In recent decades, the demographic crisis observed in highly developed countries, including Russia, has been combined with a progressive decline in the reproductive potential of human populations, which raises the question of the underlying causes of this phenomenon. In different regions of the world, adverse secular trends in male reproductive health has been observed, which are expressed in poor semen quality, an increase in the proportion of male factor in infertile couples and the growth of congenital anomalies of the male reproductive system, resulting to infertility (1–4). In addition to a reduction of semen volume, sperm concentration and motility and an increase in sperm morphological abnormalities, several epidemiological studies have indicated a population-level decline in testosterone, suggesting that all these facts may be interrelated (5–7).

Since adverse changes in male fertility have occurred in a short period of time and in parallel in different parts of the world, the decline in semen quality is probably due to environmental rather than genetic factors (4). In recent years, it has been well documented that adverse changes in male fertility seem to be primarily associated with primarily anthropogenic environmental pollution, occupational exposure and individual lifestyle (8–11). Lifestyle, especially bad habits such as smoking, alcohol consumption, physical inactivity or excessive nutrition, have a negative impact not only on general health, but also reduce sperm quality and can lead to male sub-fertility or infertility (11–13). Some authors believe that the worldwide decline in men’s reproductive potential might be due, at least in part, to lifestyle factors (4, 8, 9).

Tobacco smoking is considered among the most significant lifestyle factors affecting male reproductive function. Smoking is still widespread all over the world, including Russia. According to the World Health Organization, 22.3% of the global population, 36.7% of men and 7.8% of women, used tobacco in 2020 (14). Its prevalence in the United States is about 21-22% among men of reproductive age (9, 15). The Russian Public Opinion Research Center (VCIOM) reports that the proportion of smokers in Russia has remained unchanged over the past five years and today it is a third of total number of citizens (33%), while smoking men are much more than women (47% vs 21%) (16).

It is generally recognized that tobacco smoking has a negative impact on overall health and is one of the leading preventable causes of death. Tobacco smoking is associated with more than 27 diseases, including respiratory and cardiovascular diseases, lung, kidney, urinary bladder, pancreas cancer, type 2 diabetes and other medical conditions (17, 18). Despite the fact that smoking is a well-established cause of many hard diseases, the mechanism by which smoking causes such a wide range of diseases is not fully elucidated. Although many are aware of the dangers of tobacco use, smoking continues to be a public health concern. Only recently, due to the growth of male infertility and subfertility, increased attention has been paid to the effects of smoking on men’s reproductive health (19). Cigarette smoking is a potential risk factor for impaired male fertility and the relationship between smoking and male infertility is actively being discussed. A decrease in semen volume, sperm concentration, viability, motility and normal morphology has been shown in heavy tobacco smokers (8, 9, 19–22). Moreover, in ART programs, chronic smoking of male partner was related to poor embryo quality and the reduced success of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) (8, 23–25). Although many studies have reported a decrease in one or more conventional semen parameters or altered sperm functions in tobacco smokers, some studies have not established or shown very weak negative changes in semen quality, especially in infertile and sub-fertile men (20, 21, 23, 26, 27).

Based on studies that evaluated semen parameters in accordance with the WHO guidelines (28), it can be concluded that the effects of tobacco smoking lead to a more pronounced poor semen quality in healthy men or men from the general population (19, 29–32) than in infertile men or men from infertile couples (33–37). Moreover, it has been shown that tobacco smoking can affect the conventional semen parameters in a dose-dependent way, since the impairment of semen quality was more pronounced in heavy smokers then in moderate or mild smokers, which indicates more severe reproductive consequences with longer or intensive smoking (19, 29, 30, 38, 39).

More than 7,000 chemicals and toxins have been identified in tobacco smoke including polycyclic aromatic hydrocarbons, of which many are proven carcinogens and mutagens (8, 20, 22, 40, 41). The main pharmacological hazardous substances of tobacco – the alkaloids nicotine and its metabolite cotinine - can penetrate the blood-testis barrier and subsequently affect the process of spermatogenesis, reducing sperm concentration, total sperm count, and sperm motility (19–23). Smoking has been shown to increase genetic and epigenetic aberrations in spermatozoa, including oxidative DNA damage, chromatin packing abnormalities, chromosomal alterations, mutations, polymorphisms, epigenetic alterations such as DNA methylation and dysregulation of mRNA expression, and all of that can influence sperm functions and men’s fertility (8, 20, 22, 37, 41–46). Recent research data suggest that genetic and epigenetic changes in spermatozoa that are caused by the genotoxic components of tobacco can be transmitted to offspring (19, 20, 22, 40–43).

Chemical compounds with which smoking might affect the sperm DNA integrity include cigarette smoke components such as cadmium, lead, arsenic, carbon monoxide, nicotine and cotinine (a metabolite of nicotine), which are directly absorbed into the systemic circulation and accumulated in seminal plasma, initiate oxidative stress and dramatically affect seminal parameters, including sperm DNA fragmentation (22, 40–43). Oxidative damage is the most common cause of sperm DNA fragmentation, which can impair sperm functions, leading to a decrease in male fertility (20). Some authors have suggested using the degree of DNA integrity as an additional parameter of semen quality and a prognostic factor of male fertility (42). In 2021, the WHO laboratory guidelines (47) also recommended the analysis of sperm DNA fragmentation as a new functional test for an extended study of sperm quality. In addition to tobacco smoking, other lifestyles are associated with increased sperm DNA fragmentation, including excessive alcohol consumption, obesity, advanced age, urological diseases, type 2 diabetes (42).

The effect of tobacco smoking on semen parameters can be mediated not only by direct toxic effects, but also by changes in hormonal secretion and metabolism (20, 22, 48, 49). Tobacco smoke can induce a dysregulation of the endocrine system, which leads to problems with male fertility. To date, research assessing the correlation between cigarette smoke exposure and reproductive hormones has shown inconsistent results. Some studies revealed significantly higher levels of testosterone and/or free testosterone in smokers compared to non-smokers with a dose-response pattern (13, 32, 38, 49–52). On the contrary, others reported a decrease in testosterone concentrations in tobacco smokers (29, 53) or did not find any differences in the hormonal level between smokers and non-smokers (54, 55). Although many studies have found increased testosterone levels among smokers, the mechanism by which smoking increases testosterone levels remains unclear. The increase in testosterone levels in smokers may be due to a change in the ability of SHBG to bind testosterone (56, 57). Zhao et al. (49) suggested that cotinine (a metabolite of nicotine) can inhibit the testosterone degradation, which leads to the testosterone accumulation in smokers. Moreover, higher total testosterone levels in smokers than in non-smokers were positively associated with number of cigarettes smoked, but with age the positive relationship between smoking and testosterone weakened (58). Interestingly, higher testosterone concentrations in smokers were associated with higher sexual activity, although long-term experience of smoking has a higher risk of erectile dysfunction (20, 59, 60).

Few studies have investigated the relationship between tobacco smoking and pituitary hormones. Differences in FSH and LH levels between non-smokers and smokers were very often insignificant suggesting that there is no effect of tobacco smoking on the hypothalamic-pituitary axis (13, 32, 38, 61). Meanwhile, Blanco-Muñoz with colleagues showed that active smokers had higher LH and testosterone levels, although they were unable to detect any changes in estradiol, inhibin B and FSH levels (62). One recent study found that FSH levels were higher in smokers than in non-smokers, while testosterone levels were lower and LH levels were no different (53). Furthermore, Mitra with colleagues found that serum FSH and LH levels were higher in smokers compared to non-smokers, while testosterone levels decreased significantly with increased smoking (63).

Several studies have documented the damaging effects of tobacco smoking on the antioxidant defense system due to increased oxidative stress (20, 22, 40, 41, 64). Cigarette smoke contains high concentrations of nitric oxide, peroxynitrite, free radicals, and can potentially induce the production of reactive oxygen species (ROS) in the human body. Oxidative stress occurs when the antioxidant system can no longer counteract the ROS formation. Excessive generation of ROS damages the structural and functional parameters of sperm, including polyunsaturated fatty acids, proteins, nucleic acids, which are potential targets of ROS. Antioxidants, including superoxide dismutase, glutathione peroxidase, catalase, play an important defensive role in neutralizing ROS. The trace element zinc is an essential element as more than 300 enzymes require zinc for their function, and plays an important role in functioning the male reproductive system, including sperm physiology and the antioxidant defense mechanism (65–67). Several studies have reported that seminal zinc levels were closely associated with semen parameters in men, so zinc deficiency may be an important risk factor for lowered semen quality (65–69). Zinc has antioxidant properties that counteract the excessive production of ROS, in particular, it is a necessary component of superoxide dismutase, the most active antioxidant enzyme in the semen. Zinc depletion or deficiency (particularly the actual levels of seminal zinc) is proved to be a serious side effect of cigarette smoking (69, 70). Taha with colleagues observed that fertile non-smokers demonstrated higher progressive sperm motility, seminal zinc level, lower levels of sperm DNA fragmentation and seminal ROS compared to fertile smokers (64). Similarly, in infertile couples, it was found that the seminal zinc concentration and semen parameters (sperm concentration, motility and morphology) were lower in smokers than in non-smokers, indicating that seminal zinc may be involved in the mediating effects of tobacco smoking on semen parameters (70). In a large cohort of men from infertile couples, it was observed that sperm progressive motility, abnormal head rates, sperm viability, seminal zinc concentration were significantly reduced, as well as the DNA fragmentation rate was markedly increased in heavy smokers compared with non-smokers (35). When comparing groups of male volunteers, the smoking group compared with non-smokers showed a lower sperm concentration, the proportion of actively motile and morphologically normal sperm. Seminal zinc levels were also reduced in smokers compared to non-smokers, although there was no significant difference in serum zinc levels between smokers and non-smokers (69). The above studies clearly show the negative impact of tobacco smoking on the seminal zinc level, which may contribute to the pathogenesis of the adverse effects of smoking on semen parameters.

Tobacco smoking appears to negatively affect lipid, glucose and protein metabolism. Studies have shown an adverse effect of tobacco smoking on the lipid profile (lower HDL, higher LDL and triglycerides), while advanced age and higher BMI significantly enhance this relationship (71). Nicotine suppresses appetite and can reduce food intake and body weight, so smokers tend to be thinner and have a lower BMI than non-smokers, at that the lipolytic effect of smoking is attributed to the nicotine component through the release of catecholamines (71). Smoking can be associated with impaired in insulin action and glucose regulation, increasing risk for type 2 diabetes (71). Some men smoke because they consider their habit as a method of weight control, however, there is evidence that a part of heavy smokers has a higher BMI and a larger waist circumference than non-smokers, while the combination of smoking with abdominal fat accumulation increases the risk of metabolic syndrome, type 2 diabetes and cardiovascular diseases (72).

Most of the current researches concerning the effect of tobacco smoking on male reproductive function were carried out on specially selected small groups, in most cases with a history of infertility and/or long-term smoking experience, and very few studies have examined the effect of tobacco smoking in population-based cohorts. At the same time, a larger sample size increases its external validity and allows to control numerous potential confounding variables. Moreover, a large-scale population study can clarify previously unresolved issues, in particular, to identify new relationships due to ethnic origin and traditions. Although the exact associations tobacco smoking with the male fertility parameters are still insufficiently studied, our knowledge about its reproductive effects in Russian residents is even more limited, especially in men of active reproductive age. Thus, the purpose of this study was 1) to investigate whether tobacco smoking is associated with the semen quality parameters (total sperm count, concentration, motility, morphology, DNA integrity), the reproductive hormone and zinc levels, metabolic status in Russian men of reproductive age from the general multi-ethnic population and 2) to find out the ethno-specific effects of smoking when comparing different ethnic groups.

Our study is a rather large-scale observational investigation of the effects of tobacco smoking on semen quality, hormonal, zinc and metabolic parameters in men from Russia. According to our data, the prevalence of smoking among Russian men with a median age of 23 years was 36.8% and coincided with WHO data (14) for the world population (36.7%). In the first part of our study, we studied the effect of cigarette smoking in the multi-ethnic study population, whereas in the second part we focused on the reproductive consequences of smoking in selected ethnic groups. The first part of the study included two categories of cigarette smoking (moderate smokers and heavy smokers) to examine the dose-dependent associations between cigarette smoking and the indicators studied. The most important consequences of cigarette smoking were lower semen parameters, including semen volume, total sperm count, sperm concentration and progressive motility, increased sperm DNA fragmentation and teratozoospermia. Our results on men from the general population are consistent with conclusions of many other studies in infertile men (21, 33–36, 81–83), in men from the general population (32, 51, 54, 84, 85) or in case-control studies (29–31, 38, 39). Although the mechanisms by which smoking may be linked to detrimental effects on semen parameters remain to be fully elucidated, they are due to a direct exposure of the toxic content of cigarette smoke (mutagens, carcinogens, nicotine and its metabolites, heavy metals) (12, 20, 22, 81). An additional way may be associated with the direct effect of tobacco components, including nicotine, on the function of accessory glands (seminal vesicles and prostate gland), which alter the semen volume and the functional properties of sperm (12, 19, 20, 81, 86). Moreover, oxygen deficiency produced by cigarette smoking affects spermatogenesis, resulting in impaired sperm production (9, 81).

In our study, we also investigated how smoking affects sperm DNA fragmentation in men. It was shown that DFI% increased by about 75% in heavy smokers compared to non-smokers. These findings are consistent with the results of previous reports (43, 64, 87, 88) and confirm the conclusions of recent reviews (12, 20, 22, 41, 42, 81). Most studies have shown that the negative effect of smoking on sperm DNA can mainly be caused by excessive production of reactive oxygen species (ROS) (20, 22, 39, 43, 81), assuming that the increase in sperm DNA fragmentation in heavy smokers in our study may also be caused by an imbalance between antioxidant and pro-oxidant factors.

In our multi-ethnic study population, tobacco smoking did not cause an imbalance in the LH, FSH, testosterone and estradiol levels involved in the control of spermatogenesis, with the exception of inhibin B. These results are consistent with previously reported (21, 54, 55, 61). The hormonal results of our study are in line with the results of two population-based studies, which did not reveal a significant effect of smoking on the levels of testosterone, estradiol, inhibin B, sex hormone binding globulin, LH and/or FSH in young men (54, 55). However, as a rule, studies show contradictory associations between smoking and reproductive hormones (48, 49), and both decreased (38, 53, 63) and increased (50–52, 56, 58, 62) testosterone, LH and/or FSH levels have been described in smokers as compared to non-smokers.

The results of our study showed that cigarette smoking had an adverse effect on the metabolic health of smokers (an increase in triglycerides and uric acid, as well as a decrease in the HDL-C), which corresponds to the data obtained in other studies (71, 89–91). A meta-analysis conducted by Kauss and co-authors demonstrated that smoking was also associated with a decrease in the levels of apolipoproteins AI and AII and an increase in the level of apolipoprotein B, thereby confirming the negative impact of smoking on lipid metabolism (92). A possible mechanism for how cigarette smoking can alter the lipid profile has been proposed (92, 93). During cigarette smoking, nicotine stimulates the secretion of catecholamines and cortisol, activating adenylcyclase in adipose tissue, which leads to both lipolysis and the release of free fatty acids into the circulation, as well as to an increase in the lipoprotein production, including the HDL concentration. But, on the other hand, the release of catecholamines leads an increase in heart rate, blood pressure and cardiac output and increases the risk of cardiovascular disease. In our study, the combination of an altered lipid profile and increased body weight, waist circumference and BMI, which we found in heavy smokers, may further increase the risk of cardiovascular diseases.

It is considered proven that many of the harmful consequences of cigarette smoking are due to smoking-induced increase in the production of reactive oxygen species, leading to oxidative stress (9, 13, 20, 22, 39, 43, 64, 94, 95). The data obtained in our study correspond to the idea that smoking-induced oxidative stress appears to be the main mediating mechanism of the damaging effects of tobacco on male reproductive function, leading to the reduced semen quality, sperm DNA fragmentation, as well as an imbalance of lipid profile, which ultimately weakens male fertility.

Despite a fairly large amount of data on the impact of cigarette smoking on men’s reproductive health, often authors receive conflicting data, the interpretation of which is ambiguous and does not reach consensus. Probably, the magnitude of the negative effect depends on environmental and climatic conditions, ethnic origin and cultural traditions of the population, and, most likely, the effect will manifest itself with pronounced and prolonged exposure. There are also some uncertainties regarding the methodical questions, in particular, the dose-dependent effects of smoking on male reproductive health, which may partially explain the inconsistency of the semen and hormonal results. For example, some researchers classify smokers as those who smoke from 1 to 15 cigarettes per day, while others take into account smoking experience and include in the group of male smokers only those who regularly smoke during the year (23).

In the second part of our study, we tried to find out how smoking affects reproduction-related parameters in various ethnic groups. Men of three ethnic groups - Buryats, Slavs and Yakuts - were selected from our multi-ethnic study population. Our previous studies have demonstrated significant reproductive differences between these ethnic groups on semen quality, hormonal levels, and genetic factors (73, 74). In the present study, we have shown that the proportion of smokers was the highest in the Buryat group and the lowest among the Slavs, the Yakuts occupied an intermediate position, but the smoking intensity (cigarettes per day) was the highest among the Slavs and moderate among the Buryats and Yakuts, who did not differ in this parameter. The data obtained suggest a greater tobacco addiction in Slavs than in both Asian groups. Moreover, the most pronounced negative effects of cigarette smoking were found in men of Slavic ethnicity, weaker in Yakuts, and no negative effects were found in Buryats. The main harmful impact of smoking on reproductive function was noted among Slavs, including semen quality, sperm DNA fragmentation, seminal zinc content and metabolic profile.

In our previous study, we found that semen parameters were close and positively related to seminal zinc content, while seminal zinc deficiency was associated with a decrease in semen quality (68). In this study, we showed that in the entire study population and in a separate Slavic group, smokers had both a lower seminal zinc content and impaired semen parameters, including increased sperm DNA fragmentation, compared with non-smokers. Previously published studies have also indicated the adverse impact of smoking on the seminal zinc level (64–67, 69, 70). Our current zinc data coupled with previous published data suggest that seminal zinc is involved in the effect of cigarette smoking on semen parameters. Zinc is a cofactor for various proteins and part of the antioxidant defense, playing an important role in the inactivation of excess reactive oxygen species (ROS) in seminal plasma, as reported by recent reviews (96, 97). It seems obvious that cigarette smoking reduces the seminal zinc level due to increased ROS production and leads to depletion of the semen antioxidant defense.

The ethno-specific effects of cigarette smoking identified in this study may be associated with higher cigarette consumption and, consequently, with more harmful effect of nicotine and other tobacco compounds in the Slavic group compared to both Asian groups, in which smoking did not lead to severe damage. Moreover, taking into account the smoking-induced oxidative stress as the main damaging mechanism, it can be assumed that the ethno-specific effects of smoking may be due to a different genetic background and are connected with genetic differences in the activity of antioxidant enzymes. The seminal plasma possesses an antioxidant system capable of counteracting the harmful effects of ROS, but smoking alters the balance between total antioxidant capacity and ROS production, increasing lipid peroxidation and reducing antioxidant activity, including seminal zinc, vitamin C, and enzymes (superoxide dismutase containing zinc, glutathione peroxidase, catalase) and others antioxidants (20, 95–97). In our previous study, we compared the level of lipid peroxidation and antioxidants in the seminal plasma of young Buryats and Slavs (98). The results showed a more active functioning of the antioxidant system in the Buryats compared to the Slavs. In the present study, a decrease in the seminal zinc level of smoking Slavs compared to non-smokers is an additional evidence of a decline in semen antioxidant defense. Our study highlighted the role of the genetic background in the reproductive effects of smoking, presumably genes involved in antioxidant defense. One study showed that the combination of smoking and one functional polymorphism of the antioxidant gene NRF2 (erythroid 2-related factor 2) led to a significant decrease in semen quality, a fact that is in line with our above assumption (99).

Further research is needed to elucidate the ethno-specific molecular pathways by which cigarette smoking can affect spermatogenesis, which could also explain the conflicting data on the detrimental effects of cigarette smoking on semen quality. Returning to the underlying lifestyle causes of adverse temporal trends in the male reproductive potential, which were first reported more than half a century ago, we can assume, based on the facts obtained in this study, a significant contribution of tobacco smoking to this phenomenon, taking into account its ethno specific features.

A strength of the study is the rather large cohort of men with detailed demographic characteristics, ethnic origin, lifestyle factors, including smoking, past and current diseases for each participant. In the study, we evaluated a wide range of 30 anthropometric, seminal, hormonal, zinc and metabolic parameters in order to more comprehensively analyze the reproductive consequences of cigarette smoking. In addition, we included in the study young men from the general population, who were quite similar in social status; used a standardized recruitment protocol and questionnaire; all samples were collected and processed by the same scientific team using the same laboratory methods, equipment and supplies. The large study population allowed us to adjust the data for known confounders when comparing groups. Given the wide diversity of nationalities in Russia, we took this opportunity to clarify the ethno-specific impact of cigarette smoking on reproduction-related indicators, which can deepen existing knowledge about the reproductive consequences of smoking and have obvious advantages when integrating the results into clinical practice and research.

The study has some limitations. Our study is retrospective and observational in nature, which did not allow us to draw exhaustive conclusions about mediating mechanisms. In addition, cigarette smoking status was self-reported, resulting in risk of bias. It was difficult to accurately determine the time frame for starting smoking, since the participants, as a rule, gradually and intermittently became involved in tobacco addiction. Furthermore, this study did not analyze antioxidants and antioxidant enzymes other than zinc to determine the extent of damage of the antioxidant system, especially in heavy smokers. Finally, the number of Buryat and Yakut smokers, as well as their data on sperm DNA fragmentation, have been limited and should be expanded in future studies.

Our study showed the negative impact of cigarette smoking habits on male reproductive potential in adult Russian men from the general multi-ethnic population. In smokers, who smoked more than 10 cigarettes a day, we observed reduced semen volume, total sperm count, sperm concentration, progressive motility, seminal zinc levels, increased sperm DNA fragmentation and teratozoospermia, as well as metabolic imbalance. When investigating the effects of smoking in the selected ethnic groups, the most pronounced negative effects were found in Slavs, weaker effects in Yakuts, and no effects in Buryats. Reduced semen quality and seminal zinc content, increased sperm DNA fragmentation and teratozoospermia, serum levels of triglycerides and fasting glucose were detected in Slavic smokers, while Yakut smokers demonstrated only a slight decrease in testosterone and inhibin B levels as well as an increase in triglycerides. The data obtained indicate the ethno-specific effect of cigarette smoking on the male reproductive potential and suggest that ethnic differences may be due to a genetic background, presumably genes regulating the antioxidant system, taking into account smoking-induced oxidative stress as the main mediating mechanism.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by Research Institute of Clinical and Experimental Lymрhology – Branch of the Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences (RICEL- Branch of IC&G SB RAS). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

LO: Conceptualization, Funding acquisition, Methodology, Supervision, Writing – original draft, Writing – review & editing. MK: Formal Analysis, Investigation, Writing – original draft. AO: Conceptualization, Data curation, Methodology, Resources, Software, Writing – original draft.