A Survey of Biochemical Parameters in Fertile Smokers- A Clinical Study

 

Sridharan TB¹*, Viswapriya V S¹, Moumita Basak¹, Parameswari R¹, Kamini A Rao²,

¹Gene Cloning and Technology Lab, Vellore Institute of Technology, India-14.

²Milann Fertility Hospital Bangalore, India.

 

ABSTRACT:

Cigarette smoking causes decrease in sperm density, total sperm count, total number of motile sperm and citrate concentration. Only those spermatozoa that possess adequate mechanical stimulation, chemical activation, functional and structural properties reach the female genitalia to complete its course of action. These require optimal levels of a number of biochemical and clinical parameters for it to fulfil its apposite function.Materials and Methods: A clinical study was carried on the fertile smoker samples. Semen and biochemical parameters (Triglycerides, Total protein, Calcium, Potassium, Zinc, Magnesium, and Sodium) were assayed. Statistical analysis was done using SPSS (version 21.10) and one-way ANOVA was used.Results: However, the results showed though the levels of biochemical parameters were altered they were not radical enough to bring upon any effect on fertility of men. Conclusion: Hence, from this survey we get to know that though the clinical factors are marginally varied they retain their significance upon regulating the sperm characteristics in fertile smokers. Further prominent deductions can be derived in future by including parameters of infertile smoker samples.

 

 

 

INTRODUCTION:

Smoking is inhalation and exhalation of fumes generated from burning of tobacco. These fumes contains various components that are harmful for health, which places smoking as the reason for majority of deaths in many countries across the globe [2]. Smoking affects social behavior. Also it is seen that increasing negative mood variability is a risk for future smoking upsurge and that its mood stabilizing effects may strengthen and maintain quotidian cigarette use among youths [3]. The utmost integrals that influence the health are nicotine, tar, in the particulate phase and carbon monoxide in gaseous phase. Cigarette smoking causes drop in sperm density, overall sperm count, overall number of motile sperm and citrate concentration. [5]. Nicotine has an influence on sperm morphology and sperm count [6].

 

After their production in seminiferous tubules, sperm cells are collected and stored in the cauda of the epididymis. Here with the help of several proteins the sperm cells mature and only mature spermatozoa are capable of motility. During ejaculation, the sperm cells are pushed through deferent duct and urethra. At this time a large volume of secretions from various glands mix in and is known as seminal plasma which, makes up to 90% of the ejaculate (the rest 10% being spermatozoa). Post this step only those spermatozoa that possess adequate mechanical stimulation, chemical activation, functional and structural properties reach the female genitalia to complete its course of action. These require optimal levels of a number of biochemical and clinical parameters for it to fulfil its apposite function [1].

 

D-Aspartic acid has an influence in male fertility, it also induces the release of testosterone [7], and it occurs in human seminal plasma and spermatozoa[22]. Aspartic acid presents in spermatozoa as arginine-glycine-aspartic acid (RGD) sequence and that antibody to OPN (Osteopontin), a phosphoprotein that contains the RGD sequence, it has the capability to reduce sperm egg binding before fertilization and also in in vitro fertilization [23].Smoking has inimical outcome on sperm motility, viability, DNA fragmentation, seminal Zinc levels and semen reactive oxygen species levels, also in fertile men and it is directly corresponds with cigarette quantity and smoking period [8]. Fatty acid improves such as triglyceride improves the sperm motility and the average path velocity [9]. Among nine biological trace elements zinc, copper and selenium are paramount in reproduction in males and females. Zinc content is high in adult testis and the prostate has a higher concentration of zinc than any other parts of the body. Zinc inadequacy initially diminish angiotensin converting enzyme (ACE) activity and this in turns leads to exhaustion of testosterone and hindrance of spermatogenesis [10]. Magnesium, calcium, copper and zinc plays an important role in spermatogenesis and fertility on men [11]. Deficiency of zinc caused hypogonadism and inadequate growth of secondary sex character in human [12]. High content of zinc in seminal plasma is link with high degree of sperm cell motility [13]. High positive correlation lies between magnesium and zinc in seminal plasma [14]. Calcium is chief for sperm physiology, which include motility [15], metabolism [16], acrosome reaction [18] and fertilization [17].

 

There are reports that have some different results which have shown positive effects to male fertility due to smoking, it concluded as smokers had remarkably higher total testosterone and free testosterone levels contrast to non-smokers even after stratification as per age, BMI, triglycerides and alcohol consumption, both total testosterone and free testosterone levels were negatively correlated to the total of tobacco subjection and it even stated that smoking was an independent influencing factor for the levels of both total testosterone and free testosterone [24]. Based on above listed this study is aims to findbiochemical parameters influence in fertile smokers - a clinical study

 

MATERIALS AND METHODS:

Ethics:

This study is, minor part of major research work and it was approved by institutional review board of VIT University, with Ref. No. VIT/UHEC-3/NO.11; based on which informed consent form was obtained and maintained confidentially.

 

Exclusion Criteria and Inclusion Criteria:

Patients with sexually transmitted diseases, genital trauma or testicular torsion, testicular varicocele, genital infection, systemic and chronic diseases and medication affecting lipid metabolism were excluded. Subjects were leading normal life with fertile condition (23-45 years of age) only screened for this research. Also subjects with smoking and alcohol habits were included in this study.

 

Semen Sample Collection:

Semen samples were collected from n=40 fertile smoker subjects in the Andrology Department of BACC Healthcare Private Ltd. All the samples were collected in a sterile container and further semen analysis was done with computer assisted semen analysis (CASA) based on the standard world health organization (WHO) protocols.

 

Sperm fractionation:

It includes three steps of centrifugation i.e. samples were gone through centrifugation for 3500 rpm for 10 minutes. The supernatant was separated from the pellet. The pellets were preserved for further experiments which were the sperm cells. The supernatant was used in next centrifugation process. The supernatant was extracellular protein which was a bit preserved for further experiment. From the above process the supernatant gone through the second stage of centrifugation in which 100 µl PBS buffer was added. Again centrifugation at 3500 rpm for 10 minutes was done. The pellet was discarded and a bit of supernatant was preserved and other part of supernatant was used for last centrifugation process. The supernatant from the above process was again used for centrifugation at 12,000-14,000 rpm for 10 minutes by discarding the pellet, the supernatant was preserved for experiments. The supernatant was seminal plasma.Some amount from each centrifuge should be stored separately for assay.

 

Biochemical Parameters Analysis in Seminal Plasma:

To check the sperm quality of smokers, D-Aspartic acid estimation was performed using Potassium Phosphate dibasic (K₂HPO₄) and acetonitrile as mobile phase for HPLC at wavelength 250nmet al [21]. For Zinc and Magnesium estimation in the seminal plasma, Atomic Absorption Spectroscopy (AAS) was performed [11].For protein estimation in semen sample by Lowry’set al [26]. Kits were used for other biochemical parameters such as triglyceride analysis in semen sample, kit (Sigma Aldrich India; accession no.-TR0100)[27], sodium and potassium estimation in the semen sampleskit (Thermo Fischer; accession No. P-3000MP)[19]. Calcium estimation in semen sample kit (Sigma Aldrich India; accession No. MAK022) [20].

 

Statistics:

The correlation between semen parameters and biochemical parameters of the fertile smokers was calculated by one-way ANOVA using SPSS version 21.0.

 

RESULTS:

Table 1: Assessment of Semen parameters in fertile smokers

Semen Parameters	Fertile smokers (n=40)	p value
Volume (mL)	2.5±0.5	0.01
pH	7.2-7.4	-
Sperm Concentration (millions/mL)	39.26±0.74	0.001
Sperm morphology (%)	45.71±0.19	0.001
Total motility (%)	49.35±0.72	0.01
Rapid motility (%)	42.21±0.9	0.001
Slow motility (%)	6.9±0.2	-0.01
Head and tail defects (%)	4.32±0.02	0.01

 

Values are represented as mean± standard deviation. This table gives the correlation values for semen parameters when compared with biochemical parameters.

 

Table 2:Assessment of Biochemical and Clinical parameters in fertile smokers

 

Values are represented as mean± standard deviation. This table gives the correlation values for biochemical parameters when compared with semen parameters.

 

 

Figure 1: Assessment of total protein and triglyceride levels in whole semen

 

Values are represented here Mean ± Standard Deviation. TGE-Extra cellular triglyceride;TGP- Prostosomal triglyceride; TGS- Seminal triglyceride; PE- Extracellular protein; PS- seminal protein; PP- prostasomal protein. The figure shows that the amount of triglycerides present in the semen samples is higher than that of total protein.

 

 

Figure 2: HPLC data of standard D-aspartic acid.

 

This figure gives the peak value of standard D-Aspartic Acid at 250nm. The sample data was compared to the standard data and results were arrived at. The major conventional semen parameters were in Table 1. The biochemical and clinical parameters that were analyzed are listed in Table 2. Based on the semen parameters in Table 1 the sperm morphology of fertile smokers was45.71±0.19 (in%). Sperm Concentration, motility (total motility, slow motility, rapid motility), viability, pH and viscosity are affected by variations of seminal plasma zinc. The biochemical and clinical parameters such as zinc, calcium, magnesium etc. in table 2 are correlated with the semen parameters in table 1 (zinc affects motility and count, calcium affects motility). These correlations have a p-value of less than 0.05 indicating that in a fertile sperm sample all these are essential for maintaining normal sperm morphology, motility, concentration and volume. Potassium and Sodium levels which impacts motility and concentration are negatively correlated with semen parameters in fertile smokers. Their low levels are positively correlated to fertile spermatozoa. The triglycerides and total protein concentrations are represented in graph 1. High triglyceride concentration leads to decreased sperm motility. However, a p-value of 0.01 for total sperm motility and a very less triglyceride value (represented in nmol/10⁶) indicates their negative correlation.

 

DISCUSSIONS:

During ejaculation from male’s epididymis only those spermatozoa that possess adequate mechanical stimulation, chemical activation, functional and structural properties reach the female genitalia to complete its course of action. These require optimal levels of a number of biochemical and clinical parameters for it to fulfil its apposite function [1].

 

The current study discusses the effect of various biochemical parameters such as total protein, aspartic acid, triglycerides and metal ions are correlated with semen parameters namely sperm motility, morphology, concentration and head and tail effects. Since the study is done on samples of smokers that are fertile, though the clinical parameters are little varied from the normal levels they still do not affect the semen parameters, which rules the fertility character of a sperm. Aspartic acid plays a major role as an amino acid triad which when binds with zinc helps the binding of sperm with an egg [22]. Thus, aspartic acid is essential for fertility and the p-value in table 2 indicates the positive correlation of aspartic acid on the sperm. Optimum levels of triglycerides are very important for sperm motility, as high levels will retard their motion towards the egg. Here though the level of triglycerides is marginally high, the correlation values indicate that the sperm motility is still intact. Among the trace elements, zinc is one of the most important metal ion. Apart from activating the amino acid triad, zinc plays a vital role in sperm motility, morphology and concentration [13].

 

Here, though the amount of zinc is comparatively low, the semen parameters that are regulated by zinc are unharmed and the fertility is maintained. Zinc is positively correlated with magnesium [14].Similarly, calcium also affects the motility and concentration of sperm and its p-value of 0.001 points out that it positively regulates these semen parameters. Potassium affects the semen concentrations and lower levels of potassium point to increased motility of the spermatozoa while the reverse is true for Sodium [25].

 

Though this study states the effect of biochemical parameters on fertile smokers, correlation needs to be done between infertile smokers, fertile smokers, and the effect of these factors on both. This will yield a comparative study and will help in understanding the importance of these parameters on male fertility and the impact of smoking upon male fertility.

CONCLUSION:

This study concludes that though the clinical factors are marginally varied they retain their significance upon regulating the sperm characteristics in fertile smokers. Further studies along with infertile smoker’s parameters and a larger sample size will help elucidate the prominence of these clinical parameters upon regulating the semen parameters.

 

ACKNOWLEDGEMENTS:

We thank Bangalore Assisted Conception Centre (BACC) for providing us with the semen samples. We thank Vellore Institute of Technology (VIT), Vellore, Tamilnadu, for management and financial support.

 

CONFLICT OF INTEREST:

None

 

REFERENCES:

1.     World Health Organization. (2010). WHO laboratory manual for the Examination and processing of human semen.. Switzerland.

2.     YGOY Inc. What is smoking?.http://smoking.ygoy.com/what-is-smoking/. (Last accessed: March 2017)

3.     Weinstein, S. M., Mermelstein, R., Shiffman, S., and Flay, B. (2008). Mood variability and cigarette smoking escalation among adolescents. Psychology of Addictive Behaviors, 22(4), 504.

4.     Hammond, D., Fong, G. T., Cummings, K. M., O'Connor, R. J., Giovino, G. A., and McNeill, A. (2006). Cigarette yields and human exposure: a comparison of alternative testing regimens. Cancer Epidemiology and Prevention Biomarkers, 15(8), 1495-1501.

5.     Künzle, R., Mueller, M. D., Hänggi, W., Birkhäuser, M. H., Drescher, H., and Bersinger, N. A. (2003). Semen quality of male smokers and nonsmokers in infertile couples. Fertility and sterility, 79(2), 287-291.

6.     Oyeyipo, I. P., Yinusa, R., ObukowhoEmikpe, B., and FolashadeBolarinwa, A. (2011). Effects of Nicotine on Sperm Characteristics and Fertility Profile in Adult Male Rats: A Possible. Journal of Reproduction and Infertility, 12(3), 201-207.

7.     D'Aniello, A., Di Fiore, M. M., D'Aniello, G., Colin, F. E., Lewis, G., and Setchell, B. P. (1998). Secretion of D‐aspartic acid by the rat testis and its role in endocrinology of the testis and spermatogenesis. FEBS letters, 436(1), 23-27.

8.     Taha, E. A., Ez-Aldin, A. M., Sayed, S. K., Ghandour, N. M., and Mostafa, T. (2012). Effect of smoking on sperm vitality, DNA integrity, seminal oxidative stress, zinc in fertile men. Urology, 80(4), 822-825.

9.     Lahnsteiner, F., Mansour, N., McNiven, M. A., and Richardson, G. F. (2009). Fatty acids of rainbow trout (Oncorhynchusmykiss) semen: composition and effects on sperm functionality. Aquaculture, 298(1), 118-124.

10.   Bedwal, R. S., and Bahuguna, A. (1994). Zinc, copper and selenium in reproduction. Experientia, 50(7), 626-640.

11.   Wong, W. Y., Flik, G., Groenen, P. M., Swinkels, D. W., Thomas, C. M., Copius-Peereboom, J. H., and Steegers-Theunissen, R. P. (2001). The impact of calcium, magnesium, zinc, and copper in blood and seminal plasma on semen parameters in men. Reproductive toxicology, 15(2), 131-136.

12.   Prasad, A. S. (1991). Discovery of human zinc deficiency and studies in an experimental human model. The American journal of clinical nutrition, 53(2), 403-412.

13.   Stanković, H., and Mikac-Dević, D. (1976). Zinc and copper in human semen. ClinicaChimica Acta, 70(1), 123-126.

14.   Sørensen, M. B., Bergdahl, I. A., Hjøllund, N. H. I., Bonde, J. P. E., Stoltenberg, M., and Ernst, E. (1999). Zinc, magnesium and calcium in human seminal fluid: relations to other semen parameters and fertility. Molecular human reproduction, 5(4), 331-337.

15.   Morton, B., Harrigan-Lum, J., Albagli, L., and Jooss, T. (1974). The activation of motility in quiescent hamster sperm from the epididymis by calcium and cyclic nucleotides. Biochemical and biophysical research communications, 56(2), 372-379.

16.   Peterson, R. N., and Freund, M. (1976). Relationship between motility and the transport and binding of divalent cations to the plasma membrane of human spermatozoa. Fertility and sterility, 27(11), 1301-1307.

17.   Maleszewski, M. (2004). Mechanisms of fertilization in mammals. Acta BiologicaCracoviensia. Series Botanica. Supplement, 46(1).

18.   Yanagimachi, R., and Usui, N. (1974). Calcium dependence of the acrosome reaction and activation of guinea pig spermatozoa. Experimental cell research, 89(1), 161-174.

19.   Morisawa, M., Suzuki, K., and Morisawa, S. (1983). Effects of potassium and osmolality on spermatozoan motility of salmonid fishes. Journal of Experimental Biology, 107(1), 105-113.

20.   Fraire-Zamora, J. J., and González-Martínez, M. T. (2004). Effect of intracellular pH on depolarization-evoked calcium influx in human sperm. American Journal of Physiology-Cell Physiology, 287(6), C1688-C1696.

21.   Meeker, J. D., Ehrlich, S., Toth, T. L., Wright, D. L., Calafat, A. M., Trisini, A. T., and Hauser, R. (2010). Semen quality and sperm DNA damage in relation to urinary bisphenol A among men from an infertility clinic. Reproductive toxicology, 30(4), 532-539.

22.   D’Aniello, G., Ronsini, S., Guida, F., Spinelli, P., and D’Aniello, A. (2005). Occurrence of D-aspartic acid in human seminal plasma and spermatozoa: possible role in reproduction. Fertility and sterility, 84(5), 1444-1449.

23.   Goncalves, R. F., Wolinetz, C. D., and Killian, G. J. (2007). Influence of arginine-glycine-aspartic acid (RGD), integrins (α V and α 5) and osteopontin on bovine sperm–egg binding, and fertilization in vitro. Theriogenology, 67(3), 468-474.

24.   24.Wang, W., Yang, X., Liang, J., Liao, M., Zhang, H., Qin, X., ...and Mo, Z. (2013). Cigarette smoking has a positive and independent effect on testosterone levels. Hormones (Athens), 12(4), 567-577.

25.   Hamad, A. W. R., Al-Daghistani, H. I., Shquirat, W. D., Dayem, M. A., and Al-Swaifi, M. (2014). Sodium, Potassium, Calcium and Copper Levels in Seminal Plasma are Associated with Sperm Quality in Fertile and Infertile Men. Biochem Pharmacol, 3(141), 2167-0501.

26.   Peterson, G. L. (1977). A simplification of the protein assay method of Lowry et al. which is more generally applicable. Analytical biochemistry, 83(2), 346-356.

27.   Lahnsteiner, F., Berger, B., Weismann, T., and Patzner, R. A. (1996). Physiological and biochemical determination of rainbow trout, Oncorhynchusmykiss, semen quality for cryopreservation. Journal of Applied Aquaculture, 6(4), 47-73.

 

 

 

Accepted on 24.12.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(2):559-563.