tympanometry measures the middle ear function by assessing middle ear admittance . a probe tone frequency of 226 hz distinguishes a healthy middle ear from one presenting conducting hearing impairment . when an individual has a high impedance pathology such as otitis media with effusion ( ome ) , the 226 hz admittance tympanogram is frequently flat ; therefore , it is not possible to measure the tympanometric width and peak pressure . in these cases , a higher frequency admittance probe at 1000 hz can capture the changes not identified by the standard 226-hz tympanogram . with a 1000-hz probe , frequently , the peak compensated static acoustic admittance is doubled - shaped ( double - peak ) . because the middle ear resonance is approximately 1000 hz [ 24 ] , it is plausible that the 1000-hz admittance probe might offer more specific information about the condition of the middle ear and detect subclinical alterations . according to zhiqi et al .   and abou - elhamd et al . , the probe tone frequency of 1000 hz better discriminates middle ear status in otitis media than the 226-hz frequency . shahnaz   suggests that higher probe tone frequencies ( in relation to 226 hz ) are more accurate for detecting otosclerotic ears , but they considered a probe tone frequency of 630 hz to be more sensitive in the pathologies they examined . many authors have studied the influence of the middle ear on distortion product  otoacoustic emissions ( dpoae ) measurements [ 812 ] . dpoaes are generated by non - linear cochlear processes , when the ear is stimulated simultaneously by 2 pure tones ( stimuli f1 and f2 ) . dpoaes can detect outer hair cell ( ohc ) function and their active mechanism of vibration . in clinical terms , the most robust dpoae response is located at the 2f1f2   frequency . dpoaes can be measured either by changing the frequency and having the stimulus amplitude l1 and l2 fixed ( dp - gram ) , or fixing the frequency and changing the stimulus amplitude ( dpoae input / output function , i / o ) . for the latter , the most accepted relationship between the amplitudes of the 2 tones ( code - named as the scissor paradigm ) is defined as l1=0.4l2 + 39 db [ 1518 ] with a f2/f1=1.22 . data in the literature have suggested that it is possible to extrapolate dpoae i / o functions to estimate hearing threshold . in this context , the compressive characteristics of the basilar membrane can be studied in a healthy or damaged cochlea . however , some criteria must be considered , such as the presence of dpoae responses at l2 levels=65 db spl and normal middle ear conditions . the non - inclusion of these criteria is a result of the great variability inherent in this method , which has high specificity but low sensitivity . studied dpoae i / o functions in pigmented guinea pigs with induced middle and inner ear alterations . they found a dpoae amplitude reduction in the presence of middle ear alterations , but without modification of the slope . they assumed that this information could be useful to distinguish middle and inner ear alterations by considering that inner ear alterations usually present steeper slopes in dpoae i / o functions . pathologies that alter the impedance of the middle ear consecutively modify the dpoae amplitude because the stimulus to the inner ear is drastically altered . for a better diagnostic evaluation , the aim of this study was to provide more information on the relationship between clinical procedures that offer detailed descriptions of the middle ear . data from 2 dpoae protocols ( dp - gram , dp input / output functions ) were compared with tympanometric data from patients assessed with a 1000-hz probe . the data were compared in the context of elucidating whether the tympanometric pattern , caused by a modification of the middle ear resonance , could have any effect on the dpoae data . subjects were assigned into 2 groups according to their 1000 hz tympanometry patterns : 20 subjects ( mean age 22.64.02 y ) presenting double - peak 1000 hz tympanograms were assigned to the double - peak group ( dpg ) , and 16 subjects ( mean age 23.53.93 y ) presenting single - peak tympanograms were assigned to the single - peak group ( spg ) . exclusion criteria ( for both groups ) were : alcohol and drug dependence , vertigo , and treatment with salicylates . in addition , ears which did not present a single or a double tympanometry peak were not considered . after the application of the exclusion criteria , only 31 ears were assessed in each group . all participated subjects presented : normal audiometric thresholds at 250 , 500 , 1000 , 2000 , 3000 , 4000 , 6000 , and 8000 hz ( 20 db hl ) ; type  a  226 hz tympanometry patterns and tympanometric peak pressures from 50 to + 50 dapa ; acoustic reflexes of 70100 db hl at 1000 hz ; dpoae responses with signal / noise ratios 6 db at 2002 , 3174 , and 4004 hz . the details of the testing procedures were explained to all subjects who provided written informed consent . each subject was assessed for approximately 1 h , following this protocol : medical history compilation ; tympanometry measurements to assess middle ear status using the gsi33 middle ear analyzer ( v2 ; grason - stadler , madison , wi ) with a probe tone of 226 hz and 1000 hz ; determination of the acoustic reflex thresholds at 1000 hz ; pure - tone audiometry with a gsi 61 audiometer ( grason - stadler , madison , wi ) at frequencies of 1000 hz , 2000 hz , 3000 hz , 4000 hz , 6000 hz , 8000 hz , 500 hz , and 250 hz , with a 5-db hl resolution ; dpoaes ( dp - grams ) were measured in an audiometric cabin using the ilo 92 system ( version 6 , otodynamics ltd , hatfield , uk ) at 1000 , 1501 , 2002 , 3174 , 4004 , and 6384 hz ( f2 ) . the l1 was set=65 db spl and the l2=55 db spl , the f1/f2 ratio=1.22 and the noise floor was set to 5 db ; dpoae i / o functions were estimated at 1501 , 2002 , 3174 , 4004 , and 6384 hz . ( 1998 )  l1=(0.4l2)+39 db spl  was used , with l2 starting at 75 db spl and decreasing in 5-db spl steps until reaching 30 db spl . subjects were assigned into 2 groups according to their 1000 hz tympanometry patterns : 20 subjects ( mean age 22.64.02 y ) presenting double - peak 1000 hz tympanograms were assigned to the double - peak group ( dpg ) , and 16 subjects ( mean age 23.53.93 y ) presenting single - peak tympanograms were assigned to the single - peak group ( spg ) . exclusion criteria ( for both groups ) were : alcohol and drug dependence , vertigo , and treatment with salicylates . in addition , ears which did not present a single or a double tympanometry peak were not considered . after the application of the exclusion criteria , only 31 ears were assessed in each group . all participated subjects presented : normal audiometric thresholds at 250 , 500 , 1000 , 2000 , 3000 , 4000 , 6000 , and 8000 hz ( 20 db hl ) ; type  a  226 hz tympanometry patterns and tympanometric peak pressures from 50 to + 50 dapa ; acoustic reflexes of 70100 db hl at 1000 hz ; dpoae responses with signal / noise ratios 6 db at 2002 , 3174 , and 4004 hz . the details of the testing procedures were explained to all subjects who provided written informed consent . each subject was assessed for approximately 1 h , following this protocol : medical history compilation ; tympanometry measurements to assess middle ear status using the gsi33 middle ear analyzer ( v2 ; grason - stadler , madison , wi ) with a probe tone of 226 hz and 1000 hz ; determination of the acoustic reflex thresholds at 1000 hz ; pure - tone audiometry with a gsi 61 audiometer ( grason - stadler , madison , wi ) at frequencies of 1000 hz , 2000 hz , 3000 hz , 4000 hz , 6000 hz , 8000 hz , 500 hz , and 250 hz , with a 5-db hl resolution ; dpoaes ( dp - grams ) were measured in an audiometric cabin using the ilo 92 system ( version 6 , otodynamics ltd , hatfield , uk ) at 1000 , 1501 , 2002 , 3174 , 4004 , and 6384 hz ( f2 ) . the l1 was set=65 db spl and the l2=55 db spl , the f1/f2 ratio=1.22 and the noise floor was set to 5 db ; dpoae i / o functions were estimated at 1501 , 2002 , 3174 , 4004 , and 6384 hz . the stimulus paradigm proposed by kummer et al . ( 1998 )  l1=(0.4l2)+39 db spl  was used , with l2 starting at 75 db spl and decreasing in 5-db spl steps until reaching 30 db spl . figure 1 shows boxplots with the distribution of the pure - tone thresholds data from the 2 groups . a higher threshold variability was observed at frequencies 3000 hz in the double - peak group . in comparison , in the single - peak group , significant between - group differences were observed at 3000 , 6000 , and 8000 hz ( p=0.040 and 0.038 , p=0.047 , respectively ) . the subjects assigned to the 1000 hz double - peak group presented higher values of ear canal volume , peak compensated static acoustic admittance ( in mmho ) , and peak pressures ( dapa ) than in the single - peak group . figure 2 shows the boxplot data with the distribution of the dpoae amplitudes for the 2 tested groups . overall , the double - peak group presented higher mean dpoae values ( except at 6384 hz ) . the dpoae amplitude in the double - peak group showed a decreasing pattern with increasing frequency in comparison to the single - peak group . for example , at 1001 and 1501 hz , the double - peak group had a dpoae amplitude 8.86 db and 4.68 db higher than in the single - peak group . at the next 3 tested frequencies ( 2002 , 3174 , and 4004 hz ) , the differences become smaller . the dpg group presented dpoae amplitudes 0.14 , 1.17 , and 1.18 db higher than the spg . at the frequency of 6384 hz , the subjects of the spg group showed a mean dpoae amplitude 2.39 db higher than the corresponding dpg value . the dpoae i / o functions were analyzed with reference to the l2 stimulus values . at low stimuli ( 30 and 35 db ) and at all tested frequencies , the spg s dpoae amplitudes were higher . at the frequency of 1501 hz , the spg presented higher amplitudes at inputs from 40 to 75 db , but significant differences were observed at 65 , 70 , and 75 db ( p=0.05 , 0.01 , and 0.03 , respectively ) . at 2002 hz , the input intensities of 45 , 50 , 55 , and 75 db demonstrated higher amplitudes in the spg . at 3174 hz , higher amplitudes in the spg were observed at input intensities between 30 to 55 db and 65 db . at 4004 hz , the spg demonstrated higher values from 30 to 60 ( input intensities ) . finally , the frequency of 6384 hz demonstrated higher responses to all input intensities tested . figure 3 show the means of dpoae i / o functions of both groups . despite the amplitude differences between groups , the significant differences were observed only in 4 of the 50 tested points ( 5 tested frequencies  10 steps per frequency ) . the objective of this study was to evaluate the middle ear information as derived from 2 different clinical procedures . in this context , we have evaluated the effect of 2 distinct 1000-hz tympanogram patterns ( single and double ) on the dpoae amplitude and i / o functions . tympanometric data from table 1 show that the 2 groups of tested subjects are characterized by a significant difference in admittance ( y ) . this could suggest that there is an association between the double - peak data and the middle ear s increased mobility ( higher admittance ) . a probable contribution might be related to a normal physiological variability , but the sequelae of high - impedance pathologies ( such as otitis media ) should also be considered because they might alter admittance at high frequencies . in terms of audiometric thresholds , the double - peak group presented higher threshold variability ( distribution spread ) at frequencies > 3000 hz and the opposite at frequencies 500 hz . statistical differences were observed at 3000 , 6000 , and 8000 hz . at 4000 hz , a borderline difference ( p=0.06 ) was also observed . at frequencies 500 hz , the dpg group presented lower threshold values than the spg , but the differences were not significant . surprisingly , the data showing threshold patterns reported in previous studies were not from normal subjects , but rather from patients presenting middle - ear complications , probably because the subjects in this study were classified ( to a double- or single - peak group ) according to their 1000 hz tympanometry pattern profiles . examined types of otitis media in a nigerian population , reporting higher thresholds at high frequencies in ears with chronic suppurative otitis media and higher thresholds at low frequencies in ears with otitis media with effusion . they suggested that the middle ear may , through the contraction of the tympanic muscles , change its own resonance to increase its admittance at high frequencies , primarily in noisy environments . the reduction in transmission of high frequencies may modify this pattern and deteriorate this function in relation to speech comprehension . job and nottet , who studied dp - grams and history of otitis media , reported that the group with middle ear antecedents presented lower audiometric thresholds at 500 hz ; however , compared with the control group ( without antecedents ) , as the frequency increased , the thresholds increased as well . the 1000 hz tympanometric pattern was shown to influence dpoaes , but not by the same degree , as observed in the threshold differences . depending on the dpoae stimulus paradigm ( dp - grams or dp i / o functions ) different aspects were observed . the dpoae amplitudes of the dp - gram represent a quasi - linear context because it is assumed that at 65 and 55 db the cochlear oae generators are almost saturated . lower stimuli are connected with active non - linear dpoae generators ; therefore , different relationships were observed . dpoae amplitudes were lower in the dpg only at 6006 hz , but the amplitude difference between the dpg and spg groups decreased as the frequency increased ( see data in figure 2 ) . statistical differences were observed only at 1001 hz and a borderline difference at 1501 hz ( p=0.06 ) . this pattern has been reported in a study by garner et al . , who found great variability at low frequencies ( 500 and 1000 hz ) and at high frequencies ( 5656 and 8000 hz ) , suggesting that the low frequencies suffered from a noise effect and that the variability at high frequencies could be a consequence of middle ear transmission characteristics . dpoae i / o functions presented a different pattern than the dp - grams . at low frequency stimuli ( 35 db and 30 db ) and across all tested frequencies ( with the exception of 1501 hz ) , the spg presented higher dpoae amplitudes . sun and shaver   studied the effect of negative middle ear pressure on dpoae , and they concluded that negative middle ear pressure substantially decreases dpoae levels at low frequencies and some mid - frequencies , but tends to increase dpoae levels at high frequencies . because negative pressure induces middle ear stiffness , the stiffness modifies the admittance at high frequencies and increases the impedance of low frequencies . our findings agree with these conclusions , assuming that the decrease in stiffness considerably alters the dpoae measures . the data from this study agree with results of gorga et al .   who reinforced the need to consider a middle ear condition when predicting hearing thresholds from extrapolated dpoae i / o functions . the data suggest that alterations of the middle ear resonant properties ( as revealed by the admittance differences between the 1000 hz pattern groups ) are reflected in the attenuation of the recorded dpoaes . similar data from human and animal studies have been widely reported in the literature [ 8,15,17,2831 ] . nevertheless , the totality of factors influencing the induced changes of the middle ear still remain to be defined . differences in dpoaes between the 2 groups were significant only at 1001 hz and for high - amplitude stimuli ( 65 db spl ) . it is important to emphasize that despite the observed threshold differences at 3000 , 6000 , and 8000 hz , the dpoae data did not verify these differences . the dpoae amplitudes showed an erratic / alternating behavior between the dp - grams in comparison to the data from the i / o curves , but no significant differences were detected . in the first case ( differences at 1001 hz ) , one might assume that the tested subjects present subclinical threshold alterations , but in the second case the data offer no explanation . it is well documented that dpoaes are more sensitive indicators of alterations in the cochlear function and gain [ 2830 ] than the corresponding behavioral measurements . the only plausible hypothesis is that the observed behavioral differences have a retrocochlear origin , but the available data do not confirm this . to elucidate this clinical finding , further studies must be conducted . as new data in the literature support the efficacy of middle ear - power - analysis ( mepa ) mepa measures wideband power reflectance , which is an index of middle ear inefficiency , as a function of frequency . mepa measurements in parallel with dpoae and high - frequency tympanometry can more accurately describe the relationships observed . the 1000 hz tympanometric pattern ( single or double ) significantly affects the structure of dpoae responses only at 1001 hz . in this context , changes in the resonance properties in the middle ear ( as detected by the 1000 hz tympanometric probe ) can be considered as prime candidates for the observed variability in the dp - grams and the dp i / o functions . nevertheless , the data can not reveal the mechanism connecting the double - peak tympanometric pattern to the higher middle ear admittance .
backgroundpathologies that alter the impedance of the middle ear may consequently modify the dpoae amplitude . 
 the aim of this study was to correlate information from 2 different clinical procedures assessing middle ear status . 
 data from dpoae responses ( both dp - gram and dp i / o functions ) were correlated with data from multi - component tympanometry at 1000 hz.material/methodsthe subjects were divided into a double - peak group ( dpg ) and a single - peak group ( spg ) depending on 1000 hz tympanogram pattern . 
 exclusion criteria ( described in the methods section ) were applied to both groups and finally only 31 ears were assigned to each group . 
 the subjects were also assessed with traditional tympanometry and behavioral audiometry.resultscompared to the single - peak group , in terms of the 226 hz tympanometry data , subjects in the dpg group presented : ( i ) higher values of ear canal volume ; ( ii ) higher peak pressure , and ( iii ) significantly higher values of acoustic admittance . 
 dpoae amplitudes were lower in the dpg group only at 6006 hz , but the difference in amplitude between the dpg and spg groups decreased as the frequency increased . 
 statistical differences were observed only at 1001 hz and a borderline difference at 1501 hz . in terms of dpoae 
 i / o functions , significant differences were observed only in 4 of the 50 tested points.conclusionsthe 1000-hz tympanometric pattern significantly affects the structure of dpoae responses only at 1001 hz . in this context 
 , changes in the properties of the middle ear ( as detected by the 1000 hz tympanometry ) can be considered as prime candidates for the observed variability in the dp - grams and the dp i / o functions .