- Email: [email protected]
Noise induced hearing loss in fetal sheep
Hearing Research 74 (1994) 221-230
ELSEVIER
Noise induced hearing loss in fetal sheep Scott K. Griffiths
ayb**,Linda L. Pierson a7b,Kenneth J. Gerhardt Aemil J.M. Peters ’
’
a,b, Robert
M. Abrams
b,c,
aDepartment of Communication
Processes and Disorders, 435 Dauer Hall, University of Florida, Gainsviile, FL 32611, USA b fnstitute for the Advanced Study of the Comm~icat~n Processes ’ De~a~rne~ts of Obstetrics and Gynecology, and Pediatrics ~~~versi~ of FIorida, Ga~~vi~le, FL 32611, USA
(Received 6 April 1993;Revision received 22 September 1993;Accepted 17 November 1993)
Abstract The auditory brainstem response (ABR) was recorded in utero from chronically instrumented fetal sheep prior to and following exposure of pregnant ewes to intense broadband noise (120 dB SPL for 16 h). ABRs were elicited by clicks and tone bursts (0.5, 1, 2, and 4 kWz) delivered through a bone oscillator secured to the fetal skull. Latency-intensity functions for most of the four vertex-positive waves (labelled I-IV) were prolonged and AJ3R thresholds were temporarily elevated by an average of 8 dB following the noise exposure. Results show that exogenous sounds can penetrate the uterus and result in alterations of the fetal ABR.
Key words: Auditory brainstem
response; Fetal sheep; Fetal hearing; Noise-induced
1. Int~duction There continues to be an interest in the extent to which the fetus can detect sounds originating outside the mother. Recent studies of sound attenuation through the maternal abdomen and fluids show very littIe reduction (5 to 10 dB) for Iow frequencies ( < 1000 Hz) and somewhat greater reduction (20 to 30 dB) for higher frequencies (> 1000 Hz) Wince et al., 1985; Gerhardt et al., 1990; Richards et al., 1992; Peters et al., 1993b). Given that external sounds undergo less
attenuation in penetrating to the fetal head than had been previousiy reported (Bench, 1968; WaIker et al., 19711, the possibility that these sounds might produce changes in fetal hearing warrants consideration. The overall aim of this study was to evaluate the effects of intense noise exposure on the fetal auditory brainstem response. Sheep were selected as experimental animals for the foI~owing reasons: 1) they are a widely recognized animal model for human fetal physi* Corresponding author. Fax: (904) 392-4955. ’ Portions of this paper were presented at the annual convention of the American Speech-~nguage-Hearing Association in Atlanta, GA, November, 1991. 0378-5955/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0378-5955(93)E0202-M
hearing loss
oiogy and development (Dawes, 1968; Jones and NathanieIsz, 198.5); 2) sound transmission through maternal tissue and fluids in sheep is similar to that of humans (Gerhardt, 1990); 3) ovine and human hearing sensitivities are similar (Wollack, 1963); and, 4) the survival rates of both the instrumented fetus and ewe are good. Smaller, more commonly used animals, for example the guinea pig, have been used for fetal noise-induced hearing loss (NIHL) studies (Cook et al., 1982). Cook et al. reported that it is possible for NIHL to occur in utero in mammals whose auditory maturation is nearly complete before birth. But, the dramatic differences in the size of the pregnant abdomen obviate any generalizations to human pregnancy. Dunn et al. (1981) studied the effect of a repeated broadband noise exposure (130 dB SPL for 4 h per day, 5 days per week) on the ABR and morphology of the cochleae of lambs whose mothers were exposed for the duration of pregnancy. The ABR did not differ between the exposed group and a nonexposed group. Exposed cochleae exhibited greater and more frequent histologic anomalies than did the non-exposed cochleae. Auditory brainstem response measurements are easily obtained from fetal sheep both in utero and ex
222
S. K. Grifiiths et al. /Hearing
utero (Cook et al., 1987;Wolfson et al., 1990; Woods and Plessinger, 1985; Woods et al., 1983; Dawes, 1968). The ovine ABR is similar in morphology to that in humans, although the literature on the specific number of vertex-positive waves is inconsistent; some authors (Wolfson et al., 1990) report four (waves I-IV) and others (Woods et al., 1983) report five (waves I-V) detectable components. The mammalian fetus develops in an environment filled with sounds which originate both inside and outside the mother. External sounds are transmitted across the maternal abdominal wall, uterus and intrauterine fluids to the fetal ear (Querleu et al., 1981, 1988). Extra-abdominal, low-frequency sounds penetrate these tissues and fluids more effectively than high-frequency sounds (Gerhardt et al., 1992). Enhancement of sound pressure level (SPL) for frequencies below 0.25 kHz has been measured (Peters et al., 1993a; Gerhardt, 1990; Armitage et al., 1980; Vince et al., 1982). In addition to exogenous sounds, maternal intestinal, respiratory and cardiovascular sounds are identifiable within the uterus (Gerhardt et al., 1990; Armitage et al., 1980; Baidwin et al., 1983). Maternal vocalizations are easily transmitted to the amniotic fluid and represent the most intense sound normally present in the uterus (Gerhardt, 1989; Vince et al., 1985). Sounds present within the uterus stimulate the fetal inner ear and central auditory pathways, even though the normal route through the ossicular chain is likely to be rendered less efficient due to the fluids which fill the external canal and middle ear cavity (Gerhardt et al., 1992). Both airborne and vibroacoustic sounds from extra-abdominal sources increase fetal metabolic activity in most central auditory structures including the auditory cortex (Abrams et al., 1989; Horner et al., 1987), evoke gross body movements (Gelman et al., 1982; Patrick and Gagnon, 19891, produce accelerations in heart rate (Grimwade et al., 1971; Richards et al., 1988) and alter fetal breathing patterns (Gagnon et al., 1986). While it has been well documented that external sounds stimulate the mammahan fetus, the impact that exogenous, intense noises may have on the fetal auditory mechanism has not been identified. Therefore, the purpose of this study was to evaluate the effect of an intense noise exposure on fetal auditory brainstem response.
2. Methods Subjects
Surgery and all subsequent care of animals conformed to the guidelines approved by the University of Florida. Nine pregnant ewes carrying fetuses with ges-
Research 14 (1994) 221-230
tational ages ranging from 124 to 129 days at the time of surgery served as subjects. Gestational age of the fetus at the time of the noise exposure ranged from 126 to 134 days. The ewes were housed outdoors in individual 1.2 X 2.4 m stalls for at least 5 days before surgery to allow them to acclimate to the environment. Following surgery and each test condition, the ewes were returned to these stalls.
Surgery. The ewes were fasted for 24 h prior to sterile surgery, brought into the surgery room, anesthetized with halothane and prepared for abdominal surgery. The fetal head and forelimb were exteriorized through a midline abdominal incision. The fetal skull was exposed and stainless steel screw-electrodes were secured on the snout just anterior to nasion (ground), at the vertex (non-inverting) and at both mastoids (inverting), at or near the head of the styloid process. To provide additional stability and insulation, the electrodes were covered with methyl methacrylate. A bone oscillator (Radioear B70A) was secured to the fetal skull on the occipital bone as far caudal as possible. Two rows of three screws each were fixed in the skull and the oscillator secured between them with surgical thread. The incision in the fetal scalp was closed over the electrodes and around the bone oscillator. In one animal a hydrophone (Bruel and Kjaer Model 8103, Marlborough, MA) was sutured to the neck of the fetus just inferior to the pinna. A catheter was placed in a fetal axillary artery, Ampicillin, 0.5 mg, was introduced into the amniotic fluid and peritoneal cavity. The fetus was then returned to the uterus, and the uterus and abdomen of the ewe were closed. A catheter was placed in a maternal femoral vein. Catheters were used to monitor maternal and fetal health and to administer the barbiturate euthanizing agent at the end of the experiment. The electrode leads, wires and catheters were passed under the maternal skin, out through an incision in the maternal flank, and were stored in a pouch sutured to the maternal flank. Auditory Brainstem Response Testing. For ABR testing, the ewe was placed in a cart and wheeled into a sound-treated booth (Industrial Acoustics Co. model GDC-IL; Bronx, NY). The fetal electrode and bone oscillator leads were connected to a Tracer Northern TN 3000 evoked potential averaging unit and responses were recorded to tone-bursts and clicks transduced by the bone oscillator. Electrical input from the electrodes was differentially recorded, amplifjed, filtered (0.1-3.0 kHz), digitized over a 10 ms (for the click stimulus) or 18 ms (for tone-burst stimuli) window, averaged over 2000 trials, and stored on disk for later analysis. Latency-intensity functions (LIFs) were completed in IO dB steps beginning at the upper limit of bone oscillator linearity and decreasing until the response disap-
SK
Crifiths
et al. /Hearing
peared. Once at a level which produced no clear response, the stimulus intensity was increased in 5 dB steps until a clear response was apparent to the investigator. Threshold responses were replicated. A recording was obtained with the bone oscillator wire disconnected from the evoked potential unit for comparison to recordings made at low stimulus levels. Broadband clicks (0.1 ms duration) and tone-bursts (1, 2, and 4 kHz) with a two cycle rise/fall time and one cycle plateau were presented at a rate of 21 per second. In addition, a 0.5 kHz tone-burst with a two cycle rise/fall time, and a 0.01 ms plateau was pre-
10 d%
Research
74 (1994)
221-230
223
sented. The bone oscillator was calibrated with an accelerometer (Bruel and Kjaer model 8001, Marlborough, MA) fixed directly to its vibrating surface. Accelerometer output was directed to a real time spectrum analyzer (Bruel and Kjaer model 2123, Marlborough, MA). The amplitude and spectral characteristics of the bone oscillator were compared for each of the five stimuli used to evoke the ABR. Fig. 1 shows stimulus waveforms and spectra as recorded from the accelerometer. In addition, stimulus level was referenced to normal young adult human behavioral thresholds (dB &IL)
lo-
dB_
I cide-
I d
Fig. 1. Spectra and time waveforms for ABR stimuli recorded using accelerometer (see text). Stimuli include: 0.5 kHz tone burst (upper left), 1 kHz tone burst (upper right), 2 kHz tone burst (middle left), 4 kHz tone burst (middle right), and click (lower right). Time waveforms are shown with a 40 ms span. Spectra are in one-twelfth octave bands.
224
S.K. Griftithset al. /Hearing Research 74 (1994) 221-230
with the bone oscillator placed on the mastoid. Exact stimulus levels for the fetus can only be estimated because of the placement of the bone oscillator on the occipital bone as well as the presence of a fluid-filled environment during testing. Noise Exposure. Following pre-exposure ABR testing, the ewe’s ear canals were packed with hearing protection material to reduce possible discomfort caused by the noise. Loud-speakers were placed above, behind, and on both sides of the cart. The centers of the loudspeakers on the side of the ewe were adjusted to the same height as the center of the ewe’s flank. In order to measure the uniformity of the sound field within which the ewe was placed, a microphone (Bruel and Kjaer Type 4165, Marlborough, MA) was positioned at several locations within a field which measured 60 cm in height, width and depth. The SPL varied by less than 3 dB. During the exposure, the abdomen of the ewe was located in this sound field. The sides of the cart were open from the bottom of the abdomen to above the back, thus minimizing sound obstruction. The ewe was exposed for 16 h to broadband noise at 120 dB SPL. Fig. 2 contains time-averaged spectra of the noise in air and in utero. While the sound pressures recorded in these two conditions are similar, the sound intensity in utero would differ by approximately 30 dB due to the passage from an air to a fluid medium (Oliver, 19891. Fetal ABR testing was initiated 10 min after cessation of the exposure. Duration of the post-exposure measurements did not exceed one hour, and the sequence of stimulus presentations was randomized. In order to assess possible recovery, subsequent ABR measures were obtained from six of the nine animals between 24 and 96 h after the cessation of the noise exposure.
Fig. 2. Time averaged spectra of the broadband noise recorded via microphone in air (solid line) and via hydrophone in utero (dotted line).
ence in the amplitude values at wave IV and trough IVn. Means and standard deviations for these measures from pre-exposure ABRs evoked by stimuli at 21 dB nHL are provided in Table 1. Pre- and post-exposure latency means are plotted against stimulus level for each of waves I through IV in Fig. 4. Pre- to post-exposure latency shifts can be observed as a vertical difference between the pre-exposure (solid lines) and post-exposure (dashed lines) LIFs. As is common for these functions, the latency of each wave increases as the stimulus level is decreased. The LIFs reveal an increase in wave latencies for all waves at all click levels following the noise exposure. The pre- to post-exposure latency shift was derived as the post-exposure latency minus the pre-exposure latency for each animal, wave and click level. The same type of subtraction was applied to the wave IV ampli-
3. Results Click-ecoked ABR latencies
Examples of the click-evoked ABR for a range of stimulus levels are shown in Fig. 3. The pre-exposure bone-conduction ABR in the fetal sheep consisted of four vertex-positive waves, labelled in Fig. 3 with roman numerals as waves I through IV. The wave latenties clearly lengthen as stimulus intensity decreases. In addition, wave IV is the only identifiable wave in the response to the least intense stimuli, as is observed for wave V in the human ABR. The latencies of the waves I, II, III, and IV, and the latencies of the trough following wave IV (IVn) were measured from the onset of the click. The measured time difference between waves I and IV was defined as the inter-peak latency (IPL). In addition, the peak-topeak amplitude of wave IV was measured as the differ-
Fig. 3. Examples of click-evoked ABR waveforms. Stimulus levels used were (from top to bottom) 41, 31, 21, 11, 1, and -9 dB nHL.
S.K. Gr#ths
et al. /Hearing
Table 1 Means and standard deviations for click-evoked AEtR latencies and amplitudes at a single stimulus level (21 dB nHL) Amplitude
Latencies (ms)
Research
Table 2 Mean differences between pre-exposure and post-exposure latencies and wave IV amplitude in the click-evoked ABR (post-exposure minus pre-exposure) ABR component latencies and amplitude
Stimulus level
Wave
1
II
III
IV
IVn
I-IV
$V’
Mean SD.
2.312 0.275
3.133 0.244
4.202 0.383
5.019 0.332
6.014 0.311
2.702 0.270
0.928 0.378
tude. The values of these shifts are presented in Table 2, with statistically significant differences marked with asterisks (Student’s t-test; (Y= 0.05, two-tailed). Virtually all of the shifts in latency are positive, while the majority of the amplitude shifts are negative, indicating a lengthening of latency and reduction in amplitude following noise exposure. Shifts in these directions are consistent with NIHL or with a reduction in the effective level of the stimulus in the post-exposure condition. Statistical significance was reached for wave IV at all levels, wave III at all but one level, and wave II at the three highest stimulus levels. No significant change
225
74 (1994) 221-230
dBnHL
I
II
III
IV
IVn
Amp IV
41 31 21 11 1
0.06 0.10 0.16 * 0.08 0.00
0.14 * 0.15 * 0.27 * 0.17 0.11
0.12 0.19 * 0.24 * 0.28 * 0.17 *
0.17 * 0.14 * 0.19 * 0.27 * 0.45 *
0.03 0.05 0.17 * 0.13 0.28 *
-0.16 - 0.07 - 0.04 0.01 0.05
* P < 0.05.
in the I-IV IPL was noted between the pre-and postexposure conditions. The amplitude changes following noise-exposure were not significant. Tone-burst-evoked ABR latencies
Examples of ABR waveforms obtained to 2 kHz tone-burst stimuli for a range of levels are presented in Fig. 5. It should be noted when comparing Figs. 5 and 5.00 7 0
WAVE
\
IV
4.80 1
\
\ ‘\
WAVE
III
WAVE
I
q
PRE-EXPOSURE aocO.POST-EXPOSURE
4.20 J WAVE
4.00
9
_3.80 E3.60
\
\
‘Y -\
\
& 2.25
\
\
2
\
!ii
\
-20
I
-10
1
&
I
2.00 -I
\
4
\
\ PRE-MPOSURE POST-EXPOSURE
STIMULUS ‘&I~ Fig. 4.
.
b
2.80 2.60
\
\
*...a
*
\
& 3.40 lz t- 3.20 4 3.00
II
b
1.75
1 m
1.50
4 .a.#
\
2 I
a I (iii
-
30
ntL)
1 I
-20
PRE-EXPOSURE POST-EXPOSURE
I
-10
1
d
1 I
STIMULUS ‘kV$?_
I
I
I I , . , 30 40 50 (dB nHL)
Click-evokedABR latency-intensityfunctions for waves I through IV in the pre- and post-exposure test conditions.
SK. Griffith et al. /Hearing
226
Fig. 5. Examples of ABR waveforms evoked by the 2 kHz tone-burst. Stimulus levels used were (from top to bottom) 45, 35, 25, 1.5, 5, and -5 dB nHL.
3 that the time windows are different; the tone-burst recordings were made over an 18-ms window instead of the lo-ms window used for the click-evoked recordings. Waves II through IV can be recognized here, while wave I was typically not identifiable due to stimulus artifact. The morphology of the 4 kHz tone-burst ABR was similar to that of the click-evoked ABR. All four vertex-positive waves were identifiable even at low stimulus levels. The waveforms to 0.5 and 1 kHz tonebursts were quite different from those to higher frequency stimuli. No easily identifiable waves were present to 0.5 kHz tone-bursts and consequently only response thresholds could be ascertained. Only wave IV was consistently detectable from the waveforms to 1 kHz tone-bursts. Table 3 contains the means and standard deviations for all of the pre-exposure ABR latency and amplitude measures for the 4, 2 and 1 kHz tone-bursts at moderate stimulus levels (21 to 26 dB nHL). Values are only
Table 3 Means and standard deviations for tone-burst-evoked
Research 74 (1994) 221-230
reported when the means were based on five or more animals. Latencies can be noted to increase as stimulus frequency decreases. Fig. 6 contains pre- and post-exposure LIFs for wave IV for the 4, 2, and 1 kHz tone-bursts. Pre- to post-exposure latency shifts, which can be observed as a vertical difference between the pre-exposure (solid lines) and post-exposure (dashed lines) LIFs, are noticeably smaller than those for the click stimulus. This reduction is also reflected in the absence of significance (significance being indicated by asterisks) in Table 4, which contains the mean pre- to post-exposure shifts in latency and amplitude for the tone-burst stimuli. Significant shifts were observed only for wave IV to the 4 kHz stimulus at the higher stimulus levels. As was the case for the click stimuli, the I-IV IPL in response to 4 kHz tone-bursts did not change significantly from the pre- to post-exposure test conditions.
Recovery Following the recovery period of 24 to 96 h, absolute ABR wave latencies to all stimuli (clicks and tonebursts) decreased from post-exposure values. In most cases, latencies measured after recovery returned to pre-exposure values. Repeated measures analysis of variance (ANOVA) on the latency of wave IV revealed a significant effect for test condition (pre-exposure, post-exposure and recovery) and no significant interactions. Post-hoc pairwise comparisons demonstrated the post-exposure wave IV latencies to be significantly longer than both the pre-exposure (F = 20.669; PI 0.001) and the recovery (F = 7.632; P 5 0.01) latencies. Latencies for earlier waves in the recovery condition were not significantly different from either the pre-exposure or the post-exposure values. No significant changes were observed in the I-IV IPL across test conditions.
ABR latencies and amplitudes Amplitude CpV)
Latencies (ms) 4 kHz 21 dB nHL I Wave Mean 2.712 S.D. 0.282
II 3.617 0.347
III 4.775 0.412
IV 5.521 0.364
IVn 6.478 0.476
I-IV 2.812 0.313
IV 0.448 0.226
2 kHz 25 dB nHL I Wave Mean 3.022 S.D. 0.383
II 3.912 0.535
III 4.739 0.592
IV 5.637 0.444
IVn 6.679 0.437
I-IV 2.572 0.222
IV 0.541 0.225
II _
III
IV 6.207 0.262
IVn 7.177 0.438
I-IV _ _
IV 0.470 0.310
1 kHz 26 dB nHL I Wave _ Mean S.D. _
Research 74 (2994) 221-230
227
6.75 -;;;s.so
E
4 kt-lz
-
TONE
2 kHz
BURST
TONE
BURST
T6.25 2 6.00 S
\
\
\
5.75
t; 5 5.50
b
\
\
\
\
~_
25. G
‘B\
\
\
5.25
\
c- 5.50 4 5.25
‘c
-
5.00
PRE-EXPOSURE : ooooe POST-EXPOSURE , I I * , -10 0
STlMULtOS
L&L
\
5.00
. 1-7--7m-l
50 3&
75
PRE-EXPOSURE OOODOPOST-EXPOSURE
1
1
L
.
I
6
8
*
I
’
I
’
i 50
AL)
9.00 3
9
/;;a.50
1 kHz
E
TONE
BURST
-8.00
t;
6.50
Z,_ 6.00 4
5.50 PRE-EXPOSURE @ocwPOST-EXPOSURE
Fig. 6. Pre- and Post-exposure ABR latency intensi~ functions for wave IV in response to the 4,2, and 1 kHz
Detection thresholds far the ARR were determined independently by two different observers. Each observer used as criterion for threshold the lowest level at which a response was observed which also could be discriminated from recordings made from the same animal with no stimulus. Repeated measures ANOVA indicated that while there was no significant difference between the thresholds determined by the two observers, both the condition (pre-exposure, post-exposure and recovery) and the stimuius type (clicks, and the 4, 2, 1, and OS kHz tone-bursts) si~ifican~y affected ABR thresholds (Condition: F = 21,626; P I 0,001; Stimulus: F = 32.601; P s 0.001). Response thresholds varied significantly (P s 0.05) across test conditions for all stimulus types except the 1 kHz stimulus. Post hoc pairwise comparisons indicated that the post-exposure thresholds were significantly higher than both the pre-exposure (F = 39.783; PI 0.001) and the recovery (F = 21.107; P I 0.001) thresholds. Recovery thresholds did not differ significantly from the
CLICK Deeeo4 kliz 2 ktiz 1 ktiz *Cl.5 kHz
z
_ ,,J
POST
P:
TEST
RECOVERY
CONDITION
Fig. 7. Mean ABR thresholds to clicks and 4, 2, 1, and 0.5 kHz tone bursts recorded during pre-exposure, post-exposure, and recovery test conditions.
228
S.K. Griffiths et al. /Hearing Research 74 (1994) 221-230
Table 4
Mean differencesbetween pre-exposure and post-exposure latencies and wave IV amplitude in the tone-burst-evoked posure minus pre-exposure) Stimulus level
ABR (post-cx-
ABR component latencies and amplitude I
II
III
IV
IVn
Amp
4 kHz 41 31 21 11 1
0.00 0.04 0.06 0.05 _
0.09 0.13 0.25 0.14 _
0.22 0.22 0.14 * 0.13 _
0.18 * 0.14 * 0.16 * - 0.04 - 0.03
0.06 0.02 - 0.05 0.18 0.02
- 0.06 -0.11 - 0.02 - 0.01 0.03
2 kHz 45 35 25 15 5
_ _ _ _ _
0.02 0.01 0.08 _ _
0.26 0.25 0.34 _ _
0.10 0.04 0.04 0.12 0.00
0.06 0.00 0.04 0.02 0.01
- 0.01 - 0.02 - 0.06 0.07 0.03
_
-
_
_ _
_ _ _
0.09 - 0.06 0.19 0.24 0.28
0.25 0.36 0.19 0.20 0.20
- 0.32 0.10 0.01 - 0.05 - 0.08
1 kHz 46 36 26 16 6
_
* P < 0.05. - Component not identifiable in a majority of animals.
pre-exposure thresholds. Mean ABR thresholds to all stimuli (clicks, and the 4, 2, 1, and 0.5 kHz tone-bursts) are plotted by condition (pre-exposure, post-exposure, and recovery) in Fig. 7. The average pre- to post-exposure threshold shift across animals and stimuli was 8 dB. The same average shift from the post-exposure to the recovery condition was 5 dB. Thus, hearing (measured in terms of ABR wave latencies and thresholds) was altered in fetuses whose mothers were exposed to intense broadband noise. The sizes of both the latency and threshold increases were small but statistically significant. Small but significant shifts toward shorter latencies and lower ABR thresholds were also noted in comparing the recovery data to the post-exposure data. This result suggests that the NIHL induced in this experiment was temporary. That the NIHL occurred within the peripheral auditory mechanism is supported by the observation of the latency shifts in all waves (although significantly only in later waves) but not in the I-IV IPL. Whether or not the shifts in auditory function are accompanied by histopathologic changes in the cochlea is currently being evaluated.
4. Discussion Alterations in fetal auditory function following exogenous noise exposure of the pregnant sheep were documented in this study. This finding is consistent
with the data extant on transmission of sound pressure through the maternal abdominal wall and uterus. Fig. 2 includes spectra from one animal prepared with a hydrophone implanted in the uterus. The overall intrauterine sound pressure level was approximately 8 dB less than the externally recorded sound pressure. The least amount of attenuation (difference between the l/3-octave band levels recorded simultaneously in air and in utero) occurred for the low frequencies (< 500 Hz) and the greatest amount of attenuation occurred from 500 to 8000 Hz. These observations are similar to those reported in studies of sound attenuation in both sheep (Vince et al., 1985; Gerhardt et al., 1990; Peters et al., 1993a,b) and humans (Richards et al., 1992). Although the sound pressure levels in the uterus are similar to those recorded in air at the maternal flank, one cannot conclude that the effect on the fetus will be similar to that of a sheep similarly exposed in air. The route taken by the acoustic signal to the fetal inner ear is still unclear. Two possible pathways are through the fluid-filled ear canal and middle ear cavity to the inner ear or through the skull via bone conduction. In a fetal sound isolation study conducted by Gerhardt et al. (1992) the fetus was found to be protected from external sounds by approximately 10 dB for 125 Hz and 250 Hz and up to 40-45 dB for 1000 Hz and 2000 Hz. Thus, the energy produced by the broadband noise exposure used in the present study is thought to be significantly filtered as it passes from air through the tissues and fluids of the abdomen and uterus and into the inner ear. The filter characteristics described by Gerhardt et al. (1992) suggest that the noise exposure at the fetal inner ear is dominated by low frequency sound pressures. The intense 16-hour exposure used in the current study resulted in significant shifts in ABR thresholds and latencies. The auditory brainstem response arises primarily from fibers associated with the basal region of the cochlea (Stapells et al., 1985). The noise exposure which was assumed to be dominated by low frequency energy at the fetal inner ear resulted in elevated ABR thresholds for all eliciting stimuli. This finding suggests that the responses from basal fibers as well as those from more apical fibers stimulated by the lower frequency tone bursts were influenced by the exposure. An earlier investigation of noise induced hearing loss occuring during fetal life was reported by Dunn et al. (1981). They studied the ABR in two groups of lambs, one of which was noise-exposed during intrauterine life, the second of which was not similarly exposed. No differences in the ABR were noted between groups. Several methodological differences may explain the apparent conflict between results of the present study and those reported by Dunn et al. First, the noise exposure used in the present study may be
S.K. Grifiths et al. /Hearing
inducing temporary shifts in the ABR that are completely recovered over the course of a few days. Dunn et al. exposed fetuses to noise, delivered the animals, and 30 to 40 days later recorded ABR thresholds. Second, the animals in the present study served as their own control because pre-exposure thresholds and latencies were compared to post-exposure thresholds and latencies. This was a different design than Dunn et al. employed. Third, the exposures were quite different and administered using a different protocol. This study used a l&hour 120 dB SPL exposure, whereas Dunn et al. described exposing animals ‘4 h/day (5 days/week) to broadband noise at 130 dB SPL during the last 5 months of pregnancy’ (The length of gestation in sheep is approximately 145 days). Several researchers have used ABR latencies and thresholds to assess temporary noise-induced changes in animals with a fully developed auditory system (Attias and Pratt, 1985, 1986; Attias et al., 1990; Sohmer et al., 1991). Attias and Pratt (1985, 1986) found latency changes in the human click-evoked ABR at a level of 75 dB HL after a 15-min, 95 dB HL noise exposure. Attias et al. (1990) found no latency shifts for wave I or the I-V IPL, even though mean ABR thresholds in noise-exposed rats (115 dB SPL for 2 h) shifted by an average of 24 dB. After a 2-hour noise exposure (115 dB SPL white noise) to rats, Sohmer et al. (1991) found small latency shifts in waves I through IV accompanied by a mean ABR threshold shift of 38 dB. The present data in sheep fetuses reveal: small noise-induced latency shifts in waves I through IV, no significant change in the I-IV IPL, and, small but significant threshold shifts (8 dB). The findings of fetal ABR threshold and latency shifts following noise exposure cannot be generalized to humans. More information is needed regarding the effects of noise exposures on mature sheep ABR before an assessment can be made about species susceptibility. It should be pointed out, however, that two studies have reported hearing loss in children whose mothers had been exposed to high levels of industrial noise during pregnancy (Laciak and MajcherskaMatuchniak, 1968; Lalande et al., 1986). Both research teams concluded that there was a higher incidence of hearing loss in these children than in children whose mothers were not noise exposed. Although both studies found relatively small degrees of hearing loss and lacked adequate control groups (Henderson et al., 1993), the possibility of fetal hearing loss produced by intense noise exposure was suggested. Temporary effects on auditory function in the fetal sheep whose mother was exposed to noise has been documented in this study. Clearly, the noise exposure was of a magnitude not experienced in normal working conditions and the effects were small (approximately 8 dB). Additional information about the possible perma-
Research 74 (1994) 221-230
229
nent effects on fetal hearing are needed as well as an assessment of mature sheep susceptibility to noise before statements can be made related to human risk factors.
5. Acknowledgements The authors are grateful for the able assistance of Isabelle Williams and Jackie Combs. This study was supported by N.I.H. grant number HD20084.
6. References Abrams, R.M., Hutchison A.A. and Gerhardt, K.J. (1989) Effects of high-intensity sound on local cerebral glucose utilization in fetal sheep. Dev. Br. Res. 48, l-10. Armitage, S.E., Baldwin, B.A. and Vince, M.A. (1980) The fetal sound environment of sheep. Science 208, 1173-1174. Attias, J. and Pratt, H. (1985) Auditory-evoked potential correlate of susceptibility to noise-induced hearing loss. Audiology 24, 149156. Attias, J. and Pratt, H. (1986) Follow-up of auditory-evoked potentials and temporary threshold shift in subjects developing noiseinduced permanent hearing loss. Audiology 25, 116-123. Attias, J., Sohmer, H., Gold, S., Haran, I. and Shahar, A. (1990) Noise and hypoxia induced temporary threshold shifts in rats studied by ABR. Hear. Res. 45, 247-252. Baldwin, J.N., Toner, J.N., Vince, M.A. and Weller, C. (1983) Recording the fetal lamb’s sound environment using an implantable radio hydrophone. Physiol. Sot. 131. Bench, R.J. (1968) Sound transmission to the human foetus through the maternal abdominal wall. J. Genet. Psychol. 113, 85-87. Cook, C.J., Williams, C. and Gluckman, P.D. (1987) Brainstem auditory evoked potentials in the fetal sheep, in utero. J. Dev. Physiol. 9, 429-439. Cook, R.O., Konishi, T., Salt, A.N., Hamm, C.W., Lebetkin, E.H. and Koo, J. (1982) Brainstem evoked responses of guinea pigs exposed to high noise levels in utero. Dev. Psychobiol. 15,95-104. Dawes, G.S. (1968) Fetal and neonatal physiology: A comparative study of changes at birth. Yearbook Medical Publishers, Chicago. Dunn, D.E., Lim, D.J., Ferraro, J.A., McKinley, R.L. and Moore, T.J. (1981) Effects on the auditory system from in utero noise exposures in lambs. Abstr. Assoc. Res Otolaryngol. 61. Gagnon, R., Hunse, C., Carmichael, L., Fellows, F. and Patrick, J. (1986) Effects of vibratory acoustic stimulation on human fetal breathing and gross fetal body movements near term. Am. J. Obstet. Gyn. 155, 1227-1230. Gelman, S.R., Wood, S., Spellacy, W.N. and Abrams, R.M. (1982) Fetal movements in response to sound stimulation. Am. J. Obstet. Gynecol. 143, 484-485. Gerhardt, K.J. (1989) Characteristics of the fetal sheep sound environment. Sem. Perinatol. 13, 362-370. Gerhardt, K.J. (1990) Prenatal and perinatal risks of hearing loss. Sem. Perinatol. 14, 299-304. Gerhardt, K.J., Otto, R., Abrams, R.M., Colle, J.J., Burchfield, D.J. and Peters, A.J.M. (1992) Cochlear microphonics recorded from fetal and newborn sheep. Am. J. Otolaryngol. 13, 226-233. Gerhardt, K.J., Abrams, R.M. and Oliver, CC. (1990) Sound environment of the fetal sheep. Am. J. Obstet. Gynecol. 162,282-287. Grimwade, J.C., Walker, D.W., Bartlett, M., Gordon, S. and Wood, C. (1971) Human fetal heart rate change and movement in
230
S.K. Griffiths et al. /Hearing Research 74 (1994) 221-230
response to sound and vibration. Am. J. Obstet. Gynecol. 109, 86-90. Henderson, D., Subramaniam, M. and Boettcher, F.A. (1993) Individual susceptibility to noise-induced hearing loss; An old topic revisited. Ear Hear. 14, 152-168. Horner, KC., Serviere, J. and Granier-Deferre, C. (1987) Deoxyglucase demonstration of in utero hearing in the guinea-pig foetus. Hear. Res. 26, 327-333. Jones, C.T. and Nathanielsz, P.W. (1985) The physiological development of the fetus and newborn. Academic Press, New York. Laciak, J. and Majcherska-Matuchniak, B. (1968) Hearing in children whose mothers are working in noise. In: Meleck, Baradin and Pamietnik (Eds.), XXVII Zjasdu Otolaryngologow Polskich. W Katowicach, Warsaw. Lalande, N.M., Hem, R. and Lambert, J. (1986) Is occupational noise exposure during pregnancy a risk factor of damage to the auditory system of the fetus? Am. J. Ind. Med. 10, 427-435. Oliver, CC. (1989) Sound and Vibration in tissues. Semin. Perinatol. 13, 354-361. Patrick, J. and Gagnon, R. (1989) Fetal breathing movements and fetal rest activity patterns. In: R. Creasy and R. Resnik (Eds.), Maternal-fetal Medicine: Principles and Practice, Saunders, Philadelphia, pp. 268-287. Peters, A.J.M., Abrams, R.M., Gerhardt, K.J. and Griffiths, S.K. (1993a). Transmission of airborne sound from 50-20,000 Hz into the abdomen of sheep. J. Low Freq. Noise Vib. 12, 16-24. Peters, A.J.M., Gerhardt, K.J., Abrams, R.M. and Longmate, J.A. (1993b) Three dimensional intraabdominal sound pressures in sheep produced by airborne stimuli. Am. J. Obstet. Gynecol. Querleu, D., Renard, X. and Crepin, G. (1981) Perception auditive et reactivite foetale aux stimulations sonores. J. Gyn. Obstet. Biol. Repr. 10, 307-314. Querleu, D., Renard, X., Versyp, F., Paris-Delrue, L. and Crepin, G. (1988) Fetal Hearing. Eur. J. Obstet. Gynecol. Reprod. Biol. 29, 191-212.
Richards, D.S., Cefalo, R.C., Thorpe, J.M., Salley, M. and Rose, D. (1988) Determinants of fetal heart rate response to vibroacoustic stimulation in labor. Obstet. Gynecol. 71, 535-540. Richards, D.S., Frentzen, B., Gerhardt, K.J., McCann, M.E. and Abrams, R.M. (1992) Sound levels in the human uterus. Obstet. Gynecol. 80, 186-190. Sohmer, H., Freeman, S., Friedman, I. and Lidan, D. (1991) Auditory brainstem response (ABR) latency shifts in animal models of various types of conductive and sensori-neural hearing losses. Acta Otolaryngol. 111, 206-211. Stapells, D.R., Picton, T.W., Perez-Abalo, M., Read, D. and Smith, A. (19851 Frequency specificity in evoked potential audiometry. In: J.T. Jacobson (Ed.), The Auditory Brainstem Response. College-Hill Press, San Diego, CA, pp. 147-177. Vince, M.A., Armitage, S.E., Baldwin, B.A. and Toner, J. (1982) The sound environment of the foetal sheep. Behavior 81, 296-315. Vince, M.A., Billing, A.E., Baldwin, B.A., Toner, J.N. and Weller, C. (1985) Maternal vocalisations and other sounds in the fetal lamb’s sound environment. Early Hum. Dev. 11, 179-190. Walker, D., Grimwade, J. and Wood, C. (1971) Intrauterine noise: A component of the fetal environment. Am. J. Obstet. Gynecol. 109, 91-95. Wolfson, M.R., Durrant, J.D., Tran, N.N., Bhutani, V.K. and Shaffer, T.H. (1990) Emergence of the brain-stem auditory evoked potential in the premature lamb. EEG Neurophysiol. 75, 242-250. Wollack, C.H. (1963) The auditory acuity of the sheep (Qvis aries). J. Aud. Res. 3, 121-132. Woods, J.R. Jr, Plessinger, M.A. and Mack, C.E. (1983) Fetal auditory brainstem evoked response (ABR). Ped. Res. 18, 83-85. Woods, J.R. Jr and Plessinger, M.A. (1985) The fetal auditory brain stem response: Serial measurements at two stimulus intensities. Otolaryngol. Head Neck Surg. 93, 759-764.
ELSEVIER
Noise induced hearing loss in fetal sheep Scott K. Griffiths
ayb**,Linda L. Pierson a7b,Kenneth J. Gerhardt Aemil J.M. Peters ’
’
a,b, Robert
M. Abrams
b,c,
aDepartment of Communication
Processes and Disorders, 435 Dauer Hall, University of Florida, Gainsviile, FL 32611, USA b fnstitute for the Advanced Study of the Comm~icat~n Processes ’ De~a~rne~ts of Obstetrics and Gynecology, and Pediatrics ~~~versi~ of FIorida, Ga~~vi~le, FL 32611, USA
(Received 6 April 1993;Revision received 22 September 1993;Accepted 17 November 1993)
Abstract The auditory brainstem response (ABR) was recorded in utero from chronically instrumented fetal sheep prior to and following exposure of pregnant ewes to intense broadband noise (120 dB SPL for 16 h). ABRs were elicited by clicks and tone bursts (0.5, 1, 2, and 4 kWz) delivered through a bone oscillator secured to the fetal skull. Latency-intensity functions for most of the four vertex-positive waves (labelled I-IV) were prolonged and AJ3R thresholds were temporarily elevated by an average of 8 dB following the noise exposure. Results show that exogenous sounds can penetrate the uterus and result in alterations of the fetal ABR.
Key words: Auditory brainstem
response; Fetal sheep; Fetal hearing; Noise-induced
1. Int~duction There continues to be an interest in the extent to which the fetus can detect sounds originating outside the mother. Recent studies of sound attenuation through the maternal abdomen and fluids show very littIe reduction (5 to 10 dB) for Iow frequencies ( < 1000 Hz) and somewhat greater reduction (20 to 30 dB) for higher frequencies (> 1000 Hz) Wince et al., 1985; Gerhardt et al., 1990; Richards et al., 1992; Peters et al., 1993b). Given that external sounds undergo less
attenuation in penetrating to the fetal head than had been previousiy reported (Bench, 1968; WaIker et al., 19711, the possibility that these sounds might produce changes in fetal hearing warrants consideration. The overall aim of this study was to evaluate the effects of intense noise exposure on the fetal auditory brainstem response. Sheep were selected as experimental animals for the foI~owing reasons: 1) they are a widely recognized animal model for human fetal physi* Corresponding author. Fax: (904) 392-4955. ’ Portions of this paper were presented at the annual convention of the American Speech-~nguage-Hearing Association in Atlanta, GA, November, 1991. 0378-5955/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0378-5955(93)E0202-M
hearing loss
oiogy and development (Dawes, 1968; Jones and NathanieIsz, 198.5); 2) sound transmission through maternal tissue and fluids in sheep is similar to that of humans (Gerhardt, 1990); 3) ovine and human hearing sensitivities are similar (Wollack, 1963); and, 4) the survival rates of both the instrumented fetus and ewe are good. Smaller, more commonly used animals, for example the guinea pig, have been used for fetal noise-induced hearing loss (NIHL) studies (Cook et al., 1982). Cook et al. reported that it is possible for NIHL to occur in utero in mammals whose auditory maturation is nearly complete before birth. But, the dramatic differences in the size of the pregnant abdomen obviate any generalizations to human pregnancy. Dunn et al. (1981) studied the effect of a repeated broadband noise exposure (130 dB SPL for 4 h per day, 5 days per week) on the ABR and morphology of the cochleae of lambs whose mothers were exposed for the duration of pregnancy. The ABR did not differ between the exposed group and a nonexposed group. Exposed cochleae exhibited greater and more frequent histologic anomalies than did the non-exposed cochleae. Auditory brainstem response measurements are easily obtained from fetal sheep both in utero and ex
222
S. K. Grifiiths et al. /Hearing
utero (Cook et al., 1987;Wolfson et al., 1990; Woods and Plessinger, 1985; Woods et al., 1983; Dawes, 1968). The ovine ABR is similar in morphology to that in humans, although the literature on the specific number of vertex-positive waves is inconsistent; some authors (Wolfson et al., 1990) report four (waves I-IV) and others (Woods et al., 1983) report five (waves I-V) detectable components. The mammalian fetus develops in an environment filled with sounds which originate both inside and outside the mother. External sounds are transmitted across the maternal abdominal wall, uterus and intrauterine fluids to the fetal ear (Querleu et al., 1981, 1988). Extra-abdominal, low-frequency sounds penetrate these tissues and fluids more effectively than high-frequency sounds (Gerhardt et al., 1992). Enhancement of sound pressure level (SPL) for frequencies below 0.25 kHz has been measured (Peters et al., 1993a; Gerhardt, 1990; Armitage et al., 1980; Vince et al., 1982). In addition to exogenous sounds, maternal intestinal, respiratory and cardiovascular sounds are identifiable within the uterus (Gerhardt et al., 1990; Armitage et al., 1980; Baidwin et al., 1983). Maternal vocalizations are easily transmitted to the amniotic fluid and represent the most intense sound normally present in the uterus (Gerhardt, 1989; Vince et al., 1985). Sounds present within the uterus stimulate the fetal inner ear and central auditory pathways, even though the normal route through the ossicular chain is likely to be rendered less efficient due to the fluids which fill the external canal and middle ear cavity (Gerhardt et al., 1992). Both airborne and vibroacoustic sounds from extra-abdominal sources increase fetal metabolic activity in most central auditory structures including the auditory cortex (Abrams et al., 1989; Horner et al., 1987), evoke gross body movements (Gelman et al., 1982; Patrick and Gagnon, 19891, produce accelerations in heart rate (Grimwade et al., 1971; Richards et al., 1988) and alter fetal breathing patterns (Gagnon et al., 1986). While it has been well documented that external sounds stimulate the mammahan fetus, the impact that exogenous, intense noises may have on the fetal auditory mechanism has not been identified. Therefore, the purpose of this study was to evaluate the effect of an intense noise exposure on fetal auditory brainstem response.
2. Methods Subjects
Surgery and all subsequent care of animals conformed to the guidelines approved by the University of Florida. Nine pregnant ewes carrying fetuses with ges-
Research 14 (1994) 221-230
tational ages ranging from 124 to 129 days at the time of surgery served as subjects. Gestational age of the fetus at the time of the noise exposure ranged from 126 to 134 days. The ewes were housed outdoors in individual 1.2 X 2.4 m stalls for at least 5 days before surgery to allow them to acclimate to the environment. Following surgery and each test condition, the ewes were returned to these stalls.
Surgery. The ewes were fasted for 24 h prior to sterile surgery, brought into the surgery room, anesthetized with halothane and prepared for abdominal surgery. The fetal head and forelimb were exteriorized through a midline abdominal incision. The fetal skull was exposed and stainless steel screw-electrodes were secured on the snout just anterior to nasion (ground), at the vertex (non-inverting) and at both mastoids (inverting), at or near the head of the styloid process. To provide additional stability and insulation, the electrodes were covered with methyl methacrylate. A bone oscillator (Radioear B70A) was secured to the fetal skull on the occipital bone as far caudal as possible. Two rows of three screws each were fixed in the skull and the oscillator secured between them with surgical thread. The incision in the fetal scalp was closed over the electrodes and around the bone oscillator. In one animal a hydrophone (Bruel and Kjaer Model 8103, Marlborough, MA) was sutured to the neck of the fetus just inferior to the pinna. A catheter was placed in a fetal axillary artery, Ampicillin, 0.5 mg, was introduced into the amniotic fluid and peritoneal cavity. The fetus was then returned to the uterus, and the uterus and abdomen of the ewe were closed. A catheter was placed in a maternal femoral vein. Catheters were used to monitor maternal and fetal health and to administer the barbiturate euthanizing agent at the end of the experiment. The electrode leads, wires and catheters were passed under the maternal skin, out through an incision in the maternal flank, and were stored in a pouch sutured to the maternal flank. Auditory Brainstem Response Testing. For ABR testing, the ewe was placed in a cart and wheeled into a sound-treated booth (Industrial Acoustics Co. model GDC-IL; Bronx, NY). The fetal electrode and bone oscillator leads were connected to a Tracer Northern TN 3000 evoked potential averaging unit and responses were recorded to tone-bursts and clicks transduced by the bone oscillator. Electrical input from the electrodes was differentially recorded, amplifjed, filtered (0.1-3.0 kHz), digitized over a 10 ms (for the click stimulus) or 18 ms (for tone-burst stimuli) window, averaged over 2000 trials, and stored on disk for later analysis. Latency-intensity functions (LIFs) were completed in IO dB steps beginning at the upper limit of bone oscillator linearity and decreasing until the response disap-
SK
Crifiths
et al. /Hearing
peared. Once at a level which produced no clear response, the stimulus intensity was increased in 5 dB steps until a clear response was apparent to the investigator. Threshold responses were replicated. A recording was obtained with the bone oscillator wire disconnected from the evoked potential unit for comparison to recordings made at low stimulus levels. Broadband clicks (0.1 ms duration) and tone-bursts (1, 2, and 4 kHz) with a two cycle rise/fall time and one cycle plateau were presented at a rate of 21 per second. In addition, a 0.5 kHz tone-burst with a two cycle rise/fall time, and a 0.01 ms plateau was pre-
10 d%
Research
74 (1994)
221-230
223
sented. The bone oscillator was calibrated with an accelerometer (Bruel and Kjaer model 8001, Marlborough, MA) fixed directly to its vibrating surface. Accelerometer output was directed to a real time spectrum analyzer (Bruel and Kjaer model 2123, Marlborough, MA). The amplitude and spectral characteristics of the bone oscillator were compared for each of the five stimuli used to evoke the ABR. Fig. 1 shows stimulus waveforms and spectra as recorded from the accelerometer. In addition, stimulus level was referenced to normal young adult human behavioral thresholds (dB &IL)
lo-
dB_
I cide-
I d
Fig. 1. Spectra and time waveforms for ABR stimuli recorded using accelerometer (see text). Stimuli include: 0.5 kHz tone burst (upper left), 1 kHz tone burst (upper right), 2 kHz tone burst (middle left), 4 kHz tone burst (middle right), and click (lower right). Time waveforms are shown with a 40 ms span. Spectra are in one-twelfth octave bands.
224
S.K. Griftithset al. /Hearing Research 74 (1994) 221-230
with the bone oscillator placed on the mastoid. Exact stimulus levels for the fetus can only be estimated because of the placement of the bone oscillator on the occipital bone as well as the presence of a fluid-filled environment during testing. Noise Exposure. Following pre-exposure ABR testing, the ewe’s ear canals were packed with hearing protection material to reduce possible discomfort caused by the noise. Loud-speakers were placed above, behind, and on both sides of the cart. The centers of the loudspeakers on the side of the ewe were adjusted to the same height as the center of the ewe’s flank. In order to measure the uniformity of the sound field within which the ewe was placed, a microphone (Bruel and Kjaer Type 4165, Marlborough, MA) was positioned at several locations within a field which measured 60 cm in height, width and depth. The SPL varied by less than 3 dB. During the exposure, the abdomen of the ewe was located in this sound field. The sides of the cart were open from the bottom of the abdomen to above the back, thus minimizing sound obstruction. The ewe was exposed for 16 h to broadband noise at 120 dB SPL. Fig. 2 contains time-averaged spectra of the noise in air and in utero. While the sound pressures recorded in these two conditions are similar, the sound intensity in utero would differ by approximately 30 dB due to the passage from an air to a fluid medium (Oliver, 19891. Fetal ABR testing was initiated 10 min after cessation of the exposure. Duration of the post-exposure measurements did not exceed one hour, and the sequence of stimulus presentations was randomized. In order to assess possible recovery, subsequent ABR measures were obtained from six of the nine animals between 24 and 96 h after the cessation of the noise exposure.
Fig. 2. Time averaged spectra of the broadband noise recorded via microphone in air (solid line) and via hydrophone in utero (dotted line).
ence in the amplitude values at wave IV and trough IVn. Means and standard deviations for these measures from pre-exposure ABRs evoked by stimuli at 21 dB nHL are provided in Table 1. Pre- and post-exposure latency means are plotted against stimulus level for each of waves I through IV in Fig. 4. Pre- to post-exposure latency shifts can be observed as a vertical difference between the pre-exposure (solid lines) and post-exposure (dashed lines) LIFs. As is common for these functions, the latency of each wave increases as the stimulus level is decreased. The LIFs reveal an increase in wave latencies for all waves at all click levels following the noise exposure. The pre- to post-exposure latency shift was derived as the post-exposure latency minus the pre-exposure latency for each animal, wave and click level. The same type of subtraction was applied to the wave IV ampli-
3. Results Click-ecoked ABR latencies
Examples of the click-evoked ABR for a range of stimulus levels are shown in Fig. 3. The pre-exposure bone-conduction ABR in the fetal sheep consisted of four vertex-positive waves, labelled in Fig. 3 with roman numerals as waves I through IV. The wave latenties clearly lengthen as stimulus intensity decreases. In addition, wave IV is the only identifiable wave in the response to the least intense stimuli, as is observed for wave V in the human ABR. The latencies of the waves I, II, III, and IV, and the latencies of the trough following wave IV (IVn) were measured from the onset of the click. The measured time difference between waves I and IV was defined as the inter-peak latency (IPL). In addition, the peak-topeak amplitude of wave IV was measured as the differ-
Fig. 3. Examples of click-evoked ABR waveforms. Stimulus levels used were (from top to bottom) 41, 31, 21, 11, 1, and -9 dB nHL.
S.K. Gr#ths
et al. /Hearing
Table 1 Means and standard deviations for click-evoked AEtR latencies and amplitudes at a single stimulus level (21 dB nHL) Amplitude
Latencies (ms)
Research
Table 2 Mean differences between pre-exposure and post-exposure latencies and wave IV amplitude in the click-evoked ABR (post-exposure minus pre-exposure) ABR component latencies and amplitude
Stimulus level
Wave
1
II
III
IV
IVn
I-IV
$V’
Mean SD.
2.312 0.275
3.133 0.244
4.202 0.383
5.019 0.332
6.014 0.311
2.702 0.270
0.928 0.378
tude. The values of these shifts are presented in Table 2, with statistically significant differences marked with asterisks (Student’s t-test; (Y= 0.05, two-tailed). Virtually all of the shifts in latency are positive, while the majority of the amplitude shifts are negative, indicating a lengthening of latency and reduction in amplitude following noise exposure. Shifts in these directions are consistent with NIHL or with a reduction in the effective level of the stimulus in the post-exposure condition. Statistical significance was reached for wave IV at all levels, wave III at all but one level, and wave II at the three highest stimulus levels. No significant change
225
74 (1994) 221-230
dBnHL
I
II
III
IV
IVn
Amp IV
41 31 21 11 1
0.06 0.10 0.16 * 0.08 0.00
0.14 * 0.15 * 0.27 * 0.17 0.11
0.12 0.19 * 0.24 * 0.28 * 0.17 *
0.17 * 0.14 * 0.19 * 0.27 * 0.45 *
0.03 0.05 0.17 * 0.13 0.28 *
-0.16 - 0.07 - 0.04 0.01 0.05
* P < 0.05.
in the I-IV IPL was noted between the pre-and postexposure conditions. The amplitude changes following noise-exposure were not significant. Tone-burst-evoked ABR latencies
Examples of ABR waveforms obtained to 2 kHz tone-burst stimuli for a range of levels are presented in Fig. 5. It should be noted when comparing Figs. 5 and 5.00 7 0
WAVE
\
IV
4.80 1
\
\ ‘\
WAVE
III
WAVE
I
q
PRE-EXPOSURE aocO.POST-EXPOSURE
4.20 J WAVE
4.00
9
_3.80 E3.60
\
\
‘Y -\
\
& 2.25
\
\
2
\
!ii
\
-20
I
-10
1
&
I
2.00 -I
\
4
\
\ PRE-MPOSURE POST-EXPOSURE
STIMULUS ‘&I~ Fig. 4.
.
b
2.80 2.60
\
\
*...a
*
\
& 3.40 lz t- 3.20 4 3.00
II
b
1.75
1 m
1.50
4 .a.#
\
2 I
a I (iii
-
30
ntL)
1 I
-20
PRE-EXPOSURE POST-EXPOSURE
I
-10
1
d
1 I
STIMULUS ‘kV$?_
I
I
I I , . , 30 40 50 (dB nHL)
Click-evokedABR latency-intensityfunctions for waves I through IV in the pre- and post-exposure test conditions.
SK. Griffith et al. /Hearing
226
Fig. 5. Examples of ABR waveforms evoked by the 2 kHz tone-burst. Stimulus levels used were (from top to bottom) 45, 35, 25, 1.5, 5, and -5 dB nHL.
3 that the time windows are different; the tone-burst recordings were made over an 18-ms window instead of the lo-ms window used for the click-evoked recordings. Waves II through IV can be recognized here, while wave I was typically not identifiable due to stimulus artifact. The morphology of the 4 kHz tone-burst ABR was similar to that of the click-evoked ABR. All four vertex-positive waves were identifiable even at low stimulus levels. The waveforms to 0.5 and 1 kHz tonebursts were quite different from those to higher frequency stimuli. No easily identifiable waves were present to 0.5 kHz tone-bursts and consequently only response thresholds could be ascertained. Only wave IV was consistently detectable from the waveforms to 1 kHz tone-bursts. Table 3 contains the means and standard deviations for all of the pre-exposure ABR latency and amplitude measures for the 4, 2 and 1 kHz tone-bursts at moderate stimulus levels (21 to 26 dB nHL). Values are only
Table 3 Means and standard deviations for tone-burst-evoked
Research 74 (1994) 221-230
reported when the means were based on five or more animals. Latencies can be noted to increase as stimulus frequency decreases. Fig. 6 contains pre- and post-exposure LIFs for wave IV for the 4, 2, and 1 kHz tone-bursts. Pre- to post-exposure latency shifts, which can be observed as a vertical difference between the pre-exposure (solid lines) and post-exposure (dashed lines) LIFs, are noticeably smaller than those for the click stimulus. This reduction is also reflected in the absence of significance (significance being indicated by asterisks) in Table 4, which contains the mean pre- to post-exposure shifts in latency and amplitude for the tone-burst stimuli. Significant shifts were observed only for wave IV to the 4 kHz stimulus at the higher stimulus levels. As was the case for the click stimuli, the I-IV IPL in response to 4 kHz tone-bursts did not change significantly from the pre- to post-exposure test conditions.
Recovery Following the recovery period of 24 to 96 h, absolute ABR wave latencies to all stimuli (clicks and tonebursts) decreased from post-exposure values. In most cases, latencies measured after recovery returned to pre-exposure values. Repeated measures analysis of variance (ANOVA) on the latency of wave IV revealed a significant effect for test condition (pre-exposure, post-exposure and recovery) and no significant interactions. Post-hoc pairwise comparisons demonstrated the post-exposure wave IV latencies to be significantly longer than both the pre-exposure (F = 20.669; PI 0.001) and the recovery (F = 7.632; P 5 0.01) latencies. Latencies for earlier waves in the recovery condition were not significantly different from either the pre-exposure or the post-exposure values. No significant changes were observed in the I-IV IPL across test conditions.
ABR latencies and amplitudes Amplitude CpV)
Latencies (ms) 4 kHz 21 dB nHL I Wave Mean 2.712 S.D. 0.282
II 3.617 0.347
III 4.775 0.412
IV 5.521 0.364
IVn 6.478 0.476
I-IV 2.812 0.313
IV 0.448 0.226
2 kHz 25 dB nHL I Wave Mean 3.022 S.D. 0.383
II 3.912 0.535
III 4.739 0.592
IV 5.637 0.444
IVn 6.679 0.437
I-IV 2.572 0.222
IV 0.541 0.225
II _
III
IV 6.207 0.262
IVn 7.177 0.438
I-IV _ _
IV 0.470 0.310
1 kHz 26 dB nHL I Wave _ Mean S.D. _
Research 74 (2994) 221-230
227
6.75 -;;;s.so
E
4 kt-lz
-
TONE
2 kHz
BURST
TONE
BURST
T6.25 2 6.00 S
\
\
\
5.75
t; 5 5.50
b
\
\
\
\
~_
25. G
‘B\
\
\
5.25
\
c- 5.50 4 5.25
‘c
-
5.00
PRE-EXPOSURE : ooooe POST-EXPOSURE , I I * , -10 0
STlMULtOS
L&L
\
5.00
. 1-7--7m-l
50 3&
75
PRE-EXPOSURE OOODOPOST-EXPOSURE
1
1
L
.
I
6
8
*
I
’
I
’
i 50
AL)
9.00 3
9
/;;a.50
1 kHz
E
TONE
BURST
-8.00
t;
6.50
Z,_ 6.00 4
5.50 PRE-EXPOSURE @ocwPOST-EXPOSURE
Fig. 6. Pre- and Post-exposure ABR latency intensi~ functions for wave IV in response to the 4,2, and 1 kHz
Detection thresholds far the ARR were determined independently by two different observers. Each observer used as criterion for threshold the lowest level at which a response was observed which also could be discriminated from recordings made from the same animal with no stimulus. Repeated measures ANOVA indicated that while there was no significant difference between the thresholds determined by the two observers, both the condition (pre-exposure, post-exposure and recovery) and the stimuius type (clicks, and the 4, 2, 1, and OS kHz tone-bursts) si~ifican~y affected ABR thresholds (Condition: F = 21,626; P I 0,001; Stimulus: F = 32.601; P s 0.001). Response thresholds varied significantly (P s 0.05) across test conditions for all stimulus types except the 1 kHz stimulus. Post hoc pairwise comparisons indicated that the post-exposure thresholds were significantly higher than both the pre-exposure (F = 39.783; PI 0.001) and the recovery (F = 21.107; P I 0.001) thresholds. Recovery thresholds did not differ significantly from the
CLICK Deeeo4 kliz 2 ktiz 1 ktiz *Cl.5 kHz
z
_ ,,J
POST
P:
TEST
RECOVERY
CONDITION
Fig. 7. Mean ABR thresholds to clicks and 4, 2, 1, and 0.5 kHz tone bursts recorded during pre-exposure, post-exposure, and recovery test conditions.
228
S.K. Griffiths et al. /Hearing Research 74 (1994) 221-230
Table 4
Mean differencesbetween pre-exposure and post-exposure latencies and wave IV amplitude in the tone-burst-evoked posure minus pre-exposure) Stimulus level
ABR (post-cx-
ABR component latencies and amplitude I
II
III
IV
IVn
Amp
4 kHz 41 31 21 11 1
0.00 0.04 0.06 0.05 _
0.09 0.13 0.25 0.14 _
0.22 0.22 0.14 * 0.13 _
0.18 * 0.14 * 0.16 * - 0.04 - 0.03
0.06 0.02 - 0.05 0.18 0.02
- 0.06 -0.11 - 0.02 - 0.01 0.03
2 kHz 45 35 25 15 5
_ _ _ _ _
0.02 0.01 0.08 _ _
0.26 0.25 0.34 _ _
0.10 0.04 0.04 0.12 0.00
0.06 0.00 0.04 0.02 0.01
- 0.01 - 0.02 - 0.06 0.07 0.03
_
-
_
_ _
_ _ _
0.09 - 0.06 0.19 0.24 0.28
0.25 0.36 0.19 0.20 0.20
- 0.32 0.10 0.01 - 0.05 - 0.08
1 kHz 46 36 26 16 6
_
* P < 0.05. - Component not identifiable in a majority of animals.
pre-exposure thresholds. Mean ABR thresholds to all stimuli (clicks, and the 4, 2, 1, and 0.5 kHz tone-bursts) are plotted by condition (pre-exposure, post-exposure, and recovery) in Fig. 7. The average pre- to post-exposure threshold shift across animals and stimuli was 8 dB. The same average shift from the post-exposure to the recovery condition was 5 dB. Thus, hearing (measured in terms of ABR wave latencies and thresholds) was altered in fetuses whose mothers were exposed to intense broadband noise. The sizes of both the latency and threshold increases were small but statistically significant. Small but significant shifts toward shorter latencies and lower ABR thresholds were also noted in comparing the recovery data to the post-exposure data. This result suggests that the NIHL induced in this experiment was temporary. That the NIHL occurred within the peripheral auditory mechanism is supported by the observation of the latency shifts in all waves (although significantly only in later waves) but not in the I-IV IPL. Whether or not the shifts in auditory function are accompanied by histopathologic changes in the cochlea is currently being evaluated.
4. Discussion Alterations in fetal auditory function following exogenous noise exposure of the pregnant sheep were documented in this study. This finding is consistent
with the data extant on transmission of sound pressure through the maternal abdominal wall and uterus. Fig. 2 includes spectra from one animal prepared with a hydrophone implanted in the uterus. The overall intrauterine sound pressure level was approximately 8 dB less than the externally recorded sound pressure. The least amount of attenuation (difference between the l/3-octave band levels recorded simultaneously in air and in utero) occurred for the low frequencies (< 500 Hz) and the greatest amount of attenuation occurred from 500 to 8000 Hz. These observations are similar to those reported in studies of sound attenuation in both sheep (Vince et al., 1985; Gerhardt et al., 1990; Peters et al., 1993a,b) and humans (Richards et al., 1992). Although the sound pressure levels in the uterus are similar to those recorded in air at the maternal flank, one cannot conclude that the effect on the fetus will be similar to that of a sheep similarly exposed in air. The route taken by the acoustic signal to the fetal inner ear is still unclear. Two possible pathways are through the fluid-filled ear canal and middle ear cavity to the inner ear or through the skull via bone conduction. In a fetal sound isolation study conducted by Gerhardt et al. (1992) the fetus was found to be protected from external sounds by approximately 10 dB for 125 Hz and 250 Hz and up to 40-45 dB for 1000 Hz and 2000 Hz. Thus, the energy produced by the broadband noise exposure used in the present study is thought to be significantly filtered as it passes from air through the tissues and fluids of the abdomen and uterus and into the inner ear. The filter characteristics described by Gerhardt et al. (1992) suggest that the noise exposure at the fetal inner ear is dominated by low frequency sound pressures. The intense 16-hour exposure used in the current study resulted in significant shifts in ABR thresholds and latencies. The auditory brainstem response arises primarily from fibers associated with the basal region of the cochlea (Stapells et al., 1985). The noise exposure which was assumed to be dominated by low frequency energy at the fetal inner ear resulted in elevated ABR thresholds for all eliciting stimuli. This finding suggests that the responses from basal fibers as well as those from more apical fibers stimulated by the lower frequency tone bursts were influenced by the exposure. An earlier investigation of noise induced hearing loss occuring during fetal life was reported by Dunn et al. (1981). They studied the ABR in two groups of lambs, one of which was noise-exposed during intrauterine life, the second of which was not similarly exposed. No differences in the ABR were noted between groups. Several methodological differences may explain the apparent conflict between results of the present study and those reported by Dunn et al. First, the noise exposure used in the present study may be
S.K. Grifiths et al. /Hearing
inducing temporary shifts in the ABR that are completely recovered over the course of a few days. Dunn et al. exposed fetuses to noise, delivered the animals, and 30 to 40 days later recorded ABR thresholds. Second, the animals in the present study served as their own control because pre-exposure thresholds and latencies were compared to post-exposure thresholds and latencies. This was a different design than Dunn et al. employed. Third, the exposures were quite different and administered using a different protocol. This study used a l&hour 120 dB SPL exposure, whereas Dunn et al. described exposing animals ‘4 h/day (5 days/week) to broadband noise at 130 dB SPL during the last 5 months of pregnancy’ (The length of gestation in sheep is approximately 145 days). Several researchers have used ABR latencies and thresholds to assess temporary noise-induced changes in animals with a fully developed auditory system (Attias and Pratt, 1985, 1986; Attias et al., 1990; Sohmer et al., 1991). Attias and Pratt (1985, 1986) found latency changes in the human click-evoked ABR at a level of 75 dB HL after a 15-min, 95 dB HL noise exposure. Attias et al. (1990) found no latency shifts for wave I or the I-V IPL, even though mean ABR thresholds in noise-exposed rats (115 dB SPL for 2 h) shifted by an average of 24 dB. After a 2-hour noise exposure (115 dB SPL white noise) to rats, Sohmer et al. (1991) found small latency shifts in waves I through IV accompanied by a mean ABR threshold shift of 38 dB. The present data in sheep fetuses reveal: small noise-induced latency shifts in waves I through IV, no significant change in the I-IV IPL, and, small but significant threshold shifts (8 dB). The findings of fetal ABR threshold and latency shifts following noise exposure cannot be generalized to humans. More information is needed regarding the effects of noise exposures on mature sheep ABR before an assessment can be made about species susceptibility. It should be pointed out, however, that two studies have reported hearing loss in children whose mothers had been exposed to high levels of industrial noise during pregnancy (Laciak and MajcherskaMatuchniak, 1968; Lalande et al., 1986). Both research teams concluded that there was a higher incidence of hearing loss in these children than in children whose mothers were not noise exposed. Although both studies found relatively small degrees of hearing loss and lacked adequate control groups (Henderson et al., 1993), the possibility of fetal hearing loss produced by intense noise exposure was suggested. Temporary effects on auditory function in the fetal sheep whose mother was exposed to noise has been documented in this study. Clearly, the noise exposure was of a magnitude not experienced in normal working conditions and the effects were small (approximately 8 dB). Additional information about the possible perma-
Research 74 (1994) 221-230
229
nent effects on fetal hearing are needed as well as an assessment of mature sheep susceptibility to noise before statements can be made related to human risk factors.
5. Acknowledgements The authors are grateful for the able assistance of Isabelle Williams and Jackie Combs. This study was supported by N.I.H. grant number HD20084.
6. References Abrams, R.M., Hutchison A.A. and Gerhardt, K.J. (1989) Effects of high-intensity sound on local cerebral glucose utilization in fetal sheep. Dev. Br. Res. 48, l-10. Armitage, S.E., Baldwin, B.A. and Vince, M.A. (1980) The fetal sound environment of sheep. Science 208, 1173-1174. Attias, J. and Pratt, H. (1985) Auditory-evoked potential correlate of susceptibility to noise-induced hearing loss. Audiology 24, 149156. Attias, J. and Pratt, H. (1986) Follow-up of auditory-evoked potentials and temporary threshold shift in subjects developing noiseinduced permanent hearing loss. Audiology 25, 116-123. Attias, J., Sohmer, H., Gold, S., Haran, I. and Shahar, A. (1990) Noise and hypoxia induced temporary threshold shifts in rats studied by ABR. Hear. Res. 45, 247-252. Baldwin, J.N., Toner, J.N., Vince, M.A. and Weller, C. (1983) Recording the fetal lamb’s sound environment using an implantable radio hydrophone. Physiol. Sot. 131. Bench, R.J. (1968) Sound transmission to the human foetus through the maternal abdominal wall. J. Genet. Psychol. 113, 85-87. Cook, C.J., Williams, C. and Gluckman, P.D. (1987) Brainstem auditory evoked potentials in the fetal sheep, in utero. J. Dev. Physiol. 9, 429-439. Cook, R.O., Konishi, T., Salt, A.N., Hamm, C.W., Lebetkin, E.H. and Koo, J. (1982) Brainstem evoked responses of guinea pigs exposed to high noise levels in utero. Dev. Psychobiol. 15,95-104. Dawes, G.S. (1968) Fetal and neonatal physiology: A comparative study of changes at birth. Yearbook Medical Publishers, Chicago. Dunn, D.E., Lim, D.J., Ferraro, J.A., McKinley, R.L. and Moore, T.J. (1981) Effects on the auditory system from in utero noise exposures in lambs. Abstr. Assoc. Res Otolaryngol. 61. Gagnon, R., Hunse, C., Carmichael, L., Fellows, F. and Patrick, J. (1986) Effects of vibratory acoustic stimulation on human fetal breathing and gross fetal body movements near term. Am. J. Obstet. Gyn. 155, 1227-1230. Gelman, S.R., Wood, S., Spellacy, W.N. and Abrams, R.M. (1982) Fetal movements in response to sound stimulation. Am. J. Obstet. Gynecol. 143, 484-485. Gerhardt, K.J. (1989) Characteristics of the fetal sheep sound environment. Sem. Perinatol. 13, 362-370. Gerhardt, K.J. (1990) Prenatal and perinatal risks of hearing loss. Sem. Perinatol. 14, 299-304. Gerhardt, K.J., Otto, R., Abrams, R.M., Colle, J.J., Burchfield, D.J. and Peters, A.J.M. (1992) Cochlear microphonics recorded from fetal and newborn sheep. Am. J. Otolaryngol. 13, 226-233. Gerhardt, K.J., Abrams, R.M. and Oliver, CC. (1990) Sound environment of the fetal sheep. Am. J. Obstet. Gynecol. 162,282-287. Grimwade, J.C., Walker, D.W., Bartlett, M., Gordon, S. and Wood, C. (1971) Human fetal heart rate change and movement in
230
S.K. Griffiths et al. /Hearing Research 74 (1994) 221-230
response to sound and vibration. Am. J. Obstet. Gynecol. 109, 86-90. Henderson, D., Subramaniam, M. and Boettcher, F.A. (1993) Individual susceptibility to noise-induced hearing loss; An old topic revisited. Ear Hear. 14, 152-168. Horner, KC., Serviere, J. and Granier-Deferre, C. (1987) Deoxyglucase demonstration of in utero hearing in the guinea-pig foetus. Hear. Res. 26, 327-333. Jones, C.T. and Nathanielsz, P.W. (1985) The physiological development of the fetus and newborn. Academic Press, New York. Laciak, J. and Majcherska-Matuchniak, B. (1968) Hearing in children whose mothers are working in noise. In: Meleck, Baradin and Pamietnik (Eds.), XXVII Zjasdu Otolaryngologow Polskich. W Katowicach, Warsaw. Lalande, N.M., Hem, R. and Lambert, J. (1986) Is occupational noise exposure during pregnancy a risk factor of damage to the auditory system of the fetus? Am. J. Ind. Med. 10, 427-435. Oliver, CC. (1989) Sound and Vibration in tissues. Semin. Perinatol. 13, 354-361. Patrick, J. and Gagnon, R. (1989) Fetal breathing movements and fetal rest activity patterns. In: R. Creasy and R. Resnik (Eds.), Maternal-fetal Medicine: Principles and Practice, Saunders, Philadelphia, pp. 268-287. Peters, A.J.M., Abrams, R.M., Gerhardt, K.J. and Griffiths, S.K. (1993a). Transmission of airborne sound from 50-20,000 Hz into the abdomen of sheep. J. Low Freq. Noise Vib. 12, 16-24. Peters, A.J.M., Gerhardt, K.J., Abrams, R.M. and Longmate, J.A. (1993b) Three dimensional intraabdominal sound pressures in sheep produced by airborne stimuli. Am. J. Obstet. Gynecol. Querleu, D., Renard, X. and Crepin, G. (1981) Perception auditive et reactivite foetale aux stimulations sonores. J. Gyn. Obstet. Biol. Repr. 10, 307-314. Querleu, D., Renard, X., Versyp, F., Paris-Delrue, L. and Crepin, G. (1988) Fetal Hearing. Eur. J. Obstet. Gynecol. Reprod. Biol. 29, 191-212.
Richards, D.S., Cefalo, R.C., Thorpe, J.M., Salley, M. and Rose, D. (1988) Determinants of fetal heart rate response to vibroacoustic stimulation in labor. Obstet. Gynecol. 71, 535-540. Richards, D.S., Frentzen, B., Gerhardt, K.J., McCann, M.E. and Abrams, R.M. (1992) Sound levels in the human uterus. Obstet. Gynecol. 80, 186-190. Sohmer, H., Freeman, S., Friedman, I. and Lidan, D. (1991) Auditory brainstem response (ABR) latency shifts in animal models of various types of conductive and sensori-neural hearing losses. Acta Otolaryngol. 111, 206-211. Stapells, D.R., Picton, T.W., Perez-Abalo, M., Read, D. and Smith, A. (19851 Frequency specificity in evoked potential audiometry. In: J.T. Jacobson (Ed.), The Auditory Brainstem Response. College-Hill Press, San Diego, CA, pp. 147-177. Vince, M.A., Armitage, S.E., Baldwin, B.A. and Toner, J. (1982) The sound environment of the foetal sheep. Behavior 81, 296-315. Vince, M.A., Billing, A.E., Baldwin, B.A., Toner, J.N. and Weller, C. (1985) Maternal vocalisations and other sounds in the fetal lamb’s sound environment. Early Hum. Dev. 11, 179-190. Walker, D., Grimwade, J. and Wood, C. (1971) Intrauterine noise: A component of the fetal environment. Am. J. Obstet. Gynecol. 109, 91-95. Wolfson, M.R., Durrant, J.D., Tran, N.N., Bhutani, V.K. and Shaffer, T.H. (1990) Emergence of the brain-stem auditory evoked potential in the premature lamb. EEG Neurophysiol. 75, 242-250. Wollack, C.H. (1963) The auditory acuity of the sheep (Qvis aries). J. Aud. Res. 3, 121-132. Woods, J.R. Jr, Plessinger, M.A. and Mack, C.E. (1983) Fetal auditory brainstem evoked response (ABR). Ped. Res. 18, 83-85. Woods, J.R. Jr and Plessinger, M.A. (1985) The fetal auditory brain stem response: Serial measurements at two stimulus intensities. Otolaryngol. Head Neck Surg. 93, 759-764.