CANNABIS SIGNIFICANTLY ALTERS DNA METHYLATION OF THE HUMAN OVARIAN FOLLICLE IN A DOSE-DEPENDENT MANNER.

Cannabis-derived phytocannabinoids are increasingly consumed by women of childbearing age. The objective of this study was to determine the epigenetic effects of cannabis on cells comprising the follicular niche and supporting the growing oocyte. Matched case-control study. Cannabis consumption was determined by measuring five phytocannabinoids in follicular fluid by liquid chromatography mass spectrometry. Seven positive patients were matched by baseline characteristics (age, BMI, stimulation parameters, etc.) with 7 patients who were negative for all assayed phytocannabinoids. Granulosa cell DNA methylation was assessed using the Illumina TruSeq Methyl Capture EPIC kit. Differential methylation analysis was conducted with Methylkit, pathway analysis was conducted with g:Profiler, and dose-dependent correlations were identified by linear regression analysis. Phytocannabinoids alter the epigenome of granulosa cells. There were 3679 differentially methylated sites, with two-thirds affecting coding genes. Further analysis defined a differentially methylated hotspot region on chromosome 9, associated with two genomic features, a zinc-finger protein (ZFP37) and a long non-coding RNA (FAM225B). Of the 2214 differentially methylated genomic features identified in this study, 19 have been previously annotated in the context of cannabis-related epigenetic modifications in other models or organ systems. Pathway analysis showed enrichment in GPCR signaling, cellular transport, immune response, and cellular proliferation. By applying stringent criteria for a sliding window, we identified 71 differentially methylated regions, none of which were previously annotated in this context. Notably, correlation analysis revealed 16 unique genomic features affected by cannabis consumption in a dose-dependent manner. Of these, the histone methyltransferase SMYD3 was hypomethylated following exposure to Δ9-THC, possibly implicating histone modifications following exposure to cannabis. Cannabis significantly alters the epigenome of granulosa cells in women undergoing IVF treatment. These changes are dose-dependent and target critical pathways involved in GPCR signalling, cellular transport, immune response, and cellular proliferation.