IN-VITRO TREATMENT OF HUMAN OOCYTES WITH CANNABIS ALTERS GENES INVOLVED IN CHROMOSOMAL ORGANIZATION AND SEGREGATION - A SINGLE CELL RNASEQ STUDY

Cannabis legalization is increasing worldwide and was legalized in Canada in 2018. Tetrahydrocannabinol (THC) has been shown to reach the follicular niche and affect supportive granulosa cells in humans. Thus, knowledge of its effects on female reproduction is of utmost importance. Here, we investigated the effect of in-vitro THC exposure on oocyte maturation and the transcriptomic changes induced by THC treatments. GV oocytes were collected from 39 consenting patients (223 oocytes) undergoing IVF-ICSI. GV oocytes were split into three groups: control (n=74), THC1 (n=74) (physiological concentration of Δ9-THC) and THC2 (n=75) (supraphysiological concentration), and cultured for 24h in Sage OneStep+Menopur. To reduce inter-sample variability, we selected patients with similar demographics and stimulation parameters that also had at least 1 MII oocyte for each treatment group. cDNA from 37 oocytes (10 patients) was synthesized using SmartSeq v4. Libraries were constructed (NexteraXT) and sequenced (NovaSeq S1 2x150bp). Bioinformatics (Partek Flow) and pathway analysis were conducted to identify key pathways altered by THC treatment. Differentially expressed genes (DEG) were defined as -2>FC>2, p-value<0.05. Our results showed increased maturation rate when oocytes were treated with a supraphysiological concentration of THC (39.2% for Ctrl, 37.9% for THC1 and 49.3% for THC2), however it did not reach significance. When assessing the oocyte transcriptome; principal component analysis (PCA) clustered based on oocyte maturation level (PC1 33.3%), but not based on cannabis treatment. Of oocytes that matured to metaphase-II, there were 189 differentially expressed genes (DEGs) in THC1 vs Ctrl; 66 genes were upregulated, and pathway analysis revealed an enrichment in mitochondrial respiration and response to toxic substances. The 123 downregulated genes were associated with regulation of chromosome segregation and duplication and cell development. Furthermore, 6 genes previously associated with oocyte maturation were differentially expressed (ACTG1, ANK2, PWWP2A, LIN7C, TM4SF1, and KLHL28). In oocytes exposed to even higher (supraphysiologic) concentrations of cannabis, upregulated genes (n=6) were associated with transcription initiation and activation, while the downregulated genes (n=36) were associated with nuclear DNA replication and DNA methylation. This is the first study investigating the impact of cannabis on human oocytes. We demonstrated a significant change in ooplasm mRNA, potentially altering meiotic spindle formation/organization and cytoplasmic maturation. With cannabis legalization increasing worldwide, further investigation into its consequences is critical for clinical counselling and legalization guidelines.