Cessation of chronic delta-9-tetrahydrocannabinol use partially reverses impacts on male fertility and the sperm epigenome in rhesus macaques

Abstract

Objective: To determine whether discontinuation of delta-9-tetrahydrocannabinol (THC) use mitigates THC-associated changes in male reproductive health using a rhesus macaque model of daily THC edible consumption.

Design: Research animal study.

Setting: Research institute environment.

Patient(s): Adult male rhesus macaques (age, 8-10 years; n = 6).

Intervention(s): Chronic daily THC edible administration at medically and recreationally relevant contemporary doses followed by cessation of THC use.

Main outcome measure(s): Testicular volume, serum male hormones, semen parameters, sperm deoxyribonucleic acid (DNA) fragmentation, seminal fluid proteomics, and whole genome bisulfite sequencing of sperm DNA.

Result(s): Chronic THC use resulted in significant testicular atrophy, increased gonadotropin levels, decreased serum sex steroid levels, changes in seminal fluid proteome, and increased DNA fragmentation with partial recovery after discontinuation of THC use. For every increase of 1 mg/7 kg/day in THC dosing, there was a significant decrease in the total testicular volume bilaterally by 12.6 cm³ (95% confidence interval [CI], 10.6-14.5), resulting in a 59% decrease in volume. With THC abstinence, the total testicular volume increased to 73% of its original volume. Similarly, with THC exposure, there were significant decreases in the mean total testosterone and estradiol levels and a significant increase in the follicle-stimulating hormone level. With increasing THC dose, there was a significant decrease in the liquid semen ejaculate volume and weight of coagulum; however, no other significant changes in the other semen parameters were noted. After discontinuing THC use, there was a significant increase in the total serum testosterone level by 1.3 ng/mL (95% CI, 0.1-2.4) and estradiol level by 2.9 pg/mL (95% CI, 0.4-5.4), and the follicle-stimulating hormone level significantly decreased by 0.06 ng/mL (95% CI, 0.01-0.11). Seminal fluid proteome analysis revealed differential expression of proteins enriched for processes related to cellular secretion, immune response, and fibrinolysis. Whole genome bisulfite sequencing identified 23,558 CpGs differentially methylated in heavy-THC vs. pre-THC sperm, with partial restoration of methylation after discontinuation of THC use. Genes associated with altered differentially methylated regions were enriched for those involved in the development and function of the nervous system.

Conclusion(s): This is the first study demonstrating that discontinuation of chronic THC use in rhesus macaques partially restores adverse impacts to male reproductive health, THC-associated sperm differentially methylated regions in genes important for development, and expression of proteins important for male fertility.

Keywords: Male fertility; THC; cannabis; delta-9-tetrahydrocannabinol; marijuana.

Figure 1.. (A) Study design overview.

Adult male rhesus macaques (n=6) were used. Prior to THC induction, baseline semen collection, scrotal ultrasound (US) and blood and urine sampling were performed. THC induction occurred over ~30 weeks (~7 months) per published medical marijuana acclimation guidelines. The nonhuman primate (NHP) spermatogenic cycle is ~64 days (~10 weeks), so the THC dose was increased every 10 weeks to accommodate 1 cycle at each THC dose until the highest THC dose was reached (2.5mg/7kg/day, equivalent to a heavy human medical cannabis dose). At the end of each THC dosing period and after THC was discontinued, all males underwent serial plasma and semen collection in addition to scrotal US. Testicular sperm extraction (TESE) was performed for histologic assessment at baseline (pre-THC), during THC induction (heavy-THC), and after THC was discontinued for 140 days. (B) Total testicular volume significantly decreases with increasing THC dosing with partial recovery of total testicular volume after discontinuing THC. Individual (symbols) and average fixed effect (lines) testicular volume (cm³) in response to increasing oral THC dosage (0 to 2.5 mg/7kg/day) resulted in a 59% decrease in volume after 210 days. THC was then discontinued over 140 days with partial recovery to 73% of the original testicular volume in 6 rhesus macaques (p<0.001). (C) Reduced seminiferous tubule diameter and decreased germ cell layers with THC exposure. Representative rhesus macaque testicular histopathology from the same animal pre-THC exposure (a,c) and after THC exposure (b,d). Seminiferous tubules with reduced diameter and decreased germ cell layers as indicated by the asterisks (*), were observed in all animals. Scale bar for a,b = 200 µm, for c,d =20µm.

Figure 2.

(A) Heatmap represents all 1,395 quantifiable proteins with mean per group log transformed and centered. Red color indicates higher mean expression and blue indicates lower expression relative to mean of all groups. (B) Seminal fluid proteome module-trait relationships. WGCNA was used to find correlation networks of co-expressed proteins. Each module (x-axis) containing multiple correlated proteins was reduced to a single eigenvalue (the first principal component) and then correlated with anatomical, seminal, hormonal, and dosing measurements (y-axis). The more positively correlated the module and the trait, the more red the square; the more negatively correlated, the more blue (* unadjusted p<0.05). (C) IPA merged network for proteins differentially expressed with either dose of THC (red= increased expression, blue = decreased expression) overlaid with top 3 most represented canonical pathways (CP). Orphan (disconnected) proteins were removed for visual clarity. (D) Top differentially expressed seminal fluid proteins with THC exposure. Boxplots represent all proteins with FDR p-value <=0.1 with either dose of THC (mod- or heavy-) relative to pre-THC. The top row contains proteins restored post-THC and the bottom row contains proteins not-restored, including 2 proteins with nominal dose-dependent association with THC exposure.

Figure 3.

(A) Distribution of mean methylation across DMRs. (B) THC-exposed rhesus vs. human cannabis users. Rhesus pre-THC vs. heavy-THC DMRs which overlap genes nearest DMCs in human cannabis users vs control (Left). Post-THC DMR genes in rhesus macaques overlap with Post-cannabis DMC genes in humans (Right). (C) Identification of DMRs pre-THC versus heavy-THC restored with THC washout (Left) and persistent DMRs (Right). Heatmaps represent the log transformed and mean centered average methylation values per DMR which intersect DMRs between pre-THC and heavy-THC. Hierarchical clustering demonstrates overall similarity in means between groups. (D) Significant overlap of genes annotated to THC DMRs with candidate autism genes. Venn diagrams showing the number of genes that are differentially methylated in sperm after heavy-THC versus pre-THC which overlap (Left) genes included on the SFARI autism candidate list with homologs in rhesus macaques, and (Right) genes with DMRs in sperm from fathers of children with autism versus without (27).