Does Chewing Tobacco Change The Taste Of Semen
J Hum Reprod Sci. 2014 Apr-Jun; 7(2): 136–142.
Prevalence of abnormal spermatozoa in tobacco chewing sub-fertile males
Priyadarsini Sunanda
Department of Obstetrics and Gynaecology, Heart for Human Reproduction, Institute of Medical Sciences and SUM Hospital, Bhubaneswar, Odisha, Bharat
Babita Panda
Department of Obstetrics and Gynaecology, Center for Human Reproduction, Found of Medical Sciences and SUM Hospital, Bhubaneswar, Odisha, Republic of india
Chidananda Dash
Department of Obstetrics and Gynaecology, Center for Human Reproduction, Institute of Medical Sciences and SUM Hospital, Bhubaneswar, Odisha, Republic of india
Priyadarshi Chiliad. Ray
oneNano-Medicine Laboratory, Plant of Life Sciences, Nalco Foursquare, Bhubaneswar, Odisha, India
Rabindra Northward. Padhy
2Primal Research Laboratory, Institute of Medical Sciences and SUM Infirmary, Siksha O Anusandhan University, Bhubaneswar, Odisha, Bharat
Padmanav Routray
3Aquaculture Product and Environment Division, Key Institute of Freshwater Aquaculture, Bhubaneswar, Odisha, Bharat
Received 2013 Nov seven; Revised 2014 Jan 14; Accustomed 2014 Feb 26.
Abstract
AIM:
The aim of the post-obit study is to detect out the prevalence of abnormal spermatozoa and associated functional parameters in clinical semen samples of sub-fertile males with the tobacco chewing habit.
SETTINGS AND Blueprint:
Retrospective study was conducted at infertility unit of a third health intendance eye, in a catamenia of 3 years.
MATERIALS AND METHOD:
Semen of 642 males were analyzed; of them 194 men (30.two%) were tobacco chewers and they were grouped according to their intensity of chewing (<ten and ≥ 10 packets/day). Counts, movement, vitality, and morphology of sperms were analyzed.
RESULTS:
In tobacco chewers, 66% of subjects were oligozoospermic, 85% asthenozoospermic and 28% teratozoospermic. Sperm counts (odds ratio [OR] =2.2; 95% confidence interval [CI]: 1.five-3.09), motility (OR = 3.2; 95% CI: 2.05-4.9), and normal morphology (OR = 8.iv; 95% CI: iv.nine-fourteen.half dozen) were significantly affected (P = 0.001) in tobacco chewers than the not-chewing group. Further, in comparison to the intensity of tobacco chewing, patients with the intensive practice of using ≥10 packets/24-hour interval had a significant upshot on sperm morphology (P = 0.003, OR = two.seven; 95% CI = 1.41-5.08) merely. Structural defects in head (P = 0.001) and cytoplasmic residues (P = 0.001) were found to be positively correlated with the intensive chewing, but no pregnant changes were found in anomalies in mid-piece and tail.
Conclusion:
The adverse touch on of tobacco chewing on semen parameters was evident even with balmy chewers, but with the intensive chewing practice, phenotypes of sperms, mainly defects in the caput and cytoplasmic residue were severely affected.
Key WORDS: Cytoplasmic residues, head defect, motility, sperm count, tobacco chewing
INTRODUCTION
Increasing incidence of infertility has been a global health as well as a social problem, where 35-forty% of male partners are solely responsible.[1] Apart from the well-known conventional causes, i.east., disturbance in the endocrine system, anatomy, genetic makeup, varicocele or torsion, occurrence of diabetes and subtle unknown infections, chronic exposure to toxic chemicals and differential unhygienic lifestyle patterns besides contribute to male infertility.[two] Tobacco consumption is ane of the lifestyle factors that is ofttimes detrimental to human wellness every bit a whole.[3] Comparison depositions of nicotine in reports of International Agency for Research on Cancer (IARC, France) demonstrated that the consumption of smokeless tobacco 8-10 times versus 30-40 normal cigarettes a twenty-four hours atomic number 82 to the deposition of almost equal amount of nicotine; in improver, it leads to the incidence of mouth cancer.[four] Notwithstanding, other manifestations of homo wellness were to the lowest degree focused in the study due to tobacco consumption.
Despite strong regulations and campaign, people practice consume tobacco products that impact several physiological systems including the reproductive organisation.[5] Recently, many states of Bharat have banned the sale of chewing tobacco, merely practically it has been no effect on consumption in public. Smokeless tobacco products are available in Indian market with many forms, khaini, gutkha, and betel containing lime, catechu and dry leaves of tobacco. The general ingredients of these products vary according to unlike products.
Tobacco contains more than thirty mutagenic agents amid which, nicotine is regarded as the most important component, in the particle phase. Nicotine is chop-chop captivated through the respiratory tract, mouth mucosa and peel.[vi] Further, of the total nicotine entering the body, an lxxx-ninety% is metabolized by the liver and a fraction of nicotine and its degraded products were detected in serum, urine, saliva, milk and seminal plasma.[7] Nicotine affects the sperm plasma membrane and genetic integrity by their powerful oxidizing deportment.[8] In animal studies, the impairment of testicular histology and a reduction in diameter of seminiferous tubules as well equally, a subtract in the index of Sertoli cells, following 1.5 h of exposure to tobacco, per day (6 days, a week) every bit cigarette smoke for 10 weeks, were reported.[ix] Indeed, the reduced fertility in males with impaired spermatogenesis, sperm motility, deleterious effect on germ cells and embryo development in tobacco exposure were recorded.[x,11] However, the threshold level that damages the integrity of male person reproductive system is still obscure. A higher incidence of teratozoospermia in tobacco chewers was demonstrated,[8] but related information on details of morphological anomalies of sperms is withal scarce. Moreover, the intracytoplasmic sperm injection technique helps the treatment of astringent teratozoospermia by microinjection of sperm to metaphase-Ii oocyte; even so, failures in many cases due to delayed fertilization, abnormal cleavage rates and spontaneous abortions were known,[12] which could be, a priory, due to defective sperm characteristics, those impeding the normal event of fertilization.[13,14]
As the tobacco chewing habit is quite prevalent amidst South-East Asia population (53.5%),[xv] this retrospective study was undertaken to assess the effect of chewing tobacco on semen quality and specific sperm morphological defects in sub-fertile males undergoing an infertility handling. This study should assistance a pertinent analysis of the problem of infertility treatment of male person partners, as well every bit help public awareness in refraining from far-reaching this and other health concerns due to tobacco chewing. This study from the nation of S-eastern asia with a sizable ghetto of urban slums and uneducated villagers gives an exemplary mirror of a public health problem of male infertility linked to such a health polluting habit.
MATERIALS AND METHOD
Subjects
Patients, attention the infertility unit of a third health intendance center, from 2009 to 2012 for infertility treatment, were taken as subjects. Seminal fluid analyses were washed for 642 male partners. History of tobacco consumption and other lifestyle patterns were noted. Elapsing of tobacco chewing in the cohort of subjects was betwixt two and 18 years of age. Patients were balkanized accordingly to the habit and the intensity of chewing, i.due east., less and more than 10 packets/solar day into 2 groups. Subjects were selected on the basis of the following inclusion and exclusion criteria: Inclusion criteria were male partners having the complain of infertility with age, 20-40 years and patients should have 3-5 days of sexual abstinence; exclusion criteria were males with a known/unknown blood borne infectious diseases, patient undergoing an antibiotic treatment, diabetes mellitus, hydrocele, hernia and varicocele, azoospermia cases and addictions other than tobacco chewing.
Sample collection and semen analysis
Semen samples were collected by masturbation into broad-rima oris plastic container, in a room close to the andrology laboratory. Semen parameters were analyzed, co-ordinate to the (World Health Organization [WHO]) standard criteria, i.e., volume ≥two ml, concentration ≥20 one thousand thousand/ml, total count ≥40 million, progressive motility ≥ 50%, vitality ≥75% and normal morphology >15% (Kruger'due south criteria).[16] To determine the percentage of motile sperms, an aliquot of 10 μl of gently mixed liquefied semen was observed at ×400 magnification. At least 200 sperms were counted, and the mean value from duplicate measurements was represented. Sperm counts were done by using Neubauer'southward hemocytometer with requisite dilutions (1:ii, i:5, 1:9, 1:20), as per the WHO criteria.[xvi] Sperm morphology was assessed in Papanicolau-stained smears (Hematoxylene, orange-Grand and EA-50 stain) using calorie-free microscopy, nether the oil immersion at ×1000 magnification. For a few semen samples, sperm morphology was likewise assessed by diminutive forcefulness microscopy (AFM), for which the standardization was done by fixation of sperms in ii.five% glutaraldehyde in phosphate buffer saline (PBS), for i h and the mixture was centrifuged at 200-300 thousand for ten min. Sperm pellet was washed with PBS and smeared on a clean slide with appropriate dilution. AFM images were taken past using the JPK NanoWizard Ii (Germany), at intermittent contact (air), which is called as borer manner of operation with an ultrasharp silicon cantilever (thousand = 40 N/yard, resonant frequency = 300 kHz).[17]
Vitality of sperms was estimated by the hypo-osmotic-swelling examination by mixing equal volumes of semen and hypo-osmotic solution, prepared from 7.35 g sodium citrate and thirteen.5 1000 fructose in 1000 ml distilled water. The mixture was incubated for 30 min at 37°C, from which an aliquot of 10 μl was immediately examined at the ×400 magnification. The percentage of swollen (vital) sperm was assessed by counting a minimum of 200 spermatozoa.
Statistical analysis
Using SPSS, version xx.0 (IBM, USA), logistic regression analysis by odds ratios (ORs) and 95% confidence intervals (CIs) were computed basing on dichotomized value of semen parameters, specified by WHO standard criteria. Pearson'southward correlation coefficient, r was determined for the intensity of tobacco chewing and sperm anomalies.
RESULTS
From the questionnaire, thirty.2% (194/642) subjects were tobacco chewers, from whom, l.five% of subjects chew gutkha, 27.ix% and 21.6% of subjects chew khaini and dried tobacco leaves in betel, respectively. A comparison of semen parameters between tobacco chewers (Group I) and normal subjects, without the habit of tobacco chewing (Group II) is presented [Table 1]. In all parameters tested, Group Ii subjects had college hateful values than Grouping I subjects. Further, with respect to sperm count, the mean values declined in 18.nine% in Group I subjects; and subtract in mean values for motion, vitality and for normal morphology were 23.4, 8.4 and 28.four%, respectively.
Table 1
Comparison of semen parameters between tobacco chewers, Group I and not-chewers, Group II with 95% CI*
OR values of different semen parameters confirmed meaning difference at P = 0.001 between the two groups, I and 2 [Table 1]. The probability of oligozoospermia in Group I subjects were 2.2 times higher odds than that of Group II subjects (OR = 2.2; 95% CI: i.5-3.09). Similarly, probability of asthenozoospermia in Group I subjects was 3.2 times higher odds (OR = 3.ii; 95% CI: 2.05-4.9) and teratozoospermia 8.four times was higher odds (OR = 8.4; 95% CI: 4.9-xiv.half dozen) than those of Group II subjects. However, no pregnant deviation was observed for sperm vitality between the two groups (OR = 1.6; 95% CI: 0.739-3.6).
Further, event of intensity of tobacco chewing on semen quality was analyzed by dividing the Group I subjects into two sub-groups. Sub-group IA was subjects using more ten packets of tobacco per solar day (intensive chewers) and sub-group IB was subjects using less than x packets of tobacco per day (non-intensive chewers). Here, only normal morphology of sperm was significantly affected in samples from intensive chewers: With 2.7 times higher odds than the non-intensive chewers (P = 0.003, OR = 2.7; 95% CI = 1.41-5.08). Only, no significant difference was found for the full sperm count (P = 0.534, OR = 0.81; 95% CI: 0.441-1.v) and movement values (P = ane.0, OR = 1.1; 95% CI: 0.478-2.5) [Table 2].
Tabular array 2
Comparison of semen parameters between intensive tobacco chewers, sub-group IA and nonintensive tobacco chewers, sub-group IB*
Among tobacco chewers, 28.four% cases were oligoas-thenoteratozoospermic, 59.four% oligoasthenozoospermic and xi.2% were normozoospermic. Hateful value of the semen parameters in oligoasthenoteratozoospermia cases were presented in Table 3. The presence of normozoospermic, which contributes to higher mean values as shown in Tables 1 and 2.
Table iii
comparison of semen parameters in oligoasthenoteratozoospermia cases between intensive tobacco chewers, sub-group IA and non-intensive tobacco chewers, sub-group IB*
As OR value represented viii.4 times higher to be of teratozoospermics in tobacco chewers than not-chewers, this study intensified specific morphological anomalies due to tobacco chewing [Table 4]. Deciphering the sperm morphology, many abnormal features were observed in head, mid-piece and tail [Figure i]. Double head, pin caput, tapered head, bent neck, curved tail and tailless sperms were the several anomalous features abundantly marked. An initial attempt for sperm morphological report by AFM also showed pregnant aberrant features amidst tobacco chewers [Figure two]. Notwithstanding, mean of head defects and cytoplasmic residues were higher in the sub-group IA having an increasing trend from the control group to intensive chewers.
Table four
Comparing of morphological abnormalities of spermatozoa between intensive tobacco chewers, sub-group IA and non-intensive tobacco chewers, sub-grouping IB, in respect to normozoospermic control subjects (% values)*
Sperms with different morphological features in studied semen samples: (a) Double head; (b) Pyriform head without acrosome; (c) Abnormal caput with irregular acrosome; (d) Aptitude necked; (east) Cytoplasmic residues with tapered head; (f) Cytoplasmic residues and minor acrosome; (1000) Circular head with aberrant mid-piece; (h) Long amorphous caput; (i) Young spermatozoa; (j) Aberrant mid-piece; (k) Double tailed; (l) Normal spermatozoa
Aamplitude images of sperm by atomic force microscopy. The two-dimensional images represent length and width in 10 and Y-centrality, respectively. Each small division represents 2 μ: (a) Aberrant acrosome; (b) Aberrant mid-piece; (c) Aptitude neck with irregular acrosome; and (d) Double head with abnormal mid-piece
Awarding of Pearson's correlation likewise presented the strong positive correlation of caput defect (r = 0.400) and cytoplasmic droplet (r = 0.611) with increasing use of the number of chewing tobacco packets (P = 0.001). But no significant modify in mid-piece (P = 0.122) and tail defects (P = 0.411) were seen betwixt the three groups [Table 4]. Although cytoplasmic residue was a mid-piece defect,[16] to present the specific defects of sperms, here cytoplasmic remainder was analyzed, separately. Hence, the full results depicted the findings on sperm morphological features in addition to clinically assigned semen parameters to find their effects among tobacco chewers.
Word
The present retrospective study recorded a pass up in quality of semen amongst the tobacco chewers. Of 642 subjects, 194 (thirty.2%) had a significant decrease in sperm count, movement and dumb morphology. In tobacco chewers, 66%, 85.5% and 28.4% of subjects were establish be below the WHO standard criteria for sperm count, motility and morphology respectively. This depicted significant event of tobacco chewing as a lifestyle habit leading to several types anomaly in semen quality, consequently male infertility.
Indeed, among the two habits, chewing of tobacco and smoking most go addicted to the quondam, a priory, due to inebriating role of nicotine. Tobacco either in smoke or chewing forms has a harmful upshot on human health causing gum recession, leukoplakia, cardiovascular illness and cancer of the rima oris or larynx or throat.[eighteen] In addition, it has negative effects on male person reproductive system in thwarting the production of competent sperms, for the successful fertilization.[7,19,20] These results were corroborated by another similar report on tobacco causing adverse effect on semen quality in Mumbai, India.[viii]
Animal studies predicted the concentration-dependent upshot of nicotine on function of male reproductive organs, spermatogenesis, litter size, and level of endocrine hormones, somewhen fecundity.[21] Nicotine is known to disturb the hypothalamus-pituitary axis by disrupting the testicular microcirculation.[22] As testosterone acts on seminiferous tubules to initiate and maintain spermatogenesis,[23] reduction of this sex hormone level either past impaired Leydig cell function or disturbance in the androgen/estrogen ratio could be a cause of decreased sperm counts.[21,24,25] Further, in vitro studies indicated, nicotine at the concentration ane mM significantly declined the move of sperm, and the concentration of 70 ng/ml acquired a subtract in the kinematics of sperms.[26,27] Nicotine and other chemicals in tobacco probably crusade either damage to mitochondrial genome or/and mitochondrial enzymatic activities or an damage of function of the seminal vesicle affecting sperm movement. There was no significant effect of chewing tobacco on the function of accessory gland, seen with a pocket-sized number of 29 patients in a study, just a larger sample size could notice a definitive conclusion on the damaging effects of tobacco.[28] However, seminal plasma of not-smokers could call back motion of sperms from smokers.[29]
In intensive tobacco chewers, normal morphology was affected more than than sperm count and motion, corroborated elsewhere.[8] Fifty-fifty though sperms with irregular acrosome, aptitude cervix and coiled tail were observed in not-intensive tobacco chewers, additional bibelot of head defects and cytoplasm residues were found to be of college caste in intensive chewing cases. Aberrant development of Golgi proacrosomic vesicles and their improper zipper to nucleus leads to the formation of defective head, either with irregular acrosome or head without acrosome by the elimination of the Golgi body through residual cytoplasm.[30] Here, it could be that sure chemical components interfere the secretory or normal activity of Golgi body during spermiogenesis.
Excess cytoplasmic residues of circular spermatids are phagocytosed past Sertoli cells leaving a small-scale cytoplasmic droplet for osmotic residual and terminal tail elongation during epididymal transit.[31] Epididymal dysfunction due to tobacco chewing may be a directly cause of retaining cytoplasmic residues, by preventing the timely loss of droplet and the development of secondary abnormalities in sperms. Excess cytoplasmic residues are besides the source of reactive oxygen species which can cause impairment to sperm membrane, proteins and deoxyribonucleic acid (Deoxyribonucleic acid).[32,33,34]
Written report on ultrastructure as well revealed the presence of Sertoli cells with polymorphic mitochondria with irregular cristae, spermatids with excess cytoplasm, abnormal acrosomes in nicotine treated rats.[35] Another report was also found with curved tail, both bent and curved mid-piece in nicotine treated rats.[21] Moreover, this preliminary written report with AFM revealed minute abnormal features of sperm head and cervix past representing incandescent resolution and topographic view. However, it takes more time to scan the structure through its cantilever. So, a more intensive written report could highlight significant morphological features of sperms to correlate clinically.
Nicotine is absorbed in mouth mucosa 3-4 times faster in chewing cases than smoking. Nicotine levels increment gradually with intensive do of chewing and remainder chemicals remain for a prolonged period than in smoking cases.[36] Only, the threshold concentration affecting male reproductive system is yet to be defined. Again the quantity of other chemicals, their specific effects on male person reproductive organisation and pathological concentrations in unlike individuals is largely unknown. The presence of pesticides, maleic hydrazide, chlordane, dichlorodiphenyl-trichloroethane, dichlorodiphenyldichloroethylene, dieldrin, endrine, heptachlor in smokeless tobacco might exist causing these endocrinological disorders and harm of sperm Deoxyribonucleic acid. Carcinogenic compounds in gutkha, khaini and betel with accessories are represented [Table 5].[iv] A pictorial delineation of tobacco affecting male reproductive physiology causing infertility is too presented [Effigy iii].
Table 5
Concentration ranges of nicotine and carcinogenic compounds in commercial smokeless chewable tobacco products*
A pictorial depiction of tobacco smoking and chewing affecting male reproductive physiology in causing infertility
In the present study, 38% people were used to chew tobacco intensively, more than than ten packets/24-hour interval (10-l), which is enough to impact the sperm function. In some other signal, 13.7% subjects have been chewing tobacco more than 10 times/24-hour interval since 8 years, still they were normozoospermic; simply, the numbers of morphological abnormality cases were college. Normozoospermic subjects in intensive chewing cases indicated that some unknown protective mechanism is acting in those tobacco exposure cases. Hence, farther studies can answer whether, it is the antioxidant level only or any other factors that protects the man male reproductive system from tobacco toxicity during spermatogenesis.
CONCLUSION
It was evident that chewing tobacco had a meaning negative effect on the process of spermatogenesis, ultimately affecting sperm count, motility and morphology. Farther, intensive chewing alters the normal morphology of sperm either to crusade infertility or to cause irreversible epigenetic changes in futurity offspring. In this context, clinicians and fertility counselors need to exist more focused to control male infertility by intimating the sensation of this addiction to enhance the fertility potential; that will be more appealing than waiting for policy determination and implementation.
ACKNOWLEDGEMENTS
SP is supported past a research fellowship from SOA Academy. Authors are thankful to Prof. DK Roy, Dean for consequent encouragement. Authors are too grateful to Er. Gopabandhu Kar, Managing Member, SOA University and Vice-Chancellor, SOA Academy for the financial back up. The authors thank to Dr. Rupesh Dash, Scientist, Dr. Sanjeeb K. Sahoo, Scientist, Establish of Life Sciences, Bhubaneswar for providing facility for AFM study.
Footnotes
Source of Support: The present work is the in-house grant of Siksha O Anusandhan Academy, Bhubaneswar, for the partial fulfillment of the PhD work
Conflict of Involvement: None declared.
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