Medical interest in folic acid (vitamin B9) dates back to the 1920s and 1930s, when it was realised that the brewers’ yeast extract (or anecdotally, marmite) can be used effectively against megaloblastic anaemia. The chemical compound — pteroyl-L-glutamate — remained unknown until its successful isolation in 1941 and artificial synthesis in 1945. The chemical was named after the Latin word <em>folium</em> ie, leaf, once it was discovered that it was present in abundance in green leafy vegetables.
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<strong>Dr Bartlomiej Kuczera, Specialist Fertility Consultant</strong>
In the following years, approximately 150 folic acid derivative compounds were identified in the foods and now exist under the common name of ‘folates’. Due to the abundance of active forms, the latter term ‘folates’ is currently preferred rather than ‘folic acid’, referring to supplements which are not restricted by pharmaceutical regulations. The original term ‘folic acid’ is reserved for the synthetic form. That said, each of these forms has its own absorption rate, activity and conversion rate to folic acid. The bioavailability of folic acid comes from a mixed diet and is approximately 50 per cent, according to German studies from the late 1980s.
Biochemical activity of folic acid is based on its ability to transfer methyl groups once activated to tetrahydrofolate, and its turnover to 5-methyltetrahydrofolate orchestrated by MTHF reductase and B-complex vitamins. This is utilised to convert homocysteine to methionine and to produce substrate methyl group donor for DNA production, phospholipid and neurotransmitter synthesis, folate-B12-Iron complex in haemoglobin metabolism and epigenetic regulatory function, according to more recent studies (Pauwels et al).
The MTHFR-dependent metabolism of folic acid shows different dynamics, depending on the enzyme mutations. The most common mutation is MTHFR 677C<T, resulting in the substitution of alanine with valine, resulting in decreased homocysteine-methionine conversion and increased concentrations of homocysteine. The enzyme deficiency can mimic folic acid deficit, and vice versa.
The mutations affecting MTHFR activity are very common and affect approximately 50 per cent of the Caucasian population, often resulting in higher serum homocysteine levels and observed and internal medicine. The degree of enzyme activity depends on whether the mutation is homozygous (10 per cent of the Caucasian population) or heterozygous (approximately 40 per cent of the Caucasian population). The efficiency of homocysteine metabolism can be restored by supplementation with active 5-methyltetrahydrofolate instead of folic acid.
Folic acid deficits and/or increased homocysteine concentrations, and incidence of MTHFR 677T mutation, were observed in different pregnancy complications, including recurrent miscarriage, intrauterine growth retardation and pre-term delivery. The spontaneous dichorionic twin rate was also correlated with MTHFR mutation incidence in various ethnic groups, as seen by Hasbargen and colleagues, with the highest rate observed in Africans (30/1000; MTHFR 677T mutation below detection limits) and the lowest in Asians.
Most famously, in the late 1960s, folic acid deficiency was linked with neural tube defects (NTDs) in embryos. Studies that followed proved that supplementation around conception and in the first trimester significantly reduced the incidence of the NTD by one-third, or even up to 62 per cent, depending on the study (Goh et al, Blencowe et al).
However, the daily dose of folic acid was different between the studies and multivitamin preparations were permitted. Various national strategies were implemented in different countries to reduce the numbers of neural tube defects, including mandatory cereal food fortification or tablet supplementation to improve obstetric outcomes. The recommended daily dose varied between the countries (typically between 200µg for food fortification to 1mg for over-the-counter tablets) and the time of supplementation differed, depending on either voluntary or mandatory staple foods fortification (permanent throughout pregnancy) or dietary/tablet supplementation, or both.
Folic acid supplementation differed depending on the socioeconomic status of the pregnant woman. The homocysteine-lowering effect was, however, best proven at the tablet dose of 0.8mcg/d by Nelen et al in the recurrent miscarriages related to MTHFR 677T (the most common) mutation. Current World Health Organisation (WHO) recommendations suggest 400µg/dose for women attempting pregnancy.
Interesting effects of folic acid and B-complex vitamin supplementation were observed in assisted reproduction and subfertility. The Nurses’ Health Study II (Chavarro et al) revealed that regular multivitamin users suffered significantly less from oligo-anovulation than irregular and non-users. The serum concentrations of different B-vitamins including folate were analysed and the latter was correlated with a reduction of ovulatory infertility, giving a viable low-cost intervention option for women wanting to conceive.
However, this study could potentially be biased, as stated by the authors, as a generally healthier lifestyle profile of regular users compared to the rest of the study group in terms of alcohol and caffeine consumption, physical activity, and smoking, for example, could also be factors.
The success rates of IVF/controlled ovarian stimulation also correlate with folic acid and B-complex vitamin supplementation. Both single pregnancy rates (Broxmer et al) and dichorionic twin rates (ie, presence of a second top-quality embryo, Haggerty et al) in IVF/ICSI cycles were higher among women with high daily folate and B12 intake, high follicular fluid folic acid concentrations and low homocysteine levels, across all age groups.
The MTHFR 677T mutation was also proven to affect the <em>in vitro</em> outcomes, lowering oestradiol production and reducing the number of retrieved oocytes (Thaler, 2006; Hecht et al, 2009). The same effect was discussed in a natural menstrual cycle, pointing out possible limited stimulating effects of FSH pulses in the mutation carriers, resulting in decreased fertility. The reverse effect of high folic acid intake was seen both in the <em>in vitro</em> study as well as in the Hungarian family planning programme pregnancy rate. Similar effects were seen in animal models. On the other hand, the studies by Thaler and colleagues revealed that anti-mullerian hormone levels in MTHFR 677T mutation carriers can be falsely increased by 20 per cent, especially when commencing folic acid supplementation, thus having clinical implications for IVF treatment planning.
Recently, the focus in folic acid studies was placed on its epigenetic regulatory function, ie, selective gene activation depending on the DNA methylation. The initial clinical and population studies date back to the Dutch Hunger Winter of 1944-45, when extreme food shortages affected the entire population of the Netherlands at the end of WWII. Apart from immediate population effects, it was revealed tens of years later the different patterns of obesity and related diseases in the people conceived and exposed to food shortages <em>in utero</em>. Different obstetric outcomes were seen in individuals exposed to famine <em>in utero</em> in early pregnancy (ie, low birth weight and obesity in adulthood) compared to impaired glucose tolerance when affected at later stages of pregnancy (Roseboom and colleagues).
In the animal models, insufficient folic acid supplementation influences the phenotype of the offspring and includes metabolic programming towards obesity, hyperinsulinaemia and susceptibility to cancer (Dolinoy, 2006). Both the Dutch Hunger Winter study and animal models suggest different effects of shortage versus supplementation at different stages of pregnancy. The proposed biochemical effect is based on DNA methyltransferases, especially DNAMT3a and 3b specific for embryogenesis and potentially affected by methyl donors (including folic acid) shortage.
Such an effect on the insulin growth factor-2 DNA methylation was seen by Heijmans et al in the individuals conceived during famine, ie, exposed at peri-conception stage, as opposed to their same-sex siblings not being exposed to food shortage <em>in utero</em> or exposed in mid or late pregnancy. Similar observations referred to LEP1 gene methylation encoding leptin, a hormone regulating hunger and food uptake. The difference, however, was that the programming could occur either in early or late pregnancy rather than being limited to a short period of time.
The recently-published <em>Maternal Nutrition and Offspring’s Epigenome</em> (MANOE) study by Pauwels et al revealed different methylation patterns of the selected genes (including metabolic ones, like IGF2 and LEP) in umbilical blood and infant buccal swabs, depending on the timing of the methyl donor supplementation during pregnancy, with its possible epigenetic regulatory effect.
Recently, it was noted that dynamic growth in childhood allergies was parallel time-wise with the implementation of the folic supplementation strategies of the early 1990s. The study by Mullins et al in Australia correlates the dynamics of child food anaphylaxis hospital admissions, with the development of the folic acid supplementation strategy from 1989 to 2012. Other papers report a link between high plasma folic levels and increased incidence of respiratory wheeze in children (Haberg et al, 2009), atopic dermatitis (Kiefte-De Jong et al, 2012) or allergies to cow’s milk (Tuokkola et al, 2016).
All the studies mentioned, along with similar studies, however, remain observational and are often based on retrospective patient-declared use of folic supplementation and with a focus on specific age groups reporting no long-term effects. There are confusing reports on folic acid related decreased atopic dermatitis in South Korea, and another blood study finding no correlation between maternal folic acid levels and common allergies (Magdelijns et al, 2010).
More detailed studies, however, focusing on documented use (serum folate levels) in the second or third trimester, showed no clear evidence of folic acid-induced atopy in the children. On the other hand, the animal models of folic acid-induced asthma and colitis are well known and even suggest transgeneration effect. The weakness of observational studies in humans is a likely bias resulting from dietary habits, other immune-modulating supplements (like vitamin D3) or different local strategies in voluntary or mandatory food fortification and also the socioeconomic status of the patients.
The position of folic acid supplementation in peri-conception based on the WHO’s recommendations is stable. The same applies for the positive effect of folic acid supplementation in assisted reproduction, maximising the chance for success. However, prolonged supplementation, ie, beyond first trimester, remains a matter of study and particular attention for practitioners, especially when confronted with an increasing tendency towards voluntary dieting, use of multiple supplements and uncontrolled para-pharmaceuticals, as seen in patients getting fertility advice.