Folic acid use in pregnancy and embryo selection

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1 DOI: /j x General obstetrics Folic acid use in pregnancy and embryo selection P Haggarty, a,b DM Campbell, b S Duthie, a K Andrews, a G Hoad, a C Piyathilake, c I Fraser, b G McNeill d a Nutrition and Epigenetics Group, Rowett Research Institute, Aberdeen, UK b Department of Obstetrics and Gynaecology, Aberdeen University, Aberdeen, UK c School of Medical Sciences, Division of Nutritional Biochemistry and Genomics, University of Alabama, Birmingham, AL, USA d Department of Environmental and Occupational Medicine, Aberdeen University, Aberdeen, UK Correspondence: Dr P Haggarty, Nutrition and Epigenetics Group, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK. p.haggarty@abdn.ac.uk Accepted 3 March Objective Folic acid supplement use is recommended in pregnancy to reduce the risk of neural tube defect but concerns have been raised that increasing folic acid intake may select for embryos with genotypes that increase disease risk in the offspring. Our aim was to test for this effect. Design Observational prospective cohort study. Setting Aberdeen Maternity Hospital. Population or Sample Women born before the introduction of folic acid advice ( ) and carrying singleton pregnancies (n = 1234) and their offspring (n = 1083) born after ( ). Methods We measured the genotype (MTHFR C677T and A1298C, MTR A2756G, MTRR A66G and TCN G776C) of mothers and their offspring, maternal supplement intake, intake of folate and vitamin B12 from natural foods and maternal blood folate and B12 status at 19 weeks of gestation. Main outcome measures B vitamin related genotype of the offspring. Results There were no significant differences in any of the five genotype frequencies between mothers and their babies. There was no deviation from Hardy Weinberg equilibrium in either generation and no change in the frequency of doubly homozygous MTHFR variants (677 TT/1298 CC). The genotype of the offspring was not related to maternal periconceptual supplement use, folate intake from foods or plasma and red cell folate measured at 19 weeks of gestation. Conclusions We found no evidence to support the concern that folic acid fortification or supplement use in pregnancy results in selection of deleterious genotypes. Keywords Embryo, fertility, folic acid, MTHFR, selection. Please cite this paper as: Haggarty P, Campbell D, Duthie S, Andrews K, Hoad G, Piyathilake C, Fraser I, McNeill G. Folic acid use in pregnancy and embryo selection. BJOG 2008;115: Introduction Folic acid supplement use is recommended in pregnancy to reduce the risk of neural tube defect (NTD [MIM ]), 1,2 and some countries have introduced the additional measure of mandatory fortification of the diet with folic acid to address the same problem: in the USA, mandatory fortification of enriched grain products with folic acid resulted in an estimated mg/day increase in the intake of folates 3 and a 144% increase in plasma folate concentration in women. 4 The efficacy of folic acid in reducing the incidence of NTD is not in doubt, but its possible role in increasing the survival of embryos with deleterious genotypes continues to be widely cited as a possible argument against folic acid fortification of the diet and possibly even the use of folic acid supplements during pregnancy. 5 7 This concern arises from a report of an increase in the frequency of mutant alleles in the folate metabolising methylenetetrahydrofolate reductase gene (MTHFR: C677T and A1298C) in babies born in Spain following the recommendation of folate supplement use in that country. 8,9 The interpretation of this finding is not that folic acid causes de novo mutations but that it may result in the survival of embryos carrying these mutations, which would normally perish. If correct, this would be a significant public health concern as the general consensus is that the MTHFR 677 TT genotype is deleterious to health: it is thought to increase the risk of many diseases including ischaemic heart disease, deep vein thrombosis, pulmonary embolism and stroke and even the risk of NTD. 13 The latter observation raises the unfortunate prospect that fortification with folate could reduce the incidence of NTD in one generation but increase the genetic propensity to develop NTD in the next, potentially requiring ever increasing levels of fortification with each new generation. ª 2008 The Authors Journal compilation ª RCOG 2008 BJOG An International Journal of Obstetrics and Gynaecology 851

2 Haggarty et al. We report here the genotype frequencies for five B-vitamin-related variants in mothers, born before the widespread use of folic acid supplements, and their offspring, born in the UK after the introduction of advice on folic acid. We also analysed the genotype of the offspring in relation to maternal intake of folate and vitamin B12 from natural foods and supplements, and maternal blood folate and B12 status. Materials and methods The study was approved by Grampian Research Ethics Committee, and all women in the study gave informed written consent to take part. Women with diabetes, carrying multiple pregnancies, who conceived as a result of fertility treatment or in whom clinical data were not available were excluded. Advice on folic acid supplement use equivalent to that in Spain, and many other countries worldwide, was introduced in the UK in The mothers in this study were born before the introduction of this advice ( ) and their babies after ( ). The advice is that women intending to become pregnant in the UK should take 400 mg/day of supplementary folic acid prior to conception and up to 12 weeks of gestation to reduce the risk of having a baby with NTD. 1 The energy adjusted intake of folate and vitamin B12 from natural foods was determined by self-administered food frequency questionnaire 14 provided at recruitment. This questionnaire is designed to provide an estimate of habitual diet, and it has previously been validated in women from a similar population against weighed intakes for these nutrients. 15 Additional questions relating to the use of food supplements around the time of conception amount per day, timing and duration of consumption were linked to a database of approximately 300 supplement products. 14 Women also provided a blood sample and mouthwash sample at approximately 19 weeks of gestation. Cord blood corresponding to each maternal sample was collected at term for genotype analysis, unless the placenta had been discarded prior to collection or the blood had clotted. Total plasma folate and plasma vitamin B12 were determined by radioimmunoassay using the Simultrac Radioassay Kit Vitamin B12 [ 57 Co]/Folic acid [ 125 I] supplied by MP Biomedicals (Irvine, UK). Total red blood cell folate was measured using the 96-well plate adaptation of the Lactobacillus casei microbiological assay. 16 Plasma folate and plasma vitamin B12 were measured in duplicate and red cell folate in triplicate. Results were discarded if the internal standard coefficient of variation for the assay exceeded 15% for total plasma folate. Individual samples were excluded if the coefficient of variation between replicates exceeded 10% for folate and 30% for vitamin B12. Samples were anonymised prior to genotyping. DNA was isolated from a 10-ml saline mouthwash solution from the mother and from cord blood, quantified by RNaseP assay (Applied Biosystems, Warrington, UK) and genotyped as described elsewhere. 14 Five polymorphisms in four genes involved in B-vitamin metabolism were determined by allelic discrimination assay using TaqMan Ò MGB probes labelled with 6-FAMÔ and VIC Ò on an ABI 7700 Sequence Detection System (Applied Biosystems). These were methylenetetrahydrofolate reductase (chr 1, EC , MTHFR C677T and A1298C), methionine synthase (chr 1, EC , MTR A2756G), methionine synthase reductase (chr 5, EC , MTRR A66G) and transcobalamin (chr 22, a plasma globulin, TCN G7761C). The genotypes of the control DNA for the five variants were confirmed by sequencing on a Beckman Coulter CEQ 8000 (Beckman, High Wycombe, UK). A mother/baby discrepancy in any of the five genotypes studied resulted in all the genotype data for that pair being excluded from the analysis (n = 9). Statistical analysis was carried out using STATA/SE version 9.0 (Stata Corp, College Station, TX, USA), SPSS version 13 and SamplePower version 2.0 (SPSS, Chicago, IL, USA). Results are presented as means and SDs unless the parameter deviated from normality, in which case, the median and interquartile range (IQR) are presented. The main statistical analysis carried out was logistic regression to assess the influence of nutrient intake and status on offspring genotype with adjustment for maternal genotype. Multiple linear regression was used for continuous dependent variables and Pearson s chi-square test to compare frequency distributions. This study was able to detect with a power of 90% a change in the MTHFR 677 TT genotype frequency of 5% in the offspring of mothers consuming 400 mg/day folic acid (a = 0.05; twotailed test). It was also able to detect a 5% change in MTHFR 677 TT genotype frequency between generations with a power of 90% (a = 0.05; two-tailed test). These changes are considerably less than the effect previously reported. 9,17 Results Of 1461 eligible women, 1277 were enrolled at Aberdeen Maternity Hospital when attending for ultrasound scan. A further 184 women were recruited later in pregnancy when a mouthwash but no blood sample was collected; these women were included only in the nutrient intake analysis, and there were no significant differences in offspring genotype frequencies compared with the main group. The average age of the mothers at delivery was 30.4 (SD 5.3) years (Table 1). The babies were 53% male and 47% female, with an average birthweight of 3452 (SD 559) g. Comparisons of genotype frequencies for all five variants studied showed no significant differences between mothers and babies and no deviation from Hardy Weinberg equilibrium in either generations (Table 2). The previously reported genotype frequency changes associated with folic acid use related to the MTHFR 677 TT and MTHFR 1298 CC mutant genotypes. 9,17 The frequency of these polymorphisms was 852 ª 2008 The Authors Journal compilation ª RCOG 2008 BJOG An International Journal of Obstetrics and Gynaecology

3 Folic acid and embryo selection Table 1. Pregnancy characteristics (n = 1461) Maternal characteristics Mean age in years at delivery (SD) 30.4 (5.3) Mean height in cm (SD) (6.7) Primiparous (%) 48.9 Smoker (%) 15.2 Pregnancy outcome Baby sex: male (%) 53.3 Mean gestational age at delivery in weeks (SD) 39.4 (1.9) Mean birthweight in g (SD) 3452 (560) Pregnancy loss, n Stillbirth 2 Neonatal death in first week 4 Miscarriage 1 essentially the same in the babies and their mothers (11.8 versus 12% for MTHFR 677 TT and 9.0 and 9.1% for MTHFR 1298 CC, respectively). There was no difference between mothers and babies in terms of combined MTHFR C677T and A1298C genotypes. In both generations, double homozygous mutants (677 TT/1298 CC) were very rare; only one case in over two thousand individuals (Table 3). A more direct test of the effect of folic acid exposure may be obtained by investigating the effect of maternal supplement use and dietary intake of folate on the genotype of the offspring. In the study group, seven pregnancies (0.5%) resulted in loss of the baby either before birth or in the first week of postnatal life; the overwhelming majority of recognised pregnancies (99.5%) resulted in a surviving baby. Because nonsurvivors accounted for such a small proportion of all pregnancies, even if all of these had homozygous mutant genotypes, it would make no significant difference to the genotype frequencies in the whole birth cohort or the maternal nutrient exposure groups they came from. Only failed oocyte fertilisation or early embryo loss (i.e. prior to establishment of pregnancy) is potentially large enough to have an effect on offspring genotype of the order described by Munoz- Moran et al.; therefore, only variations in periconceptual maternal folate/folic acid intake could be expected to have an effect on offspring genotype frequency. Periconceptional use of folic acid was reported in 42% of women with 39% taking at least the recommended 400 mg/day. The energy adjusted mean intake from normal foods was 353 (SD 78) mg/day for folate and 6.3 (SD 2.8) mg/day for vitamin B12. These values were similar to those measured in nonpregnant women of similar age and from the same geographical area. 15 Corroboration of the intake data was provided by blood folate status at 19 weeks of gestation; median plasma folate = 11.1 ng/ml, IQR ng/ml; median plasma B12 = pg/ml, IQR pg/ml; median red cell folate = 203 ng/ml, IQR ng/ml. Dietary habits change little with stage of pregnancy, but changes in folic acid supplement use may modulate the strength of the relationship between periconceptual intake and blood status at 19 weeks, particularly for plasma folate rather than red cell folate which better reflects long-term status. However, the relationship between folate intake from natural foods and periconceptional supplement use and blood status remained significant; log red blood cell folate (P < for both natural foods and supplements) and log plasma folate (P < for both natural foods and supplements). Analysis of offspring genotypes in relation to periconceptual B12 intake and status was also carried out as some of the genes of interest use B12 as a cofactor or are involved in B12 metabolism. The plasma B12 concentration was related to the intake of vitamin B12 from Table 2. Genotype numbers and frequencies in mothers and babies Polymorphism MTHFR C677T MTHFR A1298C MTR A2756G MTRR A66G TCN2 C776G Mothers Homozygote wild type (%) Heterozygote (%) Homozygote mutant (%) Total number Babies Homozygote wild type (%) Heterozygote (%) Homozygote mutant (%) Total number There were no significant differences between mother and baby genotype distributions by chi-square test. There was no significant deviation from Hardy Weinberg equilibrium for any genotypes by chi-square test. ª 2008 The Authors Journal compilation ª RCOG 2008 BJOG An International Journal of Obstetrics and Gynaecology 853

4 Haggarty et al. natural foods (P < 0.001) but not the periconceptual intake of vitamin B12 from supplements (P = 0.521), which was much less common than folic acid use. Logistic regression was used to test for an effect of dietary and supplement intake on the frequency of the mutant genotype in the offspring with adjustment for maternal genotype (Table 4). There was no evidence of an effect of maternal folate/folic acid or B12 intake (from foods or supplements or foods and supplements combined) on the frequency of the mutant genotypes in the offspring. In addition to providing corroboration of the validity of folate intake, the blood status data were also evaluated for an effect on the offspring genotype. None of the measures of blood folate and B12 status was associated with offspring mutant genotype frequency for any of the five variants measured. We carried out a number of additional analyses (data not presented) including comparison of offspring genotype in only mothers taking folate at or above the recommended 400 mg/day with those taking no folate, coding folic acid use as a categorical variable, analysing the effect on allele frequencies rather than genotypes, etc. but were unable to detect any significant effect of intake on embryo genotype. Discussion Munoz-Moran et al. reported a doubling of the frequency of the MTHFR 677 TT genotype (13 26%) in a Spanish population over a period of only 20 years. 17 In a subsequent analysis in a larger group, they reported a change from 12% in those older than 25 years to 22% in those lesser than 24 years. 9 The change in A1298C genotype was less obvious, but there was an increase in the number of individuals with both mutations. They also estimated from extrapolation of the calculated selection pressure that the mutant homozygote MTHFR 677 TT frequency could reach 90% within five generations, with 90% of the population carrying at least two mutations for the C677T and A1298C variants in only three generations. These are very large effects and, if they arose as a result of folic acid use in pregnancy, would be of serious concern in relation to public health. The best study design to test this observation would be a randomised controlled trial of folic acid supplements in women intending to become pregnant. However, the evidence for a beneficial effect of folic acid on NTD is such that the inclusion of a placebo group would be unethical; therefore, an observational study such as that reported here is the only ethically acceptable design. The original embryo selection observation was based on an analysis of secular changes in genotype with age and therefore potentially subjected to confounding due to factors such as immigration/emigration and population stratification on the basis of ethnicity. The study design employed here, comparing mothers and their babies, is more robust as potential problems such as population stratification and changes in immigration should be negligible; in our population, these factors could only be relevant to the fathers. There was no change in genotype frequency, for any of the five polymorphisms studied, between the mothers (born before the supplement advice) and their babies (born after the supplement advice). The introduction of novel factors that change the balance of selection pressures may manifest in deviation of the genotype frequency distribution from Hardy Weinberg equilibrium as the population moves to a new equilibrium point. The fact that both mother and baby frequency distributions were in Hardy Weinberg equilibrium tends to argue against a new selection pressure acting on these polymorphisms between the time the mother was born and the baby was born. The original report demonstrated secular changes in combined MTHFR C677T and A1298C mutant genotypes. However, in this study, we found that double homozygous mutants (677 TT/1298 CC) were very rare and that the overall MTHFR C677T/A1298C frequency distributions were the same in the mothers and babies. Around 40% of the study group were taking folic acid supplements periconceptionally, but there was no evidence of an effect of maternal folate/folic acid or B12 intake (from foods or supplements or foods and supplements combined) on the Table 3. Frequencies of combined MTHFR C677T/A1298C genotypes Mothers Babies MTHFR C677T MTHFR A1298C Total MTHFR C677T MTHFR A1298C Total AA AC CC AA AC CC CC 160 (13.2%) 284 (23.4%) 108 (8.9%) 552 CC 149 (14.2%) 233 (22.2%) 91 (8.6%) 473 CT 266 (21.9%) 247 (20.4%) 2 (0.2%) 515 CT 232 (22.1%) 220 (20.9%) 3 (0.3%) 455 TT 146 (12.0%) 0 (0%) 1 (0.1%) 147 TT 123 (11.7%) 0 (0%) 0 (0%) 123 Total Individual counts are shown for each frequency combination together with the percentage of the total for each cell. 854 ª 2008 The Authors Journal compilation ª RCOG 2008 BJOG An International Journal of Obstetrics and Gynaecology

5 Folic acid and embryo selection Table 4. Effect of nutrient intake and status on baby genotype (homozygous mutant versus heterozygote and homozygous wild type) Parameters (OR units) MTHFR C677T MTHFR A1298C MTR A2756G MTRR A66G TCN2 C776G OR (95%CI) P value OR (95%CI) P value OR (95%CI) P value OR (95%CI) P value OR (95%CI) P value Intake from supplements Folic acid (100 mg/day) 1.02 ( ) ( ) ( ) ( ) ( ) Vitamin B12 (mg/day) 1.00 ( ) ( ) ( ) ( ) ( ) Intake from natural foods Folates (100 mg/day) 0.99 ( ) ( ) ( ) ( ) ( ) Vitamin B12 (mg/day) 1.02 ( ) ( ) ( ) ( ) ( ) Intake from natural foods and supplements Folates and folic acid (100 mg/day) 1.02 ( ) ( ) ( ) ( ) ( ) Vitamin B12 (mg/day) 1.01 ( ) ( ) ( ) ( ) ( ) Maternal B-vitamin status Plasma folate (100 nmol/l) 0.56 ( ) ( ) ( ) ( ) ( ) RBC folate (100 nmol/l) 0.95 ( ) ( ) ( ) ( ) ( ) Plasma B12 (10 nmol/l) 1.00 ( ) ( ) ( ) ( ) ( ) Logistic regression was used to test for an effect of dietary and supplement intake on the frequency of the mutant genotype in the offspring after adjustment for maternal genotype. Odds ratios are reported together with the 95% CI and P value. The units for the odds ratios were selected to represent a meaningful proportion of the range. frequency of the mutant genotypes in the offspring. Blood folate and B12 status at 19 weeks of gestation was predictive of folate intake, but again, neither of these was associated with offspring mutant genotype frequency for any of the five variants measured. However, the data were analysed and we found no evidence of an effect of periconceptual folate/folic acid or B12 intake and status on embryo genotype. For folic acid to have an effect on genotype selection, there would have to be a differential risk of conceptus loss on the basis of MTHFR genotype, which can be partially or completely overcome by increasing the folic acid exposure. There is a plausible mechanism for such an effect in that spontaneously aborting fetuses have a greater risk of carrying one or more mutant MTHFR alleles (677T and 1298C) 18,19 and combinations of these with other B-vitamin-related genotypes such as TCN2. 20 Poor B-vitamin status and increased homocysteine concentrations have been linked to early pregnancy loss, 21,22 and sequence variations in the genes involved in B- vitamin metabolism have also been studied in relation to pregnancy loss. 23,24 However, a very high rate of attrition would be required to produce a change in genotype frequency similar to that observed by Munoz-Moran et al. Taking the actual MTHFR C677T genotype distribution in the mothers studied here (CC 45%; CT 43%; TT 12%) and an extreme assumption that all the CC genotypes are replaced by TT genotypes in the nonsurvivors (CC 0%; CT 43%; TT 57%), an attrition rate of 22 26% would be necessary to produce the reported change in TT frequency. 8,9,17 The nonsurvival rate in this study group was 0.5%. This would typically be higher in twins and higher order multiples, but the frequency of these conditions means that the sum of recognised pregnancy losses from all causes miscarriage, stillbirth, etc. typically found in developed countries could not explain the effect observed. The proportion of embryos that are lost after fertilisation, or zygotes which fail to fertilise, is much greater, and a differential effect on this parameter has the potential to significantly influence genotype frequency in the offspring. One possible mechanism that could act on fertility is the use of reproductive technologies as it has been reported that particular genotypes such as MTHFR A1298C may be preferentially passed on to the offspring as a result of the effect of the mother s genotype on her response to in vitro fertilisation (IVF). 14 However, IVF births currently account for only 1% of all births in the UK and most other European countries; 25 therefore, the use of reproductive technologies in Spain around 1992 could not explain the magnitude of the observed change in genotype. It is estimated that there are between 700 and 900 NTDaffected pregnancies in the UK each year, resulting in miscarriage, termination or lifelong disability for those born with this condition. In the USA, the postfortification NTD rate is around 7 8 per live births. 2 Folic acid is known to be effective in reducing the incidence of this condition, but concern over possible effects on embryo selection continue to ª 2008 The Authors Journal compilation ª RCOG 2008 BJOG An International Journal of Obstetrics and Gynaecology 855

6 Haggarty et al. be cited as a potential reason for not fortifying the diet with folic acid and reservations about the use of folic acid supplements. We found no evidence to support these concerns. Conflict of interest P.H. is a member of the UK Scientific Advisory Committee on Nutrition that provides advice on folic acid fortification to the UK Food Standards Agency and Department of Health. Contribution to authorship P.H.: study design, data collection and statistical analysis and preparation of the manuscript; D.M.C.: study design, clinical database and recruitment and manuscript revision; S.D.: plasma folate and homocysteine and manuscript revision; I.F.: recruitment, collation of clinical and nutritional information; K.A.: sample processing, collation of nutrient and supplement information and genotyping; G.H.: sample processing, supplement database and genotyping; C.P.: red cell folate and manuscript revision; G.Mc.N.: study design, dietary questionnaire and manuscript revision. Acknowledgements This study was funded by the Food Standards Agency (grant no: N05040). P.H., S.D. and G.H. acknowledge the support of the Scottish Government Rural and Environment Research and Analysis Directorate. We are grateful to A Skene who assisted with sample processing and analysis. j References 1 Committee on Medical Aspects of Food and Nutrition Policy. 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