Neural tube defects (NTDs) are a group of malformations that result from the failure of the neural tube to close within 28 days after conception. NTDs are an important cause of perinatal mortality, and infants who survive often have life-long disabilities.1,2
Folate deficiency during the periconceptional period increases a woman's risk of having an NTD-affected pregnancy,3 and periconceptional supplementation with folic acid can reduce this risk by as much as 85%.4–6 During folate metabolism, the inactive form of folate 7,8-dihydrofolate is reduced to the active form of folate (5,6,7,8-tetrahydrofolate) by the enzyme dihydrofolate reductase. This active form can be transformed into 5,10-methylenetetrahydrofolate, which can be used directly for thymidylate synthesis, reduced to 5-methyltetrahydrofolate in the methionine synthesis cycle, or used in purine synthesis.7,8 Inhibition of dihydrofolate reductase leads to the depletion of intracellular reduced folate pools for the biosynthesis of purine and thymidine, which in turn results in the inhibition of nucleic acid biosynthesis. Recent laboratory studies have shown that tea catechins can inhibit dihydrofolate reductase in bovine and chicken livers, and folate depletion increases the sensitivity of mouse lymphoma cell line L1210 to the effects of tea catechin.9,10 In addition, in vitro studies using Caco-2 cells have shown that tea catechins may inhibit the uptake of intestinal folate, resulting in decreased bioavailability of folate to cells.11,12
In healthy male volunteers, green and black tea, effectively reduced serum folate concentrations.12 Pregnant Japanese women who drank 4 or more cups of green tea per day were more likely to have a low serum folate concentration.13 In a case-control study, Correa et al14 found an association between prenatal tea consumption and an increased risk of spina bifida (odds ratio [OR] = 2.3 [95% confidence interval (CI) = 1.2-4.4]), and this risk increased with the number of cups of tea consumed per day (ORs = 2.1 and 2.8 for 1-2 cups/d and for 3 or more cups/d, respectively). Tea originates from China, and tea drinking is very popular in some areas of the country. The aim of the present study was to explore the association between tea drinking and the risk of NTDs in an area with a high prevalence of NTDs15 and poor folate nutritional status among pregnant women.16
This case-control study was conducted from June 2002 to December 2007 in 4 rural counties (Pingding, Xiyang, Taigu, and Zezhou) in Shanxi Province, China, where the prevalence of NTDs has been reported as high as 13.9 per 1000 births.15 A population-based birth defects surveillance program was implemented in the 4 counties, and a total of 83,962 births were registered during the study period. Cases with an NTD or other major external structural malformations were ascertained from live births, stillbirths, and elective terminations. Obstetricians and midwives were trained in diagnosing malformations through newborn physical examinations. A color atlas of birth defects was provided to aid diagnosis. Trained pediatricians at our institute made the final diagnosis by reviewing the local diagnosis, the case report form, and, when the mother consented, photos of the newborn. When a fetus or a newborn with an NTD or other major malformation was identified, a singleton term newborn without any congenital malformations was selected as a control from the newborns in the same hospital following the identification of the case. The control was of the same sex as the case, had a mother residing in the same county as that of the case, and had a date of conception as close as possible to that of the case.
NTD Case Definition and Classification
In this study, an NTD was defined as anencephaly, spina bifida, or encephalocele. Spina bifida occulta was not included in the study. We classified anencephaly as occurring either in isolation or in combination with other congenital malformations of various systems; spina bifida as occurring either in isolation or in combination with congenital malformations other than anencephaly; and encephalocele as occurring either in isolation or in combination with congenital malformations other than anencephaly or spina bifida.
Trained local healthcare workers conducted face-to-face interviews with mothers of cases and controls. Ninety-two percent of interviews were performed within the first week after delivery or termination of a pregnancy. A structured questionnaire was used to collect information on the mother's sociodemographic characteristics, lifestyle, reproductive history, maternal illness, medication exposure, use of folic acid supplements, and tea drinking from 1 month before through 2 months after conception.
Tea drinking was classified as daily (at least one drink per day), weekly (fewer than one drink per day, but at least one drink per week), and none (fewer than one drink per week).
We first analyzed the association between tea drinking and the risk of NTDs in aggregate. We then analyzed the association by NTD subtypes, that is, anencephaly, spina bifida, and encephalocele. Risk was estimated by the use of ORs, and the precision of each OR was assessed by its 95% CI. Multivariate logistic regression analysis was used to estimate the adjusted OR associated with tea drinking while controlling for confounding factors. Potential confounding factors included maternal age (<25, 25–29, ≥30 years), maternal educational level (primary school or lower, junior high school, high school or above), maternal occupation (farmer/nonfarmer), and periconceptional folic acid supplementation (yes/no). Data analysis was carried out using SPSS 11.5 (SPSS, Chicago, IL).
The study was approved by the institutional review board of Peking University. All participants provided written informed consent.
During the study period, 637 mothers of NTD cases were interviewed. Of these cases, 288 (45%) had anencephaly, 300 (47%) spina bifida, and 49 (8%) encephalocele. A total of 866 controls were enrolled. Information on tea drinking was missing for 6 cases (0.9%) and for 9 controls (1.0%), leaving 631 cases and 857 controls in the final analysis.
Table 1 shows demographic and obstetric characteristics of the case and control mothers. Compared with control mothers, case mothers were more likely to be older, to have less education, and to have a history of birth defect-affected pregnancy. In addition, case mothers were more likely than control mothers to be multigravidas and multiparas. About 63% of current pregnancies in the case group were planned compared with 72% in the control group. More than 30% of case mothers reported an episode of flu or fever over 38.5°C during early pregnancy compared with only 8% of control mothers. Cigarette smoking was infrequent in both groups of mothers. However, about 70% of case mothers reported passive smoking during early pregnancy, compared with about 57% of control mothers. Fewer case mothers (11%) than control mothers (14%) had taken folic acid supplements during the periconceptional period.
Daily and weekly tea drinking during the periconceptional period was more common among case mothers than control mothers (Table 2). As compared with mothers who did not drink tea, mothers who drank tea every day were over 3 times more likely to have an NTD-affected pregnancy. ORs were 1.1 and 3.1 for weekly and daily tea drinking, respectively.
When maternal age, educational level, occupation, and periconceptional folic acid supplementation were included in the multivariate logistic regression model, the estimates were slightly stronger: the OR was 1.2 (95% CI = 0.9–1.6) for weekly tea drinking and 3.4 (1.4–8.3) for daily tea drinking, with no tea drinking as the referent (Table 2).
The magnitude of risk in association with tea drinking was similar for anencephaly and spina bifida, each having a 3-fold increased risk in association with daily tea drinking (Table 3). Daily tea drinking was more weakly associated with encephalocele.
A possible joint effect of tea drinking and folic acid supplementation was explored by analyzing the association between tea drinking and the risk of NTDs while stratifying by periconceptional folic acid supplementation. As shown in Table 4, among those who took folic acid supplements during the periconceptional period, no case mother or control mother drank tea every day, and weekly tea drinking was not associated with an elevated risk of NTDs. Among those who did not take periconceptional folic acid, daily tea drinking was associated with a greater than 3-fold increased risk of NTDs (OR = 3.6 [95% CI = 1.5–8.7]), whereas weekly tea drinking was not associated with NTDs (1.1 [0.8–1.4]).
Table 5 shows factors associated with tea drinking. Women under 30 years of age, or those who had an educational level of junior high school, or high school or above were more likely to be weekly tea drinkers. Women working in nonagricultural occupations were more likely to drink tea, both daily and weekly. In addition, women who did not take folic acid supplements during the periconceptional period were more likely to be daily and weekly tea drinkers.
In this case-control study, daily tea drinking during the periconceptional period was associated with a 3-fold elevated risk of NTDs. The association remained after adjusting for other known risk factors for NTDs. In addition, the association was present for all 3 major subtypes of NTDs, that is, anencephaly, spina bifida, and encephalocele. The association of tea drinking with all 3 subtypes of NTDs is somewhat different from an earlier case-control study conducted in Atlanta, GA, in which tea drinking was associated with spina bifida (OR = 2.3 [95% CI = 1.2–4.4]) but not anencephaly (0.9 [0.5–1.5]).14
Folate, as a major one-carbon donor, is required for purine nucleotide and thymidylate synthesis and is essential for RNA and DNA production. Folate is also needed for the synthesis of methionine, a precursor of S-adenosylmethionine, which is required for methylation of DNA, histones, lipids, and neurotransmitters.17 Due to these biologic functions, folate is important for cells and tissues that rapidly divide, and therefore is crucial to the fast developing embryo. Any interference with folate metabolism could result in impairment in DNA and RNA synthesis, impairment in translation of genes involved in neurulation, and failure of the neural tube to close, although the exact mechanisms remain unknown.8
Due to its importance in folate metabolism, dihydrofolate reductase is the target enzyme for antifolate drugs such as the anticancer drug methotrexate and the antibacterial drug trimethoprim.18 Tea contains 4 major catechins: (-)-epigallocatechin gallate, (-)-epigallocatechin, (-)-epicatechin gallate, and (-)-epicatechin.19 Because of their similarity in molecular structure to folic acid, tea catechins competitively inhibit dihydrofolate reductase in cellular folate metabolism pathways, in a way similar to those of the antifolates.10,20 High levels of green tea catechins can decrease serum 5-methyltetrahydrofolate concentrations in rats.21 In addition, tea catechins may interact with folate transporters (reduced folate carriers or proton-coupled folate transporters), and decrease the availability of folate to cells.12 In humans, a dose of 0.3 g of either green or black tea extracts reduced maximum serum folate concentration by 39% when administered with 0.4 mg folic acid.12 Pregnant Japanese women who drank 4 or more cups of green tea per day were less likely to have a normal serum folate concentration (OR = 0.47 [95% CI = 0.22–0.99]).13 Therefore, tea drinking during the periconceptional period may increase a woman's risk of conceiving a fetus affected with an NTD due to decreased availability of folate or impaired folate metabolism.
The finding that daily but not weekly tea drinking was associated with an elevated risk of NTDs suggests that heavy tea drinking may be harmful. In a case-control study,22 only women who consumed 3 or more cups of tea per day showed an elevated risk for an anencephalic stillbirth. A person who drinks 2 cups of tea per day with about 200 mL each cup will ingest about 40–140 mg of total catechins.23 In the present study, detailed information on cups of tea per day was not collected; we were unable to estimate the amount of catechins consumed per day.
The inhibition effect of tea catechins on dihydrofolate reductase is dependent on folate nutritional status. In vitro studies using mouse lymphoma cell line L1210 demonstrated that the ability of tea catechins to inhibit cell growth by dihydrofolate reductase inhibition was dependent on the folate concentration in the medium; cell growth was more sensitive to tea catechins when folate was depleted.10 In vivo human studies showed a smaller decrease in peak serum folate concentration when the same dose of catechins was administered with 5 mg of folic acid (27%) than with 0.4 mg of folic acid (39%).12 In the present study population, about 50% of women in the first 16 weeks of gestation were serum-folate deficient.16 Women in our study who did not take folic acid supplements during the periconceptional period tended to drink more tea than women who took periconceptional folic acid. Therefore, tea drinking may pose an additional risk of having a pregnancy affected by an NTD to women in this population.
There are geographical variations in types of tea consumed in China. Jasmine green tea is the most popular type in the north, including this study area.24 Concentrations of catechins may differ by types of tea and by brands. In an earlier experimental study, catechin levels in jasmine green tea from mainland China and in green tea from Taiwan were similar, but lower than catechin levels in a brand of black tea from the United States.23 In another study, jasmine green tea was found to have only one-fifteenth of the amount of catechins in green tea (the sum of 5 catechins: 16 mg/g vs. 250 mg/g).25 In a human in vivo study, green and black tea showed similar effects on serum folate levels: a 39% reduction in maximum serum folate concentration after intake of 0.4 mg folic acid and 0.3 g green or black tea extract in 250 mL water, compared with intake of 0.4 mg folic acid with 250 mL water.12
Our study has several strengths. The population-based nature decreased the likelihood of our having overestimated the effect of tea drinking resulting from selection bias. Moreover, the study population was relatively homogeneous; virtually all of the subjects were of Han nationality and lived in rural areas. Clinical descriptions and photographs of the birth defect cases were reviewed by 3 experienced pediatricians, decreasing the likelihood of misclassification of birth defects. In addition, we considered all types of pregnancies affected by NTDs, including elective terminations due to prenatal diagnosis.
An intrinsic limitation of the case-control study is recall bias. Compared with control mothers, case mothers may have had a tendency to recall certain factors that they believed contributed to their NTD-affected pregnancies. However, because tea drinking is considered part of a healthy lifestyle, and no reports on the association between tea drinking and elevated risk of NTDs are available yet in China, recall bias is likely minimal. In 2004, we conducted a survey to evaluate the accuracy and reproducibility of the case-control questionnaire data. The coincidence rate for the major variables was 93%.26 Another limitation is that information on the type of tea consumed was not collected. Although catechin content may differ by types of tea, both types of tea extracts have demonstrated similar potential to lower serum folate level in a human study.12 A third limitation is the crude quantification of tea consumption, which prevented us from further analyzing the data by more detailed categories of tea consumption. Future studies should collect information on type of tea and number of cups of tea consumed per day.
1. Davidoff MJ, Petrini J, Damus K, Russell RB, Mattison D. Neural tube defect-specific infant mortality in the United States. Teratology. 2002; 66(suppl 1):S17–S22.
2. Botto LD, Moore CA, Khoury MJ, Erickson JD. Neural-tube defects. N Engl J Med. 1999;341:1509–1519.
3. Daly LE, Kirke PN, Molloy A, Weir DG, Scott JM. Folate levels and neural tube defects. Implications for prevention. JAMA.
4. MRC Vitamin Study Research Group.Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. Lancet.
5. Czeizel AE, Dudas I. Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N Engl J Med.
6. Berry RJ, Li Z, Erickson JD, et al. Prevention of neural-tube defects with folic acid in China. China-U.S. Collaborative Project for Neural Tube Defect Prevention [erratum.] N Engl J Med.
7. Shane B. Folate and vitamin B12 metabolism: overview and interaction with riboflavin, vitamin B6, and polymorphisms. Food Nutr Bull.
8. Beaudin AE, Stover PJ. Insights into metabolic mechanisms underlying folate-responsive neural tube defects: a mini review. Birth Defects Res A Clin Mol Teratol.
9. Navarro-Peran E, Cabezas-Herrera J, Campo LS, Rodriguez-Lopez JN. Effects of folate cycle disruption by the green tea polyphenol epigallocatechin-3-gallate. Int J Biochem Cell Biol.
10. Navarro-Peran E, Cabezas-Herrera J, Garcia-Canovas F, et al. The antifolate activity of tea catechins. Cancer Res.
11. Alemdaroglu NC, Wolffram S, Boissel JP, et al. Inhibition of folic acid uptake by catechins and tea extracts in Caco-2 cells. Planta Med.
12. Alemdaroglu NC, Dietz U, Wolffram S, Spahn-Langguth H, Langguth P. Influence of green and black tea on folic acid pharmacokinetics in healthy volunteers: potential risk of diminished folic acid bioavailability. Biopharm Drug Dispos.
13. Matsuzaki M, Haruna M, Ota E, et al. Dietary folate intake, use of folate supplements, lifestyle factors, and serum folate levels among pregnant women in Tokyo, Japan. J Obstet Gynaecol Res.
14. Correa A, Stolley A, Liu Y. Prenatal tea consumption and risks of anencephaly and spina bifida [abstract.] Ann Epidemiol.
15. Li Z, Ren A, Zhang L, et al. Extremely high prevalence of neural tube defects in a 4-county area in Shanxi Province, China. Birth Defects Res A Clin Mol Teratol.
16. Ren A, Zhang L, Hao L, et al. Comparison of blood folate levels among pregnant Chinese women in areas with high and low prevalence of neural tube defects. Public Health Nutr.
17. Zhao R, Matherly LH, Goldman ID. Membrane transporters and folate homeostasis: intestinal absorption and transport into systemic compartments and tissues. Expert Reviews in Molecular Medicine.
18. Sanchez-del-Campo L, Saez-Ayala M, Chazarra S, Cabezas-Herrera J, Rodriguez-Lopez JN. Binding of natural and synthetic polyphenols to human dihydrofolate reductase. Int J Mol Sci.
19. Feng WY. Metabolism of green tea catechins: an overview. Curr Drug Metab.
20. Navarro-Peran E, Cabezas-Herrera J, Hiner AN, et al. Kinetics of the inhibition of bovine liver dihydrofolate reductase by tea catechins: origin of slow-binding inhibition and pH studies. Biochemistry.
21. Augustin K, Frank J, Augustin S, et al. Green tea extracts lower serum Epidemiology 2, Number 4, July 2011 Tea Drinking and the Risk of Neural Tube Defects folates in rats at very high dietary concentrations only and do not affect plasma folates in a human pilot study. J Physiol Pharmacol. 2009;60:103–108.
22. Fedrick J. Anencephalus and maternal tea drinking: evidence for a possible association. Proc R Soc Med.
23. Bronner WE, Beecher GR. Method for determining the content of catechins in tea infusions by high-performance liquid chromatography. J Chromatogr A.
24. Gao Y, Hu N, Han X, et al. Jasmine tea consumption and upper gastrointestinal cancer in China. Cancer Causes Control.
25. Jin Y, Jin CH, Row KH. Separation of catechin compounds from different teas. Biotechnol J.
26. Li ZW, Ren AG, Zhang L, et al. Evaluation on birth defects surveillance system in four counties of Shanxi province, China [in Chinese]. Zhonghua Liu Xing Bing Xue Za Zhi.