- Email: [email protected]
Maternal hair testing to disclose self-misreporting in drinking and smoking behavior during pregnancy
Accepted Manuscript Maternal hair testing to disclose self-misreporting in drinking and smoking behaviour during pregnancy Maria Dolores Gomez-Roig, Emilia Marchei, Sally Sabra, Francesco Paolo Busardò, Luisa Mastrobattista, Simona Pichini, Eduard Gratacós, Oscar Garcia-Algar PII:
S0741-8329(17)30832-7
DOI:
10.1016/j.alcohol.2017.08.010
Reference:
ALC 6718
To appear in:
Alcohol
Received Date: 20 July 2017 Revised Date:
10 August 2017
Accepted Date: 10 August 2017
Please cite this article as: Gomez-Roig M.D., Marchei E., Sabra S., Busardò F.P., Mastrobattista L., Pichini S., Gratacós E. & Garcia-Algar O., Maternal hair testing to disclose self-misreporting in drinking and smoking behaviour during pregnancy, Alcohol (2017), doi: 10.1016/j.alcohol.2017.08.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Maternal hair testing to disclose self-misreporting in drinking and smoking behaviour during pregnancy Maria Dolores Gomez-Roig1,2,3, Emilia Marchei4, Sally Sabra1, Francesco Paolo Busardò5, Luisa
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Mastrobattista4*, Simona Pichini4, Eduard Gratacós1,6, Oscar Garcia-Algar1,2
1. BCNatal (Barcelona Center for Maternal Fetal and Neonatal Medicine, Hospital Sant Joan de Déu and Hospital Clínic), University of Barcelona, Barcelona, Spain
2. Maternal and Child Health and Development Network II (SAMID II), Instituto de Salud Carlos
Regional Development Fund (FEDER), Madrid, Spain
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III (ISCIII),- Sub-Directorate General for Research Assessment and Promotion and the European
3. Institut de Recerca Sant Joan de Déu (IR-SJD), Barcelona, Spain
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4. National Centre on Addiction and Doping, Istituto Superiore di Sanità, Rome, Italy 5. Unit of Forensic Toxicology (UoFT), Department of Anatomical, Histological, Forensic and Orthopedic Sciences, Sapienza University of Rome, Rome, Italy
6. IDIBAPS, University of Barcelona, and Centre for Biomedical Research on Rare Diseases,
Corresponding author:
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Barcelona, Spain
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Simona Pichini National Centre on Addiction and Doping Istituto Superiore di Sanità, Rome, Italy Tel +390649906545 Fax +390649902016 Email: [email protected]
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Word count: 1970
Date: April 24, 2017
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Abstract
This study aimed to objectively verify smoking and drinking behaviour during pregnancy and to disclose self-misreporting through maternal hair analysis. A total of 153 women attending a university hospital in Barcelona (Spain) were selected and interviewed after delivery, on their smoking and drinking habits during pregnancy. A 9 cm hair strand was collected and analyzed by liquid chromatography tandem mass spectrometry for the presence of nicotine (NIC) and ethyl-
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glucuronide (EtG) as biomarkers of tobacco and alcohol consumption. Concentrations of EtG < 7 pg/mg hair and ≥30 pg/mg hair in the 0–3 cm hair segment have been used to assess respectively total abstinence and chronic excessive consumption in the previous three months, with repetitive moderate drinking lying in the interval 7–30 pg EtG per mg hair. Hair NIC less than 1 ng/mg hair
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indicates non exposure to tobacco smoke while hair NIC indicates daily active smoking. In the interview, 28.1% women declared to have smoked occasionally during gestation, while only 2.6%
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stated to have consumed alcohol in more than one occasion during pregnancy. Hair testing of smoking biomarkers disclosed that 7.2% women remained active smokers during the whole pregnancy (hair NIC: 3.21-56.98 ng/mg hair), 16.3% were passive non-smokers or occasional smokers(hair NIC: 1.04-2.99 ng/mg hair), while 76.5 % were not exposed to any cigarette smoke (hair NIC
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while 62.7% drunk a non-negligible amount of alcohol during pregnancy ( hair EtG: 7.06-26.57 pg/mg hair) and 2% were chronic excessive drinkers (hair EtG: 35.33- 47.52 pg/mg hair). Maternal hair analysis has shown to be significantly more sensitive than the interviews in revealing an alarming misreported prevalence of alcohol use during pregnancy. These findings stress the need to
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use objective measures to assess alcohol exposure and to consider the inclusion of targeted actions
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to reduce alcohol consumption in maternal-child health policies.
Keywords: hair testing; pregnancy, nicotine, ethyl glucuronide, smoking habit, drinking behaviour.
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INTRODUCTION
The most commonly psychoactive substance consumed during pregnancy is tobacco, followed by ethyl alcohol (Forray, 2016). Tobacco smoking and alcohol drinking throughout pregnancy have both been associated with serious public health problems and adverse effects on the growth, cognitive development, and behaviour of the exposed child (Forray, 2016; Cornelius and Day, 2000). In the specific case of alcohol, gestational use can cause irreversible disabilities such as
of exposure to toxic substances should be planned both,
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foetal alcohol spectrum disorders and foetal alcohol syndrome (Memo et al, 2013).3 The reduction before conception and during
pregnancy(Crighton et al, 2016). Motivational interventions and behavioural counselling to stop toxic habits in pregnant women have been shown to have only moderate success (Forray, 2016;
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Joya et al, 2016). Nevertheless, whereas some success has been obtained in gestational smoking cessation, many women continue to consume ethanol in various degrees while pregnant, despite
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increasing public awareness of the harmful effects of drinking during pregnancy (Joya et al, 2016; Reynolds et al, 2017).
A substantial number of studies have assessed (Joya et al, 2016; Manich et al, 2012; Shisler et al, 2017) smoking and drinking habits during pregnancy by maternal self-report data. However, it has been widely demonstrated that self-reports are often misleading for a variety of reasons, including social desirability bias, recall bias, and/or fear that the child may be taken away (Manich et al,
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2012). Thus, information provided from an unbiased biomarker may be essential to assess more accurately the patterns of gestational smoking and drinking behavior, especially in view of designing and monitoring future cessation interventions (Joya et al, 2016). Indeed, the use of objective measure methods is still relatively recent and only a few studies have evaluated the
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prevalence of self-misreporting in pregnant women. Maternal hair has been reported to be one of the most successful biological matrix to disclose the history of illicit drugs, medicines, tobacco and
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alcohol use during pregnancy (Shisler et al, 2017; Joya et al., 2016b; Joya et al, 2012; Llaquet et al, 2010; Pichini et al, 2003. Maternal hair testing of ethylglucuronide (EtG) can distinguish among gestational abstinence, repetitive moderate (social) drinking, and chronic heavy drinking. This can be done by using the cut-offs recently established by the Society of Hair Testing (Kintz, 2014). Concentrations of EtG < 7 pg/mg hair and ≥30 pg/mg hair in the 0–3 cm hair segment have been used to assess respectively total abstinence and chronic excessive consumption in the previous three months, with repetitive moderate drinking lying in the interval 7–30 pg EtG per mg hair. Hair testing of nicotine (NIC) and its metabolite cotinine (COT) can distinguish among non-exposure to tobacco smoke, environmental exposure and active smoke (Pichini et al, 2006). Hair NIC less than 1 ng/mg hair indicates non exposure to tobacco smoke while hair NIC higher than 3.1 ng/mg hair daily active smoking (WHO, 2013). Hair NIC between 1 and 3 ng/mg hair indicates not only 3
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passive exposure but also possible occasional (less than daily) smoking. This is because a high exposure and occasional active smoking show overlapping values in hair NIC, that sometimes render indistinguishable the two different categories (Pichini et al, 2006). Whereas in recent years many developed countries have implemented measures to reduce the exposure to tobacco during pregnancy (WHO 2013; WHO, 2014), the efforts on reducing and quitting gestational alcohol consumption have been comparatively less (WHO, 2016).
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The aim of the present study was to objectively verify smoking and drinking behaviour during pregnancy and to disclose self-misreporting in a cohort of women after delivery by using hair testing of nicotine and ethylglucuronide in the 9 cm shaft corresponding to gestational period.
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METHODS Study design and population
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A total of 153 pregnant women were enrolled for the study at BCNatal, Barcelona Center for Maternal Fetal and Neonatal Medicine, Hospital Sant Joan de Déu and Hospital Clínic-Maternitat, Barcelona, Spain. During a period of four months (February to May 2016), all women with singleton uncomplicated pregnancies, waiting for spontaneous delivery at term, were asked to participate in the study. To be included in the study a maternal hair length of minimum 9 cm at delivery was necessary to cover the whole gestational period together with a written consent to
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undergo an interview about smoking and drinking behaviour. After the interview a hair strand was cut close to the scalp and stored at ambient temperature until analysis. The study was approved by the Institutional Hospital Ethical Committee (PIC-86-14) and conducted in accordance with the
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Declaration of Helsinki.
Hair testing for NIC, COT and EtG
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Hair segments of 9 cm from the scalp representing the whole pregnancy were washed with 2.5 mL dichloromethane and 2.5 mL methyl alcohol and once dried, cut into small pieces and pulverized using a ball mill. Twenty-five milligrams of this powder were weighed in a glass tube; after the addition of 10 ng N-ethylnorcotinine and 10 pg EtG-d5 as internal standards 500 µL M3® buffer reagent (Comedical, Trento, Italy) was added to each tube for 1 h at 100 °C. Then, the treated samples were cooled at room temperature and a sample volume of 10 µL was injected into ultrahigh performance liquid chromatography tandem mass spectrometry. For EtG analysis M3 buffer reagent extract was directly injected while for NIC and COT, 100 µL of the M3 buffer reagent extract were diluted with 900 µL of water before injection. EtG, NIC and COT were detected using a previously published ultra-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) assay for EtG, revalidated for the other two biomarkers (Joya et al., 2016a). 4
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The separated analytes were detected with a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode via positive electrospray ionization. MRM transitions were: m/z 163.1 > 117.0, 163.1 > 132.6 for NIC, m/z 177.1 > 80.0, 177.1 > 98.1 for COT and m/z 190.9 > 79.9, 190.9 > 120.1 for N-ethylnorcotinine and m/z 221.1→75.0, 221.1→85.0, for EtG and m/z 226.0→75.0, 226.0→85.0 for EtG-d5 (Figures 1 and 2). The validated method showed a limit of quantification of 0.1 ng/mg and a limit of detection of
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0.03 ng/mg for NIC and COT and a limit of quantification of 7 pg/mg, and a limit of detection of 3 pg/mg for EtG. Analytical recoveries were 97.2 + 1.3 and 95.4 + 1.9% for hair NIC and COT and 92.1 + 2.0% for hair EtG.
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Statistical analyses
Associations between sociodemographics, life style characteristics of pregnant women with hair
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biomarkers for smoking and drinking behaviours were analysed by Chi-square test for dichotomous variables. Statistical significance was set at p < 0.05. Database management and statistical analysis were performed with SPSS v 14.0 (SPSS, Chicago, IL, USA).
RESULTS Participants’ characteristics
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Of the 153 post-partum women included in the study, 117 (76.5%) were Spanish, with mean (+Standard deviation, SD) age being 34.5 (± 5.2) years, 54 (35.3%) had a university degree and 29 (18.9%) primary education only; 79 (51.6%) had an unskilled job and 69 (45.1%) a skilled job (in 3.3% women the job was not given).
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There was no age-based distribution of the hair Nic and EtG in the study cohort. Forty- three (28.1%) women self-reported tobacco use sometimes during pregnancy, while only 4
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(2.6%) mothers self-declared some gestational ethyl alcohol consumption. No significant socio-demographic differences were observed between non-smoking and active smoking mothers, classified through the hair biomarkers. Conversely, hair EtG was associated with the social class, with a statistically higher number of abstinent women among those with skilled job whereas women with unskilled jobs were more prone to drink during pregnancy (p < 0.05 for Chi Square between skilled and unskilled women and drinking habit).
Hair testing for NIC, COT and EtG According to hair NIC values (
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smokers (hair NIC: 1.04-2.99 ng/mg hair) and 11 (7.2%) were active smokers (hair NIC: 3.21-56.98 ng/mg hair) . Hair COT was detected only in active smokers with a concentration range from lower than LOQ to 0.78 ng/mg hair. In active smokers, no direct correlation was found between hair NIC and hair COT and number of daily smoked cigarettes. According to hair EtG values, 54 (35.3%) women did not drink at any time during pregnancy (hair EtG: 3.89-6.73 pg/mg hair), 96 (62.7%) showed repeated moderate ethyl alcohol consumption
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during pregnancy ( hair EtG: 7.06-26.57 pg/mg hair) and 3 (2.0%) presented gestational excessive ethanol consumption (hair EtG: 35.33, 37.67 and 47.52 pg/mg hair, respectively). It was not possible to calculate a possible correlation between hair ETG values and number of drinks declared by the women, since only 4 declared some gestational ethyl alcohol consumption.
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NIC and EtG hair concentrations showed a correlation only in active smoking women (r2 = 0.75), who were also moderate or excessive drinkers. No correlations were found in the other cases
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(Figure 3).
NIC and EtG concentrations in maternal hair and self-reported gestational tobacco and ethyl alcohol consumption
The comparison between women’s self-reported data on smoking, their drinking habits and the value of hair biomarkers are reported in Table 1. The sensitivity and specificity of the findings
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demonstrate that women were reasonably conscious of their smoking status and they self -declared it with an acceptable reliability. Conversely, women were unaware or simply misreported
DISCUSSION
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gestational alcohol consumption.
The present study aimed to objectively verify smoking and drinking behaviour during pregnancy
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and to disclose self-misreporting in a cohort of Spanish post-partum women by testing their hair for NIC and EtG in the 9 cm shaft corresponding to the gestational period. From the results here obtained it can be highlighted that appropriate information and education on the negative effects of smoking during pregnancy by health professionals and the advertising campaigns concerning this issue have positively influenced women wishing to have children, to quit or significantly reduce smoking during pregnancy. Thus, in our study population the majority of pregnant women were non-smokers, who had successfully achieved to live in a smoke-free environment. Nevertheless, there was still a 26% of women misreporting their smoking status. This is in agreement with our previous studies (Pichini et al, 2003; Franchini et al, 2008), where in assessing foetal exposure to cigarette smoke through hair nicotine12and cord serum cotinine (Franchini et al, 2008) analysis, we observed that the implementation of smoke-free legislation and 6
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information campaigns against smoking, lead to a significant trend in less active smoking during pregnancy, which corresponds to self-reported data. A completely different scenario was found regarding alcohol consumption during pregnancy, suggesting several considerations. First, the high prevalence of women consuming a non-negligible amount of alcohol during their pregnancies shows that a substantial proportion of pregnant women ignore the risks of alcohol to the foetus. This is likely to reflect an absence of accurate information
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about the severe risks associated with gestational drinking, which should be addressed by active measures involving maternal-foetal health professionals and extensive information campaigns, which are still lacking, at least in the setting of our study population. Second, there was a remarkable disproportion between self-reported and objective alcohol consumption during
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pregnancy. This fact may seem to be in contradiction with the previous point, but it might illustrate a common social belief that drinking just a glass of wine or beer occasionally is not to be
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considered as “usual ethyl alcohol consumption”. In addition, it is possible that some women felt guilty or ashamed of admitting any drinking during their pregnancy.
As a limitation of the study, we acknowledge that it was conducted on a relatively small number of patients and in a specific area of Barcelona, Spain. However, we believe that the results are consistent with previous studies (Joya et al, 2016a; Joya et al., 2016b; Pichini et al, 2012). In one study conducted on a cohort of pregnant women from Barcelona, located in an urban area with a
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lower socioeconomic status and a higher percentage (more than 40%) of immigrants there was evidence of an alarming gestational alcohol consumption (Joya et al, 2016b). Similarly an Italian multicentre study which assessed prenatal exposure to ethanol by meconium analysis showed a maternal under reporting of gestational alcohol habit (Pichini et al, 2012). Another criticism can rise
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from the fact that segmental hair analysis investigating the three pregnancy trimesters was not performed, but a total 9cm hair strand was analysed, representing the whole gestational period.
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This was a choice in the initial study design dictated by the budget provided for the project andby the evidence that in previous studies on similar cohorts (Joya et al, 2016a; Joya et al., 2016b) drinking behaviour did not significantly change during gestation even when single-session motivational intervention was performed and finally because an eventual drinking cessation as soon as the woman knew she was pregnant should have resulted in a low value of hair EtG (eg. two trimesters without any drinking should have lowered the total amount of EtG in the 9 cm hair strand to a low value).This misreporting of periconception of smoking and drinking behaviours is not only typical of Mediterranean countries, but it is internationally described (Gollenberg et al, 2011; Eichler et al, 2016) . Despite public health efforts to eliminate or reduce the consumption of tobacco and alcohol during pregnancy, recent international studies indicate that still a certain number of pregnant women 7
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continue to consume tobacco and a high number of
alcoholic beverages during gestation,
worldwide (Pichini et al. 2017; Mårdby et al, 2017; Blanquet et al, 2016). Whereas prenatal exposure to tobacco can cause reversible transient alterations in exposed newborns, this is not the case of antenatal exposure to ethyl alcohol, which causes permanent disabilities (Crighton et al, 2016, Memo et al, 2013). The results of our study illustrate the importance of using objective measures to document tobacco
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and alcohol consumption during gestation and to disclose the real proportion of the phenomenon; this is in agreement with the study of Shisler S et al (Shisler et al, 2017) which considers more intensive prospective self-reporting measures and biological assays as a viable and superior alternative to single item self-report measures.
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Likewise, the findings here reported should serve as an eye opener to raise awareness among all parties concerned by increasing health policies in addressing the alarming proportion of women
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who still have toxic habits during pregnancy.
ACKNOWLEDGEMENTS
The authors thank Stefano Gentili, Michele Sciotti, Simonetta di Carlo and Antonella Bacosi for technical assistance and Massimo Gottardi for technical assistance and Comedical for providing the M3 buffer reagent, and the RETICS funded by the PN I+D+I 2008-2011 (Spain), ISCIII- Sub-
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Directorate General for Research Assessment and Promotion, and the European Regional Development Fund (FEDER), ref. RD12/0026.
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Blanquet, M., Leger, S., Gerbaud, L., Vendittelli, F, 2016. Smoking during pregnancy: a difficult problem to face. Results of a French multi-center study. J Prev Med Hyg. 57, E95-E101.
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Cornelius, M.D., Day, N.L., 2000. The effects of tobacco use during and after pregnancy on exposed children. Alcohol Res Health 24:242-9. Crighton, E., Abelsohn, A., Blake, J., Enders, J., Kilroy, K., Lanphear, B., Marshall, L., Phipps, E., Smith, G., 2016. Beyond Alcohol and Tobacco Smoke: Are We Doing Enough to Reduce Fetal Toxicant Exposure? J Obstet Gynaecol Can.38, 56-9. Eichler, A., Grunitz, J., Grimm, J., Walz, L., Raabe, E., Goecke, T.W., Beckmann, M.W., Kratz, O., Heinrich, H., Moll, G.H., Fasching, P.A., Kornhuber, J., 2016. Did you drink alcohol during pregnancy? Inaccuracy and discontinuity of women's self-reports: On the way to establish meconium ethyl glucuronide (EtG) as a biomarker for alcohol consumption during pregnancy. Alcohol 54, 39-44.
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Forray, A., 2016. Substance use during pregnancy. F1000Res. 2016 May 13;5. pii: F1000 Faculty Rev-887. doi: 10.12688/f1000research.7645.1. eCollection 2016 Franchini, M., Caruso, C., Perico, A., Pacifici, R., Monleon, T., Garcia-Algar, O., Rossi, S., Pichini, S., 2008. Assessment of foetal exposure to cigarette smoke after recent implementations of smokefree policy in Italy. Acta Paediatr. 97, 546-50. Gollenberg, A.L., Mumford. S.L., Cooney, M.A., Sundaram.,R., Louis, G.M., 2011. Validity of
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Joya X., Marchei E., Salat-Batlle J., García-Algar O., Calvaresi V., Pacifici R., Pichini S., 2016a. Fetal exposure to ethanol: relationship between ethyl glucuronide in maternal hair during pregnancy
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and ethyl glucuronide in neonatal meconium. Clin Chem Lab Med. 54, 427-35. Joya X., Mazarico E., Ramis J., Pacifici R., Salat-Batlle J., Mortali C., García-Algar O., Pichini S., 2016b. Segmental hair analysis to assess effectiveness of single-session motivational intervention to stop ethanol use during pregnancy. Drug Alcohol Depend. 158, 45-51. Kintz P., 2015. 2014 consensus for the use of alcohol markers in hair for assessment of both abstinence and chronic excessive alcohol consumption. Forensic Sci Int. 249, A1-2.
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Llaquet H., Pichini S., Joya X., Papaseit E., Vall O., Klein J., Garcia-Algar O., 2010. Biological matrices for the evaluation of exposure to environmental tobacco smoke during prenatal life and childhood. Anal Bioanal Chem. 396,:379-99.
Manich, A., Velasco, M., Joya, X., García-Lara, N. R., Pichini, S., Vall, O., Garcia-Algar, O.,
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Mårdby A.C., Lupattelli A., Hensing G., Nordeng H., 2017. Consumption of alcohol during pregnancy-A multinational European study. Women Birth. S1871-5192(17)30005-7 Memo L., Gnoato E., Caminiti S., Pichini S., Tarani L., 2013. Fetal alcohol spectrum disorders and fetal alcohol syndrome: the state of the art and new diagnostic tools. Early Hum Dev. 89 Suppl 1, S40-3.
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Pichini S., Garcia-Algar O., Muñoz L., Vall O., Pacifici R., Figueroa C., Pascual J.A., Diaz D., Sunyer J., 2003. Assessment of chronic exposure to cigarette smoke and its change during pregnancy by segmental analysis of maternal hair nicotine. J Expo Anal Environ Epidemiol.13,14451. Pichini S., Marchei E., Vagnarelli F., Tarani L., Raimondi F., Maffucci R., Sacher B., Bisceglia M., Rapisardi G., Elicio M.R., Biban P., Zuccaro P., Pacifici R., Pierantozzi A., 2012. Morini
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World Health Organization Regional Office for Europe. 2016. Prevention of harm caused by alcohol exposure in pregnancy.
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World Health Organization Regional Office for Europe. 2013. WHO recommendations for the
Figures legend
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prevention and management of tobacco use and second-hand smoke exposure in pregnancy.
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Figure 1. UHPLC–MS/MS chromatogram of: blank hair sample (A), hair sample spiked with 10 ng/mg nicotine, 1 ng/mg cotinine and 10 ng/mg N-ethylnorcotinine (B) and a real hair sample containing 16.5 ng/mg nicotine, 0.72 ng/mg cotinine and 10 ng/mg N-ethylnorcotinine (C).
Figure 2. UHPLC–MS/MS chromatogram of: blank hair sample (A), hair sample spiked with 10 pg/mg Ethylglucuronide (EtG) and 20 pg/mg EtG-d5 (B) and a real hair sample containing 18.5 pg/mg EtG and 20 pg/mg EtG-d5 (C). Figure 3. Correlation between Nicotine and ethylglucuronide (EtG) hair concentration in non exposed non smoker, exposed non smoker and active smoker women and in abstinent women and moderate alcohol drinker women.
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Table 1. Agreement between maternal self-reported data and hair NIC and EtG values.
142
3.2 – 56.9
106(TN)
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Hair EtG range (pg/mg) Abstinent Women
7(TP)
54
63.6
74.6
36(FP)
4(FN)
Agreement to self-reported data
3.8-6.9
Specificity %
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LOQ – 3
Sensitivity %
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Active smoking Women
No.
Agreement to self-reported data
Sensitivity %
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Non smoking Women
Hair NIC range (ng/mg)
2(FP)
52(TN)
2.0
99
2(TP)
97(FN)
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7.0-47.5
96.3
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Gestational alcohol consumers
Specificity %
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A 100
0
0
Cotinine m/z 177.1 > 80.0
%
0
0
N-ethylnorcotinine m/z 190.9 > 120.12
100
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100
Cotinine m/z 177.1 > 80.0
100
%
0
0
100
Cotinine m/z 177.1 > 80.0
%
0
100 N-ethylnorcotinine m/z 190.9 > 120.12
N-ethylnorcotinine m/z 190.9 > 120.12
%
%
0
0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Nicotine m/z 163.1 > 117.0
%
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%
100
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%
Nicotine m/z 163.1 > 117.0
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%
100
C
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Nicotine m/z 163.1 > 117.0
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MS/MS relative abundance
100
B
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Time (min)
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
A
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B
100
100
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100
C
%
0
0
%
%
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100 EtG-d5 m/z 226.0 > 85.0
EtG-d5 m/z 226.0 > 85.0
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100
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%
0
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
EtG m/z 221.1 > 85.0
EtG m/z 221.1 > 85.0
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MS/MS relative abundance
EtG m/z 221.1 > 85.0
%
0
100
EtG-d5 m/z 226.0 > 85.0
%
0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Time (min)
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
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Smoking habit Non-exposed non smokers R² = 0.0264
R² = 0.7473
R² = 0.0007
50
50
45
45
40
40
40
35
35
30
30
25
25
20
20
15
15
10
10
5
5
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45
35 30 25
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20
0
0
0.0
0.2
0.4
0.6
0.8
1.0
0.0
1.0
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EtG concentration (pg/mg hair)
50
Active smokers
Passive smokers (occasional smokers)
2.0
3.0
15 10 5 0 4.0
0
20
40
60
Nicotine concentration (ng/mg hair)
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Ethyl alcohol consumption
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R² = 0.0099
8 7 6 5 4 3 2 1 0
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EtG concentration (pg/mg hair)
Abstinent women
Repeated moderate ethyl acohol consumers R² = 0.1663
35 30 25 20 15 10 5 0
0.0
1.0
2.0
3.0
4.0 Nicotine concentration (ng/mg hair)
0
10
20
30
40
50
ACCEPTED MANUSCRIPT • drinking and smoking behaviour were assessed during pregnancy through maternal hair analysis • ethylglucuronide and nicotine were measured in maternal hair as biomarkers • Gestational drinking was significantly more misreported than tobacco smoking Maternal hair analysis revealed an alarming misreported prevalence of alcohol use
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during pregnancy
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•
S0741-8329(17)30832-7
DOI:
10.1016/j.alcohol.2017.08.010
Reference:
ALC 6718
To appear in:
Alcohol
Received Date: 20 July 2017 Revised Date:
10 August 2017
Accepted Date: 10 August 2017
Please cite this article as: Gomez-Roig M.D., Marchei E., Sabra S., Busardò F.P., Mastrobattista L., Pichini S., Gratacós E. & Garcia-Algar O., Maternal hair testing to disclose self-misreporting in drinking and smoking behaviour during pregnancy, Alcohol (2017), doi: 10.1016/j.alcohol.2017.08.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Maternal hair testing to disclose self-misreporting in drinking and smoking behaviour during pregnancy Maria Dolores Gomez-Roig1,2,3, Emilia Marchei4, Sally Sabra1, Francesco Paolo Busardò5, Luisa
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Mastrobattista4*, Simona Pichini4, Eduard Gratacós1,6, Oscar Garcia-Algar1,2
1. BCNatal (Barcelona Center for Maternal Fetal and Neonatal Medicine, Hospital Sant Joan de Déu and Hospital Clínic), University of Barcelona, Barcelona, Spain
2. Maternal and Child Health and Development Network II (SAMID II), Instituto de Salud Carlos
Regional Development Fund (FEDER), Madrid, Spain
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III (ISCIII),- Sub-Directorate General for Research Assessment and Promotion and the European
3. Institut de Recerca Sant Joan de Déu (IR-SJD), Barcelona, Spain
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4. National Centre on Addiction and Doping, Istituto Superiore di Sanità, Rome, Italy 5. Unit of Forensic Toxicology (UoFT), Department of Anatomical, Histological, Forensic and Orthopedic Sciences, Sapienza University of Rome, Rome, Italy
6. IDIBAPS, University of Barcelona, and Centre for Biomedical Research on Rare Diseases,
Corresponding author:
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Barcelona, Spain
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Simona Pichini National Centre on Addiction and Doping Istituto Superiore di Sanità, Rome, Italy Tel +390649906545 Fax +390649902016 Email: [email protected]
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Word count: 1970
Date: April 24, 2017
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Abstract
This study aimed to objectively verify smoking and drinking behaviour during pregnancy and to disclose self-misreporting through maternal hair analysis. A total of 153 women attending a university hospital in Barcelona (Spain) were selected and interviewed after delivery, on their smoking and drinking habits during pregnancy. A 9 cm hair strand was collected and analyzed by liquid chromatography tandem mass spectrometry for the presence of nicotine (NIC) and ethyl-
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glucuronide (EtG) as biomarkers of tobacco and alcohol consumption. Concentrations of EtG < 7 pg/mg hair and ≥30 pg/mg hair in the 0–3 cm hair segment have been used to assess respectively total abstinence and chronic excessive consumption in the previous three months, with repetitive moderate drinking lying in the interval 7–30 pg EtG per mg hair. Hair NIC less than 1 ng/mg hair
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indicates non exposure to tobacco smoke while hair NIC indicates daily active smoking. In the interview, 28.1% women declared to have smoked occasionally during gestation, while only 2.6%
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stated to have consumed alcohol in more than one occasion during pregnancy. Hair testing of smoking biomarkers disclosed that 7.2% women remained active smokers during the whole pregnancy (hair NIC: 3.21-56.98 ng/mg hair), 16.3% were passive non-smokers or occasional smokers(hair NIC: 1.04-2.99 ng/mg hair), while 76.5 % were not exposed to any cigarette smoke (hair NIC
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while 62.7% drunk a non-negligible amount of alcohol during pregnancy ( hair EtG: 7.06-26.57 pg/mg hair) and 2% were chronic excessive drinkers (hair EtG: 35.33- 47.52 pg/mg hair). Maternal hair analysis has shown to be significantly more sensitive than the interviews in revealing an alarming misreported prevalence of alcohol use during pregnancy. These findings stress the need to
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use objective measures to assess alcohol exposure and to consider the inclusion of targeted actions
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to reduce alcohol consumption in maternal-child health policies.
Keywords: hair testing; pregnancy, nicotine, ethyl glucuronide, smoking habit, drinking behaviour.
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INTRODUCTION
The most commonly psychoactive substance consumed during pregnancy is tobacco, followed by ethyl alcohol (Forray, 2016). Tobacco smoking and alcohol drinking throughout pregnancy have both been associated with serious public health problems and adverse effects on the growth, cognitive development, and behaviour of the exposed child (Forray, 2016; Cornelius and Day, 2000). In the specific case of alcohol, gestational use can cause irreversible disabilities such as
of exposure to toxic substances should be planned both,
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foetal alcohol spectrum disorders and foetal alcohol syndrome (Memo et al, 2013).3 The reduction before conception and during
pregnancy(Crighton et al, 2016). Motivational interventions and behavioural counselling to stop toxic habits in pregnant women have been shown to have only moderate success (Forray, 2016;
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Joya et al, 2016). Nevertheless, whereas some success has been obtained in gestational smoking cessation, many women continue to consume ethanol in various degrees while pregnant, despite
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increasing public awareness of the harmful effects of drinking during pregnancy (Joya et al, 2016; Reynolds et al, 2017).
A substantial number of studies have assessed (Joya et al, 2016; Manich et al, 2012; Shisler et al, 2017) smoking and drinking habits during pregnancy by maternal self-report data. However, it has been widely demonstrated that self-reports are often misleading for a variety of reasons, including social desirability bias, recall bias, and/or fear that the child may be taken away (Manich et al,
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2012). Thus, information provided from an unbiased biomarker may be essential to assess more accurately the patterns of gestational smoking and drinking behavior, especially in view of designing and monitoring future cessation interventions (Joya et al, 2016). Indeed, the use of objective measure methods is still relatively recent and only a few studies have evaluated the
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prevalence of self-misreporting in pregnant women. Maternal hair has been reported to be one of the most successful biological matrix to disclose the history of illicit drugs, medicines, tobacco and
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alcohol use during pregnancy (Shisler et al, 2017; Joya et al., 2016b; Joya et al, 2012; Llaquet et al, 2010; Pichini et al, 2003. Maternal hair testing of ethylglucuronide (EtG) can distinguish among gestational abstinence, repetitive moderate (social) drinking, and chronic heavy drinking. This can be done by using the cut-offs recently established by the Society of Hair Testing (Kintz, 2014). Concentrations of EtG < 7 pg/mg hair and ≥30 pg/mg hair in the 0–3 cm hair segment have been used to assess respectively total abstinence and chronic excessive consumption in the previous three months, with repetitive moderate drinking lying in the interval 7–30 pg EtG per mg hair. Hair testing of nicotine (NIC) and its metabolite cotinine (COT) can distinguish among non-exposure to tobacco smoke, environmental exposure and active smoke (Pichini et al, 2006). Hair NIC less than 1 ng/mg hair indicates non exposure to tobacco smoke while hair NIC higher than 3.1 ng/mg hair daily active smoking (WHO, 2013). Hair NIC between 1 and 3 ng/mg hair indicates not only 3
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passive exposure but also possible occasional (less than daily) smoking. This is because a high exposure and occasional active smoking show overlapping values in hair NIC, that sometimes render indistinguishable the two different categories (Pichini et al, 2006). Whereas in recent years many developed countries have implemented measures to reduce the exposure to tobacco during pregnancy (WHO 2013; WHO, 2014), the efforts on reducing and quitting gestational alcohol consumption have been comparatively less (WHO, 2016).
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The aim of the present study was to objectively verify smoking and drinking behaviour during pregnancy and to disclose self-misreporting in a cohort of women after delivery by using hair testing of nicotine and ethylglucuronide in the 9 cm shaft corresponding to gestational period.
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METHODS Study design and population
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A total of 153 pregnant women were enrolled for the study at BCNatal, Barcelona Center for Maternal Fetal and Neonatal Medicine, Hospital Sant Joan de Déu and Hospital Clínic-Maternitat, Barcelona, Spain. During a period of four months (February to May 2016), all women with singleton uncomplicated pregnancies, waiting for spontaneous delivery at term, were asked to participate in the study. To be included in the study a maternal hair length of minimum 9 cm at delivery was necessary to cover the whole gestational period together with a written consent to
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undergo an interview about smoking and drinking behaviour. After the interview a hair strand was cut close to the scalp and stored at ambient temperature until analysis. The study was approved by the Institutional Hospital Ethical Committee (PIC-86-14) and conducted in accordance with the
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Declaration of Helsinki.
Hair testing for NIC, COT and EtG
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Hair segments of 9 cm from the scalp representing the whole pregnancy were washed with 2.5 mL dichloromethane and 2.5 mL methyl alcohol and once dried, cut into small pieces and pulverized using a ball mill. Twenty-five milligrams of this powder were weighed in a glass tube; after the addition of 10 ng N-ethylnorcotinine and 10 pg EtG-d5 as internal standards 500 µL M3® buffer reagent (Comedical, Trento, Italy) was added to each tube for 1 h at 100 °C. Then, the treated samples were cooled at room temperature and a sample volume of 10 µL was injected into ultrahigh performance liquid chromatography tandem mass spectrometry. For EtG analysis M3 buffer reagent extract was directly injected while for NIC and COT, 100 µL of the M3 buffer reagent extract were diluted with 900 µL of water before injection. EtG, NIC and COT were detected using a previously published ultra-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) assay for EtG, revalidated for the other two biomarkers (Joya et al., 2016a). 4
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The separated analytes were detected with a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode via positive electrospray ionization. MRM transitions were: m/z 163.1 > 117.0, 163.1 > 132.6 for NIC, m/z 177.1 > 80.0, 177.1 > 98.1 for COT and m/z 190.9 > 79.9, 190.9 > 120.1 for N-ethylnorcotinine and m/z 221.1→75.0, 221.1→85.0, for EtG and m/z 226.0→75.0, 226.0→85.0 for EtG-d5 (Figures 1 and 2). The validated method showed a limit of quantification of 0.1 ng/mg and a limit of detection of
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0.03 ng/mg for NIC and COT and a limit of quantification of 7 pg/mg, and a limit of detection of 3 pg/mg for EtG. Analytical recoveries were 97.2 + 1.3 and 95.4 + 1.9% for hair NIC and COT and 92.1 + 2.0% for hair EtG.
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Statistical analyses
Associations between sociodemographics, life style characteristics of pregnant women with hair
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biomarkers for smoking and drinking behaviours were analysed by Chi-square test for dichotomous variables. Statistical significance was set at p < 0.05. Database management and statistical analysis were performed with SPSS v 14.0 (SPSS, Chicago, IL, USA).
RESULTS Participants’ characteristics
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Of the 153 post-partum women included in the study, 117 (76.5%) were Spanish, with mean (+Standard deviation, SD) age being 34.5 (± 5.2) years, 54 (35.3%) had a university degree and 29 (18.9%) primary education only; 79 (51.6%) had an unskilled job and 69 (45.1%) a skilled job (in 3.3% women the job was not given).
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There was no age-based distribution of the hair Nic and EtG in the study cohort. Forty- three (28.1%) women self-reported tobacco use sometimes during pregnancy, while only 4
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(2.6%) mothers self-declared some gestational ethyl alcohol consumption. No significant socio-demographic differences were observed between non-smoking and active smoking mothers, classified through the hair biomarkers. Conversely, hair EtG was associated with the social class, with a statistically higher number of abstinent women among those with skilled job whereas women with unskilled jobs were more prone to drink during pregnancy (p < 0.05 for Chi Square between skilled and unskilled women and drinking habit).
Hair testing for NIC, COT and EtG According to hair NIC values (
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smokers (hair NIC: 1.04-2.99 ng/mg hair) and 11 (7.2%) were active smokers (hair NIC: 3.21-56.98 ng/mg hair) . Hair COT was detected only in active smokers with a concentration range from lower than LOQ to 0.78 ng/mg hair. In active smokers, no direct correlation was found between hair NIC and hair COT and number of daily smoked cigarettes. According to hair EtG values, 54 (35.3%) women did not drink at any time during pregnancy (hair EtG: 3.89-6.73 pg/mg hair), 96 (62.7%) showed repeated moderate ethyl alcohol consumption
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during pregnancy ( hair EtG: 7.06-26.57 pg/mg hair) and 3 (2.0%) presented gestational excessive ethanol consumption (hair EtG: 35.33, 37.67 and 47.52 pg/mg hair, respectively). It was not possible to calculate a possible correlation between hair ETG values and number of drinks declared by the women, since only 4 declared some gestational ethyl alcohol consumption.
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NIC and EtG hair concentrations showed a correlation only in active smoking women (r2 = 0.75), who were also moderate or excessive drinkers. No correlations were found in the other cases
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(Figure 3).
NIC and EtG concentrations in maternal hair and self-reported gestational tobacco and ethyl alcohol consumption
The comparison between women’s self-reported data on smoking, their drinking habits and the value of hair biomarkers are reported in Table 1. The sensitivity and specificity of the findings
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demonstrate that women were reasonably conscious of their smoking status and they self -declared it with an acceptable reliability. Conversely, women were unaware or simply misreported
DISCUSSION
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gestational alcohol consumption.
The present study aimed to objectively verify smoking and drinking behaviour during pregnancy
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and to disclose self-misreporting in a cohort of Spanish post-partum women by testing their hair for NIC and EtG in the 9 cm shaft corresponding to the gestational period. From the results here obtained it can be highlighted that appropriate information and education on the negative effects of smoking during pregnancy by health professionals and the advertising campaigns concerning this issue have positively influenced women wishing to have children, to quit or significantly reduce smoking during pregnancy. Thus, in our study population the majority of pregnant women were non-smokers, who had successfully achieved to live in a smoke-free environment. Nevertheless, there was still a 26% of women misreporting their smoking status. This is in agreement with our previous studies (Pichini et al, 2003; Franchini et al, 2008), where in assessing foetal exposure to cigarette smoke through hair nicotine12and cord serum cotinine (Franchini et al, 2008) analysis, we observed that the implementation of smoke-free legislation and 6
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information campaigns against smoking, lead to a significant trend in less active smoking during pregnancy, which corresponds to self-reported data. A completely different scenario was found regarding alcohol consumption during pregnancy, suggesting several considerations. First, the high prevalence of women consuming a non-negligible amount of alcohol during their pregnancies shows that a substantial proportion of pregnant women ignore the risks of alcohol to the foetus. This is likely to reflect an absence of accurate information
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about the severe risks associated with gestational drinking, which should be addressed by active measures involving maternal-foetal health professionals and extensive information campaigns, which are still lacking, at least in the setting of our study population. Second, there was a remarkable disproportion between self-reported and objective alcohol consumption during
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pregnancy. This fact may seem to be in contradiction with the previous point, but it might illustrate a common social belief that drinking just a glass of wine or beer occasionally is not to be
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considered as “usual ethyl alcohol consumption”. In addition, it is possible that some women felt guilty or ashamed of admitting any drinking during their pregnancy.
As a limitation of the study, we acknowledge that it was conducted on a relatively small number of patients and in a specific area of Barcelona, Spain. However, we believe that the results are consistent with previous studies (Joya et al, 2016a; Joya et al., 2016b; Pichini et al, 2012). In one study conducted on a cohort of pregnant women from Barcelona, located in an urban area with a
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lower socioeconomic status and a higher percentage (more than 40%) of immigrants there was evidence of an alarming gestational alcohol consumption (Joya et al, 2016b). Similarly an Italian multicentre study which assessed prenatal exposure to ethanol by meconium analysis showed a maternal under reporting of gestational alcohol habit (Pichini et al, 2012). Another criticism can rise
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from the fact that segmental hair analysis investigating the three pregnancy trimesters was not performed, but a total 9cm hair strand was analysed, representing the whole gestational period.
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This was a choice in the initial study design dictated by the budget provided for the project andby the evidence that in previous studies on similar cohorts (Joya et al, 2016a; Joya et al., 2016b) drinking behaviour did not significantly change during gestation even when single-session motivational intervention was performed and finally because an eventual drinking cessation as soon as the woman knew she was pregnant should have resulted in a low value of hair EtG (eg. two trimesters without any drinking should have lowered the total amount of EtG in the 9 cm hair strand to a low value).This misreporting of periconception of smoking and drinking behaviours is not only typical of Mediterranean countries, but it is internationally described (Gollenberg et al, 2011; Eichler et al, 2016) . Despite public health efforts to eliminate or reduce the consumption of tobacco and alcohol during pregnancy, recent international studies indicate that still a certain number of pregnant women 7
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continue to consume tobacco and a high number of
alcoholic beverages during gestation,
worldwide (Pichini et al. 2017; Mårdby et al, 2017; Blanquet et al, 2016). Whereas prenatal exposure to tobacco can cause reversible transient alterations in exposed newborns, this is not the case of antenatal exposure to ethyl alcohol, which causes permanent disabilities (Crighton et al, 2016, Memo et al, 2013). The results of our study illustrate the importance of using objective measures to document tobacco
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and alcohol consumption during gestation and to disclose the real proportion of the phenomenon; this is in agreement with the study of Shisler S et al (Shisler et al, 2017) which considers more intensive prospective self-reporting measures and biological assays as a viable and superior alternative to single item self-report measures.
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Likewise, the findings here reported should serve as an eye opener to raise awareness among all parties concerned by increasing health policies in addressing the alarming proportion of women
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who still have toxic habits during pregnancy.
ACKNOWLEDGEMENTS
The authors thank Stefano Gentili, Michele Sciotti, Simonetta di Carlo and Antonella Bacosi for technical assistance and Massimo Gottardi for technical assistance and Comedical for providing the M3 buffer reagent, and the RETICS funded by the PN I+D+I 2008-2011 (Spain), ISCIII- Sub-
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Directorate General for Research Assessment and Promotion, and the European Regional Development Fund (FEDER), ref. RD12/0026.
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Blanquet, M., Leger, S., Gerbaud, L., Vendittelli, F, 2016. Smoking during pregnancy: a difficult problem to face. Results of a French multi-center study. J Prev Med Hyg. 57, E95-E101.
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Cornelius, M.D., Day, N.L., 2000. The effects of tobacco use during and after pregnancy on exposed children. Alcohol Res Health 24:242-9. Crighton, E., Abelsohn, A., Blake, J., Enders, J., Kilroy, K., Lanphear, B., Marshall, L., Phipps, E., Smith, G., 2016. Beyond Alcohol and Tobacco Smoke: Are We Doing Enough to Reduce Fetal Toxicant Exposure? J Obstet Gynaecol Can.38, 56-9. Eichler, A., Grunitz, J., Grimm, J., Walz, L., Raabe, E., Goecke, T.W., Beckmann, M.W., Kratz, O., Heinrich, H., Moll, G.H., Fasching, P.A., Kornhuber, J., 2016. Did you drink alcohol during pregnancy? Inaccuracy and discontinuity of women's self-reports: On the way to establish meconium ethyl glucuronide (EtG) as a biomarker for alcohol consumption during pregnancy. Alcohol 54, 39-44.
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Forray, A., 2016. Substance use during pregnancy. F1000Res. 2016 May 13;5. pii: F1000 Faculty Rev-887. doi: 10.12688/f1000research.7645.1. eCollection 2016 Franchini, M., Caruso, C., Perico, A., Pacifici, R., Monleon, T., Garcia-Algar, O., Rossi, S., Pichini, S., 2008. Assessment of foetal exposure to cigarette smoke after recent implementations of smokefree policy in Italy. Acta Paediatr. 97, 546-50. Gollenberg, A.L., Mumford. S.L., Cooney, M.A., Sundaram.,R., Louis, G.M., 2011. Validity of
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Joya X., Marchei E., Salat-Batlle J., García-Algar O., Calvaresi V., Pacifici R., Pichini S., 2016a. Fetal exposure to ethanol: relationship between ethyl glucuronide in maternal hair during pregnancy
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and ethyl glucuronide in neonatal meconium. Clin Chem Lab Med. 54, 427-35. Joya X., Mazarico E., Ramis J., Pacifici R., Salat-Batlle J., Mortali C., García-Algar O., Pichini S., 2016b. Segmental hair analysis to assess effectiveness of single-session motivational intervention to stop ethanol use during pregnancy. Drug Alcohol Depend. 158, 45-51. Kintz P., 2015. 2014 consensus for the use of alcohol markers in hair for assessment of both abstinence and chronic excessive alcohol consumption. Forensic Sci Int. 249, A1-2.
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Llaquet H., Pichini S., Joya X., Papaseit E., Vall O., Klein J., Garcia-Algar O., 2010. Biological matrices for the evaluation of exposure to environmental tobacco smoke during prenatal life and childhood. Anal Bioanal Chem. 396,:379-99.
Manich, A., Velasco, M., Joya, X., García-Lara, N. R., Pichini, S., Vall, O., Garcia-Algar, O.,
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Mårdby A.C., Lupattelli A., Hensing G., Nordeng H., 2017. Consumption of alcohol during pregnancy-A multinational European study. Women Birth. S1871-5192(17)30005-7 Memo L., Gnoato E., Caminiti S., Pichini S., Tarani L., 2013. Fetal alcohol spectrum disorders and fetal alcohol syndrome: the state of the art and new diagnostic tools. Early Hum Dev. 89 Suppl 1, S40-3.
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Pichini S., Garcia-Algar O., Muñoz L., Vall O., Pacifici R., Figueroa C., Pascual J.A., Diaz D., Sunyer J., 2003. Assessment of chronic exposure to cigarette smoke and its change during pregnancy by segmental analysis of maternal hair nicotine. J Expo Anal Environ Epidemiol.13,14451. Pichini S., Marchei E., Vagnarelli F., Tarani L., Raimondi F., Maffucci R., Sacher B., Bisceglia M., Rapisardi G., Elicio M.R., Biban P., Zuccaro P., Pacifici R., Pierantozzi A., 2012. Morini
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10.1007/s11845-017-1575-2
Shisler S., Eiden R.D., Molnar D.S., Schuetze P., Huestis M., Homish G., 2017. Smoking in
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World Health Organization Regional Office for Europe. 2016. Prevention of harm caused by alcohol exposure in pregnancy.
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World Health Organization Regional Office for Europe. 2013. WHO recommendations for the
Figures legend
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prevention and management of tobacco use and second-hand smoke exposure in pregnancy.
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Figure 1. UHPLC–MS/MS chromatogram of: blank hair sample (A), hair sample spiked with 10 ng/mg nicotine, 1 ng/mg cotinine and 10 ng/mg N-ethylnorcotinine (B) and a real hair sample containing 16.5 ng/mg nicotine, 0.72 ng/mg cotinine and 10 ng/mg N-ethylnorcotinine (C).
Figure 2. UHPLC–MS/MS chromatogram of: blank hair sample (A), hair sample spiked with 10 pg/mg Ethylglucuronide (EtG) and 20 pg/mg EtG-d5 (B) and a real hair sample containing 18.5 pg/mg EtG and 20 pg/mg EtG-d5 (C). Figure 3. Correlation between Nicotine and ethylglucuronide (EtG) hair concentration in non exposed non smoker, exposed non smoker and active smoker women and in abstinent women and moderate alcohol drinker women.
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Table 1. Agreement between maternal self-reported data and hair NIC and EtG values.
142
3.2 – 56.9
106(TN)
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Hair EtG range (pg/mg) Abstinent Women
7(TP)
54
63.6
74.6
36(FP)
4(FN)
Agreement to self-reported data
3.8-6.9
Specificity %
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LOQ – 3
Sensitivity %
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Active smoking Women
No.
Agreement to self-reported data
Sensitivity %
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Non smoking Women
Hair NIC range (ng/mg)
2(FP)
52(TN)
2.0
99
2(TP)
97(FN)
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7.0-47.5
96.3
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Gestational alcohol consumers
Specificity %
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A 100
0
0
Cotinine m/z 177.1 > 80.0
%
0
0
N-ethylnorcotinine m/z 190.9 > 120.12
100
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100
Cotinine m/z 177.1 > 80.0
100
%
0
0
100
Cotinine m/z 177.1 > 80.0
%
0
100 N-ethylnorcotinine m/z 190.9 > 120.12
N-ethylnorcotinine m/z 190.9 > 120.12
%
%
0
0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Nicotine m/z 163.1 > 117.0
%
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%
100
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%
Nicotine m/z 163.1 > 117.0
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%
100
C
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Nicotine m/z 163.1 > 117.0
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MS/MS relative abundance
100
B
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Time (min)
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
A
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B
100
100
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100
C
%
0
0
%
%
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100 EtG-d5 m/z 226.0 > 85.0
EtG-d5 m/z 226.0 > 85.0
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100
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%
0
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
EtG m/z 221.1 > 85.0
EtG m/z 221.1 > 85.0
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MS/MS relative abundance
EtG m/z 221.1 > 85.0
%
0
100
EtG-d5 m/z 226.0 > 85.0
%
0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Time (min)
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
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Smoking habit Non-exposed non smokers R² = 0.0264
R² = 0.7473
R² = 0.0007
50
50
45
45
40
40
40
35
35
30
30
25
25
20
20
15
15
10
10
5
5
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45
35 30 25
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20
0
0
0.0
0.2
0.4
0.6
0.8
1.0
0.0
1.0
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EtG concentration (pg/mg hair)
50
Active smokers
Passive smokers (occasional smokers)
2.0
3.0
15 10 5 0 4.0
0
20
40
60
Nicotine concentration (ng/mg hair)
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Ethyl alcohol consumption
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R² = 0.0099
8 7 6 5 4 3 2 1 0
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EtG concentration (pg/mg hair)
Abstinent women
Repeated moderate ethyl acohol consumers R² = 0.1663
35 30 25 20 15 10 5 0
0.0
1.0
2.0
3.0
4.0 Nicotine concentration (ng/mg hair)
0
10
20
30
40
50
ACCEPTED MANUSCRIPT • drinking and smoking behaviour were assessed during pregnancy through maternal hair analysis • ethylglucuronide and nicotine were measured in maternal hair as biomarkers • Gestational drinking was significantly more misreported than tobacco smoking Maternal hair analysis revealed an alarming misreported prevalence of alcohol use
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during pregnancy
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•