Fetal discrimination of low pitched musical notes

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J. P. Lecanuet* C. Graniere-Deferre A.-Y. Jacquet Laboratoire Cognition et De´veloppement, CNRS (UMR-8605), Universite´ de Paris V, France

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Fetal Discrimination of LowPitched Musical Notes

A. J. DeCasper Department of Psychology University of North Carolina at Greensboro (UNCG), Greensboro, NC 27410

Received 26 January 1999; accepted 8 July 1999 ABSTRACT: Cardiac responses of 36- to 39-week-old (GA) fetuses were tested with a nodelay pulsed stimulation paradigm while exhibiting a low heart rate (HR) variability (the HR pattern recorded when fetuses are in the 1f behavioral state). We examined whether fetuses could discriminate between two low-pitched piano notes, D4 (F0 ! 292 Hz/292–1800 Hz) and C5 (F0 ! 518 Hz/518–300 Hz). Seventy percent of all fetuses reacted to the onset of the first note (D4 or C5) with the expected cardiac deceleration. After heart rate returned to baseline, the note was changed (to C5 or D4, respectively). Ninety percent of the fetuses who reacted to the note switch did it with another cardiac deceleration. Control fetuses, for whom the first note did not change, displayed few cardiac decelerations. Thus, fetuses detected and responded to the pulsed presentation of a note and its subsequent change regardless of which note was presented first. Because perceived loudness (for adults) of the notes was controlled, it seems that the note’s differences in F0 and frequency band were relevant for detecting the change.

Fetuses’ ability to discriminate between spectra that lay within the narrow range of voice F0 and F1 formants may play an important role in the earliest developmental stages of speech perception. ! 2000 John Wiley & Sons, Inc. Dev Psychobiol 36: 29– 39, 2000 Keywords: fetus; auditory stimulation; musical notes; decelerative cardiac response; low heart rate variability state

This study of fetal auditory perception is the latest in a series from our laboratory that uses the autonomic cardiac deceleration, an attentional response if not an orienting response, as a measure of whether near-term fetuses can detect relatively subtle changes in their auditory environment. Here we looked for the cardiac reaction when we changed the spectral properties of

low-pitched musical notes: D4 and C51 using a nodelay pulsed paradigm.

Near term [over 35 – 36 weeks gestational age (GA)]2 fetal heart rate responses to sound show the same characteristics as those observed in newborns.

They are largely dependent on stimulus parameters such as sound pressure level (SPL), frequency, band-

*Present address: Laboratoire Cognition et De´veloppement, Institut de Psychologie, 71 Avenue Edouard Vaillant, 92774 Boulogne Billancourt, Cedex, Paris, France Correspondence to: J.-P. Lecanuet

1 D4 and C5 refer to the piano keyboard notation, numbers correspond to octave rank (from 0 to 8 starting on a C note.

2 Gestational age.

! 2000 John Wiley & Sons, Inc.

CCC 0012-1630/00/010029-11

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width, and on subjects’ parameters such as behavioral states (Granier-Deferre, Lecanuet, Cohen, & Busnel, 1985; Lecanuet, Granier-Deferre, Cohen, Le Houezec, & Busnel, 1986; Schmidt, Boos, Gniers, Auer, & Schulze, 1985). Acceleration responses are reliably evoked with broadband stimuli at or above 105-dB SPL (ref: 20 !Pa), and their frequency of occurrence is significantly enhanced as sound pressure level increases (for a review see Kisilevsky & Low, 1998; Lecanuet & Schaal, 1996). With less intense pressure level (80- to 90-dB SPL) stimulation, such as low or medium frequency noises, music, and speech, there is a shift from cardiac acceleration to deceleration. This change corresponds (with a 15- to 20-dB lag) to the change displayed by young infants and newborns when the sound pressure level of the stimulation decreases (Berg & Berg, 1987; Eisenberg, 1976; Graham, Anthony, & Zeigler, 1983). For Graham and colleagues (Graham, 1984, Graham et al., 1983; Graham & Clifton, 1966, Graham & Jackson, 1970), it reflects a shift from startle or defense response mode (DR) to orienting response mode (OR), according to Sokolov’s (1963) adult model of information processing, transferred to the infant and the newborn. As for the newborn, deceleration responses evoked in the “end of gestation fetus” are not accompanied by movements and have a smaller average amplitude than the acceleration responses (Lecanuet, Granier-Deferre, & Busnel, 1988; Lecanuet et al., 1987).

This similarity between fetal and neonatal cardiac responsiveness to auditory stimulation led us to attempt investigations of fetal discrimination abilities with a procedure derived from the one employed by Clarkson and Berg (1983) to study newborn syllabic discrimination of [A] and [i]: the “no-delay pulsed stimulation paradigm.” This procedure, used in numerous human infant and primate speech perception studies, is sometimes called “the 20/20 paradigm.” It is based on the idea that because HR response to a long-lasting stimulus “usually returns to a steady baseline while the stimulus is still present, it is possible, by introducing a second stimulus immediately following a sufficiently long-lasting first stimulus to evoke a second orientation response to the change in stimulation” (as cited in Brown, Morse, Leavitt, & Graham, 1976, p. 1). This procedure became a “no-delay” paradigm after Leavitt, Brown, Morse, and Graham (1976) suppressed the intertrial because of the memory load it imposed to subjects.

Using this procedure with 36- to 40-week-old (GA) fetuses, we showed that decelerative cardiac responses induced by onset of 16 pulsed presentations (at a rate of 3,000 ms) of the paired syllables [bi] – [bA] (emitted

at 92-dB SPL " 2 dB) were renewed after reversal of the order of the syllables: [bA] – [bi]. This demonstrated that near-term fetuses perceived the change in the organization of the stimulus (Lecanuet et al., 1987). We used the same paradigm to demonstrate that same-age-range fetuses discriminated a male voice (F0 ! 80 – 100 Hz) and a female voice (F0 !

165 – 200 Hz) uttering the same sentence; duration, prosodic contour, and loudness for adults were equated (Lecanuet, Granier-Deferre, Jacquet, & Busnel, 1992; Lecanuet, Granier-Deferre, Jacquet, Capponi, & Ledru, 1993). It should be emphasized that in both studies cardiac deceleration occurred within the first seconds of exposure to the initial stimulus and recovery of this deceleration occurred within the first seconds of a new stimulus, confirming that for fetuses as well as for newborns only a short acoustic sample is needed for the fetal auditory system to detect an acoustically relevant change.

Since our 1987 experiment, fetal cardiac decelerative reactiveness to different versions of the [A] and [i] vowels (paired or not] has been confirmed at higher sound pressure levels between (100 – 110 dB) (Zimmer et al., 1993) and down to 80-dB SPL (Groome, Mooney, Holland, Bentz, & Atterbury, 1997). Shahidullah and Hepper (1994) demonstrated that 35-weekold fetuses could discriminate between [[bAbA] and [bibi] by habituating and dishabituating startle responses to loud, 110-dB SPL stimuli.

The [bibA]/[bAbi] experiment had been designed at first as a [bi]/[bA] study, similar to Clarkson and Berg’s (1983) study and most discrimination studies, the two syllables being emitted at the same sound pressure level in air (92-dB SPL). However, due to differences in the structure of formants of the vowels: [i] F1 ! 240 Hz, F2 ! 2160 Hz and [A]: F1 ! 680 Hz, F2 ! 1200 Hz, [bi] sounded louder than [bA]. Because we had no way to determine the role of loudness in fetal perception and discrimination of acoustical stimuli, a first attempt at testing fetal discrimination abilities was made with the paired syllables as a global stimulus. Equating loudness (for adults) of male (F0 ! 83 – 100 Hz) and female (F0 ! 165 – 200 Hz) voices in the next experiment, which resulted in a 3dB SPL difference in air between these voices was another attempt. Again, we could not be certain whether the relevant dimension of difference was stimulus intensity or fundamental frequency, frequency band, and/or timbre. We do not know whether the slight SPL difference between the voices was preserved in the amniotic fluid and/or whether fetuses’ perceived loudness functions are adultlike. We do know that the intensity of the voices suffered little

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attenuated when transmitted in utero (Gagnon, Benzaquen, & Hunse, 1992; Querleu, Renard, Boutteville, & Cre´pin, 1989; Querleu, Renard, Versyp, Paris-Delrue, & Vervoort, 1988). We also know that, except frequencies below 100 Hz, the voice stimuli were not masked by the low-frequency components of maternal and placental vascular noises (50- to 60-dB SPL) because they were presented at least 30- to 40-dB higher than the background (Abrams, Gerhardt, & Peters, 1995; Armitage, Baldwin, & Vince, 1980; Gagnon et al., 1992; Gerhardt, 1989; Peters, Abrams, Gerhardt, Burchfield, & Wasserman, 1992; Querleu, et al., 1989; Querleu, Renard, & Versyp, 1981; Vince, Armitage, Baldwin, Toner, & Moore, 1982). Thus, in order to further investigate the respective role of audio – acoustic parameters, we studied discrimination of two lowpitched musical notes electronically generated in the same scale. Similar overtone organization and emission at equal adult loudness should induce their discrimination by adults on F0 ground and orient fetal discrimination to be processed mainly on a similar basis.

We tested fetuses during episodes of low heart rate variability that could reliably be considered as heart rate patterns (FHRPA), which is one of the characteristics that define behavioral state 1F (usually considered as corresponding to quiet sleep state in the neonate) (Nijhuis, Prechtl, Martin, & Bots, 1982). Cardiac and motor reactions to sound are weaker in 1F than in 2F (active sleep), which is characterized by a highvariability heart rate pattern and frequent movementinduced heart rate accelerations. However, because fetal movements are rare, testing in 1F offers the best chance of detecting stimulus-induced, low-magnitude heart rate changes and of avoiding the spontaneous changes that could mislead our assessment of fetuses’ perceptual capacities.

METHODS Subjects The initial sample consisted of 138 healthy fetuses3 between 36 and 39 weeks GA. Their mothers, recruited from the general obstetrical population of the Baudelocque and the Port-Royal University Clinics in Paris (France), were pregnant without complications 3

Fetuses were included on the basis of careful medical followup systematically provided to mothers by the maternity hospital during the whole pregnancy. Postnatal delivery and discharge examination were checked before inclusion of subjects’ data in the sample.

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and volunteered to participate in the study. Twentytwo subjects were eliminated because at some time during the experiment, their data signal was lost for more than 2.5 s, they changed state, their heart rate failed to recover within 65 s of the first stimulation period, or their HR pattern reflected sucking movements (van Woerden, van Geijn, Caron, van der Valk, & Swartjes, 1988b). HR patterns that reflected mouthing movements (van Woerden, van Geijn, Swartjes, Caron, & Brons, 1988a) or breathing movements (Nijhuis et al., 1982) did not lead to data rejection. The final sample consisted of 116 fetuses. At birth, all fetuses were healthy with weights appropriate for gestational age.

Stimuli In order to increase auditory conspicuousness, each pulse was composed of two 500-ms emissions of the same note interrupted by 150 ms of silence (total duration ! 1150 ms). D4, the low note (L) (F0 !

292 Hz, overtones up to 1800 Hz) and C5, the high note (H) (F0 ! 518 Hz, overtones up to 3000 Hz) (see Figure 1) were generated by a Yamaha synthesizer (DX7T) set up in the no-scale piano mode to control for a potential “brightness” effect4 that could contribute to the discrimination process. The notes were recorded in synchrony on separate channels of a D70 Revox stereo tape recorder so that it was possible to switch notes at any time (via a BST switch cursor preamplifier) without disrupting their temporal pattern.

The L amplified notes (Sony TA-AX330 amplifier) were delivered at 93-dB SPL, a pressure level known to evoke a high percentage of decelerative responses; 5- to 10-dB SPL higher increases the occurrence of accelerative HR responses (Kisilevky, Muir, & Low, 1989; Lecanuet et al., 1986). Because there are — to our knowledge — no equal loudness contours for preterm infants as young as our fetuses, the notes were constructed to be equally loud to adult ears. Five normal-hearing adult subjects were asked to adjust the loudness of the two stimuli in the same acoustic conditions used during fetal testing. Sound pressure peak level of the L note was first set at 93 " 2-dB SPL (re: 20 !Pa) measured with an ACLAN audiometer (SDH80) corresponding to 91 " 2-dB SPL (ref: 4 Brightness of a note generated by an instrument is related to the spectral envelope of the note: the greater the number of high frequency overtones the greater the brightness. Brightness in the piano is not the same along the keyboard. In order to control for this potential discrimination bias, we generated our stimuli using the no-scale mode of the synthesizer.

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FIGURE 1 Frequency analyses of the two notes (Fast Fourier Transform) show the bandwidth and pressure of overtones.

20 !Pa) for the H note, at 20 cm from the loudspeaker (AUDAX HR37).

Procedure When mothers arrived for testing between 9 a.m. to 12:30 p.m., they were comfortably seated in a reclining armchair and briefed about the experiment. The loudspeaker, supported by a movable C-shaped table, was placed 20 cm above the maternal abdomen and the probe of a Doppler cardiotocograph (HewlettPackard Model 8030A) was positioned on the abdomen to pick up the best quality fetal heart rate signal.

Electric pulses, each of them corresponding to a heart beat, were recorded of the cardiotocograph at the lowest signal processing level of the device we could

access and fed into a 386 SX PC. A lab-made software package computed and saved beat-to-beat intervals in milliseconds into separated files corresponding to test periods. Fetal heart rate, in beats per minute (BPM), computed by the cardiotocograph in beats per minute (BPM) was continuously displayed in digital and analog form, and was used only by the researchers to monitor the fetuses’ cardiac activity during testing.

Two trained observers (A.-Y. J., J.-P. L.) scanned this hard copy on-line for the low variability fetal heart rate pattern. Once the fetus entered what both observers defined as a stable low-variability period, earphones (Koss Pro4/AAA) were placed on the mothers’ ears that delivered guitar music at a high but still comfortable level (75- to 80-dB SPL) (Technics M215 tape recorder) to mask the musical notes delivered to the fetus. Masking was made complete by placing a thick blanket over the loudspeaker and the lower part of the mother’s body.

Half of the fetuses were scheduled randomly to first encounter C5, the high (H) note and the others were scheduled to encounter D4, the low (L) note. Half of the fetuses in these groups were also randomly assigned to encounter a second stimulus (H or L) and the other half no-change. In this way four groups were formed: Group HL encountered the H note then the L note; Group LH encountered the L note then H note; for the control Groups HH and LL, the same note continued to be emitted. Each fetus was tested only once.

Each subject was randomly assigned to either one of the two note-change groups: One group was first stimulated with the high (H) note and then with the low (L) note (HL) and the other with the L note followed by the H note (LH). Others subjects were assigned to one of two control groups for which the emitted note would continue unchanged: One group was stimulated only with the H note (HH) and one only with the L note (LL).

The three-part testing procedure began after the low-variability heart rate pattern had been observed for 5 min.

Prestimulation Period. The prestimulation period was a silent period during which the low-variability heart rate had to persist uninterrupted for 60 consecutive s before the onset of the stimulation (Period 1).

Any spontaneous cardiac acceleration or deceleration greater than 4 BPM reset the 60-s clock.

First Stimulus Period. The first stimulus period commenced at the end of the 60-s prestimulation period.

During the first stimulus period the double H note or double L note was presented once every 3 s. This pulsed stimulation persisted until the HR returned to

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a stable prestimulus rate and at least 45 but not more than 65 s had elapsed. The initial occurrence of the note was expected to elicit a heart rate deceleration one could consider as an orienting response. The return was expected to occur as the pulsed stimulus became a salient familiar feature of the general auditory environment. If a stable heart rate was not recovered, the test was cancelled. Cardiac reactions to the notes were evaluated by comparing them to the reactions of archival data from a control group of 34 similarly aged fetuses who, in an earlier study (Lecanuet et al., 1992), experienced the same 60-s prestimulation procedure but never encountered a stimulus afterward. These control subjects were not expected to show any significant cardiac change at the end of the 60-s prestimulus period, the time when the experimental fetuses were exposed to the notes.

Second Stimulus Period. The second stimulus period began when heart rate had returned to a stable pattern.

For the HL and LH groups, the first stimulus was switched to the second stimulus without disrupting the temporal patterning of the stimuli and a key was toggled on the computer’s keyboard to record the time of stimulation change. For infants in the HH and LL groups, only the key was toggled on the computer at the time criteria for note changes were fulfilled.

Thirty-one fetuses receiving the H note and 31 receiving the L note reacted with a deceleration. Thus, they were the only ones that could meet the deceleration criteria again when the second stimulus was presented; these 62 fetuses were followed into the second stimulus period. Of the 62 fetuses that oriented to the first note, 14 were in the HL group, 20 in the LH group, 17 in the HH control group, and 11 had been assigned to the LL control group. A decelerative response was expected of fetuses in the HL and LH groups but not of fetuses in the HH and LL groups because their pulsing stimulus did not change. The second stimulus period lasted 1 min.

Data Treatment Ultrasonographic detection of heart rate, based on doppler analysis of heart contractions and dilations, is less accurate than the EKG measure: Some beats may not be detected or may be detected at the wrong time.

Thus, in order to restore in a biologically relevant way values are stored in the files, every data file was processed with software running under the MS-DOS system. (The design of which required interdisciplinary cooperation.) Basically, this software defines as erroneous interbeat intervals with values that are 6% over or under the average of the three previous ones. Re-

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jection and substitution of such intervals by an estimated value are based on several other factors (i.e., duration of the next interbeat intervals, phase shifts, for the total file mean heart rate, etc.).

Interbeat intervals were then converted to weighted heart rate (HR) measured in beats per minute per second (BPM/s). To compute BPM/s, the prestimulus period, the first stimulus period, and the second stimulus period were each divided into 1-s bins from the moment they began. The number of R-wave events within each second was counted and the proportion of an R – R intervals within that second that did not end with a beat was calculated. For example, a second that contained 2.6 R-wave events was multiplied by 60 to produce a rate of 156 BPM/s.

Because the procedure used to define the nature and amplitude of fetal responses requested a temporal continuity of heart rate data, the averaging process was computed from the last beat of a period preceding onset or change of stimulation in two opposite directions: backward through this period and forward through the period immediately following. This ended with two contiguous series of heart rate values in BPM/s, time locked on the onset of each stimulation period. Thus, the dependent variables for the prestimulus, first stimulus, and second stimulus periods were strings of heart rate in BPM/s.

Because of interfetus heart rate response differences in direction (acceleratory, deceleratory, biphasic) and in slope, it was inappropriate to analyze mean group differences. Thus, it was necessary to design a quantitatively based procedure that would determine whether an individual fetus did or did not react to a stimulus and to its change. We chose a measure that was intentionally quite conservative in order to increase its reliability and validity. Some known sources of error in this kind of research include the act of ultrasound recording of fetal cardiac activity itself, the cardiotocograph’s built-in algorithm, our corrective algorithm, and the conversion from interbeat intervals to HR (BPM/s). Our measure also took into account the ongoing level of heart rate variability, which is known to modulate the amplitude of HR reactivity in newborns (Porges, 1974) and in near-term fetuses (Lecanuet et al., 1992). This was done using the following procedure-based comparison of heart rate variability ranges (see Figure 2, Panel c) that decided whether the heat rate of a short period of time (Period B) is showing a deceleratory change, an acceleratory change, or no change with regards to the immediately preceding time period of the same duration (Period A) (see Figure 2, Panel 2c).

The first quartile (Q1), the third quartile (Q3), and the interquartile range (IQR) were computed for the

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FIGURE 2 Upper Panel 2a shows an episode of low heart rate variability as displayed on the hard copy printed by the cardiotocograph. Center Panel 2b gives an example of DC responses to the onset of Note 1 and Note 2. Lower Panel 2c schematically describes, on a DC response, the procedure used to characterize heart rate changes and measure their amplitude.

last 10 HR measures (BPM/second) of Period A and for the first 10 HR measures of Period B. A deceleration change (DC) was said to occur in Period B if: (Q1 of the first 10 s of this period) # [(HR of the last second of Period A) $ (IQR of Period A)]. Reciprocally, an acceleration change (AC) was said to occur if: (Q3 of Period B) % [(HR of the last second of Period A) & (IQR of Period A)]. If both criteria were fulfilled, a biphasic reaction occurred and the decision

was made in favor of the direction of the larger change, either AC or DC. No change (NC) occurred when neither the AC or DC criterion was fulfilled. Period A was the prestimulus period and B was the first stimulus period when analyzing the effect of the first presentation of a note; Period A was the first stimulus period and B the second when studying the effect of a second note or of the continuation of the first one. In addition, we computed the maximum cardiac decelerations

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(lowest HR of Period B $ HR of last second of Period A) and maximum cardiac accelerations (highest HR of Period B $ HR of last second of Period A).

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.011, p ! .91, nor the AC mean scores, F(1, 24) ! .007, p ! .94, varied between the H and L notes.

Second Stimulus Period RESULTS Prestimulation Period Heart rate characteristics during the last 10 s of the prestimulation period for the final sample of 116 fetuses are shown in the top panel of Table 1. Fifty-six had been assigned to the H-note group and 60 to the L-note group: Their means, medians, IQR of the last 10 measures, and the very last second of the prestimulation period did not differ. Therefore, the cardiac activity of the H-note and the L-note groups was equivalent before the first stimulus was presented.

First Stimulus Period The proportion of fetuses in the H (n ! 56) and L (n ! 60) conditions that showed a DC, an AC, or NC was equivalent, "2(2) ! 0.43, p % .05 (Table 1). H and L groups seemed equally responsive to the notes in that 73% of the fetuses exposed to the low note and 78% of those exposed to the high note displayed either a DC or AC response. However, the two kinds of HR changes were not equally likely: Thirty-one of the 44 reactions to the high note and 31 of the 44 reactions to the low note were of the DC type, "2(1) !

14.72, p # 001. Sixty-two of the 88 reactions (70%) were of the DC type. Samples of HR decelerations are shown in Figure 2, Panel b.

A nonstimulated control group of near-term fetuses (n ! 34) that had been tested with the same procedure but had not been exposed to sound during the first stimulation period displayed equally large proportions of transient spontaneous ACs (15/34) and NCs (16/ 34), but spontaneous DCs were rare (3/34; 9%) (archival data from Lecanuet et al., 1992). Here, 62/116 (53%) showed a DC response, 26/116 (22%) showed an AC response and 28/116 (24%) showed a NC response. The distribution of AC, DC, and NC is different from those occurring when low-intensity musical notes were presented "2(2) ! 21.29, p # .005. A significantly higher proportion of DC responses occurs with the onset of the musical notes than during silence, "2(1) ! 16,03, p # .001.

DC amplitudes ranged from $1.5 BPM to $9.9 BPM and AC amplitudes from 1.3 BPM to 8.9 BPM (Table 1). Neither the DC mean scores, F(1, 60) !

Of the 62 who reacted with a HR DC, 14 had been assigned to the HL group, 20 to the LH group, 17 to the HH group, and 11 to the LL group (bottom panel of Table 1). Here we assess whether HR activity of fetuses in the HL and LH groups, who encountered a stimulus change, differed from the HR activity of fetuses in the HH and LL control groups, who did not encounter a stimulus change.

Twenty-four fetuses reacted to the note change with either an AC or DC response, and 22 of them (90%) were of the DC type. In contrast, 17/28 control subjects showed a spontaneous HR change at the point when a note would have changed, but only 2/11 in subgroup HH and 3/6 in subgroup LL were of the DC type. The stimulus change condition produced a disproportionately large number of DC reactions, "2(1) ! 17.2, p # .001.

DC amplitude scores ranged from $1.5 BPM to $9.4 BPM and AC scores from 1.3 BPM to 7.5 BPM. AC changes occurred so infrequently for the HL and LH conditions that their amplitude could not be meaningfully compared to each other or to the AC amplitudes of the control conditions (HH and LL). DC mean scores of the LH stimulus-change groups did not differ, F(1, 20) ! .64, p ! 0.43.

DISCUSSION More than half of all fetuses reacted to the initial onset of musical notes with a HR deceleration but only a few nonstimulated control fetuses from an earlier study did so (Lecanuet et al., 1992). Subsequently, 90% of the fetuses who had responded to the initial onset of a musical note with a HR deceleration responded with another HR deceleration when the note changed. In contrast, the no-change control subjects who also had responded to the onset of first stimulus with a DC reaction subsequently showed very few decelerations. This difference in the frequency of DC reactions of fetuses in the experimental and control conditions indicates that near-term fetuses can discriminate between musical notes that differ primarily in their constituent frequencies. The present results are quite similar to those that we observed in the syllable and male/female voice study. The procedures of the three studies are also quite similar. Thus, we conclude with some confidence that the cardiac decelerations seen at the onset of the first stimulus and again at the

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Table 1. Top Panel Shows the Mean, Standard Deviation (SD) of the Mean, Q1–Q3 and Last Sec HR of the Last 10 HR (BPM/S) of the Prestimulation Period for the H and L groups. Middle Panel Shows the Proportion of Cardiac Decelerations, Accelerations, and No-Changes and Mean HR (SD) Amplitudes (in BPM/s) for the First 10 Sec of First Stimulus Period for the H and L Groups. The Bottom Panel Shows the Proportion of Cardiac Decelerations, Accelerations, and No-Changes and Mean HR (SD) Amplitudes (in BPM/s) for the First 10 Sec of the Second Stimulus Period for the HL, LH, HH, and LL Groups GP HL & HH LH & LL

M

Prestimulus Period Median

Q1–Q3

Last Sec

134.5 (7.9) 136.0 (8.9)

134.4 (7.8) 135.9 (8.9)

1.48 (0.73) 1.45 (0.68)

134.7 (7.76) 136.1 (8.8)

First Stimulus Period DC

GP HL & HH (n ! 56) %subjects Xamp (SD) LH & LL (n ! 60) %subjects Xamp (SD)

55.4 $4.3(2.2) 51.7 $4.2(2.1)

AC 23.2 3.6(2.4) 21.7 3.7(1.3)

Second Stimulus Period DC

GP HL (n ! 14) %subjects Xamp (SD) LH (n ! 20) %subjects Xamp (SD) HH (n ! 17) %subjects Xamp (SD) LL (n ! 11) %subjects Xamp (SD)

71.4 $4.0(1.5) 56.5 $4.6(1.9) 11.8 [$2.9(1.6)] 27.3 $3.5(1.6)

AC 0 13.0 [3.9(2.1)] 52.9 2.6(0.9) 27.3 1.1(0.3)

NC 21.4 26.7

NC 28.6 30.4 35.3 45.5

Note. Q1– Q2 ! first quartile– second quartile. BPM/s ! beats per minute per second.

onset of the second stimulus were not random heart rate changes, but were autonomic cardiac responses.

These responses could be the cardiac component of an orienting response, a point that is not fully established by these data.

We have no firm explanation why 24% of the fetuses showed no reaction at the onset of the first stimulus. This number is not far from the one obtained in the voice discrimination experiment: Twenty-three percent of subjects exposed to the female voice and 18% of those exposed to the male voice did not respond to these stimuli. Age of subjects, stimulus sound pressure level, and data processing were similar in the present experiment and in the voice one. Voices utterances were slightly longer (1.8 s) than the note stimulus (1.15 s). They also show more spectral changes and their repetition rate was slower (3.5 s) than the note pulsation rate (3.0 s). These differences could “theoretically” account for a greater responsiveness to the voices than to the notes, but the observed differences were not significant.

However, five possible factors may be relevant to explain the no-change proportion. First, because our fetuses ranged from 36 – 39 weeks of age, there may be interfetus differences in auditory threshold development (see Hepper & Shahidullah, 1994 on the ontogenesis of fetal thresholds). Second, although testing in State 1F is a methodologically sound practice, the fact is that fetuses are less reactive in quiet sleep than in active sleep. Third, State 1F is not a homogenous state. The responsiveness of the autonomic nervous system may vary within a 1F episode, which is reasonable if one considers that near-term fetal quiet sleep is the same as the newborn’s and that newborns’ quiet sleep periods are not homogenous. EEG parameter levels vary within quiet sleep and the most pronounced EEG synchronization takes place at the beginning of the quiet sleep phase (Salzarulo, Fagioli, Peirano, Bes, & Schulz, 1991). Furthermore, not all quiet-sleep phases contain slow wave periods (Bes, Schulz, & Salzarulo, 1987; Schulz, Bes, & Salzarulo, 1989). Fourth, the effective stimulus may have varied between fe-

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tuses. Because of the probability of the presence of standing waves inside the amniotic fluid, perhaps the ears of some fetuses were located in a minimal soundpressure zone during stimulation. (Lecanuet et al., 1998, Nyman, Arulkumaran, Hsu, Ratnam, Till, & Westgren, 1991; Richards Frentzen, Gerhardt, Abrams, & McCann, 1992; Vince et al., 1982; Vince, Billing, Baldwin, Toner, & Weller, 1985). Finally, the signal/noise ratio might have been disadvantageous for fetuses whose heads were near the highly vascularized placenta (Querleu et al., 1988) or the maternal aorta (Benzaquen, Gagnon, Hunse, & Foreman, 1990).

Fetal audition is not only constrained by transmission of external sounds (in particular for short wave length sound components (frequencies % 500 Hz)) through various mismatched impedance media to the amniotic fluid, but also, according to Gerhardt and colleagues (Gerhardt et al., 1992; Gerhardt et al., 1996), by activation of the cochlea through bone conduction.

They found that fetal cochlear “acoustic isolation” in the fetal sheep (defined as SPL differences required to record the same amplitude cochlear microphonic potential and outside the uterus) from an external white noise emitted in air at 90-dB SPL was less than 20 – 30 dB up to 500 Hz, but was 40- to 45-dB SPL from 500 Hz to 2000 Hz. In our studies, stimulation was delivered between 90- and 95-dB SPL in air, which should be — if sheep data can be transferred to the human species — approximately 50-dB SPL above fetal cochlear isolation for frequencies above 500 Hz. For the present study, this gradual increase in “isolation” affects more the C5 note, the highest overtones of which range up to 3000 Hz (see Figure 1), than the D4 note the 5th overtone of which peaks around 1700 Hz. This implies in utero note discrimination cannot rely on the same acoustical cues in the adult airborne situation. Because the defining low frequencies of the notes suffered less attenuation and isolation on their way to the fetal cochlea, they are good candidates to contribute to the discrimination of the notes.

Perceived loudness equalization of the notes for adults controlled for this parameter of the situation. This latest study together with our previous ones demonstrates that fetuses, while in a low heart rate variability state that quite certainly corresponds to a quiet-sleep episode, can detect differences between two low-pitched complex sounds, as they do between pure tones within the same range (250 – 500 Hz, Shahidullah & Hepper, 1994) with fundamental frequencies under 700 Hz. These results strengthen the idea that this detection is performed on F0 and low-frequency spectrum range differences between sounds.

Postnatal preference studies (Fifer & Moon, 1989;

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DeCasper & Fifer, 1980; DeCasper & Spence, 1986; Moon, Cooper, & Fifer, 1993; Satt, 1984; Spence & DeCasper, 1987) demonstrated that neonates can discriminate between voices, speech sequences, languages, and songs to which they were regularly exposed in the prenatal period. In addition, it is known that the prosodic contours of speech and many of its phonemic features are preserved in utero when voices are emitted or spoken over 60-dB SPL (Griffiths, Brown, Gerhardt, Abrams, & Morris, 1994; Querleu et al., 1988; Smith Satt, Phelan, & Paul, 1990). Querleu et al. (1988) and Benzaquen et al. (1990) found that maternal vocalizations are around 10 dB higher in utero than external male and female voices delivered at the same external SPL. Similarly, Richards et al. (1992) observed that the maternal voice is amplified by 5-dB SPL and that other external voices are attenuated only by 2 – 3 dB. The present article contributes to this body of research and some of our previous data (DeCasper, Lecanuet, Busnel, Granier-Deferre, & Maugeais, 1994; Lecanuet et al., 1987; Lecanuet et al., 1993) which clearly demonstrates that the fetal auditory system can extract enough acoustic information from sounds to perceive specific auditory patterns.

Specifically, this study confirms fetuses’ ability to discriminate between spectra that lay within the range of voice F0 and F1 formants, which may be important during the earliest developmental stages of speech perception. Highly analytical studies such as this one might yield more definitive assessments of the bases for fetal perception by studying acoustic features that are not altered by the transmission through the maternal tissues, such as the temporal pattern of complex sounds.

NOTES We express our gratitude to the mothers and to the babies involved in this study. We also thank Pr. C. Sureau and Pr. E. Papiernik, respectively, heads of the Baudelocque and the Port-Royal Cliniques Universitaires, as well as Mrs. C. Contrepas and Mrs. M. Coulon, midwives, for welcoming our group. We are also very grateful to M.-C. Busnel for her help in the experiments and are especially indebted to S. Mac Adams for devising the stimuli and to Y. Barbin and C. Kervella for the HR data treatment software.

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