of the uterus [26,32]. 5-MTHF donates its methyl group to support DNA methylation via DNA methyltransferase enzymes and 5-MTHF is directly linked to the viability of the embryo and the ability to maintain DNA methylation patterns in replicating cells [26,33]. A lack of 5-MTHF may therefore compromise DNA methylation, cause uracil misincorporation, chromosomal breaks, impaired ovarian follicle development and increased risk of pregnancy loss [32]. Folate metabolism can be impaired in people who carry the methylenetetrahydrofolate gene (MTHFR) polymorphisms, resulting in reduced enzyme activity and therefore a reduction in 5-MTHF [34][35][36]. The MTHFR gene has been linked to infertility in both men and women [37][38][39][40][41][42][43]. This is particularly pertinent given that studies in mice show that excess FA reduces MTHFR activity irrespective of MTHFR polymorphisms thereby reducing methylation capacity. Research has identified 5-MTHF as a possible alternative for FA in those with MTHFR polymorphisms as doses of 800 mcg have been shown to bypass the MTHFR enzyme [23][24][25][44][45][46][47]. Almost all the reduced folates are converted into 5-MTHF and transported to the portal vein by multidrug resistance-associated protein 3 (MRP3) to the liver and blood. Folic acid is mostly passed unchanged to the portal vein, where it needs to be metabolized in the liver to dihydrofolate (DHF) by the enzyme dihydrofolatereductase (DHFR) and to tetrahydrofolate (THF) also by DHFR and finally to 5-methyltetrahdyrofolate (5-MTHF). What is not converted will pass into the plasma. Almost all the reduced folates are converted into 5-MTHF and transported to the portal vein by multidrug resistance-associated protein 3 (MRP3) to the liver and blood. Folic acid is mostly passed unchanged to the portal vein, where it needs to be metabolized in the liver to dihydrofolate (DHF) by the enzyme dihydrofolatereductase (DHFR) and to tetrahydrofolate (THF) also by DHFR and finally to 5-methyltetrahdyrofolate (5-MTHF). What is not converted will pass into the plasma.

Folic Acid Dose
Although mandatory fortification exists in some countries, the fortification levels per 100 mg food differ across each country, with no upper limit on the number of foods that may be fortified voluntarily, easily leading to an intake level above the original estimated level of 100 mcg per day [10][11][12][48][49][50][51][52][53].
FA is widely used in supplements and food fortification as it is relatively inexpensive, stable, and freely available [7,[54][55][56]. Women are encouraged by governments to take FA supplements during preconception and pregnancy in addition to any folate they may con- sume through natural folate and fortified foods. Doses of 400-500 mcg are recommended and included in prenatal multivitamins by the Department of Health in Australia [57], whereas the United States, the Centers for Disease Control and Prevention recommends 800 mcg per day [5][6][7] and in Canada at least 1000 mcg of FA are typically found in a prenatal multivitamin [51,58,59].
Women at high risk of pregnancy loss or who had a previous pregnancy affected by a NTD, are routinely prescribed FA doses of 4-5 mg in Australia, Canada, and the United States [6,7,9,46]. FA is considered to be better absorbed and therefore a measurement, known as total food folate daily folate equivalent (DFE) (see Box 1), has been devised by the Institute of Medicine and is the recommended method to provide comparability between synthetic FA and natural food folates whereby the folate from fortified food is weighted 1.7-fold greater than naturally occurring folate [10]. For example, a serving of food from leafy greens may provide 100 mcg of folate, which equals 100 mcg DFE; however, 100 mcg of folic acid in a fortified food is estimated to be 170 mcg DFE. Total food folate DFE = mcg natural food folate mcg + synthetic folic acid from fortified foods and supplements times a bioavailability factor of 1.7 = mcg DFE/day (natural food folate) + (folic acid mcg × 1.7) [1,2] or 1 DFE equals 1 mcg food folate, 0.6 mcg synthetic folic acid consumed with food or 0.5 mcg consumed on an empty stomach with fortified food [3][4][5].

Upper Tolerable Limit of Folic Acid
Unlike 5-MTHF (and natural folate), FA has an upper tolerable limit (UL), which the US' Institute of Medicine sets at 1000 mcg due to the potential for high FA intake to mask a vitamin B12 deficiency in the user [10]. The UL does not include naturally occurring folate from food or 5-MTHF as there is no recognized UL for these forms [28]. The studies used to determine this upper limit do not consider the potential impact of elevated FA intake on folate metabolism [16]. Apart from masking a vitamin B12 deficiency, the only documented risk factor for excessive FA intake is the presence of unmetabolized folic acid (UMFA) [13,16,19,21,60,61]. UMFA is the FA that cannot be metabolized to 5-MTHF due to limited DHFR activity and, therefore, starts to appear in the circulation even with 200 mcg of FA acid intake per day [62,63]. This UMFA has been linked to adverse health effects including reduced brain function and motor and somatosensory processing in offspring [64], cleft lip [65], asthma [52,66], autism [67] anemia [68], natural killer cell cytotoxicity [61], adverse cardiac events and cancer [21] and cognitive impairment [68].
There is a risk that women may be exposed to unintended adverse health outcomes through excess FA intake; for this reason, it is imperative that preconception and pregnancy folate intake from all sources for women of childbearing age is more clearly understood.

Materials and Methods
This review aimed to assess the total all-source intake of folate in women of childbearing age and in pregnancy in high-income countries with folate food fortification programs.
This systematic review was designed in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines.
We conducted a detailed search of the following electronic databases: MEDLINE (OVID), Embase, CINAHL (EBSCO), Scopus, and BiblioMAP. The search strategy is attached (File S1, Search Strategy) and was conducted on the seventh and eighth of October 2021. It included countries identified by the World Bank as high-income countries, pregnant women or women in preconception, folate exposure and source of folate.
Studies published from 1998 until the search date of 8 October 2021 were included. We wanted to include only studies that reflected total folate intake and therefore the start date of 1998 was selected as this is the date that mandatory FA fortification began in the U.S. and other countries around the world [10,63,69]. Scientific posters and conference abstracts were not included. There were no language limits. A PICO(T) format was used to formulate the search strategy and help design the research question. The population of interest (P) was identified as women of childbearing age either in preconception or pregnancy period from high-income countries (as identified by the World Bank [69] that have a mandatory fortification policy of fortifying foods with FA. The intervention/exposure (I) was folate intake of any form, that is dietary folate from non-fortified foods, dietary intake from fortified foods and/or supplements. A comparator (C) was not included. We excluded control studies and intervention studies except where the control group was representative of the study group and nutritional/supplemental data were available. The outcome (O) measured was intake of folate in any form in women of childbearing age, whether in preconception or pregnancy. This included folate from natural food sources such as leafy green vegetables, FA from fortified foods and all folate forms in supplementary dietary products and multivitamins.
Titles and abstracts were reviewed by one reviewer (CL). Manuscripts were included if they were from epidemiological observational studies that explicitly report on folate intake in women of reproductive age-specifically in preconception and pregnancy. There were no language restrictions. Studies were excluded if they did not report on folate intake (rather than folate status), included women who were breastfeeding and were not from countries with a mandatory fortification policy.
Full texts were reviewed and assessed based on the inclusion and exclusion criteria above. If full texts could not be accessed, we contacted the authors and/or publisher and if the full text could not be located they were excluded. Any uncertainty about the inclusion of a study was discussed by the other authors (AS, VS and AM) and consensus was gained. Figure 4 presents the flowchart of study selection and inclusion. A total of 1885 studies were identified. After removing duplicates (n = 780) and excluded citations by title and abstract (n = 648), 457 full-text studies were assessed for eligibility. Of those, 421 were excluded because they were either not measuring folate intake, not from a country that had a mandatory fortification policy, not an academic paper, or had an incorrect study design or date range. This left a total of 36 studies for inclusion. Critical appraisal of the studies was conducting using the Axis tool for cross-sectional studies (Table S1). The studies were assessed for key issues such as suitability of the study  [70]. Quality Assessment of Study. * p < 0.01; ** p < 0.001. Critical appraisal of the studies was conducting using the Axis tool for cross-sectional studies (Table S1). The studies were assessed for key issues such as suitability of the study to answer the research question, and possibility of bias being introduced into the study. All studies except five [2,3,66,71,72] detailed funding sources and sources of conflict.
All studies stated clear aims and objectives and the study design was appropriate for the aim.

Natural Food Folate and Fortification
In women of childbearing age, the recommended intake of 400 mcg folate per day was not met by any study from natural folate sources (see Table 1). One U.S study found that fortified cereals and grains contributes approximately 70.8% of dietary folate [5]. The studies showed that the mean intake of folate from natural food folate ranged from 228.5 to 324.3 mcg/day after fortification [72], contributing to a 50% increase in serum folate concentrations and a 59% increase in RBC concentrations [53]. Another study showed that 43% of folate came from both natural and fortified foods [86]; however, food alone did not achieve the recommended 400 mcg/day for women in childbearing years. One study that investigated only food sources of folate found that 80% of women of reproductive age did not meet the recommended daily allowance (RDA) of 400 mcg per day [88], while another study showed that only 23% of women achieved the RDA of 400 mcg per day [83].      In Australia, two studies by Dorise and Beringer found that only 20% [74] and 55% [90] of pregnant women met the folate recommendations from diet alone (including natural food folate and fortified foods), respectively. In another study, folate was consistently below the estimated average requirement (EAR) of 520 mcg [78] particularly in the periconceptional period [80]. In Canada, 70% of women did not meet the EAR of 520 mcg of folate in first trimester of pregnancy [75] nor was the diet from food or fortification sufficient to achieve the minimum 400 mcg/day FA recommendation [50]. Food fortification and natural folates contributed approximately 303 mcg/day preconceptionally [92] and in pregnancy ranged from 282.2 mcg dietary folate equivalents (DFE)/day [83], 483 mcg DFE/day in early pregnancy [62] to 331 mcg/day in women 20-38 weeks gestation [88], and 465 mcg DFE/day in late pregnancy [62]. Table 3 outlines women's folate intake by folate source or type. Of those studies that only measured natural food folate intake, women of childbearing age consumed between 170.92 and 259 mcg per day and pregnant women consumed between 140 and 483 mcg DFE. The average intake of folate from natural food folate was 205.48 mcg [1,4,5,33,89] in these studies. n/a 265.9 mcg n/a n/a n/a no NIL Jun 2020 [79] n/a n/a n/a 375 mcg n/a yes, no food NIL Marchetta 2016 [81] n/a n/a n/a 381 mcg n/a yes NIL  [84] n/a n/a n/a n/a n/a no NIL
The combined food folate of natural and synthetic folic acid based on the conversion of folates to daily folate equivalents was as high as 500.5 mcg DFE (369.9 mcg) in women of preconception age [5] and 627.6 mcg DFE per day in pregnant women [83].

Supplementation and Upper Tolerable Limit
In women of childbearing age, supplements contributed 47.5% [53] to 57% of folate intake [85] and were the main reason why 2.4-7% of women exceeded the upper tolerable limit of 1000 mcg per day [4,53,84]. The intake of FA varied throughout the studies but was typically between 400 and 1000 mcg per day through the intake of supplements with folic acid. The percentage of women taking 400 mcg of FA per day ranged from 24% in one study [77] to approximately 78% in another [85], while women taking 1000 mcg of FA via supplements represented approximately 20% of the sample population [85,86].
The level of FA intake from vitamin supplements was much higher in pregnant women than in non-pregnant women due to the high rate of supplementation in pregnant populations. Specifically, FA supplements represented between 77% [79] and 92% [92] of FA intake in pregnant women with an estimation of supplementation contributing to 84% of FA intake in early pregnancy and 63% in late pregnancy [66]. While the intake of folic acid-based supplements was high in pregnant women, it was not in women in the preconception stage with one study reporting that 63% of women were not taking a prenatal with folate in the three months before falling pregnant [80].
It was difficult to evaluate a consistent method of overall intake of folate (food folate, fortified food, and supplements) as not all studies provide intake in DFEs and therefore only DFE levels will be reported. In women of childbearing age the total level of intake ranged from 864 mcg DFE [1] to 1778 DFE [86]. In pregnant women, total intake was much higher, ranging from 1451 mcg DFE [73] to 2181 mcg DFE [75]. The total DFE intake in pregnant women was recorded predominantly in Trimester 1 of pregnancy [50,62].
The overall intake of FA throughout pregnancy including supplements, not only caused an increased red blood cell folate [51,76] but contributed to total folate intakes up to 200% above the RDA [58], sometimes with a FA intake of up to 2948 mcg/day [66]. The studies measuring UMFA showed that it was measurable in all pregnant women [51] and even early first-trimester maternal plasma UMFA was detected in 97% of women and 93% of cord blood samples [62].

Natural Food Intake and Fortification
There is a general consensus that women of childbearing age require a minimum of 400 mcg of FA per day in addition to their normal dietary intake to reduce the risk of neural tube defects [10] and, based on studies such as the ones included in this review, it is clear that women of childbearing age, are not receiving sufficient dietary folate to protect against neural tube defects, but rather they are receiving on average, half the recommended dose of folate from food. In response to studies that supported this finding of low folate intake from the diet, mandatory fortification of food products was introduced in the United States in 1998, at a level of 140 mcg per 100 g in all cereal grain products [11]. In Australia, mandatory fortification in wheat flour commenced in 2009 with 200 mcg per 100 g of wheat flour [94]. This regulation was based on modelling studies that showed that the increase in FA was expected to be approximately 100 mcg of additional FA per day; however, it is clear from the studies in this review that the intake of FA from foods alone is closer to 239 mcg or DFE 582, with the combined folate intake from natural food folate and FA fortification reaching close to the required intake of 400 mcg in women of childbearing age 500 mcg DFE. The fortification of folic acid was introduced to prevent neural tube defects in populations that were vulnerable to low levels of folate and by all accounts FA has been successful in achieving this [95][96][97][98][99][100]. The fortification program, however, did not cap the number of foods being fortified, nor did it consider populations that may be vulnerable to large amounts of FA like children and older adults [21,30,101] or tailor the intake levels via supplementation suggested for women in preconception and pregnancy.

Folate Measurement
The assessment of folate intake from food (natural folate and fortified foods) in the studies included in this review is based on the assumption that FA has better bioavailability than natural folates. However, these assumptions were arguably based on limited research do not align with emerging evidence. Natural folates are in a reduced form and are assumed to have nearly half the bioavailability of foods fortified with FA (synthetic FA fortified foods) [10]. This assumption is based on one study [102] with only ten female participants [10,102]. Researchers have since suggested that this finding may be incorrect [30] with studies investigating the bioavailability of natural folate to be anything from 78% of that of FA in food [103], and 98% for other foods [104]. A very detailed report on folate chemistry and metabolism suggests that folate polyglutamates (natural food folate) are not less bioavailable than monoglutamates (synthetic folic acid) [27]. DFE assumes that synthetic FA has a higher bioavailability compared with natural folate [33]. The measurement of food folate is difficult as confounding factors like loss of folate due to cooking, brush border enzyme activity, pH of the intestinal lumen, certain nutrients affecting absorption, and poor compliance in studies where participants are faced with difficult interventions [27,[104][105][106], contribute to the accuracy of the study. It is important, therefore, that we gain a better understanding of folate metabolism because if we are underestimating the amount of natural folate that is bioavailable, this may have a bearing on FA fortification rates currently and future directions for many governments around the world. Further bioavailability research should also compare FA to the more active form, 5-MTHF, given studies have shown that bioavailability of both folinic acid and 5-MTHF are equivalent to FA as measured by changes in plasma folate levels [30,107].

Folate Metabolism
This review has highlighted the extent to which women of childbearing age, particularly in pregnancy, are exceeding the UL of FA, and this may be impacting on their folate metabolism. Bioavailability is very different to metabolism; although FA may be more bioavailable, it is not metabolized the same was as natural folate. Natural folate uptake by the brush boarder enzymes does depend on the type of food, the pH of intestinal tract and quality of the mucosa; however, there is no solid evidence that it is 50% less absorbed than FA and the studies show huge variation in percentage uptake of natural folates versus FA. Bioavailability is key because if FA is not metabolized sufficiently to 5-MTHF, which is the main circulating form of folate used by the placenta and brain, then the increased amount may be inhibiting the production of 5-MTHF and its re-uptake. It has been found that synthetic FA has no coenzyme activity until it can be reduced by dihydrofolate reductase (DHFR) [28] and that the DHFR enzyme is easily saturated which means that above a dose of 200-300 mcg the normal intestinal absorption of is saturated and rather than being converted by DHFR to dihydrofolate (DHF) or tetrahydrofolate (THF), we begin to see an accumulation of unmetabolized folic acid in the serum [14,62,108,109]. This then becomes an important question regarding both fortification and supplementation. If as studies in this review suggest, that the level of FA from fortification alone is reaching this saturation point for FA metabolism, and women, particularly in pregnancy are almost always exceeding this threshold, should the recommendation of further FA in preconception and pregnancy be changed to 5-MTHF to achieve the recommended folate level? 5-MTHF cannot accumulate [26,27] and is considered to be as good as FA, if not better in raising folate levels and preventing neural tube defects [23][24][25]46,110,111]. The studies in this review confirm that food-based folate is not sufficient to achieve recommended levels of folate in preconception and pregnancy; however, they do highlight that although FA may be considered to be absorbed better, it is not metabolized into 5-MTHF like natural folates and is accumulating instead.
Studies that have investigated 5-MTHF as a possible substitute for FA found that 5-MTHF at a dose of 7.5 mg every 12 h over four days rapidly increased serum total folate and has an advantage over FA as it can replete body stores in folate insufficient women within a few days [110]. 5-MTHF compared to folic acid was found to increase plasma folate levels more effectively [111]. Safety studies of high-dose 5-MTHF in rats showed no adverse events [112] and dosages as high as 17 mg of 5-MTHF daily for 12 weeks have been shown to have no side effects [113]. In pregnancy, there have been reported no side effects with 1.13 mg [114] and 7.5 mg and 15 mg 5-MTHF have been used without side effects in patients with depression [115,116]. 5-MTHF is more effective at improving folate status and may therefore be a better alternative to folic acid in those with infertility and recurrent pregnancy loss [24,46].

Unmetabolized Folic Acid
The fact that previous studies show people exposed to 200 mcg per day or less of FA are unlikely to have unmetabolized folic acid in serum, yet 300 to 400 mcg or above could lead to UMFA [16,109], suggest that all the women in the studies in this review in preconception or pregnancy would have levels of UMFA in their serum. Murphy [62]. UMFA has been implicated in reduced natural killer cell activity in post-menopausal women [61], various forms of cancer, cardiovascular disease [21], autism [117] and orofacial clefts [118,119]. This has serious implications, given the studies in this review show that many women in preconception and almost all women in pregnancy have the presence of UMFA and are exceeding 400 mcg of FA per day. UMFA has also been found to cause a pseudo-MTHFR deficiency in rats [120] and reduce 5-MTHF. This effect has concerning implications as low folate is associated with uracil misincorporation [121], reduced DNA methylation [120] and changes in epigenetic patterns [122]. So too, the presence of UMFA has been linked to aberrant DNA methylation [123]. Methylation and the one carbon metabolism cycle ( Figure 5) is a critical pathway required for oogenesis [124] and spermatogenesis [125,126] and is required for epigenesis and imprinting [127] and folate is required for the synthesis of purines and thymidylate for RNA and DNA synthesis [28], thus having implications for preconception, pregnancy, birth defects and the outcome of the offspring. The folic acid UTL of 1000 mcg has been set based on the potential for high folic acid intake to mask a vitamin B12 deficiency [16,28,62] and not the level at which serum UMFA appears. Based on the studies in this review, every single woman may exceed the level of FA intake where UMFA would appear. This is not the case with 5-MTHF which cannot accumulate and has a feedback loop to ensure that there is a homeostatic mechanism that prevents excessive levels of folate in tissues [28]. Previous research has shown that high-dose folic acid results in a global loss of methylation across the sperm methylome and leads to altered sperm epigenome and sperm DNA methylation, lower pregnancy rates, infertility rates and abnormal embryos [128,129].
A lack of 5-MTHF contributes to an elevation in follicular levels of homocysteine [35,130] which correlates to adverse pregnancy outcomes as folate receptor-α has a high affinity for 5-MTHF and ensures cellular uptake where the folate receptor is expressed in the ovary, oocytes and human embryonic stem cells [32]. These biochemical mechanisms may underpin the reported links between MTHFR polymorphism and infertility [37,38,40,41,131], implantation failure [22] and recurrent pregnancy loss [24,36,46,130,132]. Such mechanisms may also explain preliminary research that has found replacing FA supplements with folinic acid or MTHF supplements may increase pregnancy success in women with MTHFR polymorphisms who have experienced recurrent pregnancy loss [133].
folic acid results in a global loss of methylation across the sperm methylome and leads to altered sperm epigenome and sperm DNA methylation, lower pregnancy rates, infertility rates and abnormal embryos [128,129]. A lack of 5-MTHF contributes to an elevation in follicular levels of homocysteine [35,130] which correlates to adverse pregnancy outcomes as folate receptor-α has a high affinity for 5-MTHF and ensures cellular uptake where the folate receptor is expressed in the ovary, oocytes and human embryonic stem cells [32]. These biochemical mechanisms may underpin the reported links between MTHFR polymorphism and infertility [37,38,40,41,131], implantation failure [22] and recurrent pregnancy loss [24,36,46,130,132]. Such mechanisms may also explain preliminary research that has found replacing FA supplements with folinic acid or MTHF supplements may increase pregnancy success in women with MTHFR polymorphisms who have experienced recurrent pregnancy loss [133].

Upper Tolerable Limit
The studies in this review confirm that many women preconceptionally and almost all women at some stage in pregnancy are exceeding the UL of 1000 mcg/day. The upper limit seems to be almost inconsequential as it is simply a consideration of vitamin B12 masking and does not take into account the consideration of metabolism. If metabolism is a concern, then all women in pregnancy are exceeding the threshold and most women taking a prenatal multivitamin most certainly are as well. Given the documented concerns regarding the presence of UMFA, this upper tolerable limit needs to be re-evaluated.

Conclusions
Natural folates may not deliver the optimal amount of folate to reduce the risk of NTDs or methylate DNA sufficiently to prevent miscarriage. Women may be at risk of producing UMFA due to their intake of FA from food fortification. Any supplementation of FA over and above that has the potential to cause adverse health outcomes, reduce MTHFR activity, mask vitamin B12 deficiency, reduce methylation of DNA and therefore contribute to adverse pregnancy outcomes such as pregnancy loss. The results of this review warrant close attention from public health researchers and policy makers and may

Upper Tolerable Limit
The studies in this review confirm that many women preconceptionally and almost all women at some stage in pregnancy are exceeding the UL of 1000 mcg/day. The upper limit seems to be almost inconsequential as it is simply a consideration of vitamin B12 masking and does not take into account the consideration of metabolism. If metabolism is a concern, then all women in pregnancy are exceeding the threshold and most women taking a prenatal multivitamin most certainly are as well. Given the documented concerns regarding the presence of UMFA, this upper tolerable limit needs to be re-evaluated.

Conclusions
Natural folates may not deliver the optimal amount of folate to reduce the risk of NTDs or methylate DNA sufficiently to prevent miscarriage. Women may be at risk of producing UMFA due to their intake of FA from food fortification. Any supplementation of FA over and above that has the potential to cause adverse health outcomes, reduce MTHFR activity, mask vitamin B12 deficiency, reduce methylation of DNA and therefore contribute to adverse pregnancy outcomes such as pregnancy loss. The results of this review warrant close attention from public health researchers and policy makers and may justify a review of the current approach to ensure folate sufficiency in the general population and avoidance of UMFA levels that may influence fertility and pregnancy outcomes.