Variations in folate prescriptions for patients with the MTHFR genetic polymorphisms: A case series study.

A cumulative summary of the patient characteristics is presented in Table 2 . Patients' age ranged from 26 to more than 46 years old, with the majority of cases between 31 and 40 years of age ( n  = 8). Of the twelve cases, 83.3% of these women presented with a history of miscarriage ( n  = 10), 41.7% experienced failed IVF or ICSI ( n  = 5), 41.7% experienced fertility related issues (n = 5), 33.3% with unexplained infertility ( n  = 4). Patients presented most commonly with an MTHFR C677T heterozygous polymorphism (n = 5, 41.67%) followed by MTHFR compound heterozygous (C677T heterozygous, A1298C heterozygous) (n = 4, 33.3%), MTHFR A1298A heterozygous ( n  = 2, 16.67%) and MTHFR C677T homozygous ( n  = 1, 8.33%). The approximate time between appointments was between one and two months. A descriptive summary of details for each of the included cases is presented below. While the data for all cases was provided to the research team in a de-identified form, all cases have been allocated a pseudonym for ease of presentation. Table 2 Participant characteristics. Table 2 Characteristic N % Gender ( n  = 12)   Female 12 100   Male 0 0   Non-binary 0 0 Age ( n  = 12)   20–25 years 0 0   26–30 years 1 8.33   31–35 years 5 41.67   36–40 years 3 25.00   41–45 years 2 16.67   46 years and over 1 8.33 History of miscarriage ( n  = 12)   No 2 16.67   Yes 10 83.33 MTHFR polymorphism ( n  = 12)   MTHFR C677T heterozygous 5 41.67   MTHFR C677T homozygous 1 8.33   MTHFR A1298C heterozygous 2 16.67   MTHFR A1298C homozygous 0 0 MTHFR Compound heterozygous 4 33.33 Presenting symptoms   Previous miscarriage 10 83.33   Unexplained infertility 4 33.33   Endometriosis 2 16.67   Failed IVF or ICSI 5 41.67   Partner related issues 2 16.67 Other – including same sex partner, failed IUI, low ovarian reserve, 5 41.67 Participant characteristics. Karen (36–40 years) presented with specialist-diagnosed infertility. She had a previous miscarriage, endometriosis, low ovarian reserve and adenomyosis. Her partners sperm had been assessed by another health professional and classified as ‘normal’. She had three previous IVF attempts of which one resulted in an egg harvest. One egg was retrieved and progressed to embryo and transfer. This did not result in a pregnancy. On first presentation to the practitioner, Karen reported using an oral MTHF-based prenatal supplement and additional MTHF oral supplement. Her naturopath then prescribed phosphatidylcholine, magnesium and adenosylcobalamin and recommended Karen avoid all sources of FA fortified foods. Carol (26–30 years of age) presented with specialist-diagnosed infertility and a history of more than five previous miscarriages. Carol was taking 2.5 mg of methyl folate on presentation to the clinic. Her partner's sperm was assessed and considered normal. Her naturopath prescribed a methyl folate and folinic based prenatal multivitamin. Erica (31–35 years) presented with a history of two previous miscarriages. Erica reported three previous rounds of IUI, two embryo transfers and a pregnancy loss at 6 weeks. Her partner's sperm was assessed and considered to be normal. The patient had a history of low serum iron and essential thrombocytosis. Erica was given a supplement containing 500μg of methyl folate. The patient was advised to be mindful of FA fortified foods and avoid them where possible. Susie (41–45 years) presented with specialist-diagnosed infertility. She has no family history of fertility issues and presented with three previous pregnancies: one live birth and two miscarriages. Over the previous nine years she had completed more than five IVF and ICSI procedures, with 6–10 eggs retrieved, resulting in one pregnancy and live birth. Susie had low serum vitamin B12, low serum folate and a positive ANA on blood tests. Her practitioner prescribed a methylated prenatal multivitamin, additional methyl folate to what was in the prenatal multivitamin, herbal medicines, phosphatidylcholine among others. Susie was advised to go on a gluten free and whole foods balanced diet and be mindful of FA fortified foods and avoid where possible. Sonia, (aged 31–35 years), presented with specialist diagnosed infertility. She had three previous pregnancies, with one live birth, previous miscarriages and a 12-week growth restriction trisomy miscarriage. She was on a homocysteine lowering formula when she presented to her practitioner. Her practitioner prescribed herbs, homeopathics and a methylated prenatal formula. She was advised to follow a gluten free, dairy free, unprocessed, whole foods diet and to avoid FA fortified foods where possible. Senna (aged 36–40 years) presented with specialist diagnosed infertility. She presented to her practitioner with three recent miscarriages with one previous successful birth. She had low serum B12, low plasma zinc, low vitamin D and low protein. She was prescribed supplements including a methylated prenatal multivitamin, additional supplemental methyl folate to her prenatal multivitamin, herbal medicines for fertility. She was advised to follow a gluten free, whole foods balanced diet. Emily (aged 36–40 years) had been recently diagnosed by her GP with infertility. History of two previous IVF treatments, one IVF attempt did not result in an egg harvest, the other IVF attempt resulted in an egg harvest of 1–5 eggs. Of these, two fertilized to embryo's that resulted in a pregnancy, but she miscarried. Emily was taking a B Vitamin containing 500μg of methyl folate. Sheena (aged 41–45 years) presented with GP diagnosed infertility. She presented with unexplained infertility, endometriosis, and failed IVF. Sheena had 4 IVF attempts, two resulting in an egg harvest with 11–15 eggs retrieved and five or more progressing to embryo stage. No transfers resulted in a pregnancy. On presenting to her practitioner, she had low serum vitamin B12, low active B12, low folate, and low iodine. Her practitioner prescribed a methylated folate and folinic acid based prenatal multivitamin, vitamin B12, zinc, phosphatidylcholine, and glutathione. Sheena was advised to follow a gluten free, dairy free, unprocessed whole foods diet and to avoid all sources of FA fortified foods. Mary (aged 31–35 years) presented with GP diagnosed infertility. Mary had two previous miscarriages and has had one live birth. Her partner had a sperm analysis conducted and all markers were considered to be within range. Her practitioner prescribed herbs and advised the patient to follow a dairy free, unprocessed whole foods diet and be mindful of FA fortified foods and avoid where possible. Cassandra (an over 46-year female of Mediterranean background) presented to her practitioner with GP diagnosed infertility. She had 10 previous IVF procedures with none resulting in a pregnancy or live birth. Cassandra was on 5 mg of FA, had low zinc, low iodine, and high mercury. She was prescribed fertility-based herbs, supplements, including a methylated prenatal multivitamin and told to stop the FA-based supplement. She was asked to follow a whole food, balanced diet, avoid high mercury foods like tuna and avoid all sources of FA fortified foods. Donna (aged 31–35 years) presented to her practitioner with three previous pregnancies, two of which resulted in a live birth. She was prescribed a methylated prenatal multivitamin, zinc, magnesium, and phosphatidylcholine and advised to avoid all sources of FA fortified foods. Rebecca (aged 31–35 years) presented to her practitioner with specialist-diagnosed infertility. She had previously been prescribed 5 mg of FA. Her partner had a sperm analysis conducted and all markers were considered to be within range. She had one previous pregnancy, with the aid of IVF that resulted in a miscarriage at 12 weeks. The IVF attempt resulted in one egg harvest of 6–10 eggs of which five fertilized to embryo status. All were transferred. The patient had low serum levels of vitamin B12, folate and vitamin D. Rebecca was prescribed nutritional supplements, a methylated prenatal multivitamin and advised to stop her FA-based supplement. She was put on a gluten free diet and told to avoid all sources of FA fortified foods.

Not applicable.

The research project was approved by the Human research and ethics Committee (HREC) of Endeavour College of Natural Health (HREC #20200406-CL-1).

Six patients were taking folate-containing supplements at their initial appointment. Two of the four patients with a compound heterozygous polymorphism (Case 10 & 12) were taking high dose FA (5000 μg or greater), and their practitioner changed the form of supplemental folate to either 5-MTHF or folinic acid. See Table 3 for dosages and forms that patients were taking prior to their initial consultation. The other two patients with compound heterozygous polymorphism (Case 5 & 8)) and the patient who had a homozygous MTHFR C677T polymorphism (Case 2) were taking 5-MTHF prior to their initial consultation. Nine patients were prescribed a 5-MTHF product by their practitioner following the first consultation. In relation to dietary advice, 75% of patients ( n  = 9) were given advice in relation to FA-fortified foods; five patients were advised to avoid all sources of FA fortified foods while four were advised to ‘be mindful’ of FA fortified foods. Table 3 Forms of folate in micrograms per day prescribed by appointment and MTHFR polymorphism. Table 3 Total folate (mcg) amount the patient was taking at the conclusion of the appointment i.e.: accumulative amount if the practitioner increased the dose. n/a – there was no appointment follow up recorded by the practitioner MTHFR polymorphism Case Form of folate* Baseline V1 First follow up V2 Second follow up V3 Third follow up V4 Fourth follow up V5 Fifth follow up V6 Sixth follow up V7 Seventh follow up Maximum Total Folate per day over treatment period Pregnancy outcome Folate on presentation Initial Prescription (Initial appointment) MTHFR A1298C Heterozygous Karen- Case 1 Folic acid – – – n/a n/a n/a n/a n/a n/a Pregnancy via IVF confirmed at visit 1. Live birth Folinic acid 300 300 600 n/a n/a n/a n/a n/a n/a 600 MTHF 700 700 1400 n/a n/a n/a n/a n/a n/a 1400 FA FF Avoid all Avoid all n/a n/a n/a n/a n/a n/a Erica- Case 3 Folic acid – – – n/a n/a n/a n/a n/a n/a Pregnancy at visit 1. Live birth Folinic acid – – – n/a n/a n/a n/a n/a n/a MTHF 500 500 500 n/a n/a n/a n/a n/a n/a 500 Dietary folate Be Mindful of MTHFR C677T Heterozygous Susie- Case 4 Folic acid – – n/a n/a n/a n/a n/a n/a Successful pregnancy at appt 1. Live birth Folinic acid – – n/a n/a n/a n/a n/a n/a MTHF 1500 3500 3500 n/a n/a n/a n/a n/a n/a 3500 Dietary folate Be mindful of Senna – Case 6 Folic acid – – – n/a n/a n/a n/a n/a Pregnancy after appt 1 and live birth Folinic acid 300 450 450 n/a n/a n/a n/a n/a 450 MTHF 1700 2300 3800 n/a n/a n/a n/a n/a 3800 Dietary folate None Emily – Case 7 Folic acid – – – – – – – – Pregnant at Initial, miscarried after visit 1, fell pregnant again appt 7. Miscarried v8. Folinic acid – – – – – – – – MTHF 500 500 500 500 500 500 500 500 500 Dietary folate None Mary – Case 9 Folic Acid – – – – – – n/a n/a n/a Pregnant at V4 (12 weeks). Live birth Folinic Acid – – – 400 800 800 n/a n/a n/a 800 MTHF – – 800 1200 1600 1600 n/a n/a n/a 1600 Dietary Folate Be Mindful of n/a n/a n/a Donna- Case 11 Folic Acid – – – – n/a n/a n/a n/a n/a Pregnant at visit 1. Live birth Folinic Acid – – – – n/a n/a n/a n/a n/a MTHF 500 2500 2500 n/a n/a n/a n/a n/a 2500 Dietary Folate Avoid All MTHFR C677T Homozygous Carol – Case 2 Folic acid – – – – – n/a n/a n/a Pregnant at visit 4. 27 weeks at visit 4. Live birth Folinic acid 300 300 300 300 500 n/a n/a n/a 700 MTHF 2500 2700 2700 2700 2700 3500 Na/ n/a n/a 3500 Dietary folate Avoid All Compound Heterozygous Sonia- Case 5 Folic acid – – – n/a n/a n/a n/a n/a n/a Pregnant at visit 1 and live birth Folinic acid – – – n/a n/a n/a n/a n/a n/a MTHF 1200 2000 3800 n/a n/a n/a n/a n/a n/a 3800 Dietary folate Be Mindful of Sheena – Case 8 Folic acid – – – – – n/a n/a n/a n/a Did not fall pregnant Folinic acid 400 800 800 800 n/a n/a n/a n/a 800 MTHF 1000 1400 1800 1800 1800 n/a n/a n/a n/a 1800 Dietary folate Avoid All Cassandra- Case 10 Folic acid 5500 – – – – – – – n/a Pregnancy at visit 5. Twins born 23/7/20 via IVF Folinic acid – – – – – – – – n/a MTHF – 500 3500 5500 2500 2500 2500 2500 n/a 2500–5500 Dietary folate Avoid All Rebecca- Case 12 Folic acid 5000 – – – – – – n/a n/a – Pregnancy at visit 2. Successful live birth via IVF Folinic acid 400 400 400 – – – n/a n/a – MTHF 400 1400 1500 1500 1500 n/a n/a 1500 Dietary folate Avoid all Forms of folate in micrograms per day prescribed by appointment and MTHFR polymorphism. In this case series, the practitioners enrolled did not prescribe FA to any patients throughout treatment whereas, they all prescribed 5-MTHF. The mean maximum amount of 5-MTHF prescribed across the twelve cases was 2325μg per day. The minimum dose prescribed was 500μg and the maximum dose prescribed was 3800μg. Five patients were prescribed a combination of 5-MTHF and folinic acid and the average amount of folinic acid prescribed was 670 micrograms. The minimum dose of folinic acid was 450μg while the maximum dose was 800μg. Patients with a A1298C heterozygous polymorphism were prescribed between 500μg and 1400μg of 5-MTHF. Individuals with a C677T heterozygous polymorphism were prescribed between 500μg to 3800μg of 5-MTHF. The mean daily amount of 5-MTHF prescribed to heterozygous C677T patients was 2850μg per day. The only patient with a homozygous C677T polymorphism was prescribed 3500μg of 5-MTHF and 700μg of folinic acid. There were 4 patients with compound heterozygous polymorphisms who were prescribed amounts of 5-MTHF ranging from 1500μg to 3800μg with the mean amount of 2400μg 5-MTHF per day prescribed to this group.

C.L was primarily responsible for the authorship of this paper, providing the majority of contribution to: development of the article topic or research question; synthesis and/or analysis of data; and drafting of the manuscript and subsequent revision. A.S and J.S provided guidance to the project, type of methods required for this study and the study itself. A.S and J.S provided feedback on all drafts of the manuscript. A.S and J.S aided in interpreting the results and worked on the manuscript. All authors discussed the results and commented on the manuscript.

Not applicable.

The researcher received funding from 10.13039/100013817 Endeavour College of Natural Health for this project.

Six practitioners consented to be involved in the study and completed the screening instrument, all of whom met the eligibility criteria. The practitioners all identified as female ( n  = 6,) and worked in clinical practice in NSW ( n  = 5) and ACT ( n  = 1). Four of the practitioners self-identified as naturopaths while the remaining two identified as a nutritionist and an acupuncturist, respectively. All practitioners reported being in clinical practice for greater than 20 years. Three of the practitioners indicated they ‘always’ investigate MTHFR polymorphisms in fertility patients, while the remainder said they screen for MTHFR polymorphisms ‘when indicated’. The six practitioners submitted a combined total of 12 cases. One case was excluded from the analysis as the practitioner did not report the patient's MTHFR genotype. This patient had been diagnosed with infertility by her GP. She had one ovulation induction and IVF which did not result in an egg harvest. The cases submitted by each practitioner is presented in Table 1 . Table 1 Cases submitted by Practitioner. Table 1 Practitioner 1 Case 1 & 2 Practitioner 2 Case 3 & 7 Practitioner 3 Case 4 & 6 Practitioner 4 Case 5 Practitioner 5 Case 8, 10, 11 & 12 Practitioner 6 Case 9 Cases submitted by Practitioner.

Describe the different forms and doses of folate that practitioners prescribe to individuals with infertility and a confirmed MTHFR polymorphism diagnosis. An online case report form was sent to Australian practitioners via the MTHFR Support Australia Practitioner membership base. Invited practitioners were asked to submit cases for women aged 18–49 years of age where they had been given a diagnosis of infertility and had an MTHFR polymorphism. This case report form was a unique instrument designed by the research team to collect the relevant data regarding infertility history and supplement use. The objective was to achieve a minimum of five cases. Collecting data via Survey Gizmo allowed the researchers to achieve the aims and objectives of the study. The online case report form allowed collection of descriptive statistics relating to the cases nominated by the practitioners. It also allowed collection of clinical data and protocols which could then be described narratively. The incidence of MTHFR polymorphisms were recorded using measures of frequency and the collection of information relating to practitioner protocols, dosages and types of folate was documented and explored narratively. This retrospective case series was a descriptive study which aimed to explore the pregnancy outcomes and different forms and dosage of folate prescribed in patients accessing fertility treatments who had the MTHFR genetic polymorphisms. This is in alignment with other studies that seek to form a basis on which further research may be conducted. This study design and format was carefully considered to reduce as many confounding factors as possible to address the research aims and objectives. The target participants of the study were registered Australian based health professionals, with at least two years' clinical experience who identified as providing care to patients with infertility for at least one year and screened their patients for MTHFR genetic polymorphisms. We aimed to collect case reports from at least five practitioners to maximise diversity of treatment approach and patient populations. Recruitment of Practitioners took place via the MTHFR Support Australia Practitioner membership base which consists of approximately 1200 practitioners. This target sample of practitioners was selected as they were likely to be aware of, and regularly test for, the MTHFR genetic polymorphisms. All practitioner listed on the membership database were invited to complete an online screening questionnaire to verify their compliance with the inclusion criteria. This screening instrument was expected to take no more than two minutes and asked seven questions to the practitioner to answer based on the following: Practitioner details: gender, state, where they practice, type of practitioner and years in clinical practice, Information: number of fertility patients seen in the last year and whether they screen their patient for MTHFR polymorphisms. The online screening instrument included a consent form, participant information sheet and further information to ensure inclusion criteria were met. The practitioners who met the selection criteria were then invited to submit appropriate cases and complete a detailed online case report form to record the details of their cases that met the inclusion criteria. Participants were able to be included in the study, and therefore provide cases, if they self-reported as Australian-based practitioner with at least two years' clinical experience who provided care to patients seeking assistance with fertility in the last year. They also needed to have screened their fertility patients for the MTHFR genetic polymorphisms, MTHFR C677T and A1298C. Only Australian-based practitioners were included in the study to minimise the number and quality issues of various supplements that may be prescribed by the practitioners in addition to assays used for identification. A screening instrument administered via an online survey platform was employed to screen Practitioners to ensure they met the inclusion criteria prior to receiving access to the case report form. Participants were asked to provide information from historical cases of female patients aged 20–49 years, who had been diagnosed with infertility and had an identified MTHFR genetic polymorphisms C677T and/or A1298C. A diagnosis of infertility was defined as unsuccessfully attempting to conceive for at least the last 12 months but not longer than 2 years and where pregnancy and/or birth outcomes were reported. Data were collected via an online case report form that was shared with participants via Qualtrics online survey platform. The case report form for the initial consultation consisted of 27 questions across four domains: (1) Patient information; (2) Fertility history and fertility status of patient and partner; (3) MTHFR polymorphism ; and (4) Prescriptions. For each consecutive appointment the practitioner was required to respond to a further seven items across another three domains: (1) Supplement's prescription; (2) Dietary prescription; (3) Pregnancy and/or birth outcome. The full case report form is attached as Supplemental File 1. Data obtained from the electronic case report forms (CRFs) was analysed using descriptive statistics (STATA 16 software). 70 The data was extracted into an excel document, cleaned, and coded from the CRF and tabulated in order to summarise the results and report descriptive data. A pseudonym was given to each case to allow easier reporting and description of findings. As the case report study design was not intended to infer associations, inferential statistical analysis was not employed.

The practitioners reported that 11 patients conceived during the course of the treatment, with one patient reporting a subsequent pregnancy loss. Three pregnancies were achieved via IVF. The practitioners reported nine live births and one ongoing pregnancy at the time of data collection, with all patients who became pregnant conceived within two and four months upon commencement of non-folic acid forms of folate. Both patients with the A1298C heterozygous polymorphism conceived and had a live birth. Five of the six patients with heterozygous C677T polymorphism became pregnant and had a live birth. The one patient with homozygous C677T polymorphism successfully conceived and had a live birth. Three of the four patients with compound heterozygous polymorphism became pregnant and had live births. The fourth patient had no history of pregnancy prior to her initial appointment, nor did she fall pregnant during the treatment time. She had been prescribed 1800μg of 5-MTHF and 800μg of folinic acid. Another patient became pregnant twice in the treatment time and miscarried both times. This patient had been prescribed the lowest dose of 5 MTHF (500μg). Adherence and tolerability of the 5 MTHF was good with only one patient reporting an increase in anxiety with the higher dose. She was subsequently told to reduce her dose, which she did and had no further issues. One patient miscarried during the treatment period and there were no other adverse events. The patients did not share their perspective on the treatment as this was a retrospective case study and the researchers had no contact with the patients.

In conclusion, this case series has highlighted clinical practices that are different from the recommendations from the Australian policy for fertility in relation to the dose and form of folate and identifies areas for future research. Individuals with MTHFR polymorphisms may benefit from tailored prescribing practices but further studies are required. The predominant form of folate prescribed in this cohort is 5-MTHF and not FA as recommended. The dose of 5-MTHF that was prescribed far exceeded the recommended 400μg of FA. The practitioners that participated in this study had been recruited from an MTHFR educational site and therefore may be more aware of potential health implications of a MTHFR polymorphism. The fertility outcomes noted from these individuals with diagnosed infertility were extremely positive, with all but two women having successful pregnancies and live births. This research identifies novel insights from clinical practice that requires further investigation and raises public health implications for recommending folic acid to all women particularly in preconception and pregnancy. Given the leading prenatal multivitamin in Australia is a retail brand and contains 800μg of folic acid it is imperative for pharmacists and their pharmacy assistants to understand that different prescriptions may be required for women with MTHFR polymorphisms, and that one form of folate may not be appropriate for all women.

This study provides some novel insights into clinical management of infertile patients with MTHFR polymorphisms that may warrant closer investigation. The findings from this study suggest that these practitioners are advising their patients to actively avoid dietary intakes of FA-fortified food. This approach to clinical care may be because the folate naturally occurring in foods such as leafy green vegetables are more readily converted to the metabolically-active 5-MTHF 29 and differs from FA, as the synthetic form of folate used in food fortification. 26 , 27 , 29 , 31 There is no debate that the national public health policies to mandatorily fortify bread and cereals with FA 71 has been effective in preventing neural tube defects. 72 , 73 , 74 , 75 , 76 , 77 , 78 Practitioners in this cohort, however, may be responding to more recent research that questions the amount of FA patients are consuming due to food fortification as it far exceeds the expected intake of 100 to 200μg. 28 , 29 , 58 , 79 , 80 , 81 , 82 It is postulated that pregnant women are possibly ingesting well above 2000 nmol/l from both their diet and their prenatal supplements. 83 FA had been found to be readily absorbed, and its conversion to tetrahydrofolate is a two-step process utilizing the enzyme DHFR. 24 Human studies propose that when FA cannot be converted to THF the enzyme is saturated and instead UMFA is produced and transported around in the blood. 25 , 26 , 29 , 82 , 84 , 85 In vitro studies suggest that UMFA may act as a folate antagonist which may not only inhibit purine synthesis but the formation of 5-MTHF required for DNA synthesis and methylation. 28 This folate antagonism may contribute to a loss of DNA methylation in sperm among men with MTHFR polymorphisms 86 , 87 and affect embryo development. 88 A murine study found that when FA is low due to no fortification of foods, 5-MTHF and folinic acid levels are significantly higher in the brain which suggests that a possible diversion from 5-MTHF to THF and folinic acid,. 89 In vitro studies indicate that high doses of FA have resulted in reduced MTHF and methylation capacity. 90 Although the existence of UMFA has been extensively studied 25 , 28 , 29 , 59 , 60 , 61 , 82 , 83 , 84 , 85 , 91 , 92 , 93 , 94 , 95 , 96 , 97 its clinical impact is not clear and requires additional researcher attention. Human studies have identified that 96% of breastfeeding women had high levels of UMFA and a corresponding reduction in 5-MTHF levels, possibly due to down regulation of folate-binding protein, which is thought to regulate folate secretion. 83 Outside of fertility, in human studies, UMFA has also been linked to natural killer cell cytotoxicity in post-menopausal women, 28 cobalamin deficiency 91 and some cancers. 28 This case series also suggests practitioners from this cohort are prescribing different forms of supplemental folate to their infertility patients with MTHFR polymorphisms, than the folic acid recommended by Australian health policy. The health recommendation in Australia is that women preparing for pregnancy should take 400-500μg of supplemental FA. 21 The practitioners in this study advised their patients to discontinue their supplements containing FA and instead prescribed folinic acid and/or 5-MTHF. 5-MTHF was the most frequently prescribed form of folate and in cases where folinic acid and methyl folate were prescribed concurrently, 5-MTHF was much higher. This deviation from accepted policy guidelines regarding folic acid supplementation in pregnancy may be due to concerns that when the policy guidelines were formulated the only research on supplemental folate was examining FA. 32 , 33 , 36 , 98 The main form of folate that has been found in cord blood is 5-MTHF and is, therefore, likely to be the form of folate that is transported to the foetus 27 and generally increases mean plasma and red cell folate concentration significantly. 32 , 62 In vitro studies have found that impairment of the folate pathway can affect one carbon metabolism, and this is critical to methylation of DNA, histones and proteins 26 directly linked to the viability of the embryo and sperm. 36 , 50 , 88 As these patients have MTHFR polymorphisms, practitioners may prefer 5-MTHF based on the results of human studies indicating methyl folate may bypass the MTHFR enzyme. 27 , 32 , 33 , 36 It is important to note that of the twelve cases presented in this case series, all were told by their practitioner to discontinue their FA supplement and were prescribed 5-MTHF or a combination of 5-MTHF and folinic acid in its place. Furthermore, eleven of the women fell pregnant within two to four months, ten had successful live births. Two of the three patients that became pregnant via IVF presented to their initial appointment on high dose FA (5000μg) which is the accepted protocol for those with a high risk of miscarriage or neural tube defects. 81 , 99 All three of these cases became pregnant and had a successful live birth after starting treatment, which included replacing their FA with an alternative form of supplemental folate. The practitioners who contributed cases to this study also prescribed much higher doses of folate (mean: 2325μg per day) than the Australian recommendation, which is currently 400-500μg of FA. 100 Our study does not uncover the reasons the practitioners were using doses much higher than the recommendations, but it may be in response to emerging research indicating that MTHF may be more effective in raising red blood cell and serum folate levels, 27 , 32 is safe 101 and may be effective in the prevention and treatment of perinatal depression. 102 Furthermore, recent research regarding doses of folate suggests approximately 800μg or more of 5-MTHF may bypass the MTHFR gene polymorphism and raise plasma folate levels rapidly, resulting in repletion of folate stores within days without creating UMFA. 33 , 36 , 98 However, this possible interpretation would need to be further studied to be confirmed. These findings warrant closer attention from researchers to clarify whether different forms of folate have an impact on the clinical outcome for women with infertility. In particular, further research is needed to investigate if these alternative forms of folate given at higher than recommended doses improve outcomes for women with infertility and MTHFR polymorphisms. The absence of substantive clinical research in humans limits the transferability of this evidence to clinical practice. Furthermore, future research that examines the reasons and implications of this gap between existing policy recommendations and clinical practice behaviour is needed. Every case presented in this study included women who had been diagnosed with infertility, had MTHFR polymorphisms and had previous miscarriages or infertility. Human research has shown that MTHFR polymorphisms may contribute to infertility in women and describes the MTHFR gene polymorphisms contributing to low folate status, elevation in homocysteine which affects folliculogenesis, 11 , 46 , 52 , 85 , 94 embryo viability, 54 recurrent implantation failure 22 and may predispose those patients with MTHFR polymorphisms to recurrent pregnancy loss. 103 , 104 , 105 Although this research cannot be extrapolated to the wider community, the successful pregnancy rate for this case series, gives rise to the need for further research to investigate whether individuals with infertility and MTHFR polymorphism may require a more nuanced approach to achieve pregnancy and live births. Research has identified that MTHFR polymorphisms affect male fertility as well and men who have polymorphisms may influence recurrent pregnancy loss irrespective of the female having the MTHFR polymorphism. 106 , 107 In males, MTHFR polymorphisms are associated with oligozoospermia 34 , 108 and male infertility in general. 53 , 109 MTHFR increases the risk in pregnancy for preeclampsia 110 , 111 and outside fertility have been linked to thrombosis and coronary artery disease, 112 cancer, 113 , 114 vascular disease, depression, 115 schizophrenia, 116 renal failure and several drugs that interfere with folate metabolism such as methotrexate and 5-fluorouracil. 43 , 116 Future research should explore the potential importance of alternative forms of folate for both men and women with infertility. It should also investigate any clinical implications associated with UMFA in individuals with MTHFR polymorphisms and highlights the need for pharmacists to be aware of advancements in the research as folic acid based supplements are primarily purchased over the counter in pharmacies by consumers who may be unaware of UMFA implications particularly if they have MTHFR polymorphisms. The increasing role of the pharmasist in every day health care means that they will be required to recommend different forms and dosages of folate for preconception and pregnancy, particularly as the rate of infertility increases and consumers become more aware of genetic susceptibility to health conditions, including the MTHFR gene polymorphisms and the metabolic differences between folic acid and 5-MTHF.

Case reports and case series are considered to be the lowest level of evidence within the evidence-based medicine hierarchy, yet they can offer valuable information from clinical practice to inform more robust clinical research. 117 By their nature, case series are limited by their small sample sizes as case studies/series generally have small numbers of people or only one person which is studied 118 , 119 , 120 . There may also be bias because the primary researcher is in a group that is exposed to information about clinical management of MTHFR polymorphisms. The practitioners recruited for this study were all from the MTHFR Support Australia database because we required patients who had been screened for the MTHFR gene polymorphisms. This most likely will bias results in relation to the amount and type of folate prescribed to the patients compared to other practitioners who are not in this group. As such, these results should not be interpreted as representative of common clinical care for infertility in Australia or elsewhere. So too, some practitioners may not have participated in the study due to unsatisfactory clinical outcomes from their treatments even though recruitment material encouraged participants to share any relevant cases, whether clinical outcomes were positive or not. The type of MTHFR polymorphism testing was also not specified. Either blood or saliva testing was accepted, and no specific test was required. This may have inferred a limitation due to the variability in the specificity and sensitivity between tests. Despite these identified limitations, this study seeks not to draw causality but rather describe and view trends that practitioners may be observing when treating infertility patients who have MTHFR polymorphisms. As such this study is suited to the research aim and objectives.

Infertility is defined as the failure to conceive a child after one year of unprotected intercourse 1 and is estimated to affect 48.5 million couples worldwide. 2 , 3 , 4 In Australia, one in six couples are expected to be diagnosed with infertility each year 5 and these couples will commonly consult initially with a General Practitioner (GP) and then be referred to fertility specialists or clinics. 6 Australian rates of couples seeking assisted reproductive technologies (ART) are on the incline – a 13.9% increase was noted in 2009 compared to 2008 levels and a 48% increase on 2005 levels – and almost all (92.4%) couples are treated through Australian fertility centres. 7 , 8 Only 17.2% of the 70,541 ART treatment cycles in 2009 resulted in live births. This low rate of success has emotional and psychological 9 , 10 as well as economic (each in vitro fertilisation (IVF) cycle costs approximately $9828 AUD per cycle 3 , 11 ) impacts on couples. There are many identified possible causes of infertility, including recent research indicating a correlation between polymorphisms in folate-metabolising enzymes and individuals with infertility seeking ART. 12 Females who have been found to have deficiencies of folate or low plasma levels of folate have been linked to neural tube defects 13. , 14 , 15 , 16 , 17 and this led to Australian national health advice recommending at last 400μg of folate daily for one month before conception as a supplement or through food fortification 18 . Folic acid oral supplementation has been the traditional form of folate prescribed by Australian practitioners for preconception and pregnancy 19 and up until 2021 the only form of folate available in prenatal multivitamins at pharmacies and retail outlets was folic acid with the leading brand, Elevit containing 800μg of folic acid. Fortification of foods with FA was introduced in Australia in September 2009 to reduce neural tube defects (NTD) and has, by all accounts, been successful. 20 , 21 Folate-dependent pathways are influenced by various biochemical variations of folate and its bioavailability (see Fig. 1 ). 22 Folate is a generic term for vitamin B9 that includes several supplemental forms. All folate forms have a common structure but differ based on the whether the pteridine ring is reduced or oxidised. 23 Folic acid is the synthetic form of folate and requires the enzyme dihydrofolate reductase (DHFR) to be converted to its metabolically active form, 5-methyltetrahydrofolate (5-MTHF). 24 The DHFR enzyme has been found to be down-regulated by an oral intake of around 200-300μg of folic acid. 24 Other forms of folate naturally occur in a reduced form in foods such as leafy green vegetables. 25 This ‘natural folate’, exists in a polyglutamate form, known as tetrahydrofolate (THF), and needs to be further hydrolysed to a monoglutamate form by the intestinal lumen to be absorbed. 26 This intestinal hydrolysis and absorption are the responsibility of the brush border enzyme glutamate carboxypeptidase II 27 and occurs independent of the dihydrofolate reductase (DHFR) enzyme. 26 , 28 Folinic acid , a 5-formyl derivative of THF, is also converted to THF without requiring the DHFR enzyme. 28 5-MTHF is a reduced derivative of THF. 25 The most metabolically active form of folate found in the body is 5-MTHF (accounting for around 90% in the blood). 26 , 29 5-MTHF plays a critical role in methylation as it donates its methyl group to the methionine cycle after which it is converted to THF which in turn provides moieties for the DNA bases. 27 , 30 This methylation process is required for synthesis of DNA, RNA, neurotransmitters, and proteins and for gene imprinting. 31 , 32 , 33 This conversion of folate to 5-MTHF is dependent upon the activity of the methylenetetrahydrofolate reductase enzyme (MTHFR) encoded by the MTHFR gene. 34 , 35 Fig. 1 Folate Metabolism: UMFA-Unmetabolised folic acid, DHFR-Dihydrofolate reductase, THF-Tetrahydrofolate, MTHFR-Methylenetetrahydrofolate Reductase, 5-MTHF-5-methyltetrahydrofolate. Fig. 1 Folate Metabolism: UMFA-Unmetabolised folic acid, DHFR-Dihydrofolate reductase, THF-Tetrahydrofolate, MTHFR-Methylenetetrahydrofolate Reductase, 5-MTHF-5-methyltetrahydrofolate. The MTHFR gene is located on chromosome 1 and it is reported there are over 35 identified polymorphisms, with the most common of these the ‘C677T’ and ‘A1298C’ variants. 36 MTHFR is the enzyme required to activate all forms of folate to 5-MTHF. Specifically, MTHFR catalyses the conversion of 5,10 methylenetetrahydrofolate to 5-MTHF 37 which serves as the methyl donor for synthesis of homocysteine to methionine and is the precursor to S-adenosylmethionine (SAM). 38 MTHFR is a flavoprotein with a cofactor of flavin adenine dinucleotide (FAD) and reducing agent nicotinamide adenine dinucleotide phosphate NAD( P )H which produces the sole source of methyl folate in an irreversible reaction and therefore integral to the synthesis of SAM, the major methyl donor for numerous biosynthetic reactions. 39 , 40 MTHFR activity is regulated by SAM which acts as an inhibitor of the MTHFR activity. 40 DNA methylation that modulates gene expression and ensures genomic integrity is maintained by DNA methyltransferases. These DNA methyltransferases use SAM as the cofactor or donor of methyl groups. The polymorphisms in the MTHFR gene cause gene instability and reduce activity. Without an optimal functioning MTHFR enzyme, the synthesis of methyl folate is reduced and this in turn will reduce one carbon metabolism required for DNA synthesis and methylation of DNA, essential for fertility. 41 Some individuals have variations, or polymorphisms, in the genetic code used for MTHFR production which differs from the ‘wild type’ or normal gene. 42 , 43 The MTHFR polymorphisms C677T and A1298C result in down regulation of enzymatic activity due to an amino acid change that influences the stability of the gene 44 and affects the conversion of 5,10-methylene tetrahydrofolate to 5-MTHF. 34 , 35 This is the only mechanism for intracellular generation of 5-MTHF. 40 , 45 A reduction in folate status due to the MTHFR polymorphisms can affect one‑carbon metabolism 22 and this may directly affect the production of SAM which is responsible for DNA methylation. 46 The MTHFR C677T homozygous polymorphisms – defined as two identical copies of an allele from both parents 47 - occur in approximately 10–14% of Caucasians, 21% of Hispanics, 1–7% of Africans, and 11% of Asians. 48 These homozygous carriers have a 70% reduction in enzyme activity. 44 Heterozygous C677T frequency is approximately 20–25% in the general population 49 and reduces the activity of the enzyme by 30% whilst the homozygous A1298C carriers have a 40% reduction in enzyme activity. 44 Research has identified that MTHFR C677T homozygous individuals have lower DNA methylation than ‘wild type’ individuals. 41 One aspect of infertility that can affect both male and female has been linked to a lack of DNA methylation which can influence key functions associated with the first phases of spermatogenesis 50 and in the growing oocytes with the transition from primary to secondary follicles. 51 One carbon metabolism is not only important systemically but also directly important for the ovary 30 and DNA methylation of imprinted genes. This plays important roles in embryo development and growth. A murine study showed that a deficiency in one carbon metabolism impairs both antral follicular development and oocyte maturation and has led to speculation that the cytoplasm of the oocyte may be an important reserve of one carbon metabolites. 30 Steegers-Theunissen et al. concluded that folate deficiency in women can impair oocyte quality, reduce the success of IVF/Intracytoplasmic sperm injection (ICSI) procedures, and is associated with reproductive failure. 30 Folate is essential to both folliculogenesis 52 and spermatogenesis 53 as it results in an uracil mis incorporation into DNA and may lead to disruption in DNA synthesis, DNA repair and chromosomal errors. 54 In female infertility, the effect of the MTHFR C677T polymorphism has been associated with hyperhomocysteinemia, 55 low folate 45 and reduced oocytes quantity. 56 In males, the effect of MTHFR polymorphisms is associated with spermatogenesis issues such as oligozoospermia and azoospermia. 34 , 57 The MTHFR genetic polymorphisms have been shown to impair both folliculogenesis and spermatogenesis and therefore the association with reduced methyl folate synthesis needs to be addressed by the prescribing practitioner. Research has identified that people living in countries with folic acid fortification of food have an intake that exceeds the original intended dose. 29 , 31 , 58 Questions have been raised as to the implication of this excess folic acid in the blood, which is referred to as unmetabolised folic acid (UMFA). 25 , 28 , 29 , 59 , 60 , 61 As more research unfolds a question is being raised as to whether 5-MTHF is required for those people with MTHFR polymorphisms and infertility. 27 , 33 , 36 , 62 , 63 Studies are emerging that suggest that administration of 5-MTHF may be more effective than folic acid in improving folate status in people who have an MTHFR polymorphism 32 and it has also been suggested that 5-MTHF may bypass MTHFR polymorphisms. 36 , 64 , 65 There are only a few studies that address what dose of 5-MTHF is optimal for preconception and pregnancy in those with MTHFR polymorphisms. These studies quote 5-MTHF doses (e.g., 7.5 mg daily 32 , 33 , 36 well above the recommended daily dose of folate for women of childbearing age (400-500μg). Understanding the most effective form of folate to prescribe in infertility is essential for practitioners to assist in improving pregnancy outcomes and may be critical for public health policymakers going forward. So too, it is essential for pharmacists and pharmacy assistants to understand the nuance of folate prescriptions to be able to fulfill their critical role in providing support to consumers who may benefit from prenatal multivitamins. As a result of this limited research on dose and form of folate in infertility patients, we endeavour to understand further what forms and dosage of folate, practitioners who test for MTHFR polymorphisms, may be prescribing to achieve improved fertility outcomes. The pharmacy environment is such, that since the COVID pandemic the pharmacist is well placed to become the non-medical prescriber to support the current healthcare system. 66 What patients now want is a patient health centric model in pharmacies and the pharmacies that are thriving are those that offer every patient health solutions, recommendations, advice and services. 67 Research has shown that in most countries, including Australia, pharmacies supply complementary medicines including prenatal multivitamins most of the time 68 and that these complementary medicines prescribed in pregnancy and lactation are seen to offer benefits to their babies in utero or while being breastfed. 69

In accordance with my ethical obligation as a researcher, Carolyn Ledowsky is reporting that she is the founder of MTHFR Support Australia and as such has access to the practitioner database of whom she contacted for the purpose of this research. A. Steel and J. Schloss declare no conflicts of interest.

The data that supports the findings of this study are not openly available due to privacy reasons and are available from the corresponding author upon reasonable request.