Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk.

Milk is a highly complex, heterogeneous biological fluid that contains non-nutritive, bioactive extracellular vesicles called exosomes. Characterization of milk-derived exosomes (MDEs) is challenging due to the lack of standardized methods that are currently being used for milk pre-processing, storage, and exosome isolation. In this study, we tested: 1) three pre-processing methods to remove cream, fat, cellular debris, and casein proteins from bovine milk to determine whether pre-processing of whole milk prior to long-term storage improves MDE isolations, 2) the suitability of two standard exosome isolation methods for MDE fractionation, and 3) four extraction protocols for obtaining high quality RNA from bovine and human MDEs. MDEs were characterized via Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and western immunoblotting for CD9, CD63, and Calnexin protein markers. We also present an optimized method of TEM sample preparation for MDEs. Our results indicate that: 1) Removal of cream and fat globules from unpasteurized bovine milk, prior to long-term storage, improves the MDE yield but not purity, 2) Differential ultracentrifugation (DUC) combined with serial filtration is better suited for bovine MDE isolation compared to ExoQuick (EQ) combined with serial filtration, however both methods were comparable for human milk, and 3) TRIzol LS is better suited for RNA extraction from bovine MDEs isolated by EQ and DUC methods. 4) TRIzol LS, TRIzol+RNA Clean and Concentrator, and TRIzol LS+RNA Clean and Concentrator methods can be used for RNA extractions from human MDEs isolated by EQ, yet the TRIzol LS method is better suited for human MDEs isolated by DUC. The QIAzol + miRNeasy Mini Kit produced the lowest RNA yield for bovine and human MDEs.

The Interplay Between Prenatal Adversity, Offspring Dopaminergic Genes, and Early Parenting on Toddler Attentional Function.

Background: Few studies have explored the complex gene-by-prenatal environment-by-early postnatal environment interactions that underlie the development of attentional competence. Here, we examined if variation in dopamine-related genes interacts with prenatal adversity to influence toddler attentional competence and whether this influence is buffered by early positive maternal behavior. Methods: From the Maternal Adversity, Vulnerability and Neurodevelopment cohort, 134 participants (197 when imputing missing data) had information on prenatal adversity (prenatal stressful life events, prenatal maternal depressive symptoms, and birth weight), five dopamine-related genes (DAT1, DRD4, DRD2, COMT, BDNF), observed maternal parenting behavior at 6 months and parent-rated toddler attentional competence at 18 and 24 months. The Latent Environmental and Genetic Interaction (LEGIT) approach was used to examine genes-by-prenatal environment-by-postnatal environment interactions while controlling for sociodemographic factors and postnatal depression. Results: Our hypothesis of a three-way interaction between prenatal adversity, dopamine-related genes, and early maternal parenting behavior was not confirmed. However, consistent two-way interactions emerged between prenatal adversity and dopamine-related genes; prenatal adversity and maternal parenting behavior, and dopamine-related genes and maternal parenting behavior in relation to toddler attentional competence. Significant interaction effects were driven by the DAT1, COMT, and BDNF genotypes; prenatal stressful life events; maternal sensitivity, tactile stimulation, vocalization, and infant-related activities. Conclusions: Multiple dopamine-related genes affected toddler attentional competence and they did so in interaction with prenatal adversity and the early rearing environment, separately. Effects were already visible in young children. Several aspects of early maternal parenting have been identified as potential targets for intervention.

Beyond maternal care: The effects of extra-maternal influences within the maternal environment on offspring neurodevelopment and later-life behavior.

The early-life maternal environment has a profound and persistent effect on offspring neuroendocrine function, neurotransmitter systems, and behavior. Studies using rodent models suggest that early-life maternal care can influence the 'developmental programming' of offspring in part through altered epigenetic regulation of specific genes. The exploration of epigenetic regulation of these genes as a biological mechanism has been important to our understanding of how animals adapt to their environments and how these developmental trajectories may be altered. However, other non-maternal factors have been shown to act directly, or to interact with maternal care, to influence later-life phenotype. Based on accumulating evidence, including our research, we discuss other important influences on the developmental programming of offspring. We highlight early-life variations in temperature exposure and offspring genotype x environment interactions as prominent examples. We conclude with recommendations for future investigations on how early-life maternal care and extra-maternal influences lead to persistent changes in the brain and behavior of the offspring throughout development.

The role of interindividual licking received and dopamine genotype on later-life licking provisioning in female rat offspring.

INTRODUCTION: Rat mothers exhibit natural variations in care that propagate between generations of female offspring. However, there is limited information on genetic variation that could influence this propagation. METHODS: We assessed early-life maternal care received by individual female rat offspring, later-life maternal care provisioning, and dopaminergic activity in the maternal brain in relation to naturally occurring genetic polymorphisms linked to the dopaminergic system. We also conducted a systematic analysis of other genetic variants potentially related to maternal behavior in our Long-Evans rat population. RESULTS: While we did not find a direct relationship between early-life licking received and later-life licking provisioning, this relationship was indirectly affected by dopamine levels in the nucleus accumbens and dependent on variation in the dopamine receptor 2 gene (rs107017253). More specifically, female rat offspring with the A/G genotype showed a positive relationship between average licking received and dopamine levels in the nucleus accumbens of the maternal brain; there was no relationship with female rat offspring with the A/A genotype. The higher dopamine levels in the nucleus accumbens corresponded with higher maternal licking provisioning from postnatal days 2-9. We also discovered and validated several new variants that were predicted by our systematic analysis. CONCLUSION: Our findings suggest that genetic variation influences the relationship between early-life maternal care received and the dopaminergic system of the maternal brain, which can indirectly influence later-life maternal care provisioning.

Mothering revisited: A role for cortisol?

This selective review first describes the involvement of the maternal hypothalamic-pituitary-adrenal (HPA) axis during pregnancy and the postpartum period, and the relation between peripartum HPA axis function and maternal behavior, stress reactivity and emotional dysregulation in human mothers. To provide experimental background to this correlational work, where helpful, animal studies are also described. It then explores the association between HPA axis function in mothers and their infants, under ongoing non-stressful conditions and during stressful challenges, the moderating role of mothers' sensitivity and behavior in the mother-child co-regulation and the effects of more traumatic risk factors on these relations. The overarching theme being explored is that the HPA axis - albeit a system designed to function during periods of high stress and challenge - also functions to promote adaptation to more normative processes, shown in the new mother who experiences both high cortisol and enhanced attraction and attention to and recognition of, their infants and their cues. Hence the same HPA system shows positive relations with behavior at some time points and inverse ones at others. However, the literature is not uniform and results vary widely depending on the number, timing, place, and type of samplings and assessments, and, of course, the population being studied and, in the present context, the state, the stage, and the stress levels of mother and infant.

Maternal symptoms of depression and sensitivity mediate the relation between maternal history of early adversity and her child temperament: The inheritance of circumstance.

We examined maternal depression and maternal sensitivity as mediators of the association between maternal childhood adversity and her child's temperament in 239 mother-child dyads from a longitudinal, birth cohort study. We used an integrated measure of maternal childhood adversity that included the Childhood Trauma Questionnaire and the Parental Bonding Index. Maternal depression was assessed with the Edinburgh Postnatal Depression Scale at 6 months postpartum. Maternal sensitivity was assessed with the Ainsworth maternal sensitivity scales at 6 months. A measure of "negative emotionality/behavioral dysregulation" was derived from the Early Childhood Behaviour Questionnaire administered at 36 months. Bootstrapping-based mediation analyses revealed that maternal depression mediated the effect of maternal childhood adversity on offspring negative emotionality/behavioral dysregulation (95% confidence interval [0.026, 0.144]). We also found a serial, indirect effect of maternal childhood adversity on child negative emotionality/behavioral mediated first by maternal depression and then by maternal sensitivity (95% confidence interval [0.031, 0.156]). Results suggest the intergenerational transmission of the effects of maternal childhood adversity to the offspring occurs through a two-step, serial pathway, involving maternal depression and maternal sensitivity.

Once and Again : History of Rearing Experiences and Psychosocial Parenting Resources at Six Months in Primiparous Mothers.

Animal and human studies suggest that parenting style is transmitted from one generation to the next. The hypotheses of this study were that (1) a mother's rearing experiences (G1) would predict her own parenting resources (G2) and (2) current maternal mood, motivation to care for her offspring, and relationship with her parents would underlie this association. In a subsample of 201 first-time mothers participating in the longitudinal Maternal Adversity, Vulnerability and Neurodevelopment project, we assessed a mother's own childhood maltreatment and rearing experiences (G1) using the Childhood Trauma Questionnaire and the Parental Bonding Instrument. At 6 months postpartum, mothers completed questionnaires on parenting stress (G2), symptoms of depression, maternal motivation, and current relationship with their own parents. The sample consisted of mostly high socioeconomic status mothers recruited from Montréal (n = 135) or Hamilton (n = 66), Canada, with an age range from 18 to 43 years (M = 29.41, SD = 4.85 years). More severe maltreatment and less supportive rearing by the mother's parents (G1) predicted increased parenting stress at 6 months (G2). These associations were mediated through distinct psychosocial pathways: maltreatment (G1) on parenting stress (G2) through symptoms of depression (Z = 2.297; p = .022); maternal rearing (G1) on parenting stress (G2) through maternal motivation (Z = -2.155; p = .031) and symptoms of depression (Z = -1.842; p = .065); and paternal rearing (G1) on parenting stress (G2) through current relationship with the father (Z = -2.617; p = .009). Maternal rearing experiences predict a mother's own parenting resources though distinct psychosocial pathways, including depressed mood, maternal motivation, and social support.

Amygdala and affective responses to infant pictures: Comparing depressed and non-depressed mothers and non-mothers.

In many mammalian species, new mothers show heightened positive responsiveness to infants and their cues when they give birth. As is evident from non-human and human studies, the amygdala is a brain region implicated in both the maternal and affective neural circuitry, and is involved in processing socioemotionally salient stimuli. In humans, infants are socially salient stimuli to women, and mothers in particular. Neuroimaging studies investigating the maternal response to infant cues have identified infant-related amygdala function as an important factor in maternal anxiety/depression, in the quality of mothering and in individual differences in the motivation to mother. The present study investigated the effects of maternal status and depression on the subjective affective response and amygdala responsiveness to unfamiliar infants using functional magnetic resonance imaging. Smiling infant pictures were used in a 2 × 2 design comparing four groups of women: mothers and non-mothers, with and without depression (total of 101 women: postpartum depression [PPD] = 32, non-PPD = 25, major depression [MDD] = 15, non-MDD = 29). We undertook an anatomically defined region of interest analysis of the amygdala response for a priori defined group comparisons. We found that mothers rated infants more positively than non-mothers and non-mothers rated non-infant stimuli (scenery) more positively than mothers. In the amygdala, we found that depression elevated response to smiling unfamiliar infants in mothers but had no effect in non-mothers. Within the depressed groups, mothers (PPD) showed an elevated amygdala response to unfamiliar smiling infants compared to depressed non-mothers. Hence, our results indicate that women with PPD show an enhanced amygdala response to affectively positive infant pictures but not to affectively positive (but non-salient) pictures of scenery. Women with depression outside of the postpartum period show no change in amygdala responsiveness to either stimulus categories.

Dissecting maternal care: Patterns of maternal parenting in a prospective cohort study.

Parental care has a strong impact on neurodevelopment and mental health in the offspring. Although numerous animal studies have revealed that the parental brain is a highly complex system involving many brain structures and neuroendocrine systems, human maternal parenting as a multidimensional construct with cognitive, emotional, and behavioural components has not been characterised comprehensively. This unique multi-method analysis aimed to examine patterns of self-reported and observed parenting from 6 to 60 months postpartum in a cohort of 496 mothers (mean maternal age = 32 years). Self-report questionnaires assessed motivational components of mothering, parenting stress, parenting-related mood, maternal investment, maternal parenting style, mother-child relationship satisfaction, and mother-child bonding at multiple time points. Observed parenting variables included the Ainsworth Sensitivity Scales at 6 and 18 months, the Behavioral Evaluation Strategies Taxonomies at 6 months, an Etch-A-Sketch cooperation task at 48 months, and the Parent-Child Early Relationship Assessment at 60 months. To examine whether different latent constructs underlie these measures of maternal parenting, we conducted an exploratory factor analysis. Self-report measures of parenting correlated only weakly with behavioural observations. Factor analysis on a subsample (n = 197) revealed four latent factors that each explained from 7% to 11% of the variance in the data (32% total variance explained). Based on the loadings of the instruments, the factors were interpreted as: Supportive Parenting, Self-Enjoyment Parenting, Overwhelmed Parenting, and Affectionate Parenting. These factor scores showed specific associations with maternal education and depressive symptoms, as well as with child outcomes, including maternally reported internalising and externalising behavioural problems, school readiness, and child-reported symptoms of mental health. These findings parallel the complexity of the parental brain, suggesting that maternal parenting consists of multiple components, each of which is associated with different maternal characteristics and child outcomes.

Maternal care activates the ventral tegmental area but not dopaminergic cells in the rat.

The ventral tegmental area (VTA), together with the preoptic area, is part of a neural circuit necessary for the expression of maternal behaviour (MB); destruction of either area disrupts MB in postpartum rats. Central to the proposal of VTA activation are dopaminergic cells, for which the cell bodies lie in the VTA and project to forebrain structures. This mesolimbic system is a motivational circuit involved in rewarding behaviours such as sex and MB. Despite their recognised importance, surprisingly, unlike the preoptic area, there are no anatomical descriptions of the pattern of VTA activation or of the dopaminergic cell activation, specifically in relation to MB in the rat. In the present study, we explore the possible activation (as indicated by Fos protein via immunohistochemistry) of the anterior and medial portions of the VTA and in the dopaminergic cells in these regions, as well as in the medial preoptic area, in lactating rats, at postpartum day 7 (after a 12-hour mother/pups separation), and in dioestrous females. After 12 hours, mothers were perfused at that moment or after a 90 minutes of interaction, or not, with their pups. We found a strong significant Fos induction in both the preoptic area and in the anterior portion of VTA in dams that interacted with their pups. The number of dopaminergic cells that coexpressed Fos did not differ across groups. Additionally, we determined Fos and GABA colocalisation in the anterior part of the VTA and found dense GABAergic processes, possibly varicosities, in the area of increased Fos expression. The results of the present study support a proposed GABAergic pathway from medial preoptic area to VTA cells, critical for the expression of MB. Future experiments are warranted to explore the neurochemical identity of the Fos and no-Fos expressing cells that are recipients of GABAergic processes in the VTA, aiming to better understand the neural circuitry of the VTA in relation to MB.

Inter-individual maternal care received and genotype interactions affect dopaminergic phenotypes in female rat offspring.

Rat mothers exhibit natural variations in care and can shape offspring adult behaviour and their maternal care by affecting the dopaminergic system. We explored whether genotype and gene × environment interactions are involved in these processes in nulliparous female offspring. We assessed maternal licking/grooming toward individual female pups during the first week postpartum and dopamine-related behaviour of the offspring in adulthood. Behaviours explored included strategy shifting, impulsive action and sucrose preference. Single nucleotide polymorphisms in the dopamine receptor 2, dopamine transporter and catechol-O-methyltransferase genes were examined in relation to offspring behaviour and baseline dopamine turnover in select brain regions. Dopamine receptor 2 (RS107017253) variation moderated, or interacted with, the relationship between early-life licking received and behaviour. Specifically, offspring with the A/A genotype showed a significant correlation between early-life licking received and behaviour. Offspring with the A/G and G/G genotypes did not show this relationship. Dopamine transporter gene variation affected offspring behaviour regardless of early-life licking received. Our findings suggest that genotype can directly affect dopamine-related behaviours and alter the sensitivity of offspring to the maternal environment. This could be informative on how maternal care is transmitted between generations of female offspring.

Alternating optimization for G × E modelling with weighted genetic and environmental scores: Examples from the MAVAN study.

Motivated by the goal of expanding currently existing Genotype × Environment interaction (G × E) models to simultaneously include multiple genetic variants and environmental exposures in a parsimonious way, we developed a novel method to estimate the parameters in a G × E model, where G is a weighted sum of genetic variants (genetic score) and E is a weighted sum of environments (environmental score). The approach uses alternating optimization, an iterative process where the genetic score weights, the environmental score weights, and the main model parameters are estimated in turn, assuming the other parameters are constant. This technique can be used to construct relatively complex interaction models that are constrained to a particular structure, and hence contain fewer parameters. We present the model as a 2-way interaction longitudinal mixed model, for which ordinary linear regression is a special case, but it can easily be extended to be compatible with k-way interaction models and generalized linear mixed models. The model is implemented in R (LEGIT package) and using SAS macros (LEGIT_SAS). Through simulations, we demonstrate the power and validity of this approach even with small sample sizes. Furthermore, we present examples from the Maternal Adversity, Vulnerability, and Neurodevelopment (MAVAN) study where we improve significantly upon already existing models using alternating optimization. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Maternal perceptions of paternal investment are associated with relationship satisfaction and breastfeeding duration in humans.

This study examined potential pathways in the associations between breastfeeding and mothers' relationship satisfaction, including her satisfaction with father involvement (FI) and parity, among mothers not working outside the home at 6 months. Mothers (n = 222) completed questionnaires at 4 time-points, 3 to 24 months postpartum as part of a longitudinal cohort study. In this study, we were interested in two main outcome variables: mothers' relationship satisfaction with their partner (RS) and continuation of breastfeeding after 3 months. Our first analysis revealed that breastfeeding at 3 months postpartum predicted decreased RS at 6 months postpartum, which was mediated by mothers' dissatisfaction with FI in infant caretaking at 6 months postpartum. These associations depended on mothers' parity: Multiparous breastfeeding mothers were the most dissatisfied with FI. Second, mothers' satisfaction with FI at 6 months also predicted increased RS at 24 months through increased RS at 12 months, but not through FI at 18 months. Third, we found that high dissatisfaction with FI at 6 months was the only significant predictor for the discontinuation of breastfeeding from 3 to 6 months postpartum. Our results suggest that multiparous breastfeeding mothers might be more dissatisfied with FI in caregiving than nonbreastfeeding mothers and primiparous breastfeeding mothers. Furthermore, mothers' satisfaction with FI seems a potent predictor of overall RS up to 24 months postpartum and the continuation of breastfeeding from 3 to 6 months postpartum, regardless of parity. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Both maternal care received and genotype influence stress-related phenotype in female rats.

Rat dams differ naturally in the level of maternal care they provide to their offspring within the same litter. We explored possible mechanisms of differential maternal care focused on genetic variation. We examined single nucleotide polymorphisms in the glucocorticoid receptor, FK506-binding protein, and serotonin transporter genes in two separate cohorts, and the relationship between differential maternal care received, genotype, and offspring phenotype. Allelic variation in all three genes was significantly associated with levels of maternal care received by offspring and behavioral and endocrine stress responses in adulthood. Differences in pup behavior were also associated with allelic variation in these genes. Together, these results indicate that the dam/pup interaction is dynamic and implicate the genotype of the offspring in influencing the level of maternal care received. They further suggest that some genotypes may have a dampening effect on the impact of maternal care on stress-related phenotypes in adulthood.

Exploring the morphological and emotional correlates of infant cuteness.

Ethologists have observed that "baby schema" or infant cuteness is an adaptive protective mechanism ensuring the young's survival. Past efforts to quantify cuteness have been restricted to line measurement techniques. We developed a novel data-driven approach to quantify infant cuteness into a single metric. Using the Psychomorph program, we delineated facial elements of 72 infant pictures using 206 facial points and identified the facial components that were significantly related to subjective cuteness perceptions of the faces. 108 nulliparous females rated the pictures on cuteness and emotional dimensions. We found that cuter infants have larger cephalic curvature compared to a smaller chin, a big smile, and round chubby features among others. We also investigated the relationship between cuteness and emotional responses. Our results show that a greater degree of cuteness elicits both increased positive emotional responses and decreased negative emotional responses. Cuter infants also elicited greater feelings of alertness, interest, and the need to respond. In fact, the participants' emotional responses were predictive of both data-driven scores and subjective perceptions of cuteness.

Executive function in teen and adult women: Association with maternal status and early adversity.

The aim of this study was to determine the impact of maternal age on executive function and the moderating effects of women's maternal status and early-life experiences. Four groups of women were assessed as a function of their age (teens vs. adults) and maternal status (mothers vs. nonmothers). Participants completed executive function tests, including Spatial Working Memory (SWM), Intra-Extra-Dimensional-Set-Shift (IED), and Stockings of Cambridge (SOC). Women also completed the Childhood Trauma Questionnaire to assess their experiences of early adversity. Results showed that for the IED-task, there were main effects of age and maternal status and an interaction between the two; adults performed better than teens, mothers performed better than nonmothers, and teen nonmothers performed the least well of all groups. For the SWM-task, adults performed better than teens. Our results indicate that although age is an important factor for proper executive functioning, different tasks are affected differently and other factors such as maternity and adverse childhood experiences moderate this functioning.

The Ontario Birth Study: A prospective pregnancy cohort study integrating perinatal research into clinical care.

BACKGROUND: Pregnancy and early childhood represent critical periods that impact health throughout the life-course. The Ontario Birth Study (OBS) is a pregnancy cohort study designed as a platform for research on pregnancy complications, maternal and infant health, and the developmental origins of health and disease. METHODS: Pregnant women <17 weeks gestational age were recruited between 2013 and 2015 from antenatal clinics at Mount Sinai Hospital, Toronto, Canada. Life style and diet questionnaires, biospecimens, and clinical data were collected throughout the pregnancy and postpartum period at the time of clinical care. The OBS was integrated into clinical care to reduce participant burden, improve efficiency, and increase research potential. RESULTS: There were 3181 eligible women approached for recruitment and 1374 (43%) participated in the study. Among the 1374 participants, 1272 (93%) delivered a liveborn infant and were followed to 6-10 weeks postpartum. Of the 1272 women who completed the study, 98% had at least one pregnancy blood sample collected, 97% had vaginal swabs collected, 90% completed the prenatal life style questionnaires, and 78% completed the Diet History Questionnaire. Most women (88%) were ≥30 years of age, 55% had no previous children, 24% were overweight or obese pre-pregnancy and 78% of parents had postsecondary education. Most pregnancies were singleton (3% twins), 34% delivered by caesarean section, and 6% preterm (<37 weeks gestation). CONCLUSIONS: The OBS is a contemporary cohort with detailed data including banked biospecimens for studies of pregnancy health and the gene-environment interactions that establish developmental trajectories to health, learning, and social functioning.

The role of breastfeeding in the association between maternal and infant cortisol attunement in the first postpartum year.

AIM: To explore the role of breastfeeding as a possible link between maternal and infant cortisol attunement across the first postpartum year. METHODS: Mothers (n = 93) provided salivary samples for cortisol levels over a two-day period during mid-pregnancy and at three, six and 12 months and infants at six and 12 months postpartum. Breastfeeding status was established at these same time points. RESULTS: Among breastfeeding mothers, positive correlations were found between maternal cortisol levels during pregnancy and at three months postpartum and infant cortisol at six or 12 months postpartum. Among nonbreastfeeding mothers, these same maternal and infant cortisol relations were inverse and less pronounced. Further, in breastfeeding mothers, the relationship between maternal prenatal cortisol and infant cortisol at 12 months was mediated through maternal cortisol at three months postpartum. CONCLUSION: These results suggest that maternal cortisol levels are positively associated with cortisol levels of the infant, among mothers who breastfeed. This relationship persists over a one-year period.

Relative Abuse of Crush-Resistant Prescription Opioid Tablets via Alternative Oral Modes of Administration.

Objective: Some crush-resistant tablet formulations (CRTs) reduce prescription opioid abuse by nonoral routes of administration (ROAs), especially insufflation and injection, while oral abuse increases. Oral abuse involving product manipulation vs swallowing whole for CRTs and comparators was examined. Methods: Abuse by oral modes of administration (e.g., swallowing whole, chewing, dissolving in the mouth), was examined using the ASI-MV, a computerized, clinical interview for adults in substance abuse treatment from January 2009 to March 2015. CRTs (reformulated oxycodone extended-release [ER], reformulated oxymorphone ER, and tapentadol ER) were compared with non-CRT versions, morphine ER, and oxycodone immediate-release single entity. Analyses employed descriptive statistics and logistic regression. Results: Among 364,329 unique assessments, 18,135 patients reported oral abuse of the CRTs and comparators examined. CRTs had a higher prevalence of oral abuse involving product manipulation than comparators (P < 0.0001) among all abusers of product. Oral abuse involving product manipulation for CRTs was greater among the subset of patients reporting oral abuse and significantly higher than comparators (P < 0.003). CRTs were significantly less likely than comparators to be swallowed whole (P < 0.0001) and significantly more likely to be chewed (P < 0.003). CRTs were more likely to be dissolved in the mouth than most comparators. Conclusions: Results suggest the need for abuse-deterrent formulations designed to reduce abuse by oral administration with product manipulation, such as chewing. Advances in this area may reduce the overall abuse of prescription opioids and interrupt the progression from abuse by swallowing whole to oral administration involving product manipulation and other ROAs.