Prenatal cannabis use is associated with altered miRNA and protein expression in the developing human brain

Abstract

Background:

Cannabis is widely used during pregnancy, increasing with cannabis legalization, and has been associated with altered neurodevelopment, yet the precise molecular mechanisms remain poorly understood.

Aims:

To assess whether utilizing fetal central nervous system-derived extracellular vesicles (fCNSEVs) isolated from maternal blood reflects cannabis-associated changes in the fetal brain.

Methods:

In a matched case-control study, maternal plasma (to isolate fCNSEVs) and paired fetal cortical tissue (9–18 weeks’ gestation) from pregnancies exposed or unexposed to cannabis were collected. We quantified protein (CB1R, D2R) and miRNA expression (using µ-paraflo^TM microarray). Selected miRNAs were validated by qPCR. Bayesian Generalized Linear Models were used to assess exposure-group by sex effects.

Results:

Maternal cannabis use was associated with altered CB1R and D2R levels in the brain with opposite directional changes in fCNSEVs. 21 miRNAs were differentially expressed: 14 downregulated and 6 upregulated, following a sex-dependent pattern (males>>females), while miR-216a-5p follows the opposite pattern (females>>males). We confirmed concordant miRNA changes in fetal brain and fCNSEVs. Many of the identified molecules are associated with pathways linked to adverse neurodevelopmental outcomes.

Conclusions:

Our data suggest that fCNSEV-based assays may provide the ability to monitor fetal brain effects of maternal cannabis in ongoing pregnancies in clinical cohorts. A critical next step is to determine if alterations in fCNSEV protein and miRNA markers predict changes in fetal brain connectivity and neurodevelopment measures of executive function.

Keywords

prenatal cannabis exposure neurodevelopment CNS-derived extracellular vesicles

Introduction

Cannabis is the most commonly used illicit drug during pregnancy (Iezzi et al., 2022). The prevalence is higher among young, and socio-economically disadvantaged women, reaching a prevalence of 23% in women aged 24 years or younger (Ko et al., 2015; Schempf and Strobino, 2008; Thompson et al., 2019; Young-Wolff et al., 2024). Over 60% of pregnant women using cannabis believe there is no risk associated with regular cannabis use (Brown et al., 2017). The primary reasons for cannabis use include relief from first-trimester nausea/vomiting, as well as treatment for anxiety and depression (Allen et al., 2020; Beatty et al., 2012; Moore et al., 2010; Young-Wolff et al., 2022). Approximately 50%–60% of pregnant women continue to use cannabis beyond the first trimester, frequently using it several times per week (Haight et al., 2021; Moore et al., 2010); use in the mid and third trimesters overlaps with cortical expansion and development of cortical connection pathways. Alterations during cortical development increase the susceptibility for neurodevelopmental or psychiatric disorders (Ball et al., 2024; Won et al., 2019).

After cannabis consumption, Δ9-tetrahydrocannabinol (THC), a cannabinoid receptor agonist, interacts with the central nervous system (CNS) endogenous cannabinoid system, binding with high affinity to the Cannabinoid Receptor 1 (CB1R) (Atakan 2012; Haney, 2022; Pagotto et al., 2006; Thompson et al., 2019). THC crosses the placental barrier, reaching the fetal endocannabinoid system in both human and animal models (Thompson et al., 2019). The fetal endocannabinoid system plays a crucial role in neurodevelopment, regulating neuronal differentiation and migration, axonal migration and connectivity, and synaptogenesis (Díaz-Alonso et al., 2012; Martínez-Peña et al., 2021). CB1R also regulates neural progenitor cell proliferation and survival, and cortical interneuron migration and differentiation by regulating neurotrophic factors, such as brain-derived neurotrophic factor (Aguado et al., 2005; Berghuis et al., 2005; Díaz-Alonso et al., 2012; Watson et al., 2008). CB1R disruption alters cortical and hippocampal development, therefore prenatal cannabis exposure may alter interneuron positioning during corticogenesis. This disruption may increase the risk of neurodevelopmental disorders by altering the formation of key brain circuits. Additionally, CB1R is linked to the dopaminergic and opioid systems (Roncero et al., 2020; Wang et al., 2003), highlighting its potential role in developmental vulnerabilities. Preclinical and human studies have found that prenatal cannabis exposure is associated with long-lasting alterations in brain-reward regions, such as the mesolimbic dopamine system (Frau et al., 2019; Mulligan and Hamre, 2023). Cannabis exposure is associated with decreased expression on dopamine receptor genes in the nucleus accumbens in murine models (DiNieri et al., 2011), and with decreased dopamine receptor 2 (D2R) levels in the amygdala of second-trimester human fetuses, particularly in males (Wang et al., 2004). Additionally, studies in rodents showed that prenatal cannabinoid exposure produces sex-specific disturbances in early communication with a higher impact on male offspring (Iezzi et al., 2022), suggesting that prenatal cannabis use may adversely affect offspring’s cognitive and executive functions. Although the existing literature in humans and non-human primates is limited, chronic THC exposure in rhesus macaques showed subtle sex-dependent alterations in volumetric development of the cortical plate (Ryan et al., 2024). Despite the significant public health relevance, a critical gap in knowledge remains regarding the effects of prenatal cannabis exposure in humans. Clinicians are left without adequate data to effectively counsel their pregnant patients.

Extracellular vesicles (EVs) are heterogeneous cell-derived vesicles released into the extracellular space, regulating intercellular communication through their protein, nucleic acids and lipid cargo (Elliott and He, 2021; Willms et al., 2016). MicroRNAs (miRNAs) are very abundant in EVs, and they regulate post-transcriptional gene expression (Fekry et al., 2024; Xu et al., 2022; Zhang et al., 2019). Fetal CNS-derived EVs (fCNSEVs) are known to cross the placenta and enter the maternal bloodstream (Czernek and Düchler, 2020). A promising marker within these EVs, is Contactin-2 (CNTN2), a transient neural surface protein found in neurons and glial cells undergoing synaptic plasticity—a key developmental process guiding axonal connections and promoting the molecular organization of myelinated nerves (Masuda, 2017). CNTN2+ EVs constitute around 20% of the total set of neuronal exosomes in the plasma of women during the first and second trimester of pregnancy (Goetzl et al., 2016). The diameter of these EVs is about 134 nm ± 46.6 nm, and they are characterized by the expression of canonical EV markers (CD81), neural markers (neurofilament-light chain), and fetal markers (Sonic Hedgehog).

Therefore, in this preliminary study, we aimed to determine whether markers involved in cannabinoid pathways, including CB1R and D2R, or miRNAs affected by maternal cannabis use in fetal brain tissue, are reflected by fCNSEVs isolated from maternal blood, and to investigate the correlation between fetal brain and fCNSEV levels.

Materials and methods

Clinical subjects

A matched case-control study was performed in women with singleton fetuses undergoing elective pregnancy termination at gestational ages 9–18 weeks (see Table 1 for further details) through surgical abortion (specifically dilation and evacuation). The study included mothers who used cannabis during pregnancy (ranging from several times per month to almost daily), but were not exposed to other illicit drugs, alcohol or other CNS-active medications. Maternal cannabis exposure was determined via a face-to-face questionnaire that included self-reported questions regarding many types of drugs/medications used, as well as tobacco exposure (Goetzl et al., 2019b). Previous confirmation of self-reported drug use by High-Performance Liquid Chromatography drug levels showed >90% concordance for opioids (unpublished data). Patients with diabetes or other chronic diseases were excluded. Biological samples for analysis were selected from participants exposed to cannabis and controls who were not exposed to cannabis during pregnancy, with equal numbers of male and female fetuses in each group. Samples from cases (n = 24) and controls (n = 24) were matched for gestational age, ensuring no differences in gestational age at collection.

Table 1.

Demographic data of the study population (mean ± SEM).

Variable	Control female (n = 12)	Control male (n = 12)	Cannabis female (n = 11)	Cannabis male (n = 13)
Maternal age (years)	25.9 ± 2.1	23.4 ± 1.2	23.5 ± 1.6	23.2 ± 1.5
Gestational age (weeks)	14.9 ± 0.8	14.7 ± 1.0	14.2 ± 0.9	14.7 ± 0.9
Gestational age (n)
9–13 weeks	3 (25%)	4 (33%)	3 (27%)	5 (38%)
14–18 weeks	9 (75%)	8 (67%)	8 (73%)	8 (62%)
BMI (kg/m²)	23.7 ± 1.2	25.4 ± 1.3	26.3 ± 1.4	26.4 ± 1.3
Race (n)
African American	5 (42%)	8 (67%)	9 (81%)	8 (62%)
Asian	0 (0%)	0 (0%)	0 (0%)	0 (0%)
White	3 (25%)	3 (25%)	1 (9%)	5 (38%)
More than one	3 (25%)	1 (8%)	1 (9%)	0 (0%)
Unknown	1 (8%)	0 (0%)	0 (0%)	0 (0%)
Ethnicity (n)
Hispanic	1 (8%)	2 (17%)	0 (0%)	0 (0%)
Non-Hispanic	10 (83%)	10 (83%)	11 (100%)	13 (100%)
Unknown	1 (8%)	0 (0%)	0 (0%)	0 (0%)
Cannabis use (n)
Yes	0 (0%)	0 (0%)	11 (100%)	13 (100%)
No	12 (100%)	12 (100%)	0 (0%)	0 (0%)
Tobacco use (n)
Yes	1 (8%)	1 (8%)	1 (9%)	1 (8%)
No	11 (92%)	11 (92%)	10 (91%)	12 (92%)

This study was approved by Temple University’s Human Research Protection Program. All patients signed an informed consent form prior to participation. Following written informed consent and immediately before elective pregnancy termination, 20 ml of venous blood was drawn into 1 ml of saline with EDTA or heparin, incubated for 10 min at room temperature, and centrifuged for 15 min at 2500×g. Plasma was stored in 0.5 ml aliquots at −80°C. Immediately following elective pregnancy termination, brain tissue samples were collected by a trained study coordinator and snap-frozen in liquid nitrogen for future RNA and protein analysis. We confirmed by immunohistochemistry that the collected tissue was predominantly cortical (Darbinian et al., 2021), but the exact brain regions could not be determined from brain homogenates. All samples were stored at −80°C until further analysis. Fetal sex was determined using DNA extracts from fetal tissue utilizing Quick-DNA Miniprep Kit (Zymo Research, Irvine, CA, USA, #11-317C), and primers designed for X- and Y-chromosome-specific genes (ZFX and ZFY) (Degrelle and Fournier, 2018).

µParaflo™ microRNA microarray assay—Labeling by direct ligation

miRNA profiling from brain tissue was performed using a service provider, LC Sciences (Houston, TX, USA). 1 µg RNA was subjected to a strand-labeling reaction with control oligo-nucleotides, App-Cap3 (5’-adenylated and 3’-dideoxy oligo-adaptor, Integrated DNA Technologies, Coralville, IA, USA) and T4 RNA ligase (Rnl2tr-K227Q, New England BioLabs, Ipswich, MA, USA) in ligation buffer supplemented with PEG 8000 and RNase inhibitor (Promega, Madison, WI, USA). After a 16 hour incubation at 16°C, the reaction was stopped by adding an equal volume of hybridization buffer. The adapter sequence includes a tag segment for subsequent fluorescent dye capture during staining.

Hybridization was performed at 40°C for 16 hours using a µParaflo microfluidic chip and a micro-circulation pump (Atactic Technologies Inc., Houston, TX, USA) (Gao et al., 2004). Each probe on the chip consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://mirbase.org) or other RNA (control or customer-defined sequences) and a polyethylene glycol spacer to improve the physical accessibility. Probes were synthesized in situ using photogenerated reagent chemistry, and chemical modifications were applied to balance hydridization melting temperatures. Hybridization was performed on 100 µl 6×SSPE buffer (0.90 M NaCl, 60 mM Na₂HPO₄, 6 mM EDTA, pH 6.8) containing 25% formamide. After hybridization, tag-conjugating Cy3 dye was applied through the chip to label the adaptors. Fluorescence was detected using a GenePix4000B a laser scanner (Molecular Device, Sunnyvale, CA, USA), and images were digitized using Array-Pro software (Media Cybernetics, Silver Spring, MD, USA). First, background was subtracted from the data, and then signals were normalized using a LOWESS filter (Locally-weighted Regression) (Bolstad et al., 2003).

miRNA functional analysis

To determine differentially expressed genes, we used a cut-off threshold of 2.5 for Log-Fold Change and an adjusted p-value <0.05, using DESeq2, ggplot2, and EnhancedVolcano libraries in R (R Core Team, 2024). Then, we used miRWalk (Dweep et al., 2011) and miRPathDB 2.0 (Kehl et al., 2020) databases to predict the downstream target genes. Once we had those genes, we performed KEGG pathway analysis using GeneCodis (version 4) (Garcia-Moreno et al., 2022) web tool (Merabova et al., 2024).

Quantitative real-time PCR

Total RNA was extracted from snap-frozen fetal brain tissue and fCNSEVs using TRIzol LS Reagent (Life Technologies, Carlsbad, CA, USA) (males n = 16; females n = 14 matched for gestational age). miRNA was isolated and converted to cDNA using the miRCURY LNA RT Kit 3 (Qiagen, Cat.#39340). Then, we quantified miRNA expression of hsa-miR-374a-5p (YP00204758), hsa-miR-216a-5p (YP00204167), hsa-miR-381-5p (YP02112411), hsa-miR-301a-3p (YP00205601), hsa-miR-3197 (YP02118682), hsa-miR-6865-5p (YP02118138), hsa-miR-301b-3p (YP00204390), hsa-miR-103a-3p (Housekeeping, YP00204063) through real-time PCR, using the miRCURY LNA miRNA PCR assay and primers (Cat.#339306, Qiagen) together with miRCURY LNA SYBR Green PCR Kit (Cat.#339345, Qiagen) and QuantStudio 3 real-time PCR (qPCR) system (Applied Biosystems, Life Technologies Holdings Pte Ltd, Singapore). After RT-qPCR, the relative gene expression levels were calculated by the ΔΔCT method, and they are expressed as the relative intensity.

Isolation of fCNSEVs

fCNSEVs were purified from the maternal blood using previously published techniques (Goetzl et al., 2021; Pineles et al., 2022). Each 250 µl plasma aliquot was incubated with 100 µl of thromboplastin-D and a cocktail of protease and phosphatase inhibitors. Supernates were incubated with Exoquick, an EV precipitation solution (System Biosciences, Inc, Mountain View, CA, USA). To isolate fCNSEVs, total suspensions of EVs were incubated with 2 µg of mouse monoclonal IgG1 anti-human Contactin-2/TAG1 antibody (clone#372913, R&D Systems Inc., Minneapolis, MN, USA) that had been biotinylated (EZ-link sulfo-NHS-biotin system, Thermo Scientific, Inc., Rockford, IL, USA), and antibody-bound EVs were precipitated with Streptavidin-Plus UltraLink resin (Pierce-Thermo Scientific, Inc., Rockford, IL, USA). EVs were eluted using 0.05 mol/L glycine-HCl (pH = 3.0), and then stored at −80°C. We have previously validated the specificity of CNTN2 for the fetal brain (Goetzl et al., 2016, Goetzl et al., 2019b, Goetzl et al., 2019a; Darbinian et al., 2021; Ibarra et al., 2025) and published nanosight tracking data demonstrating the size distribution of vesicles obtained using this methodology (Goetzl et al., 2016, Goetzl et al., 2018; Goetzl et al., 2019b).

ELISA quantification of exosomal proteins

The tetraspanning exosome marker human CD81 (Cusabio, CSB-EL004960HU), neural markers Cannabinoid Receptor 1 (CB1R, Reddot Biotech Inc, RD-CNR1-Hu), and Dopamine 2 Receptor (D2R, Abebio, AE46633HU) were quantified by using commercially available ELISA kits. All samples were run in duplicate. Optical Density was measured at 450 and 540 nm using the Agilent Biotek Epoch microplate spectrophotometer (Agilent Technologies, Winooski, VT, USA), and the Biotek Gen 5 software. Intra-assay coefficients of variance were 3.32 ± 0.64% for CD81, 3.64 ± 0.44% for CB1R, and 4.73 ± 0.68% for D2R. The mean value for all determinations of CD81 in each assay group was set at 1.0, and this value was used to normalize their recovery in individual samples.

Western blotting

Brain tissue was homogenized in RIPA lysis buffer (Thermo Scientific, Rockford, IL, USA) for 15 seconds, using a motorized tissue grinder (Fisherbrand, #12-1413-61). First, samples were spun down at 10,000×g for 10 minutes to remove insoluble material. Then, we used Pierce TM BCA Protein Assay kit (#23227, Thermo Scientific) to quantify total protein levels. Protein lysates were directly solubilized in Laemmli sample buffer. 20µg of protein lysates were added to each well and separated by electrophoresis on a 4%–20% polyacrylamide gel. After transferring separated proteins into a nitrocellulose membrane, target proteins were labeled with primary and secondary antibodies (Supplemental Table 1). We finally used Clarity Max^TM Western ECL substrate (Bio-Rad Laboratories, Hercules, CA, USA; manufactured by Cyanagen Srl, Bologna, Italy) to detect proteins through enhanced chemiluminescence. Blot intensities were quantified using ImageJ (Schneider et al., 2012). We used Cyclophilin A as a housekeeping protein to quantify CB1R levels, and β-actin to quantify D2R levels. All target protein signals were normalized to their respective housekeeping protein to account for loading variability. Normalized values were analyzed statistically using Bayesian models.

Data analytic strategy

Analyses primarily relied on generalized linear modeling (GLM) to evaluate the outcome variables. Each outcome measurement was separately modeled as a lognormal function of the interaction between group (control vs. experimental) and sex (female vs. male), controlling for constituent main effects. Follow-up analyses evaluated pairwise differences between categories of both predictors. All GLMs were also tested with and without covariate adjustment for gestational age, maternal age, and BMI. The covariate-adjusted models did not provide different inferences from unadjusted models with respect to the exposure group by sex interaction; as such, results from the unadjusted models are reported.

Bayesian statistical inference was used to directly quantify the probability that model parameters were greater or less than zero, given weakly informative priors (b ~ N(µ = 0; σ² = 1)) and the data. This paradigm was chosen for its accessible view of probability (evaluating the alternative hypothesis) and for its favorable properties with small sample sizes (Rognli et al., 2023). Bayesian analytic assumptions were tested and determined to be satisfied across models by evaluating scale convergence factors (“rhat”), effective sample size, and posterior predictive checking. The median and the 95% equal-tailed interval of the posterior distribution were taken as a point estimate (b) and credible interval (95% CrI) for each regression coefficient. The percentage of the posterior distribution greater or less than a null effect (b = 0) was captured as the posterior probability (PP) that the effect exists (i.e., PP(b > 0 | b < 0)). For example, a 75% chance that a positive effect exists would be transcribed as PP(b > 0) = 75%. The PP is sometimes reconfigured as the Bayes factor (BF): the ratio of evidence in one direction to the other (e.g., 75%/25% = BF = 3.0).

There is no single conventional PP value that defines a significant effect, and the PP does not provide an inverted p-value from the traditional frequentist perspective (i.e., p = 0.05 ≠ PP = 95%). Evaluating any given PP necessitates subjective judgment with respect to determining a threshold of evidence that denotes a meaningful effect. For relatively exploratory contexts, heuristics from the literature (Andraszewicz et al., 2015; Jeffreys, 1961; Lee and Wagenmakers, 2014) have evolved to characterize various PP thresholds as providing increasingly greater evidence: none (PP = 50%), anecdotal (PP = 51%–74%), moderate (PP = 75%–90%), strong (PP = 91%–96%), very strong (PP = 97%–99%), and extreme (PP > 99%). The lower limit of the moderate level (75%) was chosen as a minimum degree of evidence to support the existence of an effect for the current analyses. This threshold was determined a priori, before data analyses were conducted, and selected as a minimum reporting criterion appropriate for the exploratory context of the present study. Statistical analyses were performed in R (R Core Team, 2024). Bayesian GLMs were modeled via the brms (for Bayesian GLM) (Bürkner, 2017) and marginaleffects (Arel-Bundock, 2024) libraries, and Bayesian correlation (ρ) testing was performed via the BayesFactor (Morey and Rouder, 2024) library.

Results

Prenatal cannabis exposure alters CB1R and DR2 levels in the developing brain in a sex-specific manner

Maternal cannabis use altered CB1R protein levels in fetal cortical tissue. The models demonstrated extreme evidence for exposure group by sex interaction (PP = 99.8%) (Figure 1(a) and (b)). Pairwise contrasts indicated downregulation of CB1R in male fetuses exposed to cannabis relative to control males (b = −0.636, PP = 99.6%), whereas female fetuses exposed to cannabis showed moderate upregulation compared to control females (b = 0.253, PP = 89.8%).

Figure 1.

Marginal group means for each GLM fitting one biomarker as a function of the exposure group × sex interaction, controlling for main effects. (a–b) CB1R levels in cortical brain tissue were lower for the cannabis-exposed, but only for males (n = 6/group). (c) CB1R levels in fCNSEVs demonstrated the opposite pattern (n = 10/group). (d–e) D2R levels in cortical brain tissue demonstrated higher values across both sexes for the cannabis-exposed, with a stronger effect for males (n = 6/group). (f) D2R levels in fCNSEVs were lower for all cannabis-exposedand males were lower than females across exposure groups (n = 10/group).

CB1R levels were then examined in fCNSEVs isolated from maternal blood. Within sex comparisons indicated higher CB1R among cannabis-exposed males relative to control males (b = 208.1, PP = 99.2%), while cannabis-exposed females did not clearly differ from control females (PP = 59.9%). Analyses supported an interaction between exposure group and fetal sex (PP = 93.9%, Figure 1(c)). However, pairwise comparisons showed weak evidence for sex differences in control subjects (b = −50.4, PP = 75.0%), whereas cannabis-exposed females had lower CB1R levels than cannabis-exposed males (b = −240.4, PP = 99.7%). We observed strong evidence for a negative correlation (ρ = −0.36, PP = 95.9%) between fetal brain and fCNSEVs.

We measured D2R protein levels in fetal cortical tissue to assess dopaminergic signaling. Analyses supported exposure group by-sex interaction (b = −0.60, PP = 95.6%; Figure 1(d) and (e)). Pairwise comparisons indicated higher D2R levels among cannabis-exposed males relative to control males (b = 1.03, PP > 99.9%). The same upregulation pattern was also observed in female fetuses, although it was less pronounced than in males (b = 0.37, PP = 98.1%).

In fCNSEVs, the exposure group by sex interaction for D2R was not supported (PP = 65.6%, Figure 1(f)). Reducing the model to main effects only, analyses indicated higher D2R levels among controls compared to cannabis-exposed fetuses (b = 0.24, PP = 93.9%) and lower D2R among males (b = −0.12, PP = 78.8%). In this case, there was a strong negative correlation between D2R levels in fetal cortical tissue and fCNSEVs (ρ = −0.28, PP = 91.4%).

Maternal cannabis use is associated with differential miRNA expression in the fetal brain

We performed an exploratory, unbiased miRNA expression analysis of brain tissue (n = 12, controls: 3 males; 2 females; cannabis-exposed: 3 males; 4 females). In male fetuses, prenatal cannabis use was associated with downregulation of 14 miRNAs (miR-6865, miR-3197; miR-381-5p, miR-3939, miR-6820-5p, miR-548h-5p, miR-7843-5p, miR-8075, miR-4486, miR-6731-5p, miR-4280, miR-3135b, miR-6860, miR-6753-5p), and upregulation of 6 miRNAs (miR-4638-5p, miR-301a-3p, miR-374a-5p, miR-301b-3p, miR-582-5p, miR-4666b) (Figure 2(a)). However, in female fetuses, prenatal cannabis use was associated with downregulation of miR-216a-5p (Figure 2(b)).

Figure 2.

In-utero cannabis exposure alters miRNA expression in fetal brain. The volcano plot shows a greater than 2.5 fold change in 20 different miRNAs with prenatal cannabis exposure in males (a), while only shows a greater than 2.5 fold change in miR-216a-5p in females (b). (c) Top significant KEGG pathways of the target genes from the differentially expressed miRNAs with cannabis exposure.

KEGG pathway enrichment analysis of their target genes suggested that differentially expressed miRNAs related to maternal cannabis use were associated with pathways involved in neural development, such as axon guidance, GABAergic synapse, dopaminergic synapse, and AGE-RAGE signaling pathways (Figure 2(c)).

Next, we validated our findings by measuring expression of the selected candidate miRNAs in fetal brain tissue by qPCR (n = 30, controls: 6 males, 7 females; cannabis-exposed: 10 males; 7 females). GLM modeled each candidate miRNA as a function of the interaction between exposure group and sex, controlling for the constituent main effects. Tables 2 and 3 provide the point estimate, 95% CrI, and PP for each miRNA, along with each pairwise contrast within group and sex. All candidate miRNAs demonstrated at least moderate evidence for exposure group by sex interaction.

Table 2.

Candidate miRNAs from fetal brain tissue.

Outcome	Effect/Subgroup	Contrast	Point estimate	Lower 95% Crl	Upper 95% Crl	PP
miR-6865-5p	Interaction Control THC-Exposed Female Male	Group × Sex Female – Male Female – Male THC-Exposed – Control THC-Exposed – Control	0.769 0.109 0.388 −0.588 −0.875	−0.413 −1.415 0.004 −2.069 −2.490	1.899 1.617 1.109 0.245 −0.215	90.4% 57.1% 97.6% 92.3% 99.7%
miR-3197	Interaction Control THC-Exposed Female Male	Group × Sex Female – Male Female – Male THC-Exposed – Control THC-Exposed – Control	1.446 0.488 0.501 −1.148 −1.174	0.088 −1.942 0.167 −4.395 −4.018	2.709 3.623 1.586 −0.048 −0.390	98.0% 70.7% 99.9% 98.1% 99.9%
miR-381-5p	Interaction Control THC-Exposed Female Male	Group × Sex Female – Male Female – Male THC-Exposed – Control THC-Exposed – Control	0.798 0.118 0.257 −0.687 −0.843	−0.479 −1.897 −0.033 −2.603 −3.109	2.030 1.953 0.990 0.051 −0.186	88.9% 57.8% 96.1% 96.7% 99.7%
miR-301a-3p	Interaction Control THC-Exposed Female Male	Group × Sex Female – Male Female – Male THC-Exposed – Control THC-Exposed – Control	−0.759 0.359 −11.808 11.001 23.922	−2.143 −8.857 −64.188 2.034 7.460	0.639 9.273 13.907 48.258 90.066	86.4% 57.5% 86.8% 99.1% 99.8%
miR-301b-3p	Interaction Control THC-Exposed Female Male	Group × Sex Female – Male Female – Male THC-Exposed – Control THC-Exposed – Control	−1.521 −0.126 −11.204 1.773 12.915	−2.524 −1.193 −21.958 0.582 7.223	−0.425 0.640 −5.333 4.519 23.981	99.5% 65.0% >99.9% 99.8% >99.9%
miR-374a-5p	Interaction Control THC-Exposed Female Male	Group × Sex Female – Male Female – Male THC-Exposed – Control THC-Exposed – Control	−1.188 −0.481 −11.529 1.961 13.060	−2.039 −1.666 −19.607 0.775 8.198	−0.304 0.349 −6.531 4.059 21.247	99.3% 87.8% >99.9% >99.9% >99.9%
miR-216a-5p	Interaction Control THC-Exposed Female Male	Group × Sex Female – Male Female – Male THC-Exposed – Control THC-Exposed – Control	−0.947 0.705 −0.001 −1.135 −0.378	−2.314 −0.495 −0.135 −3.654 −1.818	0.523 2.902 0.188 −0.413 −0.062	90.1% 90.8% 51.0% >99.9% 99.5%

PP: posterior probability.

Table 3.

Candidate miRNAs from fetal CNS-derived EVs.

Outcome	Effect/Subgroup	Contrast	Point estimate	Lower 95% Crl	Upper 95% Crl	PP
miR-6865-5p	Interaction Control THC-Exposed Female Male	Group × Sex Female – Male Female – Male THC-Exposed – Control THC-Exposed – Control	0.709 −0.554 0.086 −0.316 −0.961	−0.256 −1.889 −0.237 −1.023 −2.316	1.664 0.342 0.499 0.153 −0.306	92.5% 89.7% 70.8% 91.2% 99.9%
miR-3197	Interaction Control THC-Exposed Female Male	Group × Sex Female – Male Female – Male THC-Exposed – Control THC-Exposed – Control	0.925 −0.523 0.125 −0.483 −1.149	−0.285 −2.454 −0.142 −1.552 −3.197	2.070 0.665 0.528 0.037 −0.355	93.5% 82.8% 84.2% 96.6% 99.9%
miR-381-5p	Interaction Control THC-Exposed Female Male	Group × Sex Female – Male Female – Male THC-Exposed – Control THC-Exposed – Control	1.660 −0.190 0.229 −0.759 −1.192	0.621 −1.499 0.096 −1.809 −2.586	2.652 0.910 0.487 −0.276 −0.586	99.8% 64.7% >99.9% 99.9% >99.9%
miR-301a-3p	Interaction Control THC-Exposed Female Male	Group × Sex Female – Male Female – Male THC-Exposed – Control THC-Exposed – Control	−0.773 0.152 −2.344 1.511 4.036	−1.850 −1.222 −6.652 −0.031 1.656	0.372 1.519 0.807 4.439 8.621	91.1% 61.8% 93.9% 97.2% 99.9%
miR-301b-3p	Interaction Control THC-Exposed Female Male	Group × Sex Female – Male Female – Male THC-Exposed – Control THC-Exposed – Control	−1.077 0.463 −2.643 2.050 5.207	−1.940 −0.242 −5.938 0.693 3.154	−0.170 1.366 0.103 4.405 8.632	99.1% 90.9% 97.1% 99.8% >99.9%
miR-374a-5p	Interaction Control THC-Exposed Female Male	Group × Sex Female – Male Female – Male THC-Exposed – Control THC-Exposed–Control	−0.834 0.066 −2.253 0.879 3.224	−2.042 −1.887 −7.722 −0.695 0.706	0.463 1.818 0.790 3.897 9.186	90.1% 54.6% 93.8% 89.4% 99.2%
miR-216a-5p	Interaction Control THC-Exposed Female Male	Group × Sex Female – Male Female – Male THC-Exposed – Control THC-Exposed – Control	−0.814 0.267 −0.071 −0.852 −0.479	−2.107 −1.321 −0.343 −2.557 −2.365	0.637 1.822 0.137 −0.279 −0.038	87.4% 70.7% 82.1% 99.9% 98.5%

CNS: central nervous system; EV: extracellular vesicle; PP: posterior probability.

Within the downregulated miRNA group, miR-6865-5p, miR-3197, and miR381-5p showed lower expression with prenatal cannabis use, with greater downregulation in males (Figure 3(a)–(c); female b = −0.59, PP = 99.7%; male: b = −0.88, PP = 98.0%; female b = −1.15, PP = 98.1%; male: b = −1.17, PP = 99.9%; female b = −0.69, PP = 96.7%; male: b = −0.84, PP = 99.7%, respectively). miR-216a also showed lower expression with maternal cannabis use in both sexes, with stronger downregulation in females (female b = −1.14, PP > 99.9%; male: b = −0.38, PP = 99.5%; Figure 3(d)).

Figure 3.

Marginal group means for each GLM fitting one miRNA measured in fetal brain as a function of the exposure group × sex interaction, controlling for main effects. miR-6865-5p (a), miR-3197 (b), and miR-381-5p (c) levels were lower across sexes, with greater downregulation for males. miR-216a-5p (d) levels were lower across sexes, with greater downregulation for females. miR-374a-5p (e), miR-301b-3p (f), and miR-301a-3p (g) levels were higher across sexes, with greater upregulation for males.

Similarly, within the upregulated miRNA group, miR-301a-3p, miR301b-3p, and miR-374a-5p, showed higher expression with prenatal cannabis use, with greater upregulation in males (Figure 3(e)–(g); female b = 11.00, PP = 99.1%; male: b = 23.92, PP = 99.8%; female b = 1.77, PP = 99.8%; male: b = 12.92, PP > 99.9%; female b = 1.96, PP > 99.9%; male: b = 1.49, PP = 95.0%, respectively).

Differential fetal miRNA expression can be detected accurately in fCNSEVs

We conducted miRNA expression analysis in fCNSEVs to confirm whether EV levels could be used to accurately estimate brain tissue patterns (Table 3). Within the downregulated miRNA group, miR-6865-5p, miR-3197, and miR-381-5p, each demonstrated downregulation across sexes, with greater effects in males (Figure 4(a)–(c), female b = −0.32, PP = 91.2%; male: b = −0.96, PP = 99.9%; female b = −0.48, PP = 96.6%; male: b = −1.15, PP = 99.9%; female b = −0.76, PP = 99.9%; male: b = −1.19, PP = >99.9%, respectively). miRNA-216a also showed lower expression with prenatal cannabis exposure, with greater downregulation in females (Figure 4(d); female b = −0.85, PP = 99.9%; male: b = −0.48, PP = 98.5%).

Figure 4.

Marginal group means for each GLM fitting one miRNA measured in fetal CNS-derived EVs as a function of the exposure group × sex interaction, controlling for main effects. miR-6865-5p (a), miR-3197 (b), and miR-381-5p (c) levels were lower across sexes, with greater downregulation for males. miR-216a-5p (d) levels were lower across sexes, with greater downregulation for females. miR-374a-5p (e), miR-301b-3p (f), and miR-301a-3p (g) levels were higher across sexes, with greater upregulation for males.

Within the upregulated miRNA group, miRNA-301a-3p, miRNA-301b-3p, and miRNA374a-5p demonstrated greater upregulation among males (Figure 4(e)–(g); female b = 1.51, PP = 97.2%; male: b = 4.04, PP = 99.9%; female b = 2.05, PP = 99.8%; male: b = 5.21, PP = >99.9%; female b = 0.88, PP = 89.4%; male: b = 3.22, PP = 99.2%, respectively).

Finally, we studied the correlation between miRNA levels in fetal brain tissue and fCNSEVs. Here we observed an extreme evidence of a positive correlation for all the studied miRNAs (miR-6865-5p: ρ = 0.40, PP = 99.2%; miR-3197: ρ = 0.44, PP = 99.6%; miR-381-5p: ρ = 0.51, PP = 99.9%; miR-301a-3p: ρ = 0.44, PP = 99.6%; miR-301b-3p: ρ = 0.58, PP = >99.9% ; miR-374a-5p: ρ = 0.43, PP = 99.6%; miR-216a-5p: ρ = 0.52, PP = 99.9%).

Discussion

Several longitudinal human studies have highlighted the potential relationship between prenatal cannabinoid exposure and associated changes in the developing brain (El Marroun et al., 2016; Mulligan and Hamre, 2023; Thompson et al., 2019). Our current investigation has confirmed changes in brain CB1R and D2R levels primarily in males following prenatal cannabis exposure, along with a greater effect on male fetal cortical miRNA expression. Furthermore, we confirmed that these changes in protein and miRNA levels can be accurately estimated in fCNSEVs isolated from maternal blood, albeit utilizing a negative correlation effect in the studied proteins.

The observed effects on CB1R are consistent with previous preclinical models, where CB1R downregulation in the prefrontal cortex was observed after THC exposure in rodents (Burston et al., 2010; Ketcherside et al., 2017). Human studies also reported CB1R downregulation in postmortem brains of chronic cannabis users (Villares, 2007). Here, we found reduced CB1R levels in fetal males following maternal cannabis use, with opposing directional changes in fCNSEVs isolated from maternal blood non-invasively. The endocannabinoid receptor system is present in the fetal brain as early as 9 weeks gestation with a critical role in the regulation of neurogenesis, axon pathfinding, and postnatal development of striosome-dendron bouquets (Crittenden et al., 2022; Zurolo et al., 2010). CB1R is particularly enriched in the GABAergic circuits from the prefrontal cortex, regulating the excitatory/inhibitory neuronal activity critical for prefrontal cortex homeostasis (Crittenden et al., 2022; Heng et al., 2011; Long et al., 2012; Peerboom and Wierenga, 2021; Renard et al., 2018). In this context, reduced CB1R levels observed in fetuses exposed to cannabis in-utero, may reflect disruption of developmental processes dependent on endocannabinoid signaling involved in prefrontal cortex maturation. Additionally, increased miR-301a-3p levels in both fetal brain tissue and fCNSEVs may be relevant, as previous research has demonstrated that miR-301a-3p regulates CB1R levels under stressful conditions (Coelho et al., 2023).

As the endocannabinoid signaling pathway can modulate dopaminergic transmission through postsynaptic interactions between CB1R and dopamine receptors (Covey et al., 2017; García et al., 2016), we also explored the expression of D2R. The observed alterations in D2R align with prior preclinical studies, which reported increased D2R levels in the prefrontal cortex of offspring exposed to perinatal cannabis (Di Bartolomeo et al., 2021). In addition, dopaminergic neurons in the ventral tegmental area (VTA) of males showed an increased excitability after in-utero THC exposure (Renard et al., 2017; Sagheddu et al., 2021), which will change their molecular, cellular, and synaptic properties, increasing the risk for neurodevelopmental and psychiatric disorders later in life (Hurd et al., 2019; Roncero et al., 2020; Tirado-Muñoz et al., 2020). Dopaminergic neurons originating in the VTA project to cortical regions, where D2R+ neurons are located, as part of the mesocorticolimbic pathway (Reynolds and Flores, 2021; Xing et al., 2022). The increased D2R levels observed in our study after maternal cannabis use may therefore reflect a compensatory mechanism in response to altered mesocorticolimbic dopaminergic signaling. Cannabis use was also associated with reduced expression of miR-6865-5p and miR-3197 in both fetal brain tissue and fCNSEVs. As these miRNAs target D2R, their downregulation may contribute to the observed increase in D2R levels.

When examining our protein biomarkers, we observed negative correlations between fetal cortical tissue and fCNSEVs for both CB1R and D2R, showing opposing patterns in fetal brain tissue and fCNSEVs. Although these findings are correlational and do not establish mechanistic receptor cross-talk or extracellular vesicle-mediated receptor offloading directly, they are consistent with known functional interactions between these systems (Goetzl et al., 2016; Hirvonen et al., 2012). Importantly, these observations are specific to the proteins examined here and do not imply that all EV protein cargo follows the same pattern.

Collectively, these findings suggest that prenatal cannabis exposure in-utero, is associated with coordinated molecular alterations in cannabinoid and dopaminergic signaling systems in the developing human brain, especially in male fetuses. Our work supports the feasibility of using this subpopulation of EVs as a non-invasive approach to detect molecular changes in the developing human brain, in the context of the now known strong negative correlation.

We have also demonstrated that cannabis exposure modulates miRNA expression, consistent with the role of miRNAs in post-transcriptional gene expression regulation (Bartel, 2009; Ma et al., 2023). Concordant with our findings around protein receptors, we found a greater number of differentially expressed miRNAs in male fetal brains. The differential expression of miRNAs targeting CB1R and D2R, such as miR-301a-3p, miR-6865-5p, and miR-3197, suggests that miRNA-mediated post-transcriptional regulation may contribute to the alterations observed in cannabinoid and dopaminergic pathways (Coelho et al., 2023). We also demonstrated a strong positive correlation between brain and fCNSEV miRNA changes, again suggesting that a non-invasive approach is valid for miRNAs. This finding is consistent with publications reporting that miRNAs are robustly packaged into EVs, and EV-derived miRNA expression profiles usually reflect those from their cells of origin (Chaput and Théry, 2011; Xu et al., 2022).

Although most miRNAs followed similar directional changes across sexes, with males showing stronger effects after maternal cannabis use, miR-216a-5p showed greater downregulation in females. Previous preclinical studies also demonstrated hormone-dependent regulation of miR216a-5p, with stronger downregulation in female neonatal brains, suggesting that the interconnection between the cannabinoid system and estrogen-related pathways may underlie these sex-specific differences (Kim et al., 2023; Morgan et al., 2017). While miRNA-216a-5p has several predicted targets located on chromosome X, including NLGN3 (Quartier et al., 2019), the chromosomal location of these targets can also contribute, but is unlikely to drive the observed sex-specific response. Instead, the interaction between prenatal cannabis use and sex-hormone-dependent regulatory pathways likely contributes to the greater effect observed in females, since estradiol levels have previously been associated with changes in CB1R expression in the brain of neonatal rodents (Kim et al 2023).

Previous studies showed that multiple miRNAs are involved in the development and progression of neuroinflammatory and neurological diseases (Czernek and Düchler, 2020; Hu et al., 2010). Here, pathway analysis of target genes associated with these miRNAs revealed involvement in pathways crucial for neural development, including axon guidance, dopaminergic synapse, and GABAergic synapse. Axon guidance pathways are essential for regulating synaptogenesis, progenitor dynamics, and cell migration during brain development (Russell and Bashaw, 2018), particularly, when growing axons interact with guidance molecules to find their targets (Plachez and Richards, 2005). Dysregulation of these pathways due to prenatal cannabis exposure may disrupt processes crucial for normal brain development during the first and second trimesters. Furthermore, cannabis’ action on CB1R during neurodevelopment can impair prefrontal cortex CB1R signaling, affecting GABAergic functionality and disrupting the maturation of the prefrontal cortex (Renard et al., 2018). GABA is the primary inhibitory neurotransmitter in the mature brain, and an improper timing in GABA signaling during development has been associated with neurodevelopmental disorders (Peerboom and Wierenga, 2021).

Overall, we observed sex-specific differences and the male human brain appears to be more vulnerable to alterations in miRNA expression and CB1R and D2R signaling due to prenatal cannabis exposure. This may align with the higher prevalence of neurodevelopmental disorders such as Attention-Deficit/Hyperactivity Disorder or Autism Spectrum Disorder, observed in males after prenatal cannabis use (Corsi et al., 2020; Tadesse et al., 2024). For example, previous research found that CB1R availability is 41% higher in males than in females (Laurikainen et al., 2019). However, rodent studies showed that under stressful prenatal and postnatal conditions, CB1R binding is reduced in males, while it is increased in females (Dow-Edwards et al., 2016). Therefore, our findings align with previous literature, and suggest that maternal cannabis use may differentially impact neurodevelopmental trajectories in males and females, highlighting the importance to consider sex as a biological variable in this context.

As an exploratory endeavor, our study had some limitations including: (1) absence of detailed confirmatory histopathological analyses of brain region or histology in post termination brain homogenates, (2) absence of neurodevelopmental outcomes due to pregnancy termination, (3) the lack of THC quantification in maternal serum, and (4) sample size limitations that may have led to null findings in some comparisons. Nonetheless, given the limited literature on the effects of maternal cannabis use on neurodevelopment in humans, our study provides crucial insights from rare samples. In summary, our findings provide preliminary evidence that prenatal cannabis exposure is associated with altered miRNA levels in both fetal brain and fCNSEVs, and suggests that changes in the endocannabinoid and dopaminergic pathways may be detectable via non-invasive fCNSEV analysis during early neurodevelopment. These non-invasive techniques, can potentially provide additionally clinically useful information to clinicians caring for women with prenatal cannabis use, regarding individual fetal response to cannabis. A critical next step is to determine if alterations in fCNSEV protein and miRNA markers predict changes in fetal brain connectivity within mesolimbic brain substrates as measured by functional MRI and with neurodevelopment measures of executive function.

Supplemental Material

sj-docx-1-jop-10.1177_02698811261453833 – Supplemental material for Prenatal cannabis use is associated with altered miRNA and protein expression in the developing human brain

Supplemental material, sj-docx-1-jop-10.1177_02698811261453833 for Prenatal cannabis use is associated with altered miRNA and protein expression in the developing human brain by Lierni Ugartemendia, Baharan Fekry, Robert Suchting, Rafael Bravo, Chioma A. Ikedionwu, Katrina S. Mark, Erica Wymore, Annie B. Ouellet, Larissa Takser, Sharon Hunter, Camille Hoffman-Shuler and Laura Goetzl in Journal of Psychopharmacology

Footnotes

Acknowledgements

We thank Dr. Sara J. Bowne and Dr. Arunmani Mani for technical assistance with early experiments.

ORCID iDs

Lierni Ugartemendia

Baharan Fekry

Robert Suchting

Rafael Bravo

Chioma A. Ikedionwu

Katrina S. Mark

Annie B. Ouellet

Camille Hoffman-Shuler

Laura Goetzl

Author contributions

Study Design: LU, LG, and FB; Experiments: LU, BF, CAI; Data analysis: RS; Gene expression analysis: LU and RB; Writing – original draft: LU; Writing – Review and editing: all authors.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by Burroughs Welcome Fund: 1022519 (LG ,Co-I), NICHD: R01HD069238 (LG), and NIDA: R01DA060319 (LG).

Declaration of conflicting interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: LG has a patent for isolating fetal/neonatal neural extracellular vesicles using Contactin-2, but there is no current commercial use. The remaining authors have nothing to disclose.

Data availability statement

The data that support the findings of this study are available on request from the corresponding author.

Supplemental material

Supplemental material for this article is available online.

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