smoking was shown to result in vascular lesions and changes in some characteristics of the blood , leading to hypoxia - induced injury of various tissues , including the organ of hearing [ 13 ] . researchers from the university of washington in seattle ( united states ) and university of melbourne ( australia ) analyzed the 19802012 data on the prevalence of cigarette smoking in 187 countries . they showed that the number of smokers older than 15 years of age increased from 721 million in 1980 to roughly one billion ( 967 million ) in 2012 . also , the total number of smoked cigarettes increased , from 4.96 billion to 6.25 billion annually . currently , the population of male and female smokers is estimated at 31% and 6.2% worldwide , respectively ; 30 years earlier , these were 41% and 10% , respectively . according to the who data from 2011 , nevertheless , 27.2% of adults in poland smoke and mean cigarette consumption is 15.4 per day . the percentages of male and female smokers in poland are estimated at 33.6% and 20.5% of the adult population , respectively . the vast majority of polish smokers are individuals between 45 and 59 years of age ( 36% ) , with 62.5% having primary or vocational education and 43% currently unemployed . exposure to harmful components of cigarette smoke leads to disorders of lipid metabolism and vascular endothelial dysfunction , which is reflected by enhanced atherosclerosis and increase in blood viscosity . carbon oxide present in tobacco smoke is a substrate for carboxyhemoglobin synthesis . despite markedly higher affinity than hemoglobin the vasoconstrictive effect of nicotine results in an impairment of tissue perfusion , which may be associated with cellular dysfunction in the case of chronic exposure to tobacco smoke . according to one hypothesis , the harmful effects of tobacco smoke on hearing are associated with the toxic dysfunction of nicotinic acetylcholine receptors ( nachrs ) , a vital component of the hearing pathway . moreover , the toxic components of cigarette smoke were shown to impair the redox system , which was reflected by enhanced tissue hypoxia and injury , inter alia impairment of the active mechanisms of the outer hair cells ( ohcs ) of the cochlea [ 1315 ] . measurement of oaes is the only available non - invasive test for selective analysis of the ohc activity , enabling simple , objective , and highly sensitive functional examination of the hearing organ . the aim of this study was to analyze the sex - specific effect of cigarette smoking on the results of subjective and objective examination of hearing , namely ultra - high frequency pta , spontaneous otoacoustic emission ( soae ) , and ceoae and dpoae levels . the study included 84 healthy volunteers aged between 25 and 45 years ( mean 34 years ) , among them 41 non - smokers ( mean age 33.3 years ) and 43 smokers ( mean age 34.7 years ) . the sample comprised 46 women ( 25 non - smokers and 21 smokers ) and 38 men ( 16 non - smokers and 22 smokers ) . the group of smokers included the individuals who smoked at least 15 cigarettes per day for at least 7 years . the exclusion criteria of the study were : abnormal result of otoscopic examination , history of ear problems , conductive hearing impairment , exposure to noise and ototoxic factors , disorders of cholesterol metabolism , arterial hypertension , chronic metabolic disorders ( such as diabetes mellitus or kidney diseases ) , head injuries associated with the loss of consciousness , family history of genetic - related hearing impairment , disorders of the central nervous system , other acute or chronic systemic conditions , and abnormal body mass index ( bmi ) . mean body height of the study subjects was 172 cm ( range 155190 cm ) . the protocol of the study was approved by the local bioethics committee at the medical university of silesia ( decision no . all the experiments were conducted in accordance with the declaration of helsinki ( revision 6 , 2008 ) regarding the principles of human experimentation . written informed consents were obtained from all the participants prior to any procedure included in the study protocol . the protocol of the study included history - taking , otoscopic examination , tympanometry , pta , and the evaluation of different types of oaes . pta included air - conduction audiometry at 2508 000 hz , bone - conduction audiometry at 250400 hz , and ultra - high - frequency pta at 8 00020 000 hz . pta was performed in a sound - treated room , using an ac-40 interacoustics audiometer . all otoacoustic emission tests ( ceoaes , dpoaes , and soaes ) were conducted with an echoport ilo292 analyzer system , version 6.0 ( otodynamics ) . prior to the test , the software automatically checked the resonance of the external ear canal and the probe sealing . ceoaes were recorded in a nonlinear mode with 80-millisecond clicks presented at 853 db pspl and at a 50 per second rate . the responses to a total of 260 sets of clicks were averaged above the noise rejection level of 45 db . the ilo292 system averages into 2 alternate buffers : a and b. the signal is estimated from the ( a+b)/2 waveform , and the noise from the a the reproducibility is defined as the zero - lag correlation coefficient between the a and b buffers . ceoaes were measured within the range of 1.05.0 khz ; the overall ceoae response was analyzed . dpoaes were measured using a 2-channel probe using the same ilo292 analyzer system . for ceoaes , a soft adapter was used to provide precise adaptation of the probe to the wall of the external ear canal . the otoacoustic emissions evoked by 2 tonal signals of different frequencies ( f1 and f2 ) in a constant relation ( f2/f1=1.22 ) were recorded . the levels of primary tones were different : l1=71 db spl and l2=60 db spl ( according to neely s and gorg s formula : l1=44 + 0.45l2 ) ; the tones were delivered at a constant frequency ratio f2/f1=1.22 . the dp - grams for 2f1f2 were collected for the f2 frequencies of 842 hz to 7996 hz with the resolution of 4 points per octave . subsequently , dpoaes were tested at the following intensities of primary stimuli , and the distortion 2f1f2 was analyzed . dpoaes were tested as a function of dp - gram and the input / output function at l2=40 db spl , 45 db spl , 50 db spl , 55 db spl , 60 db spl , 65 db spl , and 70 db spl . the input / output function was analyzed at 1000 hz , 1500 hz , 2000 hz , 3000 hz , 4000 hz , 5000 hz , and 6000 hz . the ceoaes were considered present at resp 3 db spl and repro > 75% , whereas dpoaes whenever the signal - to - noise ratio ( s / n ) was higher than at 3 db , irrespective of frequency . statistical analysis was carried out with a statistica 8.0 pl package ( statsoft , united states ) . the student t - test and the mann - whitney u - test were used for the intergroup comparisons of normally and non - normally distributed / ranked variables , respectively ( non - smokers vs. smokers , female non - smokers vs. male non - smokers , female non - smokers vs. female smokers , male non - smokers vs. male smokers , and female smokers vs. male smokers ) . the bonferroni correction for the repeated measurements was applied . we did not conduct an anova ( except from ceoae  repro , resp , noise ) due to the characteristics of data distribution ( lack of normality ) and their specific character ( the measurements were taken at 5-unit intervals , up to a maximum value ) . the results of pta and ultra - high frequency pta were recorded to the nearest 5 unit of the interval scale . due to lack of normal distribution , the mann - whitney u - test was used with bonferroni correction for repeated measurements . since the results for dpoae were not distributed normally , the mann - whitney u - test was used with bonferroni correction for repeated measurements . the only variable with normal distribution was dpoae ( noise ) ; therefore , the student t - test was conducted with bonferroni correction for repeated measurements . in the case of ceoae , resp ( db ) , repro ( % ) , and noise ( db ) variables were distributed normally ; therefore , the student t - test was used to compare them . stab ( % ) was the only variable without a normal distribution ; therefore , the mann - whitney u - test was conducted . the statistical characteristics of the analyzed variables are presented as means and their standard deviations ( sd ) . smokers and non - smokers did not differ significantly in terms of their hearing thresholds at 25020 000 hz . nevertheless , the smokers presented with slightly higher hearing thresholds at all the frequencies examined ( p>0.05 ) : pta thresholds at 2508 000 ( figures 15 ) and pta thresholds at ultra - high frequencies ( figures 610 ) . the levels of ceoaes is smokers were always lower than in non - smokers , but most of these differences did not prove significant on statistical analysis . the only statistically significant differences in the overall ceoae levels were found when the results of male smokers were compared with those of male non - smokers ( p=0.026 ) and female smokers ( p=0.001 ) ( tables 15 ) . this suggests that , in contrast to smoking women , male smokers are at increased risk of functional ohc impairment . the analyzed groups did not differ significantly in terms of their dpoae levels at various frequencies , both in snr analysis and when the overall response level was considered . the only exception pertained to the level of dpoaes at f2=1 685 hz , which was significantly lower in male smokers than in male non - smokers . irrespective of the testing conditions , no significant intergroup differences were found with regards to the background noise level . aside from significant differences between smokers and non - smokers overall , we also found significant differences between male smokers and male non - smokers , as well as between male and female smokers . irrespective of the testing conditions , no significant intergroup differences were found with regards to the background noise level . smokers presented with significantly lower levels of dpoaes than non - smokers at the following frequencies ( f2 ) and stimulus intensities ( l2 ) : 1000 hz + 60 db spl and 45 db spl , 1 500 hz + 70 db spl , 65 db spl , 60 db spl and 40 db spl , 2 000 hz + 60 db spl or 55 db spl , and 3000 hz + 60 db spl , 50 db spl and 45 db spl . these findings suggest that smoking impairs both active and passive mechanisms of the cochlea , especially at lower and moderate frequencies ( 13 khz ) . compared to male non - smokers , male smokers presented with significantly lower dpoae levels in an i / o function at the following frequencies ( f2 ) and stimulus intensities ( l2 ) : 1 000 hz + 60 db spl , 50 db spl , 45 db spl and 40 db spl , 1500 hz + 70 db spl and 65 db spl , 2 000 hz + 70 db spl , 65 db spl , 60 db spl and 55 db spl , 3000 hz + 45 db spl and 40 db spl , and 4000 hz + 70 db spl . unlike in men , female smokers and non - smokers did not differ significantly in terms of their dpoae levels in an i / o function . comparative analysis of male and female smokers showed that the former presented with significantly lower dpoae levels in an i / o function at the following frequencies ( f2 ) and stimulus intensities ( l2 ) : 1000 hz + 60 db spl , 55 db spl , 50 db spl and 45 db spl , 2000 hz + 70 db spl , 65 db spl , 60 db spl and 40 db spl , and 5000 hz + 70 db spl , 60 db spl and 55 db spl . in contrast , male and female non - smokers did not differ significantly in terms of their dpoae levels in an i / o function . altogether , the aforementioned data suggest that smoking impairs the ohc function in men , but not in women . the results of soae analysis were consistent with the abovementioned data on dpoae levels in an i / o function . soaes were observed in 4.3% and 25% of male smokers and non - smokers , respectively , and in 47.6% and 60.5% of female smokers and non - smokers , respectively . smokers and non - smokers did not differ significantly in terms of their hearing thresholds at 25020 000 hz . nevertheless , the smokers presented with slightly higher hearing thresholds at all the frequencies examined ( p>0.05 ) : pta thresholds at 2508 000 ( figures 15 ) and pta thresholds at ultra - high frequencies ( figures 610 ) . the levels of ceoaes is smokers were always lower than in non - smokers , but most of these differences did not prove significant on statistical analysis . the only statistically significant differences in the overall ceoae levels were found when the results of male smokers were compared with those of male non - smokers ( p=0.026 ) and female smokers ( p=0.001 ) ( tables 15 ) . this suggests that , in contrast to smoking women , male smokers are at increased risk of functional ohc impairment . the analyzed groups did not differ significantly in terms of their dpoae levels at various frequencies , both in snr analysis and when the overall response level was considered . the only exception pertained to the level of dpoaes at f2=1 685 hz , which was significantly lower in male smokers than in male non - smokers . irrespective of the testing conditions , no significant intergroup differences were found with regards to the background noise level . aside from significant differences between smokers and non - smokers overall , we also found significant differences between male smokers and male non - smokers , as well as between male and female smokers . irrespective of the testing conditions , no significant intergroup differences were found with regards to the background noise level . smokers presented with significantly lower levels of dpoaes than non - smokers at the following frequencies ( f2 ) and stimulus intensities ( l2 ) : 1000 hz + 60 db spl and 45 db spl , 1 500 hz + 70 db spl , 65 db spl , 60 db spl and 40 db spl , 2 000 hz + 60 db spl or 55 db spl , and 3000 hz + 60 db spl , 50 db spl and 45 db spl . these findings suggest that smoking impairs both active and passive mechanisms of the cochlea , especially at lower and moderate frequencies ( 13 khz ) . compared to male non - smokers , male smokers presented with significantly lower dpoae levels in an i / o function at the following frequencies ( f2 ) and stimulus intensities ( l2 ) : 1 000 hz + 60 db spl , 50 db spl , 45 db spl and 40 db spl , 1500 hz + 70 db spl and 65 db spl , 2 000 hz + 70 db spl , 65 db spl , 60 db spl and 55 db spl , 3000 hz + 45 db spl and 40 db spl , and 4000 hz + 70 db spl . unlike in men , female smokers and non - smokers did not differ significantly in terms of their dpoae levels in an i / o function . comparative analysis of male and female smokers showed that the former presented with significantly lower dpoae levels in an i / o function at the following frequencies ( f2 ) and stimulus intensities ( l2 ) : 1000 hz + 60 db spl , 55 db spl , 50 db spl and 45 db spl , 2000 hz + 70 db spl , 65 db spl , 60 db spl and 40 db spl , and 5000 hz + 70 db spl , 60 db spl and 55 db spl . in contrast , male and female non - smokers did not differ significantly in terms of their dpoae levels in an i / o function . altogether , the aforementioned data suggest that smoking impairs the ohc function in men , but not in women . the results of soae analysis were consistent with the abovementioned data on dpoae levels in an i / o function . soaes were observed in 4.3% and 25% of male smokers and non - smokers , respectively , and in 47.6% and 60.5% of female smokers and non - smokers , respectively . the effects of isolated exposure to tobacco smoke are extremely difficult to determine because smokers are frequently co - exposed to other ototoxic factors , especially in an occupational setting [ 1820 ] . moreover , the differences in male and female physiology should be considered ; namely , the potential protective effect of hormonal factors on the female hearing organ . finally , the accurate evaluation of the hearing effects requires a group of individuals with sufficiently long and extensive exposure to the components of tobacco smoke . furthermore , the selection of an appropriate method for hearing examination is an important issue since not every test is suitable for detection of changes at a subclinical level . evaluation of otoacoustic emissions is an objective and highly sensitive method for hearing assessment [ 1618,23,24 ] . the sensitivity of this test can be improved by the use of various intensities of l1 and l2 stimuli , as well as by the implementation of an input / output function [ 2426 ] . the smokers and non - smokers participating in our study did not differ significantly in terms of their results of pta at a 25020 000 hz frequency range . however , despite the lack of statistically significant differences , the hearing threshold of smokers was slightly higher than in non - smokers . although the hearing threshold at a standard spectrum did not exceed 25 db hl in any of the subjects participating in this study , the smokers presented with a 210 db higher hearing thresholds than the controls . however , the smokers and non - smokers did not differ significantly in terms of their hearing thresholds at high frequencies . in contrast , paschoal and azevedo   found significant differences in the audiometric hearing thresholds of smokers and non - smokers at 8 khz , 12.5 khz , and 14 khz . also , oliveira and lima   showed that the individuals who smoked for at least 5 years presented with significantly higher ( albeit within a normal limit ) hearing thresholds at a standard spectrum and at high frequencies than the subjects who never smoked . however , this study involved a relatively small sample of smokers ( n=30 ) , and its authors did not provide information about the daily number of cigarettes smoked in this group . basar and belgin   examined 30 individuals with a 10-year history of smoking at least 1 package per day and 20 non - smoking controls , and found that the former presented with significantly higher hearing thresholds solely at 16 khz and 18 khz . sousa et al .   analyzed the exposure of 625 volunteers to various risk factors of hearing impairment . neither pta nor speech audiometry confirmed the role of tobacco smoking as a risk factor for this condition . the influence of tobacco smoking and noise on hearing , examined by means of tone audiometry at a standard spectrum , was also analyzed by pouryaghoub et al . ; they found that a group of 206 smokers was characterized by significantly higher hearing threshold at 4 khz when compared to 206 non - smoking controls . aside from smoking , the hearing impairment documented in some of our participants might be related to their sex , age ( up to 67 years ) , and/or exposure to noise . an important study analyzing the effect of sex on the auditory consequences of smoking was conducted by uchida et al . . they found that the results of pta at 4000 hz were significantly worse in male smokers than in male non - smokers , but a similar phenomenon was not observed in the case of female smokers and non - smokers . conducted a meta - analysis of 15 studies published between 1966 and 2003 , in order to determine the effects of cigarette smoking on the results of pta . they documented the unfavorable effect of smoking in 9 out of the 15 analyzed studies ; the lack of such an association in the remaining studies suggests that the relationship between smoking and hearing impairment is still not completely understood . previous research on click - evoked otoacoustic emissions   showed that smokers present with lower ceoae levels than non - smokers . paschoal and azevedo   did not observe ceoaes in 13.9% and 2.8% of smokers and non - smokers , respectively ( p=0.016 ) . the levels of ceoaes do not seem to be modulated by the age of smokers . interestingly , a study of ceoaes in newborns whose mothers smoked during pregnancy demonstrated not only the functional impairment of sound perception , but also the structural abnormalities of the hearing organ . in our study , male smokers presented with significantly lower ceoae levels than male non - smokers and female smokers . to the best of our knowledge , this was the first study to demonstrate sex - specific differences in smokers ceoae levels . apart from ceoaes , we evaluated otoacoustic emissions as a dp - gram function . analyzed the dp - grams obtained using high ( l1=l2=70 db spl ) and moderate ( l1=65 db spl , l2=50 db spl ) intensity of stimulation , and showed that smokers presented with significantly lower dpoae levels at all frequencies . in contrast , we used l1=71 db spl and l2 = 60 db spl stimulus intensities and did not document significant differences in dpoaes levels of smokers and non - smokers on most comparisons , also when adjusted for sex . torre et al .   did not find a significant effect of smoking on dpoae levels ( 2.38.0 khz ) , but their results might have been confounded by selection bias , since it included subjects who smoked no longer than for 1 year , which might be an insufficient exposure to tobacco smoke . furthermore , we showed that compared to male non - smokers , male long - term smokers presented with significantly lower dpoae levels at 1685 hz ; this suggests a sex - specific effect of smoking on dpoaes . the results of our analysis of dpoaes in an input / output function , being a highly sensitive test for the active and passive mechanisms of the cochlea , are consistent with the data published by negley et al . . these authors showed that a gradual increase in the stimulation intensity ( from 20 db spl to 80 db spl , at 10-db intervals ) is reflected by a f2 frequency - specific increase in the i / o emission , by 10 db , 8 db , and 5 db for 2 khz , 4 khz , and 8 khz , respectively . in our study , statistically significant differences between smokers and non - smokers were observed at 1 khz , 1.5 khz , 2 khz , and 3 khz . moreover , we found significant differences between male smokers and male non - smokers at 1 khz , 1.5 khz , 2 khz , 3 khz , and 4 khz , as well as between male and female smokers at 1 khz , 2 khz , and 5 khz . altogether , these findings suggest that smoking exerts particularly unfavorable effects on the cochlear ohc in men , but not in women . importantly , we showed that the incidence soaes in smokers was significantly lower than in non - smokers . , our study is the first to demonstrate sex - specific differences in ceoae , dpoae , and oae responses of smokers . using an objective method for otoacoustic emission analysis , we showed that the smoking - related alterations were more pronounced in men than in women . these sex - specific differences in otoacoustic emission levels might reflect the influence of genetic , hormonal , behavioral , and/or environmental factors . previous studies documented evident sex - specific differences in the ceoae levels of infants and children . cassidy and ditty   showed that compared to male newborns , female newborns present with significantly higher ceoae levels at 1.6 khz , 2.4 khz , 3.2 khz , and 4.0 khz . demonstrated that mean ceoae levels in female neonates are higher than in male neonates ( 22.1 db spl vs. 21.4 db spl ) . interestingly , the same study documented significant differences in the ceoae levels recorded in the right and left ear ( 22.4 db spl vs. 21 db spl ) . in another study , 12-year - old girls were shown to present with significantly higher ceoae levels than their male peers . however , in contrast to aidan et al . , the authors of this study did not observe a bilateral asymmetry in ceoae levels . although we did not reveal sex - specific differences in the otoacoustic emission levels of non - smokers , it can not be excluded that men present with a genetically determined ( i.e. , sex hormone - independent ) greater susceptibility of cochlear ohcs to the ototoxic components of tobacco smoke . the results of experimental studies point to a potential protective effect of female sex hormones as an explanation of sex - specific differences in hearing . estrogens , 17-estradiol , 17-estradiol , estrone , and estriol were shown to protect against gentamicin - induced outer hair cell death ; the effect of 17-estradiol is mediated by estrogen receptor ( er ) . interestingly , the expression of estrogen receptors er and er in the inner ear ( i.e. , in the nuclei of stria vascularis , outer and inner hair cells , spiral ganglion cells , vestibular ganglion cells , and vestibular dark cells ) is known to decrease with age . these findings are consistent with the results of clinical studies . for example , kilicdag et al .   demonstrated that estrogen therapy may protect against hearing loss in aging postmenopausal women . taking this evidence into account , it can be hypothesized that female sex hormones may also protect against tobacco smoke - induced hearing impairment . behavioral factors should also be considered as a potential cause of sex - specific differences in susceptibility to tobacco smoke - related hearing loss . men not only smoke more , but also use stronger cigarettes , as well as other stimulants [ 4143 ] . our study included 21 women and 22 men , who smoked at least 15 cigarettes per day for at least 7 years . we did not compare , however , the total number of cigarettes smoked by the female and male participants . consequently , our male smokers might be exposed to tobacco smoke more often and for a longer period of time than female smokers . finally , the influence of environmental chemical ototoxins should be taken into account as a confounding factor resulting in greater impairment of hearing in our male smokers . to summarize , tobacco smoke likely induces an array of subclinical changes in the organ of hearing , especially in males . therefore , appropriate strategies preventing resultant hearing loss should be implemented before it will manifest clinically and impair patient functioning . these strategies should be adjusted for these documented sex - specific differences in susceptibility to tobacco smoke - induced hearing impairment . this study showed that smoking induces an array of subclinical changes in the organ of hearing . specifically , while it does not modulate the hearing threshold determined with pta at low , moderate , and high frequencies , it causes a significant decrease in oae levels , but only in males .
backgrounda number of studies have documented the influence of cigarette smoking on hearing . 
 however , the association between sex and hearing impairment in smokers as measured by otoacoustic emissions ( oaes ) has not been clearly established . 
 the aim of this study was to analyze sex - specific effects of smoking on hearing via conventional and ultra - high - frequency pure tone audiometry ( pta ) , and oaes , specifically spontaneous oaes , click - evoked oaes , and distortion - product oaes.material/methodsthe study included 84 healthy volunteers aged 2545 years ( mean 34 ) , among them 46 women ( 25 non - smokers and 21 smokers ) and 38 men ( 16 non - smokers and 22 smokers ) . the protocol of the study included otoscopic examination , tympanometry , low- , moderate- , and ultra - high - frequency pta , evaluation of spontaneous click - evoked ( ceaoes ) and distortion - product otoacoustic emissions ( dpoaes ) , assessment of the dp - grams for 2f1-f2 ( f1 from 977 to5 164 hz ) , and input / output function at l2 primary tone level of 4070 db spl.resultssmokers and non - smokers did not differ significantly in terms of their hearing thresholds assessed with tone audiometry . 
 male smokers presented with significantly lower levels of ceaoes and dpoaes than both male non - smokers and female smokers.conclusionssmoking does not modulate a hearing threshold determined with pta at low , moderate , and ultra - high frequencies , but causes a significant decrease in oae levels . 
 this effect was observed only in males , which implies that they are more susceptible to smoking - induced hearing impairment . 
 sex - specific differences in otoacoustic emissions level may reflect influences of genetic , hormonal , behavioral , and/or environmental factors .