IJE Advance Access originally published online on June 16, 2006
International Journal of Epidemiology 2006 35(4):948-953; doi:10.1093/ije/dyl116
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Article |
Stillbirth and slow metabolizers of caffeine: comparison by genotypes
1 The Danish Epidemiology Science Centre, Department of Epidemiology, Institute of Public Health, University of Aarhus, 8000 Aarhus, Denmark.
2 Department of Environmental and Occupational Medicine, Institute of Public Health, University of Aarhus, 8000 Aarhus, Denmark.
3 Perinatal Epidemiology Research Unit, Department of Obstetrics and Paediatrics Aarhus University Hospital, Skejby, Denmark.
4 Department of Epidemiology, School of Public Health, UCLA, Los Angeles, CA, USA.
* Corresponding author. The Danish Epidemiology Science Centre, Institute of Public Health, Vennelyst Boulevard 6, Building 1260, University of Aarhus, DK-8000 Aarhus, Denmark. E-mail: bhb{at}soci.au.dk
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Abstract |
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Background Cytochrome P4501A2 (CYP1A2) and N-acetyltransferase 2 (NAT2) are key enzymes in the metabolism of caffeine. The polymorphism of these genes facilitates the detection of fast and slow metabolizers, and if caffeine is causally related to stillbirth, we expect slow metabolizers to have a higher risk of stillbirth at any given intake of caffeine. Gluthatione S-transferase
1 (GSTA1) may also be active in the metabolism of caffeine as it conjugates glutathione to aromatic amines. Our study, therefore, included analyses of the association between GSTA1 and stillbirth. Methods A nested case non-case study among women who participated in the Danish National Birth Cohort: 142 cases of singleton stillbirths and 157 controls of singleton live births.
Results Slow oxidizer status (CYP1A2), slow acetylator status (NAT2), and low activity of GSTA1 were not individually associated with the risk of stillbirth [odds ratio (OR) = 1.06, 95% confidence interval (95% CI) 0.67–1.67, OR = 0.95, 95% CI 0.60–1.51, and OR = 1.42, 95% CI 0.88–2.28, respectively]. We did, however, observe that subjects with a combination of slow CYP1A2, slow NAT2, and low GSTA1 genes had almost a 2-fold risk of stillbirth compared with subjects with other combinations of genotypes.
Conclusions We found no link between any single genotype and the risk of stillbirth. An association between a combination of genotypes and stillbirth was discovered. Caffeine may be causally related to stillbirth, but larger studies using Mendelian randomization are needed to verify this.
Keywords caffeine, CYP1A2, NAT2, GSTA1, stillbirth
Accepted 5 May 2006
Coffee contains a variety of chemical compounds, although most health researchers have focussed mainly on caffeine because of its biological effects and its possible harmful influence on DNA.1
Recent publications report an association between coffee or caffeine intake during pregnancy and the risk of spontaneous abortion and stillbirth,2–6 but very few of these associations are causal. Coffee consumption is related to many other lifestyle factors that cannot fully be adjusted for. Furthermore, the amount of coffee drunk may be modified by a coffee aversion or pregnancy nausea, which correlates with hormone levels of significance for fetal survival.7 The association may thus reflect confounding or reverse causation.
Caffeine is an aromatic amine, which is metabolized in the liver by hepatic microsomal enzymes. The initial major step of biotransformation is catalysed by cytochrome P4501A2 (CYP1A2).8 Other enzymes such as N-acetyltransferase 2 (NAT2) are also active in the metabolism of caffeine.9 According to Mendel's second law, we are all randomized at conception to be either slow or fast metabolizers, possibly influencing the amount of coffee we drink. However, given the same caffeine intake, slow metabolizers will be more exposed to high internal caffeine levels than fast metabolizers. The genotype is, thus, a propensity score for caffeine exposure, and comparing genotypes stratified on coffee intake is expected to be unconfounded and not subject to reverse causation.
Gluthatione S-transferase 1 (GSTA1) conjugates glutathione to aromatic amines and protects against oxidative stress, which also has been associated with adverse pregnancy outcomes.10 We hypothesize that because caffeine is an aromatic amine, GSTA1 may be active in the metabolism of caffeine.
Our aim was to determine whether genotypes related to caffeine metabolism and oxidative stress were associated with the risk of stillbirth. Second, we wanted to see whether slow caffeine metabolizers had a higher risk of stillbirth, taking the external coffee intake into account.
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Methods |
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Selection of subjects
We conducted a nested case non-case control study within the Danish National Birth Cohort (DNBC), which is a nationwide cohort of pregnant women and their offspring.11 Women were recruited to the DNBC by 60% of all general practitioners in Denmark, who took part in the project. The pregnant women received written information about the DNBC at the first antenatal care visit to the general practitioner, which usually takes place in gestational weeks 6–10. We estimate that
60% of the invited pregnant women participated in the study. Information on exposures during pregnancy was obtained through computer-assisted telephone interviews. Furthermore, two blood samples from the mother were taken during pregnancy, and a blood sample from the umbilical cord was taken shortly after birth.
Information on pregnancy outcomes was obtained from the Civil Registration System and the Danish National Discharge Register by linking the records with the mother's civil registration number. Failure to identify the outcome of the pregnancy in the register was limited to 1%. The information about outcome was then obtained from the women themselves. Stillbirth was defined as the delivery of a dead infant at 28 completed weeks of gestation or later. Our case–control sample was collected from the women who were enrolled in the DNBC from May 1, 1998 to April 30, 2001 (n = 63 908) and who participated in the first pregnancy interview at 16 weeks of gestation (inter-quartile range 13–19 weeks) (n = 55 432). Women with missing information on coffee intake were excluded from the study (n = 12).
Cases included all women who had a stillbirth (n = 179). We randomly sampled a similar number of control women with a singleton live birth. Cases and controls were frequency-matched on parity.
The medical records were collected to enable the classification of stillbirths according to cause of death. A total of 17 women experienced an intrapartum fetal death due to clinical causes, which we considered to be independent of the studied genotypes, and we, therefore, excluded intrapartum deaths from the primary analysis. Blood samples were missing for 12.3% of cases (n = 20) and 12.3% of controls (n = 22). The final study population consisted of 142 cases and 157 controls.
Genotyping
A 3 mm dried blood spot on filter paper for each woman was washed with 1 ml of phosphate-buffered saline +0.1% Tween for 10 min. After removing the washing buffer, we added 200 µl 5% Chelex-100 and incubated the sample at 60°C for 30 min and 100°C for 30 min. We then centrifuged the sample at 13 000 r.p.m. for 1 min and transferred the supernatant (150 µl) to a clean tube, adding 5–15 µl DNA solution to the PCR mix. The solution was refrigerated at –20°C for later use.
For CYP1A2 genotyping we used the method described by Chida et al.12 and Sachse et al.13 The PCR product was digested by the restriction enzyme Apa I, and the products were separated by 2.0% agarose gel and visualized with ultraviolet light. After DNA analyses, the women were categorized into one of three possible genotypes: A/A, A/C, or C/C.
For the genotyping of NAT2 we used the method described by Sachse et al.14 PCR products were digested by the restriction enzymes Taq, Dde, Kpn, and Bam. The PCR-plate was read in the ABI 7000 sequence detector, and the results were analysed by allelic discrimination of the sequence detection software. The women were then categorized into one of three possible genotypes: Fast/Fast, Fast/Slow, and Slow/Slow.
GSTA1 genotyping was done according to the method described by Coles et al.15 PCR products were digested with the restriction enzyme EarI, and digest patterns were determined on a 2.0% agarose gel. The wild-type allele GSTA1a and the mutant allele GSTA1b were detected. The women were categorized into one of three possible genotypes: a/a, a/b, and b/b.
Statistical analysis
The genotypes were dichotomized for the analyses. CYP1A2 genotype was grouped into fast oxidizers (A/A) and slow oxidizers (A/C and C/C). NAT2 genotype was grouped into fast acetylators (Fast/Fast and Fast/Slow) and slow acetylators (Slow/Slow), and GSTA1 genotype was grouped into high activity (a/a) and reduced activity (a/b and b/b). Unconditional logistic regression models were constructed to estimate odds ratios (OR) and 95% confidence intervals (95% CIs) for the association between maternal characteristics and genotype, and the risk of stillbirth. We stratified the analysis of genotypes on coffee intake to see whether the association between genotype and stillbirth was modified by coffee intake. The interaction was evaluated by a likelihood ratio test.
We used STATA (Version 8.0 SE) for all statistical analyses. The Scientific Ethics Committees for the county of Aarhus and the Danish Data Agency approved the study.
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Results |
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We found that 62% of controls were slow acetylators (NAT2), 47% were slow oxidizers (CYP1A2), and 59% had low activity of GSTA1. The genotypes did not statistically significantly deviate from the Hardy–Weinberg equilibrium.
Compared with controls, the cases were older, were more often obese, and belonged to a lower socio-occupational group (Table 1).
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Women with a consumption of four or more cups of coffee per day had no higher risk of stillbirth compared with non-consumers (adjusted OR = 1.04, 95% CI 0.47–2.30). We found that the controls who were both slow oxidizers and slow acetylators tended to drink more coffee than women with other combinations, although this was not statistically significant (test for trend P = 0.08) (Table 2).
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Slow acetylators or slow oxidizers had the same risk of stillbirth as fast acetylators or oxidizers, (OR = 0.95, 95% CI 0.60–1.51 and OR = 1.06, 95% CI 0.67–1.67, respectively) (Table 3). Grouping A/A and A/C as fast oxidizers and C/C as slow oxidizers did not change our results (data not shown). Women with a low activity of GSTA1 had a higher risk of stillbirth (OR = 1.42, 95% CI 0.88–2.28), but CIs included unity. Slow oxidizers and slow acetylators had a slightly higher risk of stillbirth (OR = 1.23, 95% CI 0.74–2.04), although this was not statistically significant. However, women with a combination of slow CYP1A2, slow NAT2, and low GSTA1 had almost a 2-fold risk of stillbirth compared with women without this combination (OR = 1.86, 95% CI 1.02–3.37) (Table 3). Adjusting for coffee intake or other possible confounders (parity, smoking, alcohol intake, pre-pregnancy body mass index, and socio-occupational status) did not substantially change the results (data not shown). We did not find any consistent pattern of higher risk among slow metabolizers of caffeine at any level of coffee use (Table 4).
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Additional analyses compared the genotypes in women who had experienced an intrapartum fetal death (n = 17) with the genotypes in controls. Women who were slow acetylators, slow oxidizers, or had a low activity of GSTA1 tended to have a higher risk of intrapartum fetal death, although this was not statistically significant (crude OR = 1.86, 95% CI 0.57–6.02, OR = 1.44, 95% CI 0.51–4.06, and OR = 2.98, 95% CI 0.82–10.89, respectively).
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Discussion |
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Caffeine is considered to be a fetotoxic compound. Other studies have reported that pregnant women with an intake of four or more cups of coffee per day during pregnancy faced a higher risk of stillbirth.2,16 Little et al.16 found that women who consumed five or more cups of coffee or tea per day had a slightly higher risk of stillbirth (adjusted OR = 1.37, 95% CI 1.03–1.83). Wisborg et al. found that women who consumed four to seven cups of coffee per day had a higher risk of stillbirth compared with non-consumers (adjusted OR = 1.4, 95% CI 0.8–2.5) and women who drank eight or more cups of coffee per day had a 2-fold risk of stillbirth compared with non-consumers (adjusted OR = 2.2, 95% CI 1.0–4.7).2 We found no association between coffee intake and stillbirth in our data, but this may be due to random variation in selecting controls. Among all the participants in the DNBC, the women who consumed four or more cups of coffee per day had a risk of stillbirth with an OR = 1.21 (95% CI 0.77–1.91).17
The association between coffee or caffeine intake and fetal death could be due to confounding or reverse causation. High levels of coffee intake correlate with a number of other lifestyle factors and coffee intake is influenced by pregnancy conditions. Alleles are according to Mendel's second law randomly distributed at conception. Thus, comparisons of genotypes are not likely to be confounded by lifestyle factors,18 if the factors are unrelated to the genotypes, as is expected for most lifestyle factors except coffee intake.
If caffeine has a biological effect on stillbirth, we would expect slow metabolizers of caffeine to have a higher risk of stillbirth at any given caffeine intake since the caffeine they consume will be eliminated less rapidly from the body. Furthermore, we would expect a greater effect of the genotype among those with a high consumption. We found no association between slow oxidizers (CYP1A2) or slow acetylators (NAT2) and the risk of stillbirth, when studying single nucleotide polymorphisms. However, the combination of acetylator and oxidizer polymorphisms (slow/slow) showed a moderate but not statistically significant increased risk of stillbirth. Women who possessed a combination of the slow CYP1A2, the slow NAT2, and the low GSTA1 did have a higher risk of stillbirth, and according to the hypothesis that GSTA1 may be active in the metabolism of caffeine, we would expect women who had both slow CYP1A2 and slow NAT2, and low GSTA1 had a higher risk of stillbirth if caffeine was a causal factor. Thus our findings provide some limited support for the hypothesis that caffeine is causally related to stillbirth but this finding should be tested in larger studies, where the phenotypic activity in combination with genotypes for CYP1A2, NAT2, and GSTA1 are measured.
In the stratified analysis, we found no support for an interaction between genotypes and coffee intake. However these analyses have limited power as indicated by the wide CIs. A study with 10 times as many cases and controls may be needed to explore these interactions. For the combined genotype (slow CYP1A2, slow NAT2, and low GSTA1), we found a higher risk of stillbirths (however, CIs included unity) also among non-consumers of coffee. This could be explained by the fact that coffee is not the only source of caffeine. Caffeine is also found in tea, cocoa, cola, chocolate, and certain medications. We only have information on the consumption of tea, and in our data, 64% of non-coffee drinkers do drink tea daily.
To our knowledge, this is the first study to analyse genotypes involved in caffeine metabolism and the risk of stillbirth. Previous studies on caffeine metabolism have studied the risk of spontaneous abortion. Fenster et al. found no association between enzyme activity of CYP1A2, xanthin oxidase, and NAT2, and spontaneous abortion when the phenotypes for these three enzymes were studied.19 In contrast, Signorello et al.20 found that women with a low CYP1A2 activity (phenotype) had a statistically significant lower risk of spontaneous abortion, and women who were slow acetylators (genotypes) had a somewhat higher risk of spontaneous abortion, although not statistically significant.
We found that women with the combination of slow metabolizing genotypes had a tendency to drink more coffee and dependency may play a role for the daily consumption of caffeine,21 but studies on the association between genotypes and coffee consumption are few. Signorello et al.20 found no association between NAT2 genotypes and intake of coffee, but reported that women who were slow oxidizers (CYP1A2 phenotype) had a higher coffee intake than women who were fast oxidizers.
The phenotypes of CYP1A2 and NAT2 are related to the genotypes but may be modified by other factors. We had no phenotype measurements, but other studies suggest that a correspondence between genotype and phenotypic activity for NAT2 is very high.22,23 The activity of CYP1A2 may be decreased by pregnancy,24 and increased by smoking, 25 and certain dietary products.26 A twin study found that CYP1A2 activity is mainly governed by genetic factors,27 but studies on concordance between CYP1A2 genotype and phenotype are contradictive. Some found a greater enzyme activity in A/A compared with C/A individuals,28 others found this for smokers only,29 while some found no difference in enzyme activity according to genotype.13 Pavanello et al.30 observed that increased activity of CYP1A2 in smokers was significantly related to the A-allele. This was also found by Nordmark et al.31 who studied the influence of CYP1A2 genotype on the CYP1A2 activity in pregnant women. When we grouped CC genotypes for CYP1A2 as slow metabolizers in the present study, we still found no association with stillbirth. Future studies on genes involved in caffeine metabolism should measure phenotypes to make sure that the polymorphisms of the genes studied actually express phenotypic differences.
When only studying genotypes known to be active in caffeine metabolism, the present study does not support the hypothesis that caffeine in itself causes stillbirth, but we cannot rule out that other components in coffee may have this effect. If GSTA1 is active in the metabolism of caffeine, our results provide some support for a causal link between caffeine and stillbirth, although this may be due to chance or other effects of the gene. We are aware of the limited internal exposure contrasts in this comparison. Larger studies are needed to make more conclusive statements.
KEY MESSAGES
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Acknowledgments |
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This work was supported by a grant from the Danish Centre for Environmental Health, Danish Ministry of the Interior and Health. The Danish National Research Foundation established the Danish Epidemiology Science Centre that initiated and created the Danish National Birth Cohort. The cohort is, furthermore, a result of a major grant from this Foundation. Additional support for the Danish National Birth Cohort is obtained from the Pharmacy Foundation, the Egmont Foundation, the March of Dimes Birth Defects Foundation, and the Agustinus Foundation. Thanks to Duy Ahn Dang and Karsten Henning Sørensen for technical assistance.
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