Material and Method: This study consisted of 1005 men (smokers= 599, non-smokers= 406) with suspected infertility. It evaluated sperm parame-ters, including, leucocyte count, sperm concentration, total sperm count, motility, and the total progressive motile sperm count in these men.

Results: In our study group, 59.60% were smokers and 40.40% were non-smokers. Body mass index (BMI) was higher in non-smoker males. Sperm characteristics were similar in smokers and non-smokers. We evaluated the smoker's group according to the number of cigarettes smoked per day. There was no significant difference in sperm parameters between the group that smoked up to 30 cigarettes a day and the group that smoked more than 30 cigarettes. Then, we examined the smokers in 3 groups according to the duration of smoking: 0-10 years, 11-20 years, and 20 years and over. It was observed that non-progressive motility was the lowest and immotility was the highest in smokers who had been smoking for 20 years or more.

Conclusion: This study is the most comprehensive study in Turkey examining the relationship between smoking and sperm parameters to the best of our knowledge. Our results show that the duration of smoking affects sperm functions. Our evidence indicates that men with suspected infertility should quit smoking to optimize their successful conception.

The most important diagnostic tool is semen analysis for assessing fertility, which includes parameters such as sperm concentration, sperm count, sperm motion, semen volume, and sperm motility⁵. Semen quality, including sperm density, viability, semen volume, motility, and normal morphology, is adversely affected by toxins from tobacco smoking, leading to male infertility⁶. Aside from its link to male fertility impair-ment, smoking increases aneuploidies, sperm mutations^7, DNA damage, and spermatogenic cell apoptosis⁸. The possible relationships between smoking and male infertility have been identified in different stu-dies, some of which have reported contradictory re-sults. Several studies have shown that smoking has negatively affected parameters of semen analysis and male infertility^9-12, while other studies have not found such effects. In some cases, even positive effects on sperm motility^13, and the extent of nuclear DNA damage in sperm ^14, have been found¹⁴. A study in Turkey found that cigarette and alcohol users had significantly lower follicle stimulating hormone (FSH) levels than smokers and non-smokers, and smokers had significantly decreased sperm motility¹⁵. Furthermo-re, sperm counts dramatically decreased in smokers and alcohol abusers after washing, but especially the sperm rates of smokers were shown to decrease signifi-cantly only when the semen analysis of the smokers was evaluated.

Since the literature on the effects of smoking on human fertility has inconsistencies, we aimed to investigate the effect of smoking on sperm parameters in men with suspected infertility in a Turkish population. We also examined the effect of daily smoking and smoking duration on sperm parameters.

Semen analysis
We collected the sperm samples from those who were sexually abstinent for 2-7 days without using any lubri-cant by masturbating into the sterile disposable plastic cups. WHO criteria were applied to examine the semen samples taken from the participants after liquefaction. Based on the WHO criteria regarding viscosity, in normal semen, distinct small drops fall out of the pipet-te. The drop forms a strand longer than 2 cm in length due to the abnormal viscosity. The WHO definition of leukocytospermia (million/ml) was used in this study to define leukospermia. We first homogenized the semen samples by pipetting with a Pasteur pipette. We pipet-ted about 10 μl of semen and placed it on the Makler Camera (Makler caunting chamber; Sefı Medical Inst-ruments) and sealed it with a glass lid to determine motility and the count. We counted spermatozoa in 10 squares through the light microscope's x20 lens (Olym-pus CX31), and expressed the result in millions. We evaluated the sperm parameters such as viscosity, eja-culate volume, sperm concentration, leucocyte count, motility, immotility, total sperm count, and total prog-ressive motile sperm count (TPMSC).

Statistical analysis
The Statistical Package for the Social Sciences (SPSS) 21.0 was used for statistical analysis. The Shapiro-Wilk test was used to determine whether numerical data conformed to a normal distribution. Numerical data were expressed as median (min-max), and qualitative data were expressed as frequency and percentage va-lues. In the analysis of numerical data, the Mann-Whitney U test was used to compare the two groups. Kruskal-Wallis post hoc Dunn-Bonferroni tests were used to compare multiple groups. The chi-square test was used in the analysis of categorical data. The signi-ficance level was taken as p <0.05.

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Table 1: Comparison of demographic characteristics of smokers and non-smokers.

We evaluated sperm parameters, including ejaculate volume, leucocyte count, sperm concentration, total sperm count, motility, immotility, and TPMSC, between the smoker group and non-smoker group. Both groups had similar ejaculate volume, leucocyte count viscosity, sperm concentration, total sperm co-unt, motility, immotility, and TPMSC (p ˃0.05). Sperm parameters in smokers and non-smokers are shown in table 2.

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Table 2: Comparison of sperm parameters in smokers and non-smokers.

Then we evaluated the smoker group in two groups based on the number of cigarettes smoked per day up to 30 and from 30 upwards. The results are presented in table 3.

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Table 3: Comparison of smokers' sperm parameters according to the number of cigarettes smoked per day.

There was no significant difference in sperm parameters, including ejaculate volume, leucocyte count, sperm concentration, total sperm count, motility, immotility and TPMSC, between the group that smoked up to 30 cigarettes per day and those that smoked more than 30 cigarettes (p ˃0.05).

Then, we examined the smokers in 3 groups according to their smoking duration: 0-10 years, 11-20 years, and 20 years and older. Non-progressive motility was the lowest in smokers for 20 years or more (p= 0.003). Sperm immotility was highest in those who smoked for 20 years or more (p= 0.026). Sperm parameters 0-10 years, 11-20 years, and 20 years above are shown in table 4.

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Table 4: Comparison of sperm parameters of smokers based on smoking duration.

It is interesting that there was an improvement in the parameters that were impacted by oral nicotine after 30 days of cessation, showing a component of reversibility to these impacts. In addition, a study on 210 men showed that men with higher concentrations of cotinine in the seminal plasma also had a higher percentage of abnormal sperm morphology¹⁸. Since cotinine is a nicotine metabolite, these findings support the hypot-hesis that nicotine itself may possibly lead to the adver-se effects of smoking on fertility. In a society where males dominate, females usually carry the burden of infertility, though in most cases, the male counterpart also contributes to infertility. Soares et al.^19, who reviewed the literature on the association between smoking and reproductive function, focused on strong evidence indicating that cigarette smoking negatively affected female and male fertility. Ramlau-Hansen et al.²⁰ conducted a cross-sectional analysis of 2542 healthy men during 1987–2004 and found that the semen analysis showed that cigarette smokers had lower sperm counts, semen volumes, and motile sperm percentages than the men who did not smoke. It was also suggested that the association between smoking and sperm concentration was dosedependent. In a case-control study, there was an association between smoking and impaired motility of spermatozoa, reduced semen concentration, and a higher morphology defect²¹. In addition, an article published by the Canadian Society of Clinical Chemists showed that abnormal structural defects of spermatozoa, especially round head defects, were associated with tobacco smo-king due to a lack of sufficient scavenging antioxidant enzymes and higher oxidative stress in the infertile men's seminal fluids ²². Another study that evaluated the impact of smoking on vital seminal parameters influencing fertility showed that smoking impaired motility more than impaired sperm count²³. A meta-analysis assessing human semen found that tobacco smoking negatively affected semen parameters²⁴. The analysis of 5865 infertile and fertile men found impaired motility and a lower sperm count in the semen samples of these young men. In summary, the possible mechanisms involved with the impact of smo-king on semen parameters are that the toxic contents of cigarettes harmfully affect the male germ cells and their developmental processes²⁵.

In a meta-analysis evaluating 16 studies with a total of 10823 infertile men (5 257 smokers and 5 566 non-smokers) as participants, it was found that oligozoos-permia and morphological defects of spermatozoa were more common in smokers compared with non-smokers²⁶. In a retrospective study of 296 infertile men in our country, Ozgur et al.²⁷ reported that morpholo-gic evaluation revealed better results for the nonsmo-kers than the heavy smokers in terms of tail anomalies and percent of coiled tails.

In the current study, we examined the effects of smoking on sperm parameters in 1005 men with suspected infertility. As far as we know, this study is the most extensive one conducted in the Diyarbakir region. BMI was higher in the non-smoker group than in the smoker group (Table 1). Comparing sperm parameters between smokers and non-smokers, there was no significant difference between groups (Table 2). When we examined the effects of daily smoking on sperm parameters, we found no significant difference between the group that smoked up to 30 cigarettes per day and the group that smoked more than 30 cigarettes (Table 3). We also analyzed the smoker group by separating it according to the duration of smoking. We observed that non-progressive motility was the lowest and immotility was the highest in smokers who had been smoking for 20 years or more (Table 4).

Study Limitations
Our study is not devoid of limitations. In this study, the results of individuals with a suspicion of infertility who applied to the hospital are shown. Consequently, this study's participants have male infertility problems. These results may not show the association between the individuals' smoking status in the entire population and the sperm parameters. Furthermore, one of the limitations of this study is that it may not evaluate parameters thought to affect the participants' smoking habits, such as anxiety and depression. There should be a multidisciplinary approach to identify and inform those exposed to infertility, which has an important place in society.

Ethics Committee Approval
This study was approved by the Ethics Committee of the University of Health Sciences Gazi Yaşargil Trai-ning and Research Hospital Clinical Research (date and number of approval: 2021/868).

Conflict of interests
The authors declared no conflicts of interest with re-spect to the authorship and/or publication of this arti-cle.

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