Prenatal Cannabinoid Exposure Impacts Offspring's Health in Sickle Mice
Authors: Ventura Christopher M , Kiven Stacy B , Lambrecht Nils , Gupta Kalpna 
Published: November 2020

Cannabis use is rising amongst pregnant women. An estimated 4% of pregnant women in the United States use cannabis, but, in California approximately 20% of 18-24-year-old pregnant women in a study cohort reported using cannabis (Young-Wolff et al JAMA 2017). Cannabis and cannabinoid use are relatively higher in patients with sickle cell disease (SCD) compared to the general population, perhaps due to pain. Majority of SCD patients in the Western world are on hydroxyurea (HU), which improves survival. However, HU is not prescribed to SCD patients who become pregnant, which may increase the likelihood of cannabinoid use to control pain. This may have devastating consequences as even a single dose of synthetic- (CP 55940) or phyto-cannabinoids (cannabidiol or Δ9-tetrahydrocannabinol) when administered to pregnant C57BL/6 mice on day 8 of gestation produced developmental changes in the offspring. (Fish et al Sci. Rep. 2019). Thus, we hypothesized that cannabinoid use during pregnancy will have teratogenic effects on the offspring's health in SCD. Using humanized transgenic Berkeley sickle (HbSS) and hemizygous (HbAS) mice and control mice expressing normal human hemoglobin A (HbAA), we examined the effect of chronic maternal cannabinoid exposure on the health outcomes of their offsprings.
We paired hemizygous AS Berkeley females (homozygous SS Berkeley females do not breed well) with homozygous SS Berkeley males, while AA control mice were paired with each other. Females were treated with HU (i.p., 50 mg/kg/day) for two weeks prior to pairing with a male. Female mice were then treated with CP55940 (CP; i.p., 0.3 mg/kg/day) or vehicle (Veh; 2% DMSO in sterile saline) during breeding until the pups were born (~3 weeks). A subset of females were left untreated to assess baseline measures. At birth, we recorded: [a] litter size, [b] body weight, [c] body size (crown to rump length), [d] right and left eye diameters, and [e] front- and hind-limb lengths. High-resolution digital images were acquired to quantify these parameters. At P21 pups were weighed and euthanized then their organs were collected, weighed, and fixed in formalin.
Observations at birth: Use of HU in AS females yielded a larger mean litter size than those of untreated AS females (8.5 vs 5.5 pups/litter). No difference in mean litter size of AA mice treated with HU was observed. The body weight between offspring of AA mice treated with HU+Veh and HU+CP was not significantly different. However, in both the offspring of AA mice and AS/SS mice treated with HU+CP, we observed a ~20% decrease in body weight compared to the untreated AA (p&lt;0.001) and AS/SS (p&lt;0.001) offspring. We observed a similar 20% decrease in body weight between the offspring of AA mice treated with HU+Veh and untreated AA offspring (20%, p&lt;0.001), while no decrease in body weight was observed between AS/SS offspring treated with HU+Veh and untreated AS/SS offspring. Taken together, these data show that maternal cannabinoid treatment leads to a significant decrease in the body weight of offspring.
Observations at post-natal day 21: Significant decreases in mean body size (5%, p&lt;0.01) and right eye diameters (8%, p&lt;0.01 & 7%, p&lt;0.05) were seen in the offspring of AA mice treated with HU+CP compared to HU+Veh. Offspring of AS/SS mice treated with HU+CP showed reduced mean body size (~9%, p&lt;0.001), reduced right and left eye diameters (20%, p&lt;0.001 & 19%, p&lt;0.001 respectively), and reduced front and hind limb lengths (21%, p&lt;0.001 & 16%, p&lt;0.001 respectively) compared to those treated with HU+ Veh. In addition, the offspring of AS/SS mice treated with HU+CP had about a 20% decrease in the ratio of brain to body weight compared to HU+Veh treated AS/SS mice (p&lt;0.001). No significant differences in mean litter size, body weight, front and hind limb length and brain weight to body weight ratio were observed between offspring of AA mice treated with HU+Veh and HU+CP. Our data suggest that development/growth of the offspring's body and brain may be hindered by pre-natal cannabinoid exposure and that chronic parental cannabinoid use during pregnancy has a more substantial adverse impact on offspring's health in sickle mice than in control mice. Our data raise concern for cannabinoid use during pregnancy in SCD, which may have adverse effects on the offspring's health and therefore requires monitoring in prospective clinical studies.

Disclosures
Gupta: CSL Behring: Honoraria; Tautona Group: Honoraria; Novartis: Honoraria; Grifols: Research Funding; 1910 Genetics: Research Funding; Cyclerion: Research Funding.