{"paper_id":"869f851d-6f9c-487f-8c93-454762b8b78c","body_text":"1\n1 Tobacco, nicotine, and cannabis use and exposure in an \n2 Australian Indigenous population during pregnancy: A protocol \n3 to measure parental and foetal exposure and outcomes.\n4\n5\n6 Angela Ratsch 1,2, Elizabeth A. Burmeister1,2, Aunty Veronica Bird3, Aunty Joyce \n7 Bonner 3, Uncle Glen Miller3,4, Aunty Marj Speedy3, Graham Douglas5, Stevan Ober5, \n8 Ann Woolcock 5, Sharly Blair (nee Murdoch)5, Min-Tz Weng6, Jared A. Miles6, Kathryn \n9 J. Steadman 6\n10\n11\n12 1 Wide Bay Hospital and Health Service, Hervey Bay, Australia\n13 2 Rural Clinical School, The University of Queensland, Brisbane, Australia\n14 3 Butchulla Aboriginal Corporation, Fraser Coast, Australia\n15 4 Butchulla Mens Business Association, Fraser Coast, Australia\n16 5 Galangoor Duwalami Primary Healthcare Service, Fraser Coast, Australia\n17 6 School of Pharmacy, The University of Queensland, Brisbane, Australia\n18\n19 *Corresponding author\n20 E-mail: angela.ratsch@health.qld.gov.au\n21\n22\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \nNOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.\n\n2\n23 Tobacco, nicotine, and cannabis use and exposure in an \n24 Australian Indigenous population during pregnancy: A protocol \n25 to measure parental and foetal exposure and outcomes.\n26 Abstract \n27 Background: The Australian National Perinatal Data Collection collates all live and \n28 stillbirths from States and Territories in Australia. In that database, maternal \n29 cigarette smoking is noted twice (smoking <20 weeks gestation; smoking >20 weeks \n30 gestation). Cannabis use and other forms of nicotine use, for example vaping and \n31 nicotine replacement therapy, are nor reported . The 2021 report shows the rate of \n32 smoking for Australian Indigenous mothers was 42% compared with 11% for \n33 Australian non-Indigenous mothers. Evidence shows that Indigenous babies exposed \n34 to maternal smoking have a higher rate of adverse outcomes compared to non-\n35 Indigenous babies exposed to maternal smoking.\n36\n37 Objectives: The reasons for the differences in health outcome between Indigenous \n38 and non-Indigenous pregnancies exposed to tobacco and nicotine is unknown but will \n39 be explored in this project through a number of activities. Firstly, the patterns of \n40 parental and household tobacco, nicotine and cannabis use and exposure will be \n41 mapped during pregnancy.  Secondly, a range of biological samples will be collected \n42 to enable the first determination of Australian Indigenous people’s nicotine and \n43 cannabis metabolism during pregnancy; this assessment will be informed by \n44 pharmacogenomic analysis.  Thirdly, the pharmacokinetic and pharmacogenomic \n45 findings will be considered against maternal, placental, foetal and neonatal outcomes.  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n3\n46 Lastly, an assessment of population health literacy and risk perception related to \n47 tobacco, nicotine and cannabis products peri-pregnancy will be undertaken.\n48\n49 Methods: This is a community-driven, co-designed, prospective, mixed-method \n50 observational study with regional Queensland parents expecting an Australian \n51 Indigenous baby and their close house-hold contacts during the peri-gestational \n52 period.  The research utilises a multi-pronged and multi-disciplinary approach to \n53 explore interlinked objectives. \n54\n55 Results: A sample of 80 mothers expecting an Australian Indigenous baby will be \n56 recruited. This sample size will allow estimation of at least 90% sensitivity and \n57 specificity for the screening tool which maps the patterns of tobacco and nicotine use \n58 and exposure versus urinary cotinine with 95% CI within ±7% of the point estimate.  \n59 The sample size required for other aspects of the research is less (pharmacokinetic \n60 and genomic n=50, and the placental aspects n=40), however from all 80 mothers, all \n61 samples will be collected.\n62\n63 Conclusions: Results will be reported using the STROBE guidelines for observational \n64 studies.\n65\n66 Keywords: Tobacco; nicotine; cannabis; exposure; pregnancy, pregnancy outcomes; \n67 Aboriginal and Torres Strait Islander; Australian Indigenous.\n68\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n4\n69 Forward\n70 We acknowledge the Traditional Custodians, the Butchulla people, of the lands and \n71 waters upon which this research is conducted.  We acknowledge their continuing \n72 connections to country and pay our respects to Elders past, present and emerging.\n73\n74 Notation: In this document, the terms Aboriginal and Torres Strait Islander and \n75 Indigenous are used interchangeably for Australia’s First Nations People. No \n76 disrespect is intended, and we acknowledge the rich cultural diversity of the groups \n77 of peoples that are the Traditional Custodians of the land with which they identify \n78 and with whom they share a connection and ancestry.\n79 Introduction\n80\n81 In 1957, Simpson  [1] reported a dose-response association between maternal \n82 smoking and premature birth. This observation resulted in a world-wide research \n83 agenda focusing on the impact of maternal smoking in pregnancy, the findings of \n84 which indicate that maternal tobacco smoking, and exposure to the products of \n85 tobacco combustion (i.e., secondhand smoke exposure) are the leading modifiable \n86 risk behaviours associated with adverse maternal and neonatal outcomes. Maternal \n87 exposure increases the risk for miscarriage, ectopic pregnancy, antepartum bleeding, \n88 placental abruption, placenta previa, postpartum haemorrhage  and alters maternal \n89 thyroid function [2, 3].  Counterintuitively, smoking in pregnancy is associated with \n90 decreased hypertensive disorders of pregnancy [4, 5]. For the foetus, maternal \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n5\n91 exposure increases the risk of stillbirth, premature birth and lower birthweight [6] as \n92 well as increases the risk for a number of congenital abnormalities [7, 8]. Longer-\n93 term, offspring exposed in-utero to maternal smoking have decreased cognitive \n94 achievement [9] and an increased risk for the development of attention-\n95 deficit/hyperactivity disorder (ADHD) [10]. \n96\n97 In Australia, these adverse pregnancy and foetal findings have ensured a prenatal \n98 focus on maternal smoking behaviour with the mother’s cigarette smoking status \n99 obtained during antenatal assessment and recorded in the National Perinatal Data \n100 Collection [11] twice across the nine months of pregnancy (once < 20 weeks \n101 gestation, and once >20 weeks gestation).  In that database, the 2021 rate of smoking \n102 by Australian non-Indigenous expectant mothers was reported as 11% compared \n103 with 42% by Australian Indigenous expectant mothers [12]. However, the maternal \n104 use of other tobacco and nicotine products including e-cigarettes, hookahs, chop-chop \n105 tobacco, nicotine tooth cleaning powder, chewing tobacco, and nicotine spray, mist, \n106 lozenges, gum and patches, and cannabis is not collected.  In addition, this focus on \n107 maternal cigarette smoking fails to recognise second- and third-hand maternal \n108 nicotine vape and tobacco and cannabis smoke exposure, i.e., the impact from \n109 paternal and household tobacco, nicotine and cannabis exposure on maternal and \n110 foetal outcomes is overlooked.\n111 This assessment gap results in maternal and foetal tobacco, nicotine and cannabis \n112 exposure misclassification and ramifications in the planning of care, and in the \n113 estimation of adverse maternal, placental, foetal and neonatal outcomes from \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n6\n114 tobacco, nicotine and cannabis exposure. Nevertheless, the literature indicates that \n115 Indigenous babies exposed to maternal smoking have a higher rate of adverse \n116 outcomes compared to non-Indigenous babies exposed to maternal smoking, for \n117 example, after adjusting for maternal age and other factors, smoking in pregnancy is \n118 attributable to 22% of pre-term Indigenous births compared with 5% for non-\n119 Indigenous births [13].  Gestational age impacts birthweight, and for Indigenous \n120 mothers who smoked in the first 20 weeks of pregnancy, the risk of a lower \n121 birthweight baby was 1.8 (or about 80% higher risk) than among Indigenous mothers \n122 who did not smoke in the first 20 weeks. For non-Indigenous mothers who smoked \n123 in pregnancy, this risk was 1.3 (or 30% higher risk) [14].\n124 The other significant foetal outcome attributable to maternal smoking is stillbirth.  In \n125 Australia, stillbirth is defined as foetal death prior to birth of the baby at 20 weeks \n126 gestation or more, and/or weighing 400 grams or more [15].  In 2020, the overall \n127 Australian stillbirth rate was 7.7/1000 births, with the rate for Indigenous mothers \n128 being 11.9/1000 compared with 7.4/1000 for non-Indigenous mothers.  Smoking in \n129 pregnancy is a risk factor for stillbirth; for women who smoked in pregnancy, the \n130 stillbirth rate was 12.8 stillbirths/1000 births compared to 6.9 stillbirths/1000 births \n131 for mothers who did not smoke [15]. Sub-category analysis of smoking and stillbirth \n132 for Indigenous mothers compared to non-Indigenous mothers is not published.\n133\n134 The reasons for the differences in pregnancy outcome between Indigenous and non-\n135 Indigenous pregnancies exposed to tobacco and nicotine are unknown but will be \n136 explored in this project through a number of activities.\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n7\n137 Background\n138 Nicotine: pregnancy health and the developing human\n139\n140 Currently, tobacco assessment in pregnancy is focused on ‘smoking’.  This emphasis \n141 overlooks the absorption of the pharmacologically active, dose-dependent, \n142 potentially lethal component of tobacco-which is nicotine-from other sources [16]. \n143 This study is premised on some of the actions of nicotine. In brief, nicotine binds with \n144 and activates nicotinic acetylcholine receptors (nAChR) in central and peripheral \n145 neuronal and non-neuronal tissue, at neuromuscular junctions, and in the adrenal \n146 medulla [17]. Receptor type and individual variability, including genetics and \n147 pregnancy, result in receptor up-regulation or desensitization [16, 18] and the release \n148 of neurotransmitters including acetylcholine, norepinephrine, dopamine, serotonin, \n149 vasopressin, beta-endorphin and adreno-corticotropic-hormone, thus impacting the \n150 vasculature and producing vasoconstriction, increasing heart rate and blood pressure \n151 [19].\n152\n153 In pregnancy, nicotine acts both directly on nAChRs in the developing placenta, \n154 reducing the number of nAChR receptors [20], and alters placental morphology and \n155 vascularity [21].  Nicotine also readily crosses the placenta and stimulates nAChRs in \n156 the developing foetus impacting foetal systems, albeit in an immature physiology \n157 [22].  During early foetal development (4-6 weeks of gestation), nAChRs emerge and \n158 begin to form connections throughout the body including the brain [23], with \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n8\n159 construction of axons and synaptic connections in the brain continuing after birth \n160 into childhood, adolescence and young adulthood [24]. In the foetal brain, nicotine \n161 exposure results in accelerated cell development relative to tissue and organ age, that \n162 is, there are fewer cells correctly developed for their stage and age [25]. Following \n163 nicotine exposure, changes in nAChRs and neural plasticity result in both a deficit in \n164 the number of neurons in the foetal brain and synaptic level damage to the \n165 respiratory center, with equivalent damage to the adrenal glands [26].  Foetal \n166 nicotine exposure ultimately results in a dampened response to hypoxic episodes \n167 [27]. These physiological changes are important in understanding links between \n168 maternal nicotine exposure and foetal outcomes, for example, stillbirth and Sudden \n169 Infant Death Syndrome (SIDS). \n170 Nicotine metabolism and excretion\n171\n172 Nicotine has a half-life of about two hours and is metabolised via the CYP2A6 pathway \n173 primarily in the liver, with the brain, kidneys and lungs providing minor sites [28].  \n174 This short half-life produces large nicotine serum plasma fluctuations and poses \n175 challenges for intra- and inter-person comparisons of exposure, consumption and \n176 effect. However, cotinine (the main tobacco and nicotine metabolite) has a half-life of \n177 approximately 17 hours and serum concentrations 10-fold higher than nicotine, \n178 providing a more stable biomarker of tobacco and nicotine exposure [29].  Cotinine \n179 begins to metabolise after approximately 16 hours into trans-3’-hydroxycotinine (3-\n180 OH-cotinine), nornicotine, nicotine glucuronide and nicotine-N-oxide [18].  A more \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n9\n181 accurate assessment of tobacco and nicotine exposure is achieved by measuring \n182 serum tobacco and nicotine and metabolite concentrations (total nicotine equivalents \n183 – TNE), as opposed to measuring only nicotine concentration [30]. \n184\n185 During pregnancy, nicotine metabolism is impacted both by individual variability \n186 [16] and the changes created by pregnancy.  In the expectant mother, there is a \n187 significant induction of CYP2A6 activity which increases plasma clearances of \n188 nicotine by 60% and cotinine by 140%, in addition, there is an almost 50% reduction \n189 in cotinine half-life (down from 17 hours to 9 hours [31]).  This is important in the \n190 consideration of tobacco and nicotine use in pregnancy as decreases in nicotine and \n191 cotinine measurements in late pregnancy compared with pre-pregnancy or early \n192 pregnancy may not necessarily indicate a decrease in tobacco and nicotine exposure, \n193 but rather the more rapid metabolism of nicotine [32].  In the foetus and neonate, the \n194 immature and undeveloped CYP2A6 pathway decreases their ability to metabolise \n195 nicotine and results in a much longer plasma nicotine half-life than adults (11.2 hours \n196 compared to 2 hours) whereas cotinine elimination is similar to that of adults (16.3 \n197 hour half-life compared with 17 hours) [33].\n198\n199 CYP2A6, the major nicotine‐oxidising enzyme, is measurable using the nicotine \n200 metabolite ratio (NMR; 3′hydroxycotinine:cotinine) and is a biomarker of nicotine \n201 clearance [16]  . However, NMR is highly heritable (~80%) varying with ethnicity, in \n202 part due to CYP2A6 variants.  Variation in CYP2A6 genes has not been characterised \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n10\n203 in Australian Indigenous populations and may contribute to increased/decreased \n204 pregnancy risk from tobacco and nicotine exposure.\n205\n206 Tobacco, nicotine and the metabolites are rapidly excreted by the kidneys with the \n207 rate dependent upon urinary pH, where increased urine alkalinity decreases \n208 excretion [34]. Urinary excretion is further impacted in pregnancy due to elevated \n209 creatinine potentially leading to fluctuations in nicotine and its metabolites [35]. \n210 Thus, serum provides a measure of exposure and the recency of that exposure, while \n211 urine analysis provides a measure of tobacco and nicotine metabolism and excretion. \n212 Materials and Methods\n213 Aim and Objectives\n214\n215 The aim of this study is to develop a foundation from which approaches to tobacco \n216 and nicotine assessment, health literacy, tobacco and nicotine cessation, and \n217 maternal and neonatal health care delivery for families expecting an Australian \n218 Indigenous baby is informed by contemporary evidence. Community input on the \n219 design suggested that cannabis use (with and without tobacco) is also prevalent and \n220 continues throughout pregnancy, thus cannabis use and exposure have been \n221 included.\n222\n223\n224\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n11\n225 The objectives are to:\n226 1. Accurately describe parental and close household contact(s) peri-gestational \n227 patterns of use and exposure of tobacco, nicotine and cannabis products \n228 through the creation and use of a validated assessment tool.\n229 2. Determine the pharmacokinetic and pharmacogenomic impacts and outcomes \n230 of tobacco, nicotine and cannabis exposure. This study will be the first to \n231 establish the metabolism of tobacco, nicotine and cannabis during pregnancy \n232 by using NMR as a biomarker of individual differences in nicotine and cannabis \n233 metabolism in a parental Australian Indigenous population.\n234 3. Describe maternal, paternal, placental, foetal and neonatal outcomes \n235 according to the use and exposure to tobacco, nicotine and cannabis products \n236 and biochemical and genomic analysis. \n237 4. Describe the influences and barriers to cessation for pregnant Australian \n238 Indigenous families or close household contacts to reduce or cease tobacco, \n239 nicotine and/or cannabis use in pregnancy.\n240\n241 Research Governance\n242 This project is centred around the local Australian Indigenous population in the \n243 Fraser Coast area (Queensland, Australia). The research will be conducted primarily \n244 from Galangoor Duwalami Primary Healthcare Service (the local Aboriginal and \n245 Torres Strait Islander Primary Health Service in Hervey Bay and Maryborough) and \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n12\n246 at the Hervey Bay and Maryborough Hospitals within the Wide Bay Hospital and \n247 Health Service (WBHHS).\n248\n249 Galangoor Duwalami Primary Healthcare Service were consulted in regard to which \n250 Community members would best represent the Traditional Owners of the land where \n251 this research is to be conducted. A combined leadership group was formed which \n252 included the Butchulla Aboriginal Corporation and Butchulla Men’s Business \n253 Association and this group advised, directed and oversaw the conversations around \n254 this research from its inception and arranged for discussions with the appropriate \n255 Community members. Under the Butchulla Aboriginal Corporation’s Rule Book, the \n256 principal Objective of the corporation is to: ‘Relieve poverty and disadvantage of the \n257 Butchulla People through the advancement of education, health, social or public \n258 welfare, and culture’.  Accordingly, the Elders have approved this proposal and \n259 provided a Butchulla name for the project – Ngabang (mother), Walbai (baby), Babun \n260 (father).  The project logo (Figure 1) reflects the aspirations of the Butchulla people \n261 for this project, which are to strive for a healthy pregnancy, which results in a healthy \n262 family, which maintains a healthy culture. \n263\n264\n265 Figure 1 Project logo\n266 The logo also recognises the governance of the project by the Butchulla people, \n267 Galangoor Duwalami Primary Healthcare Service, the various Australian research \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n13\n268 guidelines, and the collaborative nature of the project with other health providers \n269 and research-intensive organisations and academic partners. The project \n270 documentation, and staff and participant shirts and onesies carry this logo (Figure 2 \n271 and 3)\n272\n273 Figure 2 Participant shirt\n274 Figure 3 Participant onesie\n275 Research context\n276 Galangoor Duwalami Primary Healthcare Service generally maintain the sole care of \n277 the pregnant women until approximately 20 weeks gestation (unless they have a \n278 high-risk pregnancy). The mother then moves to a shared care model with the Hervey \n279 Bay and Maryborough Hospitals. In this project, data is collected across the entire \n280 pregnancy.  The preferred site of data collection from Galangoor Duwalami Primary \n281 Healthcare Service patients will be at Galangoor Duwalami Primary Healthcare \n282 Service for as long as possible during the pregnancy, however some data collection \n283 will need to occur at antenatal visits at the Hervey Bay or Maryborough Hospitals. At \n284 birth (mostly likely at the Hervey Bay Hospital), biological samples will be collected, \n285 and birthing data will be collected on the participant’s discharge from hospital.\n286 Inclusion and exclusion criteria and consent\n287 Potential participants who meet the following criteria will be invited to enrol in the \n288 study: pregnant mothers, aged 15 years or older at the time of enrollment, able to \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n14\n289 understand English, able to provide informed consent, and who self-identify as being \n290 pregnant with an Australian Indigenous baby. In accordance with the value of Respect \n291 outlined in the National Statement of Ethical Conduct in Human Research [36], all \n292 potential maternal participants meeting the inclusion criteria will be carefully \n293 considered by the clinic midwife or healthcare medical officer to ensure the \n294 healthcare staff believe that potential enrolment is in the best interest of the \n295 participant at this point in their pregnancy. A list of suitable potential maternal \n296 participants will then be provided to the researchers; only those participants will be \n297 provided with research information.\n298\n299 The project will strive to enrol the family unit i.e., the mother, and the foetus, and the \n300 biological father, or non-biological parent partner (the parents), or one close \n301 household contact.  For example, if an expectant mother lives with her sister or aunty \n302 or grandmother but there is no biological father in the family at that time, or there is \n303 a partner (of either gender) and that close family contact attends the antenatal visits \n304 with the maternal participant, that person will be invited to participate with the \n305 expectant mother. Family members do not have to participate; however, ‘family’ is \n306 central to Australian Indigenous people, and enrolling participants who constitute the \n307 cultural norm of family will be especially important in the translation of findings to \n308 the participant and the community.\n309\n310 Consent: Participants can choose to enrol in any or all aspects of the data and sample \n311 collection, and they can withdraw from any or all aspects of the research at any time. \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n15\n312 The consent process enables participants to choose to have their individual results \n313 provided back to them at the completion of the project; for any or all samples to \n314 returned to the participant on completion of this project; or for the de-identified \n315 samples and de-identified data to be retained for ethically approved un-identified \n316 projects in the future. All enrolled participants will receive a participant shirt (Figure \n317 2), and liveborn newborns will receive a project onesie (Figure 3). \n318\n319 A subset of information-rich tobacco, nicotine and/or cannabis use/exposed \n320 participants will be enrolled into the separate male and female qualitative aspects of \n321 the study until saturation is reached (~30 participants).  Data will be collected in \n322 response to a range of trigger questions and survey questions. Participants enrolled \n323 in the qualitative data collection will receive a $50 grocery voucher in \n324 acknowledgement of their time to the project.\n325\n326 Tobacco, nicotine and cannabis use and exposure data and biological sample collection\n327 The quantitative data and sample collection for Objectives 1-3 is designed to \n328 measure maternal tobacco, nicotine and cannabis use, and maternal and foetal \n329 exposure, metabolism and excretion at varying times throughout the pregnancy and \n330 assess the findings against the maternal and foetal outcomes.  At each antenatal \n331 appointment and at birth, tobacco, nicotine and cannabis use and exposure \n332 information will collected on a NicOTIne, tobacco, and Cannabis use and Exposure \n333 (NOTICE) Assessment Tool (Figure 4 and see Box 1 NOTICE Notes). \n334\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n16\n335 Figure 4 NOTICE: NicOTIne, tobacco, and Cannabis use and Exposure (NOTICE) \n336 Assessment Tool \n337\n338 Box 1. NicOTIne tobacco, and Cannabis use and Exposure (NOTICE) \n339 Assessment Tool – Notes.\nThe NicOTIne tobacco, and Cannabis use and Exposure (NOTICE) assessment tool \n(Figure 4) has been developed by Angela Ratsch for this study to enable a \ncomprehensive assessment of the use of smoked and smokeless tobacco and cannabis \nproducts as well as nicotine containing products. The tool also allows for the detailing \nof second-hand exposure, and is used in this protocol to inform the use and exposure \nof all products throughout the pregnancy. The recorded information facilitates a \nscoring method to determine the level of self-reported use and exposure to tobacco, \nnicotine and cannabis products (scored for the day of assessment, previous 24 hours, \nand the previous week). \nA self-use weekly score will be calculated for each assessment completed. Scoring for \nself-use will be categorized for each product as:  0 - no use; 1 – low use (1 to 70 \ncombusted or inhaled products); 2 – medium use (71-140 combusted or inhaled \nproducts); 3 – high use (141 + combusted or inhaled products). A self-use score will \nbe calculated for each weekly product combusted or inhaled with the sum of these \nscores producing a total weekly self-use score with a range of 0-30.  An exposure score \nwill be calculated for each weekly product exposed to by multiplying the quantity \nexposed to score by the exposure. Exposure is categorized as 0 - no exposure; 1- some \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n17\nlimited exposure; 2 -moderate exposure or 3 - unrestricted exposure. A total weekly \nexposure score is then calculated by summing all the exposure scores with a range of \n0-99 for each assessment completed. \nThe assessment takes about 3 minutes to complete and will be conducted at each \nantenatal visit and immediately prior to birth.\nExhaled breathe carbon monoxide (CO) assessment\nCO as a point of care (POC) assessment provides indicative information about \ncombusted tobacco/cannabis use up to 24 hours previously. Exhaled CO monitors \n(Smokerlyser®) provide a CO ppm reading ranging between 0 and 30 and a \npercentage of oxygen replaced with CO on haemoglobin (%COHb) with higher \nreadings indicating higher levels of combusted tobacco/cannabis use. The same device \ncan measure foetal exposure as foetal carboxyhaemoglobin (%FCOHb).  Readings for \nthe level of CO ppm are categorized as:\n green—for no and general environmental combusted tobacco/cannabis \nexposure, indicated by CO <3 ppm for pregnant women, and CO <6 ppm for \nnon-pregnant adults; \n orange—moderate combusted tobacco/cannabis exposure, indicated by CO 4-\n6 ppm for pregnant women, and CO 7-10 ppm in non-pregnant adults and; \n red—heavy combusted tobacco/cannabis exposure, indicated by CO >6 ppm \nfor pregnant women, and CO >10 ppm in non-pregnant adults.\nThe assessment takes about 1 minute to complete and will be conducted at each \nantenatal visit and immediately prior to birth.\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n18\nSaliva cotinine assessment\nThe use of non-combusted tobacco products and nicotine substances, for example, \nchewed tobacco, e-cigarettes, nicotine patches and gum, does not create CO.  Instead, \ncotinine saliva assessment (Oz Drug Tests®), as a POC measurement, can detect \ncotinine (the major metabolite of nicotine metabolism). The test has a cotinine cut-off \nvalue of 10 ng/mL and the expected window of detection for cotinine in saliva at 10 \nng/mL is expected to be up to 2-3 days after the last nicotine use. The assessment takes \nabout 3 minutes to complete and will be conducted at each antenatal visit and \nimmediately prior to birth.\nProcedure: The CO assessment will be conducted first, and if the CO level is within the \nyellow or red zone (high levels), a saliva test will not be conducted as these CO levels \nare indicative of combusted tobacco use and negates the requirement for saliva \ntesting. \n340\n341 Sample collection process\n342 Mother\n343 At the first antenatal appointment, maternal urine, CO and saliva samples will be \n344 collected on a NOTICE Tool. \n345 If the CO is within the orange or red zones, a cotinine saliva test will not be \n346 conducted. Also, during this first appointment, or when maternal bloods are being \n347 collected as part of standard care, maternal vein blood will be collected for this \n348 project.  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n19\n349  At each follow-up antenatal visit, maternal urine, CO and saliva (if indicated) will \n350 be collected and a NOTICE assessment completed.    \n351 Biological father\n352  At the first antenatal appointment, if the biological father is Indigenous, paternal \n353 vein blood, urine and semen will be collected and CO, saliva (if indicated), and a \n354 NOTICE assessment completed. \n355  At each follow-up visit, the biologically paternal CO and saliva (if indicated) \n356 samples will be measured and a NOTICE assessment completed.   \n357 Non-biological parent, partner or close household member\n358  At the first antenatal visit, the non-biological parent partner or close household \n359 contact will have a CO and saliva (if indicated) measured and a NOTICE \n360 assessment completed and these same tests will be conducted at each visit.\n361 Unaccompanied mother\n362  If the biological father, partner or household member is not present, the expectant \n363 mother will report her exposure to the partner’s and/or household member’s \n364 combusted tobacco or heated nicotine (e-cigarette) as second-hand exposure on \n365 the NOTICE assessment tool.\n366 At birth\n367  On presentation for birth, mothers will have a venous blood sample, urine, CO and \n368 saliva samples (if indicated) collected and a NOTICE assessment will be \n369 completed. \n370  At spontaneous or artificial rupture of membranes or caesarean birth, amniotic \n371 fluid will be collected.\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n20\n372  Following birth and the separation of the placenta from the mother and neonate, \n373 arterial and venous cord bloods will be collected from the placenta. \n374  The placenta will be weighed in grams, and measured in centimetres at two points, \n375 the widest and narrowest to estimate the area of the placenta. Placental \n376 photographs of the maternal and foetal side and the cord will be obtained.\n377  Placental samples for macro, micro-morphological and genomic examination will \n378 be obtained, rinsed in a solution of phosphate buffered saline and distilled water \n379 and then placed in a solution of RNA later.  The placenta sent for standard \n380 histology.\n381 Postnatal\n382  Following birth, neonates will have a Day 0-1 urine and meconium collected.  If \n383 the neonate remains in hospital after Day 1, a further urine sample will be \n384 collected. The urine will be collected with the use of a standard adhesive urine \n385 collection devise, and meconium will be collected from the nappy.\n386  Colostrum/breast milk will be collected when available. As active participants in \n387 the knowledge generation from this research, mothers will be encouraged to \n388 obtain their own colostrum/breast milk sample by hand/pump expression.\n389 Clinical information\n390 Data contained in the Galangoor Duwalami Primary Healthcare Service and Hospital \n391 record including demographic, maternal, and neonatal data will be included in the \n392 research database. Pregnancy, labour and birthing information will be collected from \n393 the Queensland Health Perinatal Record following birth.\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n21\n394 Biological sample analysis. \n395 After labelling, participant samples will be stored in the appropriate solution and/or \n396 temperature control manner. Placental samples in RNA later will be transferred to \n397 the University of the Sunshine Coast for storage at -80°C. Other samples (excluding \n398 the whole placenta in formalin) will be transferred to the local Sullivan and Nicolaides \n399 Pathology at standard intervals. Sullivan and Nicolaides will then transfer directly to \n400 The University of Queensland for storage at -80°C. The placenta in formalin will be \n401 transferred to Queensland Pathology. The scientists undertaking the biological \n402 examinations and analysis will be blinded to the self-reported tobacco, nicotine and \n403 cannabis status of the participant.\n404 Pregnancy elevates creatinine levels, potentially leading to fluctuations in the levels \n405 of nicotine and its metabolites and influencing cannabinoid levels [35]. To ensure the \n406 standardisation of these levels in urine samples during pregnancy, a creatinine \n407 analysis will be conducted. The biological samples will be initially analysed for \n408 tobacco, nicotine and their metabolites and cannabinoid concentration in ng/mg \n409 creatinine or nmol/mg creatinine for urine samples and ng/mL for other biological \n410 samples using standard procedures.  Following this, the samples will undergo \n411 tobacco, nicotine and cannabinoid genomic assessment in relation to tobacco, \n412 nicotine and cannabis pharmacokinetics, and other examinations including thyroid \n413 antibodies, transthyretin and sENG levels and a genomic-wide analysis of tobacco \n414 nicotine and cannabis induced alterations will be conducted. In addition, the \n415 colostrum/breast milk microbiome will be considered for the impact of tobacco, \n416 nicotine and cannabis exposure. \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n22\n417 Qualitative data collection\n418 Information-rich participants will be invited to take part in a face-to-face interview \n419 and survey.  The interview and survey will be in the form of a yarn [37-39]  which will \n420 be conducted at a place of the participants choosing and audio recorded for review.  \n421 Participants are welcome to have support people with them should they wish. Data \n422 will be responses to a range of trigger questions and survey questions to consider the \n423 barriers and influences to tobacco, nicotine and cannabis use and cessation, and to \n424 understand the population’s health literacy and risk perception related to tobacco, \n425 nicotine and cannabis products peri-pregnancy. Participant’s own language and slang \n426 will be used when possible. Data will be collected in a coded, but re-identifiable \n427 manner to enable researcher and participant follow-up. The code that will be used \n428 will be the same as for the participant’s biological and clinical data collection.  The \n429 codebook will be kept in a separate location to the recording.  During the interviews, \n430 the participant will only be identified by the unique code.  \n431 Outcome assessment\n432 Enrollment maternal blood and urine samples provide a baseline and are correlated \n433 to exhaled carbon monoxide (CO), cotinine saliva assessment, and the self-reported \n434 tobacco, nicotine and cannabis assessment tool (see Figure 4 and Notes: Table 1).\n435 At each antenatal visit, maternal urine is collected and correlated to the CO and saliva \n436 assessment and the self-reported tobacco, nicotine and cannabis assessment tool. At \n437 birth, a second maternal blood is taken to correlate to the excretion of nicotine and \n438 cannabis by the mother and neonate as measured in maternal and neonatal urine, \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n23\n439 breast milk and meconium. The transfer of nicotine and cannabis to the foetus \n440 through the placenta and return to the mother is measured by amniotic fluid, arterial \n441 and venous cord blood, and placenta samples. \n442\n443 Outcomes include: \n444 1. Parental and close household member’s patterns of self-reported use and \n445 exposure to tobacco, nicotine and cannabis using the NOTICE Tool score. Total self-\n446 use and total exposure scores will be calculated by summing the total weekly quantity \n447 and exposure scores. Scores will then be categorised into clinically meaningful \n448 categories, with those reporting no exposure or use categorised as the reference \n449 group.\n450 2. Tobacco, nicotine, cannabis and their metabolites concentration in ng/ml or \n451 nmol/ml.  Concentrations of tobacco, nicotine, trans-3’-hydroxycotinine (3-OH-\n452 cotinine), nornicotine, nicotine glucuronide and nicotine N oxide, norcotinine, NNAL, \n453 nicotelline, anabasine and anatabine [40, 41] will be summed with the total \n454 concentration in each sample recorded as total nicotine equivalents (TNE) and the \n455 nicotine metabolite ratio (NMR: 3′-hydroxycotinine/cotinine) will be calculated [42]. \n456 Similarly, delta-9-tetrahydrocannabinol-D3 (THC-D3) and delta-8 and/or delta-9 \n457 carboxy tetrahydrocannabinol-D3 (THC-COOH-D3) will be measured and summed.\n458 3. DNA methylation patterns influenced by tobacco, nicotine and cannabis use \n459 and exposure will be identified. Methylation levels will be assessed either by \n460 immunohistological analysis of methylation intermediates (5mC, 5-hmC) or \n461 calculated and expressed as β values (β = intensity of the methylated allele (M))/ \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n24\n462 (Intensity of the unmethylated allele (U) + intensity of the methylated allele (M) + \n463 100).\n464 4. Specific tobacco, nicotine and cannabis induced genomic and DNA alterations \n465 related to the use and exposure tobacco, nicotine and cannabis products in the \n466 different participant groups (maternal, paternal, foetal placental and neonatal). \n467 Genotypes influenced by tobacco, nicotine and cannabis use and exposure will be \n468 identified. Genome-wide mRNA profiles will be sequenced and expressed as read \n469 counts per mRNA.\n470 5. Identified genes and alternations (including gene expression) associated with \n471 tobacco, nicotine and cannabis metabolism and pregnancy clinical outcomes. Genes, \n472 including CYP2A6 and TCF7L2, with single nucleotide polymorphisms (SNPs) with \n473 minor allele frequency greater than 10% will be chosen for inclusion in analyses. PCR \n474 results will be reported as negative or positive.\n475 6. Semen volume (mL) and quality including sperm concentrations (million per \n476 mL), count (million), progressive motility (%), vitality (%), morphology (%), pH (0-\n477 14), leucocyte counts (millions per mL).  \n478 7. CO readings will be used as continuous scores and also categorised as: 1) \n479 negligible combusted tobacco/cannabis exposure - indicated by <6 CO ppm for non-\n480 pregnant adults and <3 CO ppm for pregnant women; 2) light combusted \n481 tobacco/cannabis exposure - indicated by 7-10 CO ppm in non-pregnant adults and \n482 4-6 CO ppm for pregnant women; 3) heavy combusted tobacco/cannabis exposure - \n483 indicated by >10 CO ppm in non-pregnant adults and >6 CO ppm in pregnant women.\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n25\n484 8. Saliva readings will be used as dichotomous scores, categorised as negative or \n485 positive\n486 9. Maternal and neonatal outcomes including miscarriage, livebirth/stillbirth, \n487 gestational age (weeks), preterm birth (<37 weeks gestation), and birth weight \n488 (grams), pregnancy induced hypertension and pre-eclampsia, gestational diabetes, \n489 thyroid autoimmune disease, maternal anaemia and other factors of interest are \n490 listed in Supplementary Table 1.\n491 Sample size\n492 Approximately 80 women expecting an Indigenous baby attend antenatal care at \n493 Galangoor Duwalami Primary Healthcare Service and the Hervey Bay and \n494 Maryborough Hospitals each year, and birth at the Hervey Bay Hospital. \n495\n496 Sample size to validate the NOTICE tool: To estimate the sensitivity and specificity of \n497 the NOTICE self-reported assessment tool for tobacco and nicotine use and exposure, \n498 assuming tobacco and nicotine exposure and use of 66% [43], a sample of 80 mothers \n499 with 5 or more assessments would allow estimation of at least 90% sensitivity and \n500 specificity for the screening tool for tobacco and nicotine use and exposure versus \n501 urinary cotinine with 95% CI within ±7% of the point estimate  [44]. The collection \n502 and validation of the additional tobacco and nicotine products increases both the \n503 sensitivity and specificity of the tool. Validation will be to POC CO, saliva cotinine, and \n504 blood and urine total nicotine equivalents [40, 41]. The collection of cannabis use and \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n26\n505 exposure measured against urinary cannabinoid concentrations provides an \n506 assessment of the tool’s broader utility in pregnancy.\n507\n508 Using multilevel linear regression modelling with urinary cotinine levels as an \n509 outcome and adjusted for tobacco and nicotine use and exposure, to estimate a \n510 statistically significant change in tobacco and nicotine metabolism during pregnancy \n511 in an Australian Aboriginal population, with a power of 80% and alpha level of 0.05, \n512 a total of 50 mothers would be required to have a minimum of five tobacco and \n513 nicotine urine measurements during pregnancy [45] assuming an intra-class \n514 correlation of 0.3 (or lower) between urinary cotinine levels for each mother during \n515 pregnancy.  With a power of 80% and alpha level of 0.05, 34 placentas would be \n516 required to be examined to estimate a 50 cm 2 difference in area between high and \n517 low tobacco and nicotine use/exposure.\n518 Data analysis\n519 Following data cleaning, checking and validation, the participants’ results from the \n520 NOTICE scores for tobacco, nicotine and cannabis use and exposure, the biochemical \n521 analysis from all biochemical samples, and the maternal and neonatal outcomes will \n522 be examined to address the research objectives as described in Table 1. All data \n523 analysis will be conducted using Stata 17 (Statacorp, Texas). Missing data and outliers \n524 will be examined and reported. Results will be reported with 95% confidence \n525 intervals (95% CI) and statistical significance at alpha 0.05.  \n526\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n27\nTable 1 Research outcomes and statistical analysis\nObjective 1: To accurately describe parental and close household contact(s) peri-gestational patterns of use and exposure to tobacco, \nnicotine and cannabis products through the creation and use of a validated assessment tool.\nOutcomes Statistical analysis\na) Record parental and close house-hold \nmember’s patterns of self-reported use \nand exposure to tobacco, nicotine and \ncannabis using the NOTICE tool.\na) Descriptive statistics of tobacco, nicotine and cannabis use and exposure self-\nreported assessment scores will be reported including frequency, means/medians \nand percentages. The score will be used as a continuous and/or ordinal categorical \nvariable in analyses.\nb) Conduct biochemical analysis of \nparental and neonatal samples for \ntobacco, nicotine and cannabis and \nmetabolites.\nb) Descriptive statistics of tobacco, nicotine and cannabis and metabolites levels from \ncollected samples will be reported including frequency, means/medians and \npercentages. The levels will be used as a continuous and/or ordinal categorical \nvariable in analyses. Participants will be categorised into faster or slower \nmetabolism groups based on their plasma NMR, which is considered the most \nreliable measure [32, 46]. To ensure the normalization of distribution between the \ntwo groups, log-transformed NMR values will be used. In this study, log urine NMR \nwill be utilized across three trimesters of pregnancy, serving as a non-invasive \nalternative to plasma NMR values. The association between plasma NMR and other \nNMR values obtained from other biological matrices will be investigated. Two \ngroups (fast and slow metabolisers) will be compared based on their tobacco and \nnicotine use and exposure behaviours (e.g., cigarettes per day) and TNE levels from \nmaternal urine samples. To assess nicotine exposure in newborn infants, TNE will \nbe calculated for various biological samples including neonate urine, meconium, \namniotic fluid, breast milk, and neonate blood samples. A Welch t-test analysis will \nbe used to determine statistically significant differences between the two groups \nbased on their NMR values. Additionally, a regression model will be utilized to \npresent the association between NMR values using different biological matrices. The \nrelationship between plasma NMR values, smoking behaviors, nicotine exposure on \nnewborn babies, and TNE levels will be analysed using a regression model.\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n28\nc) Compare maternal self-reported use \nand exposure with biochemical \nassessment of tobacco, nicotine and \ncannabis concentrations to establish \nthe specificity, sensitivity and \npredictive validity of the NOTICE tool.\nc) The sensitivity, specificity and predictive validity of the screening tool for tobacco, \nnicotine and cannabis use and exposure will be estimated by comparing self-\nreported scores with recorded cotinine saliva and CO levels at each antenatal visit.\nReliability of the NOTICE tool will also be evaluated using linear regression with \nurinary nicotine, tobacco and metabolite levels as the outcome and the predictive value \nof the self-reported scores estimated.\nObjective 2: To determine the pharmacokinetic and pharmacogenomic impacts and outcomes of tobacco, nicotine and cannabis \nexposure. \nOutcomes Data and Statistical analysis\na) Investigate specific tobacco, nicotine and \ncannabis-induced maternal, paternal, \nfoetal, placental and neonatal genomic \nalternations related to the use and \nexposure to tobacco, nicotine and \ncannabis products.\nb) Determine tobacco, nicotine and cannabis \nmetabolism by genotype \n(pharmacogenomics).\nc) Establish the pharmacokinetics of \ntobacco, nicotine and cannabis in this \npopulation.\na, b, c) Descriptive statistics will be used to report the frequency and proportion of \nparticipants with identified genetic factors related to the use of tobacco, nicotine \nand cannabis products. These factors will then be included in univariate and \nmultivariate analyses as being present or absent to estimate their effect on \nclinical and biochemical outcomes.\nObjective 3: To describe maternal, paternal, placental, foetal and neonatal outcomes according to the use and exposure to tobacco, \nnicotine and cannabis products and biochemical and genomic analysis\nOutcomes Data and Statistical analysis\na) Describe maternal, placental, foetal and \nneonatal outcomes\na) Descriptive statistics of maternal, placental, foetal and neonatal outcomes will be \nreported including frequency, means/medians and percentages. \nb) Determine correlations maternal, \npaternal, placental and neonatal \noutcomes and parental and foetal \nb) Using clinical knowledge and the literature, directed acyclic graphs (DAGs) will \nbe constructed to aid identification of causal effects/associations between 1) \nclinical outcomes, 2) self-reported tobacco, nicotine and cannabis use, 3) tobacco, \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n29\ntobacco, nicotine and cannabis \nbiochemical \nconcentrations/pharmacokinetics.\nnicotine and cannabis metabolite concentrations, and 4) identified biomarkers, \ngenetic/epigenomic/germline alterations, to determine which factors to include \nin statistical models.\nc) Determine correlations between \nmaternal, paternal, placental and \nneonatal outcomes and genomic factors.\nc) Multilevel regression modelling will be used to account for the clustering effect \n(random effects) of each individual mother, to estimate adjusted odds ratios \n(logistic regression for dichotomous or categorial outcomes) or beta coefficients \n(linear regression for continuous data outcomes) and 95% CIs for associations. A \nrange of adjustment factors will be considered as both fixed and random effects \nin the models including gestational age, infant sex, birth weight, preterm delivery, \ndelivery method, post-partum haemorrhage, preeclampsia, maternal age, height, \nbody mass index, parity, urinary tract infection, sexually transmitted infection, \ndiabetes, hypertension, placental data.\nObjective 4: To describe the influences and barriers to cessation for pregnant Australian Indigenous women and their partners or close \nhousehold contacts to reduce or cease tobacco and nicotine use in pregnancy\nOutcomes Data and Statistical analysis\na) Qualitative exploration with \ninformation rich participants to \nunderstand the barriers and \ninfluences on tobacco, nicotine and \ncannabis cessation.\nb) Understand the population’s health \nliteracy and risk perception related to \ntobacco, nicotine and cannabis \nproducts peri-pregnancy.\na & b)Data will be coded and analysis will follow established processes [47] with a \npreliminary thematic analysis assigning meaning to the data and generating \ncategories and subcategories.  The categories most often mentioned will be \nidentified and similarities and dissimilarities detailed. The categories will then \nbe further grouped, and themes and subthemes identified. These will be shared \nwith the research team for review and modification as needed. The consensus \nthemes will be organised for analysis using NVIVO 12.\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n30\n527 Ethical approval \n528 This project has been approved by the Traditional Owners of the Fraser Coast area, \n529 the Butchulla people, in conjunction with Galangoor Duwalami Primary Healthcare \n530 Service.  The project has the support of the Queensland Aboriginal and Torres Strait \n531 Islander Health Council (QAIHC) and has ethics approval from Qhealth \n532 (HREC/2021/QRBW/77758)  and the University of Queensland (2021/HE002069).\n533\n534 Discussion\n535 The overarching vision of this clinically derived, clinically driven, community based, \n536 mixed method project is to Close the Gap in Aboriginal and Torres Strait health \n537 outcomes.  This project takes a life-course epidemiological approach to health \n538 outcomes, focusing on the start of life, that is, maternal and neonatal health to \n539 improve whole-of-life health outcomes.\n540\n541 Seventy years of evidence demonstrates that maternal tobacco smoking and exposure \n542 to combusted tobacco are the leading modifiable risk behaviours associated with \n543 adverse pregnancy outcomes [48]. Currently in Australia, the assessment for tobacco \n544 and nicotine exposure during pregnancy is focused on maternal cigarette use - no \n545 information on other forms of tobacco, nicotine or cannabis use and exposure is \n546 standardly collected or considered to inform clinical care.  In addition, fathers or \n547 other household members are not asked about their tobacco, nicotine and cannabis \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n31\n548 use. This limited (or absent) tobacco, nicotine and cannabis screening fails to address \n549 the broad range of contemporary products that are used in Australia and thus has \n550 ramifications for the mother, the father, the children, the clinician, Indigenous \n551 populations, and the broader profile of Australian health.  \n552\n553 This project will be reported against the STOBE Guidelines and has purposeful and \n554 significant objectives. Firstly, the development of a validated tobacco and nicotine \n555 screening tool that can be translated to practice Australia-wide will enhance data \n556 reporting and the understanding of tobacco, nicotine and cannabis use and exposure \n557 to pregnancy outcomes. In addition, the use of a comprehensive and contemporary \n558 screening tool will provide an opportunity for women, families and health \n559 professionals to discuss tobacco, nicotine and cannabis use and reduction/cessation \n560 options. \n561\n562 Secondly, addressing the absence of literature related to the metabolism of nicotine \n563 and the influence of pharmacogenomic factors in Australian Indigenous populations \n564 will be transformative. As biotechnology has evolved, there has been an increasing \n565 recognition that genetics, epigenetics, and environmental interactions impact on \n566 health outcomes.  The role of genomics in understanding population risks and \n567 targeting prevention or intervention programs to reduce risk or to provide treatment \n568 based on genomic knowledge (i.e., a precision medicine approach) is of enormous \n569 public health benefit.  Genomic profiling allows for the understanding of different \n570 outcomes in different populations from the same exposure.  Already research exists \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n32\n571 that shows that nicotine is metabolised differently in genetically different populations \n572 [49-53] and particular risks are higher or lower in populations based on these genetic \n573 differences, however, this same level of understanding has not been established for \n574 Australian Indigenous parental populations.  A genome-wide mapping of specific \n575 biological samples from the local Australian Indigenous parental population in \n576 relation to their potential risk from tobacco and nicotine use and exposure and \n577 establishing [the start of] a pharmacogenomic profile will structure a precision \n578 medicine approach to health care [54, 55]  for this population. \n579\n580 Comprehension and appreciation of the barriers to tobacco, nicotine and cannabis \n581 cessation is an essential mechanism in supporting the decrease in tobacco, nicotine \n582 and cannabis use. Awareness these factors can lead to the construction of a range of \n583 education and support resources which can be selected by future pregnant women \n584 and the family to assist them to reduce or cease tobacco, nicotine and cannabis use in \n585 pregnancy.\n586\n587 Importantly, the findings from the tobacco, nicotine and cannabis assessment and \n588 analysis will be linked to maternal and neonatal outcomes. Using the screening tool \n589 as part of standard practice in the future will provide a predictive methodology, \n590 enabling expectant mothers, families and health services to better plan birthing and \n591 post-birthing needs in situations where tobacco, nicotine and cannabis exposure is an \n592 independent factor.\n593\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n33\n594 This research project is built on respect for the value of Indigenous perspectives and \n595 their contribution to the study.  Indigenous knowledge systems are incorporated into \n596 the research methodology thereby mutually enriching the research.  Translation to \n597 practice is an intended outcome of this project but will not be structured until findings \n598 are available. The intention is that translation will be informed by the Indigenous \n599 participants, the Aboriginal and Torres Strait Islander health service, the research-\n600 intensive organisations supporting this research and their researchers, and the chief \n601 researcher. \n602 Limitations\n603 The study consists of some strengths and limitations. One significant strength is the \n604 recording of tobacco, nicotine and cannabis use and exposure throughout early to late \n605 pregnancy and the collection of a range of biological samples that are used to \n606 measure:\n607  Recency of maternal tobacco and nicotine exposure (maternal CO, saliva, and \n608 maternal venous blood and urine), \n609  The transfer of nicotine to the foetus (venous cord blood, amniotic fluid and \n610 neonatal urine), \n611  The return of nicotine from the foetus (arterial cord blood) \n612  The longevity of exposure (placenta and meconium) \n613\n614 This approach minimises recall bias and provides a comprehensive and measurable \n615 assessment of tobacco and nicotine exposure over the duration of pregnancy. \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n34\n616 However, the study will only recruit mothers expecting an Australian Indigenous \n617 baby in the Fraser Coast area which limits the generalizability of the findings.  \n618 Additionally, being an observational study, the results will not provide the strongest \n619 evidence to establish a causal relationship between nicotine exposure or metabolism \n620 and pregnancy outcomes.\n621 Authors contributions\n622 AR: Conceptualization, design and methodology, establish collaborations and project \n623 administration, data collection, resources, writing original draft, review and editing.  \n624 EAB: Data curation, formal analysis, investigation, methodology, software, validation, \n625 writing – review & editing. VB, JB, GM, MS: Conceptualization, supervision, writing – \n626 review & editing. GD, SO: Resources, supervision, writing – review & editing. AW: \n627 Conceptualization, methodology, resources, supervision, writing – review & editing. \n628 SB: Conceptualization, methodology, supervision, writing – review & editing. M-TW: \n629 Methodology, writing – review & editing. JM Methodology, supervision, validation, \n630 writing – review & editing. KJS: Methodology, resources, supervision, validation, \n631 writing – review & editing.\n632\n633 Acknowledgements\n634 This project could not have developed without the overwhelming endorsement and \n635 governance of the Traditional Owners of the Fraser Coast area, the Butchulla people \n636 and the Butchulla Aboriginal Corporation and the Butchulla Men’s Business \n637 Association. Furthermore, this project cannot progress without the consistent and \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n35\n638 positive leadership of GD, SO and SB at Galangoor Duwalami Primary Healthcare \n639 Service and the engaged involvement of the Galangoor Duwalami teams that wrap \n640 around and support the Indigenous expectant families of the Fraser Coast area. \n641 Moreover, the cooperation and involvement of maternal services and their support \n642 teams from Wide Bay Hospital and Health Services Fraser Coast is essential in \n643 ensuring this collaborative project can achieve its aim. Fraser Coast Sullivan and \n644 Nicolaides Pathology service are sentinel in the transport of biological samples to \n645 Brisbane and the University of Queensland and are providing this service pro bono.  \n646 In terms of the design, AR conceived, designed the framework of the study, and will \n647 lead the data collection.  AW, GM, VB, JB and MS guided the data collection design with \n648 Indigenous mothers and families and consulted with their respective Indigenous \n649 organisations and community members to ensure cultural and community safety and \n650 expectations were established.  LB designed the statistical analysis and will undertake \n651 the data analysis.  M-TW will undertake the biochemical analysis as a PhD Scholar at \n652 the University of Queensland under the supervision of JM and KS. AR is partially \n653 funded under a QHealth Advancing Clinical Research Fellowship.\n654\n655 Conflicts of Interest\n656 None declared.\n657 Abbreviations\n658 ADHD – attention-deficit/hyperactivity disorder\n659 CO - carbon monoxide \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n36\n660 DAGs - directed acyclic graphs\n661 mRNA - messenger ribonucleic acid\n662 nAChR - nicotinic acetylcholine receptors\n663 NMR - nicotine metabolite ratio\n664 NRT - nicotine replacement therapy \n665 POC - point of care \n666 QAIHC - Queensland Aboriginal and Torres Strait Islander Health Council\n667 NOTICE - Ratsch Assessment of Tobacco and Nicotine \n668 SIDS - sudden infant death syndrome\n669 SNPs - single nucleotide polymorphisms\n670 TNE - total nicotine equivalents  \n671\n672\n673 Supporting Information.  Supplementary Table 1: Variables of interest for analysis \n674 extracted from standard National Perinatal Data Collection report, together with \n675 variables of interest for this project (i.e., tobacco, nicotine and cannabis use and \n676 exposure)\n677\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n37\n678 References\n679\n680 1. Simpson WJ. A preliminary report on cigarette smoking and the incidence of \n681 prematurity. Am J Obstet Gynecol. 1957;73(4):807-15.\n682 2. Avsar TS, McLeod H, Jackson L. Health outcomes of smoking during \n683 pregnancy and the postpartum period: an umbrella review. BMC Pregnancy \n684 Childbirth. 2021;21(1):254.\n685 3. Naeye RL. Abruptio placentae and placenta previa: frequency, perinatal \n686 mortality, and cigarette smoking. Obstet Gynecol. 1980;55(6):701-4.\n687 4. Marcoux S, Brisson J, Fabia J. The effect of cigarette smoking on the risk of \n688 preeclampsia and gestational hypertension. Am J Epidemiol. 1989;130(5):950-7.\n689 5. England LJ, Levine RJ, Qian C, Morris CD, Sibai BM, Catalano PM, et al. \n690 Smoking before pregnancy and risk of gestational hypertension and preeclampsia. \n691 Am J Obstet Gynecol. 2002;186(5):1035-40.\n692 6. Gould GS, Havard A, Lim LL, The Psanz Smoking In Pregnancy Expert G, \n693 Kumar R. Exposure to Tobacco, Environmental Tobacco Smoke and Nicotine in \n694 Pregnancy: A Pragmatic Overview of Reviews of Maternal and Child Outcomes, \n695 Effectiveness of Interventions and Barriers and Facilitators to Quitting. Int J Environ \n696 Res Public Health. 2020;17(6).\n697 7. Hackshaw A, Rodeck C, Boniface S. Maternal smoking in pregnancy and birth \n698 defects: a systematic review based on 173 687 malformed cases and 11.7 million \n699 controls. Hum Reprod Update. 2011;17(5):589-604.\n700 8. Yang L, Wang H, Yang L, Zhao M, Guo Y, Bovet P, et al. Maternal cigarette \n701 smoking before or during pregnancy increases the risk of birth congenital \n702 anomalies: a population-based retrospective cohort study of 12 million mother-\n703 infant pairs. BMC Med. 2022;20(1):4.\n704 9. Clifford A, Lang L, Chen R. Effects of maternal cigarette smoking during \n705 pregnancy on cognitive parameters of children and young adults: a literature \n706 review. Neurotoxicol Teratol. 2012;34(6):560-70.\n707 10. Sourander A, Sucksdorff M, Chudal R, Surcel HM, Hinkka-Yli-Salomaki S, \n708 Gyllenberg D, et al. Prenatal Cotinine Levels and ADHD Among Offspring. Pediatrics. \n709 2019;143(3).\n710 11. Australian Institute of Health and Welfare. [Internet]. Canberra: National \n711 Perinatal Data Collection (NPDC): Australian Government; 2020 [cited 2024 3 \n712 January]. Available from: https://www.aihw.gov.au/about-our-data/our-data-\n713 collections/national-perinatal-data-collection.\n714 12. Australian Institute of Health and Welfare. [Internet]. Australia's mothers \n715 and babies; 2023 [cited 2024 3 January]. Available from: \n716 https://www.aihw.gov.au/reports/mothers-babies/australias-mothers-\n717 babies/contents/about.\n718 13. Australian Institute of Health and Welfare, National Indigenous Australian \n719 Agency. [Internet]. Canberrra: Tier 2 Determinants of health 2.21 Health behaviours \n720 during pregnancy: Australian Government; 2022 [cited 2024 5 January]. Available \n721 from: https://www.indigenoushpf.gov.au/measures/2-21-health-behaviours-\n722 during-pregnancy.\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n38\n723 14. Australian Institute of Health and Welfare. [Internet]. Canberra: Pregnancy \n724 and birth outcomes for Aboriginal and Torres Strait Islander women 2016–2018. \n725 Cat. no. IHW 234: AIHW; 2021 [cited 2023 14 October]. Available from: \n726 https://www.aihw.gov.au/reports/indigenous-australians/pregnancy-birth-\n727 outcomes-indigenous-women-2016-18/contents/summary.\n728 15. Australian Institute of Health and Welfare. [Internet]. Canberra: Stillbirths \n729 and neonatal deaths in Australia 2017 and 2018: AIHW; 2021 [cited 2024 24 \n730 January]. Available from: https://www.aihw.gov.au/getmedia/4b6ff4e5-f549-42c7-\n731 96ac-e6cc221d79b8/aihw-per-115.pdf.aspx.\n732 16. Benowitz NL, Hukkanen J, Jacob P, III. Nicotine Chemistry, Metabolism, \n733 Kinetics and Biomarkers. In: Henningfield JE, London ED, Pogun S, editors. Nicotine \n734 Psychopharmacology. Handbook of Experimental Pharmacology. 192: Springer \n735 Berlin Heidelberg; 2009. p. 29-60.\n736 17. McGrath-Morrow SA, Gorzkowski J, Groner JA, Rule AM, Wilson K, Tanski SE, \n737 et al. The Effects of Nicotine on Development. Pediatrics. 2020;145(3).\n738 18. Hukkanen J, Jacob P, 3rd, Benowitz NL. Metabolism and disposition kinetics \n739 of nicotine. Pharmacol Rev. 2005;57(1):79-115.\n740 19. Tizabi Y, Getachew B, Copeland RL, Aschner M. Nicotine and the nicotinic \n741 cholinergic system in COVID-19. FEBS J. 2020;287(17):3656-63.\n742 20. Banerjee S, Deacon A, Suter MA, Aagaard KM. Understanding the Placental \n743 Biology of Tobacco Smoke, Nicotine, and Marijuana (THC) Exposures During \n744 Pregnancy. Clin Obstet Gynecol. 2022;65(2):347-59.\n745 21. Pintican D, Poienar AA, Strilciuc S, Mihu D. Effects of maternal smoking on \n746 human placental vascularization: A systematic review. Taiwan J Obstet Gynecol. \n747 2019;58(4):454-9.\n748 22. Beltrán-Castillo S, Bravo K, Eugenín J. Impact of Prenatal Nicotine Exposure \n749 on Placental Function and Respiratory Neural Network Development. In: Gonzalez-\n750 Ortiz M, editor. Advances in Maternal-Fetal Biomedicine: Cellular and Molecular \n751 Mechanisms of Pregnancy Pathologies. Cham: Springer International Publishing; \n752 2023. p. 233-44.\n753 23. Alzu'bi A, Middleham W, Shoaib M, Clowry GJ. Selective Expression of \n754 Nicotinic Receptor Sub-unit mRNA in Early Human Fetal Forebrain. Front Mol \n755 Neurosci. 2020;13:72.\n756 24. National Research Council (US) and Institute of Medicine (US) Committee on \n757 Integrating the Science of Early Childhood Development. [Internet ]. Washington \n758 (DC): From Neurons to Neighborhoods: The Science of Early Childhood \n759 Development. The Developing Brain: National Academies Press 2000 [cited 2024 29 \n760 January]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK225562/ .\n761 25. Slotkin TA. If nicotine is a developmental neurotoxicant in animal studies, \n762 dare we recommend nicotine replacement therapy in pregnant women and \n763 adolescents? Neurotoxicol Teratol. 2008;30(1):1-19.\n764 26. Buck JM, O'Neill HC, Stitzel JA. Developmental nicotine exposure elicits \n765 multigenerational disequilibria in proBDNF proteolysis and glucocorticoid signaling \n766 in the frontal cortices, striata, and hippocampi of adolescent mice. Biochem \n767 Pharmacol. 2019;168:438-51.\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n39\n768 27. Bednarczuk N, Milner A, Greenough A. The Role of Maternal Smoking in \n769 Sudden Fetal and Infant Death Pathogenesis. Front Neurol. 2020;11:586068.\n770 28. Benowitz NL. Clinical pharmacology of nicotine. Annu Rev Med. 1986;37:21-\n771 32.\n772 29. Benowitz NL, Kuyt F, Jacob P, 3rd, Jones RT, Osman AL. Cotinine disposition \n773 and effects. Clin Pharmacol Ther. 1983;34(5):604-11.\n774 30. Benowitz NL, Dains KM, Dempsey D, Yu L, Jacob P, 3rd. Estimation of nicotine \n775 dose after low-level exposure using plasma and urine nicotine metabolites. Cancer \n776 Epidemiol Biomarkers Prev. 2010;19(5):1160-6.\n777 31. Dempsey D, Jacob P, 3rd, Benowitz NL. Accelerated metabolism of nicotine \n778 and cotinine in pregnant smokers. J Pharmacol Exp Ther. 2002;301(2):594-8.\n779 32. Arger CA, Taghavi T, Heil SH, Skelly J, Tyndale RF, Higgins ST. Pregnancy-\n780 Induced Increases in the Nicotine Metabolite Ratio: Examining Changes During \n781 Antepartum and Postpartum. Nicotine Tob Res. 2019;21(12):1706-10.\n782 33. Dempsey D, Jacob P, 3rd, Benowitz NL. Nicotine metabolism and elimination \n783 kinetics in newborns. Clin Pharmacol Ther. 2000;67(5):458-65.\n784 34. Feyerabend C, Russell MAH. Effect of urinary pH and nicotine excretion rate \n785 on plasma nicotine during cigarette smoking and chewing nicotine gum. Br J Clin \n786 Pharmacol. 1978;5(4):293-7.\n787 35. Taghavi T, Arger CA, Heil SH, Higgins ST, Tyndale RF. Longitudinal Influence \n788 of Pregnancy on Nicotine Metabolic Pathways. Journal of Pharmacology and \n789 Experimental Therapeutics. 2018;364(2):238-45.\n790 36. The National Health and Medical Research Council, the Australian Research \n791 Council, and Universities Australia. National Statement on Ethical Conduct in Human \n792 Research 2007 (Updated 2018). Canberra: Commonwealth of Australia; 2018.\n793 37. Geia LK, Hayes B, Usher K. Yarning/Aboriginal storytelling: towards an \n794 understanding of an Indigenous perspective and its implications for research \n795 practice. Contemp Nurse. 2013;46(1):13-7.\n796 38. Bessarab D, Ng'andu B. Yarning About Yarning as a Legitimate Method in \n797 Indigenous Research. International Journal of Critical Indigenous Studies. \n798 2010;3(1):37-50.\n799 39. Walker M, Fredericks B, Mills K, Anderson D. \"Yarning\" as a method for \n800 community-based health research with Indigenous women: the Indigenous \n801 Women's Wellness Research Program. Health Care Women Int. 2014;35(10):1216-\n802 26.\n803 40. Benowitz NL, Bernert JT, Foulds J, Hecht SS, Jacob P, Jarvis MJ, et al. \n804 Biochemical Verification of Tobacco Use and Abstinence: 2019 Update. Nicotine Tob \n805 Res. 2020;22(7):1086-97.\n806 41. Benowitz NL, St Helen G, Nardone N, Cox LS, Jacob P. Urine Metabolites for \n807 Estimating Daily Intake of Nicotine From Cigarette Smoking. Nicotine Tob Res. \n808 2020;22(2):288-92.\n809 42. Benowitz NL, Pomerleau OF, Pomerleau CS, Jacob P, III. Nicotine metabolite \n810 ratio as a predictor of cigarette consumption. Nicotine Tobacco Res. 2003;5(5):621-\n811 4.\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n40\n812 43. Gilligan C, Sanson-Fisher R, Eades S, Wenitong M, Panaretto K, D'Este C. \n813 Assessing the accuracy of self-reported smoking status and impact of passive smoke \n814 exposure among pregnant Aboriginal and Torres Strait Islander women using \n815 cotinine biochemical validation. Drug Alcohol Rev. 2010;29(1):35-40.\n816 44. Malhotra RK, Indrayan A. A simple nomogram for sample size for estimating \n817 sensitivity and specificity of medical tests. Indian J Ophthalmol. 2010;58(6):519-22.\n818 45. McNeish DM. Modeling sparsely clustered data: design-based, model-based, \n819 and single-level methods. Psychol Methods. 2014;19(4):552-63.\n820 46. Vaz LR, Coleman T, Cooper S, Aveyard P, Leonardi-Bee J, team St. The \n821 Nicotine Metabolite Ratio in Pregnancy Measured by trans-3'-Hydroxycotinine to \n822 Cotinine Ratio: Characteristics and Relationship With Smoking Cessation. Nicotine \n823 Tob Res. 2015;17(11):1318-23.\n824 47. Braun V, Clarke V. Using thematic analysis in psychology. Qualitative \n825 Research in Psychology. 2006;3(2):77-101.\n826 48. National Center for Chronic Disease Prevention and Health Promotion (US) \n827 Office on Smoking and Health. Reproductive Outcomes.  The Health Consequences of \n828 Smoking—50 Years of Progress: A Report of the Surgeon General. Atlanta (GA): \n829 Centers for Disease Control and Prevention (US); 2014. p. 461-521.\n830 49. Benowitz NL, Perez-Stable EJ, Herrera B, Jacob P, 3rd. Slower metabolism and \n831 reduced intake of nicotine from cigarette smoking in Chinese-Americans. J Natl \n832 Cancer Inst. 2002;94(2):108-15.\n833 50. Cecil CA, Walton E, Smith RG, Viding E, McCrory EJ, Relton CL, et al. DNA \n834 methylation and substance-use risk: a prospective, genome-wide study spanning \n835 gestation to adolescence. Transl Psychiatry. 2016;6(12):e976.\n836 51. Chen X, Woodcroft KJ. Polymorphisms in metabolic genes CYP1A1 and \n837 GSTM1 and changes in maternal smoking during pregnancy. Nicotine Tob Res. \n838 2009;11(3):225-33.\n839 52. Keskitalo K, Broms U, Heliovaara M, Ripatti S, Surakka I, Perola M, et al. \n840 Association of serum cotinine level with a cluster of three nicotinic acetylcholine \n841 receptor genes (CHRNA3/CHRNA5/CHRNB4) on chromosome 15. Hum Mol Genet. \n842 2009;18(20):4007-12.\n843 53. Marceau K, Palmer RH, Neiderhiser JM, Smith TF, McGeary JE, Knopik VS. \n844 Passive rGE or developmental gene-environment cascade? An investigation of the \n845 role of xenobiotic metabolism genes in the association between smoke exposure \n846 during pregnancy and child birth weight. Behav Genet. 2016;46(3):365-77.\n847 54. Allenby CE, Boylan KA, Lerman C, Falcone M. Precision Medicine for Tobacco \n848 Dependence: Development and Validation of the Nicotine Metabolite Ratio. J \n849 Neuroimmune Pharmacol. 2016;11(3):471-83.\n850 55. Cheng YY, Nunn J, Skinner J, Rambaldini B, Boughtwood T, Calma T, et al. A \n851 Pathway to Precision Medicine for Aboriginal Australians: A Study Protocol. \n852 Methods Protoc. 2021;4(2):42.\n853\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint \n\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}