Department of Pharmacology, Center for Alcohol and Drug Abuse Studies, Brody School of Medicine and
1 Department of Psychology, East Carolina University, Greenville, NC, USA
Received 6 January 2000; in revised form 6 March 2000; accepted 24 March 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Data collected on humans suggest that most non-alcoholic women decrease their alcohol consumption during the first trimester of pregnancy, citing a distaste for alcohol as the reason for the reduction (Little et al., 1976). Another study conducted on 530 pregnant women, 90% of whom drank before and during their pregnancies, demonstrated that the proportion of drinking women decreased with advancing gestational age. Fifty per cent of the women retrospectively reported drinking after 32 weeks and only 20% reported drinking during the last week of gestation (Halmesmaki et al., 1987
). These percentages indicate that a significant number of women continue to drink during pregnancy.
The trend of decreasing alcohol consumption during pregnancy occurs in other animals that drink alcohol, including mice (Emerson et al., 1952), rats (Means and Goy, 1982
; Sandberg et al., 1982
), hamsters (Carver et al., 1953
; Morin and Forger, 1982
), and monkeys (Elton and Wilson, 1977
). The fact that this occurs in a number of different species in conjunction with the observation that a substantial amount of women acquire a taste aversion for alcohol during pregnancy (Little et al., 1976
) suggests that there is a protective mechanism for the fetus, mediated by reproductive hormones, that has evolved to cause the gravid female to reject potentially toxic substances.
To date, a limited number of studies have utilized genetic female drinking rats, and no research has been done on these animals during pregnancy. Two independent studies have shown that outbred strains of rats decreased their proportion and consumption of ethanol during pregnancy (Means and Goy, 1982; Sandberg et al., 1982
), but the ethanol solution contained saccharin and these rats were probably drinking for either caloric value or taste (Reid, 1996
). The HEP rat, on the other hand, consumes ethanol for a pharmacological effect, and will develop significant blood-alcohol concentrations (Myers et al., 1998
). The female HEP rat therefore may provide a model for the testing of behavioural and drug interventions on drinking, as has been attempted in the present study.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The rats were placed into three conditions: either impregnated by F7 male HEP rats; given injections of progesterone to mimic pregnancy; or served as controls while their drinking was monitored. The 25 rats were divided into two groups: a group of 11 and a group of 14. The difference between the two groups was the environment in which they were tested. The first group of 11 rats were housed on the stainless-steel battery. Due to the fact that three of four presumed pregnant rats in this group failed to deliver pups, it was thought that the stainless-steel cages may have been a factor in the pregnancy failures. Therefore, the second group of 14 were housed in standard plastic cages with corncob bedding which is the environment normally used by the Department of Comparative Medicine for breeding. This change necessitated two separate protocols: one for the first group of 11 rats and another for the second group of 14 rats.
Protocol 1
After a 10-day stabilization period of drinking ethanol at each rat's preferred concentration, the 11 rats were divided into three groups (pregnant, progesterone injection, or control). This division was based on each rat's daily g/kg consumption of ethanol, so that each of the three groups had approximately the same level of intake. This division resulted in four rats that were impregnated, three rats that received progesterone injections, and a group of four controls.
To impregnate the females, the alcohol tube was removed each night and a high alcohol-consuming male HEP rat was placed in the cage for co-habitation. The male rat was removed in the morning and the alcohol bottle returned. This was repeated until a sperm plug was found in the litter tray under each cage of the four rats. The day the sperm plug was observed was designated day 1 of pregnancy or gravid day 1 (GD1). The rats were then, once again, allowed 24-h access to the alcohol solution. Each day, the volume of fluids, body weight, and amount of food (Pro Lab Chow) consumed were recorded. At GD17, a paper towel was placed in each of the cages on the battery that contained a pregnant female, so that she could begin nest-building activities. In addition, a piece of screen was placed on the floor of each cage so that the newborn pups would not fall through the grid floor. Three of the females in this group did not give birth and were later reimpregnated in the absence of alcohol and kept in standard cages with corncob bedding to show that they were capable of delivering a litter.
The progesterone group of three rats received injections of progesterone in sesame oil in order to mimic the pregnant state. The sesame oil was used as a depot to slow and prolong progesterone absorption. On gravid days (GD) 1 and 2, the rats received a 1.0 mg/kg dose of progesterone in sesame oil. On GD 3 and 4, the rats received a 2.0 mg/kg dose of progesterone in sesame oil. And on GD 5 to 20, the rats received a 3.0 mg/kg dose of progesterone in sesame oil. The 3.0 mg/kg dose of progesterone is reported to inhibit cycling by females and mimic the pregnant state and this sequence mimics the rise in progesterone after impregnation (Zarrow et al., 1964). As with the impregnated dams, body weight, food intake, and fluid consumption were recorded. Vaginal smears were also performed on this group in order to confirm that progesterone inhibited cycling (Weil, 1996
).
The control group of four rats received no treatment. After data were collected on the control group, they were impregnated to show that they were capable of reproduction. Therefore, any differences in consumption between the controls and the experimental groups could not be attributed to differences in reproductive potential (Means and Goy, 1982).
Protocol 2
A second group of HEP female rats went through the standard 10-day stabilization period on the battery. It was at this point that the decision was made to alter the living environment to increase the likelihood of the females giving birth. The entire group of 14 female rats was taken off the battery and placed in individual plastic cages adapted to hold three drinking bottles on one end. Therefore, the stabilization period was repeated with the rats in their new environments.
After this second 10-day stabilization period was repeated, the 14 rats were divided into three groups (pregnant, progesterone injection, or injection control) as before. This division was based on each rat's g/kg ethanol consumption during the stabilization period, so that each group had approximately the same level of ethanol consumption. The division resulted in a group of four impregnated dams, five rats that received injections of progesterone, and a group of five controls which received injections of sesame oil. Combined with the rats from protocol 1, this resulted in a total of eight pregnant females, eight females which received injections of progesterone, and nine controls, for the entire experiment.
Impregnation of the four females was accomplished by taking them out of their standard plastic cages each night and placing them in individual cages on the stainless-steel battery with two water bottles and a high ethanol-consuming male HEP rat. The female was placed back in her standard cage with alcohol each morning and back on the battery at night. The day a sperm plug was found was designated GD1 and the female was returned to 24-h access to the alcohol solution. Each day, the volume of fluids, body weight and amount of food consumed were recorded. At GD17, bedding materials were placed in each of the four cages, so that the pregnant mothers could begin nest-building activities. None of the four females delivered, so they were re-impregnated in the absence of alcohol to show that they were capable of delivering a litter.
The injection group of five rats was administered by s.c. injections of progesterone in sesame oil to mimic the pregnant state in the same manner as in protocol 1. The only difference was that these females were in standard cages, as opposed to the cages on the battery.
The control group received injections of sesame oil only, to test for the possibility that the injections themselves or the vehicle had an effect on ethanol consumption. After data were collected for this group of controls, each of the five females was impregnated for the same reason stated in protocol 1.
![]() |
STATISTICAL METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Vaginal smears
Vaginal smears confirmed that the 3.0 mg/kg daily dosage of progesterone made the rats anoestrous. Anoestrous is the non-receptive period of the oestrous cycle, and is characterized by small, round, immature epithelial cells with large nuclei. A smeared slide typically has a few cells: two to five per low power field. Confirmation of cycling by the control rats which were re-mated and subsequently failed to deliver, and the one rat from the pregnant group which also failed to deliver after being taken off the alcohol, was also provided by vaginal smears. After removal of ethanol, oestrous in these rats was demonstrated by the large numbers of angular, anuclear, cornified epithelial cells found in clumps on smeared slides (Weil, 1996).
Consumption of ethanol
Consumption of ethanol for each of the three groups of HEP rats (pregnant, progesterone, and control) primarily ranged from 6 to 10 g/kg per day. Overall, daily consumption during the baseline period averaged 7.10 ± 0.65 g/kg for the controls, 7.69 ± 0.94 g/kg for the pregnant rats, and 7.02 ± 1.21 g/kg for the progesterone treated rats (Fig. 1). The average preferred concentration of ethanol for each group was 14.4, 17.9, and 16.0%, respectively. There was no effect of housing condition, stainless-steel vs standard plastic cages, on the amount of ethanol consumed. Drinking was remarkably steady over the 25-day testing period, with two exceptions, but ANOVA revealed differences from baseline during the experiment: for the combined control animals, F(8,24) = 2.046, P < 0.01; for the pregnant group, F(7,27) = 1.714, P < 0.05; and for the progesterone group, F(7,25) = 0.933, n.s. On GD 16, the control group's consumption (9.71 ± 1.42 g/kg) varied from baseline (P < 0.01), and on GD 17 the pregnant group's consumption (12.17 ± 3.4 g/kg) varied from their baseline (P < 0.05). The pregnant group exhibited a decrease in drinking following cohabitation on GD 1, and an increase on GD 3, neither of which was statistically significant (Fig. 1A
).
|
An increase in variance was demonstrated by the pregnant group, which began on GD 14 and continued until GD 19. The increase in variance was not due to a consistent change in one or two females, but rather all of the females exhibited an increased variance. By comparing the range of values for proportion for each rat on GD 813, the 5 days prior to the increase in variance, to the range of values on GD 1420, it was discovered that every rat had at least 1 day with a lower value for proportion, and five of the eight had at least 1 day higher. For example, one rat had values for proportion that ranged from 0.50 to 0.625 during GD 813. This same rat had values for proportion that ranged from 0.327 to 0.785 during GD 1420. These highs and lows occurred on different days for different rats. No significant increases in variance of proportion were noted in the control or progesterone-treated groups (Fig. 3A).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Weight gain by the pregnant group was considerably less than that which is normally expected by pregnant rats. The weight gain exhibited by both the control and the progesterone groups was considered normal for a 20-day time period. Pregnant SpragueDawley rats housed under the same conditions in the same vivarium without alcohol exhibited a 60% increase in body weight (an approximate gain of 0.15 kg) during pregnancy and had an average litter size of 14 pups (Henderson, 1990). This difference may be due largely to the presumed resorption of the foetuses prior to the final week of gestation when weight gain is greatest. Despite only having eight pups, the one pregnant female that did deliver still managed to gain slightly over 0.1 kg. Most of this weight gain (0.7 kg) came during the final week of pregnancy, which coincides precisely with the time that the other seven pregnant females began losing their pregnancies. Small litter size and delayed delivery are just two of many deleterious effects caused by alcohol on pregnant rats and their offspring (Abel et al., 1979
; Sanchis et al., 1986
; Streissguth, 1997
).
A general trend of increased food consumption was exhibited by the pregnant group. Although this increased consumption was not significant on any given day, it was expected, due to the fact that the demand for nutrients and calories increases during pregnancy. It is unknown why the pregnant animals significantly decreased their consumption of food immediately following cohabitation at day 1. Perhaps, the stress of mating mediated the reduction.
A marked increase in the variance for proportion, but not amount, of ethanol consumed, was shown by the pregnant group beginning at GD 14. Drinking during the last week of pregnancy greatly fluctuated. Perhaps this was mediated by the females apparently undergoing resorption. Due to the fact that the amount of ethanol consumed did not change, with the exception of GD 17 when an increase was noted, the HEP rats must have been drinking more or less water during that time period (depending on the day in question). In fact, each rat had an increased range of values for proportion during the last week of the experiment.
A very similar increase in the variance for body weight was also observed in the pregnant animals beginning on GD 13. This finding, in conjunction with the increase in variance for proportion, which both began around the same time, led to the conclusion that the pregnant females must have begun to lose their litters at various times thereafter. If the weight for the one dam which maintained its pregnancy is dropped from the data matrix, there is still a 50% increase in the standard deviation during GD 1621, compared to GD 712. Five of the seven remaining rats exhibited an unexpected weight loss, one as much as 0.014 kg from peak weight, during this last week of gestation.
The copious quantities of ethanol that the pregnant females imbibed led to an apparent high rate of foetus resorptions. Seven of the eight females in the pregnant group apparently had resorbed their foetuses. But, when these seven were removed from the ethanol and mated again, six successfully delivered litters. Clearly, ethanol was adversely affecting pregnancy outcomes. Padmanabhan and Hameed (1988) demonstrated that administration of an acute dose of ethanol (0.03 ml/g body weight of 25% v/v absolute ethanol, or approx. 6 g of ethanol/kg) on GD 16 markedly increased prenatal mortality (resorptions) in mice. This also suggests that ethanol is lethal to the developing embryo. Administration of ethanol solutions by gavage to pregnant mice from different strains demonstrated that there are also strain-specific sensitivities to the teratogenic effects of ethanol (Gilliam and Kotch, 1990; Boehm et al., 1997
).
In the same manner that fecundity decreases with age, drinking during early development has also been shown to decrease fecundity. In fact, the HEP rats were drinking large quantities of ethanol as early as 40 days of age during the first screen. Cebral et al. (1997) observed significantly decreased in vitro fertilization rates when oocytes from prepubertal and pubertal ethanol-treated female mice were inseminated with spermatozoa from adult control males. The aim of that particular study was to investigate the effects of low chronic alcohol intake on fertility. Ethanol in tap water at a concentration of 5% (a low dose) was administered to hybrid F1 mice (C57/B1 x DBA) for 4 weeks. Low chronic ingestion of ethanol sufficiently reduced the percentage of activated oocytes, and increased the number of fragmented oocytes taken from immature females, such that in vitro fertilization rates were decreased. Others have also reported that daily oral administration of ethanol solutions decreased fecundity and increased gestation time in rats (Abel et al., 1979; Sanchis et al., 1986
). Other deleterious effects of ethanol on immature female rats include: delayed vaginal opening, decreased uterine and ovarian weights, and depressed ovarian function (Gavaler et al., 1980
; Bo et al., 1982
). In addition, ovarian failure has been found to occur in rats that are fed high doses of ethanol (Van Thiel et al., 1978
). Ethanol is undoubtedly a reproductive toxin as well as a teratogen.
Contemporary studies have shown a correlation between alcohol consumption and an increased risk of spontaneous abortion in women. In the USA, women who had been clinically diagnosed as alcohol abusers were twice as likely as controls to have suffered three or more spontaneous abortions (Sokol et al., 1980). A survey of clinical literature regarding fetal alcohol syndrome (FAS) found that out of 90 women who had given birth to children with FAS, 52% had had at least one spontaneous abortion, and the average rate of spontaneous abortion per mother was 2.2 (Abel, 1990
). In spite of the plethora of studies which have been conducted on the matter of spontaneous abortion, no threshold level or critical time period of consumption leading to the occurrence of spontaneous abortion has been established. In a retrospective study, Wilsnack et al. (1984) estimated the threshold for spontaneous abortion at six or more drinks/day consumed at least three times/week. This high level of consumption would be typical of an alcoholic. Lower thresholds have been proposed, which suggest alcohol may harm the fetus not only when alcohol is abused, but also when taken in moderation. In a study of second trimester losses, which in the rat is equivalent to GD 1420 (the time period in which the females presumably lost their litters), one or two drinks daily doubled the risk of spontaneous abortion, and more than three drinks daily more than tripled the risk (Harlap and Shiono, 1980
). Windham et al. (1997) found a twofold increase in the risk of spontaneous abortion with an average consumption of seven or more drinks/week during the first trimester. A doubled risk was also found by Kline et al. (1980) for a weekly consumption of two to six drinks. In addition, risk increased an average of 25% for each additional ounce of alcohol consumed by pregnant women in a study performed by Russell and Skinner (1988).
Some 620% of women have been reported to drink heavily during pregnancy (Halmesmaki et al., 1987). Reductions in alcohol consumption in human females tend to be inversely proportional to prior consumption. Therefore, women who drink heavily prior to pregnancy (alcoholics) are most often those who continue to drink heavily throughout their pregnancies (Little et al., 1976
). Like these women, female HEP rats do not curtail their drinking during pregnancy, and could therefore be considered valid models of the severe type 2 female alcoholic. The HEP rat's high level of alcohol consumption during pregnancy led to an apparent high rate of fetal resorptions. Due to this, the HEP rat could also potentially offer a second model for alcohol research a model of alcohol-induced spontaneous abortion. More research on the effects of ethanol on fecundity and the nature of pregnancy loss will be necessary to better define this animal as a model.
Such models could make additional research possible which in turn could substantially contribute to our understanding of the relationship of dosage, developmental timing, gender differences, genetic susceptibility, and differences in tolerance elicited by hormonal changes during pregnancy. These rats could also be used for testing drug and behavioural treatments. Such research could point towards additional therapeutic approaches that could be used on women who abuse alcohol during their pregnancies. New treatments could significantly improve the quality of life of females afflicted with alcoholism. This in turn would help decrease the prevalence of completely preventable disabilities such as FAS, alcohol-related birth defects, and alcohol-related neurological defects, as well as decrease the incidence of alcohol-induced spontaneous abortions.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abel, E. L., Dintcheff, B. A. and Day, N. (1979) Effects of in utero exposure to alcohol, nicotine, and alcohol plus nicotine, on growth and development in rats. Neurobehavioral Toxicology 1, 153159.
Bo, W. J., Krueger, W. A. and Rudeen, P. K. (1982) Ethanol induced alterations in the morphology and function of the rat ovary. Anatomical Record 202, 255260.[ISI][Medline]
Boehm, S. L., Lundahl, K. R., Caldwell, J. and Gilliam, D. M. (1997) Ethanol teratogenesis in the C57BL/6J, DBA/2J, and A/J inbred mouse strains. Alcohol 14, 389395.[ISI][Medline]
Carver, W., Nash, J. B., Emerson, G. A. and Moore, W. T. (1953) Effects of pregnancy and lactation on voluntary alcohol intake of hamsters. Federation Proceedings 12, 309.
Cebral, E., Lasserre, A., Rettori, V. and De Gimeno, M. (1997) Impaired mouse fertilization by low chronic alcohol treatment. Alcohol and Alcoholism 32, 563572.[Abstract]
Cloninger, C. R. (1987) Neurogenetic adaptive mechanisms in alcoholism. Science 236, 410416.[ISI][Medline]
Cloninger, C. R., Bohman, M. and Sigvardsson, S. (1981) Inheritance of alcohol abuse: Cross-fostering analysis of adopted men. Archives of General Psychiatry 38, 861868.[Abstract]
Elton, R. H. and Wilson, M. E. (1977) Changes in ethanol consumption by pregnant pigtailed macaques. Journal of Studies on Alcohol 38, 21812183.[ISI][Medline]
Emerson, G. A., Brown, R. G., Nash, J. B. and Moore, W. T. (1952) Species variation in preference for alcohol and in effects of diet or drugs on this preference. Journal of Pharmacology and Experimental Therapeutics 106, 384.
Forger, N. G. and Morin L. P. (1982) Reproductive state modulates ethanol intake in rats: Effects of ovariectomy, ethanol concentration, estrous cycle, and pregnancy. Pharmacology, Biochemistry and Behavior 17, 321333.
Gavaler, J. S., Van Thiel, D. H. and Lester R. (1980) Ethanol: A gonadal toxin in the mature rat of both sexes. Alcoholism: Clinical and Experimental Research 4, 271276.[ISI][Medline]
Gilliam, D. M. and Kotch, L. E. (1990) Alcohol-related birth defects in long- and short-sleep mice: post-natal litter mortality. Alcohol 7, 483487.[ISI][Medline]
Halmesmaki, E., Kaivio, K. O. and Ylikorkala O. (1987) Patterns of alcohol consumption during pregnancy. Obstetrics and Gynecology 69, 594597.[Abstract]
Harlap, S. and Shiono P. H. (1980) Alcohol, smoking, and incidence of spontaneous abortions in the first and second trimester. Lancet i, 173176.
Henderson, M. G. (1990) Effects of Prenatal Exposure to Cocaine or Related Drugs on the Development of Rat Offspring. Doctoral Dissertation, East Carolina University School of Medicine.
Kline, J., Shrout, P., Stein, Z., Suzzer, M. and Warburton D. (1980) Drinking during pregnancy and spontaneous abortion. Lancet i, 176180.
Lankford, M. F., Roscoe, A. K., Pennington, S. N. and Myers, R. D. (1991) Drinking of high concentrations of ethanol versus palatable fluids in alcohol-preferring (P) rats: Valid animal model of alcoholism. Alcohol 8, 293299.[ISI][Medline]
Little, R. E., Shultz, F. A. and Mandell, W. (1976) Drinking during pregnancy. Journal of Studies on Alcohol 37, 375379.[ISI][Medline]
McGue, M., Pickens, R. W. and Svikis, D. S. (1992) Sex and age effects on the inheritance of alcohol problems: A twin study. Journal of Abnormal Psychology 101, 317.[ISI][Medline]
McMillen, B. A. (1997) Toward a definition of a valid model of alcoholism: Multiple animal models for multiple diseases. Alcohol 14, 409419.[ISI][Medline]
Means, L. W. and Goy, H. B. (1982) Reduced preference for alcohol during pregnancy and following lactation in rats. Pharmacology, Biochemistry and Behavior 17, 10971101.[ISI][Medline]
Morin, L. P. and Forger, N. G. (1982) Endocrine control of ethanol intake by rats or hamsters: Relative contributions of the ovaries, adrenals, and steroids. Pharmacology, Biochemistry and Behavior 17, 529537.[ISI][Medline]
Myers, R. D., Robinson, D. E., West, M. W., Biggs, T. A. G. and McMillen, B. A. (1998) Genetics of alcoholism: Rapid development of a new high ethanol preferring (HEP) strain of female and male rats. Alcohol 16, 343357.[ISI][Medline]
Padmanabhan, R. and Hameed, M. S. (1988) Effects of acute doses of ethanol administered at pre-implantation stages on fetal development in the mouse. Drug and Alcohol Dependence 16, 215227.
Reid, L. D. (1996) Endogenous opioids and alcohol dependence: Opioid alkaloids and the propensity to drink alcoholic beverages. Alcohol 13, 511.[ISI][Medline]
Russell, M. and Skinner, J. B. (1988) Early measures of maternal alcohol misuse as predictors of adverse pregnancy outcomes. Alcoholism: Clinical and Experimental Research 12, 824830.[ISI][Medline]
Sanchis, R., Sancho-Tello, M. and Guerri, C. (1986) The effects of chronic alcohol consumption on pregnant rats and their offspring. Alcohol and Alcoholism 21, 295305.[ISI][Medline]
Sandberg, D., Stewart, J. and Zalman, A. (1982) Changes in ethanol consumption during pregnancy of the rat. Journal of Studies on Alcohol 43, 137145.[ISI][Medline]
Sokol, R. J., Miller, S. I. and Reed, G. (1980) Alcohol abuse during pregnancy: An epidemiologic study. Alcoholism: Clinical and Experimental Research 4, 135145.[ISI][Medline]
Streissguth, A. (1997) Fetal Alcohol Syndrome: A Guide for Families and Communities, p. 10. Paul H. Brooks, Baltimore, MD.
Van Thiel, D. H., Gavaler, J. S. and Lester, R. (1978) Alcohol-induced ovarian failure in the rat. Journal of Clinical Investigations 61, 624632.[ISI][Medline]
Weil, M. A. (1996) Vaginal cytology and optimum breeding time for bitches. Veterinary Technician 17, 137141.
West, M. W., Biggs, T. A. G., Schreiber, R., De Vry, J. and Myers, R. D. (1999) Calcium channel agonist ()-BAY k 8644 suppresses free and limited access intake of alcohol in genetic drinking rats. Psychopharmacology 142, 261269.[ISI][Medline]
Wilsnack, S., Klassen, A. D. and Wilsnack, R. W. (1984) Drinking and reproductive dysfunction among women in a 1981 national survey. Alcoholism: Clinical and Experimental Research 8, 451458.[ISI][Medline]
Windham, G. C., Von Behren, J., Fenster, C., Schaefer, C. and Swan, S. H. (1997) Moderate maternal alcohol consumption and risk of spontaneous abortion. Epidemiology 8, 509514.[ISI][Medline]
Zarrow, M. X., Yochim, J. M. and McCarthy, J. L. (1964) Experimental Endocrinology, pp. 65108. Academic Press, New York.