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DEVELOPMENT OF HABITUATION AND IN THE HUMAN FETUS Van Heteren, Cathelijne Francisca -

Development of habituation and memory in the human fetus - 2001

Thesis University Nijmegen - with réf.- with summary m Dutch -136 p.

ISBN: 90-9015000-5

Print: Grafisch Bedrijf Ponsen &i Looijen BV Wageningen

Graphic Design Marie-Louise Dusée

No part of this book may be reproduced in any form without permission of the author.

This research project was financially supported by ZorgOnderzoek Nederland and the Hersenstichting Nederland.

Publication of this thesis was financially supported by ATL Nederland BV, Ferring BV, GlaxoSmithKline, Hitachi BV, Medical Dynamics, Novo Nordisk Farma BV, Organon Nederland BV, Pie Medical Benelux BV, Sanofi-Synthélabo, Schering Nederland BV. DEVELOPMENT OF HABITUATION AND MEMORY IN THE HUMAN ?-f τ'••<,

Een wetenschappelijke proeve op het gebied van de Medische Wetenschappen

Proefschrift

ter verkrijging van de graad van doctor aan de Katholieke Universiteit Nijmegen, volgens besluit van het College van Decanen in het openbaar te verdedigen op vrijdag 5 oktober 2001 des namiddags om 1.30 uur precies

door

Cathelijne Francisca van Heteren

Geboren op 11 augustus 1971 te Arnhem Promotor: Prof dr J G Nijhuis

Co-promotores: Dr Ρ F Boekkooi

Dr HW Jongsma

Manuscriptcommissie: Prof dr M van de Bor

(voorzitter)

Prof dr PG Hepper, FBPsS

(The Queen's University of Belfast)

Dr EJH Mulder

(UMC Utrecht) d'n iene di rent veur zien leave d'n andere wandelt hiel rustig vuurbeej heej zuj d'r alles vur geave en heej zet ze moge ut hebbe van meej

woar ge ok loept en wat ge ok bint niemand de zeat ow wat goed is of slecht niemand die wet wie verluust of wie wint ge komt op ut end beej ow zelf terecht

Jack Poels

Voo/· mijn ouders

Voor Roel

CONTENTS

List of abbreviations 10

Chapter 1

General introduction and objectives 11

Fetal surveillance and neurological assessment 13

Fetal responses to vibroacoustic stimulation 17

Fetal habituation to repeated vibroacoustic stimulation 21

Objectives of this thesis 27

Chapter 2

Study design 29

Chapter 3

Fetal habituation to vibroacoustic stimulation m relation to

fetal states and fetal heart rate parameters 35

Early Hum Dev 2007,67 735-45

Chapter 4

Fetal memory demonstrated by habituation testing in term 47

Lancet (research letter) 2000,356 7769-70 - Lancet (correspondence)

2001,357479

Chapter 5

Development of memory in the human fetus, demonstrated

by habituation to vibroacoustic stimulation 55

Submitted Chapter 6

Fetal habituation to vibroacoustic stimulation in uncomplicated postterm 67

Eur J Ob Cyn Reprod Biol 2001,97 '7^ 82

Chapter 7

Fetal habituation to vibroacoustic stimulation in pregnancies complicated by intrauterine growth retardation 77

Chapter 8a

Responses to repeated vibroacoustic stimulation m a fetus with trisomy 18, a case-study 87

Eur J Obstet Cyn Reprod Biol 2001,96 725-5

Chapter 8b

Responses to vibroacoustic stimulation in a fetus with an encephalocele compared to responses of normal fetuses 93

J Pennat Med 2000,28 306-8

Chapter 8c

Responses to vibroacoustic stimulation in a fetus with a hydrocephalus, a case study 99

Submitted

Chapter 9

Summary and discussion 107

Samenvatting 113

References 117

Bibliography 129

Dankwoord 131 LIST OF ABBREVIATIONS

bpm beat(s) per minute

cm centi meter(s)

FHR fetal heart rate

FHRP fetal heart rate patterns

g gram

GA

IUCR intrauterine growth retarded / retardation

LTV long term fetal heart rate variation

m meter

ms millisecond(s)

η number

pH degree of acidity

s second(s)

STV short term fetal heart rate variation

VAS vibroacoustic stimulation /

The major goal of fetal surveillance is the reduction of fetal morbidity and mortality in fetuses with an increased risk of these complications. To this end, the accurate assessment of fetal wellbemg and early detection of (neuro- developmental disorders) are essential. Neurological morbidity includes subtle to severe functional disturbances, such as motor delay, speech delay, deafness, blind­ ness, cerebral palsy, and mental retardation. A study on the birth prevalence of moderate or severe newborn encephalopathy in a normal population in Western Australian showed that this prevalence is 3.8 per 1000 term live births and the neonatal case fatality is 9.1% '.

Despite the "intensive " of the past 30 years, with increasing attention being given to , reduction of birth trauma, and the greater use of cesarean section for high-risk deliveries, the frequency of neurodevelopmental dis­ orders remains unchanged 2'7 Genetic, congenital and environmental factors can all adversely affect the developing individual any time from conception to birth. Independent risk factors before conception and in the antepartum period for new­ born encephalopathy include socioeconomic status, family history of seizures or other neurological disease, conception after infertility treatment, maternal disease, severe pre-eclampsia, bleeding m , viral illness, having an abnor­ mal , intrauterine growth retardation (IUGR), and postmaturity '. However, in the majority of children with cerebral palsy, mental retardation, and other chronic neurodevelopmental disorders, no specific cause can be identified β But prenatal factors do seem to play an important role in the development of subsequent neu­ rological and handicap'69'0

Factors leading to encephalopathy may occur in the antepartum period, imme­ diately preceding the onset of (premature) labour, in the intrapartum period, or in the neonatal period For many years it was accepted that fetal asphyxia during labour was the major cause of both neonatal encephalopathy and cerebral palsy. However, the evidence for this is surprisingly thin 6. Researchers are reassessing the factors associated with neonatal encephalopathy and cerebral palsy, with increasing emphasis on events before the perinatal period '6". Research on the causation of neurodevelopmental disorders needs to focus more on prenatal events

12 because most bram injury would seem to occur before birth. Much more under­ standing of fetal brain development and factors affecting it is therefore needed to detect neurological abnormalities in the fetus and even more importantly, to even­ tually determine the cause of such defects to facilitate the prevention of neuro­ logical morbidity.

While tests of the fetal genetic / chromosomal condition via the analysis of fetal cells, structure via ultrasound examination, and autonomic function via all contribute to the determination of fetal health, significant advances would be made in identifying and finally preventing neurodevelopmental disorders if it were possible to examine the fetal central (CNS) function. However, there is currently no precise method for the antenatal assess­ ment of fetal CNS functioning m obstetrical practice.

FETAL SURVEILLANCE AND NEUROLOGICAL ASSESSMENT

Until the 1970's, neurological assessment was only possible after the had been born. Prenatally acquired functional impairments of the CNS could not be detected earlier than some time after birth. Only indirect assessment of the fetal condition was possible, e.g. by the mother counting fetal movements or by moni­ toring the fetal heart rate (FHR). These measures are not sensitive enough for a neurological assessment of the fetus A breakthrough was achieved in the 1970's with the introduction of real-time ultrasound, and since then it has been possible to observe the fetus.

The introduction of real-time ultrasound made it possible to study the fetus A variety of fetal movements ranging from general body movements to detailed movements of the face, fingers and toes can be observed. Physiological functions such as FHR, stomach and bladder emptying can also be perceived.

Studying fetal behaviour is one of the many ways to identify fetal disorders Just as behavioural studies of the infant or adult can be used to assess the individual's present condition and predict later behaviour and functioning, so can the behaviour exhibited by the individual prior to birth also be used to assess its present functioning. Fetal behaviour represents the functioning and integrity of the CNS, therefore assessment of fetal behaviour provides a means of assessing fetal CNS functioning. At present a vast array of methods is used to assess fetal CNS, but none of them is entirely satisfactory

Antenatal cardiotocography (CTG), the non-stress test, has become widely accepted as the primary method of antenatal fetal monitoring " The test is conducted in pregnancies where fetal wellbemg is questioned, e g postterm pregnancy, reduced fetal movements, hypertensive disease, growth retardation and bleeding in pregnancy '3 CTC records the FHR (reactivity, variation, accelerations, decelerations etc.). Four randomised trials for the purpose of fetal assessment that compared

13 antenatal cardiotocography with a control group were conducted in the early 1980's when the antenatal CTG was introduced ""7. These trials concluded that the antenatal CTG has no significant effect on perinatal outcome or on interventions such as early elective delivery. The suggested effect on reduced antenatal inter­ vention is the mam reason for the current widespread use of the CTG. However, there have been no randomised trials in recent years and it is not possible to judge whether the CTG's would have been interpreted and acted upon as they would today ".

The is performed most commonly following a non-reactive (abnormal) non-stress test and is of particular value for the recognition of imminent fetal asphyxia "'. The contraction stress test has been proposed as a method for identifying fetuses that are unable to tolerate the trial of labour, in that it is highly predictive of subsequent intrapartum fetal distress. As with the non- stress test, the major drawback of the technique relates to the high occurrence of false-positive results (approximately 25%) '2!0

The first fetal behaviour variable to have any clinical relevance was the presence or absence of fetal body movements Prolonged absence of fetal movements was often the first indication of fetal compromise or fetal death. Endogenously generated fetal movements can be observed from 7 weeks gestational age (GA) ". At 15 weeks GA various distinct movement patterns can be distinguished, such as startles, general movements, breathing, hiccups, isolated arm or leg movements, jaw movements, sucking, swallowing and yawning and hand-face contact From 18 weeks GA onwards, slow eye movements are present and somewhat later rapid eye movements can be observed 2!. All these early movements closely resemble those observed in pre-term and full-term newborns2i.

Careful evaluation of the quality and quantity of movements is an integral part of the neurological examination at any age. Assessment of the , based on the subjective perceptions of the mother, represents the oldest technique available for determining fetal wellbemg. The fetal activity as charted by the mothers was diminished m fetuses with major malformations2"35 Advances in real­ time ultrasonography and the ready availability of the technique have led to a more sophisticated approach to the observation of fetal movement. The recording of specific body movements is now feasible. The number of body movements m IUGR fetuses is diminished 26, with a rate of less than 10 movements / 12 hours and involving a higher risk of fetal death27. However, observation of the number of fetal lower limb movements appears to have no diagnostic or prognostic value in fetuses with defects or other CNS anomalies2B The limitations of this method of fetal evaluation mainly involve the practical difficulties associated with prolonged recording and the lack of standardised control data regarding duration of recording and criteria for recognising normal as opposed to abnormal movements. Furthermore, the high inter- and intra-individual variation makes the sole measure­ ment of fetal movements an inappropriate tool for assessing the fetal condition.

14 The assessment of the quality of the general body movements was seen as a promising tool for assessing the functioning of the CNS, especially m infantsi9î0. However, in IUGR fetuses no abnormality of the quality of general fetal movements was found 3'. IUGR fetuses showed a reduced variability of movement patterns, but in individual cases there was an overlap with normal control fetusesi2. Even so, the quality of the movement pattern is also influenced by external factors such as the reduction of ".Abnormal general movements are present in severely abnormal fetuses such as anencephalic fetuses * and fetuses with trisomy 18 35. However, in fetuses with there is a discrepancy between the occurrence of prenatal leg movements and the spinal localisation of the meningomyelocele on the one hand, and between the occurrence of pre-and postnatal leg movements on the other hand j6.

We can therefore conclude that although a percentage incidence of the number of movements below the lower range of normality would warrant further investi­ gation, the quantitative and qualitative measurements of fetal movement patterns would not seem to be a sufficiently sensitive tool for neurological assessment of the fetus.

The study of fetal breathing movements was also thought to provide a screening test for fetal compromise IUGR fetuses manifest a decrease in fetal breathing movements " Some IUGR fetuses showed short breathing episodes with longer apnoeic intervals than normal fetuses '" Another study showed that in IUGR fetuses body and breathing movements are reduced during and directly following late FHR decelerations. Since late decelerations are presumably signs of acute hypoxemia i9, the results suggest that these reductions are caused by hypoxemia "", However, a study of the incidence of fetal breathing in 12 IUGR fetuses showed that there was no difference in incidence between IUGR and normal fetuses "'. Obser­ vation of fetal yawning in IUGR fetuses in order to assess the fetal condition would not seem to be useful since there is no difference in the yawning pattern in IUGR fetuses compared to healthy fetusesi2.

A progressive clustering occurs m rest / activity cycles m the course of pregnancy. Behavioural and physiological variables such as FHR and fetal body and eye move­ ments show evident patterns from 26-28 weeks GA onward Ai which are the fore­ runners of behavioural states. In the third trimester the episodes of fetal motor activity and rest become increasingly associated with the fetal heart rate pattern (FHRP) and fetal eye movements, finally resulting in fetal behavioural states *". A true behavioural state is defined as any period of at least 3 minutes' duration m which the variables (FHRP, body movements and eye movements) meet the criteria of the particular state and with simultaneous changes of state variables during transitions"5. Each state reflects a particular mode of neural activity.The fetal states iF through 4F ("F"for fetal) correspond to behavioural states 1 (quiet sleep), 2 (REM sleep), 3 (quiet awake) and 4 (active awake) m the full-term born neonate !>. In the normal fetus, behavioural states are fully developed from 36-38 weeks GA onwards ^,

15 The ongoing maturation of the CNS after 41 weeks GA is reflected in changes in 46 behavioural state organisation 1 similar to the neonate born at 40 weeks CA

The development and presence of fetal behavioural states are indicators of a fully functioning CNS Delayed and disturbed development of behavioural states have been described in severely hypoxic fetuses 47, m the fetuses of insulin dependent diabetic women "', in fetuses exposed to cocaine49 and to alcohol 5°, and in hydro­ cephalic fetuses 5'. Abnormal behavioural patterns of 3 fetuses were found to be correlated with lesion sites in the CNS as discovered after birth 52. Although IUGR fetuses also show impaired development of behavioural states 5's6, there is a large interfetal variability in the time interval elapsing between abnormalities of behavioural state and the occurrence of abnormal FHR tracings ".

In healthy fetuses, state transitions (i.e. time intervals between 2 different behavioural states) usually lasted less than 3 minutes, whereas IUGR fetuses dis­ played a longer duration when compared to healthy fetuses. Moreover, a significant trend in the change of state variables was evident in healthy fetuses where IUGR fetuses showed a random sequence m order of change M.

Although the assessment of the fetal behavioural state organisation and state transitions can be used to assess the functioning and development of the fetal CNS, clinical use does not appear to be feasible.The mam limitations to fetal behavioural analysis is that it is labour intensive, time consuming and subjective, and as such, remains largely a research tool Implementation m the clinical setting has not yet been achieved

IUGR fetuses show a delay m the development of behavioural states with a decrease m fetal breathing movements " Therefore, it was thought that observing the intercostal-to-phremc-mhibitory reflex (IPIR), which is a respiratory reflex induced by manually compressing the fetal thorax, might be of use as a test to assess the possible impairment m the development of the CNS m IUGR. Since there was no difference m IPIR between healthy and IUGR fetuses, this test cannot be applied in CNS assessment in IUGR fetuses without a deteriorating condition ^

Having the mother record fetal movements (kick charts), CTG recording, the bio­ physical profile score, and Doppler ultrasound to examine the feto-placental and utero-placental blood flow are currently the only methods in clinical use In the term period these observations are state-dependent. However, observation of the (ultrasound monitoring of fetal movements, fetal tone and fetal breathing, ultrasound assessment of ammoticfluid volume and assessment of FHR by CTG recording) does not have any effect on the outcome of the pregnancy in high-risk cases.The biophysical profile is based on the association between chronic fetal compromise and changes in the FHR pattern, decreased fetal body and breathing movements, and redistribution of regional blood flow leading to a reduc­ tion m fetal renal blood flow and fetal oliguria and, therefore, less amniotic fluid From 4 randomised trials 59 62 comparing fetal biophysical profiles with other forms

16 of fetal assessment m women with high-risk pregnancies it was concluded that there was no obvious effect (either beneficial or deleterious) on a range of fetal and neonatal measures6'.

Doppler ultrasound to examine the feto-placental and utero-placental circulatory dynamics in high-risk pregnancies would appear promising m helping to reduce perinatal deaths m high-risk pregnanciestA. From the results of 11 randomised trials involving nearly 7000 women, Doppler ultrasound, in high-risk pregnancy (espe­ cially those complicated by hypertension or presumed impaired fetal growth) was associated with a trend towards reducing perinatal deaths. However, it is unknown whether Doppler ultrasound prevents an adverse neonatal outcome with sub­ sequent neurodevelopmental impairment. Questions remain as to the optimal use of information derived from Doppler studies of the umbilical artery and, m parti­ cular, the best time for delivery of a fetus with absent end-diastolic velocities m the umbilical artery, no other serious evidence of immediate compromise, and a GA that is associated with a significant risk of the possible problems as a result of pre­ maturity.

At the present time, there is no ideal test to identify fetuses likely to be com­ promised neurodevelopmentally after birth An ideal method for monitoring fetal wellbeing would be the measurement of fetal CNS function, but an adequate intrauterine neurological test is not currently available. The current priority is to develop a method that is of potential clinical use in the assessment of the functioning and integrity of the fetal CNS. The aim of this thesis is to focus on fetal habituation to repeated vibroacoustic stimulation (VAS) as a promising tool for this assessment.

FETAL RESPONSES TO VIBROACOUSTIC STIMULATION

There is little doubt that dysfunction of the fetal CNS correlates with abnormal fetal behaviour However, observation of spontaneous fetal behaviour is time- consuming. Analysis requires at least 60 to 120 minutes' observation to acquire an accurate impression K A test that elicits a behavioural response would provide more information concerning the CNS as it involves both sensory and motor response and a certain degree of co-ordination between the two which requires a higher degree of CNS functioning and integrity than spontaneous behaviour 65. Furthermore, one of the major problems facing the assessment of biophysical para­ meters is that all have substantial reserve mechanisms. Therefore, any test performed in the resting state may only be of limited predictive value. It may be of greater clinical value to attempt to assess the reserve mechanisms as loss of them may indicate an abnormality much earlier in the chain of events.

The dominant opinion used to be that the fetus lived isolated from the distur­ bances of the outside world. However, pregnant women could tell that they were able to feel the baby jerk m the womb when a loud noise occurred m their vicinity

17 Furthermore, researchers became aware that it does not seem plausible that sensory functioning has a sudden inception at birth Since then, obstetricians have been studying the fetal response to extrauterine stimuli as indicated by fetal move­ ments or change in FHR.

The human fetus is exposed to acoustic stimuli, eg. maternal intestinal noises, which may reach very high levels, vascular noises, and sounds of the mother's speech External sound sources, if loud enough, may also add to the acoustic stimulation. Lower frequencies may evoke fetal tactile responses as well as auditory responses, while the high frequencies over 1500 Hz have negligible vibratory influence, and any response evoked may be considered auditory m origin <'6. It has been demonstrated that the fetus responds to sound *', light68, taste / olfaction 69 and to VAS. Many devices are used (door buzzer, mimshaker, artificial larynx, commercial stimulator) to produce a vibroacoustic stimulus VAS on the surface of the maternal abdomen is accompanied by both acoustic and vibratory exposure of the fetus 70. The intensity, frequency, and number of stimulus applications deter­ mine not only whether a response will be elicited but also the magnitude of the response. The magnitude of the response is increased by the intensity of the stimulus 7'72.

Different fetal responses have been used to define the effect of an external stimulus, but the most common are FHR accelerations and fetal body movements However, m some studies fetal eye blinking orfetal facial reactions have been used7'7" The time lag between stimulus and the expected response has varied between studies. Whereas some investigators looked only for immediate changes in fetal activity, others followed the fetus for up to 2 hours after stimulation 7576

The onset of the fetal movement response is related to the functional maturation of the CNS In 27 healthy fetuses studied every 2 weeks between 21-31 weeks CA, 2 fetuses responded at 23-24 weeks to VAS, 89% responded by 27-28 weeks, and from 30 weeks onward all fetuses responded 77 This also applies to the FHR response to VAS, which is consistently present after 30 weeks GA 7β Based on these data clini­ cians and investigators should be aware that normal fetuses of less than 30-31 weeks CA might not respond to VAS.

As yet it is not completely clear whether VAS influences the fetus by auditory or vibratory pathways. Anencephalic fetuses that lack auditory capabilities failed to respond to VAS 7',8o. It was therefore assumed that an acoustic mechanism is needed to elicit a fetal response. However, impairment of the sensory-motor circuitry can also account for the non-response m anencephalic fetuses. Sheep fetuses with bilateral cochlear ablation failed to respond to VAS 8'. These results indicate that m fetal sheep the auditory system is necessary for the detection of VAS. Observations of the response to VAS m 31 fetuses at risk of hearing impairment are in line with these findings%' The responses from 3 fetuses deviated from the other fetuses These 3 children exhibited severe hearing impairment at 3 years of age.

18 Fetal heart rate response to vibroacoustic stimulation

Many studies describe the FHR reactivity to VAS 757β8ΐβ7 immediately after the stimulus onset, a significant increase in the duration of FHR accelerations occurs from 26 weeks GA onward 78,and an increase in baseline FHR occurs from 30 weeks GA onward 85 In the term fetus, VAS during fetal quiescence resulted in a transient increase m baseline FHR (tachycardia) for up to 15 minutes758889 with an increase m the frequency and size of accelerations

The initial clinical application of the VAS technique was to improve the efficiency of antepartum FHR monitoring It has been shown that VAS reduces the duration of the non-stress test9C9', and the frequency of the equivocal non-stress test9i93 It also limits the need for a contraction stress test 9A In high-risk pregnancies, an absent or impaired fetal response to VAS was associated with intrapartum fetal distress, perinatal mortality and morbidity and with a below the 5th centile 9?93 VAS during labour has a high negative predictive value, fetuses that respond to VAS with FHR accelerations usually have a normal outcome and an arterial pH yjis 959" On the other hand, an abnormal response is not an accurate predictor of an adverse outcome Therefore, a diagnosis of fetal distress in labour cannot reliably be made based solely on the FHR response to VAS

Fetal movement response to vibroacoustic stimulation

The normal neonatal response to VAS consists of a generalized startle-like body movement This same response can also be observed m fetuses 7399'00 The first evidence - in the 1920's - of the fetal movement response to acoustic stimuli was provided by pregnant women who anecdotally reported that their fetuses some times responded to sudden loud environmental noises such as car horns and music concerts67,0,

Application of VAS results m an increase in fetal movements, head turning, mouth opening, tongue protrusion, cheek motion, hand-to-head movement, panting and fetal eye blinking n'°2 These are all thought to represent sequelae of a startle response In term fetuses, VAS during fetal quiescence results m an increase in fetal movements for up to one hour 86e'"OÎ VAS can also be used as a tool to induce fetal movements in an effort to change fetal and so improve visualisation of the fetus during diagnostic ultrasound ,04

VAS m neurological impaired neonates (3 with severe congenital hydro­ cephalus and minimal cortical tissue and 2 infants with severe perinatal asphyxia and hypoxic ischemic encephalopathy) failed to elicit a movement and a heart rate response, while 30 normal neonates responded with movements and heart rate accelerations '05 Fetal failure to respond to VAS with movements is also associated with major CNS abnormalities, such as and microcephaly 798o,°6 The absence of a fetal response to VAS indicates a serious primary hearing impairment or significant depression of the CNS from a developmental or exogenous origin It

19 would therefore seem that VAS has a potential value as a test of neurological integrity.

Safety of fetal stimulation

Despite extensive research over the past decade there is still concern as to the safety of fetal stimulation. This concern focuses on two aspects· its effect on behavioural state and the potential damage to the fetal auditory system.

The baseline sound pressure levels, measured with hydrophones placed in the uterine cavity of sheep, range from 80-90 dB "". VAS produces an increase in base­ line intrauterine sound.This increase appears to be limited to low-frequency sound, which is transmitted better through mechanical coupling of the vibrator with the maternal abdominal wall '0,'" High-frequency airborne sound attenuates across the maternal abdominal wall but VAS at lower frequencies than airborne sound can result m sound pressure levels of 100-110 dB in the uterine cavity ,°8. These sound pressure levels, although elevated, are thought to be harmless to the fetus. Baseline levels of sound, measured by an intrauterine microphone in women in labour, averages between 72-88 dB '09 Transabdominal VAS with an average output of 84 dB at 1 m in air, resulted m intrauterine peak sound levels of between 91-111 dB '09. Since the pulses of sound are brief, fetal injury seems unlikely. Because these data are all gathered after rupture of the membranes, accurate knowledge of sound levels generated m the human with intact membranes and a normal quantity of amniotic fluid is not available Furthermore, the proportion of in utero sound level that reaches the fetal internal ear is unknown because of the surrounding amniotic fluid and the fluid m the middle ear. However, we should be aware that the intrauterine sound level during VAS could be much higher than the output sound level measured in air

A study of the auditory nerve and brain-stem evoked responses in 2-day-old new­ borns after intrauterine VAS failed to document any evidence of hearing loss "2. Studies of 4-year-old infants (n = 460 and 465, respectively) who were prenatally exposed to VAS, revealed that these infants did not manifest any hearing damage or neurodevelopmental abnormalities that could be attributed to VAS "'"4. We can conclude from these studies that VAS, as applied in clinical practice, is unlikely to cause hearing impairment or neurodevelopmental delay.

Fetal distress caused by VAS is a different issue altogether Incidences of fetal brady­ cardia "5",' or fetal tachyarrhythmia with subsequent neonatal atrial flutter following VAS "7 have been reported Unfortunately, the authors of these case- reports do not describe the duration of the stimulation and whether they stimu­ lated over the fetal head or breech. Studies on the degree of fetal disturbance after VAS revealed that although VAS disrupts the feta I rest-activity cycles, this disruption only lasted a short period "8 However, one study described that VAS m normally grown term fetuses results in excessive fetal movements, prolonged tachycardia.

20 behavioural state disorganisation, and occasionally an FHR deceleration '0° The researchers used an artificial larynx with a frequency of no Hz (spectrum between 440 and 990 Hz) and a sound pressure level of 105 dB in air at a distance of 5 cm from the vibrator for 2 s on the maternal abdomen close to the fetal head They state that this may not be harmful for a normal term fetus, but could be dangerous for IUGR fetuses because the tachycardia might reflect a sudden release of fetal catecholamines and an increase of fetal blood pressure with subsequent risk of intracranial bleeding However, 2 studies found no rise m catecholamines m fetal blood shortly after VAS "9'2° These findings suggest that sympathoadrenal activation is not part of the fetal response to VAS This does not support the suggestion that VAS may be deleterious to the fetus by provoking a sudden release of catecholamines However, induced fetal movements might reduce the oxygen content temporarily as a result of an increase in oxygen consumption, which can jeopardise the IUGR fetus

Although there are concerns about the safety of using VAS, at present there is no real evidence to suggest that the use of VAS poses a risk to the healthy fetus Because we were aware of the possible fetal disturbance caused by repeated VAS, we minimised the risk of this disturbance in the studies described m this thesis by using a VAS for 1 s over the fetal legs instead of the head This stimulus was repeated every 30 s with a maximum of 24 stimuli m each habituation procedure

FETAL HABITUATION TO REPEATED VIBROACOUSTIC STIMULATION

The integrity of the fetal CNS can be studied m three ways (1) by passive obser­ vation of activity, (2) by recording the reaction of a fetus to a stimulus, and (3) by documenting fetal habituation, the decrement and eventual cessation of a response to a repeated stimulus The latter method represents a simple form of learning12"" and is the most sophisticated evaluation of fetal neurological status of the three Habituation is a measure of the ability to inhibit response and is a more mature form of behaviour than persistent response

Since the presence or absence of fetal movement following VAS provides a some­ what limited measure of CNS functioning and maturation, habituation to repeated stimulation could be a more specific test for the assessment of the integrity and functioning of the fetal CNS The orienting reflex is the very brief, initial part of an individual's response to stimulation It has both behavioural components, for example, motor movements, and physiological components, such as changes in heart rate, electro-encephalography, and soon An orienting reflex occurs when the individual has "scanned" the stimulus and "decided" that it must be paid attention to With repeated applications of the same stimulus, unchanged m any respect, the individual ceases to pay much attention to it An individual is able to screen or filter out those stimuli that are biologically important from those stimuli that are not In order to function in a given environment an individual must learn to ignore the

21 meaningless and constant stimuli that are ever present and which stimulate its sensory receptors The ability to ignore this constant stimulation and respond to novel stimuli is provided by the ability to habituate

Habituation is presumed to be an index of CN5 functioning Although the exact role played by the in habituation has yet to be determined, it is widely accepted that the cortex is important for the habituation process "s "" However, the neural pathways involved in habituation remain ill defined Animal lesion studies suggest that this behaviour may be partially mediated by structures above the brainstem '2S,27 Kandel et al worked with snails, (Aplysia califormca) because of their large nerve cells, as a modelsystem tostudy 3 learning paradigms-habituation (applying a "trivial" stimulus such as a weak touch to the tail repeatedly, so that eventually, the animal learns to ignore it), sensitisation (exposure to a noxious stimulus-eg, repeatedly applying a shock to the tail to induce learned fear), and (pairing a noxious stimulus with a neutral stimulus so that eventually the neutral stimulus also evokes a fear response) Their previous studies had shown that the synaptic potential of a single cell from aplysia was reduced by habituation, enhanced by sensitisation, and further enhanced by classical conditioning, suggesting that aplysia's synaptic responses mirrored its behavioural responses He also discovered that the whole neural circuit for a behaviour in aplysia was the same for each animal They also showed that learning can lead to alterations m synaptic strength, but also that the persistence of the synaptic change is an indication that memory is being stored, and that a single synaptic onnection can participate m different forms of learning and different types of memory storage,28 '''

Examination of the habituation performance m individuals with various CNS disorders has shown it to be different from individuals without these conditions '2"'2'S7 Individuals with brain damage,38, ,39, autism "'c, depression 'i5, hyper­ activity "", and mental retardation '", all exhibit patterns of habituation that are dif­ ferent from unaffected individuals For example, children and adults with Down's syndrome have less ability to inhibit responses to either noise or tactile stimulation than individuals of normal intelligence 'J4'42"13 Also drugs that exert an effect on the CNS, for example amphetamine "", may affect the habituation performance The infants of mothers given general anesthesia or pethidine during labour take sigmf icantly longer to habituate compared to unexposed infants when examined after birth ",5,4t' These studies demonstrate that habituation is affected by alterations in CNS functioning

Habituation paradigms have become a part of neonatal neurobehavioural assess­ ment schemes "" High-risk infants failed to habituate, and habituation time in neonates with suspected brain damage is twice as long as that m healthy control infants ,48 Furthermore, habituation has been used to study detection, discrimina­ tion and memory m infants, and the habituation performance of infants is also thought to predict cognitive functioning in childhood '49 "•'

22 Normal fetal habituation

Habituation to VAS may reflect higher CNS function. Habituation is thought to measure aspects of sensory capacity and memory and learning. If habituation represents the functioning of the fetal CNS, then conditions affecting the CNS should affect habituation. Therefore, it might be possible that differences in the rate of habituation may help to identify the compromised fetus and hence facilitate the implementation of appropriate intervention protocols.

The first study of fetal habituation, although primarily conducted to examine fetal hearing, was reported in 1925 '".This study showed that, in late pregnancy, fetuses initially moved when a car horn was sounded close to the mother's abdomen and with repeated sounds the fetal response waned and eventually ceased. Further interest m fetal habituation began in the igSo's ,"c6,5' '». Many studies have demon­ strated that repeated presentation of the same stimulus results in a decrement of 52 is6,59 fetal response to these stimuli assessed by fetal body movements "' '" i or change in FHR82'5"56'60'6'.

Studies to demonstrate fetal habituation have varied m virtually every aspect. For example, the nature of the stimulation varied: a pure tone, pure vibration or a vibroacoustic stimulus. Furthermore, studies differed in stimulus intensity, duration, stimulus interval, and fixed or variable number of stimuli. Also the scoring of fetal movement responses following the presentation of a stimulus differed between studies. Documentation of response has included: presence or absence, duration, latency, or a more qualitative description of the response. Different response criteria may measure different aspects of the habituation process The criteria used to establish habituation have also varied. For example, the number of consecutive non-responses before habituation is thought to be achieved. In contrast to studies that have presented stimuli until the fetus ceased responding, others have not stimulated the fetus until the cessation of the response but have presented a fixed number of stimuli and noted the fetal response to them Unfortunately, no consensus has been reached as to the best procedure to demon­ strate fetal habituation. The differences in stimulus type, response measures and procedural parameters will probably all affect the outcome of fetal habituation studies Due to the difference in procedures adopted by the various studies it is not really possible to make a direct comparison between the studies, and consequently it is difficult to attribute observed differences m habituation performance to one single factor. As a matter of fact, it is only possible to compare the results of fetal habituation performance from the different study groups when the same study design is used

Fetal habituation should be distinguished from effector fatigue or receptor adaptation Repeated stimulation may either physically exhaust the fetus so it can no longer respond (motor fatigue) or may tire the sensory receptor so that the receptor can no longer respond (sensory adaptation). Criteria have been outlined to

23 ensure that habituation rather than mere sensory or motor fatigue is tested "' '6i. A study on fetal habituation demonstrated that habituation is not readily explained by fatigue factors '62. Fetuses showed a decrement in response to repeated VAS, a return of response to the presentation of a novel stimulus, i.e. dishabituation, with response decrement to repetition of the novel stimulus, and a more rapid response decrement upon re-presentation of the original stimulus. Some authors suggest that when the 3 criteria cannot be met, habituation may have occurred, but response decrement is a more appropriate term for the process.

However, there is no consensus in the literature on this dishabituation. Dishabituation classically ';' refers to the recovery of a habituated response to the original stimulus following presentation of a novel stimulus. However, more recently some authors have used the term dishabituation to refer to elicitation of the previously habituated response by the introduction of a different or new stimulus into the sequence ,6i. Here if the fetus responds upon presentation of the novel stimulus this is regarded as dishabituation. Furthermore, dishabituation could be observed m only 75-79% of normal term fetuses ". It seems to be quite difficult to administer a stimulus that is entirely different to the standard, and there is also the problem that any stimulus will generalise to some degree. Any new stimulus that falls into the range of the original stimulus will not elicit a recovery of the response. Although dishabituation was tested in some habituation studies "'^ '64, the absence of dishabituation never resulted in an exclusion of the experiments in which the fetus habituated to the original stimulus but failed to dishabituate. Therefore, the presence or absence of dishabituation did not have any conse­ quences for the results. Moreover, there are even more studies on fetal habituation ( that have not tested dishabituation «*'vw»< >\ \n a pilot-study on fetal habitua­ tion we have tested this dishabituation to a novel stimulus produced by an electro- larynx Unfortunately, the fetal response to this novel stimulus was very irregular. For these reasons we opted not to test dishabituation in our studies described in this thesis.To differentiate between fetal fatigue and fetal habituation we observed the habituation pattern again 10 minutes after the initial habituation test. A more rapid habituation pattern in the second test supports the observation of mere habituation.

Some studies have explored the mter-observer agreement in scoring the fetal movement response to VAS. Doherty and Hepper found a very high inter-and mtra- observer reliability (correlation on the number of trials to habituate was 0.98 for mter-observer, and 0.99 for mtra-observer reliability ""'. Madison et αϊ,ί'2'66 found an mter-observer agreement of 92% Groome et al '6' also found a high mtra-observer agreement (r = 085, ρ < o.oooi) and an mter-observer agreement (r = 0.82, ρ < ooooi). Leader et al J1 described that the mother and the observer recorded fetal movements independently Hepper and Shahidullah ,6i reported that inde­ pendent observers confirmed the accuracy of determination of fetal response to VAS There are no studies that report that the mter-observer agreement is not

24 satisfactory. The studies presented in this thesis were performed by only one examiner, without the presence of an independent observer. However, the presence of an additional observer might have been more appropriate.

Abnormal fetal habituation

There is evidence that fetal habituation may be useful as a diagnostic tool for iden­ tifying fetuses m distress or those likely to be compromised neurodevelopmentally after birth. Leader,6e reviewed his studies on the habituation of the fetus to VAS and claimed that neural dysfunction could be reliably detected by this method neuro- logically compromised fetuses failed to habituate or habituated more rapidly compared to normal fetuses.

Studies in compromised or chromosomally abnormal fetuses revealed that the habituation pattern of these fetuses was different from that of normal fetuses 'o6,5;'5J,63,65 A study of compromised fetuses or fetuses exposed to adverse environmental conditions (IUGR, decreased growth velocity of the fetal bipanetal diameter, or mecomum-stamed amniotic fluid) revealed that these fetuses showed a different habituation performance as compared to fetuses with no obstetrical complications 'o6. Fetuses in the compromised group habituated more rapidly (< 9 stimuli) or more slowly (> 50 stimuli), compared with normal fetuses. Fetuses of diabetic mothers were found to exhibit poorer habituation performance than fetuses of non-diabetic mothers "'5. Doherty and Hepper '6s have found that fetuses of mothers with type I diabetes were slower to habituate at 28 and 32 weeks GA than were fetuses of non-diabetic mothers. Group differences were not significant at 36 weeks GA possibly because the fetuses most affected by poorer metabolic control had been delivered by this time.The authors suggest a teratogenic effect of maternal diabetes on the fetal CNS. Researchers have reported long-range correla­ tions between an environmental aberration (eg, malnutrition, drug exposure) during critical periods in brain development and subsequent below-par neuro- behavioral functioning in the offspring. For example, measures of maternal lipid and glucose metabolism in the second and third trimesters of diabetic pregnancies correlate inversely with subsequent intelligence quotient in childhood, after the effects of family socioeconomic status and race or ethnic origin have been controlled for""5.

Fetuses with Down's syndrome habituated more slowly than unaffected fetuses '6ä Moreover, the habituation of Down's syndrome fetuses differed from one another and this performance was predictive of later neonatal outcome '6' The authors suggest that fetal habituation performance may afford the opportunity for deter­ mining the severity of disorders prenatally. It is even thought that fetal habituation may predict subsequent development and hence CNS functioning after birth as the habituation performance of infants has been found to be predictive of later educa­ tional performance ,î6'50'«'66. At 4 months of age, using the Bayley Scales of Infant Development, faster habituators were found to have a significantly higher mental

25 development score '62,66 Leader et al '" found that infants who showed a normal intrauterine habituation pattern scored significantly higher on the Griffiths Mental Developmental scale at ι year of age compared with those who had an abnormal fetal habituation pattern

Testing the fetal ability to habituate to repeated VAS might also be helpful in iden­ tifying the fetus at risk of hypoxia In fetal sheep as well as in human fetuses, fetal hypoxia resulted either in more rapid habituation or a failure to habituate compared to normoxic controls 'S2'I,:, Fetuses of mothers who inspired reduced oxygen (12%) habituated more slowly than when the mothers breathed normal room air (21%) '52 These authors suggest that it is possible that the changes in habituation performance were due to reduced oxygen tension in the fetus or to the maternal or fetal secretion of catecholamines in response to hypoxia These findings were also observed in fetal sheep '55 Hypoxia was induced m 13 fetal sheep by altering the oxygen content of the inspired air to the ewe to 9% The fetuses habituated more rapidly during this hypoxia compared with control measurements

The findings of significant differences in fetal habituation performance (either a more rapid or a more slowly rate) between normal and high risk pregnancies, com plicated by conditions that are often associated with an increased incidence of neurological damage, suggest that it may be possible to identify individual fetuses within an abnormal group that are particularly at risk

Development of fetal habituation

If habituation represents CNS functioning, then CNS maturation should influence habituation Therefore it might be possible to assess CNS development by testing fetal habituation to external stimulation at various GA since the habituation rate may change with GA Studies on the development of fetal habituation can facilitate our understanding of the functional development of the CNS

Studies on fetal habituation at various GA show conflicting conclusions concerning this relationship Some studies reveal that older fetuses habituate more rapidly than their younger counterparts ,s8'67 while others l6i could not find a relationship between GA and the number of stimuli required for habituation There is no obvious reason why these studies have different conclusions In all these studies, fetuses were tested once m a cross sectional design One longitudinal study on the development of habituation was performed in fetuses from diabetic and non- diabetic mothers The subjects were tested at 28,32 and 36 weeks GA Both groups exhibited more rapid habituation with advancing GA l65 Further work is required to explore the development of habituation m more detail

Habituation and fetal memory and learning

Despite the fact that fetal sensory systems are both morphologically and functionally immature, fetuses have the capacity to perceive, respond to, habituate

26 to, and even recall stimuli encountered in utero. The ability to suppress response to insignificant or predictable stimuli indicates the perception of order in environ­ mental events or time. This kind of habituation requires short term memory and recognition of the similarity between subsequent and antecedent events or temporal intervals and is often considered to be a basic form of learning '". Habituation can, therefore, be used as an experimental approach for exploring fetal memory Repetition of habituation tests with a certain time interval might result m fetal memory and learning of the stimuli, which results m recognition of the stimuli with a subsequent more rapid habituation pattern at repeated testing.

Memory, a prerequisite for fetal learning, is essential for normal functioning and it is plausible that such an important psychological function develops before birth m some form. Prenatal memory may be important for the development of attachment and maternal recognition There is much evidence to suggest that the fetus learns the speech characteristics of its mother prenatally and prefers its mother's voice to other female voices after birth. Newborns presented with a choice between their mother's voice as it sounded in the uterus and as it sounded outside the uterus, showed a preference for their mother's voice as it sounded in utero '7° This suggests that their preference was acquired before birth This and other studies have con­ firmed that the fetus can memorise its mother's voice before birth 'v '". Infants are also able to memorise sounds other than voices, for example a tune from a particular television program, presented to them prenatally. Infants from mothers that had watched a certain TV program several times when pregnant preferred this tune after birth to other unfamiliar tunes '"'.

Therefore, testing habituation to repeated VAS in human fetuses may be a useful diagnostic tool for identifying fetuses in distress or those likely to be compromised neurodevelopmentally after birth. Habituation can be useful in assessing the CNS function in fetuses at risk of an adverse pregnancy outcome with subsequent neurologic sequelae But habituation can also be used as an experimental approach to assess the physiological functioning of the CNS such as memory and learning. Studies on fetal habituation m low-risk and high-risk pregnancies are presented and discussed m this thesis.

OBJECTIVES OF THIS THESIS

It will be clear from the above that present obstetrical practice lacks a precise method for the antenatal assessment of fetal CNS integrity and functioning and that such a measure would represent a major advance in assessing fetal wellbeing. Observation of fetal responses and habituation of these responses to repeated VAS seems to have clinical potential as a tool for the assessment of the fetal CNS How­ ever, further experimental knowledge is needed to evaluate the clinical value of fetal habituation.

27 This study sets out to answer the following questions

ι Do fetal behavioural states influence the habituation pattern m normal term fetuses (chapters)?

2 What is the intrafetal consistency m habituation pattern when habituation is repeatedly tested (chapters 4 and 5)?

3 How does fetal habituation develop in the third trimester of pregnancy and is habituation related to gestational age (chapter 5)?

4 Do postterm fetuses habituate differently from term fetuses (chapter 6)?

5 Do intrauterine growth retarded fetuses habituate differently from normal fetuses (chapter 7)?

6 What is the habituation pattern in severely abnormal fetuses (chapter 8)7

28

STUDY POPULATIONS

The study populations presented in this thesis were derived from the clinic and out­ patient clinic of the Department of Obstetrics and Gynaecology, University Medical Centre Nijmegen, The Netherlands, between October 1998 and October 2000. Thirty-seven normal fetuses between 37 and 40 weeks gestational age (CA), 21 normal fetuses beyond 26 weeks GA, 20 normal postterm fetuses beyond 41 weeks CA, 11 fetuses suspected antepartum of being growth retarded, 2 fetuses with a brain anomaly and one fetus with trisomy 18 were studied, with a total of 245 experiments The study was approved by the hospital's ethical committee and all mothers gave their written informed consent.

INCLUSION CRITERIA

For the normal fetuses: (1) GA determined using the last menstrual period or by ultrasound when dates were uncertain; (2) the absence of maternal medical or obstetric complications; (3) single fetus without apparent structural anomalies and with an expected birth weight above the 10th centile according to population-based tables adjusted for maternal parity and fetal sex; (4) normal amniotic fluid volume as assessed by ultrasound; (5) no maternal use of alcohol, drugs or medication other than vitamins and / or iron.

For the growth retarded fetuses: (1) GA determined using the last menstrual period or by ultrasound when dates were uncertain; (2) single fetus without apparent structural anomalies and with an expected birth weight below the 5th centile according to population-based tables adjusted for maternal parity and fetal sex; (3) no maternal use of alcohol, drugs or medication other than antihypertensive drugs, vitamins and / or iron.

The neonatal outcome was examined for birth weight, GA at delivery, Apgar scores, umbilical artery blood pH, and the presence of neonatal complications. Infants were followed up at 3 months after birth by telephone interviews with one of the parents and at 1 year after birth by means of a questionnaire. Because some infants reach their first birthday only after completion of this thesis it was not possible to collect

30 (..•• -< c Ό all questionnaires. For the normal fetuses a GA between 37 and 40 weeks at birth, a < th υ birth weight > 10 centile, a 5-minute Apgar score > 8, umbilical artery blood pH ... ir,. > 7.10, and no neonatal complications were considered to be a normal neonatal e· outcome. Ζ

STUDY PROTOCOL

Our research group first performed a pilot study in 42 fullterm (37 - 40 weeks GA) and 19 preterm (32 - 36 weeks GA) born neonates. From this study we know that both groups are able to habituate to repeated vibroacoustic stimulation (VAS).This led us to conclude that the protocol used in this pilot study on neonates might also be suitable in testing the habituation pattern in fetuses. A pilot study in 5 normal fetuses between 36 and 37 weeks GA led us to conclude that the protocol used was suitable for testing habituation in fetuses. Based on this protocol we developed a protocol for studying habituation to VAS in fetuses.

Figure 2.1

The test arrangement

The women were placed in a semi-recumbent position (figure 2.1). Fetal heart rate (FHR) was recorded by cardiotocography at a paper speed of 3 cm per minute (Sonicaid FM7, Oxford, UK), starting 10 minutes prior to the tests and continuing for

31 CHAPTER 2

io minutes afterwards.The fetal trunk was visualised in a parasagittal plane by real­ time ultrasound (Hitachi model EUB-525, Tokyo, Japan). A stimulus (Corometrics model i46,Wallingford, Conn.; audible sound 20 to 9000 Hz, vibrations between 67 and 83 Hz, sound level 74 dB at 1 m in air) (figure 2.2) of ι s duration was repeatedly applied every 30 s to the maternal abdomen above the fetal legs.The stimuli were applied during fetal quiescence. A general movement of the fetal trunk within 1 sof application of the stimulus was defined as a positive response. A response decrement was noted as a tendency to change from a more intense to a less intense response pattern with successive trials. A lack of response to 4 consecutive stimuli was taken to indicate habituation. We allowed a maximum of 24 stimuli in each habituation procedure. However, a minimum of 4 additional stimuli would be necessary to show habituation if a fetus was still responding to the 21" stimulus. We therefore stopped stimulating if a fetus persisted in responding to the 21''' stimulus. The habituation rate was defined as the number of stimuli applied before a fetus stopped responding. The procedure was repeated ID minutes after the first habituation procedure to investigate whether the fetus habituated more rapidly to the second series of stimuli.

Figure 2.2 '^I^KÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊIÊÊIÊÊÊÊÊÊÊÊÊÊÊÊÊIÊÊÊÊÊÊÊÊÊÊ^K

The Corometrics fetal acoustic stimulator

With this technique, problems exist that relate principally to difficulties in deter­ mining the precise significance of a particular movement. It occasionally might be unclear whether the movement represents an actual response to the stimulus or whether it represents a spontaneous movement that has occurred coincidentally Especially in instances of high frequency of spontaneous fetal movements, the relationship of the movements to the stimulus is difficult to interpret. Therefore, habituation was considered to be established if the fetus responded regularly to the repeated stimuli.The data from experiments in which the fetuses showed alter­ nate responses and non-responses to repeated stimulation were excluded from the analysis because the habituation pattern was deemed to be uninterpretable.

32 The neurological condition of all but one infant from the group that was tested longitudinally (chapter 5), of all infants that were growth retarded at birth (chapter 7), and of one infant with hydrocephalus (chapter 8c) was examined at the age of 3 months. We assessed the quality of the general movements according to Hadders-Algra et al. 30.This method shows that definitely abnormal general move­ ments are associated with a high-risk for the development of cerebral palsy, whereas mildly abnormal general movements are associated with the development of minor neurological dysfunction, attention-deficit-hyperactivity disorder, and aggressive behaviour.

The infants from the study group that were tested longitudinally from 26 weeks CA onward were also tested between the 5lh and 8lh day after birth. A group of neonates between the 5'h and 8th day after birth that were not prenatally exposed to repeated VAS served as a control group. The results of this neonatal habituation study are beyond the scope of this thesis and are therefore not described.

33

ABSTRACT

Objectives: Fetal habituation to repeated stimulation has the potential to become a tool m the assessment of fetal condition and of the function of the fetal central nervous system (CNS). However, the influence of fetal quiescence and activity on fetal habituation remains to be clarified. We studied habituation and the influence of fetal state and fetal heart rate (FHR) parameters on habituation in healthy term fetuses.

Study design: We studied habituation in 37 healthy fetuses in 2 tests with an inter­ val of 10 minutes. The vibroacoustic stimuli (VAS) were applied to the maternal abdomen above the fetal legs for a period of 1 s every 30 s A fetal trunk movement within 1 s after VAS was defined as a positive response. Habituation rate is defined as the number of stimuli applied before an observed non-response to 4 consecutive stimuli. The FHR patterns (FHRP) of the 10 minute observation period before and after the tests were visually classified. Fetal states were defined according to the FHRP. Baseline FHR, FHR variability and the number of accelerations were calculated in a subgroup of 25 fetuses.

Results: Of the 32 fetuses that responded normally during the first test, 26 habituated and 6 had persistent responses.The median habituation rate decreased significantly in the second test (p = 0.001). There was no difference m habituation rate between fetuses that where initially in a quiet state and those m an active state The FHR parameters before the first test and the difference between these FHR parameters before and after the test did not correlate with the habituation rate.

Conclusions: Although the majority of healthy fetuses was able to habituate, the interfetal variability m habituation performance is such that testing of habituation seems not to bea sensitive tool for the assessment of the fetal CNS. This variability is neither the result of differences m fetal state nor of the various FHR parameters before testing, or of the difference in change of FHR parameters arising from stimulation.

36 INTRODUCTION

Habituation is the decrease in, and ultimate cessation of, response to repeated stimulation. Habituation can be seen as a basic form of learning that requires an intact central nervous system (CNS) '2,. If habituation represents CN5 functioning, then conditions affecting the CNS would be expected to affect habituation.This has been reported in studies on adults with various disorders of the CNS ,3i,ä5'3,

Several studies on habituation in normal fetuses have shown that fetuses are able to habituate to repeated stimuli «""i8l6i"67. studies in compromised or chromoso- mally abnormal fetuses show that the habituation pattern of these fetuses is different from that of normal fetuses 'CÉ"52,6i. Therefore, fetal habituation might identify a fetus with poor fetal condition or impaired CNS functioning

o6,59 6 Although fetal habituation has been studied widely "' ' i| its clinical relevance remains unclear. This can be explained by the variety of methodologies that have been used to test fetal habituation - there still exists no standardised test. Neither is it clear whether an abnormal habituation pattern is the result of fetal distress, impaired functioning of the CNS, or of a difference in environmental or fetal physiological factors, such as fetal behavioural states.

Behavioural states have been found to affect the magnitude of the fetal response and the time between the stimulus and the response 'S7'75'76. Although one study has demonstrated that behavioural states did not significantly influence the habituation pattern to vibroacoustic stimulation (VAS) '59, another study has shown that the decrement in response is caused by a behavioural state transition rather than by true habituation '". It remains unclear whether, and to what extent, fetal behavioural states influence habituation. Because of the potential clinical relevance of fetal habituation, it is essential to know whether fetal behavioural states and state transition affect habituation.

This study was designed to test fetal habituation to repeated VAS m healthy term fetuses and to study the influence of fetal states (fetal quiescence and activity), state transition and FHR parameters on habituation.

SUBJECTS AND METHOD

Subjects

Thirty-seven healthy pregnant women were recruited from a low-risk obstetric population (' practice) at the University Medical Centre Nijmegen in the Netherlands. The study was approved by the hospital's ethical committee and all participants gave their written informed consent. Inclusion criteria were: (i) gesta­ tional age (CA) between 37 and 40 completed weeks (pregnancies were dated using the last menstrual period or by ultrasound when dates were uncertain); (2) the absence of maternal medical or obstetric complications; (3) single fetus without

37 apparent structural anomalies and with an expected birth weight above the 10th centile according to population-based tables adjusted for maternal parity and fetal sex; (4) normal amniotic fluid volume as assessed by ultrasound; (5) no maternal use of alcohol, drugs or medication other than vitamins and /or iron.

Method

All habituation tests were performed under the same conditions (the mother was not allowed to smoke, drink coffee or eat for 3 hours before testing), in the same room, and by the same examiner between 4pm and 7pm. The women were placed ina semi-recumbent position The fetal trunk was visualised in a parasagittal plane by a real-time ultrasound scanner (Hitachi model EUB-525,Tokyo, Japan) The stimuli were produced by a fetal vibroacoustic stimulator (Corometrics model 146, Wallmg- ford.CT, USA, audible sound 20 to 9000 Hz, vibrations between 67 and 83 Hz, sound level 74 dB at ι m m air) Stimuli of 1 s duration were repeatedly applied to the maternal abdomen above the fetal legs every 30 s The stimuli were applied during fetal quiescence. A general movement of the fetal trunk within 1 s of application of the stimulus was defined as a positive response Response decrement was noted as a changing from a more intense to a less intense response pattern with successive trials. Lack of response to 4 consecutive stimuli indicated habituation. We allowed a maximum of 24 stimulus applications m each test. However, a minimum of 4 addi­ tional stimuli would be necessary to show habituation if the fetus responded to the 21" stimulus We therefore stopped stimulating if a fetus still responded to the 21s1 stimulus. The habituation rate was defined as the number of stimuli applied before a fetus stopped responding. The procedure was repeated 10 minutes after the first test to investigate whether the fetus habituated more rapidly during the second series of stimuli

The fetal heart rate (FHR) was recorded continuously with a cardiotocograph (Sonicaid FM7, Oxford, UK) at a paper speed of 3 cm per minute, starting 10 minutes prior to the tests and continuing for 10 minutes afterwards.

The outcome of each pregnancy was examined for birth weight, GA at delivery, Apgar scores, umbilical artery blood pH, and the presence of neonatal complica­ tions.The infants were followed up at 3 months after birth by telephone interviews with one of the parents and at 1 year after birth by means of a questionnaire.

Data analysis

In 37 women, the FHR recordings of the 10 minute observation periods before and after the first test were visually classified into 4 FHR patterns (FHRP) in accordance with Nijhuis et al.Ai. FHRP A is defined as a stable heart rate with little variability and isolated accelerations. FHRP Β has a wider oscillation bandwidth than FHRP A with frequent accelerations. FHRP C is a stable pattern with a wider oscillation bandwidth than FHRP A and without accelerations. FHRP D is unstable, with large

38 and long-lasting accelerations, which are frequently fused into a sustained tachy­ cardia. As the incidence of FHRP C and D is relatively low, these episodes were considered to be a FHRP corresponding to an active state. Finally, the FHR recordings were divided into a FHRP corresponding to a quiet state (FHRP A) and into a FHRP corresponding to an active state (FHRP B, C and D) In our study, determination of whether the fetus was m a quiet state or an active state was based on analysis of FHRP alone, because such a distinction is possible in normal fetuses after 35 weeks GA"e

Computerised analysis of the FHR of a subgroup of 25 fetuses was carried out using a Somcaid System 8002 (Oxford Somcaid Ltd, Abingdon, UK),79. Baseline FHR, FHR variability (long term variation (LTV) and short term variation (STV)) and the num­ ber of accelerations were calculated for the observation periods before and after the first test. Post-acquisition analysis of any fraction of the recording down to a mini­ mum of 10 minutes is possible.The system reduces the data over 3.75 s (i/i6 minute epochs).The baseline FHR is defined as the mean rate averaged over all periods of low variation. If no low variation is present, it is derived from a statistical analysis A baseline is fitted to the FHR trace and LTV is subsequently measured as the range of mean pulse intervals around the baseline, calculated minute by minute, excluding data from decelerations and errors. The system averages data over the total recording time in milliseconds (ms). STV is calculated m ms as the average of sequential i/i6lh minute pulse interval differences, after exclusion of decelerations and errors. Accelerations are defined as periods with an amplitude of more than 15 beats per minute (bpm) above the baseline FHR and lasting for more than 15 s The system treats signal loss of 30% or more as a high loss, which was also an exclusion criterion for our study.

Data of tests m which the fetus responded irregularly to the stimuli, in that alter­ nation of responses and non-responses were observed, were excluded from analysis because it was unclear whether habituation was present or not Data were compared using Wilcoxon matched-pairs signed-ranks test and Mann Whithey U test, and correlated using Spearman rank correlation coefficients, with a two-tailed ρ value of < 0.05 considered significant.

RESULTS

Habituation and the influence of fetal heart rate patterm

The data of 5 fetuses were excluded because they responded irregularly in the first test, which prevented interpretation of the test. These fetuses did not differ from the other 32 fetuses m gender, presentation, birth weight, maternal abdominal wall thickness, amniotic fluid volume, GA at testing and delivery, and neonatal outcome.

In the remaining 32 fetuses in which regular responses could be observed, the median GA at time of testing was 382'7 (range 37 °'7- 395") weeks, the median GA at

39 delivery 403'7 (range 38s'7- 42 "7) weeks, the median birth weight 3385 (range 2660- 4070) g Seven mothers were delivered by cesarean section for different reasons, but not for fetal distress. All infants, 14 males and 18 females, were in good health at birth, with birth weights above the 10th centile for GA, a 5-minute Apgar score > 8 and an umbilical artery pH > 710. Follow up at the age of 3 months (all infants) and at the age of 1 year (34 infants) revealed no serious abnormalities in any infant.

Of the 32 fetuses, 26 habituated in the first test Twenty of them habituated more rapidly or did not respond at all in the second test In the second test, only 2 fetuses habituated more slowly and in 4 fetuses habituation could not be determined as they alternated between response and non-response Of the 32 fetuses, 6 failed to habituate within 21 stimuli (4 even showed no response decrement) in the first test, 3 of them also failed to habituate in the second test and 3 habituated (after 15,13 and 3 stimuli, respectively).The median habituation rate decreased significantly in the second test (9.5 vs 2, ρ = o.ooi), meaning that fetuses habituated more rapidly during the second test (figure 3.1).

We classified the FHRP of the recordings prior to the first test into FHRP A, corre­ sponding to a quiet state and into a FHRP non-Α, corresponding to an active state. Of the remaining 31 recordings (data of 5 fetuses were excluded and 1 recording was uninterpretable owing to high signal loss), FHRP A was determined in 14 FHR recordings, and an FHRP corresponding to an active state in 17. There was no signif­ icant difference in the median habituation rate of fetuses that were initially m a quiet state and of fetuses in an active state (figure 3.2).

The 10-minute FHR recordings after the first test were also visually classified. Of the remaining 31 recordings, FHRP A was observed in only 3, and an FHRP corresponding to an active state m 28. Two fetuses remained m a quiet state after the test and one showed a state transition from active to quiet From the 28 fetuses that showed an FHRP consistent with an active state after the first test, 12 were m a quiet state prior to testing. The median habituation rate of these 12 fetuses did not differ from the habituation rate of the 16 fetuses that remained man active state after testing.

Habituation and the influence of baseline FHR, FHR variability and number of accelerations

Computer analysis of the FHR was performed m a subgroupof25fetuses.lt was not possible to determine habituation in 4 fetuses because of alternate responses and non-responses, so these FHR recordings were excluded from statistical analysis. One recording was uninterpretable as a result of high signal loss Of the remaining 20 fetuses, only the baseline FHR increased significantly after the first test (p = 0.001). The LTV, STV and number of accelerations did not change significantly after the first test (table 3.1).

40 Table 3.1 FHR parameters be/ore and after test 1 (n = 20).

Before test ι After test ι ρ

Basal FHR (bpm) 133 (127.5-137) 138 (134.5-147) 0.001 LTV(ms) 50.5(28-83) 59(40-79-5) NS STV (ms) 10.1 (8-13.3) 11-2 (9-13.1) NS Number of accelerations 2(1-3.5) 2.5(0.5-4) NS (>15 bpm, > 15 s)

Data are giuen in medians and interquartile ranges and compared using Wilcoxon matched-pairs signed-ranks test. LTV- long term uariation, STV: short term uariation, NS: not signi/ïcant

There is no correlation between the habituation rate of the first test and baseline FHR, LTV, STV and number of accelerations of the observation period prior to this test Neither is there a correlation between the habituation rate of the first test and the baseline FHR prior to this first test, the difference in baseline FHR between periods prior and after this test, LTV, STV and number of accelerations between the observation periods prior to and after this test (table 3.2, figures 3.3 and 3.4).

Table 3.2 Correlation coe/jfirients of habituation rate and FHR parameters of observation period be/ore test 1 and of habituation rate and difference between FHR parameters before and after test l(n = 20). Before test 1 Δ before and after test 1

Basal FHR (bpm) 0.06 0.8 -0.2 0.4 LTV (ms) -0.2 0.3 0.2 °-3 STV (ms) -0.2 0.3 0.2 03 Number of accelerations -0.13 0.6 0.14 0.6

Data are correlated using Spearman rank correlation coe/jficient LTV: long term uariation, STV: short term uariation.

41 Figure 3.1 Habituation rate in test 1 and test 2 (10 minutes after test 1) (n = 32)

25

ï=» 20 & 3 b c O •κ 15 Μ- +-' Π Ώra (1) .a -Ω ΙΟ m Ε 3 Χ C

Testi Test 2

Data are combined in a box and luhisfeer plot (median, 25'h and 75,h centile, maximum and minimum) A habituation rate of 23 represents a persistent response * ρ = 0 001

Figure 3.2 Habituation rate in fetuses that were initially in a quiet state (n = 14) and in an active state (n = 17).

25 -,

20 X .

J c + 15

•t; Ja IO

Ouiet state Active state

' Not significant

42 Figure 3.3 Habituation rate of test 1 and the baseline FHR prior to test 1 (n = 20).

25

r=· 20 a> 3 p c t 15 - o «ί- +J Ο TOm 3 Δ IO m E I 3 C • M 5 -

1 1 1 1 1 no 120 130 140 150 160

Figure 3.4 Habituation rate of test 1 and the difference in baseline FHR be/ore and after test 1 (n = 20).

*o - • • 20 . 2 E • c •κ Ü H- 15 - +J • · O • 3ra at XI 10 _ • m E • I 3 c • • · • • • • • 5 - • • 0 _ • 1 1 ) -10 ο io 20 30 Δ Baseline FHR (bpm)

43 DISCUSSION

In this study we investigated habituation to repeated VAS m healthy term fetuses and studied the influence of fetal quiescence and activity, state transitions and FHR parameters on habituation The type of stimulus we used induced an immediate fetal movement response regardless of the fetal state. By using this stimulator and observing the immediate movement response to repeated VAS it is easy for a clinician to distinguish between a response and a non-response and to determine fetal habituation. The method we used was not adequate to determine the presence of habituation in all fetuses. Five fetuses (13.5%) responded irregularly to the repeated stimuli. These fetuses exhibited alternate responses and non- responses, which made determination of habituation impossible.

The habituation rate of the first test varied greatly in our study, with 6 fetuses failing to habituate and 6 fetuses habituating within 5 stimuli. All fetuses did well after birth. To confirm that the observed response decrement was the result of habituation rather than motor fatigue or sensory adaptation, we repeated the test within 10 minutes of the first test. Repeated stimulation may physically exhaust the fetus or it may tire the auditory or tactile receptor, which results in receptor adap­ tation. In true habituation, the original stimulus, when presented again, initially elicits a response but this response decreases more rapidly than when first presented. In our study the habituation rate decreased significantly in the second test. This can be explained by the fact that fetuses recognise the stimuli and habituate more rapidly (chapter^ '8o. Nonetheless, 3 fetuses continued to respond in the first test as in the second test and 2 fetuses habituated more slowly m the second test.

The behavioural states of neonates and fetuses have been found to affect the response to a single vibroacoustic or sound stimulus. Neonatal responses to various stimuli vary with behavioural state, with the neonate being less responsive during quiet sleep than during an active period ;:ι"". Werner et al '75 examined the motor and FHR response to a door buzzer in term fetuses. The FHR response time and movement response time (time between the stimulus and the onset of the response) was significantly longer during fetal quiescence. In contrast, Jensen ',6 described that FHR responses to sound stimulation were mainly observed in fetuses with low baseline FHR levels prior to stimulation, in other words m fetuses in a quiet state. A non-response to this stimulus was observed mainly m fetuses in an active period.

If behavioural states affect the response to a single stimulus, one might think that it also influence habituation of responses to repeated stimuli. Although one study has demonstrated that fetal states did not significantly influence the habituation pattern '59, another study has shown that the decrement in response is caused by a behavioural state transition rather than by true habituation '". Groome et al. '" observed the movement response magnitude to a fixed number of 8 VAS m fetuses

44 between 34 and 40 weeks GA. They showed that decrement of the movement response was not influenced by the variability of the FHR prior to testing. Shalev et al '" also examined the influence of fetal states, according to FHR pattern and fetal movements, on habituation to VAS. They found that fetuses in a quiet state habituated more rapidly (mean rate of 5 stimuli) compared to fetuses in an active state (mean rate of 8 stimuli). We found that the median habituation rate of fetuses that were initially in an active state, according to visual analysis of the FHRP, did not differ from that of fetuses in a quiet state. Our findings that the baseline FHR, LTV, STV and accelerations prior to the habituation test did not correlate with the habituation rate, support the conclusion that fetal habituation is not affected by the FHRP prior to testing.

Repeated stimulation alters the behavioural state in term fetuses This could suggest that fetal habituation is induced by a state transition from a quiet to an active state '57,77 Hurt et al. '" were unable to demonstrate habituation of the move­ ment response to sound stimulation in 10 neonates. Response decrement was observed only when there was a state transition from an active to a quiet state, whereas transition from a quiet to an active state was accompanied by a recovery of response. This suggests that response decrement might not be the result of habituation but rather of a state transition Although almost all fetuses showed an active FHRP after the first test, our results show that the habituation rate varied greatly. We cannot conclude that a state transition from an active to quiet or from a quiet to active state induced by repeated stimulation influenced the habituation rate. The difference between FHR parameters before and after the first test did not correlate with the habituation rate. From these results we conclude that a change in FHRP and FHR parameters resulting from stimulation did not affect the habituation rate.

Although several methodologies have been used, all studies of normal fetuses conclude that fetuses are able to habituate to repeated stimulation ""'s8'66'6? Studies in compromised or chromosomally abnormal fetuses reveal that the habituation pattern of these fetuses was different from that of normal fetuses 'o6 'S2'6^ For example, fetuses with Down's syndrome habituated more slowly than unaffected fetuses,6î

Leader et al. studied fetal habituation to VAS produced by an electric toothbrush in compromised fetuses (small for GA, mecomum-stained amniotic fluid or decreased growth velocity of the fetal bipanetal diameter) 'o6. They found that the compro­ mised fetuses habituated more rapidly (requiring 1-9 stimuli) or more slowly (> 50 stimuli) compared with normal fetuses. In our study of normal fetuses, the habitu­ ation rate m the first test varied greatly Six fetuses failed to habituate within 21 stimuli and 6 habituated within 5 stimuli. Although we followed another protocol with a different stimulator, and a maximum of 21 stimulus applications, we cannot conclude that fetuses that failed to habituate or habituated more rapidly showed fetal distress or an adverse outcome.Therefore, there are apparently normal fetuses

45 ,. Ι' ·κ \ < r "- i

that fail to habituate to repeated VAS or that habituate more rapidly than other fetuses.There are even apparently normal fetuses m which it is impossible to deter­ mine habituation due to irregular responses.

From this study we conclude that the habituation rate varied greatly in healthy term fetuses. Given the variability of the habituation rate and the small difference between the habituation rate in fetuses m a quiet state and fetuses in an active state, the influence of fetal statescan be ignored And although habituation can be clearly observed in the majority ofterm fetuses, the mterfetal variability is such that identification of an abnormal fetus will be very difficult. This means that the clinical relevance of fetal habituation has still to be elucidated.Therefore, further studies to investigate whether or not fetal habituation can be used for the assessment of fetal condition and for the assessment of fetal CNS in high-risk fetuses are in progress.

46

ABSTRACT

Objective: Habituation to repeated vibroacoustic stimulation can be used as an experimental approach to assess fetal learning and memory. We studied fetal habituation at 3 time points to assess fetal memory in utero.

Study design: Twenty-five fetuses between 37 and 40 weeks gestational age were studied 10 minutes and 24 hours after an initial habituation test.

Results: Ten minutes after the initial test, 16 of 19 fetuses habituated more rapidly to the stimuli (p = 0.002), and 24 hours after the initial test again 16 of 19 fetuses habituated more rapidly than to the initial series on the first day (p = 0.042).

Conclusions: These results suggest that fetuses are able to learn: they have a short term memory, and a long term memory of at least 24 hours.

48 INTRODUCTION

For many years, babies have been believed to be born without a functional memory'82 However, several studies on the memory of neonates that tested the infants after birth have claimed to show that infants are able to memorise acoustic stimuli in utero "0'*,. However, whether these studies prove the existence of fetal memory is not certain, because no measurement of response was done in utero

Evidence of a different neonatal response to the familiar and the unfamiliar stimuli stimulus was taken as evidence of fetal learning and memory. A different way to test fetal memory is by using fetal habituation as an experimental approach. Our aim was to test fetal memory and learning in utero. We used observations of fetal habituation to repeated vibroacoustic stimuli (VAS) at various time points to study the fetal memory of the stimuli. Habituation is the decrease, and eventual cessation, of the response that follows repeated application of the same stimulus. The ability to recognise and ignore harmless repeated stimulation is essential for individuals to function m a given environment Habituation is thought to be based on learning '" in the same way that learning is based on memory

SUBJECTS AND METHOD

Subjects

Twenty-five healthy pregnant women were recruited from a low-risk obstetric population (midwives' practice) at the University Medical Centre Nijmegen in the Netherlands. The study was approved by the hospital's ethical committee and all participants gave their written informed consent. Inclusion criteria were (i) gesta­ tional age (GA) between 37 and 40 completed weeks (pregnancies were dated using the last menstrual period or by ultrasound when dates were uncertain); (2) the absence of maternal medical or obstetric complications, (3) single fetus without apparent structural anomalies and with an expected birth weight above the iolh centile according to population-based tables adjusted for maternal parity and fetal sex; (4) normal amniotic fluid volume as assessed by ultrasound, (5) no maternal use of alcohol, drugs or medication other than vitamins and /or iron

Method

We studied habituation at 3 time points. After the initial test, we tested the fetuses 10 minutes later and again after 24 hours. All habituation tests were done under the same conditions (the mother was not allowed to smoke, drink coffee or eat for 3 hours before testing), in the same room, and by the same examiner between 4pm and 7pm The women were placed m a semi-recumbent position The fetal heart rate (FHR) was recorded continuously with a cardiotocograph (Sonicaid FM7, Oxford, UK) at a paper speed of 3 cm per minute The fetal trunk was visualised m a parasagittal plane by real-time ultrasound (Hitachi model EUB-525, Tokyo, Japan)

49 The stimuli were produced by a fetal vibroacoustic stimulator (Corometrics model i46,Wallingford,Conn; audible sound 20 to 9000 Hz, vibrations between 67 and 83 Hz, sound level 74 dB at 1 meter in air). Stimuli of 1 s duration were repeatedly applied to the maternal abdomen above the fetal legs every 30 s. Stimuli were applied du ring fetal quiescence. A genera I movement ofthe fetal trunk within 1 sof application of the stimulus was defined as a positive response. A response decrement was noted as a tendency to change from a more intense to a less intense response pattern with successive trials. A lack of response to 4 consecutive stimuli was taken to indicate habituation We allowed a maximum of 24 stimuli in each habituation procedure. However, a minimum of 4 additional stimuli would be necessary to show habituation if a fetus was still responding to the 2iil stimulus We therefore stopped stimulating if a fetus persisted in responding to the 21s1 stimulus. The habituation rate was defined as the number of stimuli applied before a fetus stopped responding.

The outcome of each pregnancy was examined for birth weight, GA at delivery, Apgar scores, umbilical artery blood pH, and the presence of neonatal complica­ tions The infants were followed up at 3 months after birth by telephone interviews with one ofthe parents and at 1 year after birth by means of a questionnaire.

Data analysis

Habituation was considered to be established if the fetus responded regularly to the repeated stimuli The data from experiments in which the fetuses showed alter­ nate responses and non-responses to repeated stimulation were excluded from the analysis because the habituation pattern was deemed to be uninterpretable. Data from the 3 tests were compared by Friedman repeated measures analysis of variance (ANOVA). If ANOVA indicated a statistical significance, the intermediate variances were analysed using a Wilcoxon matched-pairs signed-rank test. Differences were considered significant at ρ < 0.05.

Table 4.1

Population characteristics.

Total group Analysed group

Number of fetuses 25 21

GA at time of testing (week) 38," (37 '" - 39,'7) 384" (373/7 - 39,") CA at delivery (week) 40 ^ (38 "'- 42 '") 40s" (38 6/7- 42 '") Birth weight (g) 3480 (2660-4265) 3450 (2660-4070) Sex (male/female;n) 12/13 9/12 The figures represent absolute ualues, medians and ranges.

50 RESULTS

Four fetuses were excluded from analysis because irregular movement responses to the stimuli prevented interpretation of at least 2 of 3 tests. These fetuses did not differ from the other 21 fetuses in gender, presentation, birth weight, maternal abdominal wall thickness, amniotic fluid volume, CA at testing and delivery, and neonatal outcome Seven mothers were delivered by cesarean section for different reasons, but not for fetal distress. All infants were m good health at birth, with birth weights above the iolh centile for GA, a 5-minute Apgar score > 8 and an umbilical artery pH > 7.10. Follow up at the age of 3 months (all infants) and at the age of ι year (23 infants) revealed no serious abnormalities in any infant. Characteristics of the fetuses are shown m table 4.1

Figure 4.1 Median fetal habituation rate.

25 -,

20 . v ^ 2 E ... c +•> ic 2£ I——I '•£ 0 E v « _ο -Ω e ΙΟ . si ι-^ 5 - Ι , 1 Ι ' 1

ο J ' ι ' Ι

Initial test After 10 min After 24 h

Data luere combined in a box and luhisfeer plot (median, 25''' and 75"' centile, maximum and minimum) A habituation rate of 23 represents a persistent response, a rate of 0 a non- response. * ρ = 0.002; ** ρ = 0 042

In 4 out of the remaining 21 fetuses habituation could be determined in only 2 tests. Of 2 of them the second test, and in the other 2 the third test was unmterpretable due to irregular responses. Data from the 17 fetuses who had results for 3 tests are shown in figure 4.1. The Friedman test revealed statistical significance (p < 0.001). Ten minutes after the initial test, 16 of 19 fetuses habituated more rapidly to the stimuli (p = 0.002) than to the initial series or did not respond at all (which probably indicated immediate recognition of the stimuli). Two fetuses habituated more slowly and one fetus had a persistent response in both tests. Twenty-four hours

51 after the initial test,i6 out of 19 fetuses habituated more rapidly and 3 fetuses more slowly with 2 of them showing persistent responses (p = 0.042) (figure 4.2) Three of these 16 fetuses had persistent responsesonthefirst day, but all 3 habituated 24 hours later The observed baseline FHR and FHR pattern 10 minutes before the tests did not affect the habituation rate

All 25 fetuses showed movement responses m the initial test but 4 fetuses failed to respond to the second, and one to the third test.

Figure 4.2 Change in habituation rate of each fetus (n = 17).

25 -,

20 .

2 E g "-Κ '5 -

5 ai la -£ IO . Χ 3 c 5 -

ο Initial test After 10 min After 24 hrs

DISCUSSION

Despite the fact that fetal sensory systems are both morphologically and functionally immature, fetuses have the capacity to sense, respond to, habituate to, and even recall stimuli encountered in utero Memory can be short term or long term, with short term memory or being a memory that only lasts seconds to a couple of hours, and long term memory that may last hours to months. Long-lasting memory may last a life-timel8j

The results of this study suggest that fetuses have a short term memory of at least 10 minutes and a long term memory of at least 24 hours. Fetuses are able to memorise the stimuli in utero, although they may need more than one stimulus to establish recognition To our knowledge, this is the first study that demonstrated fetal memory in utero, because measurement of response was done in utero A previous study of habituation of the heart response to an auditory stimulus on 2 successive days m neonates showed that neonates who habituates to an auditory stimulus retained the habituation effect for 24 hours '8',

52 One could suggest that the presence of fetal behavioural states at this gestational period accounts for the significant decline in habituation rate in the second test after 10 minutes of the initial test. In a previous study we have found no influence from fetal rest or activity on the habituation rate (chapter 3) ,8s. This can be explained by the fact that VAS arouses the fetus, which results m a state transition to an active state during testing m the majority of the fetuses,85. If the stimulus is intense enough, it will override the effect of fetal state. In fetal sheep, responsive­ ness and the habituation performance were not influenced by the electrocortical state of the fetus 'M. Similar observations have also been made in human fetuses'S9.

In our study all fetuses responded in the initial test, but there was no response from 4 fetuses in the second test, and from one in the third test We propose the idea that this is a result of the immediate recognition of the stimulus. As yet we have only observed this fetal non-response to this stimulus m a fetus with trisomy 18 (chapter 8a) '86 and in a fetus with a severe brain anomaly (chapter 8b) ,e7.The crucial difference between non-responders with brain or chromosomal anomalies and normal fetuses that did not respond at repeated testing is that the latter all responded initially.The abnormal fetuses never responded, even when tested again after 1 or 2 weeks.

In our study, 14 out of 75 (19%) experiments were unmterpretable because fetuses showed alternate responses and non-responses The exclusion of data of these tests was for reasons of conformity with the definition of habituation that we used. One could question the validity of our stimulator. But, because the majority of the fetuses exhibited regular responses to repeated stimulation we think that this stimulator did not contribute to an irregular response. Observation of irregular responses can be attributed to difficulties in determining the precise significance of a particular movement. It occasionally might be unclear whether the movement represents an actual response to the stimulus or whether it represents a spontaneous movement that has occurred coincidentally. Especially m instances of high frequency of spontaneous fetal movements, the relationship of the movement to the stimulus is difficult to interpret.

The fact that the human fetus can learn should not appear surprising, as similar abilities have been found in other mammals'88, birds189, fish ,9°,and invertebrates'9'. Memory is essential for normal functioning and it is not astonishing that such an important psychological function is developing before birth in some form. Prenatal memory may be important for the development of attachment and maternal recognition. There is much evidence that the fetus learns the speech characteristics of its mother prenatally and prefers its mother's voice to other female voices after birth Newborns presented with a choice between their mother's voice as it sounded in utero and as it sounded outside the uterus, showed a preference for their mother's voice as it sounded in utero '7°. This suggests that their preference was acquired before birth. This and other studies have confirmed that the fetus can memorise its mother's voice before birth '7"7Î. Infants are also able to memorise

53 other sounds than voices, for example a tune of a certain television program, presented to them prenatally. Infants from mothers that had watched a certain television-program several times when pregnant preferred this tune after birth to other unfamiliar tunes "4.

Evidence has also started to accumulate that the fetus is capable of learning and remembering olfactory stimuli. Newborn infants were capable of recognising their mothers smell soon after birth Shaal and Orgeur '92 gave a pregnant woman cumin to eat during the last 12 days of her pregnancy. After birth, the infant was exposed to the smell of cumin and citral. Only the odor of cumin resulted in the newborn infant exhibiting a change in the heart rate These studies indicate that learning and memory is not specific to sound stimuli but also occurs in other sensory modalities. Fetuses are also able to detect and memorise the odor of amniotic fluid in utero. After birth, neonatal preference for the odor of amniotic fluid can be observed '9S'9" Babies exposed to amniotic fluid cried significantly less than babies who lacked this exposure,95.

We used fetal habituation as a paradigm to explore fetal memory. Fetal habituation can also be used to examine the integrity and functioning of the fetal central nervous system (CNS). There is evidence that fetal habituation may be useful as a diagnostic tool for identifying fetuses in distress or those likely to be compromised neurodevelopmentally after birth Studies in compromised or chromosomally abnormal fetuses reveal that the habituation pattern of these fetuses is different from that of normal fetuses 'O6,Î2'",6Î. The neurological compromised fetus fails to habituate or habituates more slowly or more rapidly compared to normal fetuses

However, our results show that some of the fetuses showed persistent responses in the first test as well as in the test after 24 hours. So, the absence of successful habituation does not necessarily indicate fetal compromise or an impaired functioning of the fetal CNS. The maximum number of 21 applied stimuli may contribute to unsuccessful habituation rather than the presence of an abnormally functioning CNS

It must be taken into account that fetal memory processes or underlying cerebral functions are not necessarily similar to that in infants and adults. So the memory and learning capacities of an individual in utero does not necessarily give infor­ mation about these capacities in later life. It is also unknown at present what this study indicates about the functioning of the CNS and the continuity or relationship between the memory abilities evidenced before birth and those evidenced after birth and later in life. So, we do not claim that fetuses that failed to habituate more rapidly after 10 minutes or 24 hours are not capable of memorising stimuli. Further research should focus on the clinical relevance of failure to habituate or failure to habituate more rapidly m a repeated test

54

ABSTRACT

Objective: We used habituation to external stimulation to explore the development of fetal memory in utero. Habituation is thought to be based on learning in the same way that learning is based on memory.

Study design: We studied habituation at 10 minute and two-weekly intervals m 21 fetuses from 26 weeks gestational age (GA) onwards, and compared the results with those of 37 singly tested term fetuses.The fetal trunk was visualised by means of an ultrasound scanner A vibroacoustic stimulus was repeatedly applied to the maternal abdomen above the fetal legs for a period of ι s every 30 s. A general move­ ment of the fetal trunk within 1 s of application of the stimulus was defined as a positive response. Lack of response to 4 consecutive stimuli indicated habituation. The habituation rate is defined as the number of stimuli applied before the fetus stopped responding

Results: From 28-30 weeks GA onwards, fetuses habituate more rapidly within io minutes of the initial test The median number of stimuli needed for habituation decreased between 28 and 40 weeks GA (12 vs 2, ρ = o.oi8).Term fetuses repeatedly tested habituated much more rapidly than singly tested term fetuses (p < o.oooi).

Conclusion: A short term memory of at least 10 minutes is present in fetuses from 28-30 weeks GA and a long term memory of at least 2 weeks is present in the term period.

56 INTRODUCTION

For many years it was thought the newborn infant did not posess a functioning memory, but rather that memory developed over the months and years following birth ,β2. However, although fetal sensory systems are both morphologically and functionally immature, fetuses have the capacity to perceive, react to, habituate to and even recall stimuli encountered in utero ,7'|,8o,96,9'. Habituation is the decrease, and eventual cessation, of the response that follows repeated application of a stimulus. The ability to recognise and ignore harmless repeated stimulation is essential for individuals to function in a given environment Habituation is thought to be based on learning ,;' m the same way that learning is based on memory. To date, little has been discovered about fetal habituation under repeated testing.The possible influence of fetal learning and memorisation of the stimuli on the habituation performance is of particular interest.

Habituation can be used as an experimental approach to exploring fetal memory and learning. In a previous study of the repetition of habituation testing in 25 human fetuses between 37 and 40 weeks gestational age (GA), we showed that the fetuses habituated more rapidly to vibroacoustic stimulation (VAS) both 10 minutes after and 24 hours after the initial habituation procedure (chapter 4) ,80 These results suggest that fetuses are able to recognise stimuli that were previously presented to them m utero: apparently they have a short term memory of at least 10 minutes, and a long term memory of at least 24 hours Furthermore, studies on the memory of neonates in which the infants were tested after birth have claimed to show that infants are able to memorise acoustic stimuli in utero'70'7" Studies on the retention of information m young infants have revealed that infants are capable of memorising and learning even from the first hours after birth and they show a rapid increase in their capacity to recall information ">s"39

Our aim here is to extend the study on fetal memory and learning by observation of fetal habituation to external stimulation in the third trimester of pregnancy This study is designed to examine the influence of repeated testing on the habituation rate m normal human fetuses. We studied the fetal habituation pattern longitudinally at different GA and compared this with the habituation rate of term fetuses that were tested only once.

SUBJECTS AND METHOD

Subjects

Our experiments were conducted on the fetuses of 21 women with an uncomplicated singleton pregnancy (repeated exposure group) They were studied at two-weekly intervals between 26 and 40 weeks GA, resulting m a maximum of 7 experiments per woman (each experiment consisted of 2 habituation procedures with an interval of 10 minutes).The single exposure group consisted of fetuses of 37

57 women with an uncomplicated singleton pregnancy who were studied once between 37 and 40 weeks CA. The GA was calculated from the first day of the last menstrual period and / or from an ultrasound scan early in pregnancy The study was approved by the ethical committee of the University Medical Centre Nijmegen and all participants gave their written informed consent. Inclusion criteria were: (1) GA between 37 and 40 completed weeks (pregnancies were dated using the last menstrual period or by ultrasound when dates were uncertain); (2) the absence of maternal medical or obstetric complications; (3) single fetus without apparent structural anomalies and with an expected birth weight above the 10th centile according to population-based tables adjusted for maternal parity and fetal sex; (4) normal amniotic fluid volume as assessed by ultrasound; (5) no maternal use of alcohol, drugs or medication other than vitamins and / or iron.

Method

All experiments were performed by the same examiner in the same quiet room between 4pm and 7pm The mothers were asked not to smoke, drink coffee or eat for 3 hours before testing. Fetal heart rate (FHR) was recorded by cardiotocography at a paper speed of 3 cm per minute (Sonicaid FM7, Oxford, UK), starting 10 minutes prior to the experiment and continuing for 10 minutes afterwards. The women lay in a semi-recumbent position throughout the experiment. The fetal trunk was visualised by a real-time ultrasound scanner (Hitachi model EUB-525,Tokyo, Japan). The stimuli were produced by a fetal vibroacoustic stimulator (Corometrics model i46,Wallingford, CT, USA; audible sound 20-9000 Hz, vibrations between 67 and 83 Hz, sound level 74 dB at 1 m m air). A stimulus of 1 s duration was repeatedly applied every 30 s to the maternal abdomen above the fetal legs. The stimuli were applied during fetal quiescence A general movement of the fetal trunk within 1 s of appli­ cation of the stimulus was defined as a positive response. A response decrement was noted as a tendency to change from a more intense to a less intense response pattern with successive trials. A lack of response to 4 consecutive stimuli was taken to indicate habituation We allowed a maximum of 24 stimuli in each habituation procedure However, a minimum of 4 additional stimuli would be necessary to show habituation if a fetus was still responding to the 21" stimulus. We therefore stopped stimulating if a fetus persisted in responding to the 21s1 stimulus. The habituation rate was defined as the number of stimuli applied before a fetus stopped responding The procedure was repeated 10 minutes after the first habituation procedure to investigate whether the fetus habituated more rapidly to the second series of stimuli

The neonatal outcome of each pregnancy was examined for GA at delivery, birth weight, Apgar scores, umbilical artery blood pH, and the presence of neonatal complications The neurological condition of all but one infant from the repeated exposure group was examined at the age of 3 months by assessing the quality of the general movements according to Hadders-Algra et al J° This method shows

58 that definitely abnormal general movements are associated with a high-risk of development of cerebral palsy, whereas mildly abnormal general movements are associated with the development of minor neurological dysfunction, attention- deficit-hyperactivity disorder, and aggressive behaviour. Infants from the single exposure group were followed up at 3 months of age by telephone interviews with one of the parents.

Data analysis

In the repeated exposure group the differences in habituation rate with each of the 7 GA groups in turn (26-28; 28-30; 30-32; 32-34; 34-36; 36-38; 38-40 weeks) were analysed using the Wilcoxon matched-pairs signed-ranks test. Due to the mter- dependency of the values at subsequent time points, the level of significance per comparison was taken to be < 0.05 / number of comparisons (< o 01). Each CA group of the repeated exposure group was also compared with the single exposure group using the Mann-Whithey U test. A two-tailed ρ - value of < 005 was considered statistically significant Because the data of fetuses that persisted in responding as well as of fetuses that did not respond at all are also important, we decided not to exclude these data Fetuses that persisted in responding had a habituation rate > 21, fetuses that failed to respond had a rate of o. Habituation was considered to be established if the fetus responded regularly to the repeated stimuli The data from experiments in which the fetuses showed alternate responses and non-responses to repeated stimulation were excluded from the analysis because the habituation pattern was deemed to be uninterpretable.

RESULTS

Two fetuses from the repeated exposure group were excluded, one because of a birth weight below the s"1 centile and the presence of a heart anomaly, the other because of premature birth at 33 weeks GA. In the single exposure group, the data from 5 (13.5%) experiments were excluded because it was impossible to distinguish the presence or absence of habituation because of alternate fetal responses and non-responses. The characteristics of the remaining 19 fetuses m the repeated exposure group and of the 32 fetuses m the single exposure group are shown in table 5.1.

59 Table 5.1 Population characteristics.

Repeated exposure group Single exposure group

Number of fetuses studied 19 32 0 0 CA at time of testing (weeks) 26 " - 40 " 38 "" (37°"- 39 s'7) CA at delivery (weeks) 39 4,7(37°"- 41 ^ 40 '"(38"'- 423") Birth weight (g) 3350 (2655-4255) 3385 (2660-4070) Sex (M/F) 10/9 14/18

The figures are absolute ualues, medians and ranges There were no significant differences between the two populations for any of the uanables presented, except for the gestational age at the time of testing The repeated exposure group is the longitudinally tested group and the single exposure group is the term group tested once

The pregnancies of the remaining participants in both groups were uncomplicated and resulted in live births One fetus from the repeated exposure group and 2 fetuses from the single exposure group were in breech presentation during pregnancy and at delivery All women delivered vaginally and all infants did well at birth with birth weights between the 10th and go"1 centiles according to population- based tables adjusted for maternal parity and fetal sex Neurological examination at 3 months of age revealed no abnormalities m the infants in the repeated exposure group (one infant was followed up by telephone interview only) Follow up of the infants in the single exposure group also revealed no abnormalities Follow up at the age of 1 year was unfortunately only possible in 13 of the 19 infants

In the repeated exposure group of 19 fetuses, 126 experiments (1 e, 2 habituation procedures with an interval of 10 minutes) were performed from 26 weeks GA onward For the comparison between each GA group we only used data from the first procedure of each experiment Between 26 and 28 weeks GA, only 8 out of 19 (40%) fetuses responded with movement to stimulation Two of these 8 fetuses habituated and one responded persistently In 5 of these 8 fetuses habituation could not be established because of irregular responses In view of the fact that 60% of the fetuses in this GA group did not respond, we excluded these data from further analysis Between 28 and 30 weeks GA, 2 out of 19 (10%) fetuses failed to respond (at 28 2'7 and 29 i'7 weeks GA, respectively), and 2 fetuses responded irregularly and so prevented interpretation of the habituation test Thirteen fetuses habituated, and 2 fetuses failed to habituate and responded persistently From 30 weeks GA onward, all fetuses responded with immediate movement Data on habituation rates from 28 weeks GA onward are presented in figure 5.1

60 Figure 5.1 Habituation rates of the repeated exposure group (n = 19) and the control group (n = 32).

N=i5 N=i6 N=i9 N=i7 N=i8 N=i2 N=32

* **

- r- i— — —| — —

1 28-30 30-32 32-34 34-36 36-38 38-40 Control group

Gestational age (weeks)

The data were combined in a box and whisfeer plot (median, interquartile range, maximum and minimum). A habituation rate of 23 represents a persistent response, a rate of 0 a non- response. Term data are restricted to 12 fetuses because one fetus was born before its scheduled expenment between 36-38 weeks GA and 6 fetuses were born before their scheduled experiment at 38-40 weefes GA. * ρ = 0.04 (in uiew of the mterdependency of the values at subsequent time points, the leuel of significance per companson was tafeen to be 0.01). " ρ = 0 006

In the majority of the habituation procedures between 28 and 40 weeks GA, we could establish the presence or absence of habituation. Interpretation proved impossible m only 8 of the 107 (7.5%) experiments in the repeated exposure group and in 5 of the 37 (13.5%) experiments in the single exposure group because of irregular fetal response.

In each GA group from 28 weeks onward there was a significant decline m habituation rate between the first and the second procedure after 10 minutes (p = ο 0027, Ρ = o.oooS; ρ = 0.0019; Ρ = 0.0017; Ρ = ο.οοΐ2, ρ = ο 0037. respectively) (figure 5-2) In 28% to 42% of the experiments from 28 weeks GA onward, the fetuses did not respond at all during the second procedure.

The median habituation rate of the first procedure decreased gradually between 28 and 40 weeks GAfrom 12 stimuli between 28-30 weeks GA to 2 stimuli between 38- 40 weeks GA(p = o.oi8).The decline between the consecutive GA groups 36-38 and 38-40 weeks GA is particularly marked (7.5 vs 2, ρ = ο oo6). Furthermore, an obvious

61 but insignificant decline in habituation rate can be observed between the conse­ cutive GA groups 30-32 and 32-34 weeks GA (p = 004). The habituation rate between 32-34 and 36-38 weeks GA remains stable. However, it must be noted that the inter-experiment variability m habituation rate from the repeated exposure group is substantial.

Of the remaining 32 fetuses in the single exposure group in which the habituation pattern could be analysed, 6 fetuses persisted in responding (4 showed no response decrement), 10 fetuses habituated after more than 10 stimuli, and 16 fetuses m less than 10 stimuli. The median habituation rate in this group did not differ from the rate in each GA group of the repeated exposure group, but there is a striking difference between the habituation rate of term fetuses of the single exposure group and of the repeated exposure group (9.5 vs 2, ρ < o.oooi) (figure 5.1)

Figure 5.2 Habituation rates of the initial test (test 1) and the test after 10 minutes (test 2).

25

20

15

10 Τ A 121212121212

28-3Ο 3Ο-32 32-34 34-36 36-38 38-4Ο

Gestational age (weeks) and test number

DISCUSSION

Between 26 and 28 weeks GA, only 40% of the fetuses responded with a movement to stimulation, 90% responded between 28-30 weeks GA, and beyond 30 weeks GA all fetuses responded. Our findings corroborate that fetal movement responses to VAS are present m 89% of fetuses between 27-28 weeks GA and in all fetuses beyond 30 weeks GA " These results suggest that the onset of the fetal movement response is related to functional maturation of the CNS

62 In each GA group from 28 weeks onward there was a significant decline in habituation rate between the first and the second procedure after 10 minutes with some fetuses that did not respond at all This difference m habituation rate and responsiveness is probably due to the fact that fetuses recognise the stimuli presented to them m the first procedure. Some fetuses needed a couple of stimuli before recognition, others recognised them immediately. Thus, fetuses from 28 weeks CA onward appear to be able to memorise stimuli for at least 10 minutes. These results corroborate the conclusion from a study performed within our group that human term fetuses habituate more rapidly to the VAS 10 minutes after an initial habituation procedure (chapter 4) '8c. However, one could suggest that the results of the test after 10 minutes may be explained by the fact that the fetuses habituated more rapidly or were unable to respond due to motor or sensory fatique. Shahidullah and Hepper '6i demonstrated that all normal fetuses exhibited a decrement in response to repeated presentations of a stimulus, a 250-Hz sine wave This was the result of habituation and not fatigue since fetuses recovered responding upon presentation of a new (500 Hz) stimulus and habituated faster on re-presentation of the original stimulus. They also found conclusive evidence of a short time memory. Furthermore, the existence of a long term memory of at least 24 hours as shown m chapter 4 makes the existence of a long term memory plausible. The fact that fetuses habituate more rapidly 24 hours after an initial test is unlikely to caused by fetal fatigue.

Our data demonstrate a gradual decline in the habituation rate with advanced CA and with an increased number of experiments. One might say that this decline in habituation rate with advancing CA is influenced by fetal brain development. Studies on fetal habituation at various CA show conflicting conclusions concerning this relationship. Some studies reveal that older fetuses habituate more rapidly than their younger counterparts '5β,67 while others '6i could not find a relationship between GA and the habituation rate.There is no obvious reason why these studies have different conclusions. It should be noted that in these studies fetuses were tested only once m a cross-sectional design.

Our observation that the habituation rate declined with advancing GA appears to be unrelated to maturation of the fetal brain. Term fetuses, which were tested only once, did not habituate more rapidly than their younger counterparts in the repeatedly tested group, e.g. between 28 and 30 weeks GA We recognise that fetuses in the repeated exposure group with a GA between 28-30 weeks were tested for the second time, which could have resulted in recognition of the stimuli in this experiment. But because 60% of the fetuses did not respond to the stimuli m the first experiment, we assume that this did not influence the habituation rate. Unfortunately, it is impossible to compare fetuses of the single exposure group with the fetuses between 26-28 weeks CA of the repeated exposure group because of the high incidence of non-responders in this period.

63 Furthermore, there is a striking difference between the habituation rate of term fetuses of the repeated exposure group and of the single exposure group These results indicate that the 6 previous experiments in the repeatedly tested fetuses resulted m fetal learning and long term memorisation ,8s of the stimuli The fact that 2 of the fetuses from the repeated exposure group tested for the 7'h time did not respond at all supports the idea that they recognised the stimuli immediately It is unusual for normal term fetuses to fail to respond to this type of VAS, as such behaviour is only associated with fetuses that have severe chromosomal or structural abnormalities,36,87 We therefore conclude that fetal brain maturation did not influence the habituation rate but that repetition of the habituation experi­ ments every 2 weeks resulted m fetal learning and long term memorisation of the stimuli with subsequent more rapid habituation performance with advancing number of experiments

The marked, but not statistically significant decline m habituation rate between the consecutive CA groups 30-32 and 32-34 weeks GA suggest that fetal long term memory might originate in a period earlier than the term period Because a short term memory is already present at 28 weeks GA, it seems plausible that long term memory is also starting to develop at this age Unfortunately, the use of fetal habituation as an approach to the assessment of fetal memory before 28 weeks GA is not feasible as a result of the limitations of fetal VAS before this period

These results suggest that normal maturation of the fetal CNS underlies the development of fetal memory It is known that the period around 32 weeks GAcan be considered an important period in the functional development of the fetal CNS 2002°2 A considerable change m the incidence of several distinct movement patterns was found between 28-31 weeks GA 200, and maturation of the fetal response to stimulation begins at about 26 weeks GA, increases steadily over a 6 week period, and reaches maturity at about 32 weeks GA '°2 Others found that the rate of development of FHR variability, fetal activity, state concordance and FHR responses to VAS slows significantly between 28 and 32 weeks GA "" They propose that changes m neural organisation underlie these observations and state that although development continues after 32 weeks GA, this gestational period marks a period of neurologic maturity Furthermore, macroscopic evaluation of fetal brain maturation by magnetic resonance imaging revealed that from 30 weeks GA the cortex begins to undergo infolding, and by 36 weeks CA the cortex is extensively and compactly folded M3 It is possible that this observed cortex development is linked to the development of fetal behaviour and fetal memory

The gestational period near term would appear to be an important period in the development of fetal long term memory This period is also of importance for the development of feta I behaviour because fetal behavioural states are fully developed only from 36-38 weeks GA onward 45 One could suggest that the presence of fetal behavioural states at this gestational period accounts for the significant decline m habituation rate We studied the influence of fetal quiescence and activity, based on

64 the FHR patterns prior to testing, on the habituation rate in the 37 singly tested term fetuses (chapter 3),85. From this study we concluded that there is no influence of the fetal state on habituation performance m term fetuses.

The fact that the human fetus is able to memorise and learn is not surprising, as similar abilities have been found m other mammals 'e8, birds ,β9, fish '90, and invertebrates "',. Memory, a prerequisite for fetal learning, is essential for normal functioning and it is not surprising that such an important psychological function starts to develop before birth m some form. Other functions, like breathing, suckling, swallowing, and moving are also exercised before birth. Prenatal memory may be important for the development of attachment and maternal recognition. There is much evidence that the fetus learns the speech characteristics of its mother prenatally and prefers its mother's voice to other female voices after birth. Newborns '7C presented with a choice between their mother's voice as it sounded in utero and as it sounded outside the uterus, showed a preference for their mother's voice as it sounded in utero.This suggests that their preference was acquired before birth. This and other studies have confirmed that the fetus can memorise its mother's voice before birth ',,,,J. Infants are also able to memorise sounds other than voices, for example a tune of a certain television program, presented to them prenatally '". It should be noted that this fetal memory is presumably what neuro- scientist refer to as implicit memory Implicit memory is typically thought of as unconscious memory and includes skilled motor learning, conditioning and priming !0''. Therefore, fetuses and infants are seemingly capable of forming that cannot be recalled years later.

There is convincing evidence that leads us to conclude that a short term memory of at least 10 minutes is already present m fetuses from 28-30 weeks CA and a long term memory of at least 2 weeks is clearly present in the term period. The results further suggest that fetal long term memory already develops at around 32 weeks GA. However, in view of the limited number of subjects studied, further research to explore these findings and to explore the development of fetal memory before the term period by testing a large number of fetuses at different ages and with different experiment intervals is indispensable

65

™APTER '6

TION

Ol 2007;97;ï ABSTRACT

Objective: Testing of fetal habituation to repeated vibroacoustic stimulation (VAS) might give additional information concerning the fetal condition and central nervous system (CNS) functioning in postterm pregnancies. This study is designed to investigate whether healthy postterm fetuses are able to habituate and if so, if the habituation pattern of postterm fetuses differs from that of term fetuses.

Study design: Twenty women with an uncomplicated pregnancy beyond 41 weeks gestational age (GA) participated, and 37 women with a GA between 37 and 40 weeks served as controls. The stimulus was repeatedly applied to the maternal abdomen above the fetal legs for a period of 1 s every 30 s. A fetal trunk movement within 1 s of stimulus application was considered a positive response. Lack of response to 4 consecutive stimuli indicated habituation. The habituation rate is defined as the number of stimuli applied before the fetus stopped responding. Data were compared using the Mann Whitney U test.

Results: Of the 18 postterm fetuses in which the presence or absence of habituation could be established, 14 habituated and 4 persisted m responding Twenty-six of the 32 term fetuses, m which the presence or absence of habituation could be established, habituated and 6 persisted in responding.The habituation rate varied widely in postterm (median of 8 stimuli, quartile range 6-18) as well as m term fetuses (median of 9 5 stimuli, quartile range 6-15). There was no difference in median habituation rate between postterm and term fetuses.

Conclusion: The ongoing maturation of the CNS in the last weeks of pregnancy appears not to be reflected m the fetal habituation pattern. Furthermore, the mter- fetal variability m habituation of healthy postterm fetuses is such that testing of habituation appears not to be suitable for the identification of the fetus at risk for an adverse neonatal outcome

68 INTRODUCTION

Prolongation of pregnancy beyond term is accompanied by an increase in perinatal mortality and morbidity '°im'' and adequate fetal monitoring is indicated. Assess­ ment of the biophysical profile seems to be the best way to evaluate fetal wellbemg in a postterm pregnancy because a variety of fetal parameters (fetal heart rate (FHR), amount of amniotic fluid, fetal breathing, body movements and fetal tone) are observed 2o8. The primary disadvantage of scoring the biophysical profile is the risk for a false-positive test with subsequent needless interventions as well the risk for a false-negative test result that can result in fetal morbidity or mortality Another disadvantage is the time required to perform the test and the need for an experienced sonographer Current research on the use of fetal testing has focused on the refinement of testing protocols and the application of new methods to improve the positive predictive value of fetal testing m postterm pregnancies Ι09. Testing of fetal habituation to repeated vibroacoustic stimulation (VAS) might give additional information concerning the fetal condition and functioning of the fetal central nervous system (CNS).

Habituation is the decrease, and eventual cessation, of response that follows repeated application of the same stimulus. Habituation can be seen as a basic form of learning that requires an intact functioning of the CNS '''.Studies m persons with various disorders of the CNS reveal that the habituation pattern of these persons is different from that of persons without these disorders '^ '35'î7. Pediatricians use this habituation phenomenon to assess newborns for various pathological conditions of the CNS'47

Several studies on fetal habituation have been published ""'s8'66'6' Although the methodologies used varied widely, these studies all reveal that normal fetuses are able to habituate to repeated external stimulation. Studies m compromised or chromosomally abnormal fetuses demonstrate that the habituation pattern of these fetuses is different from that of normal fetuses ,o6'6s Testing the fetal ability to habituate to repeated stimulation could also be helpful m identifying the fetus at risk for hypoxia. In fetal sheep as well as m human fetuses,fetal hypoxia resulted in either more rapid habituation or a failure to habituate compared to normoxic controls's;'5S

It is of importance to know whether habituation could be useful to identify the fetus at risk in the postterm period Before such a study can be undertaken, it is essential to gam knowledge about the habituation pattern of normal postterm fetuses This study was therefore designed to investigate whether healthy postterm fetuses are able to habituate to repeated stimulation and if so, if the habituation pattern of postterm fetuses differs from that of term fetuses.

69 SUBJECTS AND METHOD

Subjects

Twenty healthy postterm pregnant women (study group) were recruited at the University Medical Center Nijmegen in the Netherlands. All had an uncomplicated pregnancy and were sent to the hospital by their primary health care for fetal surveillance because of prolonged pregnancy Thirty-seven healthy pregnant women (control group) were recruited from a low-risk obstetric population for a study of fetal habituation m normal term fetuses Both studies were approved by the hospitals ethical committee and all participants gave their written informed consent Inclusion criteria were (i) gestational age (CA) beyond 41 completed weeks for the postterm study group and GA between 37 and 40 completed weeks for the term control group (pregnancies were dated using the last menstrual period or by ultrasound when dates were uncertain), (2) no maternal medical or obstetric com­ plications, (3) single fetus without apparent structural anomalies and with an expected birth weight above the io,h centile according to population-based tables adjusted for maternal parity and fetal sex, (4) adequate amniotic fluid volume as assessed by ultrasound, (5) no maternal use of alcohol, drugs or medication other than vitamins and / or iron

Method

All studies were performed by the same examiner m a darkened, quiet room between 4pm and 7pm The women were placed in a semi-recumbent position The fetal trunk was visualised in a parasagittal plane by a real-time ultrasound scanner (Hitachi model EUB-525, Tokyo, Japan) The FHR was recorded continuously with a cardiotocograph (Somcaid FM7, Oxford, UK) at a paper speed of 3 cm per minute The stimuli were produced by a fetal vibroacoustic stimulator (Corometrics model 146, Wallmgford, Conn , audible sound 20 to 9000 Hz, vibrations between 67 and 83 Hz, sound level 74 dB at 1 m m air) A stimulus of 1 s duration was repeatedly applied to the maternal abdomen above the fetal legs every 30 s Stimuli were applied during fetal quiescence A movement of the fetal trunk within 1 s of application of the stimulus was considered to be a positive response A response decrement was noted as a tendency to change from a more intense to a less intense response pattern with successive trials A lack of response to 4 consecutive stimuli was taken to indicate habituation We allowed a maximum of 24 stimuli in each habituation procedure However.a minimum of 4additional stimuli would be necessary to show habituation if a fetus was still responding to the 21'' stimulus We therefore stopped stimulating if a fetus persisted in responding to the 21*' stimulus The habituation rate was defined as the number of stimuli applied before a fetus stopped responding The procedure was repeated 10 minutes after the first habituation procedure to investigate whether the fetus habituated more rapidly to the second series of stimuli

70 The outcome of each pregnancy was examined for birth weight, GA at delivery, Apgar scores, umbilical artery blood pH, and the presence of neonatal complications Infants were followed up at the age of 3 months by telephone inter views with one of the parents and at the age of lyear by means of a questionnaire

Data analysis

Habituation was considered to be established if the fetus responded regularly to the repeated stimuli The data from experiments in which the fetuses showed alter­ nate responses and non responses to repeated stimulation were excluded from the analysis because the habituation pattern was deemed to be unmterpretable Data were compared using the Mann Whitney U test and the Wilcoxon signed ranks matched-pairs test A two-tailed ρ value of < 005 was considered statistically significant

RESULTS

All postterm and term fetuses responded with a movement of the fetal trunk to at least the first stimulus In the postterm group 2 (10%) fetuses and in the term group 5 03 5%) fetuses were excluded from analysis because irregular movement responses to the stimuli prevented determination of the habituation rate These fetuses did not differ from the other fetuses in their respective groups m gender, presentation, birth weight, maternal weight, amniotic fluid volume, GA at testing and delivery, and neonatal outcome Characteristics of the remaining 18 postterm and 32 term fetuses are shown m table 6.1

Table 6.1

Characteristics of the postterm and term group.

Postterm group Term group (n = 18) (n = 32)

4 Gestational age at time of testing (weeks) 41 " (41-42) 38 '"(By- 39 "T Gestational age at delivery (weeks) 42 (41 "'- 42 "^ 40 "7 (38 s"- 42 '") Birth weight (g) 3527 (3020-4595) 3385 (2660-4070) Sex (male/female,n) 11/7 14/18

Data are given in medians and ranges

From the 18 postterm fetuses m which habituation could be determined, 14 (78%) habituated in the first test Eleven of the 14 (79%) fetuses habituated more rapidly or did not respond at all m the second test, and 3 (21%) of them habituated after 1 stimulus both in the first and in the second test Four of the 18 (22%) fetuses did not habituate within 21 stimuli in the first test and persisted m responding, but all

71 showed response decrement. Although they persisted in responding in the first test, they all habituated in the second test (after 7,1,1 and 1 stimulus, respectively). The median habituation rate decreased significantly from 8 stimuli to ι stimulus in the second test (p = 0.001), meaning that fetuses habituated more rapidly in the second test (figure 6.1).

Figure 6.1 Habituation rate of postteim and term fetuses in test 1 and test 2.

25

—. 20 I +-a>) -3^ 2 E Γ +-» ο ΙΛ 15 ™ ο 3 υ 4-* IO E Τ 3 e

Test 1 Test 2 Testi Test2

Postterm group Term group

Data are combined in a box and lühisfeer plot (median, interquartile ranges, maximum and minimum). A habituation rate of 23 represents a persistent response, a rate of 0 a non- response ' ρ < 0.001 " ρ = 0.001

From the 32 term fetuses in which the presence or absence of habituation could be established, 26 (81%) habituated m the first test Twenty (77%) of them habituated more rapidly or did not respond at all in the second test In the second test only 2 fetuses habituated slower, and m 4 habituation could not be determined due to irregular responding Of the 32 fetuses, 6 (19%) fetuses did not habituate within 21 stimuli in the first test and persisted in responding (4 even showed no response decrement), 3 of them also persisted m responding in the second test and 3 habituated (after 15, 13 and 3 stimuli, respectively). The median habituation rate decreased significantly from 9 5 to 2 stimuli in the second test (p = o.ooi)(figure6.i)

There was no difference in median habituation rate in the first test as well as in the second test between postterm and term fetuses (figure 6.2).

In the group of 18 postterm fetuses in which habituation could be determined, 4 fetuses were born macrosomic with a birth weight above the gs'" centile for CA Three of these fetuses habituated (after 18, η and 4 stimuli, respectively), and 1

72 Figure 6.2 Habituation rate of test 1 and test 2 in postterm and term fetuses.

25 -,

= 20 _ •

5 -

Postterm Term Postterm Term

Testi Test 2

: not signi/ïcant

persisted in responding in the first test Fifteen women delivered vaginally without complications, 7 at home and 8 at the hospital In 3 women labour was induced because of GA beyond 42 weeks, they all delivered vaginally.Three women delivered by cesarean section, 2 because of intrapartum fetal distress and 1 due to a cephalopelvic disproportion. All infants did well after birth, with birth weights above the 10"' centile for GA, a 5-minute Apgar score > 8 and an umbilical artery pH > 7.10. Follow up after 3 months and after 1 year revealed that all infants developed normally.

In the group of 32 term fetuses in which habituation could be established, 7 (22%) were born by cesarean section for different reasons, but not for fetal distress. All infants did well after birth, with birth weights above the 10"' centile for GA, a 5-min Apgar score > 8 and an umbilical artery pH > 7.10 Follow up at the age of 3 months (all infants) andati year of age (19 infants) revealed no serious abnormalities in any infant.

DISCUSSION

In this study, we investigated fetal habituation to repeated VAS in healthy postterm and term fetuses. The type of stimulus we used induced an immediate fetal move­ ment response, to at least the first stimulus, regardless of the fetal state By using this stimulator and observing the immediate movement response to repeated stimulation, a clinician can easily distinguish between a response and a non-

73 response and so determine fetal habituation. Ten percent of the postterm and 13.5% of the term fetuses responded irregularly to the repeated stimuli. These fetuses exhibited alternate responses and non-responses, which made determination of habituation impossible. Because the majority of the postterm and term fetuses responded consistently to each stimulus, we consider the habituation pattern of fetuses with irregular responses to be non-interpretable.

To confirm that the observed response decrement was the result of habituation rather than motor fatigue or sensory adaptation, we repeated the whole procedure withimo minutes of the first test Repeated stimulation may physically exhaust the fetus so it is unable to respond or it may tire the auditory or tactile receptor which results in receptor adaptation. In real habituation the original stimulus, when presented again, initially elicits a response but this response decreases more rapidly than when first presented ,62 In our study the habituation rate decreased significantly in the second test in the postterm as well as in the term group. In fact, 5 (28%) postterm fetuses and 8 (25%) term fetuses did not respond at all to the presentation of the original stimulus within io minutes after they habituated to this stimulus This can be explained by the fact that fetuses recognise the stimuli and habituate more rapidly to the stimuli when presented again '8o

Three postterm fetuses habituated within ι stimulus in the first as well as in the second test. In these fetuses an immediate startle was clearly observed within 1 s after the stimulus accompanied by an acceleration of the FHR of at least 15 bpm following the first stimulus. In 2 fetuses FHR accelerations following consecutive stimuli were seen, whereas no movement response could be observed to these stimuli. Although FHR accelerations were observed to consecutive stimuli, we defined habituation only according to the movement response. But it is questionable whether this habituation after 1 stimulus is real habituation because there is no response decrement and no faster habituation to presentation of the original stimulus after 10 minutes.

One of the factors that may determine the habituation rate is the degree of maturation of the fetal CNS Groome et al. '67 examined the developmental trend m habituation of the fetal startle response to repeated VAS in normal fetuses between 28 and 40 weeks of . They concluded that older fetuses habituated more rapidly than younger fetuses, with the greatest change in habituation rate between 28 and 32 weeks and 32 and 36 weeks The ongoing maturation of the CNS in the last weeks of pregnancy, which is for example reflected m the change m behavioural state organisation 46, appears not be reflected m the fetal habituation pattern We were unable to establish a faster habituation pattern in postterm fetuses compared with term fetuses

Leader et al. 'o6used an electric toothbrush to study habituation in 112 compromised fetuses of different CA (small for GA, mecomum-stained amniotic fluid or decreased growth velocity of the fetal bipanetal diameter). They found that fetuses in the

74 compromised group habituated more rapidly (< 9 stimuli) or more slowly (> 50 stimuli), compared with normal fetuses Only 8 of the compromised fetuses, that showed a different habituation pattern, had a 5-minute Apgar score less than 6 Unfortunately, neonatal outcome, except for the Apgar scores, is not reported in their study

Leader and Baillie '5i also demonstrated that reducing the amount of oxygen inspired by the mother was associated with an impaired habituation by the fetus Thirteen of 18 fetuses of mothers that inspired air with 12% oxygen failed to habituate within 51 stimuli

Although we followed another protocol with a different stimulator than Leader et al used, we cannot conclude that postterm fetuses that failed to habituate or habituated within 1 stimulus showed fetal distress or adverse neonatal outcome In our study in postterm fetuses, the habituation rate varied greatly Four fetuses persisted in responding and 3 habituated within 1 stimulus Of the 4 fetuses that persisted in responding m the first test, 2 were delivered vaginally at home, 1 was born after induction of labour and 1 was delivered by cesarean section because of intrapartum fetal compromise Of the 3 fetuses that habituated within 1 stimulus, 2 were born vaginally at the hospital and ι by cesarean section because of cephalopelvic disproportion Although 4 postterm fetuses persisted in responding in the first test and 3 fetuses habituated very rapidly, all these fetuses did well after birth and revealed no abnormalities at the age of 3 months Apparently, there are normal postterm fetuses that fail to habituate or that habituate within 1 stimulus There are even normal postterm fetuses m which habituation cannot be determined due to irregular responses

From this study we conclude that, although habituation can be clearly observed m the majority of postterm fetuses, the interfetal variability m habituation perfor mance is such that testing of habituation is not suitable for the identification of the fetus at risk for an adverse neonatal outcome In a study m a homogenous group of healthy, full-term, minimally stressed, white neonates, normative data of the habituation rate were determined "" Also neonates exhibit a wide variation in the habituation performance We attempted to find normative data of fetal habituation m postterm as well as in term fetuses and also found a marked inter fetal variability in habituation performance This can be influenced by many factors, although we used a homogeneous group of white, healthy, and minimally stressed fetuses This is presumably due to the overall individual difference in fetal behaviour, like there is an individual difference in neonatal behaviour This means that fetal habituation appears not to be suitable as a technique to distinguish normal from abnormal fetuses, in uncomplicated postterm and term pregnancies, due to this variability in habituation performance Studies to investigate whether or not fetal habituation can be a tool in the evaluation of the fetal condition and functioning of the CNS in compromised fetuses are needed

75

ABSTRACT

Objective: Testing of fetal habituation to repeated vibroacoustic stimulation (VAS) may give additional information concerning the fetal condition and central nervous system functioning in fetuses that are growth restricted This study is designed to investigate whether fetuses that are growth retarded (IUCR) are able to habituate and if so, if the habituation pattern of IUGR fetuses differs from that of term fetuses.

Study design: Twelve women with a pregnancy beyond 30 weeks gestational age (GA) with a suspected IUGR were tested at 10 minute and two-weekly intervals, and 37 healthy women with a GA between 37 and 40 weeks served as controls.

Results: In 3 out of 13 (23%) experiments performed m the IUGR fetuses irregular responding prevented interpretation of the test compared to 13% of the tests m normal fetuses. The habituation rate varied greatly in IUGR as well as in normal fetuses, although in the majority of normal fetuses a more rapid habituation pattern was observed 10 minutes after the initial test. One IUGR fetus failed to respond to VAS and this fetus showed a neurodevelopmental impairment at the age of 3 months.

Conclusion: The high rate of unmterpretable test as well as the variability in habituation rate in the remaining tests in this study hampers the assessment of habituation as a tool to identify the compromised fetus. The observation of a fetal non-response to repeated stimulation might, however, be of value in predicting an impairment in the neurological development m IUGR fetuses.

78 INTRODUCTION

Growth retarded fetuses and neonates that have normal outcomes have adapted successfully to the chronic stress that affected their growth in utero The point at which adaptive changes m growth retarded (IUGR) fetuses become abnormal is not clear, but the result is decompensation and hypoxia with subsequently the risk of neurodevelopmental delay and disability m infancy 2'0'"4. Elective delivery remains the principal management option, which emphasises the need for better screening techniques for the timely detection of the compromised IUGR fetus.

Traditional non-invasive fetal monitoring procedures for the surveillance of IUGR fetuses include the biophysical profile and Doppler velocimetry. Abnormal blood flow velocity waveform patterns, reduction in amniotic fluid volume, and abnormal FHR patterns are indicative of impaired utero-placental perfusion i'52,7. However, despite the "intensive obstetrics" of the past 30 years, with increasing attention directed to prenatal care, reduction of birth trauma, and greater use of cesarean section for high-risk deliveries, the incidence of neurodevelopmental disorders remains unchanged :67. Especially the risk of for example cerebral palsy m connection with IUGR is known. This risk has has been analysed in a case-control study. The case series comprised 519 children with cerebral palsy and the control series 445 children born during the same years m the same region. The risk of cerebral palsy in small-for-gestational-age infants was significantly increased m term and moderately preterm infants 2,8. Intrapartum events appear to play only a small part in the overall incidence of this disorder, and newer methods must be introduced to determine the actual prenatal events that lead to neuro­ developmental disability.

Current research on the use of fetal testing has focused on the refinement of testing protocols and the application of new methods to improve the positive predictive value of fetal testing in IUGR pregnancies. Therefore, it would bea great improvement if an intrauterine neurological test could be developed which would give additional information concerning the fetal condition and functioning of the fetal central nervous system (CNS) in high-risk pregnancies. Testing fetal habituation to repeated vibroacoustic stimulation (VAS) seems to be a promising tool in the fetal neurological assessment.

Habituation is the decrease, and eventual cessation, of response that follows repeated application of the same stimulus. Habituation can be seen as a basic form of learning that requires an intact functioning of the CNS u'. If habituation represents the functioning of the fetal CNS, then conditions affecting the CNS should affect the habituation performance. Several studies on fetal habituation have been published ""'s8'66'6?. Although the methodologies used varied widely, these studies all reveal that normal fetuses are able to habituate to repeated external stimulation. Studies in chromosomally or compromised fetuses demonstrate that the habituation pattern of these fetuses is different from that of

79 normal fetuses 'o6'63 In fetal sheep as well as in human fetuses, fetal hypoxia resulted m either more rapid habituation or a failure to habituate compared to normoxic controls '5!'55

It is of great importance to know whether habituation could be useful to identify the compromised IUGR fetus at risk for fetal hypoxia with subsequent neurologic injury This study was designed to investigate whether IUGR fetuses are able to habituate to repeated stimulation and if so, if the habituation pattern of IUGR fetuses differs from that of normal fetuses

SUBJECTS AND METHOD

Subjects

Twelve growth retarded (IUGR) fetuses between 30 and 40 weeks gestational age (GA) were studied at two-weekly intervals (study group) IUGR was confirmed when the birth weight was less than the 5th centile for the GA according to population- based tables adjusted for maternal parity and fetal sex Inclusion criteria were (1) GA determined using the last menstrual period or by ultrasound when dates were uncertain, (2) single fetus without apparent structural anomalies and with an abdominal circumference below the 5th centile for GA (both symmetric and asymmetric forms of IUGR were included), (3) no maternal use of alcohol, drugs or medication other than antihypertensive drugs, vitamins and /or iron

Thirty-seven low-risk pregnant women (control group) were studied once between 37 and 40 weeks GA Inclusion criteria were (1) GA determined using the last menstrual period or by ultrasound when dates were uncertain, (2) the absence of maternal medical or obstetric complications, (3) single fetus without apparent structural anomalies and with an expected birth weight above the 10th centile according to population-based tables adjusted for maternal parity and fetal sex, (4) normal amniotic fluid volume as assessed by ultrasound, (5) no maternal use of alcohol, drugs or medication other than vitamins and / or iron

The study was approved by the hospital's ethical committee and all participants gave their written informed consent

Method

All habituation experiments were done by the same examiner m the same room between ipm and 7pm The women were placed m a semi-recumbent position The fetal trunk was visualised m a parasagittal plane by real-time ultrasound (Hitachi model EUB-525, Tokyo, Japan) The stimuli were produced by a fetal vibroacoustic stimulator (Corometncs model 146, Wallmgford, Conn , audible sound 20 to 9000 Hz, vibrations between 67 and 83 Hz, sound level 74 dB at ι m m air) Stimuli of 1 s duration were repeatedly applied to the maternal abdomen above the fetal legs

80 every 30 s. Stimuli were applied during fetal quiescence A movement of the fetal trunk within 1 s of application of the stimulus was considered to be a positive response A response decrement was noted as a tendency to change from a more intense to a less intense response pattern with successive trials. A lack of response to 4 consecutive stimuli was taken to indicate habituation. We allowed a maximum of 24 stimuli m each habituation procedure. However, a minimum of 4 additional stimuli would be necessary to show habituation if a fetus was still responding to the 21s1 stimulus. We therefore stopped stimulating if a fetus persisted in responding to the 21st stimulus.The habituation rate was defined as the number of stimuli applied before a fetus stopped responding The procedure was repeated 10 minutes after the first habituation test to investigate whether the fetus habituated more rapidly to the second series of stimuli.

The outcome of each pregnancy was examined for birth weight, GA at delivery, Apgar scores, umbilical artery blood pH, and the presence of neonatal complications. Infants of the control group were followed up at the age of 3 months by telephone interviews with one of the parents.

The neurological condition of all, but one, infant from the study group was examined at the corrected age of 3 months by the assessment of the quality of the general movements according to Hadders-Algra et al. ÌO"9. Definitely abnormal general movements are associated with a high-risk for the development of cerebral palsy, whereas mildly abnormal general movements are associated with the development of minor neurological dysfunction, attention-deficit-hyperactivity disorder, and aggressive behaviourJ0.

Data analysis

Habituation was considered to be established if the fetus responded regularly to the repeated stimuli. The data from experiments in which the fetuses showed alternate responses and non-responses to repeated stimulation were excluded from the analysis because the habituation pattern was deemed to be unmterpretable.

RESULTS

Growth retarded fetuses

Three fetuses from the study group were excluded from subsequent analysis. Two were born at term with normal birth weights between the iolh and 90th centile for GA, and one appeared to suffer from trisomy 18 (chapter 8a) The other 9 fetuses were born with a birth weight less than the 5,h centile for GA. Characteristics of the study group are presented m table 7.1. Five fetuses were born vaginally, 2 spontaneously and 3 after induction of labour, with 2 because of the IUGR and one because of IUGR and maternal pregnancy-induced hypertension (fetus no. 1, 3,2, 8,

81 Table 7.1 Characteristics of the study group.

Fetus Birth Centile Mode of CA at delivery Apgar score Arterial pH no. weight (g) delivery (weeks) (at 1 and 5 min.I ]

2515 <2.3 vaginally 40°" 8/10 7·3ΐ 1930 <2.3 vaginally 38'" 9/9 no value 1217 <5 vaginally 33°" 9/10 7-27 2620 <5 CS 39 '" 9/9 731 2000 <5 vaginally 36'" 9/10 725 6 990 <2.3 CS 35°" 10 /io no value 7 1415 <2.3 CS 37 "; 9/10 714 8 2110 <2.3 vaginally 38'" 10 /IO 7.22 9 1033 <2.3 CS 35'" 8/9 723

CS = cesarean section

5, respectively) Four fetuses were born by cesarean section, 3 because of an abnormal FHR pattern before labour, and one because of a footling breech presentation with a prolonged non-reassuring (fetus no. 6, 7, 9, 4, respectively).

A total of 13 experiments were done (each experiment consisted of 2 habituation tests). Six fetuses were studied once, 2 fetuses 2 times and 1 fetus 3 times. Habituation rates at different CA for each fetus are summarized in table 7.2. None of the IUGR fetuses showed any evidence of fetal distress at the time of testing. In the first experiment, 6 fetuses showed regular responses with 5 fetuses showing

Table 7.2 Habituation rates at different GA for each IUGR fetus (n = 9).

Fetus no. Habituation GA Habituation GA Habituation GA rate exp. 1 exp. 1 rate exp. 2 exp. 2 rate exp. 3 exp. 3

0 1 9 36 " 12 38°" 20 40°" 2 17 354'7 PR 37'" 3 2 32 5" 4 IR 38 "" 5 IR 36"' 6 1 32on 7 PR 33'" 5 34 "' 8 NR 36" 9 17 35 "7

GA = gestational age (weeks); exp. expenment; NR = non-response; IR = irregular response, PR = persistent response

82 habituation (after ι, 2, 9,17,17 stimuli, respectively) and 1 fetus that persisted m responding. Two fetuses showed alternate responses which prevented interpretation of the test and one fetus failed to respond.

The second experiment after 2 weeks of the first experiment revealed that 2 fetuses habituated (after 5 and 12 stimuli, respectively) and one fetus persisted in responding. In only one fetus 3 experiments were performed In all 3 experiments the fetus habituated (after 9, 12 and 21 respectively). This fetus showed a more slowly habituation rate with advancing GA. Three out of 13 (23%) initial tests and 6 out of 13 (46%) second tests were uninterpretable because of irregular responding

At the corrected age of 3 months (between 50 and 54 weeks GA), 8 infants (5 males and 3 females) were neurologically investigated by assessment of the quality of the general movements Due to transport problems one girl was not seen m the hospital, she was only followed up at 3 months after birth by telephone interviews with one of the parents. Seven infants showed normal general movements at this assessment with normal fluency, complexity and variation of these movements. These infants had developed normally so far. Only one boy showed mildly abnormal general movements with a normal variation of movemenst but with so-called cramped-synchronized movements These mildly abnormal movements are associated with the development of minor neurological dysfunction, attention- deficit-hyperactivity disorder, and aggressive behaviour i0. Because of jittery and hypertomcity the infant required physiotherapy twice a week. This infant failed to respond to VAS over the fetal legs when tested at 36 5" weeks GA, even when the observation period was prolonged to 5 s after stimulation. Only stimulating over the fetal head resulted m a movement response. By testing again after 10 minutes the fetus also showed movement responses only by stimulating over the fetal head. FHR accelerations were observed to some stimulus applications.

Normal fetuses

The data of 5 fetuses from the control group were excluded because irregular responses prevented interpretation of the tests. These fetuses did not differ from the other 32 normal fetuses m gender, presentation, birth weight, maternal abdominal wall thickness, amniotic fluid volume, GA at testing and delivery, and neonatal outcome. In the remaining 32 fetuses m which regular responses could be observed, the median GA at time of testing was 38;'7 (range 370/7- 39 5'7) weeks, the median GA at delivery 40i/7 (range 385'7- 423'7) weeks, the median birth weight 3385 (range 2660-4070) g. Seven mothers were delivered by cesarean section for different reasons, but not for fetal distress All infants, 14 males and 18 females, were in good health at birth, with birth weights above the 10th centile for GA, a 5-minute Apgar score > 8 and an umbilical artery pH > 7.10. Follow up after 3 months and 1 year revealed no serious abnormalities in any infant.

83 Of the 32 normal fetuses, 26 habituated m the first test Twenty of them habituated more rapidly or did not respond at all in the second test In the second test, only 2 fetuses habituated more slowly and m 4 fetuses habituation could not be determined as they alternated between response and non response Of the 32 fetuses, 6 failed to habituate within 21 stimuli (4 even showed no response decrement) in the first test, 3 of them also persisted in responding m the second test and 3 habituated (after 15, 13 and 3 stimuli, respectively) The median habituation rate decreased significantly in the second test (9 5 vs 2, ρ = o ooi, Wilcoxon matched-pairs signed rank test), meaning that fetuses habituated more rapidly during the second test

DISCUSSION

In this study, we investigated fetal habituation to repeated VAS m lUGR fetuses and normal term fetuses Thirteen percent of experiments of the normal fetuses and 23% of the experiments m the lUGR group were unmterpretable because of irregular fetal responding The high incidence of unmterpretable tests in the IUCR group is mainly a result of difficulties with visualizing the fetal trunk by ultrasound The decreased amount of amniotic fluid and the decreased fetal size m the study group influenced the fetal ultrasound imaging Therefore, the results of this study m lUGR fetuses are difficult to interpret

However, 6 out of 9 lUGR fetuses showed regular responses m the first experiment with 5 fetuses that habituated and one fetus that showed persistent responses The variability m habituation is marked with one fetus that needed ι stimulus for habituation and 2 fetuses that needed 17 stimuli This variability m habituation rate can also be seen in the control group with 6 fetuses that needed 5 or less stimuli for habituation and 6 fetuses that failed to habituate So, there are apparently normal fetuses that fail to habituate to repeated VAS or that habituate more rapidly than other fetuses There are even apparently normal fetuses m which it is impossible to determine habituation due to irregular responses

Our results do not corroborate the results of Leader et al 'o6 who used an electric toothbrush to study habituation m 112 compromised fetuses of different GA (small for GA, meconium-stamed amniotic fluid or decreased growth velocity of the fetal bipanetal diameter) They found that fetuses in the compromised group habituated more rapidly (< 9 stimuli) or more slowly (> 50 stimuli), compared with normal fetuses Only 8 of the compromised fetuses, that showed a different habituation pattern, had a 5-minute Apgar score less than 6 Unfortunately, neonatal outcome, except for the Apgar scores, is not reported in their study Although we followed another protocol with a different stimulator than Leader et al used, we cannot conclude that lUGR fetuses as well as normal fetuses that failed to habituate or habituated within 1 stimulus showed fetal distress or neurologic sequelae after birth

84 Leader and Baillie "'' demonstrated that reducing the amount of oxygen inspired by the mother was associated with an impaired habituation by the human fetus Thirteen of i8 fetuses of mothers that inspired air with 12% oxygen failed to habituate within 51 stimuli In our group of IUCR fetuses we could not determine an impaired habituation pattern compared to normal fetuses It should be noted that the presence of hypoxia m the IUCR fetuses seemed to be absent because no signs of fetal hypoxia, according to the FHR pattern were present during testing

To confirm that the observed response decrement was the result of habituation rather than motor fatigue or sensory adaptation, we repeated the whole procedure within 10 minutes of the first test Repeated stimulation may physically exhaust the fetus so it is unable to respond or it may tire the auditory or tactile receptor which results in receptor adaptation In habituation the original stimulus, when presented again, initially elicits a response but this response decreases more rapidly than when first presented '62 The habituation rate decreased significantly in the second test after 10 minutes in the control group Even 8 (25%) fetuses did not respond at all to the presentation of the original stimulus within 10 minutes after they habituated to this stimulus This can be explained by the fact that fetuses recognise the stimuli and habituate more rapidly to the stimuli when presented again '8o

In the study group in 6 out of 13 experiments the first as the second test were interprétable with 4 experiments that resulted m a more rapidly habituation performance in the second test and 2 that resulted in a more slowly habituation pattern Unfortunately, this small number of experiments prevents interpretation of the results with concern to the habituation pattern of IUCR fetuses at 2 time points In view of the limited data of the study group, further research to explore whether IUCR fetuses are able to memorise stimuli is needed By testing a large number of IUCR fetuses at different ages the clinical value of habituation to repeated VAS can be determined

All but one infants developed normally at the age of 3 months, although 5 infants were born with a birth weight below the 2 3"i centile and 3 below the 5"' centile Only one infant showed mildly abnormal general movements at examination with jittery and hypertonicity This infant failed to respond m utero to repeated stimulation over the fetal legs, only stimulating over the fetal head resulted m a movement response Up till now we only have seen this non-response m a fetus with trisomy 18 and m a fetus with a severe brain anomaly (chapters 8a and 8b) ,86'Β7 It should be noted that these abnormal fetuses also failed to respond when stimu­ lated over the head Furthermore, these abnormal fetuses also failed to respond with a FHR acceleration to repeated VAS, while the IUCR fetus showed a FHR acceleration within 1 s after some of the stimulus applications These results suggest that a failure to respond with a movement to VAS might be related with neurodevelopmental delay in infancy Further studies in compromised fetuses with a subsequent impaired neurological development are necessary to gam insight into the spectrum of possible responses to VAS

85 In conclusion, the high rate of uninterpretable test as well as the variability in habituation rate in the remaining tests in this study hampers the assessment of habituation as a tool to identify the compromised fetus. The observation of a fetal non-response to repeated stimulation might, however, be of value in predicting an impairment in the neurological development in IUGR fetuses.

ACKNOWLEDGEMENTS

We like to thank Dr. M. Hadders-Algra for her assistance in the assessment of the infants general movements.

86 ugb'iooz ABSTRACT

Observation of the fetal movement and fetal heart rate responses to repeated vibroacoustic stimulation might be useful as a measure to assess fetal wellbemg. Studies of abnormal fetuses are needed to gam insight in the spectrum of possible responses to stimulation. We present a case of a fetus with trisomy 18 that failed to respond to repeated stimulation.

INTRODUCTION

Observation of fetal heart rate (FHR) responses to vibroacoustic stimulation (VAS) has been shown to be effective in identifying the acidotic fetus96 and observation of a fetal movement response to VAS to be a good predictor of a reassuring biophysical profile "0. With repeated application of the same stimulus, fetal habituation can be observed. Habituation is the decrease in fetal response, and finally cessation of this response following repeated presentation of the same stimulus. Fetal movement and FHR responses, and especially habituation of these responses, to repeated VAS, might be useful as a measure of fetal wellbemgand of the integrity of the fetal central nervous system (CNS) 8o,o6,97.

We present a case of a fetus with trisomy 18 that failed to respond to repeated VAS at 31 and 33 weeks gestational age (CA)

CASE - REPORT

A 34-year-old gravida 2, para 1, was referred for further evaluation in the 27'h week of her pregnancy because of fetal growth retardation Ultrasonographic examination revealed a fetus at the 5lhcentile for growth and a normal amount of amniotic fluid. Also, an atrioventricular septum defect of the heart and a were seen. Doppler flow velocimetry of the umbilical artery revealed absent end diastolic flow. Amniocentesis was performed for chromosomal analysis, and a diagnosis of trisomy 18 (47, XX +18, ) was made Termination of pregnancy was offered but the parents decided not to intervene

88 Before the heart anomaly was seen and the result of the chromosomal analysis was known, the patient had already been asked to participate m a study on responses and habituation to repeated VAS in growth retarded (IUCR) fetuses. Ethical approval for the study had been obtained from the hospital's Ethics and Research Committee and a written informed consent was obtained from the patient. Two tests were done at 31 and 33 weeks GA. The parents and the examinator were aware of result of the abnormal chromosomal pattern at 33, but not at 31 weeks GA

Before and after repeated stimulation, a 10-minute FHR recording was made. With a fetal vibroacoustic stimulator (Corometrics model 146, Wallmgford, Conn.; audible sound 20 to 9000 Hz, vibrations between 67 and 83 Hz, sound level 74 dB at 1 m m air) stimuli were applied to the maternal abdomen above the fetal legs for a period of 1 s with an interval of 30 s General movements of the fetal trunk were visualised using real-time ultrasound. Simultaneously the FHR was recorded by cardiotoco- graphy. Computerised analysis of the FHR was made by the Sonicaid System 8002 (Oxford Sonicaid Ltd, Abingdon, England). A general movement of the trunk within ι s after stimulation was considered a positive movement response. An acceleration of at least 15 bpm per minute within 1 s after stimulation was considered a positive FHR response.

Before the start of the 2 tests, the FHR recordings were reactive, the biophysical profile scored 10, and the quality of body and breathing movements appeared to be normal.This indicates that the fetus was in good condition, and not hypoxic During the 2 tests, no movement or FHR responses were observed at all to repeated stimu­ lation, neither did the fetus respond when the observation period was prolonged to 5 s after stimulation (figure 8a.i). Finally, even stimulation over the fetal head induced no movements or FHR responses. However, spontaneous fetal movements were perceived by the mother and observed by ultrasound 10 minutes before and after repeated stimulation. In both tests the baseline FHR, the long term and short term variation did not change following repeated VAS

Follow up revealed a fetal demise at 35 weeks GA After 5 days labour was induced and a severely macerated female infant, weighing 930 g, was born. Dysmorphic signs consistent with the syndrome were clearly present but an autopsy was refused. The diagnosis of trisomy 18 was confirmed by chromosomal analysis of a skin biopsy.

89 Figure SM

Figure 8a.2

^Ί^Γ^ Ο

"Ι •'·'

Fetal heart rate recording (paper speed of 3 cm per minute) of the fetus with trisomy 18 at 33 weeks GA (8e.l), and of a normal fetus at 34 weeks GA (8a.2). The stimuli are marked with arrows.

90 DISCUSSION

In a group of 12 normal fetuses we studied the responses to repeated VAS at 31 and 33 weeks CA. We tested the fetus with trisomy 18 following the same protocol. All normal fetuses responded with general body movements and with FHR acceleration within 1 s after the stimulus (for example see figure 8a.2) In addition, they all showed habituation of the movement response to repeated VAS.

In a group of 7IUCR fetuses, with no congenital or chromosomal anomalies and all with birth weights below the 5'h centile, we also studied the responses to VAS at different CA. All responded with general body movements and with FHR acceleration within 1 s after the stimulus. Some of them showed habituation of the movement response to repeated stimulation

These preliminary data reveal that normal and IUGR fetuses respond to VAS. In this case, the absence of response to stimulation is very likely to be due to the fetal anomalies caused by trisomy 18, because no signs of fetal hypoxia were present.

Ohel et al.s° studied the response to VAS in 2 anencephalic fetuses using an electric artificial larynx applied to the maternal abdomen over the fetal head. These 2 fetuses, tested at a CA of 33 weeks, showed neither movement nor FHR responses to stimulation.

In infants with trisomy 18 developmental retardation and hypotonia are common It is also known that these infants fail to respond to "auditory attention" (a low tone sound stimulation which should result in an interruption in the crying cycle if the baby is crying). This is a test of hearing and, to some extent, of cortical function 2" However, it is still not clear in this case whether the fetal non-response is due to deafness, because functional impairment of the tactile receptors and of the auditory or tactile receptor-motor neural interconnections can also be responsible for the non-response to VAS

Up till now observation of movement and FHR responses to VAS has been shown to be effective in identifying the normoxic fetus. In 50% of the cases in which fetuses did not respond to VAS, hypoxia and acidosis were present96. Anencephalic fetuses were also unable to respond to VAS 8° Based on the observations presented here, absence of FHR and movement responses to VAS can also be seen in a fetus with a major chromosomal anomaly without signs of fetal hypoxia. More studies of structural or chromosomal abnormal fetuses are needed to gain insight in the spectrum of possible responses to VAS.

91

ABSTRACT

Objective: Observation of fetal movement and fetal heart rate (FHR) responses to repeated vibroacoustic stimulation (VAS) might be useful as a measure to assess fetal wellbemg and to assess the integrity of the fetal central nervous system (CN5). We observed the movement and FHR responses to repeated VAS of a term fetus with a serious brain anomaly as compared to responses of normal term fetuses.

Study design: In 37 normal term fetuses and m a term fetus with an encephalocele we studied the movement and FHR response to repeated VAS.

Results: All normal fetuses responded within 1 s after stimulation with a general body movement and FHR acceleration. At 36 weeks gestational age (CA), no move­ ment or FHR responses were seen in the fetus with an encephalocele. Repetition of the test m this fetus m ι week showed again no fetal responses to repeated VAS.

Conclusion: Normal fetuses showed movement and FHR responses to external stimulation.The fetus with an encephalocele did not respond to repeated VAS with a movement or FHR acceleration. Case studies in fetuses with structural anomalies of the CNS are needed to gam insight in the spectrum of possible responses to VAS.

94 INTRODUCTION

Observation of fetal heart rate (FHR) responses to vibroacoustic or sound stimulation has been shown to be effective in identifying the acidotic fetus96 and observation of a fetal movement response to vibroacoustic stimulation (VAS) appears to be a good predictor of a reassuring biophysical profile "0. Fetuses with abnormal FHR patterns who failed to respond to external sound stimulation revealed perinatal mortality or neurologic impairment '". Observation of fetal responses to external stimulation can also be useful in identifying the anencephalic fetus 7980'06. Thus, fetal movement and FHR responses to vibroacoustic or sound stimulation might be useful as a measure of fetal wellbemg and as a measure of the integrity and functioning of the fetal central nervous system (CNS).

We studied the movement and FHR responses to repeated VAS m a term fetus with a serious brain anomaly as compared to normal term fetuses.

SUBJECTS AND METHOD

In a fetus with an occipital encephalocele and in 37 normal fetuses, between 37 and 40 weeks gestational age (GA), we studied the fetal movement and FHR responses to repeated VAS The fetal trunk was visualised using real-time ultrasound.The FHR was recorded simultaneously by cardiotocography. A fetal vibroacoustic stimulator (Corometrics model 146, Wallmgford, Conn.; audible sound 20 to 9000 Hz, vibra­ tions between 67 and 83 Hz, sound level 74 dB at 1 m m air) produced the stimulus. The stimuli, with duration of 1 s, were repeatedly applied to the maternal abdomen above the fetal legs with an interval of 30 s. Each stimulus was applied during fetal quiescence. A movement of the fetal trunk within 1 s after VAS was considered a positive movement response. A FHR acceleration of at least 15 bpm within 1 s after VAS was considered a positive FHR response.The normal fetuses were recruited for a study on fetal habituation to VAS (chapter 3) 'β5, but serve in this chapter as a control group. Ethical approval for the study of normal fetuses and of fetuses with a brain anomaly was obtained from the hospital's Ethics and Research Committee. Written informed consent was obtained from each participant.

RESULTS

All normal fetuses responded with trunk movements and with FHR accelerations to repeated VAS. The fetus with an encephalocele was tested at 36 and 37 weeks GA. At both occasions no body movements or FHR responses were observed to repeated VAS, even when the observation period was prolonged to 5 s after stimulation. Moreover, also stimulation over the fetal head induced no responses Spontaneous movements were, however, perceived by the mother and observed by ultrasound 10 minutes before and after repeated VAS.

95 CHAPTER 8b

Magnetic resonance imaging after delivery of the fetus revealed an encephalocele that contained parts of the lateral ventricles, , pons and parts of the occipital and parietal lobes, and agenesis of the corpus callosum and Arnold-Chiari malformation II (figures 8b.i and 8b.2). At neurologic examination after birth the infant showed no spontaneous movements. On acoustical or optical stimulation no movements or eye blinks were seen, only on painful stimulation a facial grimace was seen. The infant died of apnoeic spells and bradycardia 3 days after delivery.

Figure 8b.l Figure 8b.2

A sagittal Tl-iueighted (8b. 1) and an axial T2-u;eighted (8b.2) magnetic resonance image of the fetus with an occipital encephalocele. (Figures are printed with permission of the parents).

96 DISCUSSION

These data reveal that a normal term fetus will always respond to VAS with body movements and FHR accelerations The absence of response to VAS m the fetus with an encephalocele is very likely due to the brain anomaly, because signs of fetal hypoxia were not present. Before the start of the test, FHR recordings were normal and a normal biophysical profile was observed, indicating this fetus was in a good condition.

On acoustical or optical stimulation after birth m the infant with an encephalocele, no movements or eye blinks were seen. Only on painful stimulation a facial grimace was observed. This absence of response to external stimulation, except for painful stimulation, may explain the non-response of the infant in utero to VAS.

Ohel et al. 8o studied the response to VAS in 2 anencephalic fetuses at a CA of 33 weeks. These fetuses demonstrated many spontaneous body movements, but showed no movement or FHR responses to repeated VAS. Park and Kim 79 studied the FHR responses of 2 term anencephalic fetuses to VAS produced by the same device we used These fetuses revealed a non-reactive FHR pattern to VAS Unfortunately, they did not study the fetal movement responses. Leader et al.,o6 did study these movement responses to repeated VAS in 68 normal and m 4 anencephalic fetuses. The normal fetuses responded with movements to VAS, but all anencephalic fetuses failed to respond.

Observation of movement and FHR responses to external stimulation has been shown to be effective in identifying the normoxic fetus In 50% of the cases m which fetuses failed to respond to stimulation, hypoxia and acidosis were present96. Based on the literature, absence of movement or FHR responses to VAS can also be seen m anencephalic fetuses without a cerebral cortex 79β°'°6. Our results show that the fetus with an encephalocele, although some cerebral cortex was present, did not respond to VAS. All normal fetuses responded with movement and FHR responses

Further studies m fetuses with more discrete brain anomalies are necessary to gam insight into the spectrum of possible responses to VAS. Although most CNS abnormalities can be seen with ultrasound examination, observation of fetal responses to repeated VAS may be helpful to assess CNS functioning as well as detecting the fetus at risk for neurologic impairment.

97

H HYD ABSTRACT

Objective: Observation of fetal movement and fetal heart rate (FHR) responses to repeated vibroacoustic stimulation (VAS) might be useful as a measure to assess fetal wellbemg and to assess the integrity of the fetal central nervous system (CNS) We observed the movement and FHR responses to repeated VAS of a term fetus with a serious brain anomaly as compared to responses of normal term fetuses

Study design: In 37 normal term fetuses and in a term fetus with hydrocephalus we studied the movement and FHR response to repeated VAS

Results: All normal fetuses responded within 1 s after stimulation with a general body movement and FHR acceleration At 36 weeks and 39 weeks gestational age, movement and FHR responses to repeated VAS were observed m the fetus with hydrocephalus Neonatal outcome and follow up at 1 year revealed no abnormalities m this infant, except for macrocephaly

Conclusion: Observation of responses to VAS m a fetus with hydrocephalus might indicate a good fetal wellbemg and neonatal outcome Case studies m fetuses with structural anomalies of the CNS are needed to gam insight m the spectrum of possible responses to VAS

100 INTRODUCTION

Observation of fetal heart rate (FHR) responses to vibroacoustic or sound stimulation has been shown to be effective m identifying the acidotic fetus96 and observation of a fetal movement response to vibroacoustic stimulation (VAS) appears to be a good predictor of a reassuring biophysical profile "° Fetuses with abnormal FHR patterns who failed to respond to external sound stimulation revealed perinatal mortality or neurologic impairment222 Observation of the fetal responses to external stimulation can also be useful in identifying the anencephalic fetus ra8ü'06. Thus, fetal movement and FHR responses to VAS may be useful as a measure of fetal wellbemg and as a measure of the integrity and functioning of the fetal central nervous system (CNS).

We studied the movement and FHR responses to repeated VAS m a term fetus with a serious brain anomaly as compared to normal term fetuses.

SUBJECTS AND METHOD

Subjects

In a term fetus with hydrocephalus and in 37 normal fetuses, between 37 and 40 weeks gestational age (GA), we studied the fetal movement and FHR responses to repeated VAS. The fetus with hydrocephalus showed at ultrasound examination a symmetric hydrocephalus with few brain structures and a head circumference of 36 cm at 36 weeks GA (95,h centile = 35 cm) and of 37.5 at 39 weeks GA (95th centile = 36.5 cm). No other structural abnormalities were seen. This hydrocephalus was already present at 25 weeks GA (figure 801).

The normal fetuses were recruited for a study on fetal habituation to VAS (chapter 3) ,85, but serve m this article as a control group. Ethical approval for the study of normal fetuses and of fetuses with a brain anomaly was obtained from the hospital's Ethics and Research Committee. Written informed consent was obtained from each participant.

Method

The fetal trunk was visualised using real-time ultrasound. The FHR was recorded simultaneously by cardiotocography. A fetal vibroacoustic stimulator (Corometrics model i46,Wallingford, Conn.; audible sound 20 to 9000 Hz, vibrations between 67 and 83 Hz, sound level 74 dB at 1 m in air) produced the stimulus.The stimuli, with duration of 1 s, were repeatedly applied to the maternal abdomen above the fetal legs with an interval of 30 s. Each stimulus was applied during fetal quiescence A movement of the fetal trunk within ι s after VAS was considered a positive move­ ment response A FHR acceleration of at least 15 bpm within 1 s after VAS was considered a positive FHR response

101 CHAPTER 8'

Figure 8c.l

Sonogram of the head of the fetus with hydrocephalus at 29 lueefes GA.

RESULTS

All normal fetuses responded with trunk movements and with FHR accelerations to repeated VAS.The fetus with hydrocephalus was tested at 36 °'7 and 39 '" weeks GA. At 36 weeks GA the fetus showed startle-like movements to repeated VAS, however, alternate responses and non-responses prevented interpretation of the habituation pattern. At 39 weeks GA the fetus persisted in responding with movements and no response decrement was seen. FHR accelerations were seen within 1 s after stimulation at both occasions (Figure 80.2).

A female infant was born at 39 "'weeks GA by cesarean section with a birth weight of 3660 g (between the 75"' and 90th centile). Apgar scores were 9 and loat 1 and 5 minutes, respectively. Umbilical cord arterial blood pH was 7.30. On physical examination, the infant was macrocephalic with a head circumference of 38.2 cm. No other abnormalities were observed. No signs of elevated intraventricular (CSF) pressure were present. The diagnosis of symmetric normal pressure hydrocephalus was made. Ultrasound examination of the cerebrum at 1,5,

102 9, 15 weeks and 6 months of age revealed severe ventricular enlargement with slightly ballooned frontal horns without other signs of cerebral malformation. Magnetic resonance imaging of the infant's head at 3 days of age revealed a hydro­ cephalus probably due to aqueduct stenosis.

At neurologic examination directly and at ι day after birth, the infant was symmetric normotonic and showed spontaneous and reactive movements on acoustical and optical stimulation. Symmetric normal reflexes were observed. The neurological condition was examined at the age of 3 months by assessment of the quality of the general movements according to Hadders-Algra et al. 3c.This method shows that definitely abnormal general movements are associated with a high-risk for the development of cerebral palsy, whereas mildly abnormal general move­ ments are associated with the development of minor neurological dysfunction, attention-deficit-hyperactivity disorder, and aggressive behaviour. At examination the infant exhibited perfectly normal general movements. No signs of neuro- developmental delay were present Follow up at the age of 8 months showed a healthy child with a non-progressive hydrocephalus with no signs of neuro- developmental delay. Because of stabilisation of the hydrocephalus, although a severe hydrocephalus, and because no signs of clinical deterioration were present, no shunting of the CSF was required.

Figure 8C.2

sflKjigA.B η

MO 200

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60 60

OCO Ι"! Fn7 ULT »zn/fìuni * TOCO-ETOCO-EXX T -orf 100 75 75 50 — 50- -25 25

Fetal heart rate recording (paper speed of 3 cm per minute) of the fetus with hydro­ cephalus at 39 weeks GA. The stimuli are marked with arroius.

103 DISCUSSION

These data reveal that normal term fetuses respond to VAS with body movements and FHR accelerations. The fetus with the serious brain anomaly also responded with movements and FHR accelerations suggesting the fetus was m good condition. Before the start of the test in this fetus, the FHR recordings were normal and a normal biophysical profile was observed, indicating a good fetal condition with no signs of hypoxia The good neonatal outcome corroborated the good fetal condition.

Although the fetus showed fetal movement responses to VAS, no habituation could be observed at 39 weeks GA. On the contrary, the fetus persisted in responding with no signs of response decrement.This failure to habituate can also be seen in normal term fetuses. Six of the 37 healthy fetuses persisted in responding with 4 showing no response decrement. All these fetuses were born healthy. So, there are normal fetuses with a normal outcome that persist in responding to repeated VAS and fail to habituate.

Furthermore, follow up of the fetus with hydrocephalus revealed that the infant developed normally with no signs of clinical deterioration. On acoustical or optical stimulation after birth the infant also showed movement responses, as we already had observed in utero. A case-study of a fetus with an encephalocele with an adverse neonatal outcome revealed that a failure to respond with movements to VAS is associated with a poor neonatal outcome (chapter 8b) "'.These results might suggest that observation of fetal movement responses to VAS could be helpful m predicting the neonatal outcome of a fetus with a serious brain anomaly However, in the infant with hydrocephalus long term follow up is not known yet, so some neurodevelopmentally delay might become present in the future. Furthermore, mental deterioration may be subtle and even unrecognised in individuals with hydrocephalus "3. Therefore, it is not correct to state that observation of fetal responses to VAS indicates a good long term outcome

However, results from other studies support the conclusion that fetuses that fail to respond to VAS are likely to have a poor neonatal outcome. Ohel et al '" studied the response to VAS m 2 anencephalic fetuses at 33 weeks CA These fetuses demonstrated many spontaneous body movements, but showed no movement or FHR responses to repeated VAS. Park and Kim 79 studied the FHR responses of 2 term anencephalic fetuses to VAS produced by the same device we used These fetuses revealed a non-reactive FHR pattern to VAS. Unfortunately, they did not study the fetal movement responses. Leader et al. 'c6 did study these movement responses to repeated VAS m 68 normal and m 4 anencephalic fetuses. The normal fetuses responded with movements to VAS, but all anencephalic fetuses failed to respond.

Linder et al. '°5 tested the response to VAS of 5 neonates with severe neurologic deficits (3 with severe congenital hydrocephalus and minimal cortical tissue and 2 infants with severe perinatal asphyxia and hypoxic ischemic encephalopathy) and

104 compared this with 30 healthy neonates within 6-22 hours following birth when the neonates were in quiet sleep. All normal neonates responded with heart rate accelerations, irregularly breathing, and body movements to the VAS that was placed for 2 s on the mastoid process. The neurologically impaired neonates failed to elicit a response in all of the parameters measured.They state that the difference in the response to VAS in healthy and neurologically impaired neonates suggest that VAS has a potential value as a test of neurologic integrity.

Observation of movement and FHR responses to external stimulation has been shown to be effective m identifying the normoxic fetus. In 50% of the cases m which fetuses failed to respond to stimulation, hypoxia and acidosis were present96 Based on the literature, absence of movement or FHR responses to VAS can also be seen in anencephalic fetuses without a cerebral cortex7'580'06.

A study on the development of behavioural states in hydrocephalic fetuses revealed that the appearance of behavioural states was delayed in comparison to healthy fetuses of equivalent GA. The incidence of FHR pattern A and Β may be normal in such fetuses while the clustering with fetal body and eye movements is extremely low resulting m a high percentage of no-coincidence5'. In this study there were also significant differences m behavioural states between hydrocephalic fetuses with a good prognosis after birth and those with a poor outcome (i.e. death or severe neurological impairment) This finding demonstrates a potential clinical usefulness of this technique m the management of such fetuses. However, at present fetal behavioural analysis has the mam limitations of being labour intensive, time consuming and subjective, and as such, remains largely a research tool. Observation of fetal response to VAS does not have these limitations and further research on a larger number of hydrocephalic fetuses must give insight m the usefulness of the VAS technique in predicting the prognosis of hydrocephalic fetuses or of fetuses with other brain anomalies.

So, further studies in hydrocephalic fetuses or m fetuses with other (more discrete) brain anomalies are necessary to gam insight into the spectrum of possible responses to VAS Although most CNS abnormalities can be seen with ultrasound examination, observation of fetal responses to repeated VAS may be helpful to assess CNS functioning as well as detecting the fetus at risk for neurologic impairment. The advantages of this test are safety, simplicity, short duration and the ability to record objective measurements of the fetal response to VAS.

105

In recent years, attention has focused on fetal habituation to repeated stimulation as a measure of fetal well-being and as a measure of the functioning and integrity of the fetal central nervous system (CNS) "!7"4. There is general agreement that habituation is a form of learning and this construct is viewed as an important measure of CNS functioning """. Habituation is defined as a decline in the magnitude of response to repeated stimuli, in the absence of peripheral receptor / effector fatigue In order to make habituation useful to clinical practice, studies of reliability must be undertaken Reliability implies that similar protocols carried out across test sites yield similar outcomes Accordingly, the relationship between habituation and gestational age (GA),the influence of fetal state, the intrafetal and interfetal consistency in habituation performance, the influence of repeated testing on habituation, and the difference between habituation in normal and abnormal fetuses or fetuses at risk of an adverse neonatal outcome should be studied to evaluate the value of habituation as a tool m the assessment of fetal wellbeing and CNS functioning. The studies described in this thesis all focused on one of the aspects mentioned above.

The study presented in chapter 3 of this thesis showed that all normal term fetuses responded to the vibroacoustic stimulation (VAS) and habituation could be clearly observed in most of fetuses The results of this study also showed that the fetal activity or quiescence, based on the fetal heart rate (FHR) pattern and FHR parameters, prior to testing did not influence the habituation pattern. This can be explained by the fact that VAS arouses the fetus, which, in most of the fetuses, results in a transition to an active state during testing Studies have shown quite clearly that fetal responsiveness is increased as the duration "5 and intensity and frequency 7"2"6 of the stimulus is increased. Therefore, if the stimulus is intense enough, it will override the effect of fetal state. In fetal sheep, responsiveness and the habituation performance were not influenced by the electrocortical state of the fetus 'M. Similar observations have also been made m human fetuses '59.

A longitudinal study of the development of habituation m normal fetuses from 26 weeks GA onward showed that between 28 and 30 weeks GA 90% responded to VAS and only from 30 weeks GA 100 % of the fetuses were able to respond

108 (chapter 5) These results corroborate previous findings 7Î7',". This means that testing fetal habituation to VAS is only sensible from 28-30 weeks CA onwards.

Some studies reveal that older fetuses habituate more rapidly than their younger counterparts '58,5β,67 while others '62 could not find a relationship between GA and the habituation rate. By comparing the habituation rate of fetuses between 28-30 weeks GA with the rate of fetuses that were tested only in the term period we were able to conclude that the habituation performance is unrelated to maturation of the fetal brain The finding that postterm fetuses did not habituate more rapidly than term fetuses supports this conclusion. Although the ongoing maturation of the CNS in the final weeks of pregnancy is reflected m the change in behavioural state organisation46, we were unable to establish a more rapid habituation pattern m postterm fetuses compared with term fetuses (chapter 6)

The study of normal term fetuses (chapter 3) and postterm fetuses (chapter 6) revealed that there was a marked interfetal variability m the habituation perfor­ mance, with some fetuses habituating very rapidly while others failed to habituate and exhibited persistent responses The interfetal variability of the initial test is such that identification of a compromised fetus will be very difficult when the habituation performance is tested only once. However, the results also showed that most of the term and the postterm fetuses habituated more rapidly when tested for the second time within io minutes of the initial test. This fact may be of impor­ tance in testing habituation at 2 time points m fetuses at risk of neurological injury. It may be possible that compromised fetuses fail to habituate more rapidly when tested for the second time.

A study of habituation m term fetuses performed at 3 time points revealed that these fetuses habituated more rapidly both 10 minutes after and 24 hours after the initial habituation procedure (chapter 4) These results suggest that repeated testing results m fetal memorisation of the stimuli with subsequently a more rapid habituation performance. Yet, we do not claim that fetuses that failed to habituate more rapidly after a certain time interval are not capable of memorising stimuli. However, the establishment of a more rapid habituation pattern at repeated testing, suggesting the existence of fetal memory, might be of value m the assess­ ment of the functioning of the CNS. Further research should focus on the clinical relevance of the failure to habituate more rapidly to repeated testing.

In a study presented in chapter 5 we extended the study on fetal memory and learning by examining the influence of repeated testing on the habituation rate m normal fetuses We studied the fetal habituation pattern longitudinally at different GA's and compared this with the habituation rate of term fetuses that were tested only once. The results of this study led us to conclude that a short term memory of at least io minutes is already present m fetuses from 28-30 weeks GA and a long term memory of at least 2 weeks is clearly present m the term period. The results further suggest that fetal long term memory already develops at around 32 weeks

109 GA However, m view of the limited number of subjects studied, further research to explore these findings and to explore the development of fetal memory before the term period by testing a large number of fetuses at different ages and with different experiment intervals is indispensable

Although the study presented m chapter 7 on the habituation performance m growth retarded (IUGR) fetuses did not bring to light whether testing the fetal ability to habituate and memorise stimuli would be helpful m identifying the com­ promised IUGR fetus (due to the high rate of unmterpretable tests and the variability m habituation rate in the remaining tests), it revealed that the observation of a fetal non-response to repeated stimulation might be of value m predicting an impairment m neurological development m IUGR fetuses

This fetal failure to respond to VAS was also seen m a fetus with trisomy 18 (chapter 8a) and m a fetus with a severe brain anomaly (chapter 8b) Based on these results, the absence of a movement and an FHR response to VAS might be indica­ tive of a structural or chromosomal abnormality with subsequent adverse neonatal outcome The case described m chapter 8c revealed that, although a severe brain anomaly was observed m this fetus, the neonatal outcome was positive with no signs of neurological impairment This fetus responded adequately to repeated VAS Therefore, although most CNS abnormalities can be detected by ultrasound examination, observation of fetal responses to repeated VAS may be helpful to assess CNS functioning Furthermore, this test may also have additional value in identifying the fetus with trisomy 18 The advantages of this test are safety, simplicity, its short duration and the ability to record objective measurements of the fetal response to VAS More studies of fetuses with brain anomalies or chromo­ somal anomalies are needed to gam an insight into the spectrum of possible responses to VAS

In the studies described in this thesis it proved to be difficult to identify the fetus at risk of neurological injury based on a single measurement of the habituation rate, due to the high mterfetal variability m habituation rate So, single measurements of habituation cannot be used to assess fetal wellbemg However, we showed that repeated measurements are useful m the assessment of the fetal ability to memorise stimuli In a commentary Rizzo stated "These experimental studies of fetal learning and memory not only extend knowledge of early human capacities and development, but may also provide the basis for early and more sensitive assessments of the integrity of the fetal CNS and ultimately help elucidate the developmental mechanisms underlying behavioural teratogenesis" "' They also may provide infor­ mation to allow earlier and more precise prenatal diagnosis and possible treat­ ment Therefore, studying fetal memory by the habituation technique might become a tool m the detection of fetuses at risk of neurological sequelae and would therefore warrant further investigation However, future research should establish and improve the reliability of the fetal habituation technique by standardising

110 procedures so studies can be more easily compared. Furthermore, it is important to refine the technique so the number of succesful tests can be increased.

Another simple, cheap and promising test that needs further investigation is the observation of solely the fetal response to VAS. Studies presented m this thesis support the idea that this test can be helpful m identifying abnormal fetuses with an adverse neonatal outcome as well as in the prediction of the neuro- developmental outcome m fetuses with a brain anomaly. But, in view of the limited number of subjects studied, further research to explore these findings is needed It is however likely that future tests for fetal wellbeing will incorporate fetal stimulation.

Ill

SAMENVATTING

Er is veel aandacht besteed aan foetale habituatie op herhaalde vibroacoustische stimulatie als test voor foetale gezondheid en meer specifiek als onderzoek naar het functioneren van de foetale hersenen. Habituatie wordt gedefinieerd als een vermindering m respons op herhaalde stimuli met uiteindelijk het verdwijnen van die respons. Habituatie resulteert in het negeren van het bekende waardoor energie overblijft om aandacht te besteden aan het informatieve onbekende. Habituatie wordt gezien als een vorm van leren.

In dit proefschrift worden onderzoeken beschreven die als doel hebben een bijdrage te leveren aan de ontwikkeling van een test waarmee het functioneren van het foetale centraal zenuwstelsel beoordeeld kan worden. Hiervoor hebben WIJ de waarde van foetale habituatie op vibroacoustische stimulatie als test voor foetale gezondheid en met name als een mogelijke vorm van mtra-uterien neurologisch onderzoek bestudeerd.

Het onderzoek vond plaats m het Universitair Medisch Centrum Nijmegen. Gezonde foetussen en foetussen met een verhoogd risico op morbiditeit en morta­ liteit werden bestudeerd. Een groep van 37 à terme foetussen werd éénmalig getest evenals een groep van 20 post-terme foetussen. Een subgroep van 25 à terme foetussen werd op twee achtereenvolgende dagen getest. Een longitudinaal onder­ zoek vond plaats bij een groep van 21 foetussen die éénmaal in de twee weken van 26 tot 40 weken zwangerschapsduur werd bestudeerd. Met dezelfde frequentie werden η foetussen gevolgd die als groeivertraagd waren gediagnostiseerd. Het onderzoek bij twee foetussen met een ernstige hersenafwijkmg als bij een foetus met een chromosomale afwijking wordt als casuïstiek in dit proefschrift beschreven In totaal werden er 245 experimenten uitgevoerd.

Elk experiment bestond uit twee series van vibroacoustische stimuli die gegenereerd werden door een commerciële stimulator Uit de literatuur zijn tal van stimulatoren bekend Voor de onderzoeken beschreven in dit proefschrift hebben WIJ gebruik gemaakt van slechts één soort stimulator De stimuli, met een duur van 1 seconde, werden met een interval van 30 seconde via het maternale abdomen toegediend over de foetale beentjes De gegeneraliseerde beweging van de foetale romp, gevisualiseerd middels echoscopie, werd genoteerd als een respons Habituatie werd gedefinieerd als het verminderen en uiteindelijk verdwijnen van de respons. Na 10 minuten werd opnieuw de habituatiesnelheid bepaald Gedurende de gehele test werd een CTG-registratie gemaakt.

In hoofdstuk 3 wordt vastgesteld dat gezonde à terme foetussen reageerden met een beweging op de aangeboden vibroacoustische stimulus De meeste foetussen bleken te habitueren bij herhaald stimuleren De foetale gedragstoestand (waken of slapen) is met van invloed op de uitkomst van het habituatiepatroon Wel is er sprake van een groot mter-foetaal verschil m habituatiesnelheid Deze variabiliteit

113 m uitkomst heeft als gevolg dat het eenmalig bepalen van de foetale habituatie met gebruikt kan worden als onderzoek naar de hersenfunctie van a terme foetussen

Onderzoek beschreven in hoofdstuk 4 laat zien dat a terme foetussen een geheugen hebben Tien minuten als ook 24 uur na de eerste test habitueerden de meeste foetussen sneller dan tijdens de eerste test Met name de snellere habituatie 24 uur na de eerste test duidt op het bestaan van een geheugen bij foetussen Aangezien geheugen een hersenfunctie is kan de uitkomst van dit onderzoek bruikbaar zijn bij het ontwikkelen van een test naar foetale hersen­ functie Het beoordelen van foetaal geheugen als test in een klinische setting zal nog verder uitgezocht moeten worden

Een longitudinale studie, beschreven m hoofdstuk 5, naar de ontwikkeling van habituatie in foetussen tussen 26 en 40 weken zwangerschapsduur laat zien dat vanaf 30 weken zwangerschapsduur alle gezonde foetussen kunnen reageren op vibroacoustische stimulatie Dit betekent dat pas vanaf deze termijn het zinvol is foetale reacties (en eventuele habituatie) op deze vorm van stimulatie te obser­ veren Deze studie toont ook aan dat er geen relatie bestaat tussen de leeftijd van de foetus en de habituatiesnelheid Deze studie werd ook uitgevoerd om de ontwikkeling van foetale habituatie en geheugen te bestuderen We kunnen concluderen dat foetussen al bij 30 weken zwangerschapsduur beschikken over een korte-termijn geheugen Een lange termijn geheugen van twee weken lijkt pas aanwezig te zijn m de a terme periode Deze resultaten motiveren zeker tot diepgaander onderzoek

In hoofdstuk 6 is onderzocht of de habituatie-techmek van waarde zou zijn bij het beoordelen van de foetale gezondheid m post terme zwangerschappen Ook hierbij bleek dat, net als bij de a terme foetussen, de inter foetale variabiliteit m habituatiesnelheid groot is Sommige foetussen habitueerden erg snel terwijl anderen traag of zelfs helemaal met habitueerden Deze variabiliteit m uitkomst heeft als gevolg dat het eenmalig bepalen van de foetale habituatie met gebruikt kan worden als onderzoek naar de foetale gezondheid of foetale hersenfunctie m post-terme foetussen

Uit hoofdstuk 7 wordt helaas met duidelijk of het testen van habituatie m groei vertraagde foetussen zinvol is Opvallend is namelijk het grote aantal tests waarin er een onregelmatig responspatroon wordt gezien waardoor de habituatie met te beoordelen is Wel wordt vastgesteld dat het afwezig zijn van een respons op de stimulatie met gebruikelijk is Het vinden van een foetale non-respons kan mogelijk van waarde zijn m het voorspellen van hersenschade bij foetussen met een groei­ achterstand

Deze non-respons werd ook gezien m een foetus met een chromosomale afwijking (hoofdstuk 8a) en in een foetus met een ernstige hersenafwijkmg (hoofdstuk 8b) Gebaseerd op deze resultaten concluderen WIJ dat het afwezig zijn van een respons

114 (foetale beweging als wel een foetale hartslagverandering) op vibroacoustische stimulatie, na een zwangerschapstermijn van 30 weken, een teken kan zijn van een ernstige structurele hersenafwijking of chromosomale afwijking bij de foetus. De casus beschreven in hoofdstuk 8C laat zien dat ondanks de hersenafwijking deze foetus adequaat reageerde op de stimulatie Dit kind ontwikkelt zich tot op heden normaal. Concluderend: het testen van foetale reacties op stimulatie kan zinvol zijn m het beoordelen van de hersenfunctie bij foetussen met een structurele afwijking. Daarnaast kan het van additionele waarde zijn in het identificeren van een foetus met een chromosomale afwijking.

De onderzoeken beschreven m dit proefschrift maken duidelijk dat het moeilijk zal zijn om de foetus met een verhoogde kans op een neurologische afwijking te identificeren op basis van een enkele habituatietest. Dit als gevolg van de grote variabiliteit m de habituatiesnelheid. Wel is duidelijk dat herhaald testen van habituatie zinvol is voor het beoordelen van het foetale geheugen Deze experimentele studies naar het foetale geheugen vergroten met alleen onze kennis betreffende vroege menselijke capaciteiten en ontwikkeling, maar ze kunnen ook de basis vormen voor de ontwikkeling van een mtra-uterien neurologisch onder­ zoek. Daarom verdient het onderzoek naar de ontwikkeling van het foetale geheugen en leervermogen een vervolg.

Een andere veelbelovende test die verder onderzoek verdient is de observatie van foetale bewegingen en de foetale hartslagveranderingen op deze vorm van stimulatie De onderzoeken beschreven in dit proefschrift ondersteunen het idee dat deze simpele test bruikbaar kan zijn zowel bij het identificeren van een foetus met een chromosomale afwijking als bij het voorspellen van de prognose bij een foetus met een structurele hersenafwijking.

115

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122 129 Hawkins RD, Cohen TE, Greene W, Kandel ER. Relationships between dishabituation, sensitization, and inhibition of the gill- and siphon-withdrawal reflex in Aplysia califomica1 effects of response measure, test time, and training stimulus. Behav Neurosci 1998,112:24-38. 130 Castellucci VF, Carew TJ, Kandel ER. Cellular analysis of long-term habituation of the gill-withdrawal reflex of Aplysia califomica. Science 1978;202 1306-8 131 Kandel ER, Schwartz JH. Molecular biology of learning, modulation of transmitter release. Science 1982;218:433-43. 132 Gandhavadi B, Melvin JL. Electrical blink reflex habituation in mentally retarded adults. J Ment Defic Res 1985;29:49-54 133 Gandhavadi B. Glabellar reflex habituation in mentally retarded adults J Ment Defic Res 1982;26:271-8. 134 Schäfer EW, Peeke HV individuals fail to habituate cortical evoked potentials. Am J Ment Defic 1982;87.332-7. 135 Lader ΜΗ, Wing L. Physiological measures in agitated and retarded depressed patients J Psychiatr Res 1969;7 89-100. 136 McCall RB, Carriger MS. A meta-analysis of infant habituation and recognition memory performance as predictors of later IQ. Child Dev 1993;64:57-79. 137 Hutt SJ, Hutt C Hyperactivity in a group of epileptic (and some non-epileptic) brain-damaged children Epilepsia 1964;5·334-51. 138 Holloway FA, Parsons OA. Habituation of the orienting reflex in brain damaged patients Psychophysiology 1971;8:623-34. 139 Taiminen T, Jaaskelainen S, Ilonen T, Meyer H, Karlsson H, Lauerma H. et al Habituation of the blink reflex in first-episode schizophrenia, psychotic depression and non-psychotic depression. Scizoph Res 2000;44:69-79. 140 Hutt SJ, Hutt C, Lee D, Ounsted C. A behavioural and electroencephalographic study of autistic children. J Psychiatr Res 1965;3:181-97 141 Tizard B. Habituation of EEG and skin potential changes in normal and severely subnormal children. Am J Ment Defic 1968;73.34-40. 142 Dustman RE, Câliner DA. Cortical evoked responses and response decrement in nonretarded and Down's syndrome individuals. Am J Ment Defic 1979,83.391-7. 143 Bamet AB, Ohlrich ES, Shanks BL. EEG evoked responses to repetitive auditory stimulation in normal and Down's syndrome infants. Dev Med Child Neurol 1971;13:321-9. 144 Key BJ Effects of chlorpromazine and lysergic acid diethylamide on the rate of habituation of the arousal response. Nature 1961;190-275-7. 145 Moreau T, Birch HG. Relationship between obstetrical general anesthesia and rate of neonatal habituation to repeated stimulation Dev Med Child Neurol 1974;16:612-9. 146 Brackbill Y, Kane J, Manmello RL, Abramson D. Obstetric premedication and infant outcome. Am J Obstet Gynecol 1974,118.377-84. 147 Als H, Tïonick E, Lester BM, Brazelton TB The Brazelton Neonatal Behavioral Assessment Scale (BNBAS). J Abnorm Child Psychol 1977;5:215-31. 148 Eisenberg RB, Coursin DB, Rupp NR. Habituation to an acoustic pattern as an index of differences among human neonates. J Audit Res 1966;6:239-48. 149 Bomstein MH, Benasich AA. Infant habituation: assessments of individual differences and short-term reliability at five months Child Dev 1986;57:87-99. 150 Bomstein MH, Sigman MD. Continuity in mental development from infancy. Child Dev 1986;57:251-74.

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124 173 DeCasper AJ, Spence MJ Prenatal maternal speech influences newborns' perception of speech sounds Inf Behav Dev 1986, 9 133-50. 174 Hepper PG. Fetal "soap" addiction Lancet 1988;11347-8 175 Werner E, Serr DM, Shalev E. Fetal motorical and heart response to sound stimulus m different behavioral states. Gynecol Obstet Invest 1989;28:141-3. 176 Jensen OH. Fetal heart rate response to controlled sound stimuli during the third trimester of normal pregnancy Acta Obstet Gynecol Scand 1984;63:193-7. 177 Hutt C, Bemuth H, Lenard HG, Hutt SJ, Prechtl HF Habituation in relation to state in the human neonate Nature 1968;220·618-20 178 Pillai M, James D The development of fetal heart rate patterns during normal pregnancy. Obstet Gynecol 1990;76:812-6. 179 Dawes GS, Lobb M, Moulden M, Redman CW, Wheeler T. Antenatal cardiotocogram quality and interpretation using computers. Br J Obstet Gynaecol 1992,99791-7 180 Van Heteren CF, Boekkooi PF, Jongsma HW, Nijhuis JG. Fetal learning and memory Lancet 2000;356:1169-70 181 Hutt SJ, Hutt C, Lenard HG, Bemuth H, Muntjewerff WJ. Auditory responsivity in the human neonate. Nature 1968;218:888-90 182 Hepper PG. Fetal memory: does it exist? What does it do? Acta Paediatr Suppl 1996,416:16-20 183 McGaugh JL. Memory-a century of consolidation. Science 2000,287:248-51 184 Keen RE, Chase HH, and Graham FK "Nventy-four hour retention by neonates of an habituated heart rate response Psychon Sci 1965;2·265-6 185 Van Heteren CF, Boekkooi PF, Jongsma HW, Nijhuis JG Fetal habituation to vibroacoustic stimulation in relation to fetal states and fetal heart rate parameters. Early Hum Dev 2001;61:135-45. 186 Van Heteren CF, Boekkooi PF, Jongsma HW, Nijhuis JG Responses to repeated vibroacoustic stimulation in a fetus with trisomy 18, a case-study Eur J Obstet Gynecol Reprod Biol 2001,96.123-5 187 Van Heteren CF, Boekkooi PF, Jongsma HW, Nijhuis JG. Responses to vibroacoustic stimulation in a fetus with an encephalocele compared to responses of normal fetuses. J Perinat Med 2000;28:306-8 188 Suzuki WA, Eichenbaum H. The neurophysiology of memory. Ann Ν Y Acad Sci 2000;911.175-91 189 Rose SP God's organism? The chick as a model system for memory studies. Leam Mem 2000,71-17 190 Schmidt R. Cell-adhesion molecules in memory formation. Behav Brain Res 1995;66:65-72. 191 Benjamin PR, Staras K, Kemenes G A systems approach to the cellular analysis of associative learning in the pond snail Lymnaea. Leam Mem 2000;7124-31. 192 Schaal Β, Orgeur Ρ Olfaction in utero, can the rodent model be generalized? QJ Exp Psychol Β 1992,44.245-78. 193 Varendi H, Porter RH, Winberg J. Attractiveness of amniotic fluid odor, evidence of prenatal olfactory learning? Acta Paediatr 1996;85:1223-7. 194 Schaal Β, Marlier L, Soussignan R Olfactory function in the human fetus evidence from selective neonatal responsiveness to the odor of amniotic fluid Behav Neurosci 1998;112:1438-49. 195 Varendi H, Chnstensson K, Porter RH, Winberg J. Soothing effect of amniotic fluid smell in newborn infants. Early Hum Dev 1998;51.47-55. 196 Zimmer EZ, Divon MY Fetal vibroacoustic stimulation. Obstet Gynecol 1993;81-451-7.

125 197 Hepper PG. Fetal habituation, another Pandora's box? Dev Med Child Neurol 1997;39:274-8 198 Dobbing J, Sands J Quantitative growth and development of human brain Arch Dis Child 1973;48:757-67. 199 Gekoski MJ, Fagen JW, Pearlman MA. Early learning and memory in the preterm infant Inf Beh Dev 1984, 7 267-76. 200 Kozuma S, Okai T, Nemoto A, Kagawa H, Sakai M, Nishina H. et al Developmental sequence of human fetal body movements in the second half of pregnancy. Am J Pennato! 1997;14 165-9 201 DiPietro JA, Hodgson DM, Cosügan KA, Hilton SC, Johnson TR. Fetal neurobehavioral development. Child Dev 1996;67:2553-67. 202 Kisilevsky BS, Muir DW, Low JA Maturation of human fetal responses to vibroacoustic stimulation. Child Dev 1992;63:1497-508. 203 Lan LM, Yamashita Y, Tang Y, Sugahara Τ, Takahashi M, Ohba T. et al. Normal fetal brain development. MR imaging with a half-Founer rapid acquisition with relaxation enhancement sequence Radiology 2000;215:205-10. 204 Nelson CA. The nature of early memory Prev Med 1998;27 172-9. 205 Naeye RL. Causes of perinatal mortality excess in prolonged . Am J Epidemiol 1978;108:429-33 206 Boyd ME, Usher RH, McLean FH, Kramer MS Obstetric consequences of postmaturity Am J Obstet Gynecol 1988,158:334-8. 207 Callenbach JC, Hall RT Morbidity and mortality of advanced gestational age. post-term or postmature. Obstet Gynecol 1979;53·721-4 208 Manning FA, Platt LD, Sipos L. Antepartum fetal evaluation- development of a fetal biophysical profile Am J Obstet Gynecol 1980,136 787-95. 209 Bobby PD, Divon MY. Fetal testing in postdates. Curr Opin Obstet Gynecol 1997,9:79-82. 210 Severi FM, Rizzo G, Bocchi C, D'Antona D, Verzuri MS, Ardumi D. Intrauterine growth retardation and fetal cardiac function. Fetal Diagn Ther 20OO;15-8-19 211 Chandran R, Serra Serra V, Sellers SM, Redman CW. Fetal cerebral Doppler in the recognition of fetal compromise Br J Obstet Gynaecol 1993,100:139-44. 212 Ferrazzi E, Vegni C, Bellotti M, Borboni A, Della Feruta S, Barbera A. Role of umbilical Doppler velocimetry in the biophysical assessment of the growth-retarded fetus. Answers from neonatal morbidity and mortality. J Ultrasound Med 1991,10:309-15. 213 Ounsted M, Moar VA, Scott A. Neurological development of small-for-gestational age babies during the first year of life. Early Hum Dev 1988;16:163-72. 214 Gortner L, Wauer RR, Stock GJ, Reiter HL, Reiss I, Jorch G. et al Neonatal outcome in small for gestational age infants, do they really better? J Perinat Med 1999,27-484-9. 215 Bekedam DJ, Visser GH, Mulder EJ, Poelmann Weesjes G. Heart rate variation and movement incidence in growth-retarded fetuses: the significance of antenatal late heart rate decelerations. Am J Obstet Gynecol 1987;157:126-33 216 Ribbert LS, Snijders RJ, Nicolaides KH, Visser GH. Relation of fetal blood gases and data from computer-assisted analysis of fetal heart rate patterns in small for gestation fetuses Br J Obstet Gynaecol 1991,98:820-3. 217 Ribbert LS, Nicolaides KH, Visser GH Prediction of fetal acidaemia in intrauterine growth retardation: comparison of quantified fetal activity with biophysical profile score Br J Obstet Gynaecol 1993;100:653-6. 218 Uvebrant P, Hagberg G. Intrauterine growth in children with cerebral palsy Acta Paediatr 1992,81-407-12 219 Hadders-Algra M. The assessment of general movements is a valuable technique for the detection of brain dysfunction in young infants. A review Acta Paediatr Suppl 1996;416:39-43

126 220 Sarinoglu C, Dell J, Mercer BM, Sibai BM. Fetal startle response observed under ultrasonography a good predictor of a reassuring biophysical profile. Obstet Gynecol 1996,88:599-602 221 Taylor Al Autosomal trisomy syndromes· a detailed study of 27 cases of Edwards' syndrome and 27 cases of Patau's syndrome. J Med Genet 1968;5:227-52. 222 Goodlin RC, Schmidt W. Human fetal arousal levels as indicated by heart rate recordings. Am J Obstet Gynecol 1972;114·613-21. 223 Vanneste JA. Diagnosis and management of normal-pressure hydrocephalus J Neurol 2000;247:5-14. 224 Hepper PG, Leader LR Fetal habituation. Fetal Matem Med Rev 1996; 109-23 225 Pietrantom M, Angel JL, Parsons MT, McClain L, Arango HA, Spellacy WN. Human fetal response to vibroacousùc stimulation as a function of stimulus duration. Obstet Gynecol 1991;78·807-11. 226 Gagnon R, Patrick J, Foreman J, West R Stimulation of human fetuses with sound and vibration. Am J Obstet Gynecol 1986;155 848-51 227 Rizzo Τ Habituation technique in study of development of fetal behaviour. Lancet 2001;357·328-9.

127

BIBLIOGRAPHY

Publications

Van Heteren CF, Boekkooi PF, Jongsma HW, Nijhuis JG. Fetal habituation to vibroacoustic stimulation in uncomplicated postterm pregnancies. Eur J Obstet Gynecol Reprod Biol 200i;97 178-82.

Van Heteren CF, Boekkooi PF, Jongsma HW, Nijhuis JG.The responses to repeated vibroacoustic stimulation in a fetus with trisomy 18 Eur J Obstet Gynecol Reprod Biol 2001;6:123-5.

Van Heteren CF, Boekkooi PF, Jongsma HW, Nijhuis JG. Fetal habituation to vibroacoustic stimulation m relation to fetal states and fetal heart rate parameters. Early Hum Dev 2001;61:135-45

Van Heteren CF, Boekkooi PF, Jongsma HW, Nijhuis JG Fetal learning and memory. Lancet 2000;356:1169-70 (Correspondence: Lancet, 2001,357.479)

Van Heteren CF, Boekkooi PF, Jongsma HW, Nijhuis JG. Responses to vibroacoustic stimulation in a fetus with an encephalocele compared to responses of normal fetuses. J Perinat Med 2000,28 (4):3o6-8

Werre A, Mulder C, Van Heteren CF, Spillenaar Bilgen E. Dilatation of benign strictures following low anterior resection using Savary-Gilliard bougies Endoscopy 2000,32 385-8

Van Heteren CF, Nijhuis JG, Semmekrot BA, Merkus JMWM Discordante foetale groei bij meerlingen: beleid afstemmen op choriomciteit. Ned Tijdschr Geneeskd 1999;143:1017-21.

Van Heteren CF, Nijhuis JG, Semmekrot BA, Mulders LGM, Van den Berg PP The risk for the surviving twin after fetal death of the co-twin in twin-twin transfusion syndrome. Obstet Gynecol 1998:92 215-9.

Van Heteren CF, Nijhuis JG, Merkus JMWM. Therapieën bij het twm-to-twin transfusion syndroom. V.V.O.G. Jaarboek 1998 101-7

Nijhuis JG, Van Heteren CF. Het transfuseur-transfusé syndroom en intra-uteriene sterfte. Meerlingen, Elsevier/De Tijdstroom, Maarssen, H W. Brumse en G H.A Visser (red.) 1997, Hoofdstuk 11,85-93.

129 Published abstracts

Van Heteren CF, Boekkooi PF, Jongsma HW, Nijhuis JG. Fetal memory demonstrated by repeated habituation testing in the course of pregnancy. J Soc Gynecol Invest 200i;8 (Suppl 2):6gq.

Van Heteren CF, Boekkooi PFJongsma HW, NijhuisJG Responses and habituation to vibroacoustic stimulation in normal fetuses and in one fetus with a serious brain anomaly. Prenat Neon Med 2000,5. F103

Nijhuis JG, Van Heteren CF, Semmekrot BA, Mulders JGM, Van den Berg PP. What is the risk for the surviving twin after death of the co-twin in twin-twin transfusion syndrome? Am J Obstet Gynecol i997;i76:Sn6.

130 DANKWOORD

Gelukkig is het doen van promotieonderzoek geen solitaire bezigheid Met de hulp van velen is het mij gelukt deze klus te klaren. Graag wil ik allen die bij deze klus betrokken zijn geweest hartelijk danken. Enkelen wil ik met name noemen.

Allereerst ben ik veel dank verschuldigd aan mijn promotor prof. dr. J.G Nijhuis. Beste Jan, door bij jou mijn wetenschappelijke stage te doen kwam ik er al snel achter dat een enthousiaste begeleider met veel heldere ideeën en een pragmatische instelling een must is in de wetenschap. Toch heb ik je na afloop van deze stage meegedeeld dat ik zeker geen trek had in het doen van promotie­ onderzoek. Gelukkig heb je me later de kans gegeven om hier op terug te komen. Ook bij dit promotieonderzoek was je een de stuwende kracht en degene die de rode draad goed vasthield. Ik heb veel van je geleerd en veel aan je te danken. Onze samenwerking zal zeker nog een vervolg krijgen.

Dr. P.F. Boekkooi, beste Focco, als co-promotor had je de fundamenten voor dit onderzoek al zeer stevig gelegd voordat ik eraan begon Met je enthousiasme en inzet hebje er verder voor gezorgd dat ik goed op koers en snelheid bleef. Dank voor je wetenschappelijke inbreng en, even zo belangrijk, voor de gezellige samen­ werking. Want naast de gesprekken over ons onderzoek had je gelukkig ook altijd tijd voor "geouwehoer" over van alles en nog wat.

Dr. H.W. Jongsma, beste Henk, als tweede co-promotor was je onmisbaar. Zeker m de laatste fase, toen Jan en Focco Nijmegen hadden verlaten, heb je je als een ware co-promotor over me ontfermd Voor methodologische en statistische problemen, maar zeker ook voor minder wetenschappelijke vragen kon ik altijd bij je terecht.

Verloskundigen, arts-assistenten en gynaecologen van het Universitair Medisch Centrum Sint Radboud in Nijmegen, zonder jullie hulp had ik nooit zo snel zo veel zwangeren kunnen werven. Dr. Bas Keijser bedankt voor je praktische en morele steun.

De studenten Annalies, Niels, Rikke en Vanessa, bedankt voor jullie hulp bij het werven en testen van zwangeren en baby's en bij het uitwerken van de resultaten.

Gerda Theumssen en Ans Bakker bedankt voor alle hand- en spandiensten. Nelleke Hamel bedankt voor je luisterend oor en je interesse inde persoon achter de onder­ zoeker.

Gedeelde smart is halve smart heb ik vele malen mogen ervaren op de werkplek. Het is erg motiverend om lotgenoten om je heen te hebben, hoewel er ook ontel­ baar vele uren "verkletst" werden. De wetenschappelijke kruisbestuiving, het geklaag en gezeur, de vreugde als er een artikel was geaccepteerd of een hoofdstuk af was, de avondjes in de kroeg, de etentjes, de laserquest of paintball spelletjes heb ik beleefd met Anne-Marie, Erik, Eva Maria, Ingeborg, Iris, Jesper, Maarten, Marcel,

131 Marieke, Mark, Michael, Pascal, Petra, René, Ron, Ruben, Sabina, Tanya, Wai Yee en Willianne. Hopelijk zal ik jullie nog met enige regelmaat tegenkomen.

Nieuwe collega's in het Sint Joseph Ziekenhuis te Veldhoven, bedankt dat jullie mij de kans geven me verder in de gynaecologie en obstetrie te bekwamen.

Vrienden en vriendinnen,jullie wil ik bedanken voor een hele hoop dingen die mijn werk met altijd ten goede kwamen Maar juist ook deze activiteiten maakten het mogelijk dat ik na een enerverend weekend, waar dan ook, weer energie had om de week in te gaan.

Speciale dank voor Carole en Mieke. Carole voor het delen van allerlei zaken waar we om de beurt voor kwamen of nog komen te staan (of het nu deoppositie, het altaar of de goal is). Als enige wist JIJ me thuis te houden van een congres m Amerika. Mieke voor alle vriendinnendmgen Gelukkig kan ik met jou mijn afwijkende gevoel voor humor delen. Fijn dat je mijn paranimf wilt zijn.

Dames 3 van Hockeyclub Union m Nijmegen verdient het ook om hier genoemd te worden. Erg prettig om al je energie en verhalen op woensdag en zondag (en andere dagen) kwijt te kunnen. En het moet gezegd: was ik blijven spelen bij jullie dan was ik de vierde doctor in het team geworden. Voordat ik jullie voorgoed verlaat gaan we nog een keer goed feesten.

Mathijs, lieve broer, misschien is mijn grootste drijfveer op het professionele vlak wel het feit dat ik ooit voor de Cito-toets een lagere score behaalde da η jij. Toch laat jouw relaxte levensstijl mij nog wel eens nadenken over die van mij Leonie, lieve zus, met jou deel ik de relativerende kijk op ons werk en leven. Heerlijk dat je nu zo dicht bij me inde buurt bent komen wonen Broer, zus en Stephan, bedankt voor het vormen van een thuis waar het heerlijk toeven is na hard werken

Papen mam, vanaf het moment dat ik kon praten wisten jullie al dat het wel goed zou komen met me. Met jullie onvoorwaardelijke liefde, rotsvast vertrouwen en immer aanwezige raad en daad hebben jullie mij m staat gesteld om heel gelukkig te worden Mam, ik ben er trots op dat jij mijn paranimf wilt zijn.

Lieve Roel, of ik nu moest e-mailen op onze huwelijksreis m Grenada, mijn dank­ woord moest schrijven op vakantie m Turkije, je vond het allemaal goed Ook al bevond je je tijdens de afrondingsfase van mijn proefschrift aan de andere kant van de oceaan, jouw overtuiging dat ik het ook wel zonder je zou kunnen maakte dat ik dat ook deed. Toch is jouw aandeel in dit werk vele malen groter dan iedereen kan vermoeden. Op naar het volgende levenswerk.

LAST BUT NOT LEAST...

132 De ouders van

Alwin Kaylee Pim

Anke Kaylee Pim

Anne Kirsten Quillermo

Annick Larissa Raziyah

Bekir Laszlo Renée

Bram Laura Rielle

Collin Laura Rixt

Damon Lennart Rob Dana Lieneke Robin

Dorieke Lisa Robin

Dries Lisha Romy

Elaine Lotte Sanne

Erik Lotte Sanne

Erwin Lyde Sigi

Evy Marijn Sjakira

Fayda Marit Steijn

Femke Marion Stein

Fleur Martijn Stijn

Floris Maud Stijn

Gaia Max Suzan

Gosling Max Sylvan

Imane Max Tessa

Indi Mees Teun

Ivana Mike Teuntje Ivar Milo Thom

Jamque Mitchel Tim

Jenna Nick Tim

Jessie Nick Vera nee Jim Nicky Willem

Joeri Nikita Yadira Judith Oscar Yom

Katja Paul

Kayla Peter BEDANKTi

133 STELLINGEN

behorende bij het proefschrift

Development of habituation and memory in human fetuses

Cathelijne van Heteren

Nijmegen, 5 oktober 2001

1 Vanaf 30 weken zwangerschapsduur is het zinvol de reactie van een humane foetus op een vibro-acoustische stimulus te bestuderen -dit proefschnfl-

2 Een foetus heeft een geheugen -dit proefschnft-

3. Door het herhaald testen van foetale habituatie op vibroacoustische stimulatie is het mogelijk de ontwikkeling van het foetale geheugen te bestuderen -dit proefschnft-

4 Het beoordelen van het foetale geheugen kan bruikbaar zijn bij het ontwikkelen van een mtra-uterien neurologisch onderzoek, -dit proefschnfl-

5 Het éénmalig testen van foetale habituatie ter bepaling van de foetale conditie is niet zinvol -dit proefschnfi-

6. Het bestuderen van de foetale reactie op een vibroacoustische stimulus lijkt zinvol bij het bevestigen van ernstige afwijkingen van het foetale centraal zenuwstelsel, -dit proefschnfl-

7. Sommige personen zijn al voor hun geboorte Oost-Indisch doof

8. Homeopathie werkt zoals toeval bestaat

9. We have to keep our minds open, but not so open that our brains fall out -A. Anderson-

10 Het frequent veranderen van de regels van het hockeyspel komt het niveau van de clubscheidsrechter met ten goede.

11. If you want to kiss the sky, better learn how to kneel -U2-

12 Blondes have more fun.

Cathelijne van Heteren, born in Arnhem (1971), graduated from Nederrijn College in Arnhem before attending medical school at the University of Nijmegen (1989-1997). During her time in medical school, she worked as a junior house officer in Sengerema,

1997-1998 she participated in a research project, "Hormone Replacement Therapy" through the Department of Obstetrics and Gynaecology, University Medical Centre Nijmegen.

From August 1998 until December 2000, she worked :he same department on the "Fetal Habituation" research project, resulting in this thesis.

On April 2001, she started training in Obstetrics and Gynaecology at the Saint Joseph Hospital in 'hoven.