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C.S. Mott Center for Human Growth and Development,4 Departments of Obstetrics & Gynecology and Anatomy & Cell Biology, Wayne State University School of Medicine, Detroit, Michigan 48201
Department of Nutritional Sciences,5 University of Wisconsin-Madison, Madison, Wisconsin 53706
Perinatology Research Branch,6 National Institute of Child Health and Human Development, NIH, DHHS, Bethesda, Maryland 20892
ABSTRACT
Maternal alcohol abuse during pregnancy can produce an array of birth defects comprising fetal alcohol syndrome. A hallmark of fetal alcohol syndrome is intrauterine growth retardation, which is associated with elevated apoptosis of placental cytotrophoblast cells. Using a human first trimester cytotrophoblast cell line, we examined the relationship between exposure to ethanol and cytotrophoblast survival, as well as the ameliorating effects of epidermal growth factor (EGF)-like growth factors produced by human cytotrophoblast cells. After exposure to 0–100 mM ethanol, cell death was quantified by the TUNEL method, and expression of the nuclear proliferation marker, Ki67, was measured by immunohistochemistry. The mode of cell death was determined by assessing annexin V binding, caspase 3 activation, pyknotic nuclear morphology, reduction of TUNEL by caspase inhibition, and cellular release of lactate dehydrogenase. Ethanol significantly reduced proliferation and increased cell death approximately 2.5-fold through the apoptotic pathway within 1–2 h of exposure to 50 mM alcohol. Exposure to 25–50 mM ethanol significantly increased transforming growth factor alpha (TGFA) and heparin-binding EGF-like growth factor (HBEGF), but not EGF or amphiregulin (AREG). When cytotrophoblasts were exposed concurrently to 100 mM ethanol and 1 nM HBEGF or TGFA, the increase in apoptosis was prevented, while EGF ameliorated at 10 nM and AREG was weakly effective. HBEGF survival-promoting activity required ligation of either of its cognate receptors, HER1 or HER4. These findings reveal the potential for ethanol to rapidly induce cytotrophoblast apoptosis. However, survival factor induction could provide cytotrophoblasts with an endogenous cytoprotective mechanism.
apoptosis, growth factors, placenta, toxicology, trophoblast
Fetal alcohol syndrome is recognized as a pattern of growth retardation, facial anomalies, and mental retardation in infants born to alcoholic women [1]. Low birth weight is perhaps the most consistent outcome for infants exposed to ethanol during gestation [2]. Interestingly, intrauterine growth retardation (IUGR) is associated with increased apoptosis of cytotrophoblast cells [3–8], which comprise a major embryonic component of the placenta. While ethanol can pass through the placental barrier to directly inhibit fetal growth and development, evidence suggests that it could also indirectly affect the fetus by altering placental function [9]. Cultured human cytotrophoblast cells exposed to ethanol reduce DNA and protein synthesis while increasing gonadotropin (hCG) and steroid (progesterone) production [10, 11]. Hormone-stimulated transport of amino acids by the trophoblast is also inhibited by ethanol [12]. While it appears that ethanol shifts the trophoblast from a state of proliferation to one of cell cycle arrest or differentiation, the mechanism of these changes is not understood. It is also unclear whether the placenta functions normally under these conditions. No information is available on the survival of human trophoblast cells after exposure to ethanol.
The effect of maternal alcohol consumption during the prenatal period on the fetus is highly variable in humans [13] and may be influenced by concurrent exposure to other toxic substances or maternal physiology. Growth factors and cytokines can operate as survival factors to protect cells against stress induced by toxins or environmental extremes. Heparin-binding EGF-like growth factor (HBEGF) is a member of the epidermal growth factor (EGF) family that promotes cell proliferation, differentiation, motility, and survival [14, 15]. The EGF family also includes EGF, transforming growth factor 
(TGFA), and amphiregulin (AREG), among others [14], which are expressed in the human placenta as well [16–18]. Human placentas express HBEGF throughout gestation, particularly in trophoblast cells of the chorionic villi and in extravillous trophoblast cells invading the decidua [19, 20]. HBEGF is down-regulated in placentas from pregnancies complicated by IUGR or preeclampsia [20]. Both disorders are associated with reduced trophoblast survival [6, 21]. It is not known whether endogenous expression of HBEGF or other survival factors in the placenta influences the outcome for fetuses exposed to alcohol in utero. Therefore, it is important to establish whether human trophoblast survival is compromised by ethanol and if survival factors produced by the placenta are able to attenuate the harmful effects of alcohol.
In the present investigation, we have examined a first trimester human cytotrophoblast cell line, HTR-8/SVneo [22], to determine whether acute ethanol exposure is capable of altering trophoblasts physiologically. Characterization of this cell line reveals similarities to primary cultures of first trimester cytotrophoblast cells with respect to expression of trophoblast marker proteins, proliferative capacity, and invasiveness [23]. The use of this cell line provided a homogeneous population of human trophoblasts for this investigation. We have determined the time- and dose-dependent effects of ethanol on apoptosis and have investigated the ability of HBEGF and other members of the EGF family to attenuate this interaction.
Culture and Ethanol Exposure of Human Cytotrophoblast Cells
The HTR-8/SVneo cell line (generously provided by Dr. Charles Graham of Queen's University, Kingston, Ontario, Canada) was cultured in DMEM/Hams F12 medium (Sigma Chemical Co., St. Louis, MO) containing 10% donor calf serum [23]. Cells were then cultured for an additional 18–24 h without serum (5 mg/ml BSA) prior to all experiments. Ethanol (Midwest Grain Co., Pekin, IL) was prepared immediately before its addition at 0, 25, 50, or 100 mM in serum-free medium. Cells were also treated for 30 min with 2 mM H2O2. In some experiments, cells were incubated for 30 min before the addition of ethanol with 0.1–10 nM recombinant human HBEGF, EGF, TGFA, AREG (R&D Systems, Minneapolis, MN), 20 µM of the caspase inhibitors (EMD Biosciences, San Diego, CA), N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), Z-DEVD-FMK, Z-IETD-FMK, Z-LEHD-FMK, or 20 µM of the negative control compound Z-FA-FMK. Treatment with 1 nM HBEGF and 100 mM ethanol was also conducted in the presence of 10 µg/ml of CRM197 (EMD Biosciences), anti-HER1 (Ab-2, clone 225), or anti-HER4 (Ab-3, clone H4.72.8) mouse monoclonal blocking antibodies (Lab Vision Corp., Fremont, CA), as well as with 20 µg/ml of nonimmune mouse IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) as a control.
Cytotrophoblast cells grown in 96-well tissue culture plates (Becton Dickinson) were processed for immunohistochemistry, as previously described [23]. Nuclei of proliferating cells were labeled with 0.55 µg/ml anti-Ki-67 monoclonal antibody (Ki-S5; DAKO, Carpinteria, CA). To visualize and quantify (gray level) antigen, an Envision System peroxidase anti-mouse/rabbit kit (DAKO) was used in conjunction with image analysis, according to our published procedure [20]. Cells labeled with antibody against Ki-67 were counterstained with DAPI and assessed for the percentage of Ki-67/DAPI-labeled nuclei as an index of cell proliferation, as previously described [24]. Goat polyclonal antibodies (R&D Systems) against human recombinant HBEGF, EGF, TGFA, and AREG (5 µg/ml) were used to label cells expressing these growth factors. Controls were incubated with 10 µg/ml nonimmune IgG (Jackson Immunoresearch). Cells were then incubated 1 h at 25°C with 0.1 µg/ml rabbit anti-goat IgG (Jackson Immunoresearch) before processing with the Envision System. Antibody labeling was quantified by digital image analysis, as previously described [24]. Values (mean gray level) obtained by image analysis with nonimmune IgG were subtracted from those obtained with specific antibody.
Cell death was assessed in cultured cells by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method, as previously described [24]. Briefly, cells grown and treated in 96-well plates were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton x 100 for 10 min and the TUNEL assay was performed using a fluorescein-based cell death detection kit (Roche Applied Science, Indianapolis, IN), counterstaining with 5 µg/ml 4',6-diamidino-2-phenylindole, HCl (DAPI; EMD Biosciences). Three or more fields in each well were imaged using a Leica (Wetzlar, Germany) DM IRB epifluoresence microscope interfaced with a Hamamatsu Orca digital camera (Hamamatsu City, Japan) to obtain a minimum of 100 cells for analysis. The fraction of TUNEL/DAPI labeled nuclei (TUNEL Index) was assessed from micrographic images using a Simple PCI (C-Imaging Corp., Cranberry Township, PA) imaging software system and adjusting the threshold function to optimize automated counting of each fluorescent nucleus.
Externalized phosphatidylserine was detected by incubating live cells for 15 min at room temperature with biotin-labeled annexin V (1:20; Molecular Probes, Portland, OR) in 10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7. Cells were then fixed and incubated overnight at 4°C with 5 µg/ml UltraAvidin-Texas Red (Leinco Technologies, St. Louis, MO) and 5 mg/ml BSA in PBS. After counterstaining with DAPI, representative images of both DAPI and Texas Red fluorescence were acquired. Annexin V binding was quantified by image analysis to determine the fluorescence intensity (gray level) over each field of cells, as previously described.
Cell necrosis was assessed by measuring lactate dehydrogenase (LDH) activity released into the culture medium. Cells grown in black, clear-bottom 96-well tissue culture plates (Corning, Inc., Corning, NY) were treated in 100 µl of modified BWW medium (Irvine Scientific, Santa Ana, CA). Culture medium was moved to separate wells and the remaining cells were washed three times with BWW medium and lysed. Both the medium and cell lysates were assayed for LDH enzymatic activity using a DHL Cell Cytotoxicity Assay kit (AnaSpec, San Jose, CA). The fluorescent reaction product was quantified with a SpectraMax M2 multiplate spectrofluorometer (Molecular Devices, Sunnyvale, CA). To calculate the LDH release index, the value of LDH activity in the medium, multiplied by 100, was divided by the value obtained from the lysed cells.
A cell-permeable, fluorigenic substrate Phi-Phi-Lux-G1D2 (OncoImmunin, Kensington, MD) was utilized to monitor caspase 3 activity, according to the manufacturer's recommendations. After a 1-h exposure to 100 mM ethanol, cytotrophoblast cells were washed to remove medium and treated with 10 µM Phi-Phi-Lux-G1D2 for 30 min before epifluorescence microscopy and digital imaging.
All assays were conducted using triplicate samples and all experiments were repeated at least three times (n 
3). The program SPSS version 12.0 (SPSS Inc., Chicago, IL) was used to determine statistical significance. Comparisons were made to vehicle-treated controls for Ki-67, TUNEL, annexin V binding, LDH release, and antibody staining intensity using ANOVA with the Dunnett t-test for post hoc analysis.
Ethanol Inhibits Trophoblast Growth and Survival
The dose-dependent effects of ethanol on the rates of proliferation and death were examined in human cytotrophoblast cells. Proliferation, determined by nuclear expression of Ki67, decreased significantly at low to moderate (25–50 mM) concentrations of ethanol (Fig. 1A). The Ki67 index after exposure for 1 h to 100 mM ethanol was about 40% of the vehicle control. At the same time, cell death rates, determined by TUNEL, increased significantly at 50–100 mM ethanol (Fig. 1B). At 50 mM, the percentage of cells dying reached 16%, representing an increase of 3.7-fold above vehicle exposed cells. Cell death increased in a linear manner during the 2 h of exposure to ethanol (Fig. 1C).
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Ethanol Specifically Induces Apoptosis
Several criteria were used to determine if cell death due to ethanol exposure was mediated through the apoptotic pathway. Ethanol-exposed cytotrophoblast cells observed by fluorescent DAPI staining contained numerous pyknotic nuclei that were also labeled by the TUNEL method that detects fragmented DNA (Fig. 2, C and D). Pyknosis and DNA fragmentation were both rare in vehicle-treated cells (Fig. 2, A and B) Treatment with ethanol for 1 h was accompanied by a dose-dependent increase in the binding of annexin V to live cells (Fig. 3), providing evidence of phosphatidylserine redistribution that typically occurs during apoptosis [25]. In the case of cell death by necrosis, the plasma membrane is disrupted and cytoplasmic proteins are released [25]. Therefore, we assessed the release of LDH from cytotrophoblast cells exposed for 2 h to ethanol (Fig. 4). While exposure to hydrogen peroxide significantly increased LDH detected in the medium compared to vehicle, exposure to 25–100 mM ethanol had no effect on LDH release, suggesting that ethanol does not kill cytotrophoblast cells by necrosis.
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The apoptotic pathway is mediated by a cascade of cysteine proteases [26], including the initiator caspases, caspases 8 and 9, and the effector caspase, caspase 3. Caspase 3 enzymatic activity was detected in live cells using a specific caspase 3 substrate, Phi-Phi-Lux-G1D2, which fluoresces upon enzymatic cleavage. Comparison of equally confluent fields of cells (Fig. 5, A and C) by fluorescence (Fig. 5, B and D) revealed elevated caspase 3 activity when cells were exposed to 100 mM ethanol for 1 h. Furthermore, we found that caspase activity was required for ethanol-induced cell death. An inhibitor of all caspases, Z-VAD-FMK, blocked the ethanol-induced elevation of TUNEL, while an inactive, structurally related compound serving as control was without effect on TUNEL (Fig. 5E). Specific inhibitors of caspases 3 and 9 also inhibited apoptosis caused by ethanol, with caspase 3 inhibitor the most effective. Caspase 8 inhibitor had no significant effect. Based on these findings, it appears that the apoptotic pathway is rapidly activated in human cytotrophoblast cells exposed to moderate to high levels of ethanol.
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Cytotrophoblasts Produce EGF Family Survival Factors When Challenged with Ethanol
Members of the EGF family may be up-regulated during periods of oxidative stress to function as survival factors through activation of their receptors, HER1–4 [27–29]. Examination of the expression of four EGF-like growth factors after exposing cytotrophoblast cells for 2 h to ethanol revealed no change in EGF or AREG levels, but significant elevation of both TGFA and HBEGF by 25 mM ethanol (Fig. 6). HBEGF is a growth factor naturally produced by the trophoblast that can protect cells from apoptosis when exposed to stress [24]. Although the HTR-8/SVneo cells normally produce high levels of HBEGF only when exposed to hypoxia [24], significantly elevated levels of HBEGF were observed in the presence of 25–50 mM ethanol (Fig. 6). However, the cells did not significantly up-regulate HBEGF in the presence of 100 mM ethanol, whereas TGFA was up-regulated by all ethanol concentrations.
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To determine if EGF-like growth factors are cytoprotective against the detrimental effects of ethanol, 1 nM (Fig. 7A) or 10 nM (Fig. 7B) of each growth factor was added to the culture medium for 30 min prior to and during treatment of cytotrophoblast cells for 1 h with 100 mM ethanol. Apoptosis and proliferation were then assessed by TUNEL and Ki65 assays, respectively. Each growth factor significantly reduced apoptosis, although AREG was only marginally effective at either concentration. EGF prevented about half of the increase in TUNEL when used at 1 nM, while TUNEL was reduced to the control level by 10 nM EGF. TGFA prevented any significant increase in apoptosis at both 1 and 10 nM. HBEGF was equal to or better than TGFA in its effectiveness, as shown by its dose response (Fig. 7C). Cell proliferation levels were restored with 2–5 nM HBEGF (Fig. 7D).
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The specificity of the protective effect of HBEGF was demonstrated using an HBEGF-specific antagonist, CRM197 [30], and blocking antibodies against its two known receptors, HER1 (the EGF receptor) and HER4. Co-treatment with 100 mM ethanol, 1 nM HBEGF, and 10 µg/ml CRM197 resulted in a significantly higher number of TUNEL-positive cells (Fig. 8A). A similar reversal of the amelioration by HB-EGF was observed when blocking antibodies against both HER1 and HER4 were present (Fig. 8B). However, addition of either antibody alone failed to attenuate the effect of HBEGF, suggesting that the two receptors operate independently to promote cytotrophoblast survival during exposure to ethanol. As a negative control, nonimmune IgG had no effect on the ability of HBEGF to prevent ethanol-induced apoptosis. In a previous study [24], we demonstrated that inhibition of HBEGF signaling by treatment with CRM197 or antibodies against HER1 and HER4 did not affect cytotrophoblast apoptosis under the control conditions.
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Our findings support the idea that alcohol consumption during pregnancy could rapidly initiate apoptosis of cytotrophoblast cells within the placenta. Physiologically relevant levels of ethanol (25–50 mM), comparable to blood alcohol concentrations of approximately 100–200 mg/dl attained during consumption of alcoholic beverages, produced a dose-dependent increase in the death of cultured human cytotrophoblast cells. At 50 and 100 mM, the increase in TUNEL-positive cells was in excess of 3-fold (P < 0.05) and reached significance within 30–60 min. At the same time, the nuclear cell proliferation protein Ki-67 was down-regulated to below half of its normal expression level. This apparent reduction in proliferation is consistent with a previous report of inhibition of DNA synthesis in cultured first trimester cytotrophoblasts and JEG-3 choriocarcinoma cells after exposure to ethanol for several days [11]. The death of HTR-8/SVneo cells appeared to be the result of apoptosis, based on the presence of condensed, pyknotic nuclei in the TUNEL positive cells and elevated (P < 0.05) binding of annexin V to live, ethanol-exposed cells [25]. Furthermore, there was no evidence of LDH release, associated with necrotic cell death. Involvement of the apoptotic proteolytic cascade was demonstrated in ethanol-treated cytotrophoblast cells by the activation of caspase 3 and a requirement for caspases 3 and 9 in mediating cell death. The absence of a significant effect of the caspase 8 inhibitor suggests that the endogenous apoptotic pathway mediated by caspase 9 [26] is the predominant mechanism induced by ethanol in human cytotrophoblast cells. Serum withdrawal 1 day prior to ethanol challenge provided a defined culture environment, although it may introduce an added stressor. While our experimental design controlled for serum deprivation and did not produce TUNEL levels exceeding those of cells cultured in serum (data not shown), the absence of serum could heighten sensitivity to ethanol.
It is not clear whether apoptosis caused by prenatal ethanol exposure would impair the placenta or lead to repair and regrowth of the damaged tissue. Additionally, annexin V binding can reflect nonapoptotic events in human trophoblast cells. Phosphatidylserine redistribution and variable progression along the apoptotic pathway is associated with cytotrophoblast differentiation and fusion with the syncytium [31]. Production of hCG and progesterone is induced by exposure of isolated term trophoblasts to ethanol for several days [10], suggesting that ethanol is capable of inducing cytotrophoblast differentiation. While our TUNEL and morphological data point to apoptosis, we cannot rule out the possibility that ethanol also causes some cytotrophoblast cells to differentiate. Our experimental paradigm focused on ethanol exposure that was relatively brief (1–2 h) compared with that examined by many other in vitro studies, focusing our attention on the immediate cellular responses to this insult. Although the exposure time was brief, proliferation and survival were significantly compromised by alcohol.
Apoptosis occurs naturally among trophoblast cells during normal development [32], but has been reported to be elevated in placentas associated with small for gestational age infants [6]. Elevated apoptosis also occurs in placentas of women with preeclampsia [21], a condition that, when severe, is often accompanied by IUGR [33]. Trophoblast cell death observed in our study was the result of a single acute episode of ethanol exposure. There is debate as to whether a single incident of alcohol intake, or binge drinking, is sufficient to cause birth defects or IUGR [2]. Alcohol dosage, gestational timing, and genetic makeup are critical factors that influence outcome for the conceptus [34]. Damage to the placenta in chronically alcoholic women would be exacerbated by repeated exposure to ethanol. Ethanol disrupts extraembryonic tissues, including the yolk sac and placenta [35–38] in rodents treated chronically with ethanol. Placental weight reduction occurs without evidence of apoptosis [39], suggesting that dead cells have been removed after an earlier, transient period of apoptosis. Hyperplasia, tissue disruption, and irregular vascularization found in alcohol-exposed placentas could be the result of apoptosis [38]. IUGR is associated with reduced placental weight [40–42] and the inhibition of cytotrophoblast proliferation by ethanol observed here and by others [11] could contribute to diminution of placental mass. Acute exposure of embryonic cells to ethanol can initiate the apoptotic pathway, leading to altered development [43, 44]. Rapid induction of apoptosis occurs during gastrulation in ethanol-exposed mouse embryos [45] and this sensitivity continues in certain cellular populations throughout embryogenesis [46]. In the perfused human placenta, ethanol increases oxidative stress, leading within 2 h to the formation of nitrotyrosine residues on proteins and DNA damage [47]. The associated reduction in nitric oxide as it reacts with oxygen free radicals could lower placental blood flow, which may contribute to IUGR [48]. In the HTR-8/SVneo cytotrophoblast cell line, ethanol stimulates cytokine production [49], a potential source of apoptosis. Emerging neural crest cells are particularly sensitive to ethanol-induced apoptosis [50, 51]. Apoptosis induced by ethanol in neural crest cells is dependent on intracellular Ca2+ that is mobilized through phosphoinositide signaling [50]. Whether similar mechanisms underlie ethanol-induced trophoblast cell death is not known.
The placenta is protected to some extent through its production of survival factors that limit the amount of cell death induced by toxic substances such as ethanol. The same may be true of other embryonic lineages. Cerebellar granule cells isolated from neonatal rats are protected by basic fibroblast growth factor and nerve growth factor when exposed for 24 h to a high concentration (400 mg/dl; 
100 mM) of ethanol [52]. In the case of insulin-like growth factor I signaling, ethanol inhibits tyrosine autophosphorylation of its receptor to neutralize anti-apoptotic activity [53]. Apoptosis of embryonic cells exposed acutely to ethanol during gastrulation in mice is significantly attenuated by exogenous application of HBEGF [45]. HBEGF is highly expressed throughout gestation by human cytotrophoblasts, both in the villi and in the basal plate [19, 20]. We have found that cytotrophoblast cells express increased levels of HBEGF and TGFA after exposure to 25–50 mM ethanol. Both growth factors had a dose-dependent ameliorating effect on apoptosis induced by 100 mM ethanol, suggesting that the detrimental effects of ethanol could be attenuated through their endogenous production by the trophoblast. Apoptosis was prevented by 1 nM HBEGF and proliferation was restored with 2–5 nM, concentrations that are within a physiologically relevant range according to measurement of HBEGF secretion by this cell line [24]. Other EGF-like growth factors, AREG and EGF, were less effective and were not up-regulated in ethanol-exposed cytotrophoblast cells. Thus, the relative effectiveness of the growth factors examined were HBEGF > TGFA > EGF > AREG. The observed failure to up-regulate HBEGF at 100 mM ethanol could compound the detrimental effects of exposure to high alcohol concentrations.
The specificity of HBEGF was demonstrated using CRM197, an antagonist based on the role of the transmembrane form of HBEGF as the diphtheria toxin receptor [30]. Inhibition of apoptosis by HBEGF was dependent on its ability to bind either of its two known receptors, HER1 or HER4. In cytotrophoblast cells exposed to hypoxia, metalloproteinases cleave the exodomain of HBEGF from the cell surface to initiate autocrine signaling through HER1 and HER4 that activates HBEGF synthesis in a positive-feedback loop, which inhibits apoptosis [24]. It is not known whether ethanol stimulates HBEGF autocrine signaling, but the magnitude of the increase in HBEGF detected by immunohistochemistry appears to be considerably smaller than that observed during hypoxia.
In addition to the direct effects of ethanol on embryogenesis, apoptosis and reduced proliferation of trophoblast within the placenta could impact the gestational outcome in pregnancies exposed prenatally to alcohol. However, the observation that survival factors expressed by cytotrophoblast cells are capable of inhibiting ethanol-induced apoptosis raises the possibility that endogenous HBEGF or TGFA expression could diminish the impact of maternal alcohol consumption. If excessive blood alcohol levels are attained, the protective effects of endogenous HBEGF might be compromised, as suggested by its failed up-regulation when ethanol exposure was increased to 100 mM. Susceptibility to the teratogenic effects of alcohol varies widely in the human population and the ability of embryonic cells to produce EGF-like growth factors or other survival factors might influence this diversity. Importantly, it remains to be established whether altered placental function contributes significantly to the etiology of fetal alcohol syndrome in humans.
ACKNOWLEDGMENTS
The authors would like to thank Michael Kruger for help with statistical analysis and Dr. U.K. Rout for conducting preliminary experiments. Dr. Charles Graham of Queen's University, Kingston, Ontario, generously provided HTR-8/SVneo cells used in this study.
FOOTNOTES
3Current address: Wellesley College, Wellesley, MA 02481. 
1Supported by grants from the National Institute on Alcohol Abuse and Alcoholism (AA12057, D.R.A; AA11085, S.M.S.) and by the Intramural Research Program of the National Institute of Child Health and Human Development, NIH, DHHS. G.S.W. was supported by a summer research internship from the Department of OB/GYN, Wayne State University.
Correspondence: 2D. Randall Armant, C.S. Mott Center for Human Growth and Development, Department of Obstetrics and Gynecology, Wayne State University School of Medicine, 275 E. Hancock Ave., Detroit, MI 48201-1415. FAX: 313 577 8554; e-mail: d.armant{at}wayne.edu
Received: 9 October 2006.
First decision: 19 November 2006.
Accepted: 15 March 2007.
REFERENCES
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