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Habituation in fetuses of diabetic mothers
Early Human Development 59 (2000) 85–93 www.elsevier.com / locate / earlhumdev
Habituation in fetuses of diabetic mothers N. Nicola Doherty PhD, Peter G. Hepper PhD* Wellcome Trust Fetal Behaviour Research Centre, School of Psychology, The Queen’ s University of Belfast, Belfast BT7 1 NN, N. Ireland, UK Received 5 August 1999; received in revised form 30 May 2000; accepted 1 June 2000
Abstract Fetuses of diabetic mothers exhibit maturational delays in their behaviour and disturbances in behavioural and intellectual functioning in childhood. This suggests an effect of maternal diabetes on the central nervous system of the fetus. The habituation technique enables the functioning of the higher central nervous system to be examined. A normal habituation pattern reflects an intact central nervous system. Previous studies have found abnormalities in the fetal central nervous system are reflected in habituation performance. This paper examined the habituation ability of fetuses of diabetic mothers and of non-diabetic mothers. The fetuses were tested at 28, 32 and 36 weeks of gestation. After 2 min of fetal inactivity a series of vibroacoustic stimuli were presented to the fetus. This continued until no response was observed on five consecutive stimulus presentations. The number of stimulus presentations to habituate at each gestational age was recorded. The results reveal that there was a highly significant main effect of group (F(1,47) 5 19.65, P , 0.001). Fetuses of diabetic pregnancies took longer to habituate. There was a significant effect of gestational age (F(2,94) 5 44.67, P , 0.0001). In both groups the number of trials to habituate decreased with advancing gestation. There was no relationship between random blood glucose levels and habituation performance. The results demonstrate that maternal diabetes affects higher aspects of central nervous system functioning in the fetus. 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Habituation; Fetuses; Maternal diabetes
*Corresponding author. 0378-3782 / 00 / $ – see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0378-3782( 00 )00089-X
86
N.N. Doherty, P.G. Hepper / Early Human Development 59 (2000) 85 – 93
1. Introduction Whilst the successful management of blood glucose levels during pregnancy has significantly reduced mortality and morbidity in the offspring of diabetic mothers, recent evidence suggests that the offspring of diabetic pregnancies exhibit disturbances in behavioural and intellectual functioning [1–4]. For example, the examination of cognitive functioning in the children of diabetic mothers has illustrated poorer performance when compared with the children of non-diabetic mothers [1]. It is possible that the poorer performance results from an effect of the altered maternal metabolic milieu on the fetal CNS [5]. The effect of maternal diabetes on the fetal CNS may be teratogenic. Observation of the behaviour of the fetus offers the opportunity to assess the functional development of the CNS [6]. Hence observation of the behaviour of the fetus may elucidate the effect of maternal diabetes on the fetal CNS. There have been few studies of behaviour in the fetuses of diabetic pregnancies. Although there are indications that maternal diabetes acts as a behavioural teratogen. The organisation of behavioural states is poorer in fetuses of diabetic mothers than non-diabetic mothers [7]. Fetal breathing movements appear earlier in fetuses of diabetic mothers than non-diabetic mothers [8]; their incidence is higher although the rate of breathing is slower [9]. All other movements show a 1–2-week delay in there emergence [8]. The development of movements, with the exception of startles, was the same for fetuses of diabetic and non-diabetic pregnancies [8]. The quality of spontaneous general movements of fetuses of diabetic pregnancies is reported as different from fetuses of non-diabetic pregnancies [10]. More recently differences were found in the fetal heart rate response to a vibroacoustic stimulus [11]. Fetuses of diabetic mothers exhibited a less mature response than fetuses of non-diabetic pregnancies. One further technique that can be used to assess CNS functioning prenatally is that of habituation [12]. Habituation provides a sensitive means to examine higher CNS functioning, and is defined as the decrement in response to repeated stimulation [13]. In the human fetus a sound stimulus has been reliably used to elicit habituation of fetal movement and heart rate [12]. It has been argued that the normal habituation pattern reflects the normal functioning of the brain, including the cortices. Indeed abnormalities in habituation have been noticed postnatally in cases of brain abnormalities (e.g., Refs. [14,15]). Fetus’s with CNS anomalies such as microcephaly [16] and anencephaly [16,17] exhibit deviant habituation performance. Similarly abnormal habituation patterns have been observed in fetuses whose mothers were experiencing hypoxic conditions [18], in fetuses of mothers who recently had a cigarette [19], in fetuses of mothers who were taking drugs [20] and in fetuses with chromosomal conditions such as Down’s syndrome [21]. Commonly, fetuses with CNS anomalies, or exposed to adverse conditions which may affect CNS functioning, take longer to habituate, or fail to habituate [12]. Habituation, not previously studied in diabetic pregnancies, is examined here to assess the presence or absence of an effect of maternal diabetes on the CNS of the developing fetus.
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2. Methodology
2.1. Subjects Forty-nine mothers in total took part in the study, 15 of whom were diabetic and 34 non-diabetic.
2.1.1. Diabetic mothers The mothers in the diabetic group were drawn from those attending the joint metabolic and obstetric clinic at the Royal Maternity Hospital, Belfast. These mothers were all previously diagnosed insulin-dependent diabetics and were treated by insulin injection. The women were White’s classes B and C and mean length of diabetes was 14.0 (68.0) years. Their diabetes was reported by the Consultant Endocrinologist to be well-controlled (mean HbA 1c levels between 5 and 8 mmol / l). Mean age of the mothers was 28.0 (65.0) years. All mothers had singleton pregnancies, for 40% this was their first pregnancy, 26.7% of the sample had experienced miscarriage. Of the mothers, 93.3% were married, 6.7% smoked and 6.7% consumed alcohol (the use of alcohol and nicotine was controlled for in all statistics).
2.1.2. Non-diabetic mothers The subjects in the non-diabetic group were drawn from women attending the regular antenatal clinics at the Royal Maternity Hospital, Belfast. Their average age was 26.9 (66.1) years. All mothers had singleton, healthy pregnancies. Miscarriage had been experienced by 41.2%, for 23.5% this was their first pregnancy and 75.9% were either married or living with their partners. Of the mothers, 41.2% smoked and 38.2% reported use of alcohol.
2.2. Apparatus The mothers’ random blood glucose was measured by the Hemocue B-Glucose Photometer (HemoCue, South Yorkshire, UK). The fetus was viewed using an Advanced Technical Laboratories (ATL) Ultramark 4plus ultrasound machine, or Dornier AI3200 ultrasound machine, with a 3.5-MHz curvilinear scan head. The sound stimulus employed was a Fetal Acoustic Stimulator, Model 146 from Corometrics Medical Systems. This hand-held vibroacoustic stimulator is self contained and battery operated. It emits sound at a frequency of 75 Hz610% with harmonics ranging from 20 to 90 Hz. When measured in air at 1 m from the stimulator the maximum recorded sound intensity was 82 dB. Presentation and timing of the stimulus for the habituation procedure was controlled by a BBC microcomputer. All scans were recorded for later analysis. The computer also superimposed a visual indication of stimulus presentation and a centisecond timer over the ultrasound picture which was recorded on the videotape over the ultrasound image.
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2.3. Procedure Ethical permission for the study was obtained from the Research Ethics Committee, Faculty of Medicine, Queen’s University. Prior to testing the women were asked about their smoking and drinking habits and previous 2 h activity. Fetal maturity, fetal measurements (BPD, AC, FL), fetal weight and position were recorded. Immediately before scanning commenced random blood glucose was taken prior to using the Hemocue B-Glucose Photometer. All mothers were scanned approximately 2 h after their last meal. The mothers lay in a semi-recumbent position on the scan couch and a longitudinal view of the fetus was obtained, using a 3.5-MHz scan head, so that the fetal head, upper body and arms could be visualised. The ultrasound scan head was moved as necessary to maintain this view throughout the procedure. The fetus was observed for 10 min to gauge activity level and the ultrasound picture and the procedure explained to the mother. In line with a previously used paradigm [21] the following procedure commenced after a 2-min period of fetal inactivity. The head of the vibroacoustic stimulator was placed on the maternal abdomen directly above the fetal head. A series of stimuli produced by the vibroacoustic stimulator of 2-s duration and 5-s inter-stimulus interval were presented to the fetus. This continued until no response was observed from the fetus on five consecutive stimulus presentations. The fetus was considered to have responded if it made an observable movement of the head, arms or upper body within 4.5 s of the onset of the stimulus. The number of trials to cease responding was documented (this does not include the five trials when no response was viewed). If the fetus had not ceased responding after 40 stimulus presentations the trial was ended and that fetus given a score of 40. Each mother was tested at 28, 32 and 36 weeks gestational age. Six mothers in the diabetic group delivered before the 36 week test and were thus not tested at this age. The number of trials to habituate (i.e., cease responding) was analysed by a 2 3 3 ANOVA for the within subject factor of gestational age (28, 32, 36) and between subject factor of group (fetuses of diabetic or non-diabetic mother). The number of trials to habituate were calculated by ND from replay of the tapes. To assess the accuracy of this, inter- and intra-observer reliability were examined using a randomly selected portion of 11 tapes. Inter-observer reliability was assessed using an individual blind to the study who independently assessed the 11 tapes. Intra-observer reliability was assessed by ND re-analysing 11 tapes, blind to their origin (diabetic / non-diabetic). Analysis was then undertaken using correlations to assess the degree of agreement which occurred between and within the observers assessing the number of trials to habituate.
3. Results Birth data pertaining to the fetuses studied are summarised in Table 1. There was a highly significant effect of group (F (1, 47) 5 19.65, P , 0.001);
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Table 1 Birth data for the diabetic and non-diabetic groups Birth measures
Diabetic
Non-diabetic
Significance
Weight (g) Gestational age (weeks) Apgar (1 min) Apgar (5 min)
3658.8 (6690.3) 37.6 (61.8)
3397.9 (6585.9) 39.8 (61.6)
8.2 (60.9) 9.3 (60.6)
8.1 (60.8) 9.0 (60.5)
ns t (45) 5 2 4.1, P , 0.01 ns ns
fetuses of diabetic mothers took longer to habituate, i.e., required more stimulations than fetuses of non-diabetic mothers (see Fig. 1). Post hoc tests revealed this difference to be significant (P , 0.05) at 28 and 32 weeks of gestation. There was also a significant effect of gestational age (F(2,94) 5 44.67, P , 0.0001); with advancing gestational age the number of trials to habituate decreased (see Fig. 1). The interaction between group and week was not significant. Although performance of the diabetic group was poorer at 36 weeks gestation this difference did not reach significance. However at this age only nine diabetics remained, six having delivered early. Comparison of the performance of those fetuses of diabetic pregnancies who delivered early and those who did not reveals no difference in their performance (number of trials to habituate) at 28 and 32 weeks of gestation. Although on both occasions those who delivered early took more trials to habituate than those undelivered at 36 weeks (28 weeks, 30 vs. 29 stimuli; 32 weeks, 28 vs. 24, respectively).
Fig. 1. The mean number of trials (6standard error) to habituate at 28, 32 and 36 weeks gestation for the diabetic and non-diabetic groups.
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3.1. Inter- and intra-observer reliability Inter- and intra-observer reliability were very high. Correlations on the number of trials to habituate was calculated at 0.98 for inter-observer, 0.99 for intra-observer reliability.
3.2. Blood glucose and habituation performance In order to examine the relationship between random blood glucose levels at the time of the habituation task and habituation performance a series of bivariate correlations were carried out. These examined random blood glucose levels at 28, 32 and 36 weeks gestation and the number of trials taken to habituate at each of these ages. Mean blood glucose for diabetic group at each of these three ages was, 6.45, 7.21 and 5.01, respectively. For the non-diabetic group, blood glucose at each of the three test times was 5.08, 5.06 and 4.91, respectively. All random blood glucose levels (and HbA 1c levels reported earlier for the diabetic group) were within the normal range for the Hospital. More importantly, there were no significant correlations between random blood glucose at time of testing (28, 32 and 36 weeks gestational age) and habituation performance (number of trials to habituate).
4. Discussion Overall the diabetic fetuses show poorer habituation performance when compared with the fetuses of non-diabetic pregnancies. The fetuses in the diabetic group required a significantly greater number of stimulus presentations than the non-diabetic group to habituate. Previous studies of fetal habituation (for a review, see Ref. [10]) have demonstrated its sensitivity to detect changes in the functioning of the fetal CNS. More generally this study adds to the growing literature indicating the value of observing the behaviour of the fetus to assess CNS functioning. For both groups fewer stimulus presentations were required to habituate as gestation advanced. This supports previous studies that the number of trials to habituate decreases with advancing gestational age [12]. Since habituation reflects the performance of the central nervous system [12], the observed difference in habituation abilities between the diabetic and non-diabetic groups suggests a difference in the functioning of their central nervous system. This difference may reflect delayed maturation of the CNS. The fetal CNS immaturity is in line with previous work on spontaneous behaviour in diabetic pregnancy [8–11]. The initial exhibition of the spontaneous movements of fetuses in diabetic pregnancies has been reported to be delayed by 1–2 weeks compared to fetuses of non-diabetic pregnancies [8]. The similar performance of both groups at 36 weeks
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gestational age would support this view in that by this stage the diabetic fetuses may have ‘caught up’. An alternative explanation is that the difference reflects a functional, as opposed to a maturational difference between the CNS of fetuses of diabetic and non-diabetic pregnancies. The reason for the ‘similarity’ of performance at 36 weeks of gestation is that the most affected fetuses have been delivered early. It may be that the healthier, least affected fetuses were able to maintain their pregnancies longer and thus their performance was more similar to that of the control, non-diabetic group. Offspring of diabetic mothers may not experience uniform, potentially adverse, effects of their altered prenatal environment (cf. offspring of mothers who consume alcohol and despite similar exposure may exhibit quite different outcomes). Some offspring may be more severely affected than others and these deliver early. Although not significantly different and acknowledging the small sample size (n 5 15) there was a suggestion that those fetuses who delivered early took longer to habituate which may support a direct effect on the functioning of the CNS. The difference in habituation ability of fetuses from diabetic and non-diabetic pregnancies is pointed. However there is difficulty in isolating the cause of this. There was no relationship between the random blood glucose at the time of the habituation test and the number of trials required to habituate. Blood glucose levels therefore do not reflect habituation performance and suggest that blood glucose levels at time of testing are not responsible for the altered habituation performance. This supports observations in previous studies where the functional differences observed between fetuses of diabetic and non-diabetic pregnancies were not related to blood glucose levels (e.g., Ref. [11]). Maternal diabetes affects not only blood glucose but also the hormonal milieu experienced by the fetus. A wide variety of other factors are also different. Whilst blood glucose control has been the subject of most study and significance in reducing mortality and gross morbidity other factors may exert a more subtle, teratogenic effect on the fetus’s CNS [5]. Insulin’s role as teratogen has received renewed support in recent papers [10,21]. It is difficult at this stage to posit what the long-term effects of this difference in prenatal habituation performance may be. Authors are united in the belief that an intact neural system is required for the normal habituation pattern [6,12] and deviance’s in habituation performance reflect abnormalities in the central nervous system functioning. The obvious question is the relationship between prenatal habituation performance observed here and the postnatal deficits in intellectual and behavioural performance after birth in childhood in other studies [1–4]. Since both reflect the integrity functioning of the CNS it is tempting to speculate on a link between the two. Further studies are underway to examine this and to test the possibility of using habituation as an early diagnostic tool in diabetic pregnancies. In summary, fetuses of diabetic pregnancies were found to exhibit poorer habituation performance than fetuses of non-diabetic fetuses. This suggests a teratogenic effect of maternal diabetes on the fetus’s CNS and perhaps provides the earliest indicator of postnatal CNS problems reflected in intellectual, educational and behavioural problems exhibited by children of diabetic mothers.
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Acknowledgements We wish to acknowledge the support of The Wellcome Trust and the Perinatal Research Trust whose funds made this research possible. We thank Dr. J.C. Dornan, Professor K. Brown and Professor W. Thompson for providing facilities for this research. Thanks also are due to Professor D. Hadden, Dr. A. Traub and the staff and patients at the Royal Maternity Hospital, Belfast.
References [1] Rizzo T, Boyd-Metzger E, Burns J, Burns K. Correlations between antepartum maternal metabolism and intelligence of offspring. New Engl J Med 1991;325:911–6. [2] Rizzo TA, Dooley S, Metzger B, Cho N, Ogata E, Silverman B. Prenatal and perinatal influences on long-term psychomotor development in offspring of diabetic mothers. Am J Obstet Gynecol 1995;173:1753–8. [3] Rizzo TA, Silverman BL, Metzger BE, Cho NH. Behavioral adjustment in children of diabetic mothers. Acta Paediatr 1997;86:969–74. [4] Yamashita Y, Kawano Y, Kuriya N, Murakami Y, Matsuishi T, Yoshimatsu K, Kato H. Intellectual development of offspring of diabetic mothers. Acta Paediatr 1996;85:1192–6. [5] Freinkel N. Banting Lecture 1980: Of pregnancy and progeny. Diabetes 1980;29:1023–35. [6] Hepper PG. The behaviour of the foetus as an indicator of neural functioning. In: Lecanuet J-P, Fifer W, Krasnegor N, Smotherman W, editors, Fetal development. A psychobiological perspective, Hillsdale, NJ: Lawrence Erlbaum, 1995, pp. 405–17. [7] Mulder EJH, Visser GHA, Bekedam DJ, Prechtl HFR. Emergence of behavioural states in fetuses of type-1 diabetic women. Early Hum Dev 1987;15:231–51. [8] Mulder EJH, Visser GHA. Growth and motor development in fetuses of women with type-1 diabetes. II. Emergence of specific movement patterns. Early Hum Dev 1991;25:107–15. [9] Mulder EJH, Visser GHA, Morssink P, de Vries JIP. Growth and motor development in fetuses of women with type-1 diabetes. III. First trimester quality of fetal movement patterns. Early Hum Dev 1991;25:117–33. [10] Kainer F, Prechtl HFR, Engele H, Einspieler C. Assessment of the quality of general movements in fetuses and infants of women with type-1 diabetes mellitus. Early Hum Dev 1997;50:13–25. [11] Allen CL, Kisilevsky BS. Fetal behavior in diabetic and non-diabetic women: an exploratory study. Dev Psychobiol 1999;35:69–81. [12] Hepper PG, Leader LR. Fetal habituation. Fetal Maternal Med Rev 1996;8:109–23. [13] Thompson RF, Spencer WA. Habituation: a model for the study of neuronal substrates of behavior. Psychol Rev 1966;73:16–43. [14] Gandhavadi B, Melvin JL. Electrical blink reflex habituation in mentally retarded adults. J Ment Deficiency Res 1985;29:49–54. [15] Hutt SJ, Hutt C, Lee D, Dunstead C. A behavioural and electroencephalographic study of autistic children. J Psychiatr Res 1965;3:181–97. [16] Leader LR, Baillie P, Martin B, Molteno C, Wynchank S. Fetal responses to vibrotactile stimulation, a possible predictor of fetal and neonatal outcome. Aust New Zealand J Obstet Gynaecol 1984;24:251– 6. [17] Park MI, Kim DS. The acoustic stimulation test in the anencephalus: preliminary results. J Perinatol Med 1989;17:329–31. [18] Leader LR, Baillie P. The changes in fetal habituation patterns due to a decrease in inspired maternal oxygen. Br J Obstet Gynecol 1988;95:664–8. [19] Leader LR. The effects of cigarette smoking and maternal hypoxia on fetal habituation. In: Maeda K, editor, The fetus as a patient, Amsterdam: Elsevier, 1987, pp. 83–8.
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[20] Leader LR, Bennett MJ. Fetal habituation and its clinical applications. In: Levene MI, Lilford RJ, Bennett MJ, Punt J, editors, Fetal and neonatal neurology and neurosurgery, 2nd ed, Edinburgh: Churchill Livingstone, 1995, pp. 45–60. [21] Hepper PG, Shahidullah BS. Habituation in normal and Down’s Syndrome fetuses. Q J Exp Psychol 1992;44:305–17.
Habituation in fetuses of diabetic mothers N. Nicola Doherty PhD, Peter G. Hepper PhD* Wellcome Trust Fetal Behaviour Research Centre, School of Psychology, The Queen’ s University of Belfast, Belfast BT7 1 NN, N. Ireland, UK Received 5 August 1999; received in revised form 30 May 2000; accepted 1 June 2000
Abstract Fetuses of diabetic mothers exhibit maturational delays in their behaviour and disturbances in behavioural and intellectual functioning in childhood. This suggests an effect of maternal diabetes on the central nervous system of the fetus. The habituation technique enables the functioning of the higher central nervous system to be examined. A normal habituation pattern reflects an intact central nervous system. Previous studies have found abnormalities in the fetal central nervous system are reflected in habituation performance. This paper examined the habituation ability of fetuses of diabetic mothers and of non-diabetic mothers. The fetuses were tested at 28, 32 and 36 weeks of gestation. After 2 min of fetal inactivity a series of vibroacoustic stimuli were presented to the fetus. This continued until no response was observed on five consecutive stimulus presentations. The number of stimulus presentations to habituate at each gestational age was recorded. The results reveal that there was a highly significant main effect of group (F(1,47) 5 19.65, P , 0.001). Fetuses of diabetic pregnancies took longer to habituate. There was a significant effect of gestational age (F(2,94) 5 44.67, P , 0.0001). In both groups the number of trials to habituate decreased with advancing gestation. There was no relationship between random blood glucose levels and habituation performance. The results demonstrate that maternal diabetes affects higher aspects of central nervous system functioning in the fetus. 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Habituation; Fetuses; Maternal diabetes
*Corresponding author. 0378-3782 / 00 / $ – see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0378-3782( 00 )00089-X
86
N.N. Doherty, P.G. Hepper / Early Human Development 59 (2000) 85 – 93
1. Introduction Whilst the successful management of blood glucose levels during pregnancy has significantly reduced mortality and morbidity in the offspring of diabetic mothers, recent evidence suggests that the offspring of diabetic pregnancies exhibit disturbances in behavioural and intellectual functioning [1–4]. For example, the examination of cognitive functioning in the children of diabetic mothers has illustrated poorer performance when compared with the children of non-diabetic mothers [1]. It is possible that the poorer performance results from an effect of the altered maternal metabolic milieu on the fetal CNS [5]. The effect of maternal diabetes on the fetal CNS may be teratogenic. Observation of the behaviour of the fetus offers the opportunity to assess the functional development of the CNS [6]. Hence observation of the behaviour of the fetus may elucidate the effect of maternal diabetes on the fetal CNS. There have been few studies of behaviour in the fetuses of diabetic pregnancies. Although there are indications that maternal diabetes acts as a behavioural teratogen. The organisation of behavioural states is poorer in fetuses of diabetic mothers than non-diabetic mothers [7]. Fetal breathing movements appear earlier in fetuses of diabetic mothers than non-diabetic mothers [8]; their incidence is higher although the rate of breathing is slower [9]. All other movements show a 1–2-week delay in there emergence [8]. The development of movements, with the exception of startles, was the same for fetuses of diabetic and non-diabetic pregnancies [8]. The quality of spontaneous general movements of fetuses of diabetic pregnancies is reported as different from fetuses of non-diabetic pregnancies [10]. More recently differences were found in the fetal heart rate response to a vibroacoustic stimulus [11]. Fetuses of diabetic mothers exhibited a less mature response than fetuses of non-diabetic pregnancies. One further technique that can be used to assess CNS functioning prenatally is that of habituation [12]. Habituation provides a sensitive means to examine higher CNS functioning, and is defined as the decrement in response to repeated stimulation [13]. In the human fetus a sound stimulus has been reliably used to elicit habituation of fetal movement and heart rate [12]. It has been argued that the normal habituation pattern reflects the normal functioning of the brain, including the cortices. Indeed abnormalities in habituation have been noticed postnatally in cases of brain abnormalities (e.g., Refs. [14,15]). Fetus’s with CNS anomalies such as microcephaly [16] and anencephaly [16,17] exhibit deviant habituation performance. Similarly abnormal habituation patterns have been observed in fetuses whose mothers were experiencing hypoxic conditions [18], in fetuses of mothers who recently had a cigarette [19], in fetuses of mothers who were taking drugs [20] and in fetuses with chromosomal conditions such as Down’s syndrome [21]. Commonly, fetuses with CNS anomalies, or exposed to adverse conditions which may affect CNS functioning, take longer to habituate, or fail to habituate [12]. Habituation, not previously studied in diabetic pregnancies, is examined here to assess the presence or absence of an effect of maternal diabetes on the CNS of the developing fetus.
N.N. Doherty, P.G. Hepper / Early Human Development 59 (2000) 85 – 93
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2. Methodology
2.1. Subjects Forty-nine mothers in total took part in the study, 15 of whom were diabetic and 34 non-diabetic.
2.1.1. Diabetic mothers The mothers in the diabetic group were drawn from those attending the joint metabolic and obstetric clinic at the Royal Maternity Hospital, Belfast. These mothers were all previously diagnosed insulin-dependent diabetics and were treated by insulin injection. The women were White’s classes B and C and mean length of diabetes was 14.0 (68.0) years. Their diabetes was reported by the Consultant Endocrinologist to be well-controlled (mean HbA 1c levels between 5 and 8 mmol / l). Mean age of the mothers was 28.0 (65.0) years. All mothers had singleton pregnancies, for 40% this was their first pregnancy, 26.7% of the sample had experienced miscarriage. Of the mothers, 93.3% were married, 6.7% smoked and 6.7% consumed alcohol (the use of alcohol and nicotine was controlled for in all statistics).
2.1.2. Non-diabetic mothers The subjects in the non-diabetic group were drawn from women attending the regular antenatal clinics at the Royal Maternity Hospital, Belfast. Their average age was 26.9 (66.1) years. All mothers had singleton, healthy pregnancies. Miscarriage had been experienced by 41.2%, for 23.5% this was their first pregnancy and 75.9% were either married or living with their partners. Of the mothers, 41.2% smoked and 38.2% reported use of alcohol.
2.2. Apparatus The mothers’ random blood glucose was measured by the Hemocue B-Glucose Photometer (HemoCue, South Yorkshire, UK). The fetus was viewed using an Advanced Technical Laboratories (ATL) Ultramark 4plus ultrasound machine, or Dornier AI3200 ultrasound machine, with a 3.5-MHz curvilinear scan head. The sound stimulus employed was a Fetal Acoustic Stimulator, Model 146 from Corometrics Medical Systems. This hand-held vibroacoustic stimulator is self contained and battery operated. It emits sound at a frequency of 75 Hz610% with harmonics ranging from 20 to 90 Hz. When measured in air at 1 m from the stimulator the maximum recorded sound intensity was 82 dB. Presentation and timing of the stimulus for the habituation procedure was controlled by a BBC microcomputer. All scans were recorded for later analysis. The computer also superimposed a visual indication of stimulus presentation and a centisecond timer over the ultrasound picture which was recorded on the videotape over the ultrasound image.
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2.3. Procedure Ethical permission for the study was obtained from the Research Ethics Committee, Faculty of Medicine, Queen’s University. Prior to testing the women were asked about their smoking and drinking habits and previous 2 h activity. Fetal maturity, fetal measurements (BPD, AC, FL), fetal weight and position were recorded. Immediately before scanning commenced random blood glucose was taken prior to using the Hemocue B-Glucose Photometer. All mothers were scanned approximately 2 h after their last meal. The mothers lay in a semi-recumbent position on the scan couch and a longitudinal view of the fetus was obtained, using a 3.5-MHz scan head, so that the fetal head, upper body and arms could be visualised. The ultrasound scan head was moved as necessary to maintain this view throughout the procedure. The fetus was observed for 10 min to gauge activity level and the ultrasound picture and the procedure explained to the mother. In line with a previously used paradigm [21] the following procedure commenced after a 2-min period of fetal inactivity. The head of the vibroacoustic stimulator was placed on the maternal abdomen directly above the fetal head. A series of stimuli produced by the vibroacoustic stimulator of 2-s duration and 5-s inter-stimulus interval were presented to the fetus. This continued until no response was observed from the fetus on five consecutive stimulus presentations. The fetus was considered to have responded if it made an observable movement of the head, arms or upper body within 4.5 s of the onset of the stimulus. The number of trials to cease responding was documented (this does not include the five trials when no response was viewed). If the fetus had not ceased responding after 40 stimulus presentations the trial was ended and that fetus given a score of 40. Each mother was tested at 28, 32 and 36 weeks gestational age. Six mothers in the diabetic group delivered before the 36 week test and were thus not tested at this age. The number of trials to habituate (i.e., cease responding) was analysed by a 2 3 3 ANOVA for the within subject factor of gestational age (28, 32, 36) and between subject factor of group (fetuses of diabetic or non-diabetic mother). The number of trials to habituate were calculated by ND from replay of the tapes. To assess the accuracy of this, inter- and intra-observer reliability were examined using a randomly selected portion of 11 tapes. Inter-observer reliability was assessed using an individual blind to the study who independently assessed the 11 tapes. Intra-observer reliability was assessed by ND re-analysing 11 tapes, blind to their origin (diabetic / non-diabetic). Analysis was then undertaken using correlations to assess the degree of agreement which occurred between and within the observers assessing the number of trials to habituate.
3. Results Birth data pertaining to the fetuses studied are summarised in Table 1. There was a highly significant effect of group (F (1, 47) 5 19.65, P , 0.001);
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Table 1 Birth data for the diabetic and non-diabetic groups Birth measures
Diabetic
Non-diabetic
Significance
Weight (g) Gestational age (weeks) Apgar (1 min) Apgar (5 min)
3658.8 (6690.3) 37.6 (61.8)
3397.9 (6585.9) 39.8 (61.6)
8.2 (60.9) 9.3 (60.6)
8.1 (60.8) 9.0 (60.5)
ns t (45) 5 2 4.1, P , 0.01 ns ns
fetuses of diabetic mothers took longer to habituate, i.e., required more stimulations than fetuses of non-diabetic mothers (see Fig. 1). Post hoc tests revealed this difference to be significant (P , 0.05) at 28 and 32 weeks of gestation. There was also a significant effect of gestational age (F(2,94) 5 44.67, P , 0.0001); with advancing gestational age the number of trials to habituate decreased (see Fig. 1). The interaction between group and week was not significant. Although performance of the diabetic group was poorer at 36 weeks gestation this difference did not reach significance. However at this age only nine diabetics remained, six having delivered early. Comparison of the performance of those fetuses of diabetic pregnancies who delivered early and those who did not reveals no difference in their performance (number of trials to habituate) at 28 and 32 weeks of gestation. Although on both occasions those who delivered early took more trials to habituate than those undelivered at 36 weeks (28 weeks, 30 vs. 29 stimuli; 32 weeks, 28 vs. 24, respectively).
Fig. 1. The mean number of trials (6standard error) to habituate at 28, 32 and 36 weeks gestation for the diabetic and non-diabetic groups.
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3.1. Inter- and intra-observer reliability Inter- and intra-observer reliability were very high. Correlations on the number of trials to habituate was calculated at 0.98 for inter-observer, 0.99 for intra-observer reliability.
3.2. Blood glucose and habituation performance In order to examine the relationship between random blood glucose levels at the time of the habituation task and habituation performance a series of bivariate correlations were carried out. These examined random blood glucose levels at 28, 32 and 36 weeks gestation and the number of trials taken to habituate at each of these ages. Mean blood glucose for diabetic group at each of these three ages was, 6.45, 7.21 and 5.01, respectively. For the non-diabetic group, blood glucose at each of the three test times was 5.08, 5.06 and 4.91, respectively. All random blood glucose levels (and HbA 1c levels reported earlier for the diabetic group) were within the normal range for the Hospital. More importantly, there were no significant correlations between random blood glucose at time of testing (28, 32 and 36 weeks gestational age) and habituation performance (number of trials to habituate).
4. Discussion Overall the diabetic fetuses show poorer habituation performance when compared with the fetuses of non-diabetic pregnancies. The fetuses in the diabetic group required a significantly greater number of stimulus presentations than the non-diabetic group to habituate. Previous studies of fetal habituation (for a review, see Ref. [10]) have demonstrated its sensitivity to detect changes in the functioning of the fetal CNS. More generally this study adds to the growing literature indicating the value of observing the behaviour of the fetus to assess CNS functioning. For both groups fewer stimulus presentations were required to habituate as gestation advanced. This supports previous studies that the number of trials to habituate decreases with advancing gestational age [12]. Since habituation reflects the performance of the central nervous system [12], the observed difference in habituation abilities between the diabetic and non-diabetic groups suggests a difference in the functioning of their central nervous system. This difference may reflect delayed maturation of the CNS. The fetal CNS immaturity is in line with previous work on spontaneous behaviour in diabetic pregnancy [8–11]. The initial exhibition of the spontaneous movements of fetuses in diabetic pregnancies has been reported to be delayed by 1–2 weeks compared to fetuses of non-diabetic pregnancies [8]. The similar performance of both groups at 36 weeks
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gestational age would support this view in that by this stage the diabetic fetuses may have ‘caught up’. An alternative explanation is that the difference reflects a functional, as opposed to a maturational difference between the CNS of fetuses of diabetic and non-diabetic pregnancies. The reason for the ‘similarity’ of performance at 36 weeks of gestation is that the most affected fetuses have been delivered early. It may be that the healthier, least affected fetuses were able to maintain their pregnancies longer and thus their performance was more similar to that of the control, non-diabetic group. Offspring of diabetic mothers may not experience uniform, potentially adverse, effects of their altered prenatal environment (cf. offspring of mothers who consume alcohol and despite similar exposure may exhibit quite different outcomes). Some offspring may be more severely affected than others and these deliver early. Although not significantly different and acknowledging the small sample size (n 5 15) there was a suggestion that those fetuses who delivered early took longer to habituate which may support a direct effect on the functioning of the CNS. The difference in habituation ability of fetuses from diabetic and non-diabetic pregnancies is pointed. However there is difficulty in isolating the cause of this. There was no relationship between the random blood glucose at the time of the habituation test and the number of trials required to habituate. Blood glucose levels therefore do not reflect habituation performance and suggest that blood glucose levels at time of testing are not responsible for the altered habituation performance. This supports observations in previous studies where the functional differences observed between fetuses of diabetic and non-diabetic pregnancies were not related to blood glucose levels (e.g., Ref. [11]). Maternal diabetes affects not only blood glucose but also the hormonal milieu experienced by the fetus. A wide variety of other factors are also different. Whilst blood glucose control has been the subject of most study and significance in reducing mortality and gross morbidity other factors may exert a more subtle, teratogenic effect on the fetus’s CNS [5]. Insulin’s role as teratogen has received renewed support in recent papers [10,21]. It is difficult at this stage to posit what the long-term effects of this difference in prenatal habituation performance may be. Authors are united in the belief that an intact neural system is required for the normal habituation pattern [6,12] and deviance’s in habituation performance reflect abnormalities in the central nervous system functioning. The obvious question is the relationship between prenatal habituation performance observed here and the postnatal deficits in intellectual and behavioural performance after birth in childhood in other studies [1–4]. Since both reflect the integrity functioning of the CNS it is tempting to speculate on a link between the two. Further studies are underway to examine this and to test the possibility of using habituation as an early diagnostic tool in diabetic pregnancies. In summary, fetuses of diabetic pregnancies were found to exhibit poorer habituation performance than fetuses of non-diabetic fetuses. This suggests a teratogenic effect of maternal diabetes on the fetus’s CNS and perhaps provides the earliest indicator of postnatal CNS problems reflected in intellectual, educational and behavioural problems exhibited by children of diabetic mothers.
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Acknowledgements We wish to acknowledge the support of The Wellcome Trust and the Perinatal Research Trust whose funds made this research possible. We thank Dr. J.C. Dornan, Professor K. Brown and Professor W. Thompson for providing facilities for this research. Thanks also are due to Professor D. Hadden, Dr. A. Traub and the staff and patients at the Royal Maternity Hospital, Belfast.
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