OBJECTIVE: To investigate the frequency of the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in women with intrauterine fetal death, preeclampsia, preterm delivery, and small for gestational age (SGA) infants.
METHODS: In a prospective cohort study, DNA from 2,000 pregnant women were analyzed for MTHFR C677T by DNA microarray (wild-type allele, C; mutant allele, T).
RESULTS: One thousand six hundred seventy-five women completed the study. Of these, 16.6% (278 women with 556 genetic alleles) developed at least one pregnancy complication and were designated study cases. There were 1,397 women (with 2,794 genetic alleles) who served as controls. MTHFR C677T allele frequencies were significantly different between cases and controls (C [wild-type]: 346 of 556 [62%]; T [mutant]: 210 of 556 [38%] compared with C: 1,911 of 2,794 [68%]; T: 883 of 2,794 [32%]; P=.005; odds ratio [OR] 1.23, 95% confidence interval [CI] 1.06–1.42). Genotype distributions were also different between cases and controls (C/T+T/T [abnormal]: 174 of 278 [63%]; C/C [normal]: 104 of 278 [37%] compared with C/T+T/T: 728 of 1,397 [52%]; C/C 669 of 1,397 [48%]; P=.002; OR 1.54, 95% CI 1.18–2.02). The clinical effect of the MTHFR C677T polymorphism was restricted to women with SGA infants (P=.05; OR 1.33, 95% CI 1.00–1.77). No significant differences in genotype distributions were observed among women with intrauterine fetal death, preeclampsia, and preterm delivery.
CONCLUSION: MTHFR C677T is a genetic marker for identifying women at increased risk of SGA infants.
LEVEL OF EVIDENCE: II
The methylenetetrahydrofolate reductase C677T polymorphism is a genetic marker for identifying women at increased risk of small for gestational age infants.
From the 1Department of Obstetrics and Gynecology, Ludwig Boltzmann Institute of Clinical Obstetrics and Gynecology, Danube Hospital/SMZ-Ost, Vienna, Austria; and 2Department of Obstetrics and Gynecology, Medical University of Vienna, Vienna, Austria.
Corresponding author: Clemens Tempfer, MD, Department of Obstetrics and Gynecology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria; e-mail: firstname.lastname@example.org.
Financial Disclosure The authors have no potential conflicts of interest to disclose.
Pregnancy complications such as preeclampsia, small for gestational age (SGA) infants, unexplained intrauterine fetal death, and preterm delivery are among the most common causes of fetal morbidity and mortality.1 Identification of women at risk of developing these conditions and early detection are important tasks of prenatal care programs. Single nucleotide polymorphisms (SNPs) modulate numerous physiological functions such as blood pressure, blood clotting, and cardiovascular dysfunction and might therefore be useful in identifying women at risk for pregnancy complications.2
Experimental data point to the folate metabolism as an important mechanism to ensure fetal growth and reproductive performance. For example, Habibzadeh et al3 demonstrated that folate deficiency induces intrauterine death in guinea pigs. In rats and sows, folate supplementation significantly increases fetal body weight, vertex-coccyx length, and litter size.4,5 In humans, various derangements of the methionine-homocysteine metabolism have been associated with reproductive performance, eg, hyperhomocysteinemia, folate deficiency, and vitamin B12 deficiency.6–9
The exact mechanism linking folate metabolism and impaired reproduction is unknown. Several hypotheses have been put forward, among them structural and neurologic effects on the fetus, placental thrombosis, and defective chorionic villous vascularization.3,4,10,11
A common polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene consists of a cytosine to thymine substitution at position 677 in exon 4 (MTHFR C677T). The variant T allele is present in 37% to 61% of white women and leads to an increased thermolability of the MTHFR gene product, resulting in decreased activity of the enzyme and elevated homocysteine levels in some individuals.12 In retrospective association studies, carriage of the mutant MTHFR T allele has been associated with thrombosis, recurrent pregnancy loss, placental vasculopathy, premature delivery, and SGA infants.9,13–17 A series of other studies, however, found no increased serum folate concentrations or mutant MTHFR alleles in women with pregnancy complications.18–23 Many reasons may account for these discrepancies, among them varying definitions of disease entities, ethnic differences, and methodological issues with respect to bias in retrospective association studies.24
To clarify whether MTHFR C677T is a risk factor for pregnancy complications such as preeclampsia, SGA, intrauterine fetal death, and preterm delivery in white women, we genotyped 2,000 pregnant women at 12 weeks of gestation and followed them through their pregnancies. We hypothesized that the mutant MTHFR C677T allele is significantly more common among women who will develop pregnancy complications.
MATERIALS AND METHODS
A prospective, controlled, open, single-center study was carried out between January 2004 and February 2005 in a population of 2,267 consecutive pregnant white women at 12 weeks of gestation, who presented to our obstetric outpatient clinic for antenatal care. Approval for this study was obtained by the University of Vienna Institutional Review Board. Exclusion criteria were nonwhite ethnicity and twin pregnancy. After informed consent, a buccal smear was obtained, dried on air at room temperature for 1 hour, and sent to the laboratory. Every sample carried a code, and only the principal investigator (F.S.) was able to decode the results at the end of the study. Gestational age was calculated according to the first day of the last menstrual period and was confirmed by first trimester ultrasonography. Discrepancies of more than 1 week were corrected according to the ultrasound result. Preeclampsia was defined according to American College of Obstetricians and Gynecologists criteria. Preterm delivery was defined as birth before 37 weeks of gestation.
Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). All oligonucleotides were synthesized by VBC-Genomics Bioscience Research GmbH (Vienna, Austria). MTHFR C677T genotypes were assessed using a polymerase chain reaction (PCR) microarray system as described previously.25,26
Differences in the frequencies of alleles and genotypes were analyzed by χ2 test. Continuous variables were compared using the Wilcoxon rank sum test. For statistical analysis of the genotype distributions, we used a dominant genotype model, ie, SNPs were considered binary variables (homozygous wild-type versus heterozygous mutant and homozygous mutant). For assessing all three genotypes separately, we used a gene-dosage model (homozygous wild-type versus heterozygous mutant versus homozygous mutant). A power calculation demonstrated that, with a sample size of 1,675, the study has a power of 90% to detect a 10% difference in genotype distribution at a significance level of .05 using the Yates correction factor based on published genotype distributions of the MTHFR C677T SNP with expected group-specific proportions of 40% MTHFR C677T C/C and 60% MTHFR C677T C/T+T/T.9,12 The odds ratio (OR) was used as a measure of the strength of the association between allele and genotype frequencies and the risk of pregnancy complications. Some women developed more than one pregnancy complication, eg, 14 women had preeclampsia and SGA infants. Regarding the primary outcome, pregnancy complications are calculated per person; in subgroup analyses, pregnancy complications are calculated separately. All P values are two-tailed, and 95% confidence intervals (CIs) were calculated. P<.05 was considered statistically significant. We performed a multivariate linear regression model, with pregnancy complications as dependent variable and MTHFR C677T and the clinical variables age, parity, gravidity, body mass index (BMI), and smoking history as independent variables. The relationship of these variables is described by the standardized β coefficient and respective P values. We used the statistical software SPSS 11.0 for Windows (SPSS Inc, Chicago, IL) for statistical analysis.
A total of 2,267 consecutive women at 12 weeks of gestation were screened; 267 women refused to participate, and 2,000 women were enrolled in the study. Of these, 325 were excluded due to failure of DNA extraction and/or failure of PCR (n=35), twin pregnancy (n=32), and loss to follow-up (n=241). Eleven women had spontaneous abortions, and six pregnancies were terminated due to chromosomal aberrations. Outcome monitoring was available for all remaining 1,675 women. Of 1,675 women, 278 (16.6%) developed at least one of the predefined pregnancy complications. Specifically, intrauterine fetal death, preeclampsia, preterm delivery at less than 34 weeks of gestation, preterm delivery at more than 34 weeks of gestation, SGA infants, less than the 3rd percentile, and SGA infants, 4th–10th percentiles, were observed in 13 (0.8%), 25 (1%), 33 (2%), 81 (5%), 40 (2%), and 125 (7%) women, respectively. These 278 women were used as cases and were compared with 1,397 women who developed none of the above mentioned pregnancy complications. Patient characteristics are given in Table 1. There were no significant differences between the 1,675 women with outcome monitoring and the 241 women who were lost to follow-up regarding age, parity, BMI, and percentage of smokers (data not shown).
The distributions of MTHFR genotypes followed the Hardy-Weinberg equilibrium in cases and controls (P=.6 and P=.2). The allele and genotype distributions of the MTHFR C677T polymorphism among cases and controls are shown in Tables 2 and 3. The allele frequencies of the MTHFR C677T polymorphism were significantly different between cases and controls (MTHFR C: 346 of 556 [62%] and MTHFR T: 210 of 556 [38%] versus MTHFR C: 1,911 of 2,794 [68%] and MTHFR T: 883 of 2,794 [32%], respectively; P=.005; odds ratio [OR] 1.23, 95% confidence interval [CI] 1.06–1.42). In accordance, we observed a statistically significant difference in MTHFR C677T genotype distribution, with mutant alleles being overrepresented among cases compared with controls (MTHFR C/T+T/T: 174 of 278 [63%] and C/C 104 of 278 [37%] versus MTHFR C/T+T/T: 728 of 1,397 [52%] and C/C 669 of 1,397 [48%], respectively, P=.002; OR 1.54, 95% CI 1.18–2.02). Using a gene-dosage model, ie, comparing all three genotypes separately, also showed a significant difference in genotype distribution (P=.006).
In a subgroup analysis, we aimed to assess whether the observed association between MTHFR C677T and predefined pregnancy complications was restricted to some of these pregnancy complications. In this respect, we observed a statistically significant difference in MTHFR C677T genotype distribution between women with SGA infants and controls (MTHFR C/T+T/T: 105 of 165 [63%] and C/C 60 of 165 [37%] versus MTHFR C/T+T/T: 728 of 1,397 [52%] and C/C 669 of 1,397 [48%], respectively, P=.05; OR 1.33, 95% CI 1.00–1.77). No significant differences in genotype distributions were observed among women with intrauterine fetal death, preeclampsia, preterm delivery at less than 34 weeks of gestation, and preterm delivery at more than 34 weeks of gestation (Table 4). Among the study population (n=1,675), 105 of 165 women with SGA infants were identified by MTHFR C677T screening, resulting in a number needed to screen of 16.
In a univariate logistic regression model, MTHFR C677T was significantly associated with pregnancy complications (P=.002; OR 1.5, 95% CI 1.2–1.9). In a multivariate logistic regression model including MTHFR C677T, age, parity, gravidity, and smoking history, MTHFR C677T (β=0.03; P=.04), age (β=0.004; P=.04), and smoking history (β=0.12; P<.001), but not the other variables, were associated with pregnancy complications.
In the present study we found that carriage of the mutant MTHFR C677T T allele at 12 weeks gestation is associated with an increased risk of developing pregnancy complications in an unselected low-risk cohort of women. Specifically, the MTHFR C677T polymorphism was a risk factor for the development of an SGA infant, but not preeclampsia and intrauterine fetal death. Carriage of MTHFR C677T significantly increased the relative risk of an SGA infant by 33%, with a number needed to screen of 16. Our findings are consistent with histological evidence of a mild effect of MTHFR C677T on the risk of placental infarction with fetal growth restriction27–31 and placental abruption and/or infarction in a meta-analysis of eight studies.32 It is reasonable to speculate that placental perfusion is impaired in women with a mutant MTHFR C677T T allele due to hyperhomocysteinemia and subsequent local vasculopathy. Vasculopathy in the placenta as well as in cardiac allografts has been demonstrated in MTHFR C677T T allele carriers.14,33
Our results are in accordance with other retrospective studies performed by our group and others in that the MTHFR SNP does not seem to be associated with preeclampsia and intrauterine fetal death. For example, in a previous retrospective case-control study involving 188 women, we found no association between MTHFR C677T and intrauterine fetal death.27 In accordance, a meta-analysis of studies on early and late fetal loss found no association between MTHFR C677T and late recurrent fetal loss and late nonrecurrent fetal loss.29 In two meta analyses of studies evaluating women with and those without preeclampsia, no association between MTHFR C677T and preeclampsia was assessed.29,30
Of note, our data are at odds with a previous retrospective Canadian case-control study of 493 newborns with a weight below the 10th percentile describing no association with MTHFR C677T. A reason for this discrepancy might be ethnic differences between the study populations. In the study by Infante-Rivard et al,34 29.5% of participants were nonwhite, as opposed to 0% in our study.
Our study has limitations. First, we recruited women at 12 weeks of gestation. Therefore, we cannot rule out selection bias due to pregnant women not presenting to the outpatient clinic after early pregnancy loss. Also, this study is adequately powered for the primary composite outcome of pregnancy complications. Thus, the results of subgroup analyses have to be interpreted with caution. In addition, this is a single-center study. Although this design ensures consistent diagnostic and management practice, selection of the hospital by pregnant women may also reflect bias, eg, social background. The strengths of our study are the size of the patient cohort and the prospective design, ensuring predefined outcomes and diagnostic criteria. Also, women in this study constitute an ethnically homogenous cohort, avoiding methodological problems found with ethnicity-based differences in allele frequencies and genetic admixture.
Smokers were overrepresented among cases compared with controls, and smoking was an independent adverse prognosticator. We therefore performed a univariate and multivariate regression model demonstrating that MTHFR C677T is an independent risk factor for pregnancy complications besides age and a positive smoking history.
Regarding the clinical implications of these results, our data have to be interpreted with caution. In our series, women without this SNP had SGA infants, and women who were carriers of this SNP did not have SGA infants. Thus, carriage of the mutant MTHFR C677T T allele is neither necessary nor sufficient for developing pregnancy complications such as SGA infants. Because there are no evidence-based prevention strategies for SGA infants available to date, identification of women at risk can, at best, improve early detection and early treatment.
In summary, the data presented in this adequately powered prospective, controlled study establish the MTHFR C677T polymorphism as a moderate risk factor of common pregnancy complications in white women. This effect seems to be restricted to the development of SGA infants, but not preeclampsia, preterm delivery, and intrauterine fetal death. Thus, MTHFR C677T is a low-penetrance genetic susceptibility marker for identifying women at increased risk of delivering SGA infants.
1. Creasy RK, Resnik R, editors. Maternal-fetal medicine: principles and practice. 3rd ed. Philadelphia (PA): W. B. Saunders; 1994. p. 609–10.
2. Tempfer CB, Schneeberger C, Huber JC. Applications of polymorphisms and pharmacogenomics in obstetrics and gynecology. Pharmacogenomics 2004;5:57–65.
3. Habibzadeh N, Schorah CJ, Smithells RW. The effects of maternal folic acid and vitamin C nutrition in early pregnancy on reproductive performance in the guinea-pig. Br J Nutr 1986;55:23–35.
4. Achon M, Reyes L, Alonso-Aperte E, Ubeda N, Varela-Moreiras G. High dietary folate supplementation affects gestational development and dietary protein utilization in rats. J Nutr 1999;129:1204–8.
5. Matte JJ, Girard CL, Tremblay GF. Effect of long-term addition of folic acid on folate status, growth performance, puberty attainment, and reproductive capacity of gilts. J Anim Sci 1993;71:151–7.
6. Wouters M, Boers G, Blom H, Trijbels F, Thomas C, Borm G, et al. Hyperhomocysteinemia: a risk factor in women with unexplained recurrent early pregnancy loss. Fertil Steril 1993;60:820–5.
7. Pietrzik K, Prinz R, Reusch K, Bung P, Mallmann P, Chronides A. Folate status and pregnancy outcome. Ann N Y Acad Sci 1992;669:371–3.
8. Bennett M. Vitamin B12 deficiency, infertility, and recurrent fetal loss. J Reprod Med 2001;46:209–12.
9. Unfried G, Griesmacher A, Weismuller W, Nagele F, Huber JC, Tempfer CB. The C677T polymorphism of the methylenetetrahydrofolate reductase gene and idiopathic recurrent miscarriage. Obstet Gynecol 2002;99:614–9.
10. Scholl TO, Johnson WG. Folic acid: influence on the outcome of pregnancy. Am J Clin Nutr 2000;71 suppl:1295–303S.
11. Nelen WL, Bulten J, Steegers EA, Blom HJ, Hanselaar AG, Eskes TK. Maternal homocysteine and chorionic vascularization in recurrent early pregnancy loss. Hum Reprod 2000;15:954–60.
12. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995;10:111–3.
13. Den Heijer M, Lewington S, Clarke R. Homocysteine, MTHFR and risk of venous thrombosis: a meta-analysis of published epidemiological studies. J Thromb Haemost 2005;3:292–9.
14. van der Molen EF, Arends GE, Nelen WL, van der Put NJ, Heil SG, Eskes TK, et al. A common mutation in the 5,10-methylenetetrahydrofolate reductase gene as a new risk factor for placental vasculopathy. Am J Obstet Gynecol 2000;182:1258–63.
15. Valdez LL, Quintero A, Garcia E, Olivares N, Celis A, Rivas F, et al. Thrombophilic polymorphisms in preterm delivery. Blood Cells Mol Dis 2004;33:51–6.
16. Kupferminc MJ, Peri H, Zwang E, Yaron Y, Wolman I, Eldor A. High prevalence of the prothrombin gene mutation in women with intrauterine growth retardation, abruptio placentae and second trimester loss. Acta Obstet Gynecol Scand 2000;79:963–7.
17. Martinelli P, Grandone E, Colaizzo D, Paladini D, Scianname N, Margaglione M, et al. Familial thrombophilia and the occurrence of fetal growth restriction Haematologica 2001;86:428–31.
18. Sutterlin M, Bussen S, Ruppert D, Steck T. Serum levels of folate and cobalamin in women with recurrent spontaneous abortion. Hum Reprod 1997;12:2292–6.
19. Jaaskelainen E, Keski-Nisula L, Toivonen S, Romppanen EL, Helisalmi S, et al. MTHFR
C677T polymorphism is not associated with placental abruption or preeclampsia in Finnish women. Hypertens Pregnancy 2006;25:73–80.
20. Goddard KA, Tromp G, Romero R, Olson JM, Lu Q, Xu Z, et al. Candidate-gene association study of mothers with preeclampsia, and their infants, analyzing 775 SNPs in 190 genes. Hum Hered 2007;63:1–16.
21. Kaiser T, Brennecke SP, Moses EK. Methylenetetrahydrofolate reductase polymorphisms are not a risk factor for pre-eclampsia/eclampsia in Australian women. Gynecol Obstet Invest 2000;50:100–2.
22. Pegoraro RJ, Chikosi A, Rom L, Roberts C, Moodley J. Methylenetetrahydrofolate reductase gene polymorphisms in black South Africans and the association with preeclampsia. Acta Obstet Gynecol Scand 2004;83:449–54.
23. Perez-Mutul J, Gonzalez-Herrera L, Sosa-Cabrera T, Martinez-Olivares R. A mutation in the 5,10-methylenetetrahydrofolate reductase gene is not associated with preeclampsia in women of southeast Mexico. Arch Med Res 2004;35:231–4.
24. Clayton D, McKeigue PM. Epidemiological methods for studying genes and environmental factors in complex diseases. Lancet 2001;358:1356–60.
25. Southern E, Mir K, Shchepinov M. Molecular interactions on microarrays. Nat Genet 1999;21 suppl:5–9.
26. Tempfer CB, Riener EK, Hefler LA, Huber JC, Muendlein A. DNA microarray-based analysis of single nucleotide polymorphisms may be useful for assessing the risks and benefits of hormone replacement therapy. Fertil Steril 2004;82:132–7.
27. Hefler L, Jirecek S, Heim K, Grimm C, Antensteiner G, Zeillinger R, et al. Genetic polymorphisms associated with thrombophilia and vascular disease in women with unexplained late intrauterine fetal death: a multicenter study. J Soc Gynecol Investig 2004;11:42–4.
28. Rey E, Kahn SR, David M, Shrier I. Thrombophilic disorders and fetal loss: a meta-analysis. Lancet 2003;361:901–8.
29. Lin J, August P. Genetic thrombophilias and preeclampsia: a meta-analysis. Obstet Gynecol 2005;105:182–92.
30. Tempfer C, Schneeberger C, Riener EK, Hefler LA, Keck C. Polymorphisms for risk assessment of pregnancy-associated disorders: review of the literature and meta-analysis. Geburtsh Frauenheilk 2004;64:1080–9.
31. van der Molen EF, Arends GE, Nelen WL, van der Put NJ, Heil SG, Eskes TK, et al. A common mutation in the 5,10-methylenetetrahydrofolate reductase gene as a new risk factor for placental vasculopathy. Am J Obstet Gynecol 2000;182:1258–63.
32. Ray JG, Laskin CA. Folic acid and homocyst(e)ine metabolic defects and the risk of placental abruption, pre-eclampsia and spontaneous pregnancy loss: a systematic review. Placenta 1999;20:519-29.
33. Elstein E, Huan C, Fitchett D, Cantarovitch M, Marpole D. C677T gene mutation in methylenetetrahydrofolate reductase as a risk factor for cardiac allograft vasculopathy. Transplant Proc. 1999;31:99.
34. Infante-Rivard C, Rivard GE, Yotov WV, Genin E, Guiguet M, Weinberg C, et al. Absence of association of thrombophilia polymorphisms with intrauterine growth restriction. N Engl J Med 2002;347:19–25.
© 2007 The American College of Obstetricians and Gynecologists
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