P-402: Fertility loss in the sperm of tobacco smokers may be reversed after washing with a cannabinoid agonist

Abstract

Human sperm carry a nicotinic acetylcholine receptor and respond to nicotine from tobacco. They also have cannabinoid (CB) receptors and respond to marijuana and endocannabinoids or synthetic CB chemicals. We previously reported that sperm from 2/3 of tobacco smokers showed a significant drop in fertilizing capacity, using the Hemizona Assay (HZA). Their sperm showed reduced binding to the zona pellucida (cover) of the human egg. In Part II, we hypothesized that treatment of the smoker’s sperm with a CB agonist would improve sperm binding capacity. We have studied the effects of a CB agonist (AM-1346; analog of the endocannabinoid anandamide). After obtaining a positive response when donor sperm were treated with nicotine plus a CB agonist, we studied the sperm from 8 chronic tobacco smokers in 24 in vitro HZA tests. Each smoker was previously tested in the HZA, where his sperm fertilizing function was compared to a non-smoking donor. Four of 8 smokers (Group I) had shown normal sperm fertilizing function (HZI > 65). The other 4 smokers (Group II) had failed the zona assay. In Part II, each new test used the sperm from one tobacco smoker. His sperm were washed with and without a CB ligand (Control had no ligand present). We tested the effects of a potent CB1 agonist, AM-1346 (n = 17; 100 pM to 100 nM). After a “swim-up,” the motile sperm fraction was used for the HZA. Here, a nonviable human oocyte was cut in half. One half was incubated with smoker’s sperm that had been treated with a CB ligand. The matching Hemizona (HZ) was placed with that smoker’s Control sperm. Post-incubation, each HZ was rinsed and counted for the number of sperm tightly bound to the outside. The ratio of [# bound for the treated sperm / # bound for the Control sperm] was calculated as the Hemizona Index (HZI). HZI = 100 indicates that the treated sperm bound to the zona as well as the Control sperm. We hypothesized that the HZI would improve with in vitro CB treatment. After exposing the sperm to AM-1346, at any concentration, we found that Group II (low fertility) responded with markedly improved zona binding, compared to the Control sperm (4 out of 8 experiments). Specifically, when semen quality was poor, Group II showed a mean HZI of 177% (77% improvement compared to the untreated sperm). However, the sperm from smokers with normal fertility (Group I) behaved differently. Here, only 1 of 9 tests showed a zona binding enhancement (p < 0.05). In Groups I & II, there were 7 experiments where the smoker was tested with a low concentration of AM-1346 (one assay) and also tested at a higher 1346 level (second assay). In 6 of 7 cases, the higher 1346 dose produced a better HZI score (improved binding). We conjecture that most smoking men would show a stimulation of zona binding if the dose of AM-1346 is high enough. Based on our previous data, and published literature, it is clear that a majority of tobacco smokers will exhibit a small or significant decline in fertility potential. Infertility clinics worldwide are trying to meet the challenge of male and female tobacco smokers who have only borderline fertility. Smoking cessation is the best solution. However, the in vitro use of synthetic cannabinoid analogs may significantly improve the fertilizing capacity of tobacco smokers who cannot quit.