Thank you very much. Good morning, everyone. Welcome to the featured session, the first session of the day, too, titled The Role of Endocannabinoid in Early Brain Development and Consequences of Fetal Cannabinoid Exposure. My name is Daoyu Wu. I'm Program Officer at NIDA, NIH. And how cannabinoids, cannabis and cannabinoid use and misuse by pregnant people and fetal cannabinoid exposure effect on the developing brain is a timely and important research area as there has been a dramatic increase in the cannabinoid and cannabinoid use by pregnant people. So our first session is presented by Dr. Jorge Manzanares, sorry, Professor at the University of Miguel Hernandez in Spain. And Dr. Manzanares is a leading investigator in the cannabinoid research in Europe. And he has published several very significant papers on cannabinoid use by pregnant people and how the consequences of the fetal cannabinoid exposure affects the developing brain. And please join me in welcoming Dr. Manzanares. Hello. Good morning to everybody. It's a nice session for being at 8 in the morning, the first day of the meeting. So thank you all to come here for the first thing. So this is the, I have to thank also NIDA and Daoyu Wu for inviting me and Jorge for inviting me. And I'm very happy to be here. I have to thank also NIDA and Daoyu Wu for inviting me and just to show you some data that actually they are unpublished today. So this is very, very, very new. Okay. I have been working in the last three, four years on the effects of the high concentration of THC that these are the ones that have been used right now on a model of fetal, whether I'm calling fetal cannabinoid syndrome. And I will show you some of the results. Maybe I will go a little fast because I don't have too much time. But we can go through the question later. The first thing of the problem is to talk a little bit about the epidemiology, the impact of the perinatal cannabinoid use. First thing is that as Daoyu was mentioning, in recent years there has been a significant increase in cannabis use primarily driven by legalization. Second thing is now 38 states in the U.S. have legalized cannabis for medical use and 70 allow it for recreational use. You can just walk from Manhattan and smell marijuana all over. And this has been happening for many, for the last of maybe three or four years. Unfortunately, these policies have led to an increase in prevalence and frequency of cannabis use during pregnancy, particularly in the first trimester between 2002 and 2017. I will show you some, quickly some graph later on. Additionally, the rate of cannabis use disorder among pregnant women has increased five-fold from 18.4, 10,000 inpatient hospital delivers in 1993 to 93.6 in 2014. This trend is linked to legalization, false perception of safety, and mice-guided recommendation to use cannabis to alleviate pregnancy symptoms such as nausea, vomiting, and morning sickness. The American College of Obstetricians and Gynecologists and the American Academy of Pediatrics have both advised against using cannabis during pregnancy and breastfeeding, discounting the means surrounding its safety. Just look at this graph on the right. And this is, by the way, a report of United Nations Office on Drug and Crime. And you're showing that pregnant women is almost 30% increase from 2002 to 2019. In the first trimester, it's even a little more difference, and still on the second and the third trimester. This is our data for women in Colorado, and it's showing also during pregnancy the increase all over the years. So this is a big problem, not only in the United States, but in many of the states where it is legalized in Canada and some other countries. So the available evidence strongly suggests that the perinatal cannabis exposure can negatively affect the mothers and their children. Maternal cannabis use may result in emotional and cognitive impairments that could significantly impact self-care during pregnancy and the care of the newborn during lactation. I won't show you the effects today on the mothers, but the mothers lose care for their pups, similarly like in an alcohol treatment during pregnancy. The prenatal cannabis use has been also linked to low birth weight, preterm birth, and a range of attentional, social, and behavioral problems that can last into early adolescence and even later on. Little is known about the effect of the varying doses of Delta-THC. Remember that 25 years ago, the content of THC is approximately 8% to 10%. Right now, it's 20% and sometimes 30%. So that makes that many of the studies that will carry on probably 25 years ago. I did some of these studies 25 years ago. Now, they are not actual, because the concentration is much, much higher. And we don't know either what happened in doing into the adolescent, and we don't know differences between the males and the females. So we don't even know the neurobiological mechanisms underlying these cannabis-induced neurodevelopmental impairments, because they are very poorly understood. So what we did is we developed this model. Dronabinol is a synthetic THC, and we administer from day 5 of gestation until the end of lactation, 10 milligrams per kilogram every 12 hours, 12 days, or a vehicle. From day 5 of gestation until the end of lactation. So mothers are receiving the THC by the placenta first, and then the children are receiving also through the milk. Then we did several behavioral studies that I will mention. And then at the end of postnatal day 60, we also did a study on vulnerability to ethanol consumption to see if the changes that the THC is producing during pregnancy and breastfeeding have consequences on the vulnerability to alcohol. And then here we did some studies on gene expression in several nuclei. We did also immunostochimies that I will show quickly. And at the end also we studied some GRT-PCR on some of the nuclei of the animals submitted to the Alcohol Self-Administration. See, these are the results on this paradigm that is analyzing the anxiety. We measured the time that the animals are in these open arms. This is the elevated plasmase. And as you can see, in males there is a decrease. That means that there is anxious behavior. And in the females also it's the same effect. Then we analyzed also this test called novelty suppressed feeding test that analyzed the latency to go to eat after being fasted. And the time that takes to go to have the food and the amount of food is reflecting on the sometime of different type of anhedonia. So you can see that the animals coming from the mothers that receive the THC take longer time to go to be feeding and the consumption is lower and exactly the same for the females. And also we analyzed the tail suspension test. This is a behavioral test that tried to simulate depressive behavior and measured the time of immobility when the animals don't want to fight to be in that situation. And when the time increased, like we see here, there is depressive behavior in both males and females. We also analyzed the cognitive behavior using this test that is called to explore two different paradigms. And we measured the index of recognition in the novel object recognition. And you can see that in the males the effect is nothing. So there is a big, big problem. And discrimination also in female is also very low. We use also this other test, the step-down inhibitory avoidance, that is using a grid as electrified. So the animal is put in this step. When it goes here, then go back immediately to the step. And then we put again the mice here one hour and 24 hours later. If it goes down in less than three minutes, it means that it doesn't remember that that was electrified. So you can see the same thing here. At one hour, they go down. At 24 hours, they go down. So the memory, short-time memory and long-term memory, are affected very much in males and females. Also, we measured the attentional deficit by using the pre-pulse inhibition that you probably know because this has been used in a very similar manner changing some of the paradigms in animals like rodents but also in humans. And what we have here is an inability to filtrate the information in males and females. So this is telling us that there is an attentional deficit associated to the animals where the mothers have been having THC. Then we studied gene expression studies. I will go a little bit fast on that. We analyzed the stress axis and we measured CRF gene expression in the paraventricular nucleus. So we're able to do by doing micro-dissected nuclei. And R2-RT-PCR very sensible. And in males, there is not too much difference. But in the females, there is a decrease of CRF gene expression. There is an increase in the glucocorticoid receptor in the male in the hippocampus but not in the female. So we're beginning to see here some differences between males and females with respect to the stress axis and with respect to the reward circuit. There is also differences basal between males and females. And here the changes are very similar in the mu-opioid receptor gene expression. And there is also completely different reaction in the TH, tyrosine hydroxylase gene expression in the ventral tegmental area. So we have some differences here too between males and females. When we study the endocannabinoid targets, we study CB1 receptor and CB2 receptor in the hippocampus and the amygdala. You will see also that there are some differences on the CB1 receptor in the hippocampus between males and females. There is no differences between males and females in the amygdala. Of course, amygdala is more linked to the emotional dysregulation that we have. And when we measured CB2 receptor, the CB2 receptor is increased in both males and females and stays the same thing for the females in the amygdala. So when we analyze the cells, here you can, this is neurons, it's labeling neurons, you can see a clear difference in males. Just look at the amount of cells in many areas, like in the ilos and in the dentate gyrus. And also in the parietal cortex, you will see that THC is doing a disorganization of the several layers of the cortex. That means that this is associated, for example, with high impulsivity, attentional deficits and some other behavioral changes. You can see that here the layers are very well defined in the vehicle. This is the 1, 1, 2, 3, 4, 5 and 6. And here you will see that this is more compacted, less neurons labeled and more disorganized, less differences between the layers. The same thing is happening in the females, very similar here. Even the disorganization is even more pronounced than in males. And you can see it very quickly. We also measure BDNF, brain-derived neural growth factor. And the label here that is in green, you can very clearly see that there is much more label here than here. This is a lot of dark, as we say. And in the ilos here, you can see all these cells. And here there is no, even almost no cells. So we are losing the ability to neurogenesis. In the female, the result... Okay. Here. The female is the same, same results that we have. And also this we can see very clearly on the ilos. Then we analyze the neurofilaments as another element of constituting of the cells. And you can see also big changes. And probably you can see it better in this image of these neurofilaments, how we are losing labeling here in the animals coming from THC, in the males and the females. Also, we analyze excitatory and inhibitory neurons labeling with the VGLU and the VGAD. And there is a decrease in several areas of the excitatory neurons and decrease in the inhibitory neurons. In the blue, probably you don't see very well. In both males and females. Here, especially in the mossy fibers and also in some areas of the ilos. Both in males and females. So what we did is we used this paradigm, alcohol self-administration. For those that you are not familiar, there is a box, very sophisticated, where you have an active lever, an inactive lever, and a receptacle with a light. When you press the active lever, there is a light and there is an acoustic stimulator and there is a drop of alcohol that comes here. And we are going to measure two different paradigms. One is the reinforcement to alcohol and the other thing is the motivation for alcohol. The motivation we measure on the reinforcement on the FIGS ratio 1. FIGS ratio 3 will be motivation and breaking point. Also, we will say motivation. So these are males. And from the beginning, on the FIGS ratio 1, you can see that the animals coming from the mothers having THC are an increased alcohol consumption. This is the number of effective responses and this is the intake of ethanol. And this effect is permanent also in the motivation of the FIGS ratio 3. And also, you can see very clearly in the progress response on the breaking point, both here and also in the ethanol intake in this situation. In females, the effect is very similar. And we continue to have probably, if you see here, the motivation here, the effect, although it's significant, is a little less pronounced than in males. So what can we conclude of all this? Well, this is the gene expression. There are also differences in the nucleus accumbens. This is CB1 receptor. On the CB2 receptor, there are increases in both situations. In the nucleus accumbens, there is also a difference in the mu-pure receptor. It goes in different directions, males and females. It also goes in different directions, tyrosine, hydroxylase in the ventral tegmental area. So what are the conclusions of this? One is that males and female mice perinatally exposed to dronabinol present significant angiotenic and depressic-like behavior and emotional hyperreactivity. Second thing is that mice that develop cognitive disturbance, problems in associative learning memory and attentional deficit. These behavioral changes were associated with gene expression alteration in different brain targets associated with stress axis, reward circuit, and endocannabinoid system. Males and females exposed to dronabinol during gestation and lactation display alterations in the number of cells, neurofilaments, neuroplasticity, gut biergic, glutamatergic, neurotransmission, hippocampus, and cortical lamination. This very pronounced disorganization in several layers of the cortex. Ethanol consumption and motivation is increased in males and females exposed to dronabinol and was associated with gene expression alteration in the endocannabinoid and the reward system. So finally the conclusion of all that will be that probably it is essential to raise awareness among healthcare professionals and the public regarding the risk associated with cannabis use during pregnancy and to discourage the use of cannabis for medical or recreational purposes by pregnant women. And I would like just to give you an example. In many beaches with sand in many countries, the city or the government is in charge to tell you that if you go to swim, you may have problems and then they have a red flag when there is a problem in the beach or they have a green flag. The problem is in many countries, including this one, the flag for this is always green. So it's not that you make a choice consciously. You make a choice that has been manipulated by politicians mainly because this is not to increase the health of the people because we have many drugs synthesized that can be used. I know countries, I travel a lot by Central America and Iberia America, I know countries where we have doctors that are advising to have a plant at home to smoke. This is super crazy. Smoking something. I've been a smoker of everything. So how can you advise to smoke something? These are doctors doing that. So at least we have to have a red flag. At least politicians have to advise about the problem. Don't they advise about the tobacco? Don't they advise about alcohol? Why don't they advise about this? This is not lettuce or potatoes or apples. It's different. We have many, many different drugs inside. And this false perception is causing many problems. And at least for many countries, we should consider that this is an investing of money in health. So this is my final say. Thank you very much for your attention. We have time for one or two questions. Thank you for that. Excellent presentation and that very compelling data. I guess the question that many of us may be asking ourselves is, we know that these animal models, we've learned a lot about addiction from them. So we know that this is a sound foundation as a research protocol. But what do we know about dronabinol dosage? You know, from a clinical perspective, what kind of a level is that compared to typical human use, if we can say anything about that? Well, that is very difficult, because this is like if you're talking about alcohol and fetal alcohol syndrome. You may have women that drinks half a bottle of whiskey every day, and they don't have any problem. You have other women that drink three glasses of wine, and they may develop fetal alcohol syndrome. So in terms of the dose, it's very complicated, because you have vulnerability in different persons. But the problem is that now, as I said, the concentration of THC in the plants is more than double than it was 20 years ago. And also, this is one reason. The second reason that I mentioned at the beginning is that there is a false perception that there is no problem. Now you have families where the children and their parents are smoking together. And then here I imagine that most of the people that are sitting here are psychiatrists. So just ask the psychiatrist how many people are going with schizophrenia and how many of them don't smoke marijuana. And if I'm not mistaken, at least 70 to 80% of the people who go now with a schizophrenia problem to the psychiatrist have a consumption of THC. Because the development of schizophrenia is related with the concentration of THC. Just look what happened with the spice of some of the drugs or with some agonists of CB1. The schizophrenia developed even more and sometimes you cannot treat it even with antipsychotics and you may know that. I saw these people in the Bellevue Hospital a few years ago and they were not able to treat it with antipsychotics. 600 people died adolescent last year in the United States for synthetic cannabinoids. Unfortunately, it's not in all the countries right now. But it's beginning to be in the north of Mexico, in the United Kingdom, and in some others. So it should be a problem. So the concentration is also a problem. I have one question. The cytology and histology is really impressive. So from your research, would you like to comment or predict the difference between male and female to cannabinoid? Is it due to different influence of hormonal result to the brain or would you like to say it's a more structural difference that results in that kind of difference? Well, this is coming from different reasons. One is that females apparently are slightly more protected than the males for different type of paradigms, but not for all. And this is also related with the different concentration of the endogenous cannabinoids and cannabinoid receptor in different areas between males and females. Females have more, for example, CB2 receptor, more density of CB2 receptor apparently is protecting for that. So genetic expression is already... Probably is this related by the hormone estrogen circulating? Probably. In that very early stage already. This has not been studied. That's wonderful. Thanks. Thank you. Thank you very much. Our next speaker is Dr. Jamie Low, Associate Professor at Oregon Health and Science University. Dr. Low is a reproductive biologist, physician, scientist with a research focus on understanding the effects of environmental exposures, particularly substance use on reproductive health. She has done many very impressive research on the fetal development using non-human primates as a model system. Dr. Low. Awesome. Thank you for that kind introduction. Well, thank you for the invitation to speak to you guys today about our group's work. A lot of this research was really inspired by a few years ago in the clinic because I'm a high-risk obstetrician. A lot of my patients I was noticing in Oregon were telling me that they were using cannabis for a lot of their symptoms in pregnancy, especially nausea, which was interesting because they said it's more natural, it's plant-based, and first-line recommendations by the American College of Obstetrics and Gynecologists is actually using B vitamins. Which is also very natural. So it really inspired me to kind of look into this area. And when I did, I realized there wasn't a lot of guidance at that point, probably about four or five years ago for my patients. And so it led me to focus in this area. I have no relevant disclosures or conflicts of interest. So as Dr. Manzanares had indicated, there's been significant rise in prevalence of use in pregnancy, in part due to the increased availability and perceived safety. Most commonly what I see in the clinic is people use it for morning sickness, sleep, stress, and pain. Usually the prevalence is about two to five percent in most populations, but can be up to 30 percent in certain demographics, usually younger, more urban, lower socioeconomics. The figure on the right is very similar to the figure earlier, which just highlights the increasing rates of cannabis use in pregnancy across all trimesters of pregnancy, especially compared to two decades ago. And then interestingly during COVID, because of some of the increased stress people were having and trying to combat that, we saw a rise in about up to 25 percent in certain areas of the U.S. of use of cannabis in pregnancy. So as we know, cannabis in the plant contains hundreds of different chemicals, including compounds called cannabinoids. Delta-9-THC is the main psychoactive cannabinoid. It's small, highly lipophilic. It can pass through the placenta and is rapidly distributed to the brain in areas that are kind of fattier. So cannabinoids mediate the medicinal and psychoactive properties of cannabis. They bind to endogenous receptors in the endocannabinoid system. This is a widespread neuromodulatory system that I'm highlighted by the figures here. So on the left you can see, this is in green, it's showing the distribution of the cannabinoid receptor 1, or CB1. And then it's expressed throughout the body. It's most abundant in endocrine tissues and the nervous system. And then on the right, shown in blue, this is the CB2 receptor, which is more enhanced in immune-related cells and the gastrointestinal system. So as you can see here by the both figures, there's receptors of both CB1, CB2 expressed in the male and female reproductive tracts. It's also found in the placenta and major fetal organs, suggesting that the endocannabinoid system plays a role in regulating reproduction and development. Currently, the existing literature supports that prenatal cannabis use is associated with adverse fetal outcomes. These include stillbirth, preterm birth, growth restriction, small for gestational age, increased NICU emissions, and altered neurodevelopment. These findings then continue on into early childhood and adolescence, where offspring are found to be less attentive and are more impulsive and hyperactive. Adolescents are also at increased risk for substance use disorders and autism spectrum disorder. Ultimately, to date, as highlighted, there's really no amount of cannabis that I can recommend to my patients that's been proven safe to use in pregnancy. So the research on cannabis can be really challenging for many reasons, and especially in pregnancy, especially because there's increased stigma despite even the recent legalization trends. Existing studies focused on preconception or prenatal cannabis use are often retrospective or observational in study design. They often are not able to address mechanistic underpinnings and are smaller in sample size. Additionally, a lot of studies lack quantification of use or timing of exposure. And as we know, whether they use it in the first trimester, second, or third, it really does matter, especially in the first when the fetus is most vulnerable to environmental exposures. And even if the studies do have biological testing performed, they can't determine the timing, the dose, the frequency, or duration of use. Often, a lot of the studies are also confounded by concurrent tobacco use or other substance use, nutrition, sociodemographic factors. And a large portion of the literature still is published back in the 1980s when a lot of the products that were used was just smoking, not the many different modes of delivery that we have today. And also, as Dr. Manzanaris had highlighted, potency was two to three times less. So, as I mentioned, this really inspired our group to study the effects of cannabis exposure using a rhesus macaque model. We selected edibles as our mode of delivery just because it's the second most common mode of use amongst all users, especially reproductive age individuals. And it's one of the most popular methods among pregnant individuals. An animal model allows us to study the direct effects of THC only. It avoids and overcomes the challenges of confounding from other toxins or other substances that are seen in human cohorts. It also allows us for standardization when administered using a weight-based dosing method. It also allows in pregnancy studies for repeated measurements by ultrasound and MRI across the pregnancy window, and then collection of placental and fetal tissues at delivery from molecular analysis, trying to get at maybe potential underlying mechanistic pathways. The use of the rhesus macaque model is ideal because it recapitulates typical human use. Also, there's a lot of similarities between rhesus and humans, including the genome, developmental ontogeny, reproductive biology, and function. They also have a 28 to 29-day menstrual cycle and also have a similar plasma THC metabolism. So as I mentioned, we developed a rhesus macaque model to look at a prenatal cohort that was using female rhesus macaques that underwent THC induction about four months prior to conception. This is following published medical cannabis acclimation guidelines, using the same weight-based dosing as recommended in humans. During the THC induction, we observed that plasma THC levels rose appropriately with the weight-based dosing escalation. Females reached a 2.5 mg per 7 kg per day dose, which is equivalent to a heavy medical marijuana dose of like 25 mg per 70 kg human per day. And then after they reached that dose, they underwent timated breeding and then were kept on that dose throughout pregnancy. Throughout the pregnancy, as shown in this timeline, pregnancies were then assessed using serial ultrasounds, so standard clinical ultrasound that I do in my pregnant patients, and then serial MRI as well to look at the placenta and the fetal brain. And then they were delivered by term by C-section for placental and fetal tissue collection. So this slide summarizes our initial findings from the prenatal cohort. We didn't see any significant differences in fetal biometry measurements by ultrasound. And then in the bottom left, you can see on the table, we didn't see any significant differences in fetal, maternal, or placental weight between the two treatment groups at delivery. However, we did note that THC-exposed animals demonstrated significant findings suggestive of placental insufficiency. These included decreased amniotic fluid volume, placental perfusion, and fetal oxygen availability on MRI across gestation. In addition, we noted increased microscopic infarctions and syncytiotrophilastic knotting on placental histology that was only seen in THC-exposed pregnancies, not in controls. Syncytiotrophilastic knotting of the placenta is associated with adverse pregnancy outcomes and is a marker of hypoxic injury. We also took the placental tissue and performed bulk RNA sequencing. We found dysregulated expression of genes involved in vascular development, cellular proliferation, inflammation, apoptosis, and angiogenesis. Our findings of dysregulated placental gene expression and function with THC exposure is consistent with the placental brain axis hypothesis, which postulates that neurobehavioral disorders originate from dysregulated placental function. There have been several other studies, including ones by Yasmine Hurd, that have linked adverse in utero environments with changes in placental DNA methylation and associate those with neurobehavioral outcomes. Then this combined evidence led us to investigate epigenome-wide methylation profiles in the THC-exposed pregnancies. We performed this using Illumina EPIC array on placental and fetal tissues. That included cerebellum, prefrontal cortex, heart, and lung. We noted differential methylation linked to neurodevelopment in candidate autism spectrum disorder genes that were affected in all the tissues that were tested, most predominantly in the placenta. Pathways that were involved were morphogenesis, nucleic acid metabolism, and nervous system development. We also looked at the impact of THC on fetal brain development. To study this, as I mentioned before, we performed serial in utero T2-weighted MRI across gestation to assess for differences in fetal brain structure and volume. Here, shown on the slide, is representative MRI images shown in the axial, sagittal, and coronal planes. What we did was we took different areas of the brain and we segmented it for regional analysis, as shown here, color-coded. Overall, we didn't observe a significant difference in global or regional volumetric growth of the fetal brain throughout pregnancy between control and THC- exposed animals. Interestingly, we found that prenatal THC exposure was associated with a significant age-by-sex interaction in regional brain volume, especially in the cortical plate in corpus callosum. THC-exposed males alone exhibited reduced volumes, which is consistent with the literature demonstrating that male offspring typically experience a greater effect with prenatal substance use exposure. This is also consistent with the reported findings in adults that are high-potency cannabis users and other animal models, where there's a greater disruption to the corpus callosum, in part because that region has a higher cannabinoid receptor expression. The corpus callosum plays a major role in movement control and cognitive functions, such as memory and learning. At time of C-section, fetal cerebral spinal fluid was collected for microRNA studies and fetal tissue for histologic analysis. Fetal brain histology was assessed by two neuropathologists that were blinded to the treatment groups. What they found was microscopic findings noted in all THC-exposed animals, but again not in controls, suggestive of fetal brain dysregulation and injury, including acute and chronic ischemic white matter changes. And also extracellular vesicle-associated microRNAs were identified in THC-exposed fetal cerebral spinal fluid, and pathway analysis suggests that these microRNAs are associated with dysregulated axonal guidance and netrin signaling. This data is indicative of subtle molecular changes consistent with observed histological data, suggesting a potential role for fetal microRNA regulation by THC. So then given all these in utero findings that we found, suggestive of potential neurologic impact, we extended our model to include a postnatal cohort so that we can correlate these findings with the postnatal neurodevelopment changes. So for the postnatal cohort, as shown on the slide, we followed a very similar timeline to the prenatal cohort. Similarly, animals were induced on THC and acclimated over a four-month period, again to that same weight-based 2.5 per 7 kgs per day dose, similar to heavy medical marijuana dose in humans prior to pregnancy, and then this was maintained through the pregnancy and then also in the postnatal period. The postnatal cohort was allowed to deliver a naturally at term and then with maternal rearing and infant testing that was largely occurring in their home cage, and this was done to minimize potential for infant stress. On the bottom, again, there's the timeline that just highlights when infant brain MRIs were performed and neurodevelopmental and behavioral testing. Maternal-infant interactions were observed and scored on postnatal days 1, 7, 21, 29, 60, and 90, and these included observations of the infant position on the dam, contact, grooming, rebuffing, restraining, and developmental behaviors such as gaze searching, orbital licking, and then aggression in response to tantrums. Infants underwent the primate neonatal neurodevelopmental test at 30 days of life. This was modeled after the human Brazelton neonatal behavioral assessment scale that evaluates early development and can capture known emergence of the behavioral milestones, including grasping, rooting, startle response, and visual orientation tracking that then was transformed to an ordinal scale. And then at four months after birth, infants were then transported to a satellite cage in a behavioral suite. This testing takes place in a novel environment. It's composed of an acclimation period, a human intruder test, and then a novel objects test. These tests were all live coded and then analyzed for signs of anxiety and depression as shown in the blue box on the bottom left. So what we found was a treatment effect across all measures, especially THC-exposed infants exhibited less independence during maternal infant interactions with increased nursing time and time spent on the mom compared to the controls shown in the gray bar. THC-exposed infants also exhibited decreased motor development, increased grooming and hair cortisol levels, which was suggestive of increased stress. And then also we noted increased vocalizations in THC-exposed infants during the four-month temperament test compared to their controlled counterparts. And again, because these assessments were taking place in a novel environment, we think that these increased vocalizations to the novel environment and stimuli are an indicator of increased anxiety and stress in the THC-exposed cohort. We also examined the impact of THC on infant brain development. So to study this, we performed the same serial T2-weighted MRI longitudinally across the pregnancy window in the fetus in uro, as we described earlier, and then in the infant postnatally to again look at structural differences and differences in brain volumes. And so here again is just a slide demonstrating the representative MRI images, again in the axial, sagittal, and coronal planes. Although a trend of decreased regional and global brain volumes were noted on fetal brain MRI, we didn't see any significant structural or volumetric changes between treatment groups. We didn't perform a sex-specific analysis here, as we had done in the prenatal group, just because we only had one male infant in the THC-exposed group. We also assessed for fractional anisotropy on MRI, which is a measure of white matter maturation. What we found was that offspring exposed to THC in utero demonstrated higher fractional anisotropy values, especially in the corpus callosum, as well as the centrum semiovalley, compared to offspring from controlled pregnancies. And what this suggests is that gestational THC exposure results in altered white matter developmental trajectories. This pattern of increased early developmental white matter fractional anisotropy relative to controls is really similar to findings in individuals with autism spectrum disorder who have been shown to exhibit also higher white matter fractional anisotropy in the corpus callosum and other white matter tracts during early infancy. With the behavioral changes that we observed, we tried to understand what are the potential underlying pathways. We further assessed the alterations for this and the alterations to the epigenome and transcriptome. To look at the infant epigenetic changes in the brain, we performed whole-genome bisulfite sequencing, similar to the prenatal cohort, and it showed regions differentially methylated in the cerebellum that were involved in synaptic organization in brain development and were enriched for candidate autism spectrum disorder genes and neurodevelopmental genes. And then on bulk RNA sequencing, particularly in the cerebellum, we identified differentially expressed genes at FDR significance that on pathway analysis, we identified enrichment of genes related to axon development, regulation of G-coupled protein receptor signaling, and glutaminergic synapse. So in summary, we found that THC use prior to conception can adversely impact female reproductive health and pregnancy outcomes, especially the placental function and development. And as we know, based on the placental brain axis, if you disrupt the placenta, you can also potentially impact neurobehavioral outcomes and neurodevelopment outcomes in the offspring. It can also impact the placental and fetal epigenome, resulting in altered genes involved in neurodevelopment, axonogenesis, and autism spectrum disorder. Prenatal THC exposure can also affect offspring brain development. And although we didn't see any significant structural or volumetric changes in the fetal MRI images, we did see significant sex-specific changes with the male infants, largely impacted with reduced brain volumes. We also saw histologic findings only in the THC cohort, notable for acute and chronic ischemic changes in injury. And then in addition, our preliminary postnatal behavioral testing suggests that infants prenatally exposed to THC have increased anxiety, stress, and decreased motor development. And then just kind of in summary, as highlighted by Dr. Menzinar's talk, given that prevalence of cannabis use is rising in pregnancy and potency is also significantly increasing, I hope that my talk today really drives home the urgent need for more evidence-driven information on the safety of use preconception, prenatally, and postnatally, so that we can really help develop guidelines and recommendations to help inform patient counseling and public health policies focused on cannabis use. I just want to thank you all for allowing me to present our work today. I'd like to take the time to thank my lab, our collaborators, and the Oregon National Primate Research Center staff. And of course, this project wouldn't have been feasible without generous funding support, especially from the NIH. Thank you. We have time for a couple of questions. Is there research on prenatal exposure only, as opposed to, you know, people who've used marijuana as a teenager and what it does to infants? You mean in human cohorts? Anything. Oh. Yes, there's definitely, there's animal models, the primate model, and then there's human cohorts. I've also looked at just the prenatal use and how that impacts offspring outcome. Also, you know, similar to what Dr. Manzanar has presented and what I presented today, it's just in humans there's more confounding, so it's hard to say if moms only used cannabis in the pregnancy window or if there was also, you know, cigarette smoking or alcohol use and other environmental factors. With animals, it was affected behavior. Some of them showed growth restriction. There's also a larger cardiac to fetal weight, sorry, there's a larger cardiac to fetal weight ratio in terms of, there's enlarged heart size, so cardiomyopathy. There's also, we're also seeing decreased lung volumes and signs of kind of what reactive airway disease would be, similar to nicotine smoke in pregnancy. So, more than just the brain, definitely other organs as well. I wasn't clear on how much of these effects you think might be attributed to sort of placental changes, secondary hypoxia, which, you know, can be true of many things versus effects that are specific to possible, you know, activity of cannabinoids per se. Yeah. So, I have several different animal models and one of them is actually placental insufficiency where we did alter maternal hypoxia and that was our only variable that we tweaked and did not observe the same type of behavioral changes in those offspring. We also didn't observe this in the nicotine model that I had. We did observe it in the alcohol model that I had. Slightly different behavioral changes, but definitely that, even though there was also placental insufficiency. So, I think there's definitely a component where the placenta does, you know, inform, but a lot of this, I think, is mostly the THC exposure. Thank you for the talk. Do you have any evidence or any data about the CBD on fetal neurodevelopment or any evidence show the harm for any effect of CBD on the neurodevelopment? Yeah. No, I just focused on THC first and then next steps were trying to look at a THC-CBD combo one-to-one ratio. So, thank you. Can I have one question? So, does the impairment you see in the newborn get, let's say, corrected or compensated with age in your cohort? How long can you follow them? Yeah, we've only followed for six months and it seems to be progressively more detectable that there is an alteration, but we haven't taken it quite to one year or three years and that's kind of the next steps with hopefully more funding. All right. Thank you very much. Our next speaker is Dr. Laura Stroud, professor at the Brown University. Dr. Stroud is a long-time NIDA investigator in the field of substance use and dependence and she leads multiple prenatal substance use cohort studies and she has made important discoveries in impact of maternal substance use on infant development and novel biological pathways linking these prenatal substance use and fetal development. Thank you so much, Zeyu, and thank you for inviting me here today. This is a wonderful symposium and I love how it's going from the rodent to the non-human primate and I have the honor of talking about humans. So, I just want to say I have no conflicts of interest and I want to especially acknowledge NIDA funding over a lot of years for a variety of different substances, pregnancy, and again, I want to especially thank Zeyu for being an amazing program officer and I've been fortunate to have a number of amazing program officers at NIDA, so just a special thank you. Officers can really make your life a lot better when you're lucky enough to have a great one. And just to acknowledge many, many collaborators, including some great students that will be coming to postdoc and faculty positions along the way, so look out for them. So, I run something called the BAMBAM Lab, the Behavior and Mood in Babies, Adolescents, and Mothers. So, we look at the effects of substance use, different types of substances in pregnancy and other exposures such as stress and trauma, depression, things like that during pregnancy, how it impacts fetal development. We also collect placentas and look at infant development and then ultimately follow them further. So, again, we do a lot of interviewing with pregnant mothers. We do ultrasound assessment, where I'm going to talk a teeny bit about today. We actually look at fetal behavior in the ultrasound, so we try to get at behavior before birth. And then we collect placental samples and we have a new grant to interrogate them, interrogate the genome, and then look at infant neurobehavior. So, marijuana in pregnancy, you've heard a lot about it today, so I'll try to go quickly through these. So, there are high rates of marijuana use in pregnancy, and they have been increasing tremendously in the last decades. There's been some sort of co-people thinking that legalization, especially in the U.S., that legalization is indicative of increase of safety. During the pandemic, we also saw largely increased rates. You see higher rates of cannabis use in disadvantaged, poor, young, and underserved women. So, depending who you ask, how you ask, and when you ask, rates of marijuana use in pregnancy can range from 4 to 11 percent, again, as high as 30 percent in poor and young and underserved populations. As we talked about, rates doubled from 2002 and increased about 25 percent during the pandemic. My slides are showing up for me. As we've heard before, there's inadequate messaging for pregnant people, so often pregnant people are turning to social media and dispensaries for their information. So, again, there's increased social acceptability. Legalization, medicalization, and decriminalization can be confused with safety. THC potency has increased 400 percent since 1995 and doubled in the last decade, as you heard. Many, many outcomes across development, which Dr. Loh did a fantastic job of summarizing, but you see low birth weight and prematurity, increased NICU admissions in infancy, alterations in memory, attention, impulsivity, and hyperactivity in earlier childhood, and then you see alterations in attention and inhibitory control, hyperactivity and substance use uptake as people get to adolescence, and you see a lot of altered executive function over development. Some issues with the prior studies. Many of the cohort studies are over 20 years old. THC potency has increased, so as people say, it's not your mother's marijuana, and it's increasingly available and more acceptable, so it's used differently. There's different types of products available as well. The older cohorts have lower levels of use. They often didn't have biochemical verification of use, and they weren't necessarily designed to look at marijuana. Some of them were designed to look at other types of substances. And as Dr. Liu said, inadequate measures of confounding factors. So we're trying to do the best we can, obviously, in human models. You can't randomly assign people to cannabis. It's not ethical. So we do our best to rigorously measure as many confounding factors as possible and try to select our groups to better understand effects. So in particular, there's not been a lot of human research on early behavioral development and biological mechanisms, which is where our lab focuses. As an example, this might be an early biobehavioral vulnerability marker for substance use. We're also very interested in the idea of prenatal programming, which has been applied to stress models in pregnancy. So the idea that stress may activate the hypothalamic-pituitary-adrenal axis. This may impact placental pathways and impact the infant, so you're sort of programming development throughout the lifetime during the prenatal period. And we were interested in applying some of these prenatal programming ideas to looking at substance use during pregnancy, in particular, cannabis. As we've heard, the placenta is an amazing organ. And in humans, it's discarded tissue, so it's really great. We're very excited about looking at it. So it can be called a master regulator of fetal environment, sometimes called the third brain. It causes a lot of neural receptors, et cetera. And genomic and epigenomic pathways may reveal both footprints of exposure, as well as mechanisms of how effects may be transmitted to the next generation. So I'm going to start with some published studies. And like the rest of the folks here, I have some very early preliminary studies. So definitely not, they're not published yet, and they're early. But we got some, we just got back some epigenomic data. So it's very, very preliminary, but I couldn't resist sharing a little bit today. So I'll start with the published studies. So we look at something called the NICU Network Neurobehavioral Scale. It's similar to what Dr. Lowe talked about with the Braselton exam. It's like the next generation. It's particularly designed to look at infants exposed to substances during pregnancy. So it has a withdrawal scale, signs of stress, tone, and reflexes, and then behaviors, like attending to stimuli and orienting. So here's what we found. We actually did infant neurobehavioral exams at day 0, 1, 2, 4, 5, 11, and 32 of life. So our research assistants were quite busy when the babies were born. Also collected some placentas from this study. So this was originally a tobacco study, but we were unable to recruit a tobacco sample that didn't include marijuana. So we started to look at, this is how we got interested in marijuana, is we started to look at co-use. And we found that infants who were exposed to both tobacco and marijuana were showing increased neurobehavioral deficits. So in this study, over the course of the 30 days, they showed two times less self-soothing behaviors. So we measure the need for the ENDS examiner to intervene to help the infant maintain a calm state. In tobacco-plus-marijuana-exposed infants, they needed two times more interventions to self-soothe. And this is even more so than the tobacco-exposed infants. And if you can see here, it's actually the marijuana-plus-tobacco that are driving the effect. We also saw two times greater need for external soothing, so interventions that the examiner needed to do. Again, marijuana-plus-tobacco seemed to show a great deal more of an effect than tobacco alone, which was a slightly different direction. And these, again, were pregnant people who were selected for tobacco use. So these were relatively low levels of marijuana use. So they weren't selected for marijuana, they were co-using with tobacco being their main drug. And we're still seeing some of these effects. So we're making a joke here that they perhaps are showing some signs of withdrawal. Profile increased lethargy. They had difficulty self-soothing and need for external soothing. We also saw some alterations in attention. And if you think about also the profile of someone, a pregnant person, who's using marijuana is that they may have a lot more things going on in their life. And to have an infant who's starting out a little more difficult to self-soothe could set them up for further difficulties over time. We're also very interested, because of our interest in prenatal programming, in the stress hormone cortisol, which can be measured in infant saliva. So when we did these infant neurobehavioral exams, we collected saliva before, after, 20 minutes after, and then 40 minutes after the exam. And we found, here again, that the marijuana plus tobacco-exposed infants showed diminished daily response to daily stressors. So their cortisol didn't go up as much in response to the neurobehavioral exam, which can be a mild stressor, versus the controls. And they look pretty similar to the tobacco-exposed infants here. And then we also, similar to the animal studies, we're also seeing sex differences, especially in the cortisol and the stress response. So we saw with males, we see a much bigger difference between marijuana plus tobacco, versus controls. In the female, we saw also differences between marijuana and tobacco, but not as much of a sort of dose response with the tobacco. We also looked at baseline cortisol, so the cortisol of the infants at the beginning of the exam. Overall, again, we're seeing that the marijuana plus tobacco exposed infants had slightly lower baseline, not significantly so, but definitely looks lower. And it seems to be driven in particular by the males, that they had a 36% decreased cortisol response, cortisol baseline levels. Again, females not too much of a difference between groups. So in summary, there's some evidence that prenatal marijuana exposure programs offspring stress response. Infants whose mother smoked marijuana during pregnancy may not be mounting adequate physiological responses to daily stressors. And the effects we think may be mediated by changes in expression in the placenta, which we're studying in a newly funded study. And this may help us understand how maternal substance use becomes biologically embedded. Here, this is again, this is unpublished, just looking. So we use fetal ultrasound to look at how the fetus responds to environmental stimuli. We do something called the FENS, which parallels the ENDS, the Fetal Neurobehavioral Coding System developed by my amazing colleague, Amy Salisbury, at Virginia Commonwealth University. So we can look at motor behavior and activity level. So we record the ultrasound and then code it. And then we also look at response to environmental stimuli. In our more recent study, we're looking at, we've looked at vibroacoustic stimulus. We've also been looking at response to a social stimuli. So we record mothers reading a story to their fetus. And then we also have a stranger. We record the stranger reading, and then we look at heart rate and activity level in response to the stories. And again, here, very preliminary look. But you can see, and we split the tobacco controls and the green is marijuana. But you can see that they look different from tobacco in some cases, particularly post-responding to the mother's voice during second trimester, and a little bit to a little bit more of a curve during third trimester. So again, we will be looking into this more and looking at sex differences, et cetera. And I hope to have a bigger, hope to submit a bigger grant to study this more. So proof of concept evidence that subtle alterations in behavior from maternal marijuana use may be evident during fetal development. And it's possible to identify changes in fetal behavior from maternal substance use prior to birth. Future studies are needed to understand these programming effects during the time that the fetus is getting programmed. So this is our marijuana cohort study that we started in 2018 and completed recruitment in 2022. We collected placenta again and focused on infant outcomes, just to show the sample. It was pretty well matched. We did our best to match the control group to the marijuana exposed group. They're pretty well matched on anxiety and depression and stress and age. We recruited from a pretty low income, highly diverse, racially and ethically diverse clinic for both groups. So you can see on the left that their race and ethnicity are pretty similar. Income was a little bit lower. Those are in the marijuana use group. But education was similar. Just to see some patterns of marijuana use across pregnancy. So there's pre-gestation and then over the course of pregnancy, you can actually see people are trying to cut down. And many mothers said that they were trying to quit. We see our highest levels of use during first trimester and obviously preconception. And then you can see in the postpartum period that the levels go up after babies are born. Many of you may see clinically. These are, again, these are our first set of biomarkers. But we are going to have both cannabidiol and THC. So we can a little bit tease apart, try to tease apart which ones have the biggest effect on infant development and placental outcomes. For our sample, 65% met criteria for marijuana use disorder. 89% tried to quit or cut down on average three attempts over pregnancy. 82% of moms who are smoking decreased over pregnancy. 53% tried to quit in the last month. And 42% are likely to quit, to try to quit in the next year. So again, there's a lot of interest, at least in our sample, of stopping use. So there's some message seems to be out that there's a need to quit. Quantity was an average of 1.7 grams a day. Again, this is their estimate. 77% spent one to four hours high on a typical use day. Potency, 75% did not know the THC content of what they were using. There's a good amount of tobacco co-use in our sample. 61% obtained their cannabis from a friend. 33% from a dispensary spent about $200 a month. Methods of use, we see primarily blunts and smoking and edibles, similar to what Dr. Lo was saying, being the second most. These are some reasons for use. You can see fun and relaxing, stress and anxiety and nausea, and then getting into other symptoms. And we have an early career faculty, Dr. Sokol, who has been studying pre- and post-COVID since our sample overlapped, and looking for what people were using pre- and post-COVID and their reasons for use. So pre-COVID, they were more likely to increase in smoking marijuana alone, but use of blunts, which are tobacco combined with marijuana, went down during the pandemic. Vaping stayed about the same, and edibles went up during the pandemic. And then if you look at reasons for use, we found that reasons for use shifted towards treating pregnancy and mental health symptoms during the pandemic. So you see an increase in use of marijuana for chronic sickness, pain, depressed mood, appetite, vomiting, sleep, et cetera, whereas some of the reasons, the overall reasons that people were using, didn't change as much. Just very preliminary, some of our genomic findings. So this is a new grant with my colleagues, Dr. Carmen Marcin and Todd Everson. His name has moved over at Emory University. So this is, we're doing bulk sequencing. We're going to do microRNA, mRNA, and methylation that I have some data for today in the placenta. And we're going to link it to all the phenotypic measures we have in the moms, and also neurobehavioral development in infants. This is some preliminary analyses of some of our older cohorts that a graduate student, Janet Joseph, is carrying out. So she looked initially at some candidate genes related to stress and THC metabolism, and found increased NR3C1, which is along the glucocorticoid pathway, and FKBP5 in the cannabis-exposed placentas. Also FAAH, which has been related to cannabis in prior studies, found some differences in a small sample of placentas. Again, these are preliminary. And then she's working on replicating a weeded gene co-expression network analysis. This is on the transcriptome. And here, the marijuana-exposed placenta showed higher co-expression of genes related to cell cycle regulation in the first co-expression module. And then higher expression of genes related to the extracellular matrix pathways, G protein-coupled receptors, and cell-to-cell interactions. And then this is our EWAS that was literally done this last week, so definitely don't quote us. Well, only one gene so far reached all the FDA levels of significance. And just to tell this, this is KM2A, which is previously known as MLL. It's a transcriptional co-activator that plays a crucial role in hematopoiesis in regulating gene expression at early developmental stages and control of circadian gene rhythm. In the placenta, this gene is important for sensitization of trophoblasts in the placenta. There's been some other studies that have found effects on this gene. Some are related to autism, which is somewhat similar to what Dr. Lo was talking about. But again, we have a lot more work to do with these that we want to look at pathways. And there's a lot of correlation between the methylation. We'd like to look at epigenetic age and have all kinds of other things that were planned, but that's just a little snippet. These are some other genes that came out, again. And then, looking ahead, again, we'd like to do more with looking at some of the biobehavioral development during the fetal period. We'd like to look longer term and do something called FNIRs, Functional Near Infrared Spectroscopy, is a way to measure functional brain development without having to go into a scanner. I'm very interested in the impact of individual cannabis products, especially edibles, ones, vaping, and some of the flavors and sweeteners that are being used with them. Would love to do some comparative analysis with Dr. Lo, we've talked about. And then we want to do some post GWAS studies, doing multi-omics, kind of looking at links between those. Maybe single cell, if we can. And trying to use some AI tools to look at them. And just want to thank our lab. And there's my contact information. So thanks so much. Thank you. Dr. Stroud, thank you for, well, thank all of you for really great talks. I'm a reproductive psychiatrist, so I'm dealing with pregnant women and postpartum women all day, most of my days. So one question that I have as I'm listening to all these talks is, I mean, it's pretty clear that these infants are, for the most part, they're behaviorally dysregulated from the beginning. And one of the things I'm wondering about, because I'm talking to moms about risk of postpartum depression, postpartum OCD, postpartum anxiety, is are there studies that are looking at, dyadically, what happens with the dyad with an infant? I mean, an infant that needs more soothing, more attention, that may be, and I think likely is, I think we know from my colic studies and others, that that's a setup for postpartum depression. Can any of you speak to that? Since I'm up here, I'll talk. But everyone else can also feel free to weigh in. It's hugely important. And then also think about if mom's starting to use again postpartum. So that's also sort of contributing, or maybe using more, because of these dyadic stressors. So I think it's really important. We have some data. Some of it ended up being remote. But looking at mom-infant behavioral interactions in a controlled task, similar to what Dr. Lo was doing with the animals that we're working on coding right now. But I think it's super important. And I think there's a lot of parents of younger kids as well who are, like tantrums come up, things like that, that are using more of the product in addition. So it's sort of the whole. But I think it's really, really important, and sort of parallels sort of why some of the effects may get worse over time, too. Excellent question. I don't know, Dr. Lo, if you wanted to weigh in? OK. Did you hear that? The HBCD study is, we'll also be looking closely at that. It's a large-scale study funded. More questions? OK. Yeah. Go ahead. Yeah. To help me clarify, because I think I don't fully understand whether high cortisol is good or low cortisol is good, you mentioned the low cortisol change. Now, is that negative or helpful? It's a great question. It's sort of the Goldilocks phenomenon is typical. So you don't want to be too high or too low. So too low can be associated with sort of chronic pain disorders and other kinds of sort of atypical depression, what we used to call. And high cortisol can be associated with damaged organs and sort of over the long haul. So we're kind of using our control group as a typical responder in this case, the control group of infants. But hyporesponsive HPA axis has also been associated with other maladaptive outcomes. Got it. And are you seeing any genetic associations, like maybe SLC6A4 or something like that? Genetic associations with cortisol? Cortisol level, yeah. It is. We will be looking at that. That cohort, we have some placentas, not quite as many as we did in this current study. Thank you. Thank you. Great question. We do have like 12 minutes. Do you want all the speakers coming up? We can have a discussion together. OK, great. Dr. Low and Dr. Mazzanaris, please. Okay, I can start with opening the discussion here with one question, which is that from all the presentations here, it seems that the cannabinoid exposure during development is affecting the brain more than just the brain function, brain activity, but more on the process of brain, the formation of the brain. Do you feel that this kind of influence or impairment would be long term? What do you feel how the brain is changed permanently or for a long term that affect the behavior for the later life? How about anybody can suggest from your research? Anyone would be good, yeah. Well, I think it's clear that there are changes not only functional, but morphological. So this is going to last forever. And it's going to affect the behavior forever. And we have studies where mother have been smoking marijuana 20 years after the children have problems, many different type of problems. So this is something permanent. And I think there is no question about that. No, I think you agree. This is why we have to advise about that. Any suggestion from audience would be welcome, too, if you have comments. I have a question. Like years ago when the tobacco products came about, they were marketed by celebrities and were heavily marketed. We are living in the era of marijuana used in a similar fashion. It's highly marketed, widely distributed. Do you think like 20 years from now we will be fighting marijuana the way we are fighting tobacco? Maybe. I'm hesitant. And Daiyu brought this when he brought us together for the symposium, mentioned the whole crack baby epidemic, you know, back when there was this huge fear that cocaine was going to cause all kinds of... And it turned out that the effects were kind of more subtle. So I think there's a couple messages which then makes it trickier to message the public. But I do think that in humans, it's things that are subtle and things that are confounded. And the type of folks who end up using marijuana may be different than the folks who are not using it. And there is hope, even if you are exposed, if you use marijuana during pregnancy. There's a lot that postnatal period is quite a bit longer than the prenatal period. And there's things that parents can do and quitting later on and all those things. So yes, I think it's pretty obvious that there is an impact. But I think the human model is very, very complicated. And I think we want to make sure to get a message that if you do quit, that it's sort of not this, that you're not hurting your child. Ideally, you want to quit. But I also think that it's important to make sure that the message isn't sort of how it went with cocaine. Yeah, I think as an obstetrician, similar to like alcohol or nicotine, ideal is to abstain even prior to conceiving, or at least while you're pregnant, if you can't use less. But there's not significant birth defects that are obvious in cannabis users during pregnancy, the way there is for the safe fetal alcohol syndrome. So for those on a prenatal ultrasound or immediately after delivery, there's a certain kind of physical phenotype that can be detected. Whereas for cannabis, there hasn't been really studies that's consistently shown that there are birth defects that are associated. But what we're finding is there's longer term outcome impacts to the offspring that I think is worth a conversation with the patient so that they can make an informed decision. And I think the other thing to know, at least in our sample, prior to the pandemic, there's a lot of co-use or co-administration of tobacco with marijuana. And it's tricky in the human model to separate the effects of, you know, we can measure it as best we can. But a lot of times what people call marijuana is marijuana and tobacco. And we already know the effects of tobacco. I was just curious in talking with patients that are not ready or feel like they are not capable of abstaining while pregnant, any early advice? I know a lot of studies are pending in the future, but as far as we assume it would be dose dependent, but as far as what you're going to use if we mix in some CBD and it's not just cannabis or modality, if we are going to try to do edibles as opposed to smoking something, any early thoughts on patients that won't quit, but is there harm reduction, not unlike Suboxone or Methadone? Yeah, we definitely talk a lot about harm reduction. The ultimate thing is I try to understand what they're using it for. So some of my patients will stop using it because they were substituting it for their epileptic meds. I mean, they're stopping their epileptic meds and using cannabis instead. So that one's a little different. When it's like nausea and vomiting, there's a couple of first line kind of pathways that the ACOG or American Obstetrics and Gynecologists guidelines recommend first. Like I said, start with B vitamins. If it's for sleep, there are other kind of, I think, approaches that we can take. So kind of just driving at what is it that is the underlying reason for them using can help either some other approaches or maybe they can decrease use. Like you mentioned, we're definitely seeing more of a dose kind of response effect. So the less is better. So trying to get at that. And then there's some harm reduction methods that are, I think, less consistently recommended, but like Gabapentin and other things in pregnancy as well. I think somebody mentioned the CBD. Well, let me tell you my experience in mice pregnant with CBD. CBD was killing the mice pregnant. And I have to stop the experiment. And I don't know why. And I've been using CBD in my animals and in some experiments in humans for nine years now. Because remember that when CBD was forbidden in the United States, it was never in the least one of drugs in Europe. So we were able to work with CBD very easily. I was a professor of psychiatry at the NYU for 10 years. And my friends there, they took like two years to get CBD. And I took four days to get the CBD. So I've worked a lot with CBD. And I showed that CBD in animal models was good for cocaine, for alcohol, and for cannabis, and to control. And I thought that CBD was going to have like a protective effect in the pregnant woman. And it was killing the animals. I don't know if this will happen in the human. But it was very strange. And I had to stop the experiment. And I don't know exactly why that happened. So this is very, very confusing. So the effect, remember that the CBD, that is tolerability and CBD is very high. We are talking about the compound that is active in 65 targets. And also is a cytochrome inhibitor. And we have already many results in humans that use anti-epileptics or antidepressant that is causing very adverse effects, all together with the CBD. So we have to have all this in mind, that the pharmacokinetics and interactions on cytochromes is very important when we mix CBD, even with some anti-epileptics, with antidepressants. So be careful with the CBD, although it doesn't have any problem of addiction. That was very hard to demonstrate. I had a paper, very simple, that demonstrate that. That was never published in US journals. No, yesterday received another award, published in 2018. Demonstrated that it's not producing reinforcing or withdrawal or motivation. But it has many different problems of interactions with other drugs. So just keep that in mind, because you probably will have patients that are having CBD. And CBD may work in the patients, but be careful with all the drugs, because it may be worse than CBD alone. Just an observation, because that came up before. Thank you. Thank you. I guess this is particularly for Dr. Stroud. I was struck by the rates of use in your cohort and the difficulty quitting. And I was wondering about, besides the many motivations for use we talked about, how much you're thinking about cannabis use disorder, you know, in that cohort. And, you know, to what extent, you know, are frank issues of addiction, which we know is complicated by motivation and everything else, contributing to some of the levels of use and difficulty quitting that you're seeing. For sure. And we did, we actually did an interview for marijuana use disorder, and I think a good percentage of them met criteria for marijuana use disorder. Nonetheless, a good number of them also quit over the course of pregnancy. So I'll have to go back and look at who met disorder status, but definitely. Pregnancy turns out, I mean, many, many, many people quit a lot of things over the course of pregnancy, particularly after sort of the end of, when they find out towards the end of first trimester. It's often after postpartum is when they don't stay, quit for tobacco and cannabis. The rates go right back up. Thanks, Laura. Okay, it's 930. Okay, one question, last one. Go ahead. So I was struck by the bold signal that showed that there was less oxygen available. It's well known that the P50, so the oxygen hemoglobin, is decreased in THC users. So that might play a role in that. And so when that is decreased, then it's harder for the oxygen to be released to the cells. Thank you. Appreciate that. Great. Let's thank all the speakers for their excellent presentation.

cannabinoids

THC

prenatal exposure

brain development

behavioral impairments

cognitive deficits

neurodevelopment

placental function

fetal stress

public health

cannabis safety

pregnancy risks