The aim of the study is to determine the prevalence of alcohol and drug use among Slovenian pregnant women. Alcohol and drug use during pregnancy has significant effects on the foetus and has short- and long-term health consequences. One of the most serious consequences is foetal alcohol syndrome, which is characterised by congenital anomalies, cognitive impairment and growth deficits. The prevalence of alcohol and drug use during pregnancy will be investigated in collaboration with the National Institute of Public Health, the Institute of Forensic Medicine of the Faculty of Medicine, University of Ljubljana, Institute Jozef Stefan and the Clinical Department of Neonatology of the Paediatric Clinic, University Clinical Centre Ljubljana, using two different methods. A cross-sectional study will be conducted on a nationally representative sample of meconium samples. Meconium samples will be collected from maternity hospitals in Slovenia. Biomarkers for alcohol and illicit drugs will be determined in the meconium samples.Meconium samples will be collected anonymously. National survey on the lifestyle will be conducted in Slovenian maternity hospitals, including questions on alcohol and drug use during pregnancy. The survey will be anonymous and not related to meconium sampling; participation in the survey will be voluntary. Data linkage between questionnaire responses and meconium analysis will be performed only within the additional subgroup of participants who provided informed consent. Meconium samples that will test positive for either alcohol of canabis will be analysed for microbiome. For every poisitve meconium sample, two negative samples will be analysed.

The aim of the study is to determine the prevalence of alcohol and canabis use among Slovenian pregnant women. Alcohol and drug use during pregnancy has significant effects on the foetus and has short- and long-term health consequences. One of the most serious consequences is foetal alcohol syndrome (FAS), which is characterised by congenital anomalies, cognitive impairment and growth deficits. Another health effect of prenatal alcohol exposure is foetal alcohol spectrum disorder (FASD), which is characterised by impairment of the central nervous system and leads to behavioural and cognitive problems, attention deficit disorder, executive dysfunction and memory problems. Consequently, children affected by maternal alcohol abuse have poorer school performance, lower education, psychiatric disorders, higher rates of alcohol and drug use, higher crime rates, unemployment and other problems that place a burden on individuals and society. Drug use during pregnancy affects both the mother and the foetus and is closely associated with perinatal problems, miscarriages, premature births, low birth weight, small head circumference and congenital malformations. Maternal drug use during pregnancy causes neonatal abstinence syndrome; it also affects foetal brain development, which can lead to long-term neuropsychiatric problems. The prevalence of alcohol and drug use among pregnant women in Slovenia has not been studied before.

The adult gastrointestinal tract is densely populated by microorganisms. The microbiota, which represents a taxonomic inventory of the present microbial genera and species-primarily bacteria-and, in a broader sense, the microbiome, which encompasses all microorganisms (bacteria, archaea, fungi, protozoa, viruses, mobile genetic elements) together with their functional genes and metabolites present in the intestine, varies considerably in its composition among individuals, depending on the resolution of observation. At birth, a child's gastrointestinal tract has a lower density of microbial cells, as a network of selective pressures begins to develop only upon contact with maternal secretions, food, and the environment. These pressures include microbial fermentative products derived from breast milk or infant formula, gene expression, and microbial competition, leading to the dynamic development of the microbiome. Several factors influence the development of the infant gut microbiome after birth, including mode of delivery, type of feeding, prematurity, antibiotic use, and other environmental factors. Increasing evidence also points to the influence of prenatal factors, such as gestational diabetes and obesity, infections, stress, and maternal diet. A growing number of studies further demonstrate that an altered microbiota during pregnancy and early childhood can affect brain development and behavior later in life via the gut-brain axis. The impact of alcohol consumption on the gut microbiota has been studied mainly in adults, in whom a reduction in microbiota α-diversity has been observed. Prenatal exposure to alcohol may influence the newborn's microbiota primarily through alcohol-induced alterations of the maternal microbiota and, consequently, nutrient absorption. Meconium analysis represents a unique opportunity to study exclusively prenatal factors affecting the microbiome, as postnatal factors do not influence the meconium microbiome. Wang and colleagues studied mother-newborn pairs by analyzing maternal stool samples in the third trimester and neonatal samples within the first 48 hours after birth. They demonstrated significant differences in the microbiota composition of mothers who consumed alcohol and in the microbiota of their newborns compared with mother-newborn pairs in which the mothers did not consume alcohol. There are only a few studies in animal models that have confirmed differences in the gut microbiota of adult offspring whose mothers were fed an alcohol-containing diet during pregnancy. To date, no published studies have characterized the meconium microbiome of newborns whose mothers consumed cannabis.

Methods: The prevalence of alcohol and canabis use during pregnancy will be investigated in collaboration with the National Institute of Public Health, Institute of Forensic Medicine (IFM) of the Faculty of Medicine, University of Ljubljana, Institute Jozef Stefan and the Clinical Department of Neonatology of the Paediatric Clinic, University Clinical Centre Ljubljana, using three different methods:

1. A cross-sectional study will be conducted on a nationally representative sample of meconium samples. The meconium samples will be collected from maternity hospitals in Slovenia. Biomarkers for alcohol and illicit drugs will be determined in the meconium samples. The sample size of the study will be calculated considering the population of Slovenia, the number of births per year and the estimated prevalence of alcohol and drug use during pregnancy. Meconium samples will be collected anonymously. Diapers with meconium will be collected in special waste bags by professionals caring for newborns and mothers on the first day after birth. Diapers for the meconium sampling will be randomly taken from the bags; the sample will be labelled with a serial number. Data about neonate's sex, gestational age (mature/premature), type of birth (vaginal/caesarean), antibiotic use during labour and maternal chronic diseases will be colected anonimously without any other personal or time data. There will be a special group of 100 meconium samples from one maternity hospital, where we will pair mother's questionaire and meconium sample. For this we will ask for mother's permission. Here also there will be no personal data collection, the meconium sample and mother's questionaire will be paired through code. The samples will be analysed in the IFM toxicology laboratory. For the study, a new analytical method for the examination of meconium will be introduced in Slovenia.
2. National survey on the lifestyle will be conducted in Slovenian maternity hospitals, including questions on alcohol and drug use during pregnancy. The survey will be anonymous and not related to meconium sampling; participation in the survey will be voluntary. Women will be given access to the online survey, after receiving information about the aim of the study, the method used to ensure anonymity and the collection methods.
3. Microbiome analysis: all meconium samples that will test positive for alcohol and cannabis metabolites, as well as two negative meconium samples per every positive meconimu sample, will be included in the analysis of meconium microbiome composition. In the final part of the study-the comparison of the microbiome-all meconium samples positive for alcohol or cannabis metabolites will be included. Microbiome analysis will be carried out in the final phase of the study within the framework of the Million Microbiomes from Humans Project consortium and the Slovenian Metabolomics Database (in preparation). The molecular data obtained from metagenomic sequencing will be analyzed on the Slovenian supercomputing infrastructure (Vega, Maister) using tools developed and employed on the supercomputing network by the interdisciplinary group. We will generate taxonomic matrices of bacteria, archaea, fungi, protozoa, and DNA viruses; diversity matrices (35 diversity indices); functional gene matrices (20 million groups); enzymatic reaction matrices (500,000 categories); metabolic pathway matrices (100,000 variables); and metabolite matrices (1,000 variables). The collected data and their transformation from molecular into numerical formats will serve as the basis for machine learning (standardization, normalization, evaluation of sample separation and classification performance using different algorithms and hyperparameters), identification of biomarker networks (n > 100), and nonparametric statistical analysis of the gut microbiome.

Statistical analysis: Statistical data analysis will be performed using the IBM SPSS Statistics 25 software package. Descriptive statistical methods will be used to characterize the population of postpartum women participating in the survey and to summarize the collected data. The prevalence of alcohol or cannabis use among pregnant women will be calculated based on the number of meconium samples positive for alcohol or cannabis metabolites. The prevalence of alcohol and cannabis use based on self-reporting will be calculated separately. Molecular data obtained from microbiome analysis will be converted into numerical data and used for machine learning (standardization, normalization, evaluation of sample separation and classification performance using different algorithms and hyperparameters), identification of biomarker networks (n > 100), and nonparametric statistical analysis of the gut microbiome, as conducted by the interdisciplinary group. The obtained data will then be statistically compared between the group of meconium samples positive for alcohol and cannabis metabolites and the group of meconium samples in which these metabolites were not present.

Expected results: We aims to determine the prevalence of alcohol and drug use among Slovenian pregnant women. The data on alcohol and drug metabolites obtained from meconium samples will be compared with the results of the survey, in which the prevalence of alcohol and drug use will be determined by self-report. In this way, we can assess the reliability of self-report. The results of the study will enable us to assess the prevalence of FAS and FASD in Slovenia, help us to develop a strategy to raise awareness among professionals and the public about the prevalence and consequences of alcohol and drug use during pregnancy, to plan preventive measures and interventions in the target group of pregnant women, to establish diagnostic teams for FAS and FASD. As of the microbiome, we expect a significant difference in the microbiome composition of meconium samples that test positive for alcohol or cannabis compared with samples that test negative (taking into account sex, gestational age, antibiotic exposure during delivery, and the presence of chronic diseases).

Inclusion Criteria meconium:

• meconium samples of preterm and term neonates

Exclusion Criteria meconium:

• if the neonate passes meconium intrauterine (meconium stained amniotic fluid) or during the labour

Inclusion Criteria for structured questionnaire:

- any postpatrum women

Probability sampling will be applied to the selection of meconium samples, which will be collected randomly. In contrast, postpartum women will be recruited upon invitation, representing a non-probability (convenience) sample.

• Random and anonymous meconium samples collected across Slovenian maternity hospitals during the defined study period.
• Postpartum women who voluntarily participated by completing the structured questionnaire.

Beginning 6 months and ending 3 years after the publication of results

Individual participant data (IPD) and supporting information will be made available to researchers who provide a methodologically sound proposal and justify their request. Access will be granted upon reasonable request, subject to approval by the study investigators and in accordance with applicable ethical and data protection regulations. Requests should be directed to the corresponding author.