Since the COVID-19 pandemic, the prevalence of substance use, including tobacco products, marijuana, opioids, and alcohol, has been increasing, especially among men of reproductive age^28,29. In 2021, 44% of adults surveyed believed smoking marijuana every day is safer than smoking tobacco, compared to about 37% in 2017³⁰.

It is widely accepted that unhealthy lifestyles, including consuming alcohol and using tobacco or marijuana products, can negatively affect overall health. However, the trend of increased consumption is concerning because men are often unaware of the negative impact of these lifestyle on their reproductive health. Available evidence suggests that substance use may affect spermatogenesis, secretion of reproductive hormones through the hypothalamic-pituitary-gonadal (HPG) axis, and sexual function^31–36.

To our best knowledge, this is the first study that aims to compare and determine the effects of tobacco and cannabis smoking in association with variants in the Cytochrome C Oxidase 1, 2 and 3 genes (MT-CO1, MT-CO2, and MT-CO3), on spermatozoa function and male fertility. The findings of the present study showed that normal sperm morphology was significantly lower not only in tobacco smokers but also in cannabis smokers in comparison to non-smokers (p < 0.001) (Table 1). Moreover, there was a significant reduction in normal sperm morphology in the cannabis smoker group compared to the tobacco smoker group (p = 0.002) (Fig. 1). Sperm progressive motility showed a non-significant reduction in the cannabis-smoking group compared to tobacco-smoking and the non-smoking group (14.27 ± 11.3%) (p = 0.223) (Table 1). Moreover, non-progressive motility showed a significant reduction in the cannabis-smoking group and the tobacco-smoking group compared to the non-smoking group (p < 0.001). However, the mean percentage of immotile sperm was significantly higher in the cannabis-smoking group compared to tobacco-smoking and the non-smoking group (p < 0.001) (Table 1). On the other hand, sperm concentration, and semen volume showed no significant difference between the three groups (p = 0.199; p = 0.091, respectively) (Table 1).

We investigated the sperm DNA integrity using acridine Orange (AO) assay and Chromomycin staining (CMA3). The results showed a significant increase in the AO + score in the cannabis-smoking group (28.53 ± 15.8%) compared to the non-smoking group (10.1 ± 14.2%) and the tobacco-smoking group (6.4 ± 10.2%) (p < 0.001) (Table 2). At the same time, there was no significant difference in AO + score between the tobacco-smoking and non-smoking groups (p = 0.19) as shown in Fig. 5. In addition, the CMA3 + test showed a significant increase in the CMA3 + score in the cannabis-smoking group compared to the non-smoking group (p < 0.001) and between the tobacco-smoking group compared to the non-smoking group (p = 0.003). At the same time, there was a significant increase in the CMA3 + score of cannabis-smoking men in comparison to tobacco-smoking men (p = 0.001) (Table 2; Fig. 6).

These results are in accordance with our previous study showing that smoking negatively alters the sperm standard parameters, DNA stability of sperm, and the ratio of protamine mRNA as well as downregulates the expression of H2BFWT, PRM1, and PRM2³⁶. Moreover, several studies have reported that the spermatozoa of smokers have higher levels of DNA fragmentation in comparison with non-smokers³⁶. Numerous studies have provided insights into the multiple pathways through which tobacco smoking affects sperm cells. For instance, it has been discovered that nicotine impairs spermatogenesis by inducing oxidative stress, DNA damage, and apoptosis within germ cells, leading to reduced sperm count and motility^37,38.

Also, cigarette smoking has been found to detrimentally impact sperm chromatin condensation and viability. Moreover, these negative effects correlate with both the quantity of cigarettes smoked daily and the overall duration of smoking. In a study conducted by Yu et al. (2014) it was observed that the rate of histone abnormalities was significantly lower in non-smoking men with normal sperm counts, while the highest rates were found in heavy smokers with oligospermia within the Chinese population studied³⁹. In addition, excessive ROS production can lead to oxidative stress, which in turn affects sperm nuclear DNA, sperm mitochondrial respiratory activity⁴⁰ and endocrine function, leading to a variety of male reproductive system disorders, and consequently may lead to male infertility^41,42. In addition, passive smoking effects on various sperm parameters have been studied^43–45. For example, a decrease in sperm density, motility, and possible negative effects on morphology have been demonstrated⁴⁶. Sperm concentration also decreased by an average of 22% in a dose-dependent manner⁴⁷. Although sperm concentration, motility, and/or morphology are reduced compared to nonsmokers, they generally remain within normal ranges. However, available evidence suggests that smoking may negatively affect sperm binding to the zona pellucida. These findings are based on a study using a penetration test with zona-free hamster eggs⁴⁸.

Cannabis compounds can have a significant impact on sperm motility. ur In our previous study, we have pointed out that the use of high-potency cannabis can lead to a decrease in sperm motility, which may have implications for male fertility³⁵. Our findings are consistent with previous reports from fertility centers about the effect of marijuana use on sperm morphology^11,49,50. In three studies conducted in Jamaica, the Pacific Northwest, marijuana use (past, past 3 months, and current) was associated with an increased risk of abnormal sperm morphology^11,49,50.

On the other hand, in a study of current, past ever, and never marijuana men users, no significant association with the percentage of normal morphology was found. The risk of morphological abnormalities was also lower compared with men who had never smoked marijuana. Although heavy or recent marijuana use was associated with an increased risk of “abnormal motility” in Jamaican men⁴⁹, no significant associations were found between different categories of marijuana use and total sperm motility percentage⁵¹.

Nevertheless, a pile of evidence suggested that cannabis smoke impaired male reproductive health, particularly the viability and functionality of sperm. Gundersen et al., documented a significant decrease in sperm concentration among cannabis users compared to non-users⁵².

On the other hand, genetic defects are thought to be the cause of 15–30% of male infertility cases³. Several genes associated with fertility and longevity are also associated with mitochondria²⁶. Sperm rely on the mitochondrial oxidative phosphorylation machinery (OxPhos) to generate the energy they need for movement⁵³.

SNPs or large deletions are types of mutations that affect sperm mitochondrial DNA^54,55. Therefore, mutated mitochondrial DNA in sperm can cause respiratory problems, affect energy production, and lead to reduced motility, affecting the normal activity of sperm.

Studies have shown that changes in mtDNA may affect sperm velocity and quality⁵⁶. Their study demonstrated that changes in mitochondrial DNA can have a significant impact on sperm quality and its swimming ability⁵⁶.

In the contrary, another study found no significant difference in mtDNA deletions in sperm from asthenozoospermic and normozoospermic men and concluded that these deletions do not have a role in male infertility⁵⁷.

A previous study has reported that mtDNA deletions do not affect sperm motility, as there was no significant difference in the incidence of mtDNA deletions in low-quality and high-quality sperm⁵⁸.

Recently, various researchers showed that the expression of mitochondrial genes can be modulated by lifestyle factors such as smoking and drinking, which also may lead to psychiatric disorders. These habits may affect the activity of the mitochondrial respiratory chain as well as the replication and transcriptional regulation of mitochondrial genes, leading to altered mitochondrial function and, in turn, to psychiatric disorders⁵⁹.

Therefore, this study aimed to investigate the potential genetic alterations within the genes that encode for the mitochondrial cytochrome c oxidase (Complex IV) (MT-CO1, MT-CO2, and MT-CO3) in the sperm cells of individuals who consume tobacco and cannabis. These genes are crucial for cellular respiration and energy production within the cell, and any variations could significantly impact cellular performance. Given the widespread use of tobacco and cannabis, understanding their potential impact on genetic integrity and fertility is important.

A total of 23 SNPs in MT-CO1 were identified; 15 of them were synonymous variants, while eight were missense mutations (Table 4). The percentage of men with total variants in the CO1 gene groups NS, TS, and CS were: 97.2%, 82.0%, and 86.4% respectively (Table 3). However, none of these SNPs were significantly different between the studied groups (p = 0.10).

A total of 15 genetic alterations in the MT-CO2 gene were identified; ten of them were synonymous variants and five of them were missense mutations (Table 5). The percentage of men with total variants in the CO2 gene groups NS, TS, and CS were: 18.9%, 33.3%, and 18.9% respectively (Table 3). None of these SNPs were significantly different between our groups (p = 0.23).

A total of 30 genetic variations in MT-CO3 were identified; 22 of them were synonymous variants and eight of them were missense mutations (Table 6). The percentage of men with total variants in the CO3 gene groups NS, TS, and CS were: 67.6%, 43.6%, and 45.9% respectively (Table 3). Also, none of these SNPs were significantly different between the three groups (p = 0.07).

However, other study demonstrated a significantly higher mtDNA mutation in the never-smokers compared to the current-smoker having lung cancer (p = 0.006). MtDNA mutation was significantly higher in the never-smoker Asian compared to the current-smoker Caucasian patients’ population (p = 0.026). They also observed a significant increase in mtDNA content among the never-smoker lung cancer patients (p = 0.037)⁶⁰. Moreover, the US Department of Health and Human Services pointed out that tobacco smokers undergo a substantial build-up of genetic mutations due to the complex chemical composition of tobacco smoke⁶¹.

Nevertheless, the analysis of sperm exposed to cannabis revealed significant changes in DNA methylation patterns. The hypomethylation of CpG sites observed in individuals who consume cannabis suggests a decrease in methylation of cytosine-phosphate-guanine (CpG) dinucleotides. Tetrahydrocannabinol (THC), the main psychoactive component of cannabis, exerts a variety of effects on the epigenetic properties of sperm⁶². This suggests a possible correlation with the observed differences in mitochondrial DNA (mtDNA). When sperm are exposed to THC, THC interacts with the endocannabinoid system, a complex signalling network that is critical for a variety of physiological activities¹³.

To conclude, the current research demonstrated that cannabis smoking deteriorates sperm quality and DNA integrity more than tobacco smoking. Besides, the lack of a correlation between the identified variant alleles and each of the non-smoker, tobacco smokers and cannabis smoker groups demonstrated that smoking seems unlikely to alter the nucleotide sequence of these genes rather than sperm DNA. However, an analysis of a larger sample size is critical, and would allow a better understanding of the impact of these lifestyles and genetic changes, and could shed light on the risks associated with drug use. The enduring message of this study is the importance of promoting education and awareness about tobacco and marijuana use and their profound effects not only on individual health but also on future generations. Besides, male partners of infertile couples should strictly quit cigarette and cannabis smoking at least three months before undergoing assisted reproductive treatment (ART).