Men with oligozoospermia had lower level of seminal plasma pyridoxine compared to normozoospermic men

Abstract

The contribution of pyridoxine on various human disorders has been revealed in so many studies; however, this contribution on poor semen quality has yet to be investigated. Here, we intended to measure the level of seminal plasma pyridoxine (SPP) in men with oligozoospermia compared to normozoospermic men. Thirty-three men with oligozoospermia and forty-three normozoospermic men were randomly enrolled in this study. Collected semen were tested for sperm motility, sperm concentration, semen volume, and pyridoxine status. Liquid chromatography with tandem mass spectrometry was used to measure SPP in the collected samples. There was a significant reduction (p < 0.0001) in the concentrations of SPP in men with oligozoospermia (0.79 ± 0.41 μg L⁻¹) compared to normozoospermic men (3.17 ± 0.96 μg L⁻¹). Besides, SPP was not significantly correlated (p > 0.05) with sperm motility, sperm concentration, and semen volume in both tested groups, but, independently, it was found to be positively correlated (p = 0.0154) with male age in oligozoospermic group. Men with oligozoospermia had lower level of SPP compared to normozoospermic men. These results may open a new research gate for the use of pyridoxine in the management of male infertility.

Pyridoxine; Sperm; Seminal plasma; Oligozoospermia; Normozoospermia.

1. Introduction

Pyridoxine, also recognized as vitamin B₆, is a member in the B vitamin family, which are water-soluble nutrients that play crucial functions in cell metabolism [1]. In humans, pyridoxal 5′-phosphate is distinguished as the biologically functional form of pyridoxine [2], which acts as an activator (i.e., coenzyme) for more than hundred metabolic enzymes, particularly in amino acids, carbohydrates, and lipids metabolism [3, 4]. In addition, pyridoxine was found to have a crucial function in gene expression and hemoglobin biosynthesis [5, 6]. Moreover, pyridoxine mediates the production of hydrogen sulfide (H₂S), a signaling molecule involved in metabolism, in the human body [7]. Furthermore, pyridoxine intake has been found to enhance some immune functions in pyridoxine-deficient humans as well as experimental animals [8].

Therefore, there was so many studies that have revealed the impact of pyridoxine on humans' health; for example, supplementation of pyridoxine has been found to be associated with decreased risk of diabetes [9], cardiovascular disease [10], Alzheimer's disease [11], and stroke [12]. In addition, a significant correlation was found between pyridoxine and risk of cancer, particularly gastrointestinal cancer [13]. Moreover, a systematic review conducted by Romoli et al. suggested that pyridoxine might be beneficial in alleviating levetiracetam-related neuropsychiatric adverse events among people with epilepsy [14].

Here, in particular, the effect of pyridoxine on human male infertility has not yet well-established [15]. Up till now, pyridoxine has been found to be existing in humans’ seminal fluid [16], nevertheless, the present studies in this research setting were failed to determine its impact on human semen quality measures, and thus on male infertility; however, there is indirect conclusion that decreased levels of pyridoxine may negatively influence semen quality parameters [15].

In the last two decades, there is growing evidence, direct or indirect, that poor semen quality, and hence infertility in males, may be due to nutritional factors, and dietary supplements such as vitamins may boost semen quality parameters [17, 18]. For example, recent findings, in 2019, showed that swim up medium supplemented with vitamin C had beneficial effects on sperm motion [19]. Also, a study by Hamedani et al. on rams’ semen showed that vitamin B₁₂ enhances sperm characteristics such as motility and vitality in pre- and post-cryopreservation conditions. In addition, dietary habits and drinks are considered important factors that may affect semen quality in males. For example, adoption of high-fat diets, particularly during early life, was found to be associated with irreversible alternations in lipid metabolism and content in the testes [20], which may, in later life, lead to permanent damage to the quality of sperm [21]. In addition, consumption of white tea was found to prevent prediabetes-induced metabolic dysfunction, and hence preserve sperm quality, in the testes [22].

In particular, in 2017, it was concluded that in vitro research studies as well as clinical trials that reveal the influence of pyridoxine on human semen quality are highly required [15]. Accordingly, in the first set of this demanded research context, as a long-term goal to explore basically and clinically the impact of pyridoxine on semen quality, we have shown that men with asthenozoospermia had lower SPP level compared to those with normal motility of sperm [23]. Further, in this work, as an added important contribution, we asked whether seminal plasma pyridoxine (SPP) level is different in males with oligozoospermia compared to normozoospemic males, also, using the analytically sensitive ultra-high-performance liquid-chromatography tandem-mass spectrometry (ultra-LC-MS/MS).

2. Materials and methods

2.1. Subjects and semen sample collection

Thirty-three semen samples from different men with oligozoospermia (33.0 ± 7.13 years), specifically with oligoasthenozoospermia, and forty-three samples from aged-matched (19–48 years) normozoospermic men (31.0 ± 7.12 years) were randomly collected from those who directed to the Al-Qudah Private Laboratories and the andrology laboratory at the In-vitro Fertilization Center at Jordan University of Science Technology Hospital in the North of Jordan. Semen specimens were collected during the period from March 2018 to February 2019. All samples were collected by masturbation in well-suited collecting rooms in the Al-Qudah Private Laboratories in the north of Jordan and the andrology laboratories at the University Hospital-JUST after 2–7 days of sexual abstinence. Prior to sample collection, each male who enrolled in this study filled out a pre-questionnaire. The following information were obtained from this questionnaire: family history, age, supplementation of B vitamins, the use of medications, and presence of chronic diseases. Based on this questionnaire, each eligible participant in this study should fulfil the following criteria: Taking no pyridoxine supplements, taking no medication, no family history with infertility, no chronic diseases such as cancer, diabetes, or cardiovascular diseases, and no symptoms or history of reproductive disorders such as varicocele.

2.2. Ethical approval/considerations

Procedures, protocols, and analysis of this study were approved by the ethical committee (Institutional Review Board Committee, IRB) of Jordan University of Science and Technology (ID: 2018/156). In addition, the experimental procedures and analysis were honestly explained the enrolled participants by qualified and rologists in both assigned sample-collection locations. Also, written informed consents were taken from each recruited male before sample collection.

2.3. Preliminary semen analysis and semen storage

The volume of each semen sample was determined using a sterile graduated tube following a liquefaction time of approximately 20–30 min. Then, the sample was analyzed for sperm concentration and motility (progressive and total). Then, the sample was centrifuged for 10 min at approximately 2500× g and the seminal plasma was gently separated and stored in sterile (non-toxic to sperm) freezing tubes at −20 °C to be analyzed for pyridoxine using ultra-LC-MS/MS.

2.4. Assessment of sperm motility and concentration

In this study, Makler counting chamber (Irvine Sci., Santa Ana, California, US) at a phase contrast optics of 200× magnification was used to assess sperm motility and concentration. In these assessments, approximately 5 μL from each sample were utilized. Each sample analyzed for sperm concentration and motility within 10–15 min to avoid evaporation. Sperm concentration was calculated in millions per mL of semen, while sperm motility was measured in terms of percentages.

Regardless of speed, sperm actively moving, linearly or in large circles, were considered progressive, and the rest of other patterns of sperm motion with an absence of active progression, such as the flagellar force hardly displacing the spermatozoa head and swimming in small circles, were considered non-progressive. For better accuracy, approximately 200 sperm were evaluated per each replicate. Tested fields were randomly selected to reduce the bias in the measurements. Also, the counting of sperm was performed quickly to omit any possible positive analytical error in the results [24].

2.5. Measuring concentration of SPP

2.5.1. Semen sample preparation

To increase the thinning effect of plasma, soluble proteins were precipitated by combining 0.25 mL of pure ethanol and 3.0 mL of pure chloroform with 0.25 mL of seminal plasma. Then, samples were thoroughly vortexed, for 30 min, and centrifuged, for 10 min, at 2500× g. After that, sample supernatants were separated from protein layers and transported into sterile tubes. Then, protein free-supernatants were placed in N₂ evaporator to completely evaporate the solvent. Later, sample pellets were resuspended in 0.75 mL of pure methanol and gently transferred to a high-performance liquid chromatography (HPLC) ampoule and placed in the auto-sampler.

2.5.2. Determination of pyridoxine concentration in seminal plasma

The concentration of SPP was measured using ultra-LC-MS/MS method [25]. Briefly, in this experimental setting, the used HPLC set was a 30-AD SHIMADZU Binary-LC of a 20.0 × 0.01 μmol/L filter (SHIMADZU Co., Kyoto, Japan). Also, the HPLC commercial column, which is 30.0 × 2.1-millimeter Zorbax Eclipse Plus C-18 (particle size = 1.8-micrometer), was equipped with a 5 × 2.1-millimeter Zorbax Eclipse plus C18 (particle size = 1.8 μmol/L) (SHIMADZU Co., Tokyo, Japan). The analysis was steered by injecting 10.0 μL of sample in the column. The HPLC mobile-phase was consisted of water of approximately 98.0% purity, 4.8 g L⁻¹ (NH₄)₂CO₃ of 50 mM, and 2.0% methanol of 9.5 pH. Targeted compounds were eluted iso-cratically at 0.4 mL min⁻¹ flow rate and 150 s analytical run.

The mass spectrometry (MS) analytical detection was performed on an 8030 m/s setting (SHIMADZU Co., Tokyo, Japan). The MS settings were adjusted as follows: g Temp. = 200.0 °C, nebulizer pressure = 40 psi, gas flow = 10 L/min, sheat gas heater = 350 °C, sheat gas flow = 11.0 L/min, charging voltage (+ ion mode) = 500-volt, capillary voltage = 3500-volt. To prevent contamination of the ion source with polar contaminants, the MS system was allowed to waste in the first 45.0 s.

2.6. Statistical analyses

The statistical analyses of the collected data were conducted using the computer software GraphPad Prism (5.01) (San Diego, CA). Student's t-test was used to measure the statistical differences in concentrations of SPP between males with oligozoospermia and normozoospermic men. Data represented in these differences were presented as means ± standard deviations. Associations between SPP and sperm concentrations, sperm motilities, and semen volume were figured by Pearson correlation-coefficient.

3. Results

Semianl plasma pyridoxine was measured in ejaculated semen samples with abnormal (low) count of sperm compared with samples of normal sperm count. Then, progressive and total motility, sperm concentration, and semen volume of each tested group was correlated with the measured SPP. Table 1 shows the concentrations of SPP in men with oligozoospemia compared to normozoospermic men (control) as evaluated by ultra-LC-MS/MS. As shown in the figure, there is a significant decrease (p < 0.0001) in the concentrations of SPP in men with oligozoospermia (0.79 ± 0.41 μg L⁻¹) compared to normozoospermic men (3.17 ± 0.96 μg L⁻¹). The percent reduction in the mean of SPP between the tested groups was approximatly 75%.

Table 1.

The concentration of pyridoxine and measured sperm parameters in normozospermic men versus men with oligozoospermia. Data are presented as means ± standard deviations (range).

Parameter	Normozoospermia	Oligozoospermia	P-value
Male age (Years)	30.7 ± 7.12 (19–48)	33.0 ± 7.1 (19-48)	0.167
Semen volume (mL)	3.89 ± 1.65 (1.3–7)	3.29 ± 1.51 (1.3–7)	0.108
Sperm concentration (Million/mL)	56.31 ± 20.2 (23–105)	9.74 ± 4.76 (1–13)	<0.0001
Pyridoxine level (μg/L)	3.17 ± 0.96 (1.23–5.3)	0.79 ± 0.41 (0.09–1.48)	<0.0001
Progressive motility (%)	59.4 ± 11.1 (40–80)	19.9 ± 9.65 (∼0.1–35)	<0.0001
Total motility (%)	68.2 ± 11.6 (50–100)	28.4 ± 14.3 (∼0.1–64)	<0.0001

Figure 1 illustrates the correlations between SPP in normozoospermic men (Figure 1A, C) and men with oligozoospermia (Figure 1B, D) versus progressive (Figure 1A, B) and total (Figure 1C, D) motility of sperm. As shown in the figure, no significant correlations (p > 0.05) were found between SPP in both groups versus progressive and total motility.

Figure 1.

Correlations between seminal plasma pyridoxine in normozoospermic men (A, C; n = 43) and men with oligozoospermia (B, D; n = 33) versus progressive (A, B) and total (C, D) motility of sperm.

The correlation between SPP and total sperm concentration in men with oligozoospermia (Figure 2B) compared with normozoospermic men (Figure 2A) was demonstrated in Figure 2. As illustrated in the figure, no statistically significant correlation was found between SPP and total sperm concentration in both tested groups (normozoospermic: p = 0.0118, r² = 0.4938; oligozoospermic: p = 0.2509, r² = 0.0423).

Figure 2.

Correlation between seminal plasma pyridoxine and total sperm concentration in men with oligozoospermia (B; n = 33) compared with normozoospermic men (A; n = 43).

Figure 3 demonstrates the correlation between SPP and total semen volume in men with oligozoospermia (Figure 3B) compared to normozoospermic men (Figure 3A). As shown in the figure, there was no correlation (p > 0.05) between SPP and total semen volume in both samples and controls (normozoospermic: p = 0.3471, r² = 0.0221; oligozoospermic: p = 0.8714, r² = 0.0009).

Figure 3.

Correlation between seminal plasma pyridoxine versus total semen volume in men with oligozoospermia (B; n = 33) compared to normozoospermic men (A; n = 43).

Figure 4 shows the concentration of SPP in normozoospermic men (Figure 4A) and men with oligozoospermia (Figure 4B) versus male age. As demonstrated in the figure, no statistical correlation (p = 0.1115, r² = 0.0620) were found between SPP level in normozoospermic men and male age, while there was positive statistical correlation (p = 0.0154, r² = 0.1750) between SPP level in men with oligozoospermia and male age.

Figure 4.

Seminal plasma pyridoxine in normozoospermic men (A) and men with oligozoospermia (B) versus male age.

4. Discussion

This work is the first of its kind that investigates the level of SPP in men with oligozoospermia. It represents the first phase of a long-standing goal to provide a new input to manage male factor infertility, in particular, idiopathic male infertility. In this study, we hypothesized that SPP level is lower in oligozoospermic men compared to normozoospermic. In accordance with our hypothesis, men with oligozoospermia were found to have reduced levels of SPP compared with normozoospermic men.

It has been shown that young boars administered water-soluble vitamins, including pyridoxine, had higher sperm concentrations compared with the control [26]. A newly published study on rat varicocele model concluded that vitamin B complex, including pyridoxine, administered at 6 mg kg⁻¹, is efficient to enhance the parameters of sperm, particularly count and motility [27]. Recently, in the previous work, we have shown that men with only asthenozoospermia had lower seminal pyridoxine concentration compared to those with normal motility of sperm [23]. These findings, albeit partially, are in line with our findings.

In effect, the observed beneficial influence of pyridoxine on sperm concentration can be owed to different biochemical routes such as increasing production of gonadal hormones, enhancing antioxidant defense mechanisms, particularly the testicular defense mechanisms, and decreasing homocysteine toxicity [15].

In 1981, Ebadi concluded that pyridoxine deficiency may disrupt the biosynthesis of luteinizing hormone and testosterone and alter gonadal function [28]. Later finding, in 1984, by Symes et al. revealed that pyridoxine-deficient male rats have lower levels of testosterone [29]. Another in vivo system study conducted by Hanai and Esashi in 2012 revealed that the reduction in gonadal development in male rats, kept in darkness, improved when receiving adequate amounts of thiamine (vitamin B₁) and pyridoxine, and high amounts of pantothenic acid (vitamin B₅) [30]. Therefore, together, given that gonadal hormones, mainly testosterone, are required to achieve spermatogenesis [31, 32], then the above indication suggests a vital contribution of pyridoxine in conserving normal gonadal function, and thus normal quantity of sperm.

Pyridoxine has been identified as having a potential antioxidant activity [33]. In particular, pyridoxine has been found to stabilize singlet oxygen radical (O₂•) [34]. Mechanistically, 3-hydroxypyridines, a chromophoric moieties of pyridoxine, were recognized to create unique biochemical models that embody the dynamic behavior of pyridoxine as an electron donor (reducing agent) against riboflavin-generated reactive oxygen radicals [35]. In effect, lysozyme proteins were found to be photo-preserved by pyridoxine from riboflavin-sensitized photo injury [35]. In addition, B-complex vitamins, including pyridoxine, were found to reduce the level of oxidative stress state, which is an imbalance between oxidants and antioxidants [36, 37], in patients with acute ischemic stroke [38], indicating a direct antioxidant potential to such group of vitamins [39, 40]. Consequently, given that accumulation of reactive oxygen species in the testes may lead to oxidative damage to sperm and reduce sperm concentration [41, 42, 43], then adequate testicular levels of pyridoxine may enhance the antioxidant defense mechanism and hinder the progression of this oxidative damage, which consequently maintain, albeit partially, an adequate number of ejaculated sperm.

In fact, there are two kinds of antioxidant mechanisms: endogenous and exogenous. Endogenous antioxidants have different classifications such as enzymatic (e.g., catalase, superoxide dismutase, and glutathione peroxidase) and non-enzymatic (e.g., glutathione and coenzyme Q10), lipid soluble (e.g., coenzyme Q10), and water soluble (e.g., glutathione), and small molecules (e.g., glutathione) and large molecules (e.g., superoxide dismutase). And, exogenous antioxidants are mainly the dietary antioxidants (e.g., carotenoids, polyphenols, ascorbic acid, and α-tocopherol) [44]. It is worth mentioning that endogenous and exogenous antioxidants are working synergistically to restore the redox homeostasis [44].

In addition, studies have confirmed the presence of glutathione system (glutathione, glutathione peroxidase, and glutathione reductase) in human semen [45]. In fact, glutathione system has been recognized as one of the key molecular defense mechanisms in semen that protects the sperm from oxidative damage; mechanistically, by reducing oxidative potential of hydrogen peroxide and minimizing the generation of hydroxyl radicals [46, 47]. It is renowned that low levels of pyridoxine decreases the existence of glutathione deprivation in the blood [48, 49, 50]. Another study in this research context revealed that infertile human males had lower levels of glutathione system in sperm compared to fertile [51]. Also, it is evident that reduced testicular glutathione system leads to a reduction in the ejaculated sperm number [52]. Accordingly, it can be suggested that reduced level of testicular glutathione system, and hence seminal glutathione one, may lead to a reduction in sperm concentration.

Further, pyridoxal 5′-phosphate, an active form of pyridoxine, is considered as key coenzyme for cystathionine-β-synthase, which is a multidomain enzyme catalyzes the conversion of homocysteine into cystathionine and cysteine [53, 54]. Decreased levels of pyridoxine may lead to accumulation of homocysteine and hyperhomocysteinemia (increased level of homocysteine in the blood) [55, 56]. It has been shown that deficiencies in B vitamins, including pyridoxine, is associated with hyperhomocysteinemia and altered sperm differentiation and maturation [16, 54, 57]. Moreover, a study conducted by Vujkovic (2009) found that 'Health Conscious' diet is negatively associated with seminal homocysteine and positively associated with seminal pyridoxine [58]. Collectively, the above evidence reveals that adequate levels of seminal pyridoxine appear to be crucial in maintaining normal count and function of sperm.

Besides, in the current study, no association was found between seminal SPP and progressive and total sperm motility, sperm concentration, and semen volume in men with oligozoospermia compared to normozoospermic men.

An observational study done by Boxmeers' et al. (2009) on fertile and subfertile men revealed an inverse relationship between seminal pyridoxine and semen volume, while no correlation was found between their seminal pyridoxine and sperm concentration or sperm motility [59]. In the current study, no correlation was observed between SPP and sperm concentration, sperm motility, and semen volume in men with oligozoospermia compared to normozoospermic men. The contradiction in semen volume results between our study and Boxmeers' study could be attributable to differences in the size and the nature of the studied populations as well as the methods of detection. In particular, the current study was particularly conducted on males with oligozoospermia compared to normozoospermic males, while the study by Boxmeer and co-workers was conducted on subfertile men compared to fertile. Also, in the present study, we have measured seminal pyridoxine using ultra-LC-MS/MS, while in Boxmeer's et al. study, such measurement was conducted using only HPLC.

Independently, in this study, we did not find a correlation between SPP level in normozoospermic men and male age, while a positive correlation was recognized between SPP level in men with oligozoospermia and male age. Most highly, this work is the first of its kind to probe such specific correlation, while in plasma, Rose et al. (1976) documented an inverse correlation between pyridoxine level and male age [60]. In fact, albeit partially, the positive correlation between SPP level in men with oligozoospermia and male age may be due to specific environmental factors (e.g., nutritional factors). In addition, it is worth mentioning that the age range of the recruited men with oligozoospermia was approximately (22–44 years), which is a narrow range in terms of male infertility; given that the age of man was found not to significantly alter semen quality [61]. Also, the number of recruited men could be a factor when discussing such specific correlation. Accordingly, a study that recruit wider age of men and larger population will of great importance to provide more detailed discussion to this correlation.

The sample size in this study is relatively considered a limitation. Since, increasing the sample size of the recruited men with oligozoospermia may improve the probed correlations (SPP versus sperm motility, sperm concentration, semen volume, and men age).

In conclusion, males with oligozoospermia have lower levels of SPP compared to normozoospermic males. Besides, SPP was found not to be correlated with progressive and total motility of sperm, sperm concentration, and semen volume in both tested groups. Also, a positive correlation was observed between SPP level in men with oligozoospermia and male age.

The findings from this work provides a significant support to the role and contribution of pyridoxine in maintaining adequate semen quality. And, such filled gap of knowledge adds a new tool to the management of male infertility, in particular those with idiopathic male infertility. Consequently, a long-term clinical trial in this research setting appears to be significant to confirm such pyridoxine contribution.

Declarations

Author contribution statement

Saleem A. Banihani: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Shefa’ M. Aljabali: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.

Funding statement

Professor Saleem A Banihani was supported by Deanship of Research-Jordan University of Science and Technology [193-2018].

Data availability statement

Data will be made available on request.

Declaration of interest's statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.