Tobacco, nicotine, and cannabis use and exposure in an Australian Indigenous population during pregnancy: A protocol to measure parental and foetal exposure and outcomes

1: To accurately describe parental and close household contact(s) peri-gestational patterns of use and exposure to tobacco, nicotine and cannabis products through the creation and use of a validated assessment tool. Outcomes Statistical analysis a) Record parental and close house-hold member’s patterns of self-reported use and exposure to tobacco, nicotine and cannabis using the NOTICE tool. a) Descriptive statistics of tobacco, nicotine and cannabis use and exposure self- reported assessment scores will be reported including frequency, means/medians and percentages. The score will be used as a continuous and/or ordinal categorical variable in analyses. b) Conduct biochemical analysis of parental and neonatal samples for tobacco, nicotine and cannabis and metabolites. b) Descriptive statistics of tobacco, nicotine and cannabis and metabolites levels from collected samples will be reported including frequency, means/medians and percentages. The levels will be used as a continuous and/or ordinal categorical variable in analyses. Participants will be categorised into faster or slower metabolism groups based on their plasma NMR, which is considered the most reliable measure [32, 46]. To ensure the normalization of distribution between the two groups, log-transformed NMR values will be used. In this study, log urine NMR will be utilized across three trimesters of pregnancy, serving as a non-invasive alternative to plasma NMR values. The association between plasma NMR and other NMR values obtained from other biological matrices will be investigated. Two groups (fast and slow metabolisers) will be compared based on their tobacco and nicotine use and exposure behaviours (e.g., cigarettes per day) and TNE levels from maternal urine samples. To assess nicotine exposure in newborn infants, TNE will be calculated for various biological samples including neonate urine, meconium, amniotic fluid, breast milk, and neonate blood samples. A Welch t-test analysis will be used to determine statistically significant differences between the two groups based on their NMR values. Additionally, a regression model will be utilized to present the association between NMR values using different biological matrices. The relationship between plasma NMR values, smoking behaviors, nicotine exposure on newborn babies, and TNE levels will be analysed using a regression model. . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint 28 c) Compare maternal self-reported use and exposure with biochemical assessment of tobacco, nicotine and cannabis concentrations to establish the specificity, sensitivity and predictive validity of the NOTICE tool. c) The sensitivity, specificity and predictive validity of the screening tool for tobacco, nicotine and cannabis use and exposure will be estimated by comparing self- reported scores with recorded cotinine saliva and CO levels at each antenatal visit. Reliability of the NOTICE tool will also be evaluated using linear regression with urinary nicotine, tobacco and metabolite levels as the outcome and the predictive value of the self-reported scores estimated.

2: To determine the pharmacokinetic and pharmacogenomic impacts and outcomes of tobacco, nicotine and cannabis exposure. Outcomes Data and Statistical analysis a) Investigate specific tobacco, nicotine and cannabis-induced maternal, paternal, foetal, placental and neonatal genomic alternations related to the use and exposure to tobacco, nicotine and cannabis products. b) Determine tobacco, nicotine and cannabis metabolism by genotype (pharmacogenomics). c) Establish the pharmacokinetics of tobacco, nicotine and cannabis in this population. a, b, c) Descriptive statistics will be used to report the frequency and proportion of participants with identified genetic factors related to the use of tobacco, nicotine and cannabis products. These factors will then be included in univariate and multivariate analyses as being present or absent to estimate their effect on clinical and biochemical outcomes.

3: To describe maternal, paternal, placental, foetal and neonatal outcomes according to the use and exposure to tobacco, nicotine and cannabis products and biochemical and genomic analysis Outcomes Data and Statistical analysis a) Describe maternal, placental, foetal and neonatal outcomes a) Descriptive statistics of maternal, placental, foetal and neonatal outcomes will be reported including frequency, means/medians and percentages. b) Determine correlations maternal, paternal, placental and neonatal outcomes and parental and foetal b) Using clinical knowledge and the literature, directed acyclic graphs (DAGs) will be constructed to aid identification of causal effects/associations between 1) clinical outcomes, 2) self-reported tobacco, nicotine and cannabis use, 3) tobacco, . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint 29 tobacco, nicotine and cannabis biochemical concentrations/pharmacokinetics. nicotine and cannabis metabolite concentrations, and 4) identified biomarkers, genetic/epigenomic/germline alterations, to determine which factors to include in statistical models. c) Determine correlations between maternal, paternal, placental and neonatal outcomes and genomic factors. c) Multilevel regression modelling will be used to account for the clustering effect (random effects) of each individual mother, to estimate adjusted odds ratios (logistic regression for dichotomous or categorial outcomes) or beta coefficients (linear regression for continuous data outcomes) and 95% CIs for associations. A range of adjustment factors will be considered as both fixed and random effects in the models including gestational age, infant sex, birth weight, preterm delivery, delivery method, post-partum haemorrhage, preeclampsia, maternal age, height, body mass index, parity, urinary tract infection, sexually transmitted infection, diabetes, hypertension, placental data.

4: To describe the influences and barriers to cessation for pregnant Australian Indigenous women and their partners or close household contacts to reduce or cease tobacco and nicotine use in pregnancy Outcomes Data and Statistical analysis a) Qualitative exploration with information rich participants to understand the barriers and influences on tobacco, nicotine and cannabis cessation. b) Understand the population’s health literacy and risk perception related to tobacco, nicotine and cannabis products peri-pregnancy. a & b)Data will be coded and analysis will follow established processes [47] with a preliminary thematic analysis assigning meaning to the data and generating categories and subcategories. The categories most often mentioned will be identified and similarities and dissimilarities detailed. The categories will then be further grouped, and themes and subthemes identified. These will be shared with the research team for review and modification as needed. The consensus themes will be organised for analysis using NVIVO 12. . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint 30 527 Ethical approval 528 This project has been approved by the Traditional Owners of the Fraser Coast area, 529 the Butchulla people, in conjunction with Galangoor Duwalami Primary Healthcare 530 Service. The project has the support of the Queensland Aboriginal and Torres Strait 531 Islander Health Council (QAIHC) and has ethics approval from Qhealth 532 (HREC/2021/QRBW/77758) and the University of Queensland (2021/HE002069). 533 534 Discussion 535 The overarching vision of this clinically derived, clinically driven, community based, 536 mixed method project is to Close the Gap in Aboriginal and Torres Strait health 537 outcomes. This project takes a life-course epidemiological approach to health 538 outcomes, focusing on the start of life, that is, maternal and neonatal health to 539 improve whole-of-life health outcomes. 540 541 Seventy years of evidence demonstrates that maternal tobacco smoking and exposure 542 to combusted tobacco are the leading modifiable risk behaviours associated with 543 adverse pregnancy outcomes [48]. Currently in Australia, the assessment for tobacco 544 and nicotine exposure during pregnancy is focused on maternal cigarette use - no 545 information on other forms of tobacco, nicotine or cannabis use and exposure is 546 standardly collected or considered to inform clinical care. In addition, fathers or 547 other household members are not asked about their tobacco, nicotine and cannabis . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint 31 548 use. This limited (or absent) tobacco, nicotine and cannabis screening fails to address 549 the broad range of contemporary products that are used in Australia and thus has 550 ramifications for the mother, the father, the children, the clinician, Indigenous 551 populations, and the broader profile of Australian health. 552 553 This project will be reported against the STOBE Guidelines and has purposeful and 554 significant objectives. Firstly, the development of a validated tobacco and nicotine 555 screening tool that can be translated to practice Australia-wide will enhance data 556 reporting and the understanding of tobacco, nicotine and cannabis use and exposure 557 to pregnancy outcomes. In addition, the use of a comprehensive and contemporary 558 screening tool will provide an opportunity for women, families and health 559 professionals to discuss tobacco, nicotine and cannabis use and reduction/cessation 560 options. 561 562 Secondly, addressing the absence of literature related to the metabolism of nicotine 563 and the influence of pharmacogenomic factors in Australian Indigenous populations 564 will be transformative. As biotechnology has evolved, there has been an increasing 565 recognition that genetics, epigenetics, and environmental interactions impact on 566 health outcomes. The role of genomics in understanding population risks and 567 targeting prevention or intervention programs to reduce risk or to provide treatment 568 based on genomic knowledge (i.e., a precision medicine approach) is of enormous 569 public health benefit. Genomic profiling allows for the understanding of different 570 outcomes in different populations from the same exposure. Already research exists . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint 32 571 that shows that nicotine is metabolised differently in genetically different populations 572 [49-53] and particular risks are higher or lower in populations based on these genetic 573 differences, however, this same level of understanding has not been established for 574 Australian Indigenous parental populations. A genome-wide mapping of specific 575 biological samples from the local Australian Indigenous parental population in 576 relation to their potential risk from tobacco and nicotine use and exposure and 577 establishing [the start of] a pharmacogenomic profile will structure a precision 578 medicine approach to health care [54, 55] for this population. 579 580 Comprehension and appreciation of the barriers to tobacco, nicotine and cannabis 581 cessation is an essential mechanism in supporting the decrease in tobacco, nicotine 582 and cannabis use. Awareness these factors can lead to the construction of a range of 583 education and support resources which can be selected by future pregnant women 584 and the family to assist them to reduce or cease tobacco, nicotine and cannabis use in 585 pregnancy. 586 587 Importantly, the findings from the tobacco, nicotine and cannabis assessment and 588 analysis will be linked to maternal and neonatal outcomes. Using the screening tool 589 as part of standard practice in the future will provide a predictive methodology, 590 enabling expectant mothers, families and health services to better plan birthing and 591 post-birthing needs in situations where tobacco, nicotine and cannabis exposure is an 592 independent factor. 593 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint 33 594 This research project is built on respect for the value of Indigenous perspectives and 595 their contribution to the study. Indigenous knowledge systems are incorporated into 596 the research methodology thereby mutually enriching the research. Translation to 597 practice is an intended outcome of this project but will not be structured until findings 598 are available. The intention is that translation will be informed by the Indigenous 599 participants, the Aboriginal and Torres Strait Islander health service, the research- 600 intensive organisations supporting this research and their researchers, and the chief 601 researcher. 602 Limitations 603 The study consists of some strengths and limitations. One significant strength is the 604 recording of tobacco, nicotine and cannabis use and exposure throughout early to late 605 pregnancy and the collection of a range of biological samples that are used to 606 measure: 607  Recency of maternal tobacco and nicotine exposure (maternal CO, saliva, and 608 maternal venous blood and urine), 609  The transfer of nicotine to the foetus (venous cord blood, amniotic fluid and 610 neonatal urine), 611  The return of nicotine from the foetus (arterial cord blood) 612  The longevity of exposure (placenta and meconium) 613 614 This approach minimises recall bias and provides a comprehensive and measurable 615 assessment of tobacco and nicotine exposure over the duration of pregnancy. . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint 34 616 However, the study will only recruit mothers expecting an Australian Indigenous 617 baby in the Fraser Coast area which limits the generalizability of the findings. 618 Additionally, being an observational study, the results will not provide the strongest 619 evidence to establish a causal relationship between nicotine exposure or metabolism 620 and pregnancy outcomes. 621 Authors contributions 622 AR: Conceptualization, design and methodology, establish collaborations and project 623 administration, data collection, resources, writing original draft, review and editing. 624 EAB: Data curation, formal analysis, investigation, methodology, software, validation, 625 writing – review & editing. VB, JB, GM, MS: Conceptualization, supervision, writing – 626 review & editing. GD, SO: Resources, supervision, writing – review & editing. AW: 627 Conceptualization, methodology, resources, supervision, writing – review & editing. 628 SB: Conceptualization, methodology, supervision, writing – review & editing. M-TW: 629 Methodology, writing – review & editing. JM Methodology, supervision, validation, 630 writing – review & editing. KJS: Methodology, resources, supervision, validation, 631 writing – review & editing. 632 633 Acknowledgements 634 This project could not have developed without the overwhelming endorsement and 635 governance of the Traditional Owners of the Fraser Coast area, the Butchulla people 636 and the Butchulla Aboriginal Corporation and the Butchulla Men’s Business 637 Association. Furthermore, this project cannot progress without the consistent and . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint 35 638 positive leadership of GD, SO and SB at Galangoor Duwalami Primary Healthcare 639 Service and the engaged involvement of the Galangoor Duwalami teams that wrap 640 around and support the Indigenous expectant families of the Fraser Coast area. 641 Moreover, the cooperation and involvement of maternal services and their support 642 teams from Wide Bay Hospital and Health Services Fraser Coast is essential in 643 ensuring this collaborative project can achieve its aim. Fraser Coast Sullivan and 644 Nicolaides Pathology service are sentinel in the transport of biological samples to 645 Brisbane and the University of Queensland and are providing this service pro bono. 646 In terms of the design, AR conceived, designed the framework of the study, and will 647 lead the data collection. AW, GM, VB, JB and MS guided the data collection design with 648 Indigenous mothers and families and consulted with their respective Indigenous 649 organisations and community members to ensure cultural and community safety and 650 expectations were established. LB designed the statistical analysis and will undertake 651 the data analysis. M-TW will undertake the biochemical analysis as a PhD Scholar at 652 the University of Queensland under the supervision of JM and KS. AR is partially 653 funded under a QHealth Advancing Clinical Research Fellowship. 654 655 Conflicts of Interest 656 None declared. 657 Abbreviations 658 ADHD – attention-deficit/hyperactivity disorder 659 CO - carbon monoxide . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint 36 660 DAGs - directed acyclic graphs 661 mRNA - messenger ribonucleic acid 662 nAChR - nicotinic acetylcholine receptors 663 NMR - nicotine metabolite ratio 664 NRT - nicotine replacement therapy 665 POC - point of care 666 QAIHC - Queensland Aboriginal and Torres Strait Islander Health Council 667 NOTICE - Ratsch Assessment of Tobacco and Nicotine 668 SIDS - sudden infant death syndrome 669 SNPs - single nucleotide polymorphisms 670 TNE - total nicotine equivalents 671 672 673 Supporting Information. Supplementary Table 1: Variables of interest for analysis 674 extracted from standard National Perinatal Data Collection report, together with 675 variables of interest for this project (i.e., tobacco, nicotine and cannabis use and 676 exposure) 677 . 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(which was not certified by peer review) The copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 4, 2024. ; https://doi.org/10.1101/2024.02.29.24303540doi: medRxiv preprint