Prenatal Exposure to Tobacco and Alcohol Alters Development of the Neonatal Auditory System

The purpose of this study was to determine the effects of prenatal alcohol exposure (PAE), prenatal tobacco exposure (PTE), and the combination of the two on the developing auditory system of human infants. Two objective measures used as biomarkers of auditory system function were employed. The speed and objectivity of the auditory brainstem response (ABR) [1, 2] and the transient otoacoustic emission (TEOAE) [3, 4] have established these tests as diagnostic and scientific measures of auditory system function in newborns. The measures as applied in this study have been described previously [5].

ABR is a sound-evoked, averaged neural potential, differentially recorded from surface electrodes in response to transient sounds. The surface-recorded ABR was first described by Jewett and colleagues [6] 50 years ago. ABR generators are the auditory nerve and the low brainstem auditory pathway [7, 8]. The morphology of the response from newborns is comprised of 3 of the 5 peaks seen in the mature response. ABR can be used to characterize the temporal aspects of neural transmission through the brainstem pathway when elicited by high-level (60–80 dB above hearing threshold), transient, click stimuli [2].

The TEOAE first described by Kemp [9] is reflected energy from the cochlear amplifier of the outer hair cells (OHCs). The stimulus is presented via an ear canal probe device with a transducer to deliver sound to the tympanic membrane. The sound pressure is transmitted through the middle ear and into the cochlear fluids. This motion activates the inner hair cells (IHCs) and OHCs of the cochlea. The action of the OHC is a motor-like response, physically expanding and contracting the cells. The hair cells are firmly attached to the basilar membrane and therefore the OHC-transmitted motion increases the sensitivity of the IHC by as much as 20–60 dB and dramatically sharpens the tuning, thus the term cochlear amplifier [10]. Some of this additional energy from the OHC travels back along the incoming sound path and creates a pressure waveform in the ear canal (otoacoustic emission) that is measured with a sensitive microphone in the OAE probe.

The presence of a TEOAE provides an objective measure of cochlear function, specifically OHC presence and motility. The motor process of the OHC requires no neural input. However, the TEOAE is modulated (inhibited) by activation of brainstem medial olivocochlear (MOC) efferent neurons that synapse on the OHC [11].

The TEOAE and ABR are both objective measures that are well suited to infant evaluations but provide distinct information about the auditory system. They are not used in this application to determine hearing levels, rather cochlear (TEOAE) and brainstem (ABR) function. The TEOAE can also be an indirect measure of efferent activity from the brainstem.

Prenatal exposure to cigarette smoking and alcohol is known to inflict damage on the developing human auditory system [12-14]. Both nicotine and alcohol are found in the fetal blood supply after passing through the placenta where they can have significant toxic effects on many aspects of cell development [15]. However, prior studies have not clearly identified the degree of in utero exposure associated with damage nor the extent of such damage to the developing human auditory system.

Fetal morbidities related to PTE include reduced birth weight, decreased head circumference, hypoxia, and alteration in fetal blood flow and protein metabolism [13-18]. Both prenatal [19] and environmental exposure [17, 20] to tobacco smoke have been clearly linked to increased incidence of otitis media in infants and children. One study documented that PTE results in slightly increased levels of high-frequency hearing loss and a greater percentage of unilateral hearing loss in adolescents [21]. However, no evidence of peripheral hearing loss in infants or young children following PTE has been published. Neurodevelopmental disorders are also associated with PTE [13, 14], many of which are related to auditory system function. In particular, children born with PTE have been found to show lower scores on measures of cognitive function and developmental skills [18, 22], memory [23, 24], verbal subscales of intelligence tests [24-26], and reading and phonological processing [27]. Difficulty with tasks involving auditory processing is repeatedly mentioned in children with PTE [18, 28-32]. McCartney et al. [33] found reduced performance of 6- to 11-year-old children on the SCAN test, which evaluates central auditory processing skills. Performance from the children with PTE was particularly poor on the test section involving identification of dichotic competing words. Clearly, children with PTE are at risk for auditory difficulties that can be subtle and may not involve peripheral hearing loss.

Auditory system evaluations have been previously used to assess outcomes of PTE. Kable and colleagues [34] found that the PTE level was negatively related to wave V latency and waves I–V interpeak latency intervals in 6-month-old infants. Katbamna et al. [35] compared full-term, otherwise healthy infants from a group of mothers who smoked to a control group born to nonsmoking mothers using ABRs and distortion-product otoacoustic emissions (DPOAEs). DPOAE amplitudes were reduced in the infants with PTE. The latency of ABR peak V and the interpeak interval I–V was also reduced in the exposed infants relative to controls. Korres et al. [36] enrolled pregnant women at the time of birth who were grouped as nonsmokers, and low-, moderate-, or high-level smokers based on history. Binaural TEOAEs were smaller for the PTE group, but TEOAE levels did not change with smoking group. Durante et al. [37] compared neonates of nonsmoking mothers to infants of mothers who smoked >5 cigarettes per day. The infants with PTE demonstrated reduced amplitude TEOAEs relative to controls in the right ear only. Neither TEOAEs in the left ear nor DPOAEs in either ear distinguished the groups.

Prenatal alcohol exposure may result in fetal alcohol spectrum disorder (FASD) with fetal alcohol syndrome (FAS) being the most severe condition in the spectrum. PAE can lead to oxidative injury, apoptosis, gene expression modulation, and disruption of neuronal migration, which is reflected in central nervous system dysfunction, growth deficiencies, characteristic facial abnormalities, and other malformations [38, 39]. Abnormal functioning in the areas of cognition, executive function, memory, language, adaptive functioning, auditory and visual perception, and academic performance has been described in persons with FASD [39-41]. Neuroimaging and autopsy studies of FASD and PAE have universally found evidence of reduced brain volume in response to the exposure [42-49]. Specific abnormalities in brain structure including cortical development, white matter microstructure, and functional connectivity have been associated with PAE [39].

Auditory system dysfunction is often distinguished in association with PAE although hearing loss, as defined by elevated hearing thresholds due to middle ear or cochlear dysfunction, has only been described in children with FAS. One study of visual and auditory attention compared adults with FASD to typically functioning, age-matched adults and found that the participants with alcohol exposure had more difficulty with auditory stimuli than visual on the attention task [50]. Behavioral assessments of “Central Auditory Processing” abilities have also uncovered dysfunction in children with FASD including abnormal speech perception in noise [51] and reduced incidence of the expected “right ear advantage” on dichotic speech perception tests [52].

There have been no systematic studies of ABR results on human infants or children with PAE without FASD. One study of ABR in children with FAS [53] found a high prevalence of hearing loss, which would contaminate the evaluation of ABR peak latencies and amplitudes. Standard cortical event-related potentials [54, 55] and magnetoencephalography [56] event-related potentials have found that children with PAE demonstrate abnormally long peak latencies and reduced peak amplitudes. These findings are indicative of poor processing of auditory signals at the level of the auditory cortex. At present, otoacoustic emission testing has not been applied to the study of PAE.

Auditory system outcomes, as with other neurodevelopmental outcomes [57], may be influenced by interactions between the effects of prenatal alcohol and tobacco. Kable et al. [34], when evaluating PTE effects on ABR, also found that alcohol use was related to ABR latency. Unfortunately, other evaluations of auditory system outcomes either eliminated maternal subjects with a history of the other exposure [35, 37] or did not include the other substance use in maternal assessments [36, 54, 55, 58]. Therefore, little is known about how interactions of these prenatal exposures may influence auditory system outcome.

The current study reports findings from the safe passage study, a large, international, prospective, and multidisciplinary investigation designed to examine the role of PAE and PTE in association with sudden infant death syndrome (SIDS), stillbirth, and to determine physiological outcomes from exposures in surviving children [59]. In the full study, 10,727 infants were followed for 1 year [60].

This study had the advantage of extensive prospective documentation of exposures. Other studies of ABR or TEOAE following prenatal exposures enrolled mothers at the time of birth with retrospective documentation of smoking and drinking behavior [34-37]. The limitations of historical data collection are well known. Jacobson et al. [61] studied antenatal and retrospective reporting of alcohol, smoking, and cocaine use in pregnant women and demonstrated that the antenatal interview provided the most valid information. The data presented here represent the largest database and only prospective study of serial ABRs, and TEOAEs with documented PTE and PAE available to date. The sample size, the diversity of the participants, and the extensive prenatal data collection afford a more complete representation of the PAE and PTE results. The physiologic auditory system outcomes are described here.

Participants in this study were enrolled in the safe passage study [59], a multidisciplinary, prospective study designed primarily to investigate the relationship between prenatal alcohol exposure and SIDS [60] and stillbirth. Mother-infant dyads were enrolled at 2 regional centers, the Northern Plains of the USA and South Africa. Institutional Review Board approvals were obtained from sponsoring organizations at the participating clinical sites, as well as for the Data Coordinating Center and the Physiological Assessment Center [59, 62]. Newborn and 1-month visits were conducted in North and South Dakota from 5 sites, including 2 Native American Reservations, and in Cape Town, South Africa (Tygerberg and Karl Bremer hospitals). Sites were selected for high rates of prenatal alcohol use and SIDS. In addition, these populations represent diverse communities who exemplify one culture or multiple cultures and socioeconomic status. The Dakota sites are grouped to form the Northern Plains (NP) Center and South Africa (SA) forms the second Center.

Extensive pre- and postnatal documentation and assessments of the mothers and infants were conducted including, but not limited to, exposures to alcohol and cigarette smoking. Data collected included demographics, medical and obstetric records, and information on dietary and psychosocial history including depression, resilience, traumatic and threatening experiences, anxiety, and perceived stress. Self-reported exposure (e.g., alcohol, tobacco, marijuana, and methamphetamines) and fetal physiological measurements were obtained approximately every 8 weeks during the prenatal period. Summaries of enrollment and exposure assessments have been previously published [59, 60].

Newborn participant demographics for infants in this study including gestational age (GA) at birth, hours of life at test, GA at test, delivery type, sex, and race/ethnicity are shown in Table 1. Table 2 includes exposure levels for alcohol and smoking clusters by trimester. Table 3 includes a cross-tabulation of cluster membership for participants by evaluation type. Across ages and assessments, 31.2% of subjects had no exposure, 16% were exposed to tobacco alone, 24.5% were exposed to alcohol alone, and 27.3% were exposed to both alcohol and tobacco.

Auditory system data were collected between April 2009 and August 2015. ABRs were performed on 3,892 newborns (1,567 from NP and 2,325 from SA) and again on 1,092 infants at 1 month of age (402 from NP and 690 from SA). Preterm infants were included in the analysis, and the GA at birth was used as a covariate. Data for the final ABR analysis were missing or excluded on 733 due to technical errors or uninterpretable recordings. Participant’s data were also excluded from the analysis based on the prenatal exposure to psychiatric medications at any point in pregnancy (SSRIs, antidepressants, anxiolytics, antipsychotics, mood stabilizers, stimulants, and anticonvulsants) or early infant demise. The final analysis was conducted on 2,613 newborn and 925 1-month ABRs.

TEOAEs were recorded from 2,250 newborns (825 from NP and 1,425 from SA) and 2,227 infants at 1 month of age (1,058 from NP and 1,389 from SA). TEOAEs that did not meet a criterion signal-to-noise ratio (SNR) of 4 dB in 4 of the 6 frequency bands were removed from analyses due to possible contamination from common infant middle ear conditions [5]. The final number of participants providing TEOAE data for analysis was 1,928 newborn and 1,973 1-month TEOAEs.

Cluster analyses used to characterize levels of PAE and PTE into subcategories were based on timing, duration, and amount of exposure as previously described [63, 64]. Two of the original 4 PASS alcohol clusters were collapsed for this study to create a 3-level PAE variable. The “Low Continuous” and “High or Moderate Continuous” clusters [52] were combined into 1 cluster termed “Continuous.” The final PAE clusters included “Unexposed,” “Quit Early,” and “Continuous.” The clusters used for PTE included “Unexposed,” “Low Continuous and Quit Early” (LCQE), and “High and Moderate Continuous” (HMC). The data for each exposure and cluster by trimester can be found in Table 2. The cross-tabulation of participants by smoking and alcohol clusters for each experimental group is shown in Table 3. Overall, 31.2% of mothers used neither alcohol nor tobacco, 16% smoked but did not drink alcohol, 25.5% drank alcohol but did not smoke, and 27.3% used both tobacco and alcohol.

Complete details of ABR and TEOAE procedures used in this study have been published [5]. ABRs were measured with Intelligent Hearing Systems USBLite hardware in response to 80 dB nHL clicks and TEOAEs were measured on an Interacoustics Otoread system in response to 83 dB SPL clicks. TEOAEs were obtained in response to 300 stimuli with artifact-free responses and ABRs to 2 averages, each with 1,024 clean responses. A typical ABR from a newborn is shown in Figure 1. Two initial ABRs were compared for consistency and averaged. Three peaks were marked for latency in milliseconds and amplitude from peak to following trough in microvolt. All ABR scoring was reviewed by the first author and corrected if necessary.

The TEOAE averaged waveform is split into 2 buffers created by positive and negative polarity stimuli. These buffers are subtracted to compute the background noise and added to produce the overall TEOAE response in dB SPL. TEOAE and noise spectra were sampled at 1.5k, 2k, 2.5k, 3k, 3.5k, and 4k Hz. Data used in analysis include the TEOAE signal level in dB SPL and the SNR.

Mixed-design analyses of covariance (ANOVAs) (repeated measures) were computed for ABR peak latencies and peak amplitudes, with waves I, III, and V as repeated measures and TEOAE level and SNR, with individual frequencies as repeated measures, to examine the main effect of PAE, the main effect of PTE, and the interaction between the two exposures. In addition, ANOVAs were applied to the ABR interpeak intervals of I–III, III–V, and I–V. Both ABR and TEOAE measures have been shown to be dependent upon GA at birth [65-68], sex [69, 70], and postnatal age in hours of life (HOL) at the time of assessment [66, 71, 72]. Analyses controlled for these factors and for the clinical center. In addition, the PAE analysis controlled for PTE and vice versa. The analyses of 1-month metrics followed the same procedure except that HOL was not included as a covariate because the influence of HOL has not been demonstrated beyond the first 48 hours [72].

ABR latency has been shown to be influenced by head circumference [73-76] and reduced head circumference has been associated with PTE [77]. Accordingly, the association between PTE and head circumference was analyzed.

A summary of F and p values found for repeated-measures analyses can be found in Table 4. Those found for interpeak intervals are shown in Table 5.

PTE had a significant effect on newborn ABR latency in the right (p = 0.001) and left (p = 0.026) ears as shown in Figure 2a. An interaction between PTE and PAE on newborn ABR latency was found in the left ear only (p = 0.037) as shown in Figure 3. PAE alone did not influence absolute peak latencies nor peak amplitudes in newborns. Newborn ABR interpeak I–III interval was significantly influenced by PAE (p = 0.007) and PTE (p = 0.038) in the right ear, but no interaction of exposures was found for this measure. The III-V interval in the left ear was significant for PAE (p = 0.022).

The effect of PTE on the newborn ABR was a reduction of the peak latencies as shown in Figure 2a. The significance of individual paired comparisons of latency with exposure is indicated with star symbols. In no instance was the difference between the latency of the “Unexposed” and the “LCQE” smoking conditions significant. All “Unexposed” and “HMC” comparisons were significant except for wave V in the left ear which approached but did not reach significance at p = 0.063.

A significant interaction was found between newborn alcohol and tobacco exposures regarding ABR latency for the left ear only (p = 0.037) as shown in Figure 3. This was the only instance in which PAE was shown to affect the newborn ABR absolute latencies.

At 1 month of age, PTE had a significant effect on ABR amplitude in the right (p = 0.001) and left (p = 0.004) ears as shown in Figure 2b. PTE was also found to have a significant influence on the I–V interpeak interval in the left ear (p = 0.002) at 1 month.

Analysis of absolute ABR metrics from infants with tobacco exposure at 1-month of age revealed a significant decline in peak amplitude in the left (p = 0.004) and right (p = 0.001) ears. The amplitudes are plotted by exposure in Figure 2b. Amplitude reductions were greatest in the “Unexposed” to “LCQE” comparisons. These were significant in both ears and for all peaks except for the left ear for wave III. Wave III overall showed the least change in amplitude with tobacco exposure. Also, while wave I showed a continuous decline in amplitude with exposure, wave V showed no change or a very slight increase in amplitude with the highest level of exposure when compared to the “LCQE” condition.

Neither alcohol nor tobacco exposures were associated with changes in the newborn TEOAE measures. However, a significant alcohol exposure effect was found for the TEOAE in the left ear only of infants at 1 month of age (p = 0.011). Figure 4a displays the effects of alcohol exposures on the frequency bands of the TEOAE in both ears. The reduction in the TEOAE amplitude on the left was greatest in the mid-frequency regions from 2.0 to 3.5k Hz. No similar trends in TEOAE amplitudes due to alcohol exposure were seen in the right ear. Alcohol exposure did not produce significant effects on the overall SNR of the TEOAE although the 2 measures are closely related. The SNR is the TEOAE amplitude divided by the background noise. SNR values are plotted by alcohol exposure for each ear in Figure 4b. While the overall repeated measures in the left ear were not significant (p = 0.158), 3k Hz and 3.5k Hz pairwise exposure comparisons for the SNR were significant. These 2 frequencies demonstrated the same pattern shown for the TEOAE alone, that is, the greatest reduction in amplitude in the “Continuous Alcohol” group and in the mid-frequency region. Neither the overall SNR nor any of the pairwise comparisons were significant in the right ear. This further emphasizes the TEOAE ear asymmetry in response to alcohol exposure.

TEOAEs that did not meet criterion SNR were removed before data analysis for reasons stated in the methods section. To determine if exposure was related to pass/fail status, a χ² analysis was conducted on the distributions of pass/fail for smoking groups and alcohol groups. The comparison of the proportion of newborns with a “fail” in one or both ears to those with “pass” status in both ears was significant for PTE (χ² = 8.713, p = 0.013) as shown in Figure 5. The percentage of failed TEOAEs was higher in smoking groups and lower in the nonsmoking group. There was no association between pass/fail status and PAE and no association of alcohol or smoking with pass/fail status at 1 month of age.

The effect of PTE and PAE on infant head circumference was analyzed by ANOVA. PTE was shown to have a significant effect on head circumference in the newborn stage (p = 0.002) but not at 1 month of age (p = 0.476). The pattern of effects showed a large reduction in head circumference between the “Unexposed” and “LCQE” exposures (p = 0.001). Average head circumference increased slightly between “LCQE” and “HMC,” but this was not significant (p = 0.566). The reductions in head circumference from “Unexposed” to “HMC” were also significant (p = 0.046). PAE was not found to influence head circumference (p = 0.935) and alcohol by tobacco interaction was also not significant (p = 0.650).

Statistics on the covariate factors for the conditions with significant exposure effects are shown in Table 2 Delivery GA was significant for newborn ABR measures but was not a factor in the 1-month amplitude measures. Sex and postnatal age (HOL) were both significant covariates for ABR measures as expected [70, 72, 78]. Clinical center also showed influence on most exposure effects. One possible explanation lies in the cultural diversity between the NPs and SA sites. Figure 6 demonstrates the values of smoking and alcohol consumption within each cluster across the trimesters comparing the 2 centers. Wide discrepancies are apparent in the amounts of exposure within each cluster across centers. These could account for the influence of center on the exposure effects.

In this study of the effects of PAE and PTE on the developing human auditory system, our major conclusions are as follows. First, latencies of ABR peaks elicited in both ears are reduced in newborns in response to PTE in a manner that is proportional to the exposure. Reduced latencies are apparent only with the highest level of PTE. This effect is not seen when the infants reached 1 month of age. Second, ABR peak amplitudes are reduced in response to PTE, not at birth but by 1 month of age, again bilaterally and proportional to exposure. The amplitude reduction is greatest with the lowest level of tobacco exposure. And third, an interaction of PAE and PTE on newborn ABR latencies was found only in the responses from the left ear. The pattern of interactions, as seen in Figure 3, does not show any systematic patterns. With this one exception, in one ear only, PAE did not show a significant influence on ABR measures from either age-group.

Prenatal exposures had scattered effects on ABR interpeak latencies. Both alcohol and tobacco demonstrated these effects in both age-groups. With one exception (right ear I–III interval by PTE), the absolute latencies were not influenced by the same exposures. Interpretation of interpeak intervals that do not show a corresponding overall effect on absolute latencies is questionable. The interpeak interval findings were also unilateral in all instances. Given the shared generators of the ABR elicited by each ear, any important change in neural conduction time should have been reflected on both sides [2].

Newborn TEOAEs were not significantly affected by either exposure. However, the response was reduced significantly in the left ear of the 1-month-old infants by exposure to PAE. This finding is discussed in detail below.

Although not part of the primary analysis of effects, a significant increase in poor TEOAE responses was found in newborns with PTE. This finding is in line with increased incidence of otitis media in infants with PTE [19]. Middle ear disease is known to reduce or eliminate the TEOAE regardless of the status of the cochlea [79, 80].

As has been shown in other studies [34, 35, 58], ABR latencies are shortened by PTE. This finding is consistent across ears, is seen for all ABR peaks, and occurs in a dose-dependent manner as shown in Figure 2a. Other than PTE, damage to the peripheral auditory system has previously been associated with an increase in ABR peak latencies rather than a decrease. Premature infants are known to have delayed ABR latencies [65, 66]. When used in the diagnosis of neurological diseases, including vestibular schwannoma, multiple sclerosis, Bell’s palsy, and others, increased ABR peak and interpeak latencies and reduced amplitudes have been found [2, 81]. In a preliminary description of unexposed infants from this study’s population, Sininger et al. [5] demonstrated that the latency of all ABR peaks was reduced and the amplitude increased over the first month of life. A variety of mechanisms have been postulated for the rapid decrease in ABR peak latency in the first month of life including improved middle ear transmission [82, 83]. This study found that PTE reduced latencies in all 3 ABR peaks including wave I, which is generated in the most peripheral portion of the auditory nerve [84]. Kable et al. [34] found significantly shortened latencies for wave V only but also demonstrated reduced latency for waves I and III that were proportional to exposure but did not reach statistical significance. The discrepancy between studies could have been due to differences in the sample size.

Reduced head circumference has been associated with reduced brainstem length [73] and with shortened ABR peak and interpeak latencies [73-76]. A reduction in head circumference was associated with PTE in this population. However, the pattern of effects of PTE on head circumference changes cannot predict the changes seen in latency. The lowest level of PTE results in significant reduction in head circumference but not in ABR latency reductions. Also, adding head circumference to the covariates in the smoking by latency ANOVAs did not reduce the significance of the effect. Therefore, while intriguing, head circumference reductions due to tobacco exposure cannot be the explanation for reduced ABR latencies.

A plausible candidate for the source of reduced ABR latencies is altered synaptic transmission at the IHC-spiral ganglion neuron intersection. Nicotine toxicity is the most likely source of damage from PTE and systems involving nicotinic receptors are the likely target of such involvement. Animal preparations have shown that prenatal nicotine exposure consistently leads to an upregulation of presynaptic, nicotinic ACh receptors (nAChRs) [85, 86] and such exposure impairs the ability of the receptors to modulate the release of both inhibitory and excitatory neurotransmitters [87].

The explanation for prenatal nicotine exposure effects on ABR peak latencies in early development involves the cholinergic olivocochlear system, which modulates activity in the hair cells and early segments of the auditory nerve [11, 88, 89]. The medial and lateral olivocochlear (MOC and LOC) efferent neurons make up a descending pathway originating in the superior olivary complex in the brainstem. Olivocochlear efferents contact the IHC and OHC as well as the primary portion of the auditory nerve early in development [90].

The MOC neurons are primarily, if not exclusively, cholinergic and innervate the OHCs in the mature auditory system [91, 92]. However, of particular importance to the interpretation of data from this study is the fact that cholinergic MOC fibers, during the very earliest stages of hearing development in mammals, before innervation of OHCs, transiently project directly to the IHCs communicating via α9- and α10-nAChRs [93-97]. In rodents, the direct innervation of IHC by the MOC happens just after birth and lasts through the second postnatal week [98, 99].

The role of efferent innervations to the developing hair cells is to set activity patterns in the cells and the synaptic structures. Functional nAChRs mostly of the α9 and α10 subtype are found on rodent IHCs and MOC fibers just after birth [99] where they promote synaptic maturation and stabilization during early development [100, 101]. Johnson et al. [102] studied the IHC ribbon synapse in an α9 nAChR knockout mouse model to demonstrate that the efferent system was required for normal synaptic development.

Latency reductions in ABR wave I that accompany PTE could be explained by a transient increase in synaptic efficiency at the IHC. Such effects could occur during early (third trimester) MOC innervation of the IHC, which, due to the nicotine exposure, may result in an overproduction of nAChRs. A reduction of newborn ABR latencies with PTE was not seen in the infants with “LCQE” exposures but was substantial for infants born to mothers with “HMC” smoking. Differences could relate to either the timing or degree of exposure or both. According to Moore and Linthicum [90], the MOC innervation of the IHC in humans would be expected around the beginning of the third trimester, which is after the timing of the smoking cession in the “quit early” mothers. If the temporary influence of the MOC on the IHC is implicated in reduced latencies of the ABR, infants of mothers who quit early would be expected to be sparred.

It could be argued that increased ABR peak amplitude should accompany reduced latency when synaptic efficiency increases. However, no such amplitude increase was observed. For this theory to be valid, increased synaptic activity would need to be reflected in synaptic timing rather than in numbers of nerve fibers activated. The reduced latencies in waves III and V could be a simple reflection of upstream shortened latency of wave I. Mechanisms involving other than the initial synapse might have been expected to change interpeak latencies, but no consistent or bilateral changes in interpeak latency were found.

The finding of reduced ABR peak latency with PTE was not observed at 1 month of age. This indicates that the effects of PTE on the latency of the ABR are overcome after the newborn period. Kable et al. [34] found reduced ABR latencies in PTE infants at 6 months of age, but these were only significant for wave V. It should be noted that the sample size of ABR data in this study was reduced at 1 month with 925 participants as compared to 2,613 in the newborn period. This was not due to lost participants, rather to an emphasis on TEOAE measures at 1 month. However, examination of 1-month ABR data relative to smoking clusters demonstrated a (nonsignificant) trend for latency to increase with exposure. This would argue against missing a latency reduction at 1 month due to insufficient sample size.

After 1 month of age, ABR amplitudes, particularly for peaks I and V, were found to be reduced in infants with PTE. Amplitude reductions were found in ABRs elicited from both left and right ears and were reduced in proportion to PTE as shown in Figure 2b. A study of 54 early demise fetuses and infants examined brainstem samples, which were subjected to AChR immunohistochemistry. Thirty-four of the infant’s mothers were confirmed smokers and 20 were nonsmokers. Only in the samples with tobacco exposure were high levels of α7-nAChR expression observed along with hypoplasia of some brainstem structures [103]. Perinatal nicotine exposure also has been shown to disrupt the maturation of glutamatergic inputs in the auditory brainstem, specifically delaying the developmental downregulation of functional nAChRs in a mouse model. A dramatic reduction in the synaptic input amplitude in the lateral lemniscus was also seen [104]. Any of the aforementioned mechanisms could explain the reduction of amplitude in the ABRs of the 1-month-old infants.

Unlike the smoking effects on newborn latencies, the effects seen at 1 month of age on amplitude are found with both the “LCQE” and “HMC” exposures. This indicates that all levels and timing of PTE will lead to reduced amplitudes of the auditory system brainstem activity.

Clear evidence of reduced amplitude of the TEOAEs was found in association with PAE, in the left ear only. TEOAEs are generated by the OHCs in each ear, which are known to be sensitive to effects of oxygen deprivation, and hypoxia has often been associated with PAE [105-108]. However, hypoxia would certainly affect the hair cells of both ears and therefore cannot explain reduced amplitude of the OAE in one ear only.

The major damage associated with PAE, especially during brain growth spurts in the last trimester, is massive neuron loss due to apoptosis [109, 110]. The cerebral cortex is one of the most damaged areas of the brain, and within the cortex, pyramidal neurons of layer 5 are more susceptible to the effects of alcohol than cells in other layers [111, 112]. Layer 5 of the auditory cortex is also the origin of efferent neurons that innervate the inferior colliculus and medial geniculate [113] and continue to the superior and lateral olive. Corticofugal influences on the inner ear begin in the auditory cortex and descend via the brainstem to the olivocochlear neurons. Axons descending from the human inferior colliculus to the olivocochlear neurons can be seen in staining between the 22nd and 26th weeks of gestation [90].

PAE has been shown to cause asymmetrical damage to the developing brain with greater damage to structures in the right hemisphere [114]. Specific damage to the right temporal lobe has often been reported in relation to PAE in studies using MRI. Rajaprakash et al. [115] noted a reduced cortical surface area in the right superior temporal gyrus of children aged 8–16 years with PAE as compared to unexposed controls. Chen et al. [43] using structural MRI found that young adults with PAE, when contrasted to control subjects, demonstrated reduced volume in the right inferior temporal gyrus. Lebel et al. [116] looked at longitudinal changes in cortical volume in children and youth comparing those with PAE to controls. They found that the right transverse temporal lobe regions were negatively correlated with PAE; greater alcohol exposures were associated with larger decreases in volume from MRI scans spaced 2 years apart.

Perhaps, the most convincing data on damage specifically to the right temporal lobe in response to PAE come from Sowell and colleagues [117]. They utilized cortical matching techniques to examine hemispheric asymmetry in adolescents with heavy PAE and controls and found that alcohol-exposed individuals had significantly reduced “right to left” ratios based on both surface and volumetric image analysis in the posterior temporal lobes. The authors point out that right greater than left asymmetry in the posterior temporal lobes [118] is still noted in most MRIs from participants with PAE. However, there is a significant reduction in this asymmetry associated with alcohol exposure.

The auditory efferent system connects the auditory cortex to the cochlea through a series of afferent-efferent feedback loops [119]. Damage to the right hemisphere temporal lobe will influence the left side of the peripheral auditory system via the crossed descending pathways. MOC inhibition of the OHC is a well-known phenomenon, which can be evaluated in mature auditory systems with a technique known as the Medal Olivocochlear Reflex (MOCR) [120]. Effects of activation of cortical neurons can be seen in cochlear functions including otoacoustic emissions. Removal of parts of the temporal lobe in patients with epilepsy will alter the MOCR particularly in the ear contralateral to the ablation [121]. In a study of patients scheduled for temporal ablations, direct electrical stimulation of the cortex was shown to significantly reduce the amplitude of the TEOAE in the ear contralateral to the stimulation [122]. Central damage to the auditory regions of the right hemisphere from PAE could translate to disruption of peripheral MOC innervation on the left ear due to a primarily crossed pathway.

The exact mechanism by which the reduction of the otoacoustic emission might be brought about by unilateral disruption of efferent stimulation is unclear. The left ear amplitude reduction seen in this study could reflect the partial damage to the outer hair cells or abnormal activation of the MOCR. In the developing auditory system, neither efferent stimulation nor innervation has been shown to be necessary for the OHC electromotility that produces the otoacoustic emission [123]. However, damage to the descending pathway would reduce the MOC activation at the level of the cochlea and interruption of MOC activation has been linked to increased damage to hair cells from mechanisms such as noise exposure [124] and aging [125].

The distinct asymmetry in the presentation of TEOAEs following PAE could allow this simple measure to be utilized as a biomarker for alcohol exposure. OAEs and ABRs have been explored as diagnostic indicators of other childhood disorders, primarily autism spectrum disorder (ASD). Otoacoustic emission amplitudes [126] and ABR indices, primarily latencies [127-129], have been shown to distinguish children with ASD from controls. The MOCR [130] has been shown to correlate with hyperacusis in children with ASD. However, none of these potential indices for autism have found an asymmetric response.

This study represents the largest sample of mother-infant dyads with carefully documented maternal use of tobacco and alcohol and neonatal auditory system outcomes. Prenatal tobacco use resulted in disruption of responses at the brainstem level at both the newborn timeframe (shortened ABR latencies) and the 1-month timeframe (reduced ABR amplitude). Reduced latencies may be attributable to prenatal disruption of synaptic mechanisms at the inner hair cell and the later amplitude reductions to cell damage in the brainstem. PTE was also associated with an increase in the number of infants that failed the TEOAE test. This was likely an epiphenomenon due to increased middle ear disease and not a reflection of outer hair cell damage accompanying PTE. Otherwise, no cochlear effects were found in infants exposed to tobacco; the TEOAE of infants with a robust response was not influenced by the exposure. In contrast, prenatal exposure to alcohol was found to show no effect on brainstem measures but led to a significant reduction in the cochlear response (TEOAE) in the left ear only of 1-month-old infants. This unilateral finding is felt to reflect cortical damage to the right temporal lobe, which sends information to the left cochlea via the corticofugal and olivocochlear efferent systems. This finding opens the possibility of the TEOAE test as a potential biomarker for PAE.

The PASS Network is solely responsible for the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript. The following researchers compose the PASS Network:

• PASS Steering Committee Chair (University of Texas Medical Branch): Gary D.V. Hankins, MD.

• Data Coordinating & Analysis Center (DM-STAT, Inc.): PI: Kimberly A. Dukes, PhD; Co-PI: Lisa M. Sullivan, PhD; Biostatistics: Tara Tripp, MA; Fay Robinson, MPH; and Cheri Raffo, MPH; Project Management/Regulatory Affairs: Julie M. Petersen, BA; and Rebecca A. Young, MPH; Statistical Programming/Data Management: Cindy Mai, BA; and Elena Grillo, MBA BS, BBA; Data Management/Information Technology: Travis Baker, BS; Patti Folan; Gregory Toland, MS; and Michael Carmen, MS.

• Developmental Biology & Pathology Center (Children’s Hospital Boston): PI: Hannah C. Kinney, MD; Assistant Director: Robin L. Haynes, PhD; Coinvestigators: Rebecca D. Folkerth, MD; Ingrid A. Holm, MD; Theonia Boyd, MD; David S. Paterson, PhD; Hanno Steen, PhD; Kyriacos Markianos, PhD; Drucilla Roberts, MD; Kevin G. Broadbelt, PhD; Richard G. Goldstein, MD; Laura L. Nelsen, MD; Jacob Cotton, BS; and Perri Jacobs, BS.

• Comprehensive Clinical Site Northern Plains (Sanford Research): PI: Amy J. Elliott, PhD; Co-PI: Larry Burd, PhD; Coinvestigators: Jyoti Angal, MPH; Jessica Gromer, RN; H. Eugene Hoyme, MD; Margaret Jackson, BA; Luke Mack, MA; Bradley B. Randall, MD; Mary Ann Sens, MD; Deborah Tobacco, MA; and Peter Van Eerden, MD.

• Comprehensive Clinical Site South Africa (Stellenbosch University): PI: Hendrik Odendaal, MBChB, FRCOG, MD; Co-PI: Colleen Wright, MD, FRCPath, PhD; Coinvestigators: Lut Geerts, MD, MRCOG; Greetje de Jong, MBChB, MMed, MD; Pawel Schubert, FCPath (SA) MMed; Shabbir Wadee, MMed; Johan Dempers, FCFor Path (SA); Elsie Burger, FCFor Path (SA), MMed Forens Path; and Janetta Harbron, PhD; Coinvestigator & Project Manager: Coen Groenewald, MBChB, MMed, FCOG, M. Comm.

• Physiology Assessment Center (Columbia University): Co-PIs: William Fifer, PhD; Michael Myers, PhD; Coinvestigators: Joseph Isler, PhD; Yvonne Sininger, PhD; Project Management: J. David Nugent, MA; and Carmen Condon, BA; Data Analysis: Margaret C. Shair, BA; and Tracy Thai, MA.

• NIH Project Scientists: Marian Willinger, PhD (NICHD); Dale Hereld, MD, PhD (NIAAA); Howard J. Hoffman, MA (NIDCD); and Chuan-Ming Li, MD, PhD (NIDCD).

We would like to thank Dr. Jos Eggermont for helpful suggestions regarding interpretation of results.

This research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki. Details of enrollment policies can be found in the Methods section under Participants and are published in Dukes et al. [59].

The authors have no conflicts of interest to declare.

Research reported in this publication was supported by National Institutes of Health grants U01HD055154, U01HD045935, U01HD055155, U01HD045991, and U01AA016501 funded by the National Institute on Alcohol Abuse and Alcoholism, Eunice Kennedy Shriver National Institute of Child Health and Human Development, and the National Institute on Deafness and Other Communication Disorders.

Drs Yvonne S. Sininger and William P. Fifer had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis; Yvonne S. Sininger, Michael M. Myers, Amy J. Elliott, William P. Fifer, Hein J. Odendaal, and Howard J. Hoffman were involved in study concept and design; Carmen G. Condon, Tracy Thai, Amy J. Elliott, Hein J. Odendaal, Jyoti Angal, and Deborah Tobacco assisted with data acquisition; Yvonne S. Sininger, Dwayne D. Simmons, Carmen G. Condon, Lauren C. Shuffrey, Lissete A. Gimenez, Nicolò Pini, James D. Nugent, Michael M. Myers, and William P. Fifer performed data analysis or interpretation; Yvonne S. Sininger, Carmen G. Condon, Lissete A. Gimenez, and Lauren C. Shuffrey drafted the manuscript; Michael M. Myers, Amy J. Elliott, William P. Fifer, and Hein J. Odendaal obtained funding; Carmen G. Condon, Hein J. Odendaal, Howard J. Hoffman, William P. Fifer, and Amy J. Elliott contributed administrative, technical, or material support; Carmen G. Condon, Hein J. Odendaal, Coen Groenewald, Amy J. Elliott, William P. Fifer, Jyoti Angal, and Deborah Tobacco were involved in study supervision.

The data that support the findings of this study are not publicly available due to involvement of Tribal Nations that preclude data sharing. Please contact the corresponding author if you have any questions or would like more information.