increasing incidence of infertility has been a global health as well as a social problem , where 35 - 40% of male partners are solely responsible .
apart from the well - known conventional causes , i.e. , 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 too contribute to male infertility .
tobacco consumption is one of the lifestyle factors that is often detrimental to human health as a whole . comparing 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 day lead to the deposition of almost equal amount of nicotine ; in addition , it leads to the incidence of mouth cancer .
however , other manifestations of human health were least focused in the study due to tobacco consumption . despite strong regulations and campaign , people do consume tobacco products that affect several physiological systems including the reproductive system .
recently , many states of india have banned the sale of chewing tobacco , but 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 different products . tobacco contains more than 30 mutagenic agents among which , nicotine is regarded as the most important component , in the particle phase .
further , of the total nicotine entering the body , an 80 - 90% is metabolized by the liver and a fraction of nicotine and its degraded products were detected in serum , urine , saliva , milk and seminal plasma .
nicotine affects the sperm plasma membrane and genetic integrity by their powerful oxidizing actions . in animal studies , the impairment of testicular histology and a reduction in diameter of seminiferous tubules as well as , a decrease in the index of sertoli cells , following 1.5 h of exposure to tobacco , per day ( 6 days , a week ) as cigarette smoke for 10 weeks , were reported .
indeed , the reduced fertility in males with impaired spermatogenesis , sperm motility , deleterious effect on germ cells and embryo development in tobacco exposure were recorded .
however , the threshold level that damages the integrity of male reproductive system is still obscure . a higher incidence of teratozoospermia in tobacco chewers was demonstrated , but related information on details of morphological anomalies of sperms is still scarce .
moreover , the intracytoplasmic sperm injection technique helps the treatment of severe teratozoospermia by microinjection of sperm to metaphase - ii oocyte ; nonetheless , failures in many cases due to delayed fertilization , abnormal cleavage rates and spontaneous abortions were known , which could be , a priory , due to defective sperm characteristics , those impeding the normal event of fertilization . as
the tobacco chewing habit is quite prevalent among south - east asia population ( 53.5% ) , 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 treatment .
this study should help a pertinent analysis of the problem of infertility treatment of male partners , as well as help public awareness in refraining from far - reaching this and other health concerns due to tobacco chewing .
this study from the nation of south - east 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 .
patients , attending the infertility unit of a tertiary health care center , from 2009 to 2012 for infertility treatment , were taken as subjects .
duration of tobacco chewing in the cohort of subjects was between 2 and 18 years of age .
patients were balkanized accordingly to the habit and the intensity of chewing , i.e. , less and more than 10 packets / day into two 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 .
semen samples were collected by masturbation into wide - mouth plastic container , in a room close to the andrology laboratory .
semen parameters were analyzed , according to the ( world health organization [ who ] ) standard criteria , i.e. , volume 2 ml , concentration 20 million / ml , total count 40 million , progressive motility  50% , vitality 75% and normal morphology > 15% ( kruger 's criteria ) . to determine the percentage of motile sperms ,
an aliquot of 10 l 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 's hemocytometer with requisite dilutions ( 1:2 , 1:5 , 1:9 , 1:20 ) , as per the who criteria .
sperm morphology was assessed in papanicolau - stained smears ( hematoxylene , orange - g and ea-50 stain ) using light microscopy , under the oil immersion at 1000 magnification . for a few semen samples ,
sperm morphology was also assessed by atomic force microscopy ( afm ) , for which the standardization was done by fixation of sperms in 2.5% glutaraldehyde in phosphate buffer saline ( pbs ) , for 1 h and the mixture was centrifuged at 200 - 300 g for 10 min .
sperm pellet was washed with pbs and smeared on a clean slide with appropriate dilution .
afm images were taken by using the jpk nanowizard ii ( germany ) , at intermittent contact ( air ) , which is called as tapping mode of operation with an ultrasharp silicon cantilever ( k = 40 n / m , resonant frequency = 300 khz ) .
vitality of sperms was estimated by the hypo - osmotic - swelling test by mixing equal volumes of semen and hypo - osmotic solution , prepared from 7.35 g sodium citrate and 13.5 g fructose in 1000 ml distilled water .
the mixture was incubated for 30 min at 37c , from which an aliquot of 10 l was immediately examined at the 400 magnification .
the percentage of swollen ( vital ) sperm was assessed by counting a minimum of 200 spermatozoa . using spss ,
version 20.0 ( 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 's correlation coefficient , r was determined for the intensity of tobacco chewing and sperm anomalies .
patients , attending the infertility unit of a tertiary health care center , from 2009 to 2012 for infertility treatment , were taken as subjects .
duration of tobacco chewing in the cohort of subjects was between 2 and 18 years of age .
patients were balkanized accordingly to the habit and the intensity of chewing , i.e. , less and more than 10 packets / day into two 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 .
semen samples were collected by masturbation into wide - mouth plastic container , in a room close to the andrology laboratory .
semen parameters were analyzed , according to the ( world health organization [ who ] ) standard criteria , i.e. , volume 2 ml , concentration 20 million / ml , total count 40 million , progressive motility  50% , vitality 75% and normal morphology > 15% ( kruger 's criteria ) . to determine the percentage of motile sperms , an aliquot of 10 l 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 's hemocytometer with requisite dilutions ( 1:2 , 1:5 , 1:9 , 1:20 ) , as per the who criteria .
sperm morphology was assessed in papanicolau - stained smears ( hematoxylene , orange - g and ea-50 stain ) using light microscopy , under the oil immersion at 1000 magnification . for a few semen samples ,
sperm morphology was also assessed by atomic force microscopy ( afm ) , for which the standardization was done by fixation of sperms in 2.5% glutaraldehyde in phosphate buffer saline ( pbs ) , for 1 h and the mixture was centrifuged at 200 - 300 g for 10 min .
sperm pellet was washed with pbs and smeared on a clean slide with appropriate dilution .
afm images were taken by using the jpk nanowizard ii ( germany ) , at intermittent contact ( air ) , which is called as tapping mode of operation with an ultrasharp silicon cantilever ( k = 40 n / m , resonant frequency = 300 khz ) .
vitality of sperms was estimated by the hypo - osmotic - swelling test by mixing equal volumes of semen and hypo - osmotic solution , prepared from 7.35 g sodium citrate and 13.5 g fructose in 1000 ml distilled water .
the mixture was incubated for 30 min at 37c , from which an aliquot of 10 l was immediately examined at the 400 magnification . the percentage of swollen ( vital ) sperm was assessed by counting a minimum of 200 spermatozoa .
using spss , version 20.0 ( 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 's correlation coefficient , r was determined for the intensity of tobacco chewing and sperm anomalies .
from the questionnaire , 30.2% ( 194/642 ) subjects were tobacco chewers , from whom , 50.5% of subjects chew gutkha , 27.9% 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 higher mean values than group i subjects .
further , with respect to sperm count , the mean values declined in 18.9% in group i subjects ; and decrease in mean values for motility , vitality and for normal morphology were 23.4 , 8.4 and 28.4% , respectively .
comparison of semen parameters between tobacco chewers , group i and non - chewers , group ii with 95% ci * or values of different semen parameters confirmed significant difference at p = 0.001 between the two groups , i and ii [ 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 : 1.5 - 3.09 ) .
similarly , probability of asthenozoospermia in group i subjects was 3.2 times higher odds ( or = 3.2 ; 95% ci : 2.05 - 4.9 ) and teratozoospermia 8.4 times was higher odds ( or = 8.4 ; 95% ci : 4.9 - 14.6 ) than those of group ii subjects .
however , no significant difference was observed for sperm vitality between the two groups ( or = 1.6 ; 95% ci : 0.739 - 3.6 ) .
further , effect 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 than 10 packets of tobacco per day ( intensive chewers ) and sub - group ib was subjects using less than 10 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 ) .
but , no significant difference was found for the total sperm count ( p = 0.534 , or = 0.81 ; 95% ci : 0.441 - 1.5 ) and motility values ( p = 1.0 , or = 1.1 ; 95% ci : 0.478 - 2.5 ) [ table 2 ] .
comparison of semen parameters between intensive tobacco chewers , sub - group ia and nonintensive tobacco chewers , sub - group ib * among tobacco chewers , 28.4% cases were oligoas - thenoteratozoospermic , 59.4% oligoasthenozoospermic and 11.2% were normozoospermic .
the presence of normozoospermic , which contributes to higher mean values as shown in tables 1 and 2 .
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 8.4 times higher to be of teratozoospermics in tobacco chewers than non - 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 1 ] .
double head , pin head , tapered head , bent neck , curved tail and tailless sperms were the several anomalous features abundantly marked .
an initial attempt for sperm morphological study by afm also showed significant abnormal features among tobacco chewers [ figure 2 ] .
however , 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 .
comparison of morphological abnormalities of spermatozoa between intensive tobacco chewers , sub - group ia and non - intensive tobacco chewers , sub - group 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 head with irregular acrosome ; ( d ) bent necked ; ( e ) cytoplasmic residues with tapered head ; ( f ) cytoplasmic residues and small acrosome ; ( g ) round head with abnormal mid - piece ;
( h ) long amorphous head ; ( i ) immature spermatozoa ; ( j ) abnormal mid - piece ; ( k ) double tailed ; ( l ) normal spermatozoa amplitude images of sperm by atomic force microscopy .
the two - dimensional images represent length and width in x and y - axis , respectively .
each small division represents 2  : ( a ) abnormal acrosome ; ( b ) abnormal mid - piece ; ( c ) bent neck with irregular acrosome ; and ( d ) double head with abnormal mid - piece application of pearson 's correlation also presented the strong positive correlation of head 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 change in mid - piece ( p = 0.122 ) and tail defects ( p = 0.411 ) were seen between the three groups [ table 4 ] .
although cytoplasmic residue was a mid - piece defect , to present the specific defects of sperms , here cytoplasmic residue was analyzed , separately .
hence , the total results depicted the findings on sperm morphological features in addition to clinically assigned semen parameters to find their effects among tobacco chewers .
the present retrospective study recorded a decline in quality of semen among the tobacco chewers .
of 642 subjects , 194 ( 30.2% ) had a significant decrease in sperm count , motility and impaired morphology . in tobacco chewers , 66% , 85.5% and 28.4% of subjects
were found be below the who standard criteria for sperm count , motility and morphology respectively .
this depicted significant effect 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 get addicted to the former , a priory , due to inebriating role of nicotine .
tobacco either in smoke or chewing forms has a harmful effect on human health causing gum recession , leukoplakia , cardiovascular disease and cancer of the oral cavity or larynx or pharynx .
in addition , it has negative effects on male reproductive system in thwarting the production of competent sperms , for the successful fertilization .
these results were corroborated by another similar report on tobacco causing adverse effect on semen quality in mumbai , india .
animal studies predicted the concentration - dependent effect of nicotine on function of male reproductive organs , spermatogenesis , litter size , and level of endocrine hormones , eventually fecundity .
nicotine is known to disturb the hypothalamus - pituitary axis by disrupting the testicular microcirculation . as testosterone acts on seminiferous tubules to initiate and maintain spermatogenesis , reduction of this sex hormone level
either by impaired leydig cell function or disturbance in the androgen / estrogen ratio could be a cause of decreased sperm counts .
further , in vitro studies indicated , nicotine at the concentration 1 mm significantly declined the motility of sperm , and the concentration of 70 ng / ml caused a decrease in the kinematics of sperms .
nicotine and other chemicals in tobacco probably cause either damage to mitochondrial genome or / and mitochondrial enzymatic activities or an impairment of function of the seminal vesicle affecting sperm motility .
there was no significant effect of chewing tobacco on the function of accessory gland , seen with a small number of 29 patients in a study , but a larger sample size could find a definitive conclusion on the damaging effects of tobacco .
however , seminal plasma of non - smokers could retrieve motility of sperms from smokers . in intensive tobacco chewers ,
normal morphology was affected more than sperm count and motility , corroborated elsewhere . even though sperms with irregular acrosome , bent neck and coiled tail were observed in non - intensive tobacco chewers , additional anomaly of head defects and cytoplasm residues
abnormal development of golgi proacrosomic vesicles and their improper attachment 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 . here
, it could be that certain chemical components interfere the secretory or normal activity of golgi body during spermiogenesis .
excess cytoplasmic residues of round spermatids are phagocytosed by sertoli cells leaving a small cytoplasmic droplet for osmotic balance and final tail elongation during epididymal transit .
epididymal dysfunction due to tobacco chewing may be a direct cause of retaining cytoplasmic residues , by preventing the timely loss of droplet and the development of secondary abnormalities in sperms .
excess cytoplasmic residues are also the source of reactive oxygen species which can cause damage to sperm membrane , proteins and deoxyribonucleic acid ( dna ) .
study on ultrastructure also revealed the presence of sertoli cells with polymorphic mitochondria with irregular cristae , spermatids with excess cytoplasm , abnormal acrosomes in nicotine treated rats .
another report was also found with curved tail , both bent and curved mid - piece in nicotine treated rats .
moreover , this preliminary study with afm revealed minute abnormal features of sperm head and neck by representing incandescent resolution and topographic view .
so , a more intensive study 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 increase gradually with intensive practice of chewing and residual chemicals remain for a prolonged period than in smoking cases .
but , the threshold concentration affecting male reproductive system is yet to be defined . again
the quantity of other chemicals , their specific effects on male reproductive system and pathological concentrations in different individuals is largely unknown .
the presence of pesticides , maleic hydrazide , chlordane , dichlorodiphenyl - trichloroethane , dichlorodiphenyldichloroethylene , dieldrin , endrine , heptachlor in smokeless tobacco might be causing these endocrinological disorders and damage of sperm dna .
carcinogenic compounds in gutkha , khaini and betel with accessories are represented [ table 5 ] .
a pictorial depiction of tobacco affecting male reproductive physiology causing infertility is too presented [ figure 3 ] .
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 10 packets / day ( 10 - 50 ) , which is enough to affect the sperm function . in another point , 13.7% subjects have been chewing tobacco more than 10 times / day since 8 years , still they were normozoospermic ; but , the numbers of morphological abnormality cases were higher .
normozoospermic subjects in intensive chewing cases indicated that some unknown protective mechanism is acting in those tobacco exposure cases .
hence , further studies can answer whether , it is the antioxidant level only or any other factors that protects the human male reproductive system from tobacco toxicity during spermatogenesis .
it was evident that chewing tobacco had a significant negative effect on the process of spermatogenesis , ultimately affecting sperm count , motility and morphology .
further , intensive chewing alters the normal morphology of sperm either to cause infertility or to cause irreversible epigenetic changes in future offspring . in this context ,
clinicians and fertility counselors need to be more focused to control male infertility by intimating the awareness of this addiction to enhance the fertility potential ; that will be more appealing than waiting for policy decision and implementation .
 aim : the aim of the following study is to find 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 design : retrospective study was conducted at infertility unit of a tertiary health care center , in a period of 3 years.materials and method : semen of 642 males were analyzed ; of them 194 men ( 30.2% ) were tobacco chewers and they were grouped according to their intensity of chewing ( < 10 and  10 packets / day ) . 
 counts , motility , 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 ] : 1.5 - 3.09 ) , motility ( or = 3.2 ; 95% ci : 2.05 - 4.9 ) , and normal morphology ( or = 8.4 ; 95% ci : 4.9 - 14.6 ) were significantly affected ( p = 0.001 ) in tobacco chewers than the non - chewing group . 
 further , in comparison to the intensity of tobacco chewing , patients with the intensive practice of using 10 packets / day had a significant effect on sperm morphology ( p = 0.003 , or = 2.7 ; 95% ci = 1.41 - 5.08 ) only . 
 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 significant changes were found in anomalies in mid - piece and tail.conclusion:the adverse impact of tobacco chewing on semen parameters was evident even with mild chewers , but with the intensive chewing practice , phenotypes of sperms , mainly defects in the head and cytoplasmic residue were severely affected . 
INTRODUCTION
MATERIALS AND METHOD
Subjects
Sample collection and semen analysis
Statistical analysis
RESULTS
DISCUSSION
CONCLUSION