O-074 THC reduces sperm mitochondrial membrane potential, but does not affect the acrosome reaction or in-vitro embryo developmental rates.

Abstract

Abstract Study question Does THC alter sperm function and early embryo development following in vitro fertilization with THC-exposed sperm? Summary answer THC reduces sperm mitochondrial membrane potential (MMP), mainly through cannabinoid receptor agonism, but does not alter the acrosome reaction or rates of embryo development. What is known already Cannabis is the most commonly used recreational drug among males of reproductive age. THC is the main psychoactive component of cannabis and can cross the blood-testis barrier, disrupting the endocannabinoid system (ECS) in sperm. However, the mechanisms by which THC affect sperm function and early embryo development are unclear. In sperm, endogenous cannabinoids influence the acrosome reaction and MMP, which are critical for motility, viability and, ultimately, fertilization. To date, no studies have investigated how sperm exposed to physiologically relevant doses of THC may impact in-vitro early embryo development, and cannabis use recommendations remain unclear for patients undergoing IVF procedures. Study design, size, duration Bovine sperm and cumulus oocyte complexes (COCs) were used as a translational model for humans. Cryo-thawed sperm was separated using a Percoll gradient and incubated for 6-hours in one of five treatment groups: control, vehicle (0.01% ethanol), low-THC (0.032µM), mid-THC (0.32µM), and high-THC (4.8µM) – concentrations equivalent to THC plasma levels following therapeutic and mid-high recreational use, respectively (Whan et al., 2006). A minimum of 30,000 sperm and 60 COCs were analyzed per group. Participants/materials, setting, methods Sperm were treated for 6-hours prior to all experiments. Flow cytometry using sperm (1-2x106) stained with propidium iodide (PI) and FITC-conjugated peanut agglutinin (FITC-PNA) or JC-1 was used to measure either the acrosomal reaction or MMP, respectively. COCs were aspirated from slaughterhouse ovaries and matured in-vitro for 24-hours. IVF was performed with THC-treated sperm (1x106/mL/drop) for 10-hours. Presumptive zygotes were cultured in-vitro for 8-days. Cleavage and blastocyst rates were measured 48-hours and 8-days post-fertilization, respectively. Main results and the role of chance Measuring PI and FITC-PNA fluorescence showed the percent of sperm that were acrosome-reacted and either alive or necrotic. There were no significant differences in acrosome-reacted sperm among groups (n = 9). Measuring JC-1 fluorescence showed the percentage of sperm with high MMP. Results indicate a significant reduction in sperm with high MMP (37%) following high THC (4.8µM) exposure (p = 0.002418, n = 4). No significant differences in MMP were observed among other groups. To elicit the mechanism by which THC was reducing sperm MMP, we repeated experiments using only JC-1-stained sperm exposed to high THC treated with SR141716 and SR144528, which are CB-1 and -2 antagonists, respectively. Additional groups included: SR141716 (4.8.µM), SR144528 (4.8µM), THC + SR141716 and THC + SR144528. Results indicate a significant reduction in sperm with high MMP (38%) in the THC group (4.8µM) compared to control (51%) (p = 0.0417, n = 6), vehicle (54%) (p = 0.0069, n = 6), and SR141716 (65%) (p < 0.0001, n = 6). There were no significant differences in sperm with high MMP in THC + SR141716 or THC + SR144528 groups, indicating that THC is acting agonistically, primarily at CB1 receptors. Following IVF with THC-treated sperm, there were no significant differences in cleavage or blastocyst rates among treatment groups. Limitations, reasons for caution The use of bovine instead of human cells could be considered a limitation. However, as studying effects of THC-treated sperm on IVF outcomes would not be possible using human samples, it is actually an advantage. The similarity between bovine and human gametes makes bovine an ideal translational model for humans Wider implications of the findings As cannabis use and THC concentrations increase, this research addresses the growing concern of how cannabis impacts fertility and, ultimately, pregnancy outcome. Understanding how THC affects sperm function and embryo development will provide physicians with science-based recommendations concerning cannabis use for patients trying to conceive, pregnant, or undergoing fertility treatments. Trial registration number not applicable