Italian Guidelines for the diagnosis and treatment of Fetal Alcohol Spectrum Disorders: detecting alcohol drinking during pregnancy

GIAMPIERO FERRAGUTI§1, FRANCESCA FANFARILLO§1, SIMONA NICOTERA2, SERGIO TERRACINA1, CLEMENTINA MOSCHELLA2, ALESSANDRO MATTIA2, MARIA CHIARA DAVID2, SIMONA PICHINI3, GIOVANNA CORIALE4, DANIELA FIORENTINO5, MAURO CECCANTI6, Maria grazia piccioni7, LUIGI TARANI7, MARCO FIORE8; INTERDISCIPLINARY STUDY GROUPS* SAPIENZA, ISS, ISTAT, AIDEFAD, SITAC, SIFASD, FIMMG-Lazio, SIPPS, SIMPeSV, CIPe

1Department of Experimental Medicine, Sapienza University of Rome, Italy; 2Dipartimento della Pubblica Sicurezza, Direzione Centrale di Sanità, Centro di Ricerche e Laboratorio di Tossicologia Forense, Ministero dell’Interno, Roma; 3National Centre on Addiction and Doping, Istituto Superiore di Sanità, Rome, Italy; 4CRARL Lazio, ASL Roma 1, Italy; 5ASL Rieti, Italy; 6SITAC, Società Italiana per il Trattamento dell’Alcolismo e le sue Complicanze, Rome, Italy; 7Department of Maternal Infantile and Urological Sciences, Sapienza University of Rome, Italy; 8Institute of Biochemistry and Cell Biology (IBBC-CNR), Department of Sensory Organs, Sapienza University of Rome, Italy.

Equally contributed as first authors.

Summary. Fetal Alcohol Spectrum Disorders (FASD) is an encompassing term used to describe a range of afflictions brought about by the consumption of alcohol during gestation. The detrimental effects primarily manifest in the central nervous system, growth, and distinctive facial features. Given that there are no known treatments for FASD, the meticulous screening for this condition in the earliest stages of pregnancy bears immense significance, ensuring the avoidance of the grievous consequences stemming from exposure to alcohol in utero. Screening measures for FASD encompass the assessment of alcohol biomarkers such as Phosphatidylethanol (PEth) in the maternal bloodstream, Fatty Acid Ethyl Esters (FAEEs) in the meconium, and Ethyl Glucuronide (EtG) in the meconium, maternal urine and hair. In particular, urinary EtG is highly sensitive and could be routinely used in pregnant women for detecting also occasional drinking. Questionnaire evaluations including AUDIT-C, T-ACE, and TWEAK, alongside a detailed Food Diary method to identify alcohol misuse and high-risk pregnancies, are also available. However, these questionnaires might provide an inadequate reflection of alcohol consumption in women due to their inclination to dissemble to comply with prevailing sociocultural expectations. Hence, this comprehensive review advocates for the indispensable integration of alcohol biomarkers detection in the course of pregnancy monitoring, as it constitutes a valuable tool for facilitating FASD screening.

Key words. Alcohol, EtG, EtS, FAEE, FASD, PEth.

Linee guida italiane per la diagnosi e il trattamento dei disturbi dello spettro feto-alcolico: rilevamento del consumo di alcol durante la gravidanza.

Riassunto. Il disturbo dello spettro feto-alcolico (FASD) è un termine onnicomprensivo utilizzato per descrivere una serie di disturbi causati dal consumo di alcol durante la gestazione. Gli effetti dannosi si manifestano principalmente nel sistema nervoso centrale, nella crescita e nelle caratteristiche facciali distintive. Dato che non esistono trattamenti noti per il FASD, lo screening meticoloso per questa condizione nelle prime fasi della gravidanza ha un significato immenso, limitando le gravi conseguenze derivanti dall’esposizione all’alcol in utero. Le misure di screening per la FASD comprendono la valutazione dei biomarcatori dell’alcol come il fosfatidiletanolo (PEth) nel flusso sanguigno materno, gli esteri etilici degli acidi grassi (FAEE) nel meconio e l’etilglucuronide (EtG) nel meconio, nelle urine materne e nei capelli. In particolare, l’EtG urinario è altamente sensibile e potrebbe essere utilizzato di routine nelle donne in gravidanza per rilevare anche il consumo occasionale. Sono inoltre disponibili valutazioni tramite questionari tra cui AUDIT-C, T-ACE e TWEAK, insieme a un diario alimentare per identificare l’abuso di alcol e le gravidanze ad alto rischio. Tuttavia, questi questionari potrebbero fornire un quadro inadeguato del consumo di alcol nelle donne a causa della loro inclinazione a mentire per conformarsi alle aspettative socioculturali prevalenti. Pertanto, questo lavoro evidenzia l’indispensabile integrazione del rilevamento dei biomarcatori dell’alcol nel corso del monitoraggio della gravidanza, poiché costituisce uno strumento prezioso per facilitare la scoperta precoce di un eventuale FASD.

Parole chiave. Alcol, EtG, EtS, FAEE, FASD, PEth.

Introduction

Alcohol abuse represents a risk in everyday life that may induce damage to all the organ systems, but especially affects the liver, pancreas, brain, heart, and immune system, impairing also sleeping and causing brain damage and severe cognitive issues like dementia or Wernicke-Korsakoff syndrome1-10.

About 10% of women in the general population consume alcohol during pregnancy11 and there is a higher risk of alcohol consumption during pregnancy in women of lower socio-economic status, women belonging to marginalized communities or women with substance use disorders12-17.

It has been widely demonstrated that alcohol consumption during pregnancy is related to negative pregnancy and neonatal outcomes, such as spontaneous abortion, stillbirth, pre-term birth, intrauterine growth retardation and fetal alcohol spectrum disorders (FASD), a group of conditions caused by maternal alcohol consumption during pregnancy18-25. Low levels of prenatal alcohol exposure can still be noxious for the fetus’s development and increase the risk for FASD26,27.

Screening for prenatal alcohol exposure is a relevant tool for FASD prevention and it allows to reduce the cost and service burden in healthcare globally28.

Within the body, ethanol is distributed from the blood into all tissues and fluids in proportion to their relative water content and quickly reaches equilibrium with the concentration of ethanol in plasma29-32. However, some factors influence alcohol drinking, including ethnicity, culture, religion, and family environment, while individual characteristics, such as genetic polymorphisms, can affect alcohol absorption and metabolism33,34. Ethanol and its teratogenic metabolite acetaldehyde can easily cross the placental barrier and compromise the development of the fetal nervous system35-37, thus it is strongly recommended that women totally abstain from alcohol consumption during pregnancy and nursing38-40.

The assessment of a dose-response link between the levels of prenatal alcohol exposure and the effect on fetal and neonatal outcomes remains a problem. Even if binge drinking is considered less concerning than chronic heavy alcohol consumption, adverse effects on fetus development can still occur especially during critical phases of organogenesis41,42. Moreover, alcohol metabolism is slower in the fetus than the mother and so in fetal blood, there could be a higher concentration of alcohol for a longer period9,43-47.

The brain has a high susceptibility to prenatal alcohol exposure. A wide number of studies has described the effects of alcohol exposure on behavior, cognition and mental health48,49. In individuals with prenatal alcohol exposure, it has been found the presence of neuroanatomical changes, such as grey matter reduction in specific brain regions and a global decrease in white matter volume50-52.

It is difficult to assess the prevalence of FASD; some studies estimated 0.2 to 7 individuals with FASD in every 1000 live births in specific areas of the United States53,54. FASD may be associated with neuropsychological disorders55-57, attention deficit disorder58,59, physical abnormalities60,61 and behavioral problems. FASD is irreversible and there is no treatment for it, but it can be completely prevented with abstention from alcohol consumption during pregnancy and when trying to conceive62-65.

FASD evaluation requires a multidisciplinary team, including an audiologist, cardiologist, neurologist, psychiatrist, psychotherapist, ophthalmologist, and many others. The diagnosis of the disorder begins with the estimation of prenatal alcohol exposure by documenting the mother’s reported levels of alcohol consumption up to three months before pregnancy66. The analysis during gestation of biomarkers of alcohol drinking and the administration of alcohol screening questionnaires to the pregnant women are used for this purpose. The risk of FASD is related either to the amount of alcohol used and the frequency of alcohol consumption. The severity of the symptoms is related to the phase of fetal development and to the gestational age in which alcohol consumption occurs67,68.

Materials and methods

Biomarkers of alcohol consumption

Conventional alcohol biomarkers are mainly measured in blood matrix, both on serum and plasma, but other matrices, such as urine and keratin matrix, which have gained importance in recent years, especially for ethylglucuronide (EtG), are also considered29-32,69,70. It should be noted that not all the biomarkers available for alcohol abuse detection are suitable for being used during pregnancy71.

In this work we take to count only direct biomarkers, namely that compounds created from alcohol metabolism. Direct biomarkers are: Phosphatidyl Ethanol (PEth), Fatty Acid Ethyl Esters (FAEEs), Ethyl Glucuronide (EtG) and Ethyl Sulfate (EtS). Indirect biomarkers, such as the hepatic enzyme gamma-glutamyl transpeptidase (GGT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the glycoprotein CDT, mean corpuscular volume (MCV) of the erythrocytes, are not ideal in the contest of pregnancy where a tempestive assessment of alcohol misuse is needed. MCV in example, takes 6-8 weeks of heavy drinking (more than 40 grams of alcohol/day) to significantly increase. Enzymes like AST, ALT and GGT need as well high levels of alcohol drinking to elevate, have a low level of sensitivity and a low positive predictive value. CDT levels tend to be higher in women than men, independently of their history of drinking. Furthermore, pregnancy was found to be correlated with increased CDT levels, regardless of alcohol consumption72,73 and for this reason CDT will be not discussed in this work.

Phosphatidylethanol (PEth)

PEth is not a single molecule, but a class of ethanol derivatives in the form of phospholipids. These are obtained through a trans-phosphatidylation reaction of phosphatidylcholine present in cell membranes and catalyzed by phospholipase D, which, in the presence of ethanol, selectively catalyzes the PEth-forming reaction74. Among the different molecular forms, there is a predominant homolog chosen as an analytical target: 16:0/18:1 PEth, which will be referred to from here on simply as PEth. For the detection of this biomarker of alcohol use and abuse, dried blood spot testing (DBS) is used. The spot containing about 50µL of sample is placed in a vial and submerged with 250μL of a 2:4:0.2 solution of water-isopropanol-ammonium acetate 2mM solution 0.01% formic acid; in this way, the phosphate group of phosphatidylethanol is protonated, making it extractable into the organic phase, and basic molecules in the blood are deprotonated. Subsequently, the internal standard (PEth-D5) and the extraction solvent, usually hexane, are added. The vials are then mixed on vortex shaker for a few seconds, and left for 30 min in sonicator. Next, the vials are mixed again for 10 minutes on multimixer, with the aim of maximizing the amount of analyte passing into solution. At the end of agitation, the paper spot is removed from the vial with a special hooked instrument, and the organic phase is recovered through a Pasteur pipette. The organic phase is separated and brought to dryness under nitrogen flow, and then resuspended with 50µL of acetonitrile and transferred to a conical-bottomed glass vial, capped with a pierceable autosampler cap, for analysis in UHPLC-MS/MS. PEth-DBS is a stable method, especially when stored at lower temperatures75.

Fatty Acid Ethyl Esters (FAEEs)

In the fetal circulation, ethanol can be metabolized into FAEEs, which accumulate in meconium and thus can be used as a biomarker of fetal alcohol exposure76.

For the analysis of FAEEs, an optimized and validated method previously described by Hutson et al.77 could be headspace-solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). Ethyl myristate (E14), ethyl palmitate (E16), ethyl linoleate (E18:2), ethyl oleate (E18:1) and ethyl stearate (E18) were analyzed, then 25mL of their internal standards (1 μg/mL in n-heptane) were taken and placed in a 10 mL SPME headspace vial. N-heptane was completely evaporated at 40 °C in a stream of nitrogen and 50 mg meconium and 1 ml phosphate buffer (0.1 M, pH 7.6) were added. The mixture was shaken for 30 minutes and then 500 mg NaCl was added. Finally, the vials were capped with screw caps. Ethyl myristate was then excluded from the analyses as it was observed that it was also found in the meconium of infants with abstinent mothers. On the other hand, all samples with a sum of concentrations of the other four esters (E16, E18, E18:1, E18:2) greater than or equal to 100 ng/g were considered analytically positive.

Ethyl Glucuronide (EtG) and Ethyl Sulphate (EtS)

EtG and EtS are metabolites of ethanol and are used as biomarkers of alcohol consumption. EtG can be detected in a variety of biological matrices; in this work, methods on urine, hair and infant meconium are described. EtS accumulates in hair after chronic alcohol consumption and its detection can be used as a biomarker for alcohol consumption78. The main reason for using EtG as an alcohol biomarker is its long detection time for recent drinking, if compared to breath or blood ethanol test. Blood is not usually used as a matrix for the detection of EtG because it would involve shorter detection times due to its lower concentrations in blood79.

Urinary EtG is studied by an enzyme immunoassay using specific antibodies that can detect EtG without creating cross-reactivity with other glucuronic compounds30,31. The test is based on competition between glucose-6-phosphate dehydrogenase (G6PDH)-conjugated drug and free drug in the urine sample for a fixed number of antibody-specific binding sites. In the absence of a free drug in the sample, the antibody binds to the drug conjugated with G6PDH causing a decrease in enzyme activity. This phenomenon creates a direct relationship between drug concentration in urine and enzyme activity. The active enzyme converts NAD to NADH producing an alteration in absorbance, which can be measured by spectrophotometric examination at 340 nm. In this way, we obtain qualitative and semiquantitative detection of urinary EtG, which is considered positive when concentrations are greater than or equal to 100 ng/mL70,80.

In the hair, EtG can be detected by gas chromatography (GC) or liquid chromatography (LC), coupled with mass spectrometry (MS). Before analysis, the hair is treated with organic solvents, usually methanol and dichloromethane, to remove interfering substances, and is chopped. Approximately 50mg of hair is then taken and brought into 2mL of deionized water, to which 15µL of EtG-D5 deuterated standard is added. The samples are placed in the oven at 60° overnight, and the following day the extraction is done in SPE columns.

The column is first washed with 2 mL methanol and 2 mL deionized water, then the sample is loaded and slowly flowed. The SPE column is washed with 2 mL of 0.1 mM ammonia solution in water with a slight vacuum, followed by washing with 2 mL of methanol under a high vacuum, after which elution of the species of interest is made with 2 mL of a 99:1 solution of methanol and formic acid. The collected eluate is brought to dryness under nitrogen flow and derivatized with 30µL N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA), then the sample is ready for analysis. The mass analyzers were used in selected reaction monitoring mode and m/z 405 fragments (m/z 410 for EtG-D5 internal standard) was used as the precursor ion, while m/z 405 → 359 and m/z 410 → 359 transitions were set for quantification and m/z 405 → 331 and m/z 405 → 287 for qualification/confirmation81. Depending on which chromatographic separation technique is used and the instrument conditions applied, the EtG peak comes out at different retention times. Under normal conditions, its retention time is between 7 and 12 minutes and the limit of detection is between 5 and 6 pg/mg.

EtG accumulates in the intestine (in meconium) of the fetus from the 20th week of gestation until birth. Meconium from the newborn is taken (about 1 g) in the first 2 to 24 hours after delivery and stored frozen for analysis; some studies have confirmed alcohol exposure with values of EtG present in meconium as low as 10 ng/g, up to higher values of 112 ng/g for higher exposure82. The analysis starts by taking about 200 mg of meconium, stirring for 10 seconds, and adding about 0.5 mL of methanol. After stirring again, 1 mL of hexane is added and centrifuged. Next, 1 mL of acetonitrile is added, and after centrifuging again, SPE is performed. The column is conditioned with hexane and EtG elution is done with about 1 mL of methanol. At this point, the sample is dried under nitrogen flow, imaged with water and analyzed by LC-MS/MS83.

Alcohol screening questionnaires

Table 1 shows the questions and hallmarks of the Alcohol Use Disorders Identification Test – Consumption (AUDIT-C), of the Tolerance-Worried-Eye opener-Amnesia-(K)Cut down (TWEAK test) and of the T-ACE/TACER-3, all aiming at disclose putative alcohol drinking during pregnancy84-86.




The AUDIT-C has three short questions that estimate alcohol consumption in a standard, meaningful and non-judgmental manner. The total score from these questions provides an indication of the risks to the woman’s health and can be used to guide conversations about alcohol and pregnancy. AUDIT-C has been validated for use with pregnant women87. The AUDIT-C is a shortened version88 of the 10-item AUDIT tool developed in a WHO collaborative project84 and has been extensively used and studied since. The score for each question is from 0 to 4 in a five-point scale. The score of the single items summed makes the total score. For women, a cumulative score of 3 or more may indicate alcohol misuse. The AUDIT questions/scores and a chart illustrating the approximate number of standard drinks in different alcoholic beverages are available online at https://www.drugabuse.gov/sites/default/files/files/AUDIT.pdf. Information about the sensitivity and specificity of the test can be found at: https://pubs.niaaa.nih.gov/publications/arh25-3/204-209.htm.

The TWEAK test was originally used to screen for harmful drinking and to diagnose alcoholism or heavy drinking in different populations89. In 1994 the TWEAK was then used as a screening tool for periconceptional risk drinking among obstetric outpatients; the aim was to target risky drinkers and to ameliorate the outcome of their pregnancy by reducing alcohol intake85. Questions and scoring of the test are shown in table 1; further information about the test is available at the following address: https://pubs.niaaa.nih.gov/publications/arh25-3/204-209.htm.

The T-ACE is a screening test for at-risk drinking based on the CAGE questionnaire, but modified to be used in obstetric-gynecologic practices90. It was developed by an obstetrician who observed that a question about Tolerance was better accepted by patients than the Guilt question from the CAGE. The shortness of the test (takes less than 1 minute to ask) and the fact that any score above 0 is considered positive (0/1 easy scoring) make it an attractive instrument to identify potential pregnant risk drinkers. More recently an updated version of the test (TACER-3) has been proposed and validated86,91. Questions and scoring of the test are shown in Table 1; further information about the test is available at the following address: https://pubs.niaaa.nih.gov/publications/arh25-3/204-209.htm.

A food diary30 was also used to measure dietary intake over the pregnancy period, based on the University Hospital Umberto I Rome Guidelines. The aim was to assess the intake of a variety of nutrients and alcohol. The food diary questions related to the eating habits during breakfast, lunch and dinner are as follows:

• What do you usually eat for breakfast, lunch and dinner during working days?

• What do you eat habitually for breakfast, lunch and dinner on days that differ from normality, such as the weekend?

It was also asked what kind of favorite beverage she assumes during breakfast, lunch and dinner as follows:

• With working meals what are your favorite beverages?

• With weekend meals what are your favorite beverages?

If the women assume alcohol (positive score), the investigators ask the specification of drink size for different types of alcoholic beverages. In general, it is recommended to include questions about drinking in the context of a diet diary, always to avoid social stigma92.

Results and discussion

Table 2 summarizes the detection window, clinical application and different cut-offs of the five metabolites discussed in the previous chapter. Accuracy in analysis is important because detecting the fairest concentration of the metabolite within the biological matrix makes it possible to discriminate moderate alcohol use from more persistent use.




Since there is no treatment for FASD, the screening of this condition in the earliest week of gestation has a great importance in order to prevent fetus damage.

Among the metabolites discussed in this article, blood PEth and EtG proved to be high reliable. Furthermore, the detection of EtG in urine has higher sensitivity than in other matrices making this test more reliable compared to the other methodologies. Indeed, in two Italian studies based on urinary EtG on pregnant women data showed percentage values of alcohol consumption comprised between 34.28 and 25.630,70. By contrast, in other Italian studies on pregnant women based on EtG in the hair or meconium data showed much more lower values of alcohol drinking94,96.

Collection of maternal urine samples is most likely to be done during the third month of pregnancy when maternal screening begins. The optimization of techniques such as GC-MS/MS allows many toxicology laboratories worldwide to perform analyses for EtG easily and effectively.

The global prevalence of FASD is 0.2-7.7%, with a higher prevalence of 2-5% in Europe and North America60,96, underscoring the need for increased diagnosis and treatment. However, different diagnostic systems and disagreements over criteria have slowed progress in the diagnosis and management of the disorder. Furthermore, by measuring the number of pregnant women drinking alcohol throughout the analysis of ethanol metabolites as the EtG in the urine, hair or meconium data showed high variability of consumption with values ranging from 2/3% up to values superior than 30%30,31,70,94,96,97, with the highest percentages when the EtG was measured in the urine of the pregnant women. Nonetheless, there is evidence that a combination of biomarkers, or combining biomarkers with self-report, increases diagnostic accuracy98.

It should be mentioned, however, that not all women who drink during pregnancy give birth to a child with Fetal Alcohol Syndrome; this implies a greater need for both pre- and postnatal testing.

Conclusions

This review aims to emphasize the imperative of incorporating the examination of alcohol consumption in pregnant women into primary care systems. By implementing strategies such as screening for alcohol use in women of childbearing age, referring those who exhibit alcohol-related disorders to rehabilitation programs, raising awareness about the subject to promote health maintenance, we could potentially witness a decline in the number of cases of FASD.

However, the utilization of the T-ACE and TWEAK questionnaires alone may be considered insufficient and less than completely reliable, as women might not always be inclined to disclose the full truth regarding their alcohol consumption. In contrast, the analysis of various biological samples, in particular urinary EtG for ethanol metabolites, presents an opportunity to definitively confirm or rule out alcohol consumption through tests that can be carried out by any toxicology laboratory. These analyses additionally permit non-invasive sampling techniques and offer long-term assessment potential, as exemplified by hair analysis. Moreover, in cases where these markers indicate alcohol consumption, it is worthwhile to extend the analysis to include the biological matrices of the newborn, thereby providing an initial foundation for the diagnosis of FASD.

*Interdisciplinary Study Groups: - Sapienza Università di Roma, ISTAT - Istituto nazionale di statistica, AIDEFAD - Associazione Italiana Disordini da Esposizione Fetale ad Alcol e/o Droghe, SITAC - Società italiana per il trattamento dell’alcolismo e delle sue complicanze. SIFASD - Società Italiana Sindrome Feto-Alcolica, ISS - Istituto Superiore di Sanità, SIPPS - Società Italiana di Pediatria Preventiva e Sociale, FIMMG-Lazio - Federazione Italiana dei Medici di Medicina Generale Lazio, SIMPeSV - Società Italiana di Medicina di Prevenzione e degli Stili di Vita, CIPe - Confederazione Italiana Pediatri. Adele Minutillo, Alba Crognale, Alberto Chiriatti, Alberto Spalice, Antonella Polimeni, Antonio Greco, Arianna Barzacchi, Duccio Cordelli, Francesca Tarani, Ginevra Micangeli, Giovanni Corsello, Luca Cavalcanti, Lucia Leonardi, Luigi Meucci, Marco Lucarelli, Camilla Perna, Mario Vitali, Marisa Patrizia Messina, Martina Derme, Michela Menghi, Nunzia La Maida, Roberto Paparella, Sabrina Venditti, Serafino Zangaro, Pier Luigi Bartoletti, Silvia Francati.

Conflict of interests: the authors have no conflict of interests to declare.

References

1. Rosoff DB, Charlet K, Jung J, Lee J, et al. Association of high-intensity binge drinking with lipid and liver function enzyme levels. JAMA Netw Open 2019; 2: e195844.

2. Cruise KE, Becerra R. Alexithymia and problematic alcohol use: a critical update. Addict Behav 2018; 77: 232-46.

3. Polimanti R, Gelernter J. ADH1B: from alcoholism, natural selection, and cancer to the human phenome. Am J Med Genet B Neuropsychiatr Genet 2018; 177: 113-25.

4. Szabo G, Saha B. Alcohol’s effect on host defense. Alcohol Res 2015; 37: 159-70.

5. Erol A, Karpyak VM. Sex and gender-related differences in alcohol use and its consequences: contemporary knowledge and future research considerations. Drug Alcohol Depend 2015; 156: 1-13.

6. Nutt D, Hayes A, Fonville L, et al. Alcohol and the brain. Nutrients 2021; 13: 3938.

7. Shi X, DeLucia AL, Bao J, Zhang P. Alcohol abuse and disorder of granulopoiesis. Pharmacol Ther 2019; 198: 206-19.

8. Ceccanti M, Iannitelli A, Fiore M. Italian Guidelines for the treatment of alcohol dependence. Riv Psichiatr 2018; 53: 105-6.

9. D’Angelo A, Petrella C, Greco A, et al. Acute alcohol intoxication: a clinical overview. Clin Ter 2022; 173: 280-91.

10. Ceccanti M, Coriale G, Hamilton DA, et al. Virtual Morris task responses in individuals in an abstinence phase from alcohol. Can J Physiol Pharmacol 2018; 96: 128-36.

11. Popova S, Lange L, Probst C, Gmel G, Rehm J. Estimation of national, regional, and global prevalence of alcohol use during pregnancy and fetal alcohol syndrome: a systematic review and meta-analysis. Lancet 2017; 5: 290-9.

12. Ciafrè S, Ferraguti G, Greco A, et al. Alcohol as an early life stressor: epigenetics, metabolic, neuroendocrine and neurobehavioral implications. Neurosci Biobehav Rev 2020; 118: 654-68.

13. Fraser SL, Muckle G, Abdous BB, Jacobson JL, Jacobson SW. Effects of binge drinking on infant growth and development in an Inuit sample. Alcohol 2012; 46: 277-83.

14. Montag AC. Fetal alcohol-spectrum disorders: identifying at-risk mothers. Int J Womens Health 2016; 8: 311-23.

15. Popova S, Lange S, Probst C, Parunashvili N, Rehm J. Prevalence of alcohol consumption during pregnancy and Fetal Alcohol Spectrum Disorders among the general and Aboriginal populations in Canada and the United States. Eur J Med Genet 2017; 60: 32-48.

16. McQuire C, Daniel R, Hurt L, Kemp A, Paranjothy S. The causal web of foetal alcohol spectrum disorders: a review and causal diagram. Eur Child Adolesc Psychiatry 2020; 29: 575-94.

17. Von Hinke Kessler Scholder S, Wehby GL, Lewis S, Zuccolo L. Alcohol exposure in utero and child academic achievement. Econ J 2014; 124: 634-67.

18. Micangeli G, Menghi M, Profeta G, et al. The impact of oxidative stress on pediatrics syndromes. Antioxidants 2022; 11: 1983.

19. Lebel C, Roussotte F, Sowell ER. Imaging the impact of prenatal alcohol exposure on the structure of the developing human brain. Neuropsychol Rev 2011; 21: 102-18.

20. Henriksen TB, Hjollund NH, Jensen TK, et al. Alcohol consumption at the time of conception and spontaneous abortion. Am J Epidemiol 2004; 160: 661-7.

21. Nykjaer C, Alwan NA, Greenwood DC, et al. Maternal alcohol intake prior to and during pregnancy and risk of adverse birth outcomes: Evidence from a british cohort. J Epidemiol Community Health 2014; 68: 542-9.

22. O’Connor MJ, Kogan N, Findlay R. Prenatal alcohol exposure and attachment behavior in children. Alcohol Clin Exp Res 2002; 26: 1592-602.

23. Patra J, Bakker R, Irving H, Jaddoe VWV, Malini S, Rehm J. Dose-response relationship between alcohol consumption before and during pregnancy and the risks of low birthweight, preterm birth and small for gestational age (SGA)-a systematic review and meta-analyses. BJOG An Int J Obstet Gynaecol 2011; 118: 1411-21.

24. Jones KL, Smith DW, Ulleland CN, Streissguth AP. Pattern of malformation in offspring of chronic alcoholic mothers. Lancet 1973; 301: 1267-71.

25. Jones KL, Smith DW. Recognition of the fetal alcohol syndrome in early infancy. Lancet 1973; 302: 999-1001.

26. Chambers CD, Coles C, Kable J, et al. Fetal Alcohol Spectrum Disorders in a Pacific Southwest city: maternal and child characteristics. Alcohol Clin Exp Res 2019; 43: 2578-90.

27. May PA, Blankenship J, Marais AS, et al. Maternal alcohol consumption producing fetal alcohol spectrum disorders (FASD): Quantity, frequency, and timing of drinking. Drug Alcohol Depend 2013; 133: 502-12.

28. Greenmyer JR, Klug MG, Kambeitz C, Popova S, Burd L. A multicountry updated assessment of the economic impact of fetal alcohol spectrum disorder: costs for children and adults. J Addict Med 2018; 12: 466-73.

29. Cederbaum AI. Alcohol metabolism. Clin Liver Dis 2012; 16: 667-85.

30. Ferraguti G, Ciolli P, Carito V, et al. Ethylglucuronide in the urine as a marker of alcohol consumption during pregnancy: Comparison with four alcohol screening questionnaires. Toxicol Lett 2017; 275: 49-56.

31. Ferraguti G, Merlino L, Battagliese G, et al. Fetus morphology changes by second-trimester ultrasound in pregnant women drinking alcohol. Addict Biol 2020; 25: e12724.

32. Fiore M, Petrella C, Coriale G, et al. Markers of neuroinflammation in the serum of prepubertal children with Fetal Alcohol Spectrum Disorders. CNS Neurol Disord Drug Targets 2022; 21: 854-68.

33. Wall TL, Luczak SE, Hiller-Sturmhöfel S. Biology, genetics, and environment: underlying factors influencing alcohol metabolism. Alcohol Res 2016; 38: 59-68.

34. Ferraguti G, Pascale E, Lucarelli M. Alcohol addiction: a molecular biology perspective. Curr Med Chem 2015; 22: 670-84.

35. Terracina S, Tarani L, Ceccanti M, et al. The impact of oxidative stress on the epigenetics of Fetal Alcohol Spectrum Disorders. Antioxidants 2024; 13: 410.

36. Coriale G, Ceccanti M, Fiore M, et al. Delay in the fine-tuning of locomotion in infants with meconium positive to biomarkers of alcohol exposure: a pilot study. Riv Psichiatr 2024; 59: 52-9.

37. Siqueira M, Stipursky J. Blood brain barrier as an interface for alcohol induced neurotoxicity during development. Neurotoxicology 2022; 90: 145-57.

38. Popova S, Dozet D, Akhand Laboni S, Brower K, Temple V. Why do women consume alcohol during pregnancy or while breastfeeding? Drug Alcohol Rev 2022; 41: 759-77.

39. Chu JTW, McCormack J, Marsh S, Wells A, Wilson H, Bullen C. Impact of prenatal alcohol exposure on neurodevelopmental outcomes: a systematic review. Heal Psychol Behav Med 2022; 10: 973-1002.

40. Quenby S, Gallos ID, Dhillon-Smith RK, et al. Miscarriage matters: the epidemiological, physical, psychological, and economic costs of early pregnancy loss. Lancet 2021; 397: 1658-67.

41. Dejong K, Olyaei A, Lo JO. Alcohol use in pregnancy. Clin Obstet Gynecol 2019; 62: 142-55.

42. Chung DD, Pinson MR, Bhenderu LS, Lai MS, Patel RA, Miranda RC. Toxic and teratogenic effects of prenatal alcohol exposure on fetal development, adolescence, and adulthood. Int J Mol Sci 2021; 22: 8785.

43. Ferraguti G, Terracina S, Micangeli G, et al. NGF and BDNF in pediatrics syndromes. Neurosci Biobehav Rev 2023; 145: 105015.

44. Carson G, Cox LV, Crane J, et al. Alcohol use and pregnancy consensus clinical guidelines. J Obstet Gynaecol Canada 2010; 32: S1-2.

45. Carson G, Cox LV, Crane J, et al. No. 245-Alcohol use and pregnancy consensus clinical guidelines. J Obstet Gynaecol Canada 2017; 39: e220-54.

46. Idänpään-Heikkilä J, Jouppila P, Åkerblom HK, Isoaho R, Kauppila E, Koivisto M. Elimination and metabolic effects of ethanol in mother, fetus, and newborn infant. Am J Obstet Gynecol 1972; 112: 387-93.

47. Burd L, Blair J, Dropps K. Prenatal alcohol exposure, blood alcohol concentrations and alcohol elimination rates for the mother, fetus and newborn. J Perinatol 2012; 32: 652-9.

48. Mattson SN, Bernes GA, Doyle LR. Fetal Alcohol Spectrum Disorders: a review of the neurobehavioral deficits associated with prenatal alcohol exposure. Alcohol Clin Exp Res 2019; 43: 1046-62.

49. Weyrauch D, Schwartz M, Hart B, Klug MG, Burd L. Comorbid mental disorders in fetal alcohol spectrum disorders: a systematic review. J Dev Behav Pediatr 2017; 38: 283-91.

50. Tang S, Xu S, Zhu W, Gullapalli RP, Mooney SM. Alterations in the whole brain network organization after prenatal ethanol exposure. Eur J Neurosci 2020; 51: 2110-8.

51. Moore EM, Xia Y. Neurodevelopmental trajectories following prenatal alcohol exposure. Front Hum Neurosci 2022; 15: 695855.

52. Mathews E, Dewees K, Diaz D, Favero C. White matter abnormalities in fetal alcohol spectrum disorders: focus on axon growth and guidance. Exp Biol Med 2021; 246: 812-21.

53. May PA, Gossage JP, Kalberg WO, et al. Prevalence and epidemiologic characteristics of FASD from various research methods with an emphasis on recent in-school studies. Dev Disabil Res Rev 2009; 15: 176-92.

54. Lange S, Probst C, Gmel G, Rehm J, Burd L, Popova S. Global prevalence of fetal alcohol spectrum disorder among children and youth: a systematic review and meta-analysis. JAMA Pediatr 2017; 171: 948-56.

55. Connor PD. Neuropsychological assessment of Fetal Alcohol Spectrum Disorder in adults. In: Novick Brown N (ed). Evaluating Fetal Alcohol Spectrum Disorders in the forensic context: a manual for mental health practice. Cham, CH: Springer International Publishing, 2021.

56. Mattson SN, Crocker N, Nguyen TT. Fetal alcohol spectrum disorders: neuropsychological and behavioral features. Neuropsychol Rev 2011; 21: 81-101.

57. Maya-Enero S, Ramis-Fernández SM, Astals-Vizcaino M, García-Algar Ó. Neurocognitive and behavioral profile of fetal alcohol spectrum disorder. An Pediatr 2021; 95: 208.e1-208.e9.

58. Burd L. FASD and ADHD: are they related and how? BMC Psychiatry 2016; 16: 325.

59. Peadon E. Distinguishing between attention-deficit hyperactivity and fetal alcohol spectrum disorders in children: clinical guidelines. Neuropsychiatr Dis Treat 2010; 6: 509.

60. Wozniak JR, Riley EP, Charness ME. Clinical presentation, diagnosis, and management of fetal alcohol spectrum disorder. Lancet Neurol 2019; 18: 760-70.

61. Popova S, Charness ME, Burd L, et al. Fetal alcohol spectrum disorders. Nat Rev Dis Prim 2023; 9: 11.

62. Mitchell AM, Porter RR, Pierce-Bulger M, McKnight-Eily LR. Addressing alcohol use in pregnancy. Am J Nurs 2020; 120: 22-4.

63. Rawoot IA, Scott CJ, Urban MF. Barriers and facilitators of alcohol abstinence during pregnancy. Afr J Reprod Health 2022; 26: 53-65.

64. Fuso A, Lucarelli M. CpG and non-CpG methylation in the diet-epigenetics-neurodegeneration connection. Curr Nutr Rep 2019; 8: 74-82.

65. Britton A. Alcohol consumption for women trying to conceive. BMJ 2016; 354: i4540.

66. Denny LA, Coles S, Blitz R. Fetal Alcohol Syndrome and Fetal Alcohol Spectrum Disorders. Am Fam Physician 2017; 96: 515-22.

67. Coles C. Critical periods for prenatal alcohol exposure: evidence from animal and human studies. Alcohol Health Res World 1994; 18: 22-9.

68. Vorgias D, Bernstein B. Fetal Alcohol Syndrome. 2023 May 27. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing, 2024.

69. Derme M, Piccioni MG, Brunelli R, et al. Oxidative stress in a mother consuming alcohol during pregnancy and in her newborn: a case report. Antioxidants 2023; 12: 1216.

70. Ceci FM, Fiore M, Agostinelli E, et al. Urinary ethyl glucuronide for the assessment of alcohol consumption during pregnancy: comparison between biochemical data and screening questionnaires. Curr Med Chem 2021; 29: 3125-41.

71. Pragst F. Alcohol biomarkers in hair. In: Hair analysis in clinical and forensic toxicology. Amsterdam: Elsevier, 2015.

72. Bager H, Christensen LP, Husby S, Bjerregaard L. Biomarkers for the detection of prenatal alcohol exposure: a review. Alcohol Clin Exp Res 2017; 41: 251-61.

73. Littner Y, Bearer CF. Detection of alcohol consumption during pregnancy-Current and future biomarkers. Neurosci Biobehav Rev 2007; 31: 261-9.

74. Beck O, Mellring M, Löwbeer C, Seferaj S, Helander A. Measurement of the alcohol biomarker phosphatidylethanol (PEth) in dried blood spots and venous blood: importance of inhibition of post-sampling formation from ethanol. Anal Bioanal Chem 2021; 413: 5601-6.

75. Bakhireva LN, Shrestha S, Gutierrez HL, Berry M, Schmitt C, Sarangarm D. Stability of phosphatidylethanol in dry blood spot cards. Alcohol Alcohol 2016; 51: 275-80.

76. Caughey AB. CME review article. Pediatr Emerg Care 2017; 33: 792-3.

77. Hutson JR, Aleksa K, Pragst F, Koren G. Detection and quantification of fatty acid ethyl esters in meconium by headspace-solid-phase microextraction and gas chromatography-mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 2009; 877: 8-12.

78. Cappelle D, Lai FY, Covaci A, et al. Assessment of ethyl sulphate in hair as a marker for alcohol consumption using liquid chromatography-tandem mass spectrometry. Drug Test Anal 2018; 10: 1566-72.

79. Neumann J, Beck O, Böttcher M. Phosphatidylethanol, ethyl glucuronide and ethanol in blood as complementary biomarkers for alcohol consumption. J Mass Spectrom Adv Clin Lab 2021; 22: 3-7.

80. Van De Luitgaarden IAT, Beulens JWJ, Schrieks IC, et al. Urinary ethyl glucuronide can be used as a biomarker of habitual alcohol consumption in the general population. J Nutr 2019; 149: 2199-205.

81. Mattia A, Moschella C, David MC, et al. Development and validation of a GC-EI-MS/MS method for ethyl glucuronide quantification in human hair. Front Chem 2022; 10: 858205.

82. Maschke J, Roetner J, Goecke TW, et al. Prenatal alcohol exposure and the facial phenotype in adolescents: a study based on meconium ethyl glucuronide. Brain Sci 2021; 11: 1-20.

83. Woz´niak MK, Banaszkiewicz L, Aszyk J, et al. Development and validation of a method for the simultaneous analysis of fatty acid ethyl esters, ethyl sulfate and ethyl glucuronide in neonatal meconium: application in two cases of alcohol consumption during pregnancy. Anal Bioanal Chem 2021; 413: 3093-105.

84. Saunders JB, Aasland OG, Babor TF, De la Fuente JR, Grant M. Development of the Alcohol Use Disorders Identification Test (AUDIT): WHO Collaborative Project on Early Detection of Persons with Harmful Alcohol Consumption-II. Addiction 1993; 88: 791-804.

85. Russell M. New assessment tools for risk drinking during pregnancy: T-ACE, TWEAK, and Others. Alcohol Heal Res World 1994; 18: 55-61.

86. Chiodo LM, Delaney-Black V, Sokol RJ, Janisse J, Pardo Y, Hannigan JH. Increased cut-point of the TACER-3 screen reduces false positives without losing sensitivity in predicting risk alcohol drinking in pregnancy. Alcohol Clin Exp Res 2014; 38: 1401-8.

87. Dawson DA, Grant BF, Stinson FS, Zhou Y. Effectiveness of the derived Alcohol Use Disorders Identification Test (AUDIT-C) in screening for alcohol use disorders and risk drinking in the US general population. Alcohol Clin Exp Res 2005; 29: 844-54.

88. Bush K, Kivlahan DR, McDonell MB, Fihn SD, Bradley KA. The AUDIT alcohol consumption questions (AUDIT-C): an effective brief screening test for problem drinking. Ambulatory Care Quality Improvement Project (ACQUIP). Alcohol Use Disorders Identification Test. Arch Intern Med 1998; 158: 1789-95.

89. Chan AWK, Pristach EA, Welte JW, Russell M. Use of the TWEAK Test in screening for alcoholism/ heavy drinking in three populations. Alcohol Clin Exp Res 1993; 17: 1188-92.

90. Sokol RJ, Martier SS, Ager JW. The T-ACE questions: practical prenatal detection of risk-drinking. Am J Obstet Gynecol 1989; 160: 863-70.

91. Chiodo LM, Sokol RJ, Delaney-Black V, Janisse J, Hannigan JH. Validity of the T-ACE in pregnancy in predicting child outcome and risk drinking. Alcohol 2010; 44: 595-603.

92. Zizzo N, Di Pietro N, Green C, Reynolds J, Bell E, Racine E. Comments and reflections on ethics in screening for biomarkers of prenatal alcohol exposure. Alcohol Clin Exp Res 2013; 37: 1451-5.

93. Hasken JM, Marais A-S, de Vries MM, et al. Assessing the sensitivity and specificity of phosphatidylethanol (PEth) cutoffs to identify alcohol exposed pregnancies. Curr Res Toxicol 2023; 4: 100105.

94. Pichini S, Marchei E, Vagnarelli F, et al. Assessment of prenatal exposure to ethanol by meconium analysis: results of an Italian multicenter study. Alcohol Clin Exp Res 2012; 36: 417-24.

95. Papaseit E, Muga R, Zuluaga P, Sanvisens A, Farré M. Chapter 60 - Meconium Biomarkers of Prenatal Alcohol Exposure. In: Preedy VR (ed). Neuroscience of alcohol. Cambridge, MA: Academic Press, 2019.

96. La Maida N, Di Giorgi A, Pellegrini M, et al. Reduced prevalence of fetal exposure to alcohol in Italy: a nationwide survey. Am J Obstet Gynecol MFM 2023; 5: 100944.

97. Pichini S, Busardò FP, Ceccanti M, Tarani L, Pacifici R. Unreliable estimation of prevalence of fetal alcohol syndrome. Lancet Glob Health 2017; 5: e574.

98. Howlett H, Abernethy S, Brown NW, Rankin J, Gray WK. How strong is the evidence for using blood biomarkers alone to screen for alcohol consumption during pregnancy? A systematic review. Eur J Obstet Gynecol Reprod Biol 2017; 213: 45-52.