The impact of plasma folate levels of mothers and newborns on intrauterine growth retardation and birth weight

Mutation Research 591 (2005) 302–310

The impact of plasma folate levels of mothers and newborns on intrauterine growth retardation and birth weight Radim J. Sram ∗ , Blanka Binkova, Zdena Lnenickova, Ivo Solansky, Jan Dejmek Institute of Experimental Medicine Academy of Sciences of Czech Republic, Health Institute of Central Bohemia, Videnska 1083, 142 20 Prague 4, Czech Republic Received 1 December 2004; received in revised form 11 April 2005; accepted 13 April 2005 Available online 15 August 2005

Abstract Folate plays an important role in the genomic stability of human cells. In our studies of the impact of environmental pollution on human health, we have found that air pollution can affect pregnancy outcome. As it may be also affected by nutrition, we examined the effect of plasma folate levels of mothers and newborns on intrauterine growth retardation (IUGR) and birth weight (BW) in cohorts from Prague (N = 319) and Teplice (N = 444). The lower plasma levels (<6.1 nmol/L) were observed in 7.1% of mothers in Prague, and in 9.6% of mothers in Teplice. The higher plasma levels (>36.5 nmol/L) were observed in 28.4% of mothers in Prague, and in 15.7% of mothers in Teplice. The higher plasma levels were observed in 75.4% of newborns in Prague, and 73.2% of newborns in Teplice. When comparing maternal high versus low folate plasma levels and IUGR by logistic regression, the risk of IUGR was significantly decreased for European cohort (according to ethnicity) with gestation age >36 weeks [N = 536, OR = 0.44, P = 0.026], and even more pronounced in the group of European smokers [N = 157, OR = 0.14, P = 0.015]. Using multiple regression analysis, plasma folate levels of mothers and newborns significantly affected the birth weight of newborns of smoking mothers (P < 0.05). The obtained results seem to indicate a positive effect of folate on pregnancy outcome, especially its potential to decrease the risk of IUGR in European population and lower birth weight in smoking European mothers. It would be warranted to study the effect of folate levels on pregnancy outcomes in the relationship to different environmental pollution and life styles of mothers. © 2005 Elsevier B.V. All rights reserved. Keywords: Folate; Intrauterine growth retardation; Newborns birth weight; Smoking; ETS

1. Introduction Abbreviations: BMI, body mass index; BW, birth weight; ETS, environmental tobacco smoke; FA, folate; GA, gestational age (in weeks); IUGR, intrauterine growth retardation; PAHs, polycyclic aromatic hydrocarbons; RDI, recommended dietary intakes ∗ Corresponding author. Tel.: +420 241 062 596; fax: +420 241 062 785. E-mail address: [email protected] (R.J. Sram). 0027-5107/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2005.04.015

Folate (FA) is an important micronutrient which plays a significant role in DNA metabolism [1]. It is a long established fact that FA is required for the synthesis of dTMP from dUMP. Under conditions of FA deficiency, dUMP accumulates and as a result,

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uracil instead of thymine is incorporated into DNA. This excessive incorporation of uracil into DNA leads to single and double stranded DNA breaks, chromosome breakage, and micronucleus formation [2,3]. The impact of FA on genomic stability has been advocated for a long time especially by Michael Fenech [1,4]. In conditions of deficiency, there is increased chromosome breakage and DNA hypomethylation. FA deficiency (<400 nmol/kg diet) is also related to threefold increase in fetal developmental abnormalities [5]. Folate deficiency is an important risk factor for neural tube defects [6], cancer [7–9] and cardiovascular diseases [10]. These adverse effects are seen at FA level in plasma below 5.5 nmol/L. Nevertheless, it is not known what the optimal concentration of FA in plasma should be in order to suppress the DNA damage. According to Fenech [1,4], the recommended dietary intake (RDI) of FA (=400 ␮g per day) required to minimize DNA damage are expected to result in plasma >5.0 nmol/L (based on prevention of anemia). However, this RDI is significantly lower than required intake in diet corresponding to the average FA concentration levels (47.7 nmol/L in plasma) in which the DNA damage is minimized [1]. FA plasma level <4.9 nmol/L is associated with an increased risk of early spontaneous abortions [11]. Maternal periconceptional supplementation with FA has been known for more than two decades to be effective in reducing the risk of selected human congenital malformations [12] as neural tube defects, heart defects, and craniofacial malformations [13–17]. A folate deficiency during embryogenesis can significantly alter the gene expression, demonstrating the importance of proper maternal nutrition for transcriptional competence [18,19]. This knowledge became the reason for a broad use of periconceptional supplementation with FA. The relationship between folate level and birth weight may be related to FA role in genomic stability. Both in vitro and in vivo studies of human cells clearly show that folate deficiency causes expression of chromosome fragile sites, chromosome breaks, excessive uracil in DNA, micronucleus formation, and DNA hypomethylation [1]. For example, we already observed the effect of carcinogenic PAHs (polycyclic aromatic hydrocarbons) on the induction of DNA adducts in vitro [20], in placenta in mothers [21], and IUGR [22]. Recently Perera observed the effect of PAHs to induce DNA adducts and hprt mutations in

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newborns in relation to birth weight [23]. Therefore, we questioned if such effect can be possibly ameliorated by folate supply. Insufficient folate means inhibition of nucleic acid synthesis. As nucleic acid and protein synthesis is increased during embryogenesis, the lack of FA may affect IUGR and birth weight. But no such data analyzing the relationship between folate plasma level in mothers and newborns and IUGR and/or birth weight were found in PubMed. Similarly, no studies of a possible relationship of folate and homocysteine with IUGR was listed in the same source. Therefore, the relationship between FA, homocysteine, and IUGR is not discussed further in the present communication. Higher FA level at 30 weeks gestational age was related to increased birth weight and lower risk of fetal growth retardation [24]. Similarly, Neggers et al. [25] experienced association between FA intake at 30 weeks of gestation and birth weight. Using questionnare, Mitchell et al. [26] observed the association of folate supplementation with the reduced risk of SGA (small for gestation age). Hyperhomocysteinemia in mothers with the history of preeclampsia and fetal growth restriction was corrected by FA [27]. High red cell folate was associated with a decreased risk for abruptio placentae and intrauterine growrh restriction, hyperhomocysteinemia with an increased risk [28]. De Onis et al. [29] analyzed 12 randomized control trials on nutritional interventions to reduce risk of IUGR and proposed that folate supplementation during gestation merit further research. As the accumulated evidence suggests that FA plays an important role in genomic stability, we have decided to study the level of FA in pregnant mothers and newborn children in regions where we had studied the impact of air pollution on pregnancy outcomes [21,22,30,31].

2. Materials and methods 2.1. Subjects The background sample was collected in the district of Teplice and the University hospital in Prague 2 (New Town, the City Center). The district of Teplice with about 120,000 inhabitants and 1000 births per year, lies in the brown coal basin of Northern Bohemia, polluted

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by chemical factories, surface mining, and large coal power plants [21]. In this district, we have studied the impact of air pollution on pregnancy outcome since the year 1994 [22,30]. FA level was analyzed in pregnant mothers delivering singleton in the district of Teplice and Prague in the period 2001–2002. Mothers were selected for the investigation as a nested-control study from women who were carrying a child with low birth weight (below 2500 g), prematurely born (<37 weeks), and controls. The total number of all women in the sample was 766 subjects: 444 from Teplice and 322 from Prague. Almost all mothers in Prague were of European origin (N = 314), while in Teplice they were mostly of European origin as well as Gypsies (Europeans N = 374, Gypsies N = 61). European origin is defined according to ethnicity, not including Gypsies [30]. The study was approved by the Institutional Review Board for the Protection of Human Subjects of the Regional Institute of Hygiene of Central Bohemia. The mother’s signature of a written informed consent form was the final condition of the enrollment in the study. Pregnancies for specimen collection and analysis were selected systematically: maternal and cord blood specimen were collected at delivery for every fifth pregnancy in the background sample; blood plasma was frozen immediately and stored at −70 ◦ C until analysis. 2.2. Determination of folates The CEDIA folate kit (Roche Diagnostics) was used for the determination of folates in plasma. Sorbation was measured on the ELISA Reader Spectra (TECAN) at wavelength 415 nm (with the reference wavelength 630 nm and the limit of detection 0.6 ng/ml). According to Roche Diagnostics the level of FA in healthy population corresponds to 2.7–16.1 ng/ml (6.1–36.5 nmol/l). 2.3. Statistical analysis SAS ©Logistic regression was used to estimate the impact of FA level on IUGR. Many biological and social confounders were tested. FA levels were characterized as tertiles of distribution. Statistica ©Multiple regression was used to estimate the impact of FA on birth weight. These levels of FA were transformed by square root function. Odd ratios were used for evalu-

ating binary outcome of IUGR+/− or similarly, as the result of a logistic regression estimate regresion coefficients were used to evaluate the impact of continuous values as, e.g. birth weight. Multiple regression was also used to estimate factors affecting FA levels. Confounders were the same as in logistic regression: BMI (body mass index) as difference from standard BMI value (25), ETS (environmental tobacco smoke) when mother declared exposure to passive smoking during pregnancy, and active smoking approved by the mother anytime during pregnancy, As well as primipara symptom, an increment in gender ratio as female to male children, difference from standard pregnancy duration (gestational age 40 weeks), and effect of education observed at secondary school degree. In order to observe possible differences between strongly premature and term deliveries as well as to exclude extreme cases, the estimates are broken into two variants: gestational age of more than 32 weeks, and more than 36 weeks (term births). High and middle tertile FA levels were both compared to low tertile by logistic regression. IUGR (intrauterine growth retardation, small for gestational age) was determined as the lowest tenths weight percentile of newborn children in the Czech Republic by gestational age and gender from the state statistics. IUGR correlates significantly with birthweight, but it is not the same type of value, birth weight is absolute value, but weight IUGR is related to the apropriate gestational age.

3. Results FA levels in maternal and fetal plasma are given in Table 1. The fetal plasma level is approx. twice as high as in maternal plasma. The levels of FA in maternal plasma were higher in Prague subjects than in Teplice Europeans, while the lowest levels were detected in Gypsies from Teplice. When we used the distribution of FA levels, maternal plasma (all subjects) contained lower level <6.1 nmol/L in 8.8% of cases, normal level in 70.2%, and higher level >36.5 nmol/L in 21.1%, respectively (Fig. 1, Table 2). In fetal plasma only two levels were observed: normal level of FA in 25.8% of cases, and higher level in 74.2% of cases (Fig. 2, Table 2). FA level was higher in maternal plasma of subjects using vitamin supplements during their preg-

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Table 1 Maternal and fetal folate levels in different groups (nmol/L)

Maternal Fetal Correlation

Maternal Fetal Correlation

All (766)

Europeans (688)

Europeans NS (505)

Europeans SM (183)

Gypsies (64)

Teplice (444)

Teplice Europeans (374)

24.7 ± 16.3 47.7 ± 17.0 0.50

25.4 ± 16.6 48.1 ± 17.3 0.50

26.6 ± 16.8 49.0 ± 17.7 0.49

22.0 ± 15.9 45.2 ± 15.7 0.51

17.3 ± 12.7 43.6 ± 15.4 0.39

22.5 ± 16.1 46.3 ± 16.8 0.52

23.6 ± 16.8 46.8 ± 17.0 0.54

Teplice Europeans NS (251)

Teplice Europeans SM (123)

Teplice Gypsies (61)

Prague (319)

Prague Europeans (314)

Prague Europeans NS (254)

Prague Europeans SM (60)

24.5 ± 16.8 48.4 ± 17.4 0.54

21.3 ± 16.3 43.8 ± 15.4 0.51

17.0 ± 16.3 44.0 ± 15.4 0.39

27.7 ± 16.3 49.5 ± 17.3 0.46

27.7 ± 16.1 49.5 ± 17.3 0.46

28.6 ± 16.3 49.9 ± 17.5 0.45

23.4 ± 14.8 47.9 ± 15.7 0.51

Values are means ± S.E., NS: nonsmokers; SM: smokers; correlation, maternal:fetal.

Fig. 1. Folate level: maternal (lower = <6.1 nmol/L, normal = 6.1– 36.5 nmol/L, higher = >36.5 nmol/L).

nancy (3.17, P = 0.0000) as well as in fetal plasma (1.82, P = 0.0012). This relationship was stronger in maternal versus fetal plasma, but not observed in the fetal plasma of smoking mothers. Table 2 The distribution of folate in plasma (according to tertiles) <6.1

6.1–36.5

>36.5 nmol/L

Maternal All Prague Teplice Teplice-Europeans Teplice-Gypsies

68 (8.8%) 23 (7.1%) 43 (9.6%) 32 (8.5%) 11 (18.0%)

543 (70.2%) 209 (64.5%) 334 (74.7%) 277 (73.5%) 49 (80.3%)

163 (21.1%) 92 (28.4%) 70 (15.7%) 68 (18.0%) 1 (1.6%)

Fetal All Prague Teplice Teplice-Europeans Teplice-Gypsies

– – – – –

198 (25.8%) 79 (24.6%) 119 (26.8%) 102 (27.3%) 16 (26.2%)

570 (74.2%) 242 (75.4%) 325 (73.2%) 272 (72.7%) 45 (73.8%)

Fig. 2. Folate level: fetal (normal = 6.1–36.5 nmol/L, higher = > 36.5 nmol/L).

When analyzing factors affecting FA levels, we found that maternal education is a predominating factor in European population (2.56, P = 0.02 for maternal level; 3.42, P = 0.005 for fetal level) as well as European population in Teplice (2.78, P = 0.02 for maternal level; 3.76, P = 0.005 for fetal level). In addition to secondary education, smoking in the first trimester affected fetal FA levels in Europeans in Teplice (−1.99, P = 0.002). The effect of maternal FA on IUGR was analyzed separately in the whole group (All), in Europeans, European smokers (SM) and nonsmokers (NS), Prague Europeans (as well as SM and NS), Teplice Europeans (as well as SM and NS), and in Gypsies. Tertiles of FA levels were used for the logistic regression analysis as follows: maternal plasma, lower <15.4 nmol/L, middle 15.4–28.8 nmol/L, higher >28.8 nmol/L, and fetal plasma, lower <40.2 nmol/L, middle 40.2–55.2 nmol/L, higher >55.2 nmol/L. The

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Table 3 Effect of maternal folate on IUGR: Europeans GA >32 weeks, N = 652

GA >36 weeks, N = 536

OR

95% CI

P

OR

95% CI

P

Folate Middle tertile High tertile

0.77 0.44

(0.40–1.49) (0.20–0.95)

0.43 0.037

0.76 0.38

(0.38–1.51) (0.17–0.89)

0.43 0.026

District TP/PR BMI Smoking ETS Primipara Gender (females)

0.52 0.86 1.90 1.85 0.69 0.98

(0.28–0.95) (0.78–0.95) (0.99–3.65) (0.88–3.89) (0.37–1.29) (0.55–1.76)

0.032 0.002 0.053 0.11 0.24 0.95

0.45 0.87 2.05 1.85 0.82 0.94

(0.24–0.84) (0.79–0.96) (1.04–4.04) (0.85–4.05) (0.43–1.56) (0.51–1.74)

0.013 0.007 0.039 0.12 0.55 0.85

BMI: body mass index; ETS: environmental tobacco smoke; PR: Prague; TP: Teplice.

IUGR as well as birth weight were affected in the same direction by BMI. Higher BMI decreased IUGR in Europeans (Table 3, P < 0.01) as well as in European smokers (Table 4, GA >32 weeks, P < 0.05). Analyzing the effect on birth weight, we observed that higher BMI increased birth weight in Europeans (Table 5, P < 0.001), European smokers (Table 6, GA >32 weeks, P < 0.05), and Prague smokers (Table 8, GA >36 weeks, P < 0.01). However, FA level in the maternal blood correlated negatively with BMI in European nonsmokers (−0.06, P < 0.05) and Teplice European nonsmokers (−0.06, P = 0.08). In the cord blood, a negative impact of FA levels on BMI was observed in the whole group (−0.03, P = 0.09), Gypsies (−0.16, P < 0.05), and Gypsies in Teplice (−0.19, P < 0.05). Ten cases of eclampsia and pre-eclampsia (7 and 3, respetively) occurred in our sample. The relationship between folate level in both maternal and fetal plasma

results in Europeans indicate that high tertile of maternal FA significantly decreases IUGR in the gestational age over 32 weeks (OR = 0.44, P = 0.037), as well as the gestational age over 36 weeks (OR = 0.38, P = 0.026) (Table 3). This effect was even more pronounced in Europeans-smokers: maternal FA high tercile substantially decreases IUGR in the gestational age over 32 weeks (OR = 0.24, P = 0.035), as well as the gestational age over 36 weeks (OR = 0.14, P = 0.015) (Table 4). The analysis of the impact of maternal FA on birth weight demonstrated no effect in all Europeans (Table 5), European nonsmokers as well as smokers (Table 6), and Gypsies (Table 7). However, a significant effect of smoking and ETS was observed in European mothers (Table 5). The ameliorating effect of FA on birth weight is seen only in the group of smoking mothers from Prague with gestation age over 36 weeks as the impact of maternal as well as fetal FA (Tables 8 and 9). Table 4 Effect of maternal folate on IUGR: Europeans, smokers GA >32 weeks, N = 192

GA >36 weeks, N = 157

OR

95% CI

P

OR

95% CI

P

Folate Middle tertile High tertile

0.55 0.24

(0.21–1.39) (0.06–0.90)

0.20 0.035

0.45 0.14

(0.17–1.23) (0.03–0.68)

0.12 0.015

District TP/PR BMI ETS Primipara Gender (females)

0.83 0.87 2.75 0.76 1.00

(0.33–2.13) (0.76–1.00) (0.34–22.58) (0.30–1.90) (0.42–2.39)

0.70 0.047 0.35 0.55 0.99

0.59 0.88 2.90 0.90 1.04

(0.22–1.60) (0.76–1.01) (0.34–24.79) (0.34–2.38) (0.41–2.64)

0.30 0.070 0.33 0.84 0.93

BMI: body mass index; ETS: environmental tobacco smoke; PR: Prague; TP: Teplice.

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Table 5 Effect of maternal folate on birth weight: Europeans GA >32 weeks, N = 657

Intercept Folate District TP/PR BMI Smoking ETS Primipara Gender (females) Gestation age

GA >36 weeks, N = 539

BW (g)

P

BW (g)

P

3656 3 35 18 −132 −74 −46 −184 172

0.83 0.32 0.000 0.001 0.042 0.16 0.000 0.000

3692 −4 30.3 18 −165 −67 −82 155 −182

0.82 0.44 0.000 0.000 0.12 0.034 0.000 0.0001

BMI: body mass index; BW: birth weight in g; ETS: environmental tobacco smoke; PR: Prague; TP: Teplice.

Table 6 Effect of maternal folate on birth weight: Europeans, smokers GA >32 weeks, N = 191 BW (g) Intercept Folate District TP/PR BMI ETS Primipara Gender (females) Gestation age

3451 14 15 17 −38 −56 −195 146

GA >36 weeks, N = 157 P

BW (g)

P

0.60 0.82 0.024 0.71 0.36 0.001 0.000

3400 24 27 15 −29 −106 −162 137

0.42 0.71 0.081 0.80 0.13 0.019 0.000

BMI: body mass index; BW: birth weight in g; ETS: environmental tobacco smoke; PR: Prague; TP: Teplice.

and this disease was observed with a simple t-test. In the maternal plasma it was quasi significant (6.01 versus 10.95, P = 0.0553), in fetal plasma the difference was significant (16.34 versus 21.06, P = 0.0478). Single

eclamptic or pre-eclamptic outcomes were both with a lower folate level then another subjects, but this individual folate level did not significantly differ from the all group dispersion.

Table 7 Effect of maternal folate on birth weight: Gypsies GA >32 weeks, N = 60 BW (g) Intercept Folate District TP/PR BMI Smoking ETS Primipara Gender (females) Gestation age

3442 63 117 −13 −199 −359 −123 −66 179

GA >36 weeks, N = 48 P

BW (g)

P

0.33 0.73 0.34 0.15 0.29 0.32 0.58 0.000

3171 38 134 −13 −151 – −185 −63 266

0.61 0.70 0.47 0.32 – 0.21 0.64 0.000

BMI: body mass index; BW: birth weight in g; ETS: environmental tobacco smoke; PR: Prague; TP: Teplice.

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Table 8 Effect of maternal folate on birth weight: Prague, smokers GA >32 weeks, N = 55

Intercept Folate BMI Gypsies/Europeans ETS Primipara Gender (females) Gestation age

GA >36 weeks, N = 44

BW (g)

P

BW (g)

P

3387 79 33 −223 −117 −50 −238 131

0.15 0.072 0.61 0.49 0.68 0.055 0.000

3553 167 57 58 −583 −56 −186 −8

0.004 0.003 0.88 0.004 0.62 0.12 0.85

BMI: body mass index; BW: birth weight in g; ETS: environmental tobacco smoke. Table 9 Effect of fetal folate on birth weight: Prague, smokers GA >32 weeks, N = 56

Intercept Folate BMI Gypsies/Europeans ETS First child Gender (females) Gestation age

GA >36 weeks, N = 44

BW (g)

P

BW (g)

P

3530 3 27 −337 −98 −97 −222 118

0.70 0.17 0.47 0.59 0.47 0.088 0.000

3683 18 50 −34 −535 −138 −264 15

0.051 0.012 0.93 0.013 0.27 0.033 0.76

BMI: body mass index; BW: birth weight in g; ETS: environmental tobacco smoke.

4. Discussion According to our knowledge, our study is the first one to explore the effect of FA in plasma on IUGR and birth weight in a larger population. All our results indicate a significant decrease of IUGR in European mothers when FA plasma level is higher than 28.8 nmol/L (Table 3). This effect is much more pronounced than the effect of FA on birth weight. A correlation between FA levels in maternal and in fetal blood being higher in fetal blood corresponds to the results of Dejmek et al. describing Vitamin C levels in mothers and newborns in the Teplice district [32]. Similar, approx. twice as high, difference between cord blood and maternal serum FA level was observed by Guerra-Shinohara et al. [33] in Brazil. The level of maternal FA in Teplice Europeans was approx. more than 20% higher than in Gypsies (Table 1), similarly as in the study of Vitamin C levels. Levels of maternal as well as fetal FA were affected in Europeans by education.

Surprisingly, there is no substantial difference in the fetal plasma levels of FA between the groups Prague versus Teplice Europeans versus Teplice Gypsies. We can speculate that the level of FA observed in fetal plasma corresponds to the optimal level, which is necessary for the regulation of gene expression during development. The risk of IUGR is significantly increased by smoking and this effect seems to be substantially decreased with a sufficient FA supply. When we used multiple regression to analyze the impact of FA level on birth weight, other factors as smoking or ETS were much more predictive. The effect of FA was seen only in the group of Prague smokers where birth weight was affected by maternal as well as fetal FA level. Originally, it was expected that higher supply of FA may substantially increase the birth weight of newborn. It was not observed in our sample. Our results indicate that smoking mothers had lower FA level than nonsmoking ones. The same results were observed by McDonald [34] and Ozerol et al. [35].

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IUGR as well as birth weight were significantly affected by BMI. We can hypothesize that a high BMI may be indicative of a better micronutritient status due to increased food intake. However, it contradicts the effect of FA on BMI, when an increase in the intake of FA is observed to decrease BMI in European nonsmokers. It could correspond to better lifestyle factors in this group. It may be concluded that folic acid status of mothers in the Czech Republic is surprisingly satisfactory, being higher than in Germany [36] and Italy [37]. It may be due to favorable changes in nutritional habits as well as multiple vitamin supplementation in pregnancy during the last several years. It is of interest that the fetal FA level in Czech newborns falls within the limit corresponding to Fenech idea about the levels of FA at which DNA damage is minimized: 47.7 nmol/L (range 16.6–120.3 nmol/L) for plasma [1]. We may speculate that this particular FA level represents the optimal level for the fetus. Our data together with literature may be interpreted that higher FA intake (supplementation) may be recommended to smoking mothers to prevent IUGR similarly as FA is already used to prevent developmental abnormalities and spontaneous abortions in early pregnancies. It would be warranted to study the effect of FA levels on pregnancy outcomes in the relationship to different types of environmental pollution and lifestyles of mothers together with genetic polymorphisms of genes coding proteins involved in the FA metabolism. Acknowledgements This study was supported by the Ministry of Environment of the Czech Republic (grants No. VaV/340/2/00 and VaV/740/5/03), Academy of Sciences of the Czech Republic (1QS500390506) and the Commission of the European Communities (grant QLK4-CT-2002-02198 and ICA1-CT-2000-70028). Authors thank to Dr. M. Fenech and Mrs. K. Kavka for their critical comments. References [1] M. Fenech, The role of folic acid and vitamin B12 in genomic stability of human cells, Mutat. Res. 475 (2001) 57–67.

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