Research Articles
Alcohol Use Prior to Pregnancy Recognition R. Louise Floyd, DSN, RN, Pierre Decoufle´, ScD, Daniel W. Hungerford, DrPH Background: Frequent alcohol use during the first 8 weeks of pregnancy can result in spontaneous abortion and dysmorphologic changes in the developing organ systems of the embryo, including the heart, kidneys, and brain. However, few population-based studies are available that describe the prevalence of frequent drinking (6 or more drinks per week) among women prior to and during early pregnancy (the periconceptional period), and the sociodemographic and behavioral factors that characterize these women. Such knowledge is fundamental to the design of targeted interventions for the prevention of fetal alcohol syndrome (FAS) and other prenatal alcohol-related disorders. Methods:
This cross-sectional study used survey data collected by the National Center for Health Statistics as part of the 1988 National Maternal and Infant Health Survey (NMIHS). Weighted prevalence estimates were calculated using SUDAAN, and multivariate analyses were used to determine risk factors for frequent drinking.
Results:
Forty-five percent of all women surveyed reported consuming alcohol during the 3 months before finding out they were pregnant, and 5% reported consuming 6 or more drinks per week. Sixty percent of women who reported alcohol consumption also reported that they did not learn they were pregnant until after the fourth week of gestation. Risk factors for frequent drinking during the periconceptional period included 1 or more of the following: being unmarried, being a smoker, being white non-Hispanic, being 25 years of age or older, or being college educated.
Conclusions: Half of all pregnant women in this study drank alcohol during the 3 months preceding pregnancy recognition , with 1 in 20 drinking at moderate to heavy levels. The majority did not know they were pregnant until after the fourth week of pregnancy, and many did not know until after the 6th week. Alcohol is a teratogen capable of producing a number of adverse reproductive and infant outcomes. Public health measures needed to reduce these potentially harmful exposures include alcohol assessment , education, and counseling for women of childbearing age, with referral sources for problem drinking, and family planning services for pregnancy postponement until problem drinking is resolved. Medical Subject Headings (MeSH): alcohol drinking, pregnancy, National Center of Health Statistics, teratogens, prenatal, alcohol, prevalence, fetal alcohol syndrome (Am J Prev Med 1999;17(2):101–107) © 1999 American Journal of Preventive Medicine
Introduction
N
umerous studies have documented the effects of alcohol use during pregnancy, 1–3 including the effects of exposure during the first trimester, a critical period of development for the central nervous system in the embryo and fetus.4 Studies confining exposure to early gestation have concluded that dysmorphic facial features characteristic of fetal alcohol syndrome (FAS) result specifically from ethanol expoFrom the National Center for Environmental Health, and National Center for Injury Control and Prevention, Centers for Disease Control and Prevention, Atlanta, GA Address correspondence to: Dr. R. Louise Floyd, National Center for Environmental Health, Fetal Alcohol Syndrome Prevention Section, Centers for Disease Control and Prevention, 4770 Buford Highway NE MS-F15, Atlanta, GA 30341-3724. E-mail:
[email protected]. The full text of this article is available via AJPM Online at http://www.elsevier.com/locate/ajpmonline
Am J Prev Med 1999;17(2) © 1999 American Journal of Preventive Medicine
sure during the embryonic period, which encompasses the first 3 to 8 weeks of gestation.5 Other studies have documented increased rates of spontaneous abortion,6 decreased height (length) and weight, increased craniofacial abnormalities,7–9 and neurobehavioral deficits10,11 among children born to women who consumed moderate to heavy amounts of alcohol during the first trimester. Recent analyses of the Behavioral Risk Factor Surveillance System (BRFSS) found that half (51%) of all U.S. women of childbearing age (18 – 44) reported consuming alcohol in the month prior to the survey.12 Three percent reported moderate drinking (7–14 drinks per week), 1% reported heavy drinking (more than 14 drinks per week), and 11% reported binge drinking (consumed 5 or more alcoholic drinks on at least 1 occasion) during the previous month. Given that half
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of all pregnancies in the U.S. are unplanned,13 these reports raise questions as to the number of women of childbearing age who are drinking at moderate to heavy levels while in the early stages of an unrecognized pregnancy. In 1981 the U.S. Surgeon General issued a statement advising women who were pregnant or considering pregnancy not to drink alcohol.14 More recently this advisory has been reiterated by the Secretary of Health and Human Services as part of the Dietary Guidelines for Americans.15,16 Many sexually active, women of childbearing age will become pregnant, but may not heed this advice because they are not consciously planning to become pregnant or do not see themselves as being at risk for pregnancy. Few epidemiologic studies have used population-based studies to document the national prevalence and maternal correlates of frequent alcohol use during the weeks immediately preceding and following conception, which we term the periconceptional period. Our purpose in conducting this study is to describe the prevalence of frequent drinking during early gestation, prior to pregnancy recognition, and to characterize the women who report drinking at this level.
Methods The National Maternal and Infant Health Survey (NMIHS) conducted by the National Center for Health Statistics (NCHS) included a mailed questionnaire followed up by telephone and in-person interviews of mothers of a stratified, systematic sample of 13,417 live-born infants (0.35% of all U.S. infants born alive in 1988). 17 The sampling frame was live-birth certificates filed in 48 states (Montana and South Dakota excluded), the District of Columbia, and New York City. The sample was stratified by race (black, nonblack) and birthweight (⬍1500g, 1500 –2499g, and 2500g⫹); very low and moderately low birthweight children as well as black infants were oversampled. Of the mothers of the 13,417 infants sampled, 9,953 (74%) responded to the survey. The mean time interval between delivery and interview was 17 months. The NMIHS queried women regarding their drinking patterns during 2 distinct time periods: (1) the 3-month interval just before they learned they were pregnant and (2) the time between learning they were pregnant and giving birth. This report focuses on reported alcohol use during the 3 months before respondents learned they were pregnant, which is called the periconceptional period because it includes the weeks immediately preceding and following conception. In the survey, participants were asked the question, “How many drinks did you have on average during the 3 months before you found out you were pregnant?” Participants were also asked, “How many weeks preg102
nant were you when you first found out you were pregnant?” Responses to this question allowed us to calculate the overall portion of the sample whose “3 months before you found out you were pregnant” included a critical portion of the first trimester. We analyzed sociodemographic and behavioral characteristics of women who engaged in frequent periconceptional drinking, which we defined as having had an average of 6 or more drinks per week in the 3 months before finding out they were pregnant. We also determined the cumulative proportion of women who found out they were pregnant by the gestational week they reported first finding this out. Finally, we obtained the proportion of women who consumed alcohol by maternal age at the time of delivery, maternal race/ethnicity, maternal education at time of survey, total household income for the 12 months immediately before delivery, marital status at time of survey, live birth order of the study child, average number of cigarettes smoked a day during the 3 months before pregnancy recognition, and adequacy of prenatal care. We determined the adequacy of prenatal care using the Kessner Index,18 which takes into account both the total number of prenatal visits as well as the estimated gestational age at which prenatal care began. After exclusion of records with missing data for key variables necessary for the analysis, 9,559 of the 9,953 respondents were included in the study. Data were weighted to allow for the complex sampling design and to adjust for nonresponse. We used SUDAAN19 to calculate population estimates and their standard errors. To isolate the effects due solely to a given level of any one factor, we used multivariate logistic regression to model the proportion of periconceptional drinkers as a function of the 8 study characteristics. The results are presented in the form of odds ratios. An odds ratio for a given category of a given characteristic (e.g. women aged 35 or older) is interpreted as the relative increase or decrease in the prevalence of drinking among women in that category compared with the prevalence among women in the referent group, holding the other 7 factors constant. Thus the odds ratios permit the isolation of the effects of each individual factor after the potential confounding effects of the other 7 factors have been removed. The referent groups were maternal age 25 through 29, white nonHispanic race/ethnicity, 12 years of education, family income less than $10,000, married, first-born child, nonsmoker, and adequate prenatal care.
Results Overall, 45% of respondents reported alcohol use during the periconceptional period (Table 1). Approximately 1 in 4 respondents (25.5%) reported consuming less than 1 drink per week, and 1 in 7 (14.8%) reported consuming 1 to 5 drinks per week. About 1 in
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Table 1. Prevalence of levels of alcohol use among women during the periconceptional period NMIHS - 1988 (N ⫽ 9559)
No Alcohol Use Any Alcohol Use ⱕ1 drink/month 2–3 drinks/month 1 drink/week 2 drinks/week 3–5 drinks/week 6–8 drinks/week 9–13 drinks/week ⱖ14 drinks/week
Sample number
(N ⴝ 9559) Weighted %
95% CI
5,905 3,654 1,219 834 292 426 447 206 106 124
54.8 45.2 15.3 10.2 3.7 5.1 6.0 2.4 1.2 1.3
53.4–56.2 43.8–46.6 14.3–16.3 9.4–11.1 3.2–4.3 4.5–5.7 5.3–6.7 1.9–2.8 0.9–1.5 1.0–1.6
20 respondents (4.9%) reported consuming 6 or more drinks per week. To gain a better understanding of the actual gestational time period encompassed by the phrase “3 months before you knew you were pregnant,” we plotted cumulative distribution curves for the week of pregnancy when women found out they were pregnant, by drinking status. Nondrinkers were women who reported no alcohol use, infrequent drinkers were those who reported drinking fewer than 6 drinks per week, and frequent drinkers were those reporting 6 or more drinks per week. As seen in Figure 1, at 4 weeks gestation about 40% of drinkers and about 42% of nondrinkers knew they were pregnant, leaving 60% of
Figure 1. Cumulative percent distribution of week of pregnancy in which the respondents first knew they were pregnant, by drinking status.
drinkers and 58% of nondrinkers who learned of their pregnancy later in gestation. At 6 weeks gestation almost 1 in 3 drinkers still did not know they were pregnant. By the end of the first trimester, more than 90% of women in all 3 groups knew they were pregnant. Once pregnancy recognition occurred, significant numbers of women stopped drinking entirely, and among those who continued to drink, significant reductions in the amount consumed were observed. Alcohol use overall went from a prevalence rate of 45% during the 3 months before pregnancy recognition, to 21% during pregnancy (Table 2). Of those women continuing to drink, many shifted into lower drinking categories. Prior to pregnancy recognition 56% of drinkers reported drinking less than 1 drink per week. After pregnancy recognition, 81% of drinkers fell into this category. Prior to pregnancy, 5% of all women drank 6 or more drinks per week; after pregnancy recognition, less than 1% (0.7%) drank at this level. Among the 8 maternal characteristics analyzed in this study, the crude prevalence of frequent periconceptional alcohol use was highest among women who smoked 11 or more cigarettes a day (12.4%) (Table 3). There was a 3-fold increase in frequent periconceptional drinking among women who reported smoking 1–10 cigarettes a day as compared to nonsmokers, and a 5-fold increase among those who smoked more than 10 cigarettes a day as compared to nonsmokers. Unmarried women had the next highest prevalence rate (7.2%), and were 2.5 times more likely to be frequent periconceptional drinkers than women who were married. With respect to education, a woman’s risk of frequent periconceptional drinking increased as her educational attainment increased, with those of the highest educational attainment being almost twice as likely to be frequent drinkers as those with a high school education or less. Among racial-ethnic groups, white non-Hispanic women were twice as likely as any other racial-ethnic group to be frequent periconceptional drinkers and had an overall crude prevalence rate of 6%. Women who were 25 through 29 years of age (prevalence ⫽ 5.8%) and those over 35 years (prevalence ⫽ 6.0%) were more likely to be frequent drinkers than women who were less than 25 years old. Finally, women having their first child had the highest prevalence rate (5.6%) of frequent drinking among the birth order categories analyzed, with women having their second child having the lowest prevalence rate. No significant differences in the prevalence of frequent periconceptional alcohol use were found among women of different income levels, nor did adequacy of prenatal care have any effect. In addition to defining individual risk factors associated with frequent periconceptional drinking, we also examined the prevalence rates for selected combinations of risk factors (Table 4). The highest prevalence Am J Prev Med 1999;17(2)
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Table 2. Prevalence of reported alcohol use among women during the periconceptional and intrapartum periods by level, NMIHS, 1988 (N ⫽ 9559)
No alcohol use Any alcohol use ⬍1 drink/week 1–5 drinks/week 6–8 drinks/week 9–13 drinks/week ⬎14 drinks/week a b
Periconceptionala period
Intrapartumb period
%
95% CI
%
95% CI
54.8 45.2 25.5 14.9 2.4 1.2 1.3
53.4–56.2 43.8–46.6 24.2–26.8 13.8–15.9 1.9–2.9 0.9–1.5 0.9–1.6
79.3 20.7 16.7 3.4 0.3 0.1 0.2
79.1–80.5 19.5–21.9 15.6–17.8 2.9–3.9 0.1–0.4 .02–0.1 0.1–0.4
In the 3 months before recognition of pregnancy After recognition of pregnancy
rates of frequent periconceptional drinking were found among women with 4 of the strongest risk factors identified in this study: smoker, white non-Hispanic,
ⱖ25 years old, and unmarried. One in 5 women (21%) with this combination of factors were frequent periconceptional drinkers. However, women who were smokers, white non-Hispanic, ⱖ25 years old, and married had a prevalence rate about half that of their unmarried counterparts (10.7%). The second highest prevalence rate was found among women who were smokers, non-white, ⱖ25 years of age, and unmarried (17%). As a final point of consideration we computed the weighted proportion of the population of women who gave birth to a liveborn infant in 1988 represented by these various combinations of risk factors (Table 4). While some combinations of risk factors have a high prevalence rate of frequent periconceptional alcohol use, they may represent small proportions of the overall population. For example, while the prevalence rate of frequent periconceptional drinking in women who were smokers, white non-Hispanic, ⱖ25 years of age,
Table 3. Crude prevalence (percentage) of frequent a periconceptional drinking by selected characteristics and adjusted odds ratios, NMIHS, 1988 (N ⫽ 9559) Age (years) ⬍19 20–24 25–29 30–34 ⱖ35 Race White (N-Hisp) White (Hisp) Black (N-Hisp) Black (Hisp) Other Education (years) 0–11 12 13–15 ⬎16 Household income (dollars) ⬍10,000 10,000–24,999 25,000–39,999 ⬎40,000 Marital status Married Unmarried Live birth order of study child 1st 2nd ⬎3rd or later Smoking status (average ft cigarettes per day) 0 1–10 ⬎10 Kessner index adequate prenatal care inadequate prenatal care a b
Number of Respondents
Crude % (weighted)
95% CI
1556 2756 2741 1801 705
4.1 4.1 5.8 4.3 6.0
0.5 0.5 1.0 0.7 1.1
0.3–0.8 0.4–0.8 Referent 0.5–1.0 0.7–1.8
3854 684 4566 119 336
6.0 2.0 3.1 2.8 2.2
1.0 0.4 0.4 0.5 0.5
Referent 0.2–0.8 0.3–0.6 0.2–1.5 0.2–1.2
2187 3862 2214 1296
4.3 4.5 5.4 5.4
0.9 1.0 1.4 1.8
0.6–1.4 Referent 1.0–2.1 1.2–2.9
3094 2949 1823 1693
6.0 4.0 4.0 5.7
1.0 0.8 0.7 1.1
Referent 0.5–1.1 0.5–1.1 0.7–1.7
5442 4117
4.0 7.2
1.0 2.5
Referent 1.8–3.5
4642 2610 2307
5.6 3.5 5.0
1.0 0.6 0.8
Referent 0.4–0.8 0.6–1.2
6689 1382 1488
2.5 7.1 12.4
1.0 3.2 5.4
Referent 2.2–4.8 3.8–7.7
8478 1081
4.9 4.2
1.0 0.9
Referent 0.5–1.6
Six or more drinks per week. The odds ratios for each characteristic are adjusted for the other seven characteristics.
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Adjusted ORb
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Table 4. Prevalence of frequent periconceptional drinking among women with selected combinations of risk factors
Risk factors Smoker, white non-Hispanic, ⱖ25 yrs., unmarried Smoker, non-white, ⱖ25 yrs., unmarried Smoker, white non-Hispanic, ⬍25 yrs., unmarried Smoker, white non-Hispanic, ⱖ25 yrs., married Smoker, non-white, ⬍25 yrs., unmarried Smoker, non-white, ⬍25 yrs., married Nonsmoker, white nonHispanic, ⬍25 yrs., unmarried Smoker, white nonHispanic, ⬍25 yrs., married
Crude prevalence (weighted)
Percent of women in the population (weighted)
20.8
1.8
17.1
2.1
12.0
4.6
10.7
10.1
9.4
2.3
8.1
0.8
6.9
2.9
6.1
6.7
and unmarried was 21%, this subgroup represented only about 2% of the women giving birth to a liveborn infant in 1988. On the other hand, women with all of the same risk factors except for being married exhibited a prevalence rate of 11% for frequent periconceptional drinking, but they represented 10% of women giving birth to liveborn infants in 1988.
Discussion One in 20 women who had a liveborn infant in 1988 reported frequent alcohol use during the periconceptional period. Approximately 60% of frequent drinkers in this survey did not know they were pregnant until after the fourth week of gestation, and at 6 weeks gestation, approximately 30% did not know they were pregnant. Documented cases of fetal alcohol syndrome have been found predominantly among women consuming heavy amounts of alcohol, (i.e., 10 drinks or more per day),20 although more recent studies have found measurable growth and cognitive effects among children whose mothers drank at thresholds comparable to an average of 6 to 7 drinks per week.21,22 In addition, increased risks of spontaneous abortions have been reported at this level and lower.23 This study provides an estimate of the frequency of early first trimester exposures to alcohol in a cohort of women who had a liveborn infant. Survey respondents were asked about alcohol use during the 3 months before finding out they were pregnant as opposed to the 3 months prior to pregnancy, which is a more commonly used timeframe. Few epidemiologic studies are available for comparison. Bruce and associates24
reported prevalence rates for alcohol use in the 3 months before women became pregnant from population-based surveys in 4 states using the Pregnancy Risk Assessment Monitoring System (PRAMS). Results from the 4 states combined showed that approximately 46% of women reported drinking during the 3 months prior to conception. Drinking levels among the states ranged from 31.9% to 53.8% for drinking 1 to 6 drinks per week, and from 2.2% to 3.3% for drinking 7 or more drinks per week. As cited earlier, data from the BRFSS showed that about half of all women of childbearing age drink, with approximately 3% averaging between 1 and 2 drinks per day, and approximately 2% averaging 2 or more drinks per day.12 Both reports have findings similar to ours in that 45% of women in the NMIHS reported using alcohol before learning that they were pregnant, with about 5% reporting that they consumed 6 or more drinks per week. Personal characteristics associated with frequent drinking in this study (white, non-Hispanic, college educated, unmarried, a smoker 25 years of age and older) are consistent with those seen in other surveys that described drinking patterns among women of childbearing age in general.25–27 Results from both multivariate (Table 3) and stratified analyses (Table 4) highlight the importance of the 2 strongest risk factors identified in the study, being a smoker or being unmarried. At first glance, the findings that the characteristics “being unmarried” and “older age (ⱖ25)” act as independent high-risk factors for frequent drinking might seem contradictory, since these traits may seem more incompatible than not. However, stratified analyses of these 2 factors showed prevalence rates for frequent drinking of 2.8%, 4.5%, 5.8%, and 9.8% among younger (⬍25) married women, older (ⱖ25) married women, younger unmarried women, and older unmarried women, respectively. Thus, being unmarried is the more important determinant of high risk when considering age and marital status together. Women having their first child were more likely to be frequent periconceptional drinkers than women in any of the other parity categories. Given the rate of unplanned pregnancies in the United States,13 it is likely that many of these women were not intending to become pregnant and therefore were not consciously striving to attenuate their drinking habits in light of an expectation of pregnancy. Once pregnancy recognition occurred, women having their first child were those least likely to report frequent alcohol use.28 The association between smoking and alcohol use reported in this study has been noted by others. Previous reports confirm that smokers are more likely than nonsmokers to report having consumed alcohol during the month prior to the survey (62% versus 46%),29 and are more likely than nonsmokers to be heavy alcohol users (14% versus 3.2%).26 Among pregnant women, Serdula and associates30 examined a number of factors from the Am J Prev Med 1999;17(2)
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Behavioral Risk Factor Surveillance System (BRFSS) from 1985 through 1988 and found the highest prevalence rates of alcohol use among women who were smokers (37%) and those who were unmarried (28%). Given that alcohol consumption rates among women overall showed a decline from 1983 through 1988,25 one might wonder if periconceptional alcohol use and alcohol use in general among women of childbearing age have continued to drop in the ensuing years since the 1988 NMIHS was conducted. A recent CDC study comparing alcohol use rates in 1991 and 1995 for women of childbearing age found little change in those rates, which were 49% in 1991 and 51% in 1995.12 Data compiled from the National Household Survey of Drug Abuse (NHSDA) for the years 1992 through 1994 found that rates of alcohol use during the month prior to the survey among women in the 14 to 44 age group were 49% for 1992, 51% for 1993, and 54% for 1994 (unpublished data from the National Household Survey on Drug Use 1992–1994). Similarities in prevalence rates among these studies and the current study lead one to believe that prevalence rates for periconceptional alcohol use closely mirror those of childbearing age in general. They are less like reported prevalence rates for alcohol use during pregnancy or after pregnancy recognition as illustrated in Table 2. While these data provide evidence of moderate to heavy alcohol use among women during the periconceptional period, some high-risk factors identified in this study are not those that health care providers might normally associate with risk drinking during pregnancy (for instance, higher education). However, previous studies have found a positive association between educational attainment and reported alcohol use among women in general.25,31 Previous analyses of the data set used in this study did not find that being college educated was a risk factor for frequent drinking after pregnancy recognition.28 Women who were more likely to continue frequent drinking after pregnancy recognition included those who were smokers, unmarried, other than white race/ethnicity, ⱖ35 years of age, and with annual household incomes ⬍$10,000. However, when these factors and others factors including level of periconceptional alcohol use were used in logistic modeling to determine the best predictors of alcohol use during pregnancy, the strongest predictor was level of periconceptional alcohol use. Moderate drinkers in the periconceptional period were 4 times more likely to drink during pregnancy than light periconceptional drinkers, and heavy drinkers were 6 times more likely to continue drinking in pregnancy A number of study limitations bear mentioning. We were unable to identify women whose intake pattern included binge drinking, a pattern thought to have particularly deleterious effects on pregnancy and infant outcomes.32 Because slightly less than 5% of all respondents consumed 6 or more drinks per week, we had a 106
relatively small number of frequent periconceptional drinkers available for multivariate analyses. Moreover, the heaviest drinkers may not have been accessible to the survey, participation in which required that the respondents to have an address, thus creating a possible nonrespondent bias. Among those selected for the survey, response rates were lower for teenage mothers; mothers of races other than white; and for those with 4 or more children, little prenatal care, or fewer years of education. Further, the data were obtained by selfreport of the respondents, and therefore are potentially affected by respondents’ recall biases, and the perceived social desirability of a negative or attenuated response to the question of alcohol use. Also, the outcome of the pregnancy was known at the time the mothers were surveyed. Women whose infants had a less than optimal outcome may have been less inclined to disclose levels of prenatal alcohol use accurately, especially heavier levels. Additional population-based surveys of alcohol use among women of childbearing age both pregnant and nonpregnant, are needed to correct methodologic limitations found in this and other epidemiologic studies addressing alcohol use during different stages of gestation. While significant reductions in alcohol use were reported by women in this study once pregnancy recognition occurred, many were drinking at potentially high-risk levels during the first 4 to 6 weeks of gestation. Therefore, prevention of alcohol exposures in early pregnancy will require preconceptional strategies including increased public awareness as well as targeted prevention activities by health care providers. Primary health care settings such as women’s health clinics, health facilities serving families with young children, and Women, Infant, and Children’s (WIC) clinics should become dissemination points for advice and education regarding alcohol use and its adverse effects on pregnancy. Women should also be made aware of the high rates of unintended pregnancy, and that being sexually active, a frequent alcohol user, and not using effective contraception places them at risk for having an alcohol-exposed pregnancy. Risk factors such as smoking and being unmarried can be helpful in alerting health providers who attend populations with high rates of these factors to the importance of addressing periconceptional alcohol use. Of further importance is the need to make systematic assessment of alcohol use a routine part of the health care visit for women of childbearing age. Health facilities and practices serving populations with high rates of alcohol use and risk factors for alcohol use should consider use of specific screening instruments such as the AUDIT33 and the TWEAK.34 Those who screen positive for problem alcohol use can be provided brief interventions as described by Fleming and associates,35 whose study found brief advice and counseling by primary care physicians to be very effective in reducing
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problem drinking, particularly among females. An additional approach is to encourage women of childbearing age who are unable to reduce problem drinking not only to seek treatment for alcohol abuse, but also to institute effective methods of contraception in order to avoid an unintended prenatal alcohol exposure.
17.
18.
19.
We would like to thank Shahul Ebrahim, MD, MSc, Coleen Boyle, PhD, Karrien Williams, and Michael Tully, MS, for their assistance in the preparation of this manuscript.
20. 21.
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