Effect of Smoking on Seminal Plasma Vitamin B6 in Fertile and Subfertile Men

Despite extensive documentation of the negative consequences of smoking on a wide range of diseases and disorders, the direct effect of smoking on seminal plasma vitamin B6 (vitB6) concentration is not addressed yet. Here, we attempted to examine the influence of smoking on the levels of seminal plasma vitB6 in fertile and subfertile males. One hundred and ninety-five participants were categorized into two groups: fertile men (smokers (n = 32), nonsmokers (n = 43)) and subfertile men (smokers (n = 65), nonsmokers (n = 55)). According to the World Health Organization criteria, conventional semen analysis was performed for all subjects involved in the study. The concentration of vitB6 in semen samples was measured by high-performance liquid chromatography tandem-mass spectrometry. There was no significant difference in the mean seminal plasma concentrations of vitB6 in smokers versus nonsmokers in both fertile (p=0.5096) and subfertile (p=0.5224) groups. Besides, no significant correlations were observed between seminal plasma concentration of vitB6, duration of smoking, and men's age in all tested groups. In conclusion, these findings elucidate that smoking has no significant impact on the levels of seminal plasma vitB6 in both fertile and subfertile men.

Yet, there have been only limited studies on the infuence of vitB 6 in boosting human semen parameters and hence the fertilizing capacity [12].So far, it has been documented that vitB 6 is existing in human semen, but investigations in this research approach failed to demonstrate its direct efect on semen quality and ability of fertilization, though there are intriguing inferences that depressed levels of vitB 6 in semen are linked to poor semen quality parameters [12].Our study in 2020, albeit partially, has confrmed these indirect conclusions which demonstrated that asthenozoospermic semen samples signifcantly contain less content of vitB 6 when compared to normozoospermic samples [13].Te fndings from this research provide additional support to consider that normal amounts of vitB 6 in semen are highly recommended to maintain normal semen quality characteristics.Consequently, potential factors that might afect the levels of vitB 6 in semen should be assessed and controlled.One of such important factors is smoking since it is known to be highly common in men of reproductive age [14].
According to the World Health Organization, tobacco smoking is considered as epidemic [15].Young adult men during their reproductive period were found to have the highest prevalence of smoking worldwide [14].Numerous studies have reported that smoking exerts various detrimental efects on humans' health [16,17].For example, smoking was found to increase the risk of cardiovascular diseases, lung diseases, cancers, and diabetes [18][19][20].Among other consequences, there is evidence of increased infammation, oxidative stress, and decreased levels of antioxidant vitamins [21,22].Ascorbic acid and β-carotene are antioxidant micronutrients known to be greatly afected by smoking status [23][24][25][26][27][28].However, the evidence for a link between tobacco smoking and circulating concentrations of vitB 6 is equivocal.On the one hand, some studies revealed that there is no statistically signifcant diference regarding plasma vitB 6 levels in smokers versus nonsmokers [29,30].On the other hand, other studies revealed that serum vitB 6 levels were considerably lower in smokers when compared to nonsmokers [31,32].
Consequently, even though there are several published articles that are documenting the impact of smoking on the levels of vitB 6 in blood, its efect on the levels of vitB 6 in semen ejaculates is not addressed yet.Terefore, we asked whether smoking has a direct infuence on the levels of vitB 6 in the human ejaculated semen.Here, we hypothesized that the levels of seminal plasma vitB 6 are negatively afected by tobacco smoking.To accurately test this hypothesis, we have used the high-performance liquid chromatography tandemmass spectrometry (HPLC-MS/MS) as a highly robust and reliable analytical method.

Study Population and Specimen Collection.
One hundred ninety-fve male participants, aged 19 to 47 years, were randomly recruited from the In Vitro Fertilization (IVF) Center at King Abdullah University Hospital (KAUH) and Al-Qudah Private Laboratories in Irbid, Jordan.Study subjects were categorized into two groups: fertile men (smokers (n � 32), nonsmokers (n � 43)) and subfertile men (smokers (n � 65), nonsmokers (n � 55)).Semen ejaculates were collected via masturbation after 3-7 days of sexual abstinence.
Prior to sample collection, all study subjects flled out a general questionnaire in both locations.Smoking status, duration of smoking, age, diseases, family history, and the use of medication or vitamin supplements were data extracted from the questionnaire.Te cases with varicoceles, chronic diseases, family history with infertility, and cases who are on vitamin supplementation or on medication were excluded from the study.

Ethical
Approval.Tis research study was approved by the Research Ethics Committee (#: 193-2018) at Jordan University of Science and Technology (JUST).Te study's aims and analysis were clearly explained to the recruited men by qualifed researchers at Al-Qudah Private Laboratory and the IVF Center in the JUST University Hospital, Irbid province.Prior to participation, each recruited male, at both locations, voluntarily signed an informed consent form.

Semen Analysis and Storage.
After liquefaction, semen parameters were immediately analyzed according to the World Health Organization guidelines 2010 (normal criteria for fertile men: semen volume ≥1.5 mL, progressive motility of sperm ≥32%, sperm count ≥15 million/mL, normal sperm morphology ≥4.0%) [33].Males were considered subfertile when sperm count is less than 15 million/mL and/or sperm progressive motility is less than 32% [33].For better accuracy, at least two separate analyses were performed on semen specimens.Afterwards, samples were centrifuged for 10 minutes at 2500 x g and the seminal plasma (supernatant) was isolated and stored without any additives at −20 °C for HPLC-MS/MS analysis.

Measurement of Sperm Motility and Concentration.
Sperm motility and concentration were evaluated using a phase contrast microscope and Makler counting chamber (Irvine Sci., USA).After gentle mixing, each semen sample (∼10 µL) was assessed for sperm motility and concentration in a period of 10-15 minutes after sample collection.Total and progressive motility were calculated in percentages, whereas sperm concentrations were measured in 10 6 /mL of semen.Despite the speed of spermatozoa, circularly or linearly active motile sperm were considered progressive.To achieve accurate results, a total of 200 spermatozoa were estimated in each replicate to determine the percentage of both total and progressive motility.Furthermore, to minimize the assessment's bias, all scanned sperm felds were chosen at random.Positive error in the results was avoided by counting the sperm cells quickly [34].Te sperm motility and concentration measurements were performed by highly skilled andrologists who achieve within-individual variation of less than ±5%.

Seminal Plasma Sample Preparation.
Seminal plasma specimens were thawed at room temperature and mixed on a vortex mixer.Protein precipitation was achieved by mixing 250 μL of seminal plasma with 3 mL of chloroform and 250 μL of ethanol.After that, the mixture was completely vortexed for 30 s and then centrifuged at 2,500 g for 10 min.Supernatants were transferred into a clean microfuge tube and then vaporized completely in a N 2 evaporator.Next, 750 μL of methanol was added to the pellet and the fnal suspension was subsequently placed into an autosampler vial for HPLC-MS/MS.

Measuring VitB 6 Levels by HPLC-MS/MS.
Te concentrations of vitB 6 in seminal plasma were determined using the 30AD SHIMADZU Binary HPLC system coupled with an 8,030 ESI-MS/MS system (SHIMADZU Corp.) [35].In summary, the separation analysis was mainly carried out by injecting 10 μL sample volume into the revered phase HPLC column (Zorbax Eclipse Plus C18, 1.8 μm particle size, 30 × 2.1 mm) (SHIMADZU Corp.).Te mobile phase was made up of 2% methanol, 4.8 g/L ammonium carbonate, and 98% water.Te analytes of interest were separated by an isocratic elution at a fow rate of 0.4 mL/min.
Te quantitative determination was achieved on the tandem mass spectrometer with the following conditions: gas fow � 10 L/min, sheet gas fow � 11 L/min, gas temperature � 200 °C, sheet gas heater � 350 °C, charging voltage � 50 V, capillary voltage � 3500 V, and nebulizer pressure � 40 psi.All data were obtained using positive mode electrospray ionization (ESI) and all measurements were achieved in duplicate with sensitivity of 0.01 μg L −1 .
LC-MS/MS is a highly accurate and precise method for quantifying vitB 6 , particularly pyridoxal-5′-phosphate (PLP), in biological samples.Te method demonstrates excellent intra-day and inter-day precision, with coefcients of variation typically below 10.4% [35].Te accuracy is also high, with recovery rate of approximately 100% [35].Additionally, the method ofers a very low limit of detection, around 25.9 nmol/L, making it suitable for detecting trace amounts of PLP [35].Te total imprecision for PLP ranges from 30 to 130 nmol/L [35].

Statistical Analysis.
Data were represented as mean-± standard error of the mean.Te statistical appraisals and analysis were carried out using GraphPad Prism 5.01 Computer Software (GraphPad Software Inc.).Student's t test was performed to examine the diferences in the mean seminal plasma vitB 6 levels in smokers versus nonsmokers in both fertile and subfertile groups.Pearson's correlation coefcient was used to compute the correlations between seminal plasma vitB 6 concentration, duration of smoking, and men's age in the tested groups.Results were considered statistically signifcant at "p" value <0.05.

Results
In this work, semen samples were obtained randomly from diferent male subjects (n � 195).Table 1 shows the preliminary results of conventional semen parameters and seminal plasma vitB 6 levels for the recruited samples for fertile (n � 75) and subfertile (n � 120) men.Results of semen volumes, sperm counts, sperm motilities (total and progressive), and seminal plasma vitB 6 concentrations were expressed as mean-± standard error of the mean.Males were considered subfertile when sperm count is less than 15 million/mL and/or sperm progressive motility is less than 32% [33].
Figure 1 exhibits the diference in mean seminal plasma vitB 6 levels-as evaluated by HPLC-MS/MS-in smokers (n � 32) versus nonsmokers (n � 43) in the fertile group.As displayed in the fgure, there is no statistically signifcant diference (p � 0.5096) in the concentrations of seminal plasma vitB 6 in smokers when compared to nonsmokers in this group.
Te comparison in mean seminal plasma vitB 6 concentrations in smokers (n � 65) versus nonsmokers (n � 55) in the subfertile group is illustrated in Figure 2. Statistically, there is no signifcant diference in the mean seminal plasma vitB 6 levels (p � 0.5224) in smokers versus nonsmokers in this group.
Te correlations between vitB 6 levels in seminal plasma and duration of smoking in both fertile (Figure 3(a), n � 75) and subfertile (Figure 3(b), n � 120) groups are demonstrated in Figure 3.As elucidated in the fgure, the correlations between seminal plasma vitB 6 levels and duration of smoking in fertile men (p � 0.7321, r 2 � 0.01093) and subfertile men (p � 0.1539, r 2 � 0.01715) were not statistically signifcant.

Discussion
In 2017, our systematic review has strongly suggested that vitB 6 defciency might have a negative impact on sperm parameters [12].Tis suggestion was drawn based on intriguing fndings from diferent previous research investigations.In fact, several studies have established that vitB 6 concentration is positively correlated with the levels of sex hormones, mainly testosterone [36][37][38] (Figure 5).Tis fnding endorses that vitB 6 is essential in maintaining normal gonadal function.Additionally, it is postulated that vitB 6 might protect sperm cells from possible oxidative injury since it acts as a potent antioxidant [39,40] and maintains the normal levels of reduced glutathione in blood [41,42].Moreover, due to the important role of vitB 6 in converting homocysteine, a toxic byproduct, into cysteine amino acid, it is speculated that vitB 6 supplementation might reduce the possible chemical toxicity to sperm induced by hyperhomocysteinemia [43,44].In 2020, our recent study demonstrated that asthenozoospermic semen samples signifcantly contain less content of vitB 6 than normozoospermic samples [13].Te fndings from this research provide additional support to consider that normal amounts of vitB 6 in semen are vital and highly recommended to maintain normal sperm characteristics [13].Subsequently, investigating potential factors, including smoking and men's age, that might afect the level of vitB 6 in semen should be deliberately assessed and controlled.
Here, in this work, we attempted to identify the direct efect of smoking on seminal plasma vitB 6 concentrations in men.We hypothesized that smoking has a negative impact on the seminal plasma vitB 6 levels in fertile and subfertile men.However, the fndings were not in agreement with our central hypothesis.Our data analysis has revealed that there is no diference in seminal plasma vitB 6 levels between smokers and nonsmokers in either fertile or subfertile men.Besides, it has been shown that the correlations between seminal plasma vitB 6 concentrations and duration of smoking in both studied groups are not statistically signifcant.

Journal of Nutrition and Metabolism
In 1986, Ritchie and Singkamani examined the blood plasma vitB 6 concentrations in nonsmoking and smoking females with premenstrual syndrome (PMS) and compared them with the blood plasma vitB 6 values in nonsmoking and smoking females who did not have PMS.In either group, there was no diference in plasma vitB 6 levels between nonsmokers and smokers [29].Also, a study published by Giraud et al. revealed that there were no changes in the plasma vitB 6 values of young males who smoked, chewed, or never used tobacco [30].A later research has provided, albeit partially, similar fndings when vitamin B 6 , B 9 , and B 12 were measured in 285 pregnant women at 18 and 30 weeks gestation [45].Specifcally, it was found that pregnant smokers have lower vitB 6 values in plasma when compared to pregnant nonsmokers at 18 weeks gestation, whereas no such disparity was detected at 30 weeks gestation.Moreover, no signifcant association was seen between levels of vitB 6 in plasma and thiocyanate, a biomarker of smoking, in pregnant smoker group at both 18 and 30 weeks gestation [45].Tese fndings are coherent with our fndings.
On the contrary, our fndings are not in accordance with some results in other studies.Vermaak et al. reported that serum PLP levels were lower in smokers when compared to ex-smokers and nonsmokers, although PLP levels in erythrocytes did not signifcantly difer between the tested groups.Te authors felt that the depressed levels of PLP in serum are uncertain and measurement of PLP in erythrocytes is more accurate since the PLP is an intracellular coenzyme and more liable in blood plasma [31].Another study reported similar results in which tobacco users and nonusers had diferent levels of certain B 6 vitamers (PLP and pyridoxine 5′-phosphate) in their plasma but not in their erythrocytes [46].In 1986, a research study compared the levels of plasma PLP in tobacco smoking men to those of nonsmoking controls.Te authors observed that tobacco smoking men were found to have lower concentrations of plasma PLP when compared to nonsmoking men [32].Te discrepancy between our study and these studies might be ascribed to variations in the sample type and nature of the tested subjects, as well as detection methods.Here, in the present study, we examined specifcally smoking/nonsmoking fertile and subfertile men to evaluate levels of vitB 6 by HPLC-MS/MS in semen specimens while these previous studies examined the levels of blood plasma vitB 6 in completely diferent populations and employed diferent methodologies.
Smoking accelerates the production of reactive oxygen species, leading to cellular oxidative stress.Tis cellular imbalance can deplete antioxidant reserves, including vitB 6 , and cause damage to cell components.However, the human body has adaptive mechanisms to maintain essential nutrient levels.VitB 6 , in its active form, PLP, is vital for various physiological functions, including neurotransmitter synthesis, amino acid metabolism, and immune function [47].Te body's ability to sustain PLP levels under oxidative stress may be attributed to increased dietary intake, enhanced absorption, or more efcient recycling and utilization of the  4 Journal of Nutrition and Metabolism vitamin [48].Moreover, smokers often exhibit altered antioxidant defense systems, which may include compensatory mechanisms to maintain vitB 6 levels.For example, the liver, which plays a crucial role in metabolizing and storing vitamins, might compensate by more tightly regulating vitB 6 metabolism in smokers [49].Tis could explain the lack of signifcant diferences observed between the two groups in the study.Moreover, in this study, although we recognized that seminal plasma vitB 6 concentrations and men's age were positively associated in fertile smokers and subfertile nonsmokers, these associations were not statistically signifcant.Also, this correlation was seen to be not statistically signifcant in fertile nonsmokers and subfertile smokers.In 1976, Rose et al. discovered an inverse relationship between blood plasma vitB 6 levels and male age [50].In fact, our correlation focused primarily on fertile and subfertile men who smoked or never smoked, aged from 19 to 47 years, whereas Rose et al. included community-dwelling men, aged from 18 to 90 years.Also, here, we evaluated specifcally vitB 6 in seminal plasma using HPLC-MS/MS, whereas Rose et al. evaluated vitB 6 in blood plasma using simple enzymic assay.Such age, population, and methodological diferences might explain the observed contradiction between the two studies.According to such specifc investigation, levels of vitB 6 in semen seem not to be altered by factors like men's age or smoking status, which may suggest that depressed levels of vitB 6 in semen can be mainly attributed to nutritional factors.However, this suggestion requires more research studies to be confrmed.Diet and lifestyle factors markedly infuence vitB 6 status, with recent research highlighting their complex roles.A diet rich in vitB 6 , found in foods like fsh, poultry, potatoes, and bananas, is vital for maintaining optimal concentrations of this vitamin [51].Conversely, lifestyle factors such as alcohol consumption and chronic stress, in addition to smoking, can negatively afect vitB 6 metabolism [52].For example, alcohol impairs the conversion of pyridoxine to PLP, leading to reduced levels even in subjects with sufcient dietary intake [52].Furthermore, chronic stress has been associated with an increased demand for B vitamins, including vitB 6 , due to their roles in neurotransmitter synthesis and adrenal function [52].Tis evidence underscores the importance of a balanced diet and healthy lifestyle choices in supporting adequate levels of vitB 6 , particularly for those with high-risk behaviors.
In this study, cases involving varicoceles, chronic diseases, a family history of infertility, and individuals on B vitamin supplementation or medication were excluded from all groups.However, controlling the diet of the recruited participants remains a limitation due to the random selection process and the inherent variability of dietary factors.Additionally, other limitations arose from confounding variables such as socioeconomic and environmental factors (e.g., pollution and occupational exposure), which, if controlled, could further strengthen the study's fndings.Terefore, an interventional study in this research setting is deemed essential to validate and confrm the results of this study.
In conclusion, there is no change in the level of seminal plasma vitB 6 between smokers and nonsmokers in both fertile and subfertile groups.Also, the seminal plasma vitB 6 concentrations are not correlated with either men's age or smoking duration in the studied groups.Collectively, these results manifest that smoking has no signifcant infuence on seminal plasma vitB 6 in fertile or subfertile men.

Figure 1 :Figure 2 :
Figure 1: Concentrations of seminal plasma vitB6 in fertile smokers (n � 32) compared to fertile nonsmokers (n � 43).Data are represented as mean ± standard error of the mean.

Figure 5 :
Figure 5: Possible mechanistic routes for the impact of vitB 6 on semen quality in men.

Table 1 :
Te conventional semen parameters and seminal plasma vitB6 concentrations for recruited semen samples for both fertile and subfertile men.