Neural Tube Defects in Relation to Use of Folic Acid Antagonists during Pregnancy

Sonia Hernández-Díaz1,2, Martha M. Werler1, Alexander M. Walker2 and Allen A. Mitchell1

1 Slone Epidemiology Unit, Boston University School of Public Health, Brookline, MA.
2 Department of Epidemiology, Harvard School of Public Health, Boston, MA.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Periconceptional folic acid supplementation reduces the risk of neural tube defects (NTDs). To determine whether periconceptional exposure to folic acid antagonists (FAAs) might therefore increase the risk of NTDs, the authors examined data from an ongoing case-control study of birth defects (1979–1998) in the United States and Canada. They compared data on 1,242 infants with NTDs (spina bifida, anencephaly, and encephalocele) with data from a control group of 6,660 infants with malformations not related to vitamin supplementation. Mothers were interviewed within 6 months of delivery about demographic, reproductive, medical, and behavioral factors and about medication use. The adjusted odds ratios of NTDs related to exposure to FAAs (including carbamazepine, phenobarbital, phenytoin, primidone, sulfasalazine, triamterene, and trimethoprim) during the first or second months after the last menstrual period, compared with no use in either month, were 2.8 (95% confidence interval: 1.7, 4.6) for FAAs as a group, 4.8 (95% confidence interval: 1.5, 16.1) for trimethoprim (based on five exposed cases), and 6.9 (95% confidence interval: 1.9, 25.7) for carbamazepine (six exposed cases). These results are adjusted for region, interview year, periconceptional folic acid supplementation, maternal age, weight, education, and infections early in pregnancy. These findings suggest that a number of FAAs may increase NTD risk, and they provide estimates of risk for selected drugs.

carbamazepine; case-control studies; folic acid antagonists; neural tube defects; pregnancy; teratogens; trimethoprim

Abbreviations: CI, confidence interval; FAA, folic acid antagonist; NTD, neural tube defect.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neural tube defects (NTDs), characterized by a failure of the neural tube to close properly after conception, affect about one per 1,000 livebirths in the United States (1Go). Observational and experimental studies have shown that periconceptional folic acid supplementation can significantly reduce the risk of NTDs (2Go, 3Go). These findings, together with the observation that low maternal red cell folate in early pregnancy is related to NTDs (4Go, 5Go), suggest that folic acid antagonists (FAAs) may increase the risk of NTDs.

FAAs include aminopterin, carbamazepine, methotrexate, phenobarbital, phenytoin, primidone, sulfasalazine, triamterene, trimethoprim, and valproic acid. Although it has been documented or suggested that NTD risk is associated with use in early pregnancy of certain specific FAAs (aminopterin (6Go), methotrexate (7Go), valproic acid (8Go, 9Go), and carbamazepine (8Go, 10Go)), risk has not been studied for FAAs as a group nor for specific FAAs such as trimethoprim. For carbamazepine, the magnitude of risk is unclear. The purpose of the present study was to evaluate the risk of having an infant with an NTD after periconceptional exposure to FAAs in general and, where numbers permitted, to specific FAAs.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Study population
Since 1976, the Slone Epidemiology Unit Birth Defects Study has been interviewing mothers of malformed children born in the greater metropolitan areas of Boston, Massachusetts, Philadelphia, Pennsylvania, Toronto, Canada, and, between 1983 and 1985, part of the state of Iowa (11Go). Until 1988, study subjects included only liveborn infants ascertained at birth or tertiary care hospitals. Since then, stillborn infants and fetuses therapeutically aborted because of a malformation have also been included. Study subjects are identified through review of admissions and discharges at major referral hospitals and clinics and through regular contact with newborn nurseries in community hospitals (the latter to identify infants born with defects who might not have been referred to major centers). Since 1993, a random sample of nonmalformed infants has been identified at the birth hospitals as potential controls. Physicians of study subjects confirm the diagnosis. Among eligible subjects, the mothers of 84 percent of cases, 83 percent of malformed controls, and 80 percent of nonmalformed controls contacted agreed to an interview and to provide informed consent. The present study is based on subjects ascertained between 1976 and 1998.

Cases
Cases consisted of 1,242 infants and fetuses with a diagnosis of anencephaly, spina bifida, encephalocele, or other NTDs (table 1). The relative prevalence of anencephaly and other NTDs is not representative, mainly because stillbirths and abortions were included later in the study. Infants with chromosomal or Mendelian-inherited anomalies (n = 44) or with amniotic bands, caudal regression, or twin disruption (n = 15) were excluded under the assumption that the etiologies of their NTDs were different from those of the remaining cases. NTDs complicated with other defects (but not as part of an identified chromosomal or Mendelian-inherited syndrome) were included in the general analysis and studied separately.


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TABLE 1. Selection of cases and controls, Slone Epidemiology Unit Birth Defects Study, United States and Canada, 1976–1998

 
Controls
Because mothers of malformed and mothers of nonmalformed infants may differ in their recall of antenatal drug exposure (12Go), our primary control group consisted of infants with malformations other than NTDs. Among the 13,666 infants with structural malformations, we excluded 7,006 subjects with oral clefts, urinary tract defects, limb reduction, heart defects, and conditions related to NTD (hydrocephalus, microcephalus, and other anomalies of the brain, spinal cord, or nervous system), because folic acid may play a role in the genesis of these malformations (13Go). The final control group consisted of 6,600 subjects. The major types of primary diagnosis included in the control group were hypertrophic pyloric stenosis (n = 906), indeterminate sex or pseudohermaphroditism (n = 509), musculoskeletal anomalies of the skull or face (n = 387), feet deformities (n = 296), anomalies of the diaphragm (n = 282), gastroschisis/omphalocele (n = 257), esophageal stenosis (n = 219), stenosis of the large intestine or anus (n = 216), congenital dislocation of the hip (n = 179), hypospadias (n = 150), and others. We also selected two secondary series of controls, one of nonmalformed infants (n = 1,626) and one of infants with chromosomal or Mendelian anomalies (n = 2,138).

Exposure assessment
Within 6 months of the subject's delivery, trained study nurses unaware of the hypothesis interviewed mothers of cases and controls. More than 90 percent of interviews were conducted in the subjects' homes, a setting selected to minimize anxiety, to encourage cooperation, and to maximize data collection; the remaining interviews took place at an alternative site of the mother's choice (8 percent) or by telephone (1 percent). The interview was detailed and structured and included questions on demographic characteristics, patients' medical history, an obstetric history, parents' habits and occupations, and a detailed history of the use of medication (both prescription and over-the-counter) from 2 months before conception through the entire pregnancy. Recall of medication exposures was enhanced by questions regarding indications for use. Recall of the timing of use was enhanced by using a calendar that highlighted the date of the woman's last menstrual period. Completed questionnaires were submitted to a variety of quality control procedures and entered into a computer file.

Mothers were considered exposed if they reported using an FAA any time during the 2 months after the last menstrual period; these 2 months encompass the period of neural tube development (14Go).

Data analysis
Prevalence odds ratios of having an infant with an NTD and 95 percent confidence intervals were estimated for women exposed versus women not exposed to an FAA, using unconditional logistic regression. The associations presented below were adjusted, using multivariate models, for geographic region, interview year (1976–1982, 1983–1987, 1988–1992, 1993–1998), maternal age (<=24, 25–29, 30–34, >=35 years), periconceptional folic acid supplementation (during the 2 months after the last menstrual period--never, occasionally, or daily), prepregnancy weight, years of education, and infections during the first trimester. Folic acid supplementation was also considered as a potential effect modifier.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The distributions of selected characteristics are shown for cases and malformed controls in table 2. Prevalences of any FAA use and specific FAA use are presented for cases and malformed controls in table 3. Among the 1,242 infants with NTDs, 27 (2.2 percent) were exposed to FAAs early in pregnancy, while 67 (1.0 percent) were exposed among the 6,660 malformed controls. The estimated adjusted odds ratio for women exposed to FAAs compared with women not exposed was 2.8 (95 percent confidence interval (CI): 1.7, 4.6) (table 3). When controlled for potential confounders, the findings did not differ appreciably from the crude analysis; therefore, only adjusted data are presented. Control for other factors (e.g., maternal smoking during pregnancy, alcohol intake, previous affected pregnancy, family history, birth order, or pregnancy planning) did not materially influence the odds ratio estimates.


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TABLE 2. Distribution of selected characteristics, Slone Epidemiology Unit Birth Defects Study, United States and Canada, 1976–1998

 

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TABLE 3. Use of FAAs* during early pregnancy in 1,242 cases and in 6,660 malformed controls, along with prevalence odds ratios of neural tube defects and their 95% confidence intervals, associated with use of FAAs in early pregnancy, Slone Epidemiology Unit Birth Defects Study, United States and Canada, 1976–1998

 
The odds ratio for NTDs complicated with other defects (n = 506) was 3.1 (95 percent CI: 1.5, 6.2). For NTD type, odds ratios were 6.4 (95 percent CI: 2.7, 15.3) for anencephaly, 2.7 (95 percent CI: 1.6, 4.7) for spina bifida, and 0.0 (95 percent CI: 0.0, 2.5) for encephalocele (there were no exposed cases). Restriction of the analysis to infants without a family history of NTDs yielded an odds ratio of 5.8 (95 percent CI: 2.5, 13.7).

Odds ratios for specific FAAs varied considerably; to minimize statistical instability, we limited estimating risk to those specific FAAs for which there were at least five exposed cases: carbamazepine and trimethoprim.

In our population, 0.5 percent (n = 6) of cases and 0.1 percent (n = 5) of controls used carbamazepine (as monotherapy in four cases and three controls) during the first 2 months after the last menstrual period, for an adjusted odds ratio of 6.9 (95 percent CI: 1.9, 25.7) (odds ratio was similar for monotherapy). Because carbamazepine was used throughout pregnancy, we were unable to consider effects according to month of exposure.

Overall, 0.4 percent (n = 5) of cases and 0.1 percent (n = 8) of controls used trimethoprim around the time of conception, yielding an odds ratio of 4.8 (95 percent CI: 1.5, 16.1). The prevalence of trimethoprim exposure according to lunar month of pregnancy is presented in figure 1. Trimethoprim exposure among cases was significantly higher during the first 2 months after the last menstrual period but not during the month preceding or following that period. The NTD risk for women exposed to trimethoprim during each lunar month, compared with women not exposed to trimethoprim during these months, was 1.3 (95 percent CI: 0.3, 5.6) for the month before the last menstrual period, 7.8 (95 percent CI: 2.2, 27.0) for the first month after the last menstrual period, 6.4 (95 percent CI: 1.5, 26.3) for the second month after the last menstrual period, and 0.9 (95 percent CI: 0.1, 10.7) for the third month after the last menstrual period (two, five, four, and one exposed cases, respectively).



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FIGURE 1. Trimethoprim exposure (%), according to lunar months, in neural tube defect cases and malformed controls, Slone Epidemiology Unit Birth Defects Study, United States and Canada, 1976–1998. The adjusted odds ratio estimate and its 95% confidence interval are presented for each month around the last menstrual period. NTD, neural tube defect; LMP, last menstrual period; OR, odds ratio; CI, confidence interval.

 
To assess whether folic acid supplementation modifies the risk associated with carbamazepine and trimethoprim during the 2 months after the last menstrual period, we compared use and nonuse of these drugs for users and nonusers of folic acid supplementation (table 4). In the absence of carbamazepine and trimethoprim exposure, and compared with women who took neither these drugs nor folic acid during the 2 months after the last menstrual period, daily folic acid supplementation was protective (odds ratio = 0.7; 95 percent CI: 0.5, 0.8). Women exposed to either trimethoprim or carbamazepine and not taking folic acid had an odds ratio of 13.3 (95 percent CI: 2.9, 61.4), whereas women taking one of these medications and daily folic acid had an odds ratio of 1.2 (95 percent CI: 0.1, 12.7).


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TABLE 4. Prevalence odds ratios of neural tube defects for women exposed periconceptionally to trimethoprim or carbamazepine, folic acid supplements, or both, Slone Epidemiology Unit Birth Defects Study, United States and Canada, 1976–1998

 
All trimethoprim-exposed subjects took a combination of trimethoprim and a sulfonamide, either sulfamethoxazole (n = 11) or sulfadiazine (n = 2). There were no exposures to sulfamethoxazole or sulfadiazine alone, so we could not assess their separate effects. However, for 11 cases and 42 controls exposed to sulfonamides (other than the FAA sulfasalazine), the odds ratio was 1.4 (95 percent CI: 0.7, 2.9).

Because trimethoprim was used primarily for urinary tract infections, we considered whether the observed association might be due to urinary tract infections rather than to trimethoprim. We did not find an appreciable association between urinary tract infections and NTDs (odds ratio = 1.3; 95 percent CI: 0.8, 2.2). For other antibiotics used for urinary tract infections during the first 2 months after the last menstrual period, the odds ratio was 1.2 (95 percent CI: 0.7, 2.0) for amoxicillin/ampicillin and 1.6 (95 percent CI: 0.6, 4.3) for cephalosporins. (In one subgroup analysis, cephalexin had an odds ratio of 4.0 (95 percent CI: 1.2, 12.8), based on six exposed cases.)

When cases were compared with nonmalformed controls, the odds ratios were 2.8 (95 percent CI: 1.3, 6.3) for FAAs, 2.0 (95 percent CI: 0.4, 10.1) for carbamazepine, and 7.7 (95 percent CI: 0.7, 80.2) for trimethoprim (based on 13, four, and one exposed controls, respectively). When they were compared with chromosomal disorders, the odds ratios were 3.2 (95 percent CI: 1.7, 6.1) for FAAs, 10.3 (95 percent CI: 1.2, 91.4) for carbamazepine, and 3.7 (95 percent CI: 0.8, 16.5) for trimethoprim (based on 18, one, and four exposed controls, respectively).

If the associations were causal, for drugs with odds ratios as high as 5 or 7 the excess number of NTDs would be four or six per 1,000 pregnant women exposed to these drugs early in pregnancy (assuming a prevalence of one NTD per 1,000 births among women not exposed) (1Go).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure to FAAs as a group during the first or second months after the last menstrual period more than doubled the risk of NTDs. NTD risk increased more than sixfold after carbamazepine exposure and more than fourfold after trimethoprim exposure. For trimethoprim, the effect was maximal during or shortly before the time of neural tube closure; in contrast, exposure before or after this sensitive period did not increase the risk of NTDs. As would be expected, folic acid supplementation in the absence of carbamazepine and trimethoprim reduced the risk of NTDs; though based on small numbers, such supplementation also appeared to reduce the magnitude of NTD risk associated with exposure to carbamazepine and trimethoprim.

Human studies have shown that exposure to FAAs such as aminopterin (15Go), methotrexate (16Go), and valproic acid (8Go, 9Go, 17Go) can cause NTDs. An association between carbamazepine and NTDs has also been suggested in a number of studies (8Go, 10Go, 17Go), but they have tended to be small, leaving the magnitude of risk unclear. Little has been described for trimethoprim risks, however. Clinical trials did not indicate an increased rate of birth defects, but those studies contained too few pregnancies to allow detection of a teratogenic effect (18GoGo–20Go). Epidemiologic studies have only rarely considered this drug; a decade ago, two case-control studies found a significant increase in the risk of oral clefts and hypospadias among trimethoprim users (21Go, 22Go). The present findings are the first to link trimethoprim to NTDs in humans.

Most members of the FAA "class" seem to induce NTDs. Two exceptions are phenytoin and phenobarbital, which have been associated with various congenital defects in earlier studies (9Go, 17Go, 23Go, 24Go). However, neither those studies nor the current one has linked these drugs to NTDs. Drug-specific differences in teratogenic effects among different FAAs may be due to variations in folate antagonism potency or to actions at different steps of folate metabolism (25Go). Those that are folic acid analogs (including aminopterin, methotrexate, valproic acid, triamterene, and trimethoprim) displace folate from enzymes and block the enzymatic reactions in which folate participates. For instance, the antimicrobial activity of trimethoprim results from a selective inhibition of dihydrofolate reductase in unicellular organisms (26Go). Although the concentration needed to inhibit human dihydrofolate reductase is 100,000-fold greater than that required to inhibit the bacterial enzyme, the inhibition in humans is enough to be associated with megaloblastic anemia (26Go), and enzymes in the human embryo may behave differently from those in adults. Further, folic acid supplementation reduces the clinical toxicity of these FAAs (27GoGoGo–29Go) and, in one study of rats, malformations induced by trimethoprim were prevented by administration of folinic acid or dietary supplementation with folic acid (30Go).

Our study has a number of limitations. Although we included stillborn infants and therapeutically aborted fetuses since 1988, we still are likely to be missing affected pregnancies, particularly early spontaneous abortions. However, if the NTDs produced by FAAs were more often lethal or more severe (i.e., more easily detected prenatally), our subjects would represent a survivor cohort and would lead to an underestimate of the NTD risk due to FAAs.

Use of malformed controls would have introduced bias in this study if FAAs were related to malformations other than NTDs. However, subjects with conditions potentially associated with folate supplementation were excluded. Further, since the controls include a variety of malformations, a previously undocumented effect of FAA on any one malformation would have little impact on these findings. In fact, the use of alternative control groups did not materially change the results. Moreover, under most scenarios of biased control selection, the reported risk would underestimate the true association.

Because we relied on women's ability to recall and report information, there may have been differential misclassification of past exposure between case and control mothers. Use of malformed controls should reduce this possibility. In addition, the carefully designed questionnaire, administered relatively soon after delivery by interviewers blind to the hypothesis, is likely to have substantially reduced information errors. On average, mothers of NTD cases were interviewed sooner after the last menstrual period than were mothers of malformed controls (53.4 and 58.8 weeks, respectively), mainly because of a higher number of stillbirths and therapeutic abortions among cases. However, these differences did not affect maternal recall, as reflected by the similar time intervals between the last menstrual period and interview found for exposed and unexposed controls.

Nondifferential underreporting, or misclassification of the dates of the last menstrual period, or of exposure would underestimate the true association. We did not consider food sources of folate, which have greatest impact on folate status among nonusers of folic acid supplements (31Go). However, unaccounted variations in folate intake among persons would tend to dilute the effects of folic acid.

Known confounding factors were taken into account in the statistical analysis. Trimethoprim, typically combined with sulfamethoxazole, is used primarily for urinary tract infections. Neither urinary tract infections nor other infections during the first trimester were significantly associated with NTDs, and they did not materially change the odds ratio estimate for trimethoprim. Use of other drugs commonly prescribed for urinary tract infections in pregnancy, notably ampicillin and amoxicillin, was also not associated with an increased risk nor was the use of cephalosporins as a group; in the context of multiple testing, the specific finding for cephalexin may be due to chance. We cannot separate an effect of trimethoprim alone from an effect of trimethoprim combined with sulfonamides, but we found no association with sulfonamides other than the FAA sulfasalazine. It is of note that sulfamethoxazole inhibits de novo production of folic acid only in bacteria, but trimethoprim affects transformation of folic acid into required co-enzymes in both bacteria and humans (26Go). Thus, biologic coherence would favor the effect of trimethoprim over sulfamethoxazole. For carbamazepine, as for other antiepileptic drugs, it is always difficult to disentangle a direct effect of the medication from a potential effect of epilepsy itself; however, NTDs are not consistently associated with most antiepileptic drugs.

Maternal periconceptional exposure to FAAs such as trimethoprim and carbamazepine appears to increase the risk of NTDs. The strength, time-specific effects, and biologic plausibility of the findings, together with previous studies in animals, in humans, and with other FAAs, offer support for a causal association. Fortunately, among women who use carbamazepine or trimethoprim, the absolute risk appears to be modest, because more than 99 percent of women exposed to these drugs early in pregnancy will deliver an infant unaffected by an NTD. Moreover, the prevalence of carbamazepine and trimethoprim use in our population was low, so that the number of cases potentially attributable to these drugs was small. It is unclear whether supplemental folic acid will reduce this excess risk or whether it will affect the therapeutic efficacy of these drugs. Finally, though these findings extend the number of FAAs that may cause NTDs, they are based on relatively few subjects and need to be evaluated in further studies.


    ACKNOWLEDGMENTS
 
Supported in part by the Pharmacoepidemiology Teaching and Research Fund of the Harvard School of Public Health: the National Center for Environmental Health, Centers for Disease Control and Prevention, through a grant to the Massachusetts Center for Birth Defects Research and Prevention; and by the Massachusetts Department of Public Health: the National Institute of Child Health and Human Development grant HD27697 and the National Heart, Lung, and Blood Institute grant HL50763.

Additional support for the Slone Epidemiology Unit Birth Defects Study was provided by Hoechst Marion Roussel, Inc. (Kansas City, Missouri); Pfizer, Inc. (New York, New York); the Glaxo-Wellcome Company (Research Triangle Park, North Carolina); and Rhone Pouleuc Rorer (Collegeville, Pennsylvania).

The authors thank Rachel Wilson, project coordinator; Fiona Rice, research coordinator; Rita Krolak, quality control/coder; Sally Perkins, Mary Krieger, Kathleen Sheehan, Karen Bennett Mark, Deborah Kasindorf, and Clare Coughlin, interviewers; Joan Shander, Diane Gallagher, and Valerie Hillis, research assistants; Thomas Kelley, research pharmacist; and Nastia Dynkin and John Farrell, programmers, for their assistance.

The authors also are indebted to the following institutions that generously provided them access to patients: Boston area: Baystate Medical Center, Beth Israel Deaconess Medical Center, Boston Medical Center, Brigham & Women's Hospital, Brockton Hospital, Cambridge Hospital, Children's Hospital, Charlton Memorial Hospital, Deaconess Waltham Hospital, Emerson Hospital, Falmouth Hospital, Franciscan Children's Hospital, Good Samaritan Medical Center, Haverhill Municipal-Hale Hospital, Holy Family Hospital, Jordan Hospital, Kent County Memorial Hospital, Lawrence General Hospital, Lowell General Hospital, Melrose-Wakefield Hospital, UMASS Memorial Health Care (Memorial Campus), Metro West Medical Center (Framingham Campus), Mt. Auburn Hospital, New England Medical Center, Newton-Wellesley Hospital, North Shore Medical Center, Rhode Island Hospital, Saints Memorial Medical Center, South Shore Hospital, Southern New Hampshire Regional Medical Center, St. Elizabeth's Medical Center, St. Luke's Hospital, St. Vincent Hospital, and Women & Infants' Hospital; Philadelphia area: Abington Memorial Hospital, Albert Einstein Medical Center, Alfred I. duPont Institute, Bryn Mawr Hospital, Chester County Hospital, Children's Hospital of Philadelphia, Christiana Care Health Services, City Avenue Hospital, Community General Hospital, Crozer-Chester Medical Center, Doylestown Hospital, Frankford Hospital (Torresdale Division), Hospital of the University of Pennsylvania, Lancaster General Hospital, Lehigh Valley Hospital, Nanticoke Memorial Hospital, Pennsylvania Hospital, Reading Hospital & Medical Center, Sacred Heart Hospital, St. Mary Medical Center, Temple University Hospital, and Thomas Jefferson University Hospital; and Toronto area: Cambridge Memorial Hospital, Grand River Hospital, Guelph General Hospital, Hospital for Sick Children, Humber River Regional Hospital, Joseph Brant Memorial Hospital, London Health Sciences Center, McMaster University Medical Center, Mt. Sinai Hospital, North York General Hospital, Oakville Trafalgar Memorial Hospital, Lakeridge Health Corporation, Peel Memorial Hospital, Scarborough General Hospital, St. Joseph's Health Centre (London), St. Joseph's Health Centre (Toronto), St. Joseph's Hospital (Hamilton), St. Michael's Hospital, Toronto East General Hospital, Toronto Hospital (General Division), Trillium Health Center, Women's College Hospital, York County Hospital, and York Central Hospital.


    NOTES
 
Reprint requests to Sonia Hernández-Díaz, Slone Epidemiology Unit, Boston University School of Public Health, 1371 Beacon Street, Brookline, MA 02446 (e-mail: shernan{at}bu.edu).

Presented in part at the 15th International Conference on Pharmacoepidemiology, Boston, MA, August 1999.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication April 21, 2000. Accepted for publication September 18, 2000.