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Maternal Tobacco Exposure and Cotinine Levels in Fetal Fluids in the First Half of Pregnancy

JAUNIAUX, ERIC MD, PhD; GULBIS, BEATRICE MD, PhD; ACHARYA, GANESH MD; THIRY, PHILIPPE PhD; RODECK, CHARLES DsC

ORIGINAL RESEARCH
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Objective To evaluate the distribution of cotinine in fetal fluids and serum during the first half of pregnancy, and compare the fetal and maternal cotinine levels in passive and active smokers.

Methods Maternal smoking status was determined by questionnaire in 85 pregnant women requesting abortion for psychosocial reasons between 7 and 17 weeks' gestation. Coelomic and amniotic fluid samples were collected between 7 and 11 weeks and fetal blood and amniotic fluid between 11 and 17 weeks. Cotinine levels were measured by radioimmunoassay.

Results Women classified themselves as nonsmokers in 40 cases, passive smokers in 19 cases, and voluntary smokers in 26 cases. Five nonsmokers, 16 passive smokers, and all smokers had cotinine levels above the detection limit of the assay. Cotinine was invariably found in coelomic, amniotic, and fetal serum when maternal serum and urine cotinine levels exceeded 25 and 250 ng/mL, respectively. Higher cotinine levels were found in fetal fluids and serum than in maternal serum. Positive linear correlations were found between maternal urine and amniotic fluid cotinine concentrations (r = .75), between maternal urine cotinine concentration and number of cigarettes smoked per day (r = .66), and between maternal and fetal serum cotinine concentrations (r = .97).

Conclusion Cotinine accumulates in the fetal compartments as early as 7 weeks' gestation in both active and passive smokers. Women should be advised to give up smoking from conception and avoid environmental tobacco smoke exposure.

Cotinine accumulates in fetal fluids early in gestation in active and passive smokers, and fetal and maternal concentrations are related.

Academic Department of Obstetrics and Gynaecology, University College London, United Kingdom, and the Department of Clinical Chemistry, Academic Hospital Erasme, Université Libre de Bruxelles, Belgium.

Address reprint requests to: Eric Jauniaux, MD, Academic Department of Obstetrics and Gynaecology, University College London Medical School, 86–96 Chenies Mews, London WC1E 6HX, United Kingdom; E-mail: e.jauniaux@ucl.ac.uk

This work was supported by a grant from the David and Alice Van Buuren Foundation (Université Libre de Bruxelles, Brussels, Belgium) and a grant from the Fonds National de la Recherche Scientifique (No. 9.4523.97).

Received April 22, 1998. Received in revised form June 22, 1998. Accepted July 9, 1998.

Maternal cigarette smoking around conception and during pregnancy is associated with two well-established effects: increased miscarriage rate1 and fetal growth restriction (FGR).2–4 These effects are linked to alterations in placental structure and function induced by tobacco smoke compounds.5–7 The effect of smoking on fetal growth is related to the level of cigarette consumption,4,8 and women who give up smoking in early pregnancy have infants with birth weights comparable to those of nonsmokers.4

Many epidemiologic studies8–10 have suggested recently that a 10–100 g decrease in birth weight occurs in infants of women exposed to environmental tobacco smoke. This finding remains controversial11,12; however, the fact that some tobacco carcinogens are produced in greater amounts in sidestream than mainstream smoke,12 and are associated with an increased risk of childhood cancer,13 is also of serious concern to pregnant women exposed to tobacco smoke.

Nicotine, the main alkaloid found in tobacco, is a lipid-soluble molecule with a half-life of 1–2 hours, which easily crosses biological membranes and is primarily metabolized by the liver and eliminated by the kidney.14,15 Fetal exposure to nicotine can be indirectly evaluated by measuring the levels of the main metabolite, cotinine, in maternal urine, serum, or saliva. Because of its longer half-life, cotinine provides a better index of tobacco exposure than nicotine.11,14,15

Two studies14,16 evaluated cotinine levels in third-trimester placental tissue, fetal cord blood, or amniotic fluid of active smokers.14,16 The aim of the present study was to compare the distribution of cotinine in fetal fluids and serum during the first half of pregnancy in passive and active smokers, and to evaluate the relationship between maternal and fetal cotinine levels and maternal smoking habits. We also investigated the relationship between maternal smoking and fetal exposure to cannabinoids in early pregnancy.

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Patients and Methods

Samples of maternal and fetal blood, maternal urine, coelomic fluid and amniotic fluid were collected over a 6-month period from 85 women with apparently normal first- and second-trimester pregnancies requesting termination under general anaesthesia for psychosocial reasons. The study population was composed of 70% white, 20% black, and 10% Asian women, all from an urban area around the hospital. Women with vaginal bleeding were excluded from the study. In each case, a detailed transvaginal ultrasound confirmed gestational age and assessed fetal heart activity and anatomy.

Written consent was obtained from each woman after receiving full information on the procedure. Women who consented were asked to complete a demographic questionnaire including their smoking habits (number of cigarettes per day) or exposure to tobacco smoke within their social environments, mainly at home and work. This study was approved by the University College London Hospitals Committee on the Ethics of Human Research.

At 7–11 weeks' gestation, coelomic (n = 23) and amniotic (n = 25) fluid samples were retrieved by transvaginal puncture inserting a 20-gauge needle, under ultrasonographic guidance, into the corresponding cavities. Coelomic fluid was aspirated first, and within 10 seconds another needle was inserted into the amniotic cavity and fluid aspirated. Matched series of samples were taken from 21 cases. Between 11 and 17 weeks, amniotic fluid (n = 58) was taken by transabdominal puncture and fetal blood (n = 46) was taken by transabdominal cardiac puncture, with a 22-gauge needle.

Cotinine was analyzed by a double antibody liquidphase radioimmunoassay using reagents from Diagnostic Products Corporation (Los Angeles, CA), in which iodine-125–labeled cotinine competes with cotinine and other nicotine metabolites present in the sample tested for antibody sites. All samples were assayed in duplicate. Intra- and interassay coefficients of variations were less than 10% and the lower limit of sensitivity of the assay was 25 ng/mL. A semiquantitative reagent system was used to detect cannabinoids in the same series of samples. These measurements were from a fluorescence polarization immunoassay technique using an automated instrument (Axsym system, Abbott Laboratories, Chicago, IL). The National Institute on Drug Abuse recommended cut-off value for screening assays was used.

Data are presented as means and 95% confidence intervals (CI). Individual correlations between concentrations of cotinine in different compartments and number of cigarettes per day were calculated by the least-squares method, and their slopes tested for significance by the F ratio test. A biomedical data processing statistical package (Statgraphics, Manugistics, Rockville, MD) was used for this analysis, and results were considered statistically significant at P < .05.

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Results

Analysis of the questionnaires showed that women evaluated their exposure to tobacco as follows: nonsmokers with no or limited social exposure (n = 40), passive smokers with chronic social exposure (n = 19), and active smokers (n = 26). The voluntary smokers admitted daily consumption of 5–25 cigarettes (mean standard error of the mean [SEM] 13 (0.2), 95% CI 10, 15). Maternal urine and serum analysis revealed cotinine levels above the limit of the assay in five of the nonsmokers, in 16 of the passive smokers, and in all 26 active smokers. Nonsmokers and passive smokers with cotinine in urine or serum were combined into one subgroup called involuntary smokers (n = 21).

Cotinine was not detected in fetal fluid or serum if not found in maternal serum or urine. Cotinine levels above 25 ng/mL in maternal serum and above 250 ng/mL in maternal urine were invariably associated with detectable cotinine levels in fetal compartments. In matched maternal and fetal samples from six first-trimester pregnancies, cotinine was found in both amniotic and coelomic fluid (Table 1), whereas in second-trimester pregnancies it was found in both amniotic fluid and fetal serum (Table 2). In samples from first-trimester pregnancies and involuntary smokers, mean cotinine levels were higher in fetal than maternal serum and lower in fetal fluids than maternal urine. In active smokers, maternal cotinine levels were higher than fetal fluid or serum. In this subgroup, positive linear correlations were found between maternal urine and amniotic fluid cotinine concentration (r = .75; SEM = 48.6; F = 26.3; n = 22; P < .001), between maternal urine cotinine concentration and number of cigarettes smoked per day (r = .66; SEM = 1511; F = 14.8; n = 26; P < .005), and between maternal and fetal serum cotinine concentrations (r = .97; SEM = 39; F = 59; n = 25; P < .001). No other correlations were observed.

Table 1

Table 1

Table 2

Table 2

Eight of 65 women tested positive for cannabinoids, with urine or serum concentrations up to five times over the corresponding National Institute on Drug Abuse cut-off value of 25 μg/mL, and all eight were active smokers. In four of these cases, similar levels of cannabinoids were found in amniotic fluid, but not detected in fetal serum. None of these women reported use of cannabinoids in the questionnaire or during the preoperative medical assessment.

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Discussion

The present study provides direct proof that cotinine accumulates in the early fetal circulation and fluids in measurable concentrations. It has been estimated that at term only a small fraction of the circulating nicotine crosses the trophoblastic membrane and reaches the unborn child.15 Cotinine levels in neonates born to women smoking fewer than 10 cigarettes per day were below the detection limit of the corresponding assay,16 suggesting that the placenta might be a relatively effective barrier, preventing nicotine metabolites from reaching the fetus. Inulin concentrations are similar in coelomic fluid and maternal serum within 20 minutes after injecting the mother, indicating the permeability of the placenta is greater in early pregnancy than at term.17 Higher mean and individual cotinine concentrations in fetal fluid or serum than in maternal serum support this hypothesis.

Consistent with previous studies14,16 investigating samples collected at term, this study suggests a positive linear correlation between maternal and fetal serum cotinine levels. It also provides information for a strong linear relationship between cotinine concentration in maternal urine and amniotic fluid. From the 4th to 11th week of gestation, the fetus is surrounded by two distinct fluid cavities: the amniotic cavity and the exocoelomic cavity (Figure 1). The latter forms inside the extraembryonic mesoderm, adjacent to the placental chorionic plate, and is believed to be an important transfer interface and reservoir of nutrients for the embryo.18 The accumulation of placental protein inside the exocoelomic cavity suggests that turnover of coelomic fluid is slow. The secondary yolk sac floats inside the exocoelomic cavity and has a well-demonstrated effect on hematopoiesis and protein biosynthesis. We recently showed that the yolk sac membrane is also an important zone of transfer between the extraembryonic and embryonic compartments, and that the main flux of molecules occurs from outside the yolk sac, ie from the exocoelomic cavity into the lumen, and subsequently into the embryonic gut and circulation.19 The fetal skin, which only becomes keratinized around midgestation, is highly permeable to fluids and small dissolved molecules, such as most toxic tobacco compounds. The fetal oral and anal membranes open around 8 weeks' gestation and the metanephros, or permanent kidney, begins to produce urine around 10 weeks (Figure 2). From this stage in pregnancy, the three different sources of fetal exposure, ie maternal circulation, skin absorption, and gastrointestinal reabsorption of urine, render the fetus susceptible to chronic toxic exposure.

Figure 1

Figure 1

Figure 2

Figure 2

Tobacco carcinogens are transformed into metabolites that might react with DNA and induce subsequent cellular damage in human tissues and mouse skin.20 This damage at the genetic level might contribute to the impairment of fetoplacental development21 and explain the possible teratogenic effect of cigarette smoking.22,23 The average placental tissue/maternal serum nicotine ratio is around 2.6, suggesting that the placenta concentrates nicotine.14 Histomorphometric analysis of term placental tissue from smokers has demonstrated a reduced capillary volume fraction and an increased thickness of the villous membrane.5 Some of the villous changes related to maternal cigarette smoking occur very early during pregnancy.6 In particular, an increased incidence of syncytiotrophoblastic necrosis is found in first-trimester villous tissue samples from heavy smokers. The maternal and fetal responses to nicotine include increased maternal blood pressure and heart rate, decreased uteroplacental blood circulation, elevated middle cerebral artery resistance to blood flow, and lower fetal heart rate reactivity.14,24 However, blood gases and pH are unchanged in fetuses of active smokers. Women who chew tobacco have a higher incidence of stillbirths and small-for-gestational-age fetuses.14 These findings suggest that placental anatomical changes associated with maternal tobacco smoke inhalation are due to direct cellular damage rather than chronic uteroplacental hypoxia. High cotinine levels in coelomic fluid indicate that tobacco genotoxic compounds might react with fetal DNA as early as the first week after implantation.

Evaluating tobacco smoke exposure based on women's self-reports is notoriously imprecise,8–11 possibly because of guilt. Smoking mothers are reluctant to honestly report their tobacco use. The present data also indicate that these mothers are unlikely to report the use of other drugs, in particular cannabis. We found a positive linear correlation between the number of cigarettes smoked per day by mothers and the concentration of cotinine in maternal urine. Maternal or fetal serum cotinine concentration was independent of the number of cigarettes smoked per day and might be influenced by other factors, such as the time between last maternal intake and the sampling and nicotine content of the cigarette brand. These findings suggest that urine and amniotic cotinine levels can be used to study the epidemiology of maternal-fetal tobacco exposure during pregnancy.

Evaluation of the effects of environmental tobacco smoke exposure during pregnancy on fetal growth has generated conflicting reports.8–12 Differences in study design, sample size and population studied might have had major influence on these results. The fact that, in involuntary smokers, fetal serum and amniotic fluid cotinine levels reached 30 and 44% of the corresponding levels in active smokers, respectively, supports the evidence that passive smoking could have a substantial deleterious effect on the fetus. Five of 40 women who originally reported as nonsmokers were found to have urine or serum cotinine levels well above the detection limit of the assay. Although these women did not live with a smoking partner, their exposure to tobacco smoke, presumably at work, was sufficient to generate cotinine levels in both their circulations and those of their fetuses, equivalent to those found in passive smokers. Thus, our results further support antismoking advice, suggesting that women should not only stop cigarette consumption before conception, but also avoid environmental tobacco smoke exposure during pregnancy.

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