Delay in the fine-tuning of locomotion in infants with meconium positive to biomarkers of alcohol exposure: a pilot study

Giovanna Coriale1, Mauro Ceccanti2, Marco Fiore3, Francesca Tarani4, Ginevra Micangeli4, Michela Menghi4, Adele Minutillo5, Paolo Berretta5, Giampiero Ferraguti6, Angela Iannitelli7, Giovanni Parlapiano4, Roberto Paparella4, Marisa Patrizia Messina4, Mario Vitali8, Daniela Fiorentino9, Simona Pichini5, Luigi Tarani4

1CRARL Lazio, ASL Roma 1, Italy; 2SITAC, Società Italiana per il Trattamento dell’Alcolismo e le sue Complicanze, Rome, Italy; 3Institute of Biochemistry and Cell Biology (IBBC-CNR), Department of Sensory Organs, Sapienza University of Rome, Italy; 4Department of Maternal Infantile and Urological Sciences, Sapienza University of Rome, Italy; 5National Centre on Addiction and Doping, Istituto Superiore di Sanità, Rome; 6Department of Experimental Medicine, Sapienza University of Rome, Italy; 7Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, Italy; 8ASUR Marche, AV4, Ancona, Italy; 9UOC Patologia da Dipendenza, ASL Rieti, Italy.

Summary. Introduction. Prenatal alcohol exposure causes a variety of impairments to the fetus called Fetal Alcohol Spectrum Disorders (FASD). Since it is very difficult to identify women that consume alcohol during pregnancy, different methods have been studied to evaluate alcohol exposure. Ethyl Glucuronide (EtG) and Fatty Acid Ethyl Esters (FAEEs) are commonly used to measure alcohol consumption in individuals at-risk for alcohol abuse, including pregnant women. Materials and methods. We conducted a study of two cohorts of 1.5 year-old infants (of mothers without a history of alcohol abuse) with or without meconium samples positive to both EtG and FAEEs and we evaluated their cognitive-behavioral development by the Griffiths Mental Developmental Scale (GMDS) method. Our protocol included 8 infants with meconium positive to alcohol metabolites (EtG and FAEEs) and 7 with meconium negative to alcohol metabolites. Results. None of the 8 alcohol metabolites positive meconium infants exhibited distinctive facial features and growth retardation of severe FASD, showing that other factors may contribute to the FASD onset but elevations in EtG and FAEEs in the meconium were significantly associated with disrupted neurodevelopment and adaptive functions within the first year and a half of life. Indeed, we found out that infants with meconium positive for both EtG and FAEEs, although without displaying any FASD morphological features, had a delay in the fine regulation of their own locomotory capabilities. Conclusions. Further analyses and larger studies are needed to estimate the right link between prenatal alcohol exposure and the different range of disorders connected but this study provides an additional step in the field of FASD in order to suggest early treatments for at-risk newborns and infants.

Key words. Alcohol, EtG, FAEE, FASD, infants, meconium, neurodevelopment.

Ritardo nella regolazione del movimento fine in neonati con meconio positivo ai biomarcatori di consumo alcolico materno durante la gravidanza: uno studio pilota.

Riassunto. Introduzione. L’esposizione prenatale all’alcol provoca una serie di problematiche al feto chiamate disturbi dello spettro alcolico fetale (FASD). Poiché è molto difficile identificare le donne che consumano alcol durante la gravidanza, sono stati studiati diversi metodi per valutare l’esposizione all’alcol. L’etilglucuronide (EtG) e gli esteri etilici degli acidi grassi (FAEE) sono comunemente usati per misurare il consumo di alcol in soggetti a rischio di abuso di alcol, comprese le donne in gravidanza. Materiali e metodi. Abbiamo condotto uno studio su due coorti di neonati di 1,5 anni (di madri senza storia di abuso di alcol) con o senza campioni di meconio positivi sia a EtG che a FAEE e abbiamo valutato il loro sviluppo psicomotorio mediante la Griffiths Mental Developmental Scale (GMDS). Il nostro protocollo includeva 8 neonati con meconio positivo ai metaboliti dell’alcol (EtG e FAEE) e 7 con meconio negativo ai metaboliti dell’alcol. Risultati. Nessuno degli 8 neonati positivi al meconio per i metaboliti dell’alcol ha mostrato caratteristiche facciali distintive e ritardo della crescita causata da FASD grave, dimostrando che altri fattori possono contribuire all’insorgenza della FASD. Tuttavia, la presenza di EtG e FAEE nel meconio è risultata significativamente associata a uno sviluppo neurologico alterato. Infatti, i bambini con meconio positivo sia per EtG che per FAEE, pur non mostrando alcuna caratteristica morfologica FASD, avevano un ritardo nella regolazione fine delle proprie capacità locomotorie. Conclusioni. Sono necessarie ulteriori analisi e studi più ampi per stimare il giusto legame tra l’esposizione prenatale all’alcol e la diversa gamma di disturbi connessi, ma questo studio fornisce un ulteriore passo avanti nel campo della FASD per suggerire trattamenti precoci per neonati e bambini a rischio.

Parole chiave. Alcol, EtG, FAEE, FASD, meconio, neonati, neurosviluppo.

Introduction

The identification of the teratogen effects of drinking alcohol during gestation is referred back to 19731, and since then, numerous research focused on the long-term effects of prenatal alcohol exposure indicating the importance of abstaining from alcohol during pregnancy and while breastfeeding2.

Prenatal alcohol exposure can in fact lead to a plethora of consequences for the fetus called Fetal Alcohol Spectrum Disorders (FASD)3. Fetal alcohol syndrome (FAS) is the most severe form of FASD and relies on the presence of distinct facial anomalies and other physical dysmorphologies4 associated with behavioral, cognitive and social disruptions5-7.

The most devastating alterations affect the developing brain fetus, resulting in neurobehavioral deficits, including overall intellectual performance, executive function, learning and memory, language, visual-spatial ability, motor function, attention, and activity levels as well as behavioral problems including adaptive dysfunction, academic difficulties, and increased rates of psychiatric disorders8-10.

This causes, among other things, a significant social cost: the annual expenditure expected in America for the support of all those affected by FASD was estimated, in 2018, between 1.6 and 5.4 billion dollars11; while the cost of treating a single FASD patient is about $2 million12. A systematic review of published literature on the cost of FASD reported that in the United States, one case of FASD can be prevented for a cost between $20,200 and 47,615, showing that the price of a single FASD prevention would be considerably less expensive than the cost of care for a single FASD case13.

Despite numerous guidelines suggest abstaining from consumption of alcohol during pregnancy, about 10% of women in the general population consume alcohol while pregnant, even though the European region has the highest rate of consumption (up to 25%)14. The estimated prevalence of FAS in the general population is 14.6 per 10,000 people. The greatest consumption of alcohol in Europe is reflected in having the highest prevalence of FAS: 2.6 times higher than the global average (Ireland, Belarus, Denmark, the United Kingdom and Russia have the world’s highest rates of alcohol consumption during pregnancy)15.

Thus, the early identification and diagnosis of affected children is crucial for the appropriate interventions16; this could be very challenging, because of the lack of confirmation of prenatal alcohol exposure, lack of clinical expertise, and many possible confounding variables (i.e. children not in the care of their biological mothers, maternal death, or social stigma)8. Even when exposure is known, several factors may affect diagnosis: it is essential to assess the maternal prenatal alcohol intake, measured by the quantity of alcohol consumed per occasion (standard drinks per drinking day), frequency of consumption (eg, daily, times per week), and timing during gestation, because different timing of significant exposure can produce different physical and neurobehavioral phenotypes9,10.

One of the main problems, because of the possible stigmatization associated with prenatal alcohol use, is estimating the maternal prenatal intake and it has been shown that self-declarations are often not reliable17-21.

Hereafter, the consequence of identifying women who drink during gestation becomes even greater22,23. To overcome these difficulties and to obtain a reliable picture of the alcohol behavior in pregnant women, specific methodologies and strategies based on the analyses of ethanol metabolites in biological matrix such as meconium and maternal urine and hair have been established24-26. Indeed, Ethyl Glucuronide (EtG) and Fatty Acid Ethyl Esters (FAEEs) are commonly used to measure alcohol consumption in individuals at-risk for alcohol abuse, including pregnant women24-27. EtG and FAEEs are products of the non-oxidative metabolism of ethanol: EtG is an ethanol metabolite forming in the body by glucuronidation following exposure to alcohol, usually from drinking alcoholic beverages; FAEEs are products of the non-oxidative metabolism of ethanol which are formed by the esterification of ethanol with endogenous free fatty acids or with acyl-CoA28. The esterification process is catalyzed by two enzymes: FAEEs synthetase (present in the liver and pancreas) and acyl-CoA transferase29.

Thus, the aim and novelties of the present study were to evaluate in two cohorts of 17 months-old infants (of mothers without a history of alcohol abuse) with or without meconium samples positive to both EtG and FAEEs, the cognitive-behavioral development by the Griffiths Mental Developmental Scale (GMDS)30 and the presence physical abnormalities, such as facial dysmorphism and growth retardation, characteristics of FASD. We predicted that prenatal alcohol exposure, according to positive EtG and FAEEs in the meconium, could affect the infant’s fine movement leading to a delay in neurodevelopment.

Materials and methods

Infants’ recruitment

The original sample consisted of a total of 21 infants (12 females and 11 males) in follow-up at the Department of Pediatrics of the Sapienza University Hospital “Policlinico Umberto I” of Rome, Italy. As previously shown31, the main exclusion criteria to avoid any bias in the selection for the infants’ (and mothers’) enrolment included a mean age for the infants comprised between 16 and 19 months for the GDMS neurobehavioral analysis, ongoing pediatric pathologies, inflammatory, endocrine and autoimmune disorders, diagnosed cardiovascular pathologies and the use of drugs or chemicals such as antidepressants, anti-inflammatory and immunosuppressants.

Afterwards, according to the above-mentioned recruitment criteria, the protocol included 8 age-matched infants with meconium positive to alcohol metabolites (EtG and FAEEs) (6 females, 2 males; mean age in months ± SE: 17.25 ± 0.55) and 7 with meconium negative to alcohol metabolites (EtG and FAEEs) (3 females, 4 males; mean age in months ± SE: 17.77 ± 0.57). The meconium, collected within the first 24 to 48 hours of newborn life, was placed into a plastic tube, homogenized, and immediately stored in aliquots at -20 °C. The aliquots were sent to the Istituto Superiore di Sanità in Rome to be analyzed for EtG and FAEEs. Samples were received anonymously because they were identified by a code with a progressive number assigned by the neonatology ward.

All the information on mother-infant dyads, identified by the same code were also provided. A structured questionnaire on cigarette smoking and drugs consumption during the 3 trimesters of pregnancy was completed by the mothers. Additional information about the mothers and the newborns was obtained from hospital records. The children enrolled in the study underwent a clinical evaluation and neurodevelopmental evaluation. The clinical evaluation was carried out by pediatricians with experience in the field of genetic and/or malformation syndromes and in particular on FASD32-34. This allowed us to have the most accurate auxological and dysmorphological measurements possible. The administration of GDMS was instead performed by clinical psychologists with experience in the field of pathologies of neurocognitive development in children35.

The study was approved by the Sapienza University Hospital ethical committee (Ref. 5825); an informed consent was signed by each parent of the children and all the study procedures were under the Helsinki Declaration of 1975, as revised in 1983, for human experimentation.

EtG and FAEEs determinations in neonatal meconium

Fully validated liquid chromatography-tandem mass spectrometric (LC-MS/MS) methods were used for the determination of EtG and the seven FAEEs (palmitic, palmitoleic, stearic, oleic, linoleic, linolenic and arachidonic acid ethyl esters), respectively, in the collected meconium samples27,36-40. The established cut-off of 2 nmol of the total amount of the seven FAEEs and of 2 nmol for EtG per gram of meconium were used to consider whether infants were exposed or not to gestational ethanol intake.

Physical features measurement and dysmorphology exams to disclose FASD

As for the clinical diagnosis, FASD may result in a combination of facial malformations, growth retardation that may involve both length and weight and neurocognitive retardation together with a confirmed history of maternal alcohol intake during pregnancy41. The facial clinical features of FAS include short palpebral fissures, a low nasal implant with a short nose, a long nasal philtrum, and thinning of the upper lip39.

To search for these anomalies in our sample, each child was examined by two clinicians, in double-blind data collection. The doctors evaluated the children measured, height, weight, occipitofrontal circumference (OFC), length of the filter, interpupillary distance, intracanthal distance (ICD) and palpebral fissure length (PFL) in each child. Measurements were taken using a rigid ruler, marked in millimeters, with examiners seated directly in front of the subject.

Each subject’s upper lip and philtrum conformation was assessed using the lip-philtrum guide which provides a numerical interpretation of the qualitative aspects42. Independent scores were assigned for the thinness of the upper lip and the prominence or flatness of the philtral ridges. Scores of 4 or 5 allowed for the diagnosis of FASD. All children enrolled in the study then received then a total dysmorphology score that allowed them to be assigned to one of three categories: established FASD, another non-alcohol-related diagnosis, or no diagnosis of FASD43.

Neurodevelopment assessment (Griffiths Mental Developmental Scale)

For the assessment of neurocognitive development, all children in the study underwent the Griffiths Mental Developmental Scale (GMDS)30,44,45. The GMDS was created to assess the cognitive and motor development of children from birth to six years of age. The scale is currently in its third edition which was revised in 2015 and is regarded as the gold standard for assessing neuromotor development in children44-46.

The extended Griffiths scale evaluates 5 different domains such as basics of learning (A), language and communication (B), hand-eye coordination (C), personal-social-emotional (D) and gross-motor skills (E)47. Scale A aims to explore developmental potential and discoveries that foster innate future learning. The items administered include activities such as: manipulating objects (cubes and slots), building with bricks, repeating numbers, comparing quantities, putting in series, rearranging images to tell a story, building puzzles and activities that require associating objects and remembering30. Scale B evaluates the child’s development of language and speech and the ability to use these skills to initiate social interactions with others. The items propose activities such as: naming objects and images, defining objects according to their function, telling a story, following instructions, saying the opposite and making similarities. The level of development and the content of the language is also assessed48. The C scale evaluates the early stages of the child’s development with regard to visual perception, visual directionality and object manipulation. Items in this scale include activities such as following the movement of objects, grasping toys of different sizes, building cubes, stringing beads, cutting paper with scissors, copying shapes, connecting dots, and performing more complex construction activities46. The D scale assesses areas of a child’s personal, social and emotional development. These areas are characterized as being of fundamental importance for an adequate evolution of the child’s development, learning and well-being. The administered items are organized according to the child’s increasing level of emotional regulation, development of empathy, and recognition of morals. Were also included items to measure the early ability to put yourself in the shoes of others and the theory of mind30. The E scale evaluates the early development of postural control, general body motor coordination, and visual-spatial coordination. The items administered in this scale include activities such as observing the child’s movements in space, the ability to jump on two feet and on one foot, running, playing ball, maintaining balance and jumping by alternating feet. With younger children, spontaneous body movement behaviors can be noted, regardless of the pre-established order of administration48.

This gives total raw scores for each scale and an overall raw development score. The raw score will be converted into “equivalent age” in months, “weighted score”, “developmental quotient”, “confidence interval”, “stanine” and “percentile”49:

• the equivalent age measure establishes, for each subject in each subscale examined, the chronological age to which the performances presented by the subject are referred;

• the weighted score indicates the relative position of the subject in the normative sample and provides comparable measures which make it possible to immediately compare the results achieved by the same subject in different tests;

• the development quotient indicates the level of development achieved by the child in the particular area being considered;

• the confidence interval is a set of values defined as a range of plausible values, with respect to the parameter that has been measured, which gives greater reliability with respect to the result obtained;

• the stanine is a unit on a nine-level scale used to group test results;

• the percentile is a measure used in statistics to indicate the minimum value below which a given percentage of the other elements under observation falls.

It should be noted that since the age of the sample studied was less than 2 years, the general quotients obtained from the subscales were used50.

Data analysis

According to methods previously described51, neurobehavioral data were evaluated by two-way analysis of variance (ANOVA) (positive/negative meconium and sex effect). Since no effects of sex were disclosed during the statistical investigation (except for body weight) this main factor was pooled out from the description of the results. Post hoc comparisons were analyzed by Tukey’s HSD test. The Spearman Nonparametric test was utilized in infants to correlate the FASD features with the infants’ age, BMI, weight and height. Data normal distribution was also analyzed. All data were analyzed by JASP (the University of Amsterdam, version 0.14.1 for the Mac).

Results

Physical and dysmorphological evaluations

Table 1 shows the body weight, height, BMI and head circumference of the infants characterized by meconium positive of not to ethanol metabolites.




As shown by ANOVA no differences between groups were found. Furthermore, according to the data obtained from the statistical analysis, there were no statistically significant differences between the two groups of infants for the physical and dysmorphological parameters characterizing FASD. Indeed, OFC, ICD, PFL, philtrum, vermilion, honey stick and epicanthus were comparable between the two groups (data not shown). Furthermore, no Spearmen correlations were found between FASD features and body weight, height, BMI and head circumference. None of the 8 children who tested positive and then exposed to alcohol during intrauterine life had facial abnormalities attributable to severe FASD52. Growth percentile measurements were within the normal age ranges in all infants.

Neurodevelopment assessment (Griffiths Mental Developmental Scale)

In order to evaluate the effects in terms of psychomotor skills in the children enrolled in the study the GMDS test was used. Figure 1 shows the developmental quotients of the main Griffiths subscales and the total developmental quotients expressed as the sum of the 5 main subscales.




In particular, no significant differences induced by gestational alcohol exposure were revealed by ANOVA for the GMDS domains dealing with learning, language and communication, hand-eye coordination, and personal-social-emotional. However, the outcomes regarding the general motor coordination (figure 2) disclosed a delay in the fine regulation of locomotion expressed as equivalent age, developmental quotient, and percentile [F(1,13)=8.97, 9.98, 11.90; ps<0.05; respectively].




Discussion

To the best of our knowledge, this is the first study to demonstrate a significant delay in general motor coordination expressed as fine locomotion in a year and a half-infants with meconium positive to ethanol metabolites as EtG and FAEEs but without displaying any morphological feature of severe FASD. Notably, EtG and FAEEs in the meconium are biomarkers with high specificity for prenatal alcohol exposure in infants as previously mentioned.

According to the results of the present study, elevations in EtG and FAEEs in the meconium were significantly associated with a lower degree of neurodevelopment and adaptive functions within the first year and a half of life. None of the 8 alcohol metabolites positive meconium infants exhibited distinctive facial features and growth retardation of FAS or partial FAS. These findings suggest that other factors may contribute to the FAS onset such as paroxysmal alcohol drinking by the mother, genetic and epigenetics, unappropriated nutrition by the mother, and concomitant presence of both other substance abuse and gestational associated pathologies53-56. Indeed, animal model studies clearly showed that the supplementation in the diet of natural antioxidants may counteract the damage induced by alcohol abuse57-64. Nonetheless, the recruited alcohol-exposed infants of the present investigation may only show subtle fetal alcohol effects at birth but manifest neurodevelopmental abnormalities at a later time16,65.

Our report showed a delay in fine locomotor skills as assessed by GMDS in infants with meconium positive for FAEEs and EtG. Indeed, the group not exposed to alcohol had scores above the medium range, with an average quotient of 102.3 in locomotion ability, while the group of children exposed to alcohol was placed in the low range, with a mean score of 87.7 (SD= 2.4). Motor function impairment has been observed in children with FAS, but also in children exposed to alcohol in utero who did not meet FAS criteria66. A previous study showed that high and frequent alcohol consumption during pregnancy was related to abnormal walking and balance, and grasping and motor coordination at 1 year of age67. In another study investigating the performance of children whose mothers abused alcohol and drugs heavily during pregnancy, it was shown that the developmental performance of preschool children exposed to alcohol and drugs prenatally was, on average, substantially lower than expected for age regardless of the study group68. However, it should be noted that to evaluate the accuracy of motor assessment and cognition tools several factors should be taken into account such as diagnostic accuracy of motor assessment tools and subtests, accuracy of alternate cut-offs and accuracy of multiple subtests versus total scores66.

In other studies investigating the relationship between meconium FAEEs and neurodevelopment in infants by using the Bayley’s Mental Developmental Index (MDI) and Psychomotor Developmental Index (PDI) scores it was found that increased FAEEs were significantly associated with poorer mental and psychomotor development during the first 2 years of age69,70.

At the same time, neurodevelopmental delays with cognitive retardation compared with the general population and behavioral alterations were found in children exposed to alcohol during pregnancy with positive meconium for EtG or FAEEs71. Indeed, it was found that children with greater concentrations of FAEEs in the meconium were associated with lower Wechsler Intelligence Scales for Children-Fourth Edition Verbal Comprehension Index, Working Memory Index, and Full-Scale IQ scores72. Similarly, primary-school-age children with the meconium positive for EtG: i) allocated fewer attentional resources than controls to the go/no-go task; ii) had reduced IQ; and iii) and displayed ADHD-related behavior70,73,74. Furthermore, in a recent study, it was found that higher concentrations of FAEEs in the meconium are potential markers for children at risk for aggressive and delinquent behaviors related to the effects of prenatal alcohol exposure75.

This study has clearly some limitations. The number of the recruited infants was small (but this depends also on the relatively restricted inclusion/exclusion criteria of the study and because the outcomes of this investigation derive from a single University hospital), so some biases could have happened. Besides, the absence of longer clinical info from additional follow-ups could be considered a study limit.

Conclusions

In conclusion, this study clearly discloses that infants with meconium positive for both EtG and FAEEs, although without displaying any FASD morphological features, had a delay in the fine regulation of their own locomotory capabilities. Furthermore, these findings once again emphasize: i) the importance, for women during before and during pregnancy, but also for the father76, of carrying on the gestation in the healthiest mode possible, avoiding any kind of superfluous stress, which involves smoking, the intake of alcohol, and obviously, the consumption of any kind of drugs and ii) the importance of postpartum intensive hospital controls and monitoring in newborns and infants when FASD is questioned77. Of course, further analyses are needed, however, this investigation provides an additional step in the field of FASD in order to suggest early treatments for at-risk newborns and infants. These data could be of interest to experts involved in the study of prenatal alcohol exposure-associated disorders too.

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

References

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

2. Clarren SK, Salmon A. Prevention of fetal alcohol spectrum disorder: proposal for a comprehensive approach. Expert Rev Obstet Gynecol 2010; 5: 23-30.

3. May PA, Baete A, Russo J, Elliott AJ, Blankenship J, Kalberg WO, et al. Prevalence and Characteristics of Fetal Alcohol Spectrum Disorders. Pediatrics 2014;134:855-66.

4. Hoyme HE, Kalberg WO, Elliott AJ, Blankenship J, Buckley D, Marais AS, et al. Updated clinical guidelines for diagnosing fetal alcohol spectrum disorders. Pediatrics 2016;138:e20154256-e20154256.

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

6. Jacobson JL, Akkaya-Hocagil T, Ryan LM, Dodge NC, Richardson GA, Olson HC, et al. Effects of prenatal alcohol exposure on cognitive and behavioral development: findings from a hierarchical meta-analysis of data from six prospective longitudinal U.S. cohorts. Alcohol Clin Exp Res 2021; 45: 2040-2058.

7. Burd L, Carlson C, Kerbeshian J, Burd L, Kerbeshian J. Fetal alcohol spectrum disorders and mental illness. Int J Disabil Hum Dev 2007;6:383-96.

8. 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: 1046-1062.

9. May PA, Gossage JP, Marais AS, Hendricks LS, Snell CL, Tabachnick BG, et al. Maternal risk factors for fetal alcohol syndrome and partial fetal alcohol syndrome in South Africa: A third study. Alcohol Clin Exp Res 2008;32:738-53.

10. Sebastiani G, Borrás-Novell C, Casanova MA, et al. The effects of alcohol and drugs of abuse on maternal nutritional profile during pregnancy. Nutrients 2018; 10: 1008.

11. Andersson E, Elliott E. Economic costs of Fetal Alcohol Specturm Disorder (FASD). J Paediatr Child Health 2018; 54: 7-7.

12. Lupton C, Burd L, Harwood R. Cost of Fetal Alcohol Spectrum Disorders. Am J Med Genet C Semin Med Genet 2004; 127C: 42-50.

13. Greenmyer JR, Popova S, Klug MG, Burd L. Fetal alcohol spectrum disorder: a systematic review of the cost of and savings from prevention in the United States and Canada. Addiction 2020; 115: 409-17.

14. Garcia-Algar O, Kulaga V, Gareri J, et al. Alarming prevalence of fetal alcohol exposure in a Mediterranean city. Ther Drug Monit 2008; 30: 249-54.

15. 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.

16. Bertrand J, Floyd LR, Weber MK; Fetal Alcohol Syndrome Prevention Team, Division of Birth Defects and Developmental Disabilities, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention (CDC). Guidelines for identifying and referring persons with fetal alcohol syndrome. MMWR Recomm Rep 2005; 54: 1-14.

17. 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.

18. 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.

19. 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.

20. Sobell LC, Agrawal S, Annis H, et al. Cross-cultural evaluation of two drinking assessment instruments: Alcohol timeline followback and inventory of drinking situations. Subst Use Misuse 2001; 36: 313-31.

21. Floyd RL, Decouflé P, Hungerford DW. Alcohol use prior to pregnancy recognition. Am J Prev Med 1999; 17: 101-7.

22. Lange S, Rovet J, Rehm J, Popova S. Neurodevelopmental profile of Fetal Alcohol Spectrum Disorder: a systematic review. BMC Psychol 2017; 5: 22.

23. 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.

24. Pichini S, Morini L, Marchei E, et al. Ethylglucuronide and ethylsulfate in meconium to assess gestational ethanol exposure: preliminary results in two Mediterranean cohorts. Can J Clin Pharmacol 2009; 16: e370-e375.

25. Gomez-Roig MD, Marchei E, Sabra S, et al. Maternal hair testing to disclose self-misreporting in drinking and smoking behavior during pregnancy. Alcohol 2018; 67: 1-6.

26. Joya X, Marchei E, Salat-Batlle J, et al. Fetal exposure to ethanol: relationship between ethyl glucuronide in maternal hair during pregnancy and ethyl glucuronide in neonatal meconium. Clin Chem Lab Med 2016; 54: 427-35.

27. Morini L, Marchei E, Tarani L, et al. Testing ethylglucuronide in maternal hair and nails for the assessment of fetal exposure to alcohol: comparison with meconium testing. Ther Drug Monit 2013; 35: 402-7.

28. Kaphalia BS, Cai P, Khan MF, Okorodudu AO, Ansari GAS. Fatty acid ethyl esters: markers of alcohol abuse and alcoholism. Alcohol 2004; 34: 151-8.

29. Berrigan P, Andrew G, Reynolds JN, Zwicker JD. The cost-effectiveness of screening tools used in the diagnosis of fetal alcohol spectrum disorder: a modelled analysis. BMC Public Health 2019; 19: 1746.

30. Luiz DM, Foxcroft CD, Stewart R. The construct validity of the griffiths scales of mental development. Child Care Health Dev 2001; 27: 73-83.

31. Tarani L, Carito V, Ferraguti G, et al. Neuroinflammatory markers in the serum of prepubertal children with Down Syndrome. J Immunol Res 2020; 2020: 6937154.

32. May PA, Fiorentino D, Phillip Gossage J, et al. Epidemiology of FASD in a province in Italy: prevalence and characteristics of children in a random sample of schools. Alcohol Clin Exp Res 2006; 30: 1562-75.

33. Tarani L, Micangeli G, Rasio D, et al. Clinical and genetic approach to the dysmorphic child. Biomed Rev 2018; 29: 37-46.

34. Tarani L, Rasio D, Tarani F, et al. Pediatrics for disability: a comprehensive approach to children with syndromic psychomotor delay. Curr Pediatr Rev 2021; 18: 110-20.

35. Guerri C, Bazinet A, Riley EP. Foetal Alcohol Spectrum Disorders and alterations in brain and behaviour. Alcohol Alcohol 2009; 44: 108-14.

36. La Maida N, Di Trana A, Mannocchi G, Zaami S, Busardò FP. Sensitive and reliable gas chromatography tandem mass spectrometry assay for ethyl glucuronide in neonatal meconium. J Pharm Biomed Anal 2019; 175: 112743.

37. American Academy of Forensic Science. Standard Practices for Method Validation in Forensic Toxicology, 2019.

38. 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.

39. Carito V, Parlapiano G, Rasio D, et al. Fetal alcohol spectrum disorders in pediatrics. FASD and the pediatrician. Biomed Rev 2018; 29: 27.

40. Messina MP, D’Angelo A, Battagliese G, et al. Fetal alcohol spectrum disorders awareness in health professionals: implications for psychiatry. Riv Psichiatr 2020; 55: 79-89.

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

42. Astley S. Diagnostic Guide for Fetal Alcohol Spectrum Disorders. The 4-Digit Diagnostic Code (Third Editon). Third-200. Seattle, Washington: FAS Diagnostic and Prevention Network, University of Washington, 2004.

43. 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.

44. Green E, Stroud L, Bloomfield S, et al. Griffiths Scales of Child Development (3rd ed.). Oxford: Hogrefe, 2016.

45. Green EM, Stroud L, Marx C, Cronje J. Child development assessment: practitioner input in the revision for Griffiths III. Child Care Health Dev 2020; 46: 682-91.

46. Luiz DM, Foxcroft CD, Povey JL. The Griffiths Scales of Mental Development: a factorial validity study. South African J Psychol 2006; 36: 192-214.

47. Chaudhary T, Walch E, Herold B, et al. Predictive and concurrent validity of standardized neurodevelopmental examinations by the Griffiths scales and Bayley scales of infant development II. Klin Padiatr 2013; 225: 9-13.

48. Luiz DM, Foxcroft CD, Tukulu AN. The Denver II Scales and the Griffiths Scales of Mental Development: a correlational study. J Child Adolesc Ment Heal 2004; 16: 77-81.

49. Conn P. The relations between Griffiths Scales assessments in the pre‐school period and educational outcomes at 7+ years. Child Care Health Dev 1993; 19: 275-89.

50. Granziera F, Guzzardi MA, Iozzo P. Associations between the mediterranean diet pattern and weight status and cognitive development in preschool children. Nutrients 2021; 13; 3723.

51. Fiore M, Minni A, Cavalcanti L, et al. The impact of alcohol consumption and oral microbiota on upper aerodigestive tract carcinomas: a pilot study. Antioxidants 2023; 12: 1233.

52. Davies L, Dunn M, Chersich M, et al. Developmental delay of infants and young children with and without fetal alcohol spectrum disorder in the Northern Cape Province, South Africa. Afr J Psychiatry 2011; 14: 298-305.

53. Ciafrè S, Carito V, Ferraguti G, et al. How alcohol drinking affects our genes: an epigenetic point of view. Biochem Cell Biol 2019; 97: 345-56.

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

55. Smith SM, Virdee MS, Eckerle JK, et al. Polymorphisms in SLC44A1 are associated with cognitive improvement in children diagnosed with fetal alcohol spectrum disorder: an exploratory study of oral choline supplementation. Am J Clin Nutr 2021; 114: 617-27.

56. Gutherz OR, Deyssenroth M, Li Q, et al. Potential roles of imprinted genes in the teratogenic effects of alcohol on the placenta, somatic growth, and the developing brain. Exp Neurol 2022; 347: 113919.

57. Carito V, Ceccanti M, Ferraguti G, et al. NGF and BDNF alterations by prenatal alcohol exposure. Curr Neuropharmacol 2019; 17: 308-17.

58. Petrella C, Carito V, Carere C, et al. Oxidative stress inhibition by resveratrol in alcohol-dependent mice. Nutrition 2020; 79-80: 110783.

59. Carito V, Ceccanti M, Cestari V, et al. Olive polyphenol effects in a mouse model of chronic ethanol addiction. Nutrition 2017; 33: 65-9.

60. Fiore M, Messina MP, Petrella C, et al. Antioxidant properties of plant polyphenols in the counteraction of alcohol-abuse induced damage: impact on the Mediterranean diet. J Funct Foods 2020; 71: 104012.

61. Kumar A, Singh CK, LaVoie HA, DiPette DJ, Singh US. Resveratrol restores Nrf2 level and prevents ethanol-induced toxic effects in the cerebellum of a rodent model of fetal alcohol spectrum disorders. Mol Pharmacol 2011; 80: 446-57.

62. Muralidharan P, Connors CT, Mohammed AS, et al. Turmeric extract rescues ethanol-induced developmental defect in the Zebrafish Model for Fetal Alcohol Spectrum Disorder (FASD). J Food Sci 2017; 82: 2221-5.

63. Almeida-Toledano L, Andreu-Fernández V, Aras-López R, García-Algar Ó, Martínez L, Gómez-Roig MD. Epigallocatechin gallate ameliorates the effects of prenatal alcohol exposure in a fetal alcohol spectrum disorder-like mouse model. Int J Mol Sci 2021; 22: 1-24.

64. Farhadi L, Hojati V, Khaksari M, Vaezi G. Neuroprotective effects of crocin against ethanol neurotoxicity in the animal model of Fetal Alcohol Spectrum Disorders. Neurochem Res 2022; 47: 1001-11.

65. Little BB, Snell LM, Rosenfeld CR, Gilstrap LC, Gant NF. Failure to recognize Fetal Alcohol Syndrome in newborn infants. Am J Dis Child 1990; 144: 1142-6.

66. Johnston D, Branton E, Rasmuson L, Schell S, Gross DP, Pritchard-Wiart L. Accuracy of motor assessment in the diagnosis of fetal alcohol spectrum disorder. BMC Pediatr 2019; 19: 171.

67. Jacobson JL, Jacobson SW, Sokol RJ, Martier SS, Ager JW, Kaplan‐Estrin MG. Teratogenic effects of alcohol on infant development. Alcohol Clin Exp Res 1993; 17: 174-83.

68. Kartin D, Grant TM, Streissguth AP, Sampson PD, Ernst CC. Three-year developmental outcomes in children with prenatal alcohol and drug exposure. Pediatr Phys Ther 2002; 14: 145-53.

69. Peterson J, Kirchner HL, Xue W, Minnes S, Singer LT, Bearer CF. Fatty acid ethyl esters in meconium are associated with poorer neurodevelopmental outcomes to two years of age. J Pediatr 2008; 152: 788-92.

70. Koren G, Cohen R. Quantifying fetal alcohol exposure by meconium fatty acid ethyl esters (FAEE): association with adverse fetal outcomes and population estimates of fetal alcohol exposure. Drug Metab Rev 2019; 51: 524-32.

71. Kalberg WO, Provost B, Tollison SJ, et al. Comparison of motor delays in young children with fetal alcohol syndrome to those with prenatal alcohol exposure and with no prenatal alcohol exposure. Alcohol Clin Exp Res 2006; 30: 2037-45.

72. Min MO, Singer LT, Minnes S, Wu M, Bearer CF. Association of fatty acid ethyl esters in meconium and cognitive development during childhood and adolescence. J Pediatr 2015; 166: 1042-7.

73. Eichler A, Hudler L, Grunitz J, et al. Effects of prenatal alcohol consumption on cognitive development and ADHD-related behaviour in primary-school age: a multilevel study based on meconium ethyl glucuronide. J Child Psychol Psychiatry Allied Discip 2018; 59: 110-8.

74. Grimm J, Stemmler M, Golub Y, et al. The association between prenatal alcohol consumption and preschool child stress system disturbance. Dev Psychobiol 2021; 63: 687-97.

75. Singer LT, Min MO, Momotaz H, Powers G, Minnes S, Bearer CF. Association of fatty acid ethyl esters in meconium with behavior during childhood. Drug Alcohol Depend 2021; 218: 108437.

76. Terracina S, Ferraguti G, Tarani L, et al. Transgenerational abnormalities induced by paternal preconceptual alcohol drinking. Findings from humans and animal models. Curr Neuropharmacol 2021; 19: 1158-73.

77. Williams L, Jackson CPT, Choe N, Pelland L, Scott SH, Reynolds JN. Sensory-motor deficits in children with Fetal Alcohol Spectrum Disorder assessed using a robotic virtual reality platform. Alcohol Clin Exp Res 2014; 38: 116-25.