Recent studies in human populations have associated self-reported prenatal cannabis exposure with increased risk of offspring mortality (Gretchen Bandoli et al., 2023; Crosland et al., 2024; Shi et al., 2021; Varner et al., 2014), pre-term delivery (Crosland et al., 2024; Luke et al., 2022; Petrangelo et al., 2019; Shi et al., 2021), and Sudden Infant Death Syndrome (SIDS; (Scragg et al., 2001)). Our data indicate that the link with perinatal mortality and preterm birth is likely to be causal. Importantly, we observed elevated mortality through the pre-pubertal period, an outcome that needs to be assessed in human populations as well. We also found that the impact of prenatal cannabinoid exposure on offspring mortality was further exacerbated by simultaneous alcohol exposure, resulting in fewer live births and reduced litter sizes. This is significant, since polysubstance use in the context of risky alcohol consumption is increasingly common. There are a few pre-clinical studies on the combined effects of alcohol and cannabinoids, and our results are largely consistent with those studies. For example, an early investigation showed that combined prenatal alcohol (2 g/kg) and Δ9-tetrahydrocannabinol (THC; 100 mg/kg) exposure from GD 1-15 resulted in significant increases in fetotoxicity among mice and rats compared to single-drug-exposed and drug-free offspring (Abel, 1985).

More recently, Breit and colleagues (2019a) demonstrated greater lifetime mortality in offspring following co-exposure to alcohol (5.25 g/kg/day) and cannabinoids (CP55940; 0.4 mg/kg/day) from PD 4-9 compared to single-drug-exposed and drug-free offspring. Collectively, these and our study show that prenatal co-exposure to alcohol and cannabinoids may confer additional risks for delivery complications and offspring mortality, independent of the developmental window for exposure (prenatal vs early postnatal) or type of cannabinoid compound (naturally occurring vs synthetic). Notably, alcohol exposure alone in our model did not significantly affect offspring viability or litter size. While some clinical studies have linked prenatal alcohol exposure to pre-term delivery and SIDS (Beth A Bailey & Robert J Sokol, 2011), others report no association between alcohol exposure and delivery complications, indicating that these effects may depend on the dose and timing of exposure (Lundsberg et al., 2015; Pfinder et al., 2013).

Prenatal exposure to alcohol and cannabinoids, both individually and in combination, can also result in significant growth deficits and adverse physical development. Prenatal alcohol exposure is well-documented to cause persistent growth deficits, affecting offspring weight, height, and head circumference, with delays extending into adolescence (Carter et al., 2013; Day et al., 1989; Day et al., 1994; Pielage et al., 2023). These outcomes, while dose-dependent, can occur following even low levels of alcohol consumption during pregnancy (Day et al., 2002).

Similarly, prenatal cannabinoid exposure has been associated with growth restrictions and an increased likelihood of being born small for gestational age (Crosland et al., 2024; Huizink, 2014; Natale et al., 2020). In co-exposure models, more severe growth deficits have sometimes been observed in co-exposed offspring compared to single-drug-exposed offspring (Breit et al., 2019a, 2019b). However, other studies have not replicated these findings (Breit et al., 2020), indicating variability in outcomes. Interestingly, in our study, no growth restrictions were observed in viable offspring subjected to prenatal substance exposure. Instead, ALC+CB offspring demonstrated initial weight gains by PD 21. These weight increases may reflect the smaller litter sizes in ALC+CB groups, which could potentially increase access to maternal care and nutrition among surviving offspring. However, this early advantage did not persist; by PD 40 and PD 80, the weights of ALC+CB offspring were comparable to those of all other exposure groups.

A lack of weight differences among drug-exposed offspring may reflect a survival bias, as no offspring born prematurely survived to term. Underdeveloped animals may have died, skewing our results to only the most resilient litters. This possibility is supported by our physical examinations of non-viable offspring, with consistent physical malformations observed across offspring from six cannabinoid or dual-exposed litters, revealing hypodevelopment of the face, abdomen, and limbs. Independently, prenatal alcohol exposure is well-documented to produce craniofacial dysmorphologies characteristic of Fetal Alcohol Spectrum Disorder (FASD). These anomalies include alterations in the midface, nose, lips, and eyes, often resulting in a general recession of the midface and superior displacement of the nose (Muggli et al., 2017; Suttie et al., 2013). Studies using rodent models have further demonstrated that alcohol exposure during critical periods of development can produce significant craniofacial malformations. [see review: (Petrelli et al., 2018)]. Prenatal cannabinoid exposure has similarly contributed to the development of microphthalmia, iridial colobomas, facial clefts, and holoprosencephaly (Gilbert et al., 2016), as well as limb reduction, musculoskeletal deficiencies, and cardiovascular issues (Reece & Hulse, 2019). Moreover, limited studies on co-exposure suggest synergistic effects, such as enhanced craniofacial defects mediated by disruptions in cannabinoid receptor 1 (CNR1) signaling and the Sonic Hedgehog pathway (Boa-Amponsem et al., 2019; Fish et al., 2019).

Unfortunately, as our morphometric analyses were limited to litters with complete mortality, that were predominantly those exposed to cannabinoids, we could not evaluate whether combined exposure exacerbates outcomes relative to single-drug exposure in this cohort.

Notably, non-viable cannabinoid-exposed offspring exhibited evidence of intra-abdominal hemorrhage and preterm fetuses exhibited failure of abdominal wall closure. One potential cause is prenatal inflammation, which is strongly associated with preterm birth and fetal hemorrhaging. For example, lipopolysaccharide (LPS) exposure in utero induced liver hemorrhaging in ∼70% of fetuses, while control fetuses remained unaffected (Ernst et al., 2010). Alternatively, exposure to alcohol and cannabinoids may dysregulate the mechanistic target of rapamycin (mTOR) signaling pathway, which governs vertebrate growth, embryonic development, and organogenesis (Hwang et al., 2008). Acute exposure to both alcohol and THC can increase mTORC1 signaling (Neasta et al., 2014), and uterine mTOR hyperactivation has been implicated in preterm birth in mice (Hirota et al., 2011). While prenatal alcohol exposure has been shown to alter mTOR activity in the fetal hippocampus (Lee et al., 2020), cerebellum, and skeletal muscle (Sawant et al., 2020), the potential role of mTOR dysregulation in drug-induced uterine or systemic fetal outcomes warrants further investigation. Importantly, the relationship between intrauterine inflammation and compromised fetal health may be mediated by mTOR signaling, as LPS administration has been associated with acute elevations in mTOR signaling in the fetal brain (Dong et al., 2020).

Additional mechanisms for increased fetal mortality and abdominal bleeding include deficiencies in blood coagulation factors. Deficiencies in blood coagulation Factor X (Dewerchin et al., 2000) and prothrombin clotting factor 2 (Xue et al., 1998) have been associated with embryonic lethality and fatal neonatal bleeding, while Factor XIII A subunit-deficient mice experience severe uterine bleeding and spontaneous miscarriage (Koseki-Kuno et al., 2003).

However, the effects of prenatal alcohol and cannabinoid exposure on coagulation factors are currently poorly understood. Finally, fetal hypoxia and compromised blood flow/ischemia can contribute to vascular stress and breakdown, leading to internal bleeding. We recently demonstrated that both alcohol and cannabinoid exposures reduce fetal cerebral blood flow in utero (Rouzer et al., 2024), with combined exposure augmenting blood flow deficits through the internal carotid artery. These reductions corresponded with instances of intrauterine growth restriction, emphasizing the need for further research to determine whether recovering losses in fetal blood flow could mitigate embryonic lethality and developmental malformations associated with prenatal drug exposure.

Significant sex differences emerged when assessing coordination abilities in prenatally exposed young adult offspring. Male offspring prenatally exposed to cannabinoids required significantly more trials to habituate to the Rotarod task on the first day, aligning with prior findings that THC-exposed adolescent offspring required more trials to achieve their first success performance in a parallel bar test (Breit et al., 2022). Among control offspring, females consistently outperformed their male siblings, balancing longer on the Rotarod, although both sexes improved across testing trials. This observation is consistent with reports that B6 mouse strains exhibit motor learning through repeated testing (McFadyen et al., 2003), and B6J female mice can balance longer on the rotarod than male counterparts (Ashworth et al., 2015). Analysis of exposure effects within sexes revealed distinct patterns. In males, all prenatal exposure groups showed reduced balancing time on the Rotarod compared to unexposed controls, with the most pronounced deficits occurring in the final trial. In contrast, females exhibited impairments exclusively in single-drug exposure groups, with the most significant deficits observed during the first trial, suggesting that drug-exposed females displayed “catch-up” performance with repeated testing. These findings are consistent with previous studies on prenatal single-drug exposure, which report impairments in motor learning and coordination (Breit et al., 2019a; Breit et al., 2022; Connor et al., 2006; Reekes et al., 2016; Thomas et al., 2000). Interestingly, the deficits observed in ALC males resemble a study of six-month-old male mice, where moderate prenatal alcohol exposure did not affect initial performance but reduced improvement across testing days (Reekes et al., 2016). However, additional studies of prenatal alcohol or cannabinoid exposure report no significant coordination impairments following single-drug exposure (Breit et al., 2019a; Breit et al., 2022; Heaton et al., 2022).

These inconsistent outcomes in coordination deficits extend to preclinical investigations of ALC+CB exposure. For example, in a parallel bar test, co-exposure to CP-55940 and alcohol amplified adolescent (∼PD 30) coordination deficits compared to single-drug exposure, particularly in female offspring (Breit et al., 2019a). However, the same research group found that alcohol co-exposure did not exacerbate THC-induced deficits, with THC alone driving reduced coordination (Breit et al., 2022). In our study, co-exposure did not augment outcomes from single-drug exposure. Co-exposed offspring generally exhibited impairments comparable to single-drug exposed groups, except for ALC+CB females, who performed similarly to controls. Notably, ALC+CB females also had the lowest survival rate in our cohort (n=6 from 13 litters), suggesting their strong performance may reflect a survival bias favoring resilient individuals.

Variability in reported outcomes within this field of investigation likely arises from differences in the type of cannabinoid used, dosage, route of administration, and timing of gestational exposure, among other experimental variables. These findings underscore the importance of studying co-exposures alongside comparable single-drug exposures, accounting for experimental factors that influence behavioral outcomes.

In contrast to coordination assessments, sex differences were minimal in open field activity measures. In control offspring, males did spend more time in the center of the OF than females; otherwise, no sex differences were observed in open field measures. Furthermore, unlike coordination measures, single-drug exposures did not alter open field behaviors. However, ALC+CB exposure significantly reduced center time in males compared to controls and CB-only offspring. Notably, ALC+CB offspring entered the center more frequently than ALC or CB groups, but this likely reflects polysubstance-induced hyperactivity, as co-exposed offspring also exhibited significantly increased speed and distance travelled. Overall, ALC+CB offspring displayed a thigmotaxic behavioral phenotype, characterized by faster movement along the periphery of the open field.

The open field test is a validated tool for assessing hyperactivity and anxiety-like behaviors in rodents (Kraeuter et al., 2019). Hyperactivity and inattention are common symptoms expressed by individuals with prenatal exposure to alcohol (Dodge et al., 2023; Han et al., 2015; Infante et al., 2015) or cannabinoids [reviewed in (Ikeda et al., 2022)] throughout the lifespan. In the open field test, avoidance of, and reduced time spent in the center of the open field may reflect increased anxiety-like behavior. In rodents, avoidance of the center reflects increased anxiety-like behavior, aligning with the high prevalence of anxiety disorders in individuals with prenatal alcohol exposure (Burgess & Moritz, 2020; Hellemans et al., 2010), who may first demonstrate symptoms in early childhood (O’Connor & Paley, 2009). Similarly, prenatal cannabinoid exposure can also heighten the risk for offspring anxiety disorders (Grant et al., 2018; Nashed et al., 2021).

When reviewing existing preclinical studies, it’s noteworthy that assessments of effects of ALC exposure on open field activity have produced mixed results, with some researchers reporting no effects of prenatal exposure (Allan et al., 2003; Downing et al., 2008), while others report alcohol-induced hyperactivity in the open field (Randall et al., 1985), or hypoactivity (Rouzer et al., 2017; Schambra et al., 2016). Similarly, preclinical investigations of prenatal cannabinoid exposure have uncovered reduced time in the center of the open field (Cupo et al., 2024; Newsom & Kelly, 2008), and increased anxiety-like behaviors and attention deficits following THC exposure (Navarro et al., 2024). However, findings vary depending on the type and dose of cannabinoid, route of administration, rodent strain, and timing of exposure [as reviewed in (Higuera-Matas et al., 2015)]. Factors of offspring age and sex (Higuera-Matas et al., 2015; Osterlund Oltmanns et al., 2022; Schambra et al., 2016; Andrew F Scheyer et al., 2019) further mediate the effects of prenatal substance exposure on attention, hyperactivity, and anxiety-like behaviors.

It is notable that within our own study, individual exposures to alcohol or cannabinoids did not alter open field behaviors, but simultaneous prenatal exposure produced a phenotype of hyperactivity and anxiety-like behavior consistent with FASD. Our findings align with prior research demonstrating augmented hyperactivity following co-exposure. For instance, Ornelas et al (2024) reported increased adolescent hyperactivity levels in co-exposed males compared to cannabinoid-exposed-only offspring, at CP-55940 doses <50% lower than those used in our study. Separately, zebrafish co-exposed to ethanol and a cannabinoid receptor 1 agonist demonstrated significantly increased swimming activity, an effect that was absent following equivalent single-substance exposures (Boa-Amponsem et al., 2019). Similarly, third-trimester-equivalent co-exposure to alcohol and CP-55940 (0.4mg/dL) augmented locomotor activity beyond individual substance effects (Breit et al., 2019b). Collectively, these studies highlight hyperactivity as a robust phenotype of prenatal co-exposure, supporting the need for further research into the distinct and synergistic effects of co-exposures compared to single-drug exposures.