Journal of Physiology (1992), 445, pp. 713-723 713With 5 figuresPrinted in Great Britain

FETAL BREATHING, SLEEP STATE AND CARDIOVASCULARADAPTATIONS TO ANAEMIA IN SHEEP

BY KAZUHIRO MATSUDA, CHARLES DUCSAY AND BRIAN J. KOOSFrom the Division of Perinatal Biology, Departments of Physiology and Obstetrics

and Gynecology, School of Medicine, Loma Linda University, Loma Linda,CA 92324, USA

(Received 3 April 1991)

SUMMARY

1. In unanaesthetized fetal sheep (> 0-8 term) prolonged anaemia initiallyreduced the incidences of low-voltage electrocortical activity, rapid eye movementsand breathing activity; but the incidence of each returned to normal within 4-7 h.

2. Anaemia induced a persistent rise in fetal heart rate and plasma concentrationsof adrenaline, noradrenaline and cortisol.

3. After 16 h the fetal haematocrit was returned to normal. Isocapnic hypoxiainduced < 1 h later also inhibited eye and breathing activity.

4. After 1 h fetal arterial Po, (P, 02) was returned to normal. This rise in 02 tensionwas associated with an elevation in the incidence of low-voltage electrocorticalactivity, eye movements and breathing. Breathing movements also occurred duringhigh-voltage electrocortical activity.

5. It is concluded that the brain Po2 set-point for hypoxic inhibition adapts rapidlyto alterations in 02-carrying capacity and is probably due to changes in theconcentration and/or receptor affinity of a central neuromodulator. Secondly, a risein brain Po2 at birth may contribute to the onset of continuous breathing.

INTRODUCTION

Fetal breathing movements normally occur in episodes and are present about30-50% of the time. In sheep (> 0-8 term) this breathing activity is associated withrapid eye movements and low-voltage electrocortical activity, a behavioural statesimilar to phasic rapid eye movement sleep after birth (Dawes, Fox, Leduc, Liggins& Richards, 1972). Although hypoxia normally stimulates respiration after birth,acute reductions of arterial 02 tensions in the fetus inhibit fetal breathing (Boddy,Dawes, Fisher, Pinter & Robinson, 1974), presumably as the result of lowered 02tensions in the brain stem (Dawes, Gardner, Johnson & Walker, 1983; Koos,Matsuda & Power, 1990).

If the hypoxic stimulus is extended for several hours, adaptation occurs and theincidence of fetal breathing returns to normal, provided tissue hypoxia is not toosevere (Bissonnette & Hohimer, 1987; Koos, Kitanaka, Matsuda, Gilbert & Longo,1988). Little is known regarding the mechanism(s) of the return of breathing duringMS 9272

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K. MATSUDA, C. DUCSAY AND B. J. KOOS

sustained 02 deprivation, but several factors could contribute to this interestingphenomenon. For example, the progressive acidaemia associated with hypoxia mightinduce breathing by exciting the central chemoreceptors. Brain Po2 might also riseover time, despite low arterial P02 (Pal 02)' as a result of increase in brain blood flow,or by an induction of an 02 carrier which would facilitate 02 diffusion within braincells.

If brain Po0 remains low, the return of fetal breathing could be due to changes inthe activity of an 02-sensitive neuromodulator, and such a mechanism would beexpected to adjust relatively quickly to changes in central Po2. Therefore, this studywas carried out to determine whether fetal breathing adapts rapidly to anaemia-induced 02 deficiency.

METHODS

Seven pregnant sheep were operated on at 121-127 days gestation (- 0-8 term). Under halothaneanaesthesia, catheters were placed in a brachial artery, carotid artery, external jugular vein,and trachea of the fetus and in the amniotic sac. Bipolar stainless-steel electrodes were implantedon a lateral and medial orbital ridge to record eye movements and electrocortical activity (Daweset al. 1983). Catheters were also inserted into a femoral artery and vein of the ewe. Trachealpressure, arterial pressure, heart rate, electrocorticogram and electrooculogram were recorded onan eight-channel Gould polygraph. All pressures were measured with Cobe pressure transducers(Lakewood, CO, USA) and were referenced to amniotic fluid pressure. Fetal heart rate and bloodpressure were analysed on-line using an IBM-AT computer. The signals were sampled every 0-1 s,and minute averages were stored on disc.Blood gas tensions and pH were determined with Radiometer electrodes (ABL-2). Blood 02

content was calculated from haemoglobin concentration and 02 saturation (OSM Hemoximeter,Radiometer, Copenhagen). Haematocrit of arterial blood was measured in triplicate usingmicrohaematocrit techniques (Brace, 1983).

Experiments were started on the fourth day after surgery. Fetal isocapnic hypoxaemia wasinduced for t h by having the ewe breathe for t h a hypoxic gas mixture (9% 02, 3% C02, 88%N2) from a plastic bag as previously described (Koos, Sameshima & Power, 1987 a). Thehypoxaemia was initiated at the start of a breathing episode. This procedure determined whetherhypoxaemia inhibited breathing and eye movements.At 14.00-15.00 h on the fifth postsurgical day, fetal anaemia was induced by infusing maternal

plasma into the jugular vein while simultaneously withdrawing fetal blood at the same rate fromthe carotid artery. At 09.00-10.00 h on the following day, the fetal haematocrit was returned tonormal by infusing packed fetal red cells in exchange for whole blood. After correcting the anaemia,breathing and eye movement responses to hypoxaemia were again tested by having the ewebreathe the hypoxic gas mixture for a second time using the same protocol.

Because of the circadian variation in fetal breathing activity (Boddy, Dawes & Robinson, 1973),the incidences of fetal breathing, eye movements and low-voltage electrocortical activity were alsodetermined on a day before or after anaemia was induced. These control observations were madeat the same time of day as the anaemia studies.

Preductal arterial blood was collected in syringes containing EDTA and glutathione. The bloodsamples were cooled immediately on ice and centrifuged at 4 'C. The separated plasma was storedat -70 "C until analysed.Plasma noradrenaline and adrenaline concentrations were measured by HPLC using a modified

Hjemdahl (Hjemdahl, Daleskog & Kahan, 1979) technique. Dihydroxybenxylamine was added tothe plasma as an internal standard. Catecholamines were concentrated by alumina extraction, anddesorption from the alumina was performed by 0-1 M-acetic acid. Catecholamine analysis wascarried out using a Waters 460 electrochemical detector and pump system with a Resolve C18column (Waters, Milford, MA, USA). The intra-assay coefficient of variation was less than 7% foreach catecholamine, and the method had a sensitivity of 10-15 pg.Plasma cortisol was measured using an antiserum (a gift of J. Challis) which has been

characterized previously (Challis, Patrick, Cross, Workewych, Manchester & Power, 1981). The

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ADAPTATIONS TO FETAL ANAEMIA

intra-assay and inter-assay coefficients of variation were 7 and 13 %, respectively. The sensitivityof the assay was 02 ng/ml.The incidences of breathing, eye movements and low-voltage electrocortical activity during

anaemia were compared to the 4 h averages recorded before anaemia was induced. The incidencesof breathing and rapid eye movements during hypoxic hypoxia after red cell volume was restoredwere compared to those during hypoxic hypoxia prior to the induction of anaemia. The data arepresented as means+ S.E.M. Significant differences (P < 005) were determined by two- and three-way analysis of variance with Duncan's test.

RESULTS

AnaemiaOne exchange transfusion was carried out in each of seven fetal sheep. The volume

of plasma infused was 262 +24 ml, and the average time for this exchange processwas 87 + 7 min. The exchange transfusion decreased the mean fetal arterialhaematocrit from 284+ 0-8 to 13-5 + 0-6% during the first hour of anaemia. This53% reduction in packed red cell volume persisted throughout the next 16 h.

After 16 h of anaemia, fetal red cell volume was restored by infusing packed fetalred cells in exchange for whole blood. This process took about 30 min. The bloodcollected during the exchange process was centrifuged, and the packed erythrocytes( 10 ml) were infused over 10 min. This procedure increased the fetal haematocritto 28-0+1 1/%.

Blood gases and pHThe mean brachial P, °2 decreased slightly (- 3 Torr) during the first 2 h of

anaemia, but significant reductions (> 4 Torr) were present after more than 6 h ofanaemia (Fig. 1). Mean arterial CO2 tensions were little affected, although a slight risein PalC2 ( 2 Torr) was present after 16 h of anaemia. Compared to control values,mean arterial pH was maximally reduced after 3-6 h of anaemia, but thereafter theaverage pH increased towards normal values.Compared with control mean, the average jugular venous Po2 was reduced

significantly by 2'8 Torr after 1 h of anaemia (Fig. 1). Venous Po2 (PV 02) decreasedfurther with time and was 4-9 Torr lower than control values after 16 h of anaemia;however, the average pH had returned to near normal values after 16 h of anaemia.Mean Pv co, was not significantly affected by anaemia.

Electrocortical activityDuring the first 3 h of anaemia, the average incidence of low-voltage electrocortical

activity was significantly less than the control value of 37+ 6-0 min/h (Fig. 2).Thereafter, the mean incidence returned to normal. Compared to the control mean(20+ I 0 min/h), the incidence of high-voltage electrocortical states was significantlygreater during the first (27 + 2-4 min/h) and third (28 ± 2-2 min/h) hours of anaemia.The average time for a cycle of low- and high-voltage electrocortical activity

during the control period was 30+1-7 min. During the first hour of anaemia, themean value fell significantly to 19 + 2-3 min and subsequently increased to26+2-7 min during the fifth hour of study.

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K. MATSUDA, C. DUCSAY AND B. J. KOOS

Eye movements

During the first hour of anaemia, the average incidence of rapid eye movementswas reduced by about 69 % compared to the control mean. Over the next 10 h theincidence of eye movements gradually returned to normal. The amplitude of eye

30

Haematocrit

(mi/dl)

pH, *

50 0 E

(Torr) 4

30

P02. .-

(Torr) 10Tm

L1' 1 1 ' ' ~~I I IJo-2 0 2 4 6 16

Time (h)

Fig. 1. Haematocrit, blood gases and pH for brachial arterial (@) and jugular venousblood (0). The shaded vertical bar shows the average time for exchange transfusion.Values are means+ S.E.M. *P < 005 compared to control mean.

movements was depressed during the first 4 h of anaemia, and thereafter theamplitude increased towards normal values over the subsequent 4-6 h.

Breathing movementsFigure 2 shows that the incidence of fetal breathing was reduced by about 70%

during the first 2 h of anaemia. As with eye movements, the incidence of breathingincreased over time and was similar to control values after 10 h of anaemia. Theamplitude of breathing was not affected by anaemia as judged by visual inspection.

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ADAPTATIONS TO FETAL ANAEMIA

Cardiovascular effectsMean arterial pressure averaged 42 + 2-6, 43 + 3-0, 41+241 and 38+19 mmHg

after 1, 3, 6 and 16 h of anaemia, respectively. These values were not significantlydifferent than the control mean of 40+ 1-9 mmHg. Mean fetal heart rate increased by13-22 % during the last 10 h of the experiment (Fig. 3).

45

Low-voltageactivity(min/h)

35 1

2540

Eyemovements

(mi n/h)

Breathing(min/h)

O L-

-6-. . . I . a

-4 -2 0 5 10 15

Time (h)Fig. 2. Effects of anaemia (thick lines) on the incidence of low-voltage electrocorticalactivity, rapid eye movements and breathing activity. The shaded vertical bar representsthe average time for exchange transfusion. The thin lines show mean values for the samefetuses on a day before or after anaemia study. Values are means + S.E.M. *P < 005compared to the 4 h control mean.

HormonesPlasma catecholamine concentrations also rose during anaemia (Fig. 3). Peak

levels occurred within 6 h after anaemia was established and were about threefoldgreater than the control mean for adrenaline and fivefold greater than the controlvalue for noradrenaline.Plasma cortisol concentrations averaged 27 + 3-9 ng/ml during the control period.

During anaemia, mean plasma concentrations rose significantly to 58+ 7-4 ng/ml at1 h, 63+ 5-9 ng/ml at 3 h, 72 + 10 ng/ml at 6 h and 60±+I ng/ml at 16 h.

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718 K. MATSUDA, C. DUCSAY AND B. J. KOOS

220

EE180-

I.-0140-

600 *'

Cj-300~

0 2000 dC~~~~~~~~~Es 1000- *

-00

r- r- r r-2 0 2 4 6 16

Time (h)Fig. 3. Effects of anaemia on fetal heart rate and plasma catecholamine concentrations.

*P < 005 compared to control value.

TABLE 1. Brachial arterial blood gases and pH with hypoxaemiaBefore After

n anaemia anaemia

pHControl 7 7-316 + 0-014 7-327 + 0022Hypoxia 7 7 314+ 0014 7 306+ 0017

PCO,(Torr)Control 7 4430+ 12 45 1+16Hypoxia 7 42-0+1-2 44-6+P12

P0,(Torr) Control 7 26-1+1 1 22-8+1-2Hypoxia 7 16-3+1.1* 14-4+1-4*

*P < 005 compared to control value. n, number of fetuses.

HypoxaemiaFetal breathing and eye movement responses to hypoxia were tested twice: before

lowering fetal haematocrit and immediately after restoring fetal 02-carryingcapacity. Acute isocapnic hypoxaemia was induced for 1 h by having the ewe breathea hypoxic gas mixture, which reduced fetal Pa 02 by about 10 Torr (Table 1). Thisacute hypoxaemia inhibited fetal eye movements and breathing activity in a similarmanner, whether the hypoxaemia was induced before anaemia or less than 1 h afterthe haematocrit was restored to normal (Fig. 4).

Table 2 shows catecholamine and cortisol concentrations after 60 min of hypoxia.Hypoxia significantly increased fetal plasma concentrations of noradrenaline,adrenaline and cortisol when the experiments were performed on a day before theinduction of anaemia. When hypoxia was carried out within 1 h of restoring O2-

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ADAPTATIONS TO FETAL ANAEMIA 719

60Low-

voltage 50 ~ TT 1activity40 ...I...~~~~~~~~~~~~......

(min/h) 30 L<

movements 30 F :...I I(mmn/h) 20F

60-

50~ ~ ~ ,

Breathing *

(mm1/h) 4030 f20-

-4 -2 0 2 4 6

Time (h)

Fig. 4. Effects of hypoxia (shaded vertical bar) on low-voltage electrocortical activity, eyemovements and breathing. The thin lines show values when hypoxia was induced at aboutthe same time on a day before anaemia study. Incidences during anaemia (dotted lines),during restoration of fetal haematocrit (dashed lines), and following the return Of 02-carrying capacity (thick lines) are also shown. *P < 005 compared to mean values at thesame time after hypoxia before lowering fetal haematocrit (thin lines).

TABLE 2. Hormone concentrations during hypoxia

n Control HypoxiaBefore anaemia

Noradrenaline (pg/ml) 4 248+ 132 985+ 189*Adrenaline (pg/ml) 4 146+33 308+73*Cortisol (ng/ml) 4 23+2-5 76+ 12*

After anaemiaNoradrenaline (pg/ml) 6 559+ 122 1061+254Adrenaline (pg/ml) 5 217+60 407+ 113Cortisol (ng/ml) 6 40+8-1 88+9.9*

*P < 005 compared to control value. n, number of fetuses.

carrying capacity, the hormone concentrations reached values similar to thoseattained during hypoxia before anaemia. Because baseline hormone levels wereraised, a significant increase in this case was observed only for cortisol.

RecoveryThe incidences of low-voltage electrocortical activity, eye movements and

breathing were significantly greater during the recovery phase after fetal haematocritand Pa 02 had been returned to normal (Fig. 4). This rebound effect was also unusual

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K. MATSUDA, C. DUCSAY AND B. J. KOOS

in that breathing movements occurred at times during high-voltage electrocorticalactivity (Fig. 5) in all seven fetuses studied. The mean lag time for the onset ofbreathing during high-voltage states was 2 + 0-7 h (range: 0-2-4-8 h) after thetermination of fetal hypoxia. During the 16 h after fetal Pa °2 was restored to normal,

Before anaemia After anaemia

amIinIhgsaMWI.I-O 4000

w

t 400[0w

E -10[E

0..,

10 minFig. 5. Fetal electrocorticogram (ECoG), electrooculogram (EOG) and tracheal pressure(P,) recordings during the control period before anaemia and after fetal haematocrit wasrestored to normal.

fetal breathing during high-voltage activity totalled 44+14 min, compared to nilduring 16 h of the control period before anaemia was induced (P < 0 05). Breathingduring high-voltage states also did not occur following the first hypoxia study.

DISCUSSION

The fetus responds to hypoxia in ways which are generally thought to enhancefetal survival. An example is hypoxic inhibition of breathing (Boddy et al. 1974) andother muscular activity (Natale, Clewlow & Dawes, 1981) which is associated with afall in fetal 02 consumption of up to 20% (Rurak & Gruber, 1983; Rurak, Cooper &Taylor, 1986). Along with the redistribution of blood flow (Cohn, Sacks, Heymann& Rudolph, 1974), this reduction in fetal 02 consumption helps increase 02availability to critical organs such as the brain and heart during periods of 02deprivation. Therefore, the return of breathing movements is somewhat surprising inview of the persisting reduction in 02 availability to fetal tissues and of the assumedcontinuing need for minimizing fetal 02 consumption.The present study provides new information regarding the adaptation of breathing

activity to 02 deficiency. First, jugular venous Po2 remains depressed at a time whenthe incidence of breathing movements rises towards normal values. In short-term

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ADAPTATIONS TO FETAL ANAEMIA

studies, the incidence of fetal breathing during anaemia or hypoxia is directly relatedto brain end-capillary Po2, as calculated using jugular venous Po0 and a correction forred cell spacing in capillaries (Koos et al. 1990). Because jugular venous PO2 andhaematocrit remained low, the restoration of breathing during prolonged anaemia isnot likely due to an increase in brain end-capillary (and consequently brain) Po2, suchas might occur with a progressive increase in brain blood flow. Second, breathingadaptation to anaemia appears to be short-lived once fetal 02-carrying capacity isrestored. Although 4-7 h are required for breathing to return once anaemia isestablished, this resistance to 02 deficiency is lost less than 1 h after the haematocritis returned to normal values.The time course for these breathing responses indicates that the Po2 set-point for

hypoxic inhibition quickly adjusts once tissue 02 tensions are altered. This responsewould appear to be too rapid to be accounted for by changes in a hypotheticalintracellular 02 carrier. Metabolic responses and receptor affinity can change overthis time, and it is likely that hypoxic adaptation involves alterations inconcentrations and/or receptor affinity of a central neuromodulator. Adenosine is anobvious candidate because it inhibits fetal breathing (Koos & Matsuda, 1990) and itsconcentration in tissues is determined by metabolic rate and 02 availability(Bardenheuer & Schrader, 1986).

In sheep (> 0X8 term), fetal breathing movements normally do not occur duringhigh-voltage electrocortical activity. Therefore, an unexpected finding was thepresence of fetal breathing during high-voltage states after fetal 02-carrying capacityand Pa °2 were restored. It cannot be determined from these studies whether thisbreathing response was induced by anaemia alone or whether hypoxaemia is alsorequired. Because both forms of 02 deficiency reduce calculated brain end-capillary02 tensions (Koos et al. 1990), it is reasonable to assume that the response is relatedto the rise in fetal brain Po2 following prolonged 02 deprivation. Such a mechanismhas important physiological significance because it could contribute to the onset ofcontinuous breathing at birth, when newborn P" 02 rises with the first breath.As with fetal breathing, rapid eye movements normally occur in episodes during

low-voltage electrocortical activity, and they are inhibited by hypoxia in a dose-dependent manner (Koos et al. 1987 a). In the present study with anaemia, the timecourse of adaptation for eye movements was virtually the same as that for breathingactivity. Thus, low 02 tensions probably depress breathing and eye activity througha similar mechanism.Hypoxia (A fetal Pa ° 10 Torr) has been reported to decrease the incidence of

low-voltage electrocortical activity (Boddy et al. 1974; Bocking, Harding &Wickham, 1986; Martin, Voermans & Jongsma, 1987). However, this effect has notbeen found by other investigators when 02 deficiency has been induced by loweringmaternal P. 0, (Adamson, Patrick & Challis, 1984; Koos et al. 1987 a) or by inducingfetal anaemia (Bissonnette & Hohimer, 1987; Koos, Sameshima & Power, 1987 b),methaemoglobinaemia (Koos et al. 1990) or carboxyhaemoglobinaemia (Koos,Matsuda & Power, 1988). In the present study, the incidence of low-voltageelectrocortical activity was significantly reduced during the first 3 h of anaemia butwas normal for the rest of the study.

Short-term fetal anaemia (Koos et al. 1987 b) and methaemoglobinaemia (Koos et

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K. MATSUDA, C DUCSA Y AND B. J. KOOS

al. 1990) are associated with a rise in fetal heart rate. The present work withprolonged anaemia shows that this tachycardia is progressive and is associated withelevations in plasma concentrations of noradrenaline and adrenaline. As a result, therise in fetal heart rate is most likely due to an increase in plasma catecholamineconcentrations and probably sympathetic tone. No attenuation was seen in theseresponses over the 16 h of study.

In conclusion, fetal responses to prolonged anaemia vary. Some, such as elevationsin fetal heart rate, plasma catecholamines and cortisol, were maintained throughoutthe 16 h of study. Others, such as electrocortical activity, eye movements andbreathing activity, adapt to anaemia even though jugular venous (and probablybrain) Po2 remains low. The time course of these changes and the rapid recovery ofhypoxic inhibition following the restoration of fetal haematocrit are consistent withthe possibility that fetal adaptations to 02 deficiency result from alterations in theconcentration and/or receptor affinity of a central neuromodulator. Finally, theincrease in breathing incidence following prolonged 02 deprivation suggests that therise in brain PO at birth may contribute to the onset of continuous breathing.

We thank A. Van Varick for his technical assistance and S. Whitson, R. Tanen, J. Inamine andS. Allan for help in typing the manuscript. This study was supported in part by National Instituteof Child Health and Human Development grants HD-18478 and HD-22865 and by Basil O'ConnorStarter Grant 5-515 from the March of Dimes Birth Defects Foundation.

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BISSONNETTE, J. M. & HOHIMER, A. R. (1987). Acute anemic hypoxemia produces a transientdepression in fetal respiratory activity. Journal of Applied Physiology 63, 1942-1946.

BOCKING, A. D., HARDING, R. & WICKHAM, P. J. D. (1986). Effects of reduced uterine blood flowin electrocortical activity, breathing, and skeletal muscle activity in fetal sheep. AmericanJournal of Obstetrics and Gynecology 154, 655-662.

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BODDY, K., DAWES, G. S. & ROBINSON, J. (1973). A 24-hour rhythm in the foetus. In Foetal andNeonatal Physiology: Proceedings of the Sir Joseph Barcroft Centenary Symposium, ed. CROSS, K.WV., DAWES, G. S., COMLINE, R. & NATHANIELSZ, P., pp. 63-66. Cambridge University Press,Cambridge.

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HJEMDAHL, P., DALESKOG, M. & KAHAN, T. (1979). Determination of plasma catecholamines byhigh performance liquid chromatography with electrochemical detection. Comparison with aradioenzymatic method. Life Science 25, 131-136.

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Koos, B. J., SAMESHIMA, H. & POWER, G. G. (1987a). Fetal breathing, sleep state, andcardiovascular responses to graded hypoxia in sheep. Journal of Applied Physiology 62,1033-1039.

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MARTIN, C. B., VOERMANS, T. M. G. & JONGSMA, H. W. (1987). Effects of reducing uteroplacentalblood flow on movements and on electrocortical activity of fetal sheep. Gynecological andObstetric Investigation 23, 34-39.

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